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[Cancer Research 64, 659-664, January 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Predictive Impact of Urokinase-Type Plasminogen Activator

Plasminogen Activator Inhibitor Type-1 Complex on the Efficacy of Adjuvant Systemic Therapy in Primary Breast Cancer

Peggy Manders1,2, Vivianne C. G. Tjan-Heijnen2, Paul N. Span1, Nicolai Grebenchtchikov1, John A. Foekens3, Louk V. A. M. Beex2 and C. G. J. (Fred) Sweep1

1 Departments of Chemical Endocrinology, and
2 Medical Oncology University Medical Centre Nijmegen, Nijmegen, and
3 Department of Medical Oncology, Rotterdam Cancer Institute and University Hospital Rotterdam, Rotterdam, the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system. This system comprises of, among others, the urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-1). In this study we investigated whether the uPA:PAI-1 complex is associated with the responsiveness of patients with primary breast cancer to adjuvant systemic therapy.

Quantitative enzyme-linked immunosorbent assays were used to assess the levels of uPA, PAI-1, and uPA:PAI-1 complex in 1119 tumors of patients with primary invasive breast cancer. These patients were followed for a median follow-up time of 59 months (range, 2–267 months) after the primary diagnosis. Correlations with well-known clinicopathological factors, and univariate and multivariate survival analyses were performed.

High uPA:PAI-1 complex levels were correlated with an adverse histological grade, and inversely associated with negative estrogen and progesterone receptor status. High tumor levels of uPA:PAI-1 complex predicted an early relapse in the univariate relapse-free survival analysis (P < 0.001). The multivariate analysis showed that high uPA:PAI-1 complex levels were associated with a decreased relapse-free survival time (P = 0.033), independently of age, tumor size, number of lymph nodes affected, progesterone receptor status, uPA, adjuvant endocrine, and chemotherapy. More important, it was demonstrated that there is a larger benefit from adjuvant chemotherapy for patients with higher versus lower tumor levels of uPA:PAI-1 complex.

The results of this study imply that the expression of uPA:PAI-1 complex independently predicts the efficacy of adjuvant chemotherapy in patients with primary breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Different protease systems are involved in cancer progression, especially in invasion and the development of metastases. One of these systems is the plasminogen activation system in which the serine protease urokinase-type plasminogen activator (uPA) plays a central role (1, 2, 3) . Generally, this protease is released from producer cells as an inactive single-chain proenzyme, convertible to its active two-chain form by limited proteolysis (4 , 5) . uPA can be regarded as a multifunctional protein that is involved in both proteolysis and signal transduction. The activity of uPA can be neutralized by two main specific inhibitors, plasminogen activator inhibitor (PAI) type-1 and type-2, which form inactive complexes of 1:1 stoichiometry with the plasminogen activators (6 , 7) .

The markers uPA and PAI-1 can be used to separate patients into low-risk and high risk-groups in terms of the probability of recurrence, and to focus the treatment efforts on patients at high-risk. In this respect, uPA and PAI-1 have been shown to be of prognostic value (8, 9, 10, 11) . In addition, it is interesting to know whether the high-risk patients, as classified by these parameters of the plasminogen activation system, also benefit the most from adjuvant systemic therapy. In other words, whether these prognostic factors also are of predictive value with respect to the efficacy of adjuvant systemic therapy.

A recent study focused on the predictive value of uPA and PAI-1 in patients treated with neo-adjuvant chemotherapy (12) , and another had addressed the efficacy of systemic therapy in patients with advanced breast cancer (13) . The results of those studies may not be simply translated to the adjuvant setting. Harbeck et al. (14 , 15) performed two studies on the predictive value of uPA and PAI-1 for adjuvant treatment in patients with primary breast cancer, showing that patients with high tumor tissue levels of both uPA and PAI-1 benefited more strongly from conventional adjuvant systemic therapy, particularly from chemotherapy, than those with low levels (15) .

To our knowledge, there are no data available on the predictive value of the uPA:PAI-1 complex with regard to the efficacy of adjuvant systemic therapy in patients with primary breast cancer. The complex between uPA and PAI-1 selectively reflects the amount of uPA that has been activated from proenzyme uPA and subsequently inactivated by PAI-1. Only the active form of PAI-1 is able to form complexes with active uPA. The tumor levels of this uPA:PAI-1 complex can, thus, be considered as a special manifestation of the previously active form of both the activator uPA and the inhibitor PAI-1 (16) . Therefore, it can be hypothesized that the tumor levels of uPA:PAI-1 complex may provide valuable predictive information in patients with primary breast cancer. In the current study we focused on the clinical relevance of the complex in the soluble, nonmembrane bound fraction, because in an earlier study it was shown that the complex of tissue-type plasminogen activator:PAI-1 as measured in the soluble fraction has clinical relevance in terms of relapse-free survival (RFS) and overall survival (OS), whereas levels in pelleted (membrane) fractions were substantially less relevant (17) .

So, the primary goal of this present study was to assess retrospectively whether tumor levels of uPA:PAI-1 complex were related to the efficacy of adjuvant endocrine therapy and chemotherapy in patients with primary breast cancer. Furthermore, the secondary aim of the present study was to investigate whether the predictive value of the separate components, uPA and PAI-1, for the efficacy of adjuvant chemotherapy could be established. For this purpose, we included a series of 1119 breast cancer patients in this study, consisting of a group of patients treated with adjuvant systemic therapy and a group of patients not treated with adjuvant systemic therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
A total of 1119 patients with invasive primary breast cancer was included in this study. This group of patients underwent resection of their primary tumor between January 1987 and December 1996. The patients were without evidence of distant metastases at the time of diagnosis and had no evidence of disease within 1 month after primary surgery. The patients also had no previous diagnosis of carcinoma (with the exception of basal cell skin cancer) and had no bilateral breast cancer.

Patients underwent a modified radical mastectomy (n = 762) of whom 359 received complementary radiotherapy, or a breast conserving lumpectomy with axillary lymph node dissection (n = 357) plus complementary radiotherapy (n = 349). The median age was 58 years (range, 28–90 years). Additional characteristics of the patients are listed in Table 1Citation .


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  		Table 1 Correlations of uPA,a PAI-1, and uPA:PAI-1 complex levels in 1119 cytosols with patient and tumor characteristics

 
Adjuvant Systemic Treatment.
Two hundred and forty-nine patients were treated with adjuvant endocrine therapy alone. This therapy consisted of tamoxifen, 40 mg once daily for (at least) 2 years. In total, 100 patients received adjuvant chemotherapy only. Fifty-nine patients were premenopausal at diagnosis and remained premenopausal after the adjuvant chemotherapy. In 72 patients, adjuvant chemotherapy consisted of the classical cyclophosphamide, methotrexate, and 5-fluorouracil schedule for six cycles. Twenty-eight patients received adjuvant chemotherapy with five cycles of 5-fluorouracil, epirubicin/Adriamycin, and cyclophosphamide. There were also 83 patients who were treated with both endocrine therapy and chemotherapy. All of these patients were treated with the classical cyclophosphamide, methotrexate, and 5-fluorouracil schedule as adjuvant chemotherapy. Forty-two of these patients became postmenopausal caused by this adjuvant chemotherapy or subsequent ovarian ablation, which was considered to be an endocrine treatment. The other 41 patients received additional tamoxifen. The frequency of prescriptions of adjuvant endocrine therapy or chemotherapy in subgroups defined by lymph node status are listed in Table 2Citation .


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  		Table 2 Adjuvant endocrine therapy and chemotherapy by lymph node status

 
Clinical Follow-Up.
Patients were seen (history, physical examination, and routine laboratory investigations) once every 3 months during the first 2 years, once every 6 months for 5 years, and once a year thereafter. Once a year an X-ray mammography was made. The median follow-up time of patients was 59 months (range, 2–267 months). During follow-up, 324 patients (29%) had a recurrence (locoregional, distant metastases, or both), and 162 patients (15%) died due to breast cancer, whereas 41 died (4%) without evidence of disease at last follow-up. Contralateral breast cancer or second malignancies were not considered as recurrent disease.

Tumor Tissue Processing.
Tumor specimens were drawn from a pool of frozen specimens (stored in liquid nitrogen) originally submitted to our laboratory for steroid-hormone receptor analysis. Processing of the tumors was performed as described previously (18) .

Steroid Hormone Receptor and Total Protein Assays.
Estrogen receptor (ER) and progesterone receptor (PgR) levels were determined with ligand binding assay in soluble tissue fraction as described earlier (19) . The cutoff level used to classify tumors as ER or PgR positive or negative was 10 fmol/mg protein. The protein was measured according to Lowry et al. (20) with BSA as a standard.

Determination of the Antigen Levels of uPA, PAI-1, and uPA:PAI-1 Complex.
We used ELISAs developed by our Department of Chemical Endocrinology essentially as described earlier for uPA and PAI-1 (21) , and for the uPA:PAI-1 complex (22) . The uPA:PAI-1 complex assay was modified for the use of ß-galactosidase as a marker enzyme with fluorescent detection to increase the sensitivity of the assays. To this end, a goat antirabbit IgG Biotin conjugate (Sigma Chemical Co., St. Louis, MO) was used for detection antibody. After washing the plates were incubated (2 h at ambient temperature) with streptavidin-labeled ß-galactosidase (Roche Diagnostics, Almere, The Netherlands), followed by an incubation of 1 h with 4-methylumbelliferyl-ß-D-galactopyranoside substrate (Sigma Chemical Co.). The reaction was stopped by addition of 0.1 N NaHCO3/Na2CO3 (pH 10.5), and fluorescence was measured with a fluorometric plate reader (Fluoroskan; Lab Systems, Helsinki, Finland) using 355-nm excitation and 460-nm emission filters.

The analytical sensitivities, defined as the amount of uPA, PAI-1, or uPA:PAI-1 complex giving a signal in the corresponding ELISAs >1.96 times the SD above the blank values, were 9 pg/ml, 10 pg/ml, and 8 pg/ml for uPA, PAI-1, and uPA:PAI-1, respectively. The functional sensitivities for uPA, PAI-1, and uPA:PAI-1 were 11 pg/ml, 22 pg/ml, and 7 pg/ml, respectively.

To check between-assay variability and to monitor overall long-term performance of the assays a lyophilized quality control sample prepared from human breast cancer xenograft tissue (marked 101094 for uPA and PAI-1) and a cytosol from a pool of human breast tumor biopsies (for uPA:PAI-1 complex) were included in each plate (23) . The between-assay variation was found to be <12% for all three of the ELISAs. The within-assay variation of samples measured in duplicate was always <5%.

Data Analysis.
To analyze interrelations among uPA, PAI-1, and uPA:PAI-1 complex and various clinicopathological parameters, Spearman rank correlations (rs) were calculated for continuous variables and the Kruskal-Wallis test for ordered variables, followed by a Wilcoxon-type test for trend if appropriate. The traditional clinicopathological parameters consisted of age, tumor size, histological grade, number of lymph nodes involved, and ER and PgR status.

Cox univariate regression analysis was used in the analysis of the associations between the different variables and RFS time (defined as the time from surgery until the diagnosis of recurrent disease; Ref. 24 ). RFS was considered to be more relevant with respect to efficacy of adjuvant systemic therapy than OS that is also influenced by systemic therapy for metastatic disease or other causes of death. To study the possible relationship of uPA, PAI-1, and uPA:PAI-1 complex with RFS, the levels of the three markers were divided into four groups (Q1 to Q4 by their respective quartiles). In addition, these analyses were also performed with uPA, PAI-1, and uPA:PAI-1 complex as log-transformed continuous variables.

Multivariate Cox regression analysis for RFS with stepwise removal of nonsignificant factors was used to assess the contributions of the clinicopathological factors as well as uPA, PAI-1, and uPA:PAI-1 complex levels, adjuvant endocrine therapy, chemotherapy, and radiotherapy to RFS time. This analysis resulted in a basic model to which interaction terms were added to establish whether uPA, PAI-1, and/or uPA:PAI-1 complex had a predictive value for adjuvant systemic therapy success according to an earlier reported method (15 , 25) . These interaction terms consisted of endocrine therapy or chemotherapy with uPA, PAI-1, or uPA:PAI-1 complex. Furthermore, an interaction term for endocrine by chemotherapy was added to investigate a possible additive effect of this treatment combination. The prognostic impact of nodal status is such that the (additional) value of the factors under investigation is likely to differ between patients with or without nodal involvement. Therefore, we also included interaction terms between lymph node status and uPA, PAI-1, or uPA:PAI-1 complex. The aforementioned analyses result in a final model consisting of the basic model and the interaction variables that reached statistical significance. Subsequently, multivariate regression analysis was performed on treatment effects within subsets if the appropriate interaction had been found statistically significant (25) . All of the computations were done with the SPSS statistical package (release 10.0.5, November 1999). Two-sided Ps < 0.05 were considered to be statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of uPA, PAI-1, and uPA:PAI-1 Complex Levels.
The antigen levels of uPA, PAI-1, and uPA:PAI-1 complex were determined in nonmembrane bound tissue fractions, derived from 1119 primary breast tumors, using ELISAs. The levels of uPA ranged from 0.00 to 11.60 ng/mg protein (median 0.45 ng/mg protein). A wide range of concentrations of PAI-1, ranging from 0.00 to 48.01 ng/mg protein, was observed. The median PAI-1 level was 1.49 ng/mg protein. The levels of uPA:PAI-1 complex ranged from 0.00 to 2.415 ng/mg protein, with a median value of 0.048 ng/mg protein.

Correlations.
The correlation between the uPA levels and the PAI-1 levels was statistically significant (rs = 0.508; P < 0.001). Significant correlations were also found between the levels of uPA:PAI-1 complex and uPA levels (rs = 0.424; P < 0.001), and uPA:PAI-1 complex and PAI-1 levels (rs = 0.569; P < 0.001).

The uPA:PAI-1 complex levels showed a significant correlation with age, with the highest tumor levels in patients of <=40 years (Table 1)Citation . The tumor levels of uPA, PAI-1, and uPA:PAI-1 complex were correlated significantly with tumor size, with the highest levels in tumors with a size of 2–5 cm (pT2 tumors). The tumor levels of all three of the markers showed also a significant association with histological grade, i.e., 54–63% of poorly differentiated tumors had uPA, PAI-1, and uPA:PAI-1 complex levels above their median values, whereas this was only the case in 33–45% of well-differentiated tumors. Similar differences were seen in ER and PgR status, with higher uPA, PAI-1, and uPA:PAI-1 complex levels in ER- and PgR-negative tumors (54–71% of tumors had values above median) compared with ER- and PgR-positive tumors (43–49% above median; Table 1Citation ).

Univariate Analysis of RFS.
Table 3Citation shows the results of the Cox univariate regression analysis for RFS in all 1119 patients whether or not they were treated with adjuvant systemic therapy for primary breast cancer. In this analysis, younger age, larger tumor size, poorer differentiated tumors, more lymph nodes affected, and negative ER and PgR status were all significantly associated with a poor RFS. Increasing levels of uPA, PAI-1, and uPA:PAI-1 complex were also related with a worse RFS in univariate analysis (Table 3)Citation . The 5-year probability of RFS was 79% for patients with low uPA:PAI-1 complex levels (Q1) and 59% for those with the highest uPA:PAI-1 complex levels (Q4, an absolute difference of 20%).


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  		Table 3 Cox univariate regression analysis of relapse-free survival in all patients (n = 1119)

 
There was not only a significant association between higher uPA, PAI-1, and uPA:PAI-1 complex levels and poor RFS when these markers were used as categorized variables (Table 3, QCitation 1 to Q4 by their respective quartiles), but also when used as log-transformed continuous variables (P = 0.003, P < 0.001, and P < 0.001, respectively).

Analysis of Predictive Impact of uPA, PAI-1, and uPA:PAI-1.
Because uPA, PAI-1, and uPA:PAI-1 complex levels were shown to be associated significantly with RFS in univariate analysis, a Cox multivariate analysis was performed with these factors and included all of the established clinicopathological factors. The combined value of the established prognostic clinicopathological factors (age, tumor size, histological grade, lymph node status, ER, and PgR), as well as uPA, PAI-1, and uPA:PAI-1 complex levels, adjuvant endocrine therapy, chemotherapy, and radiotherapy, were assessed in a basic model by Cox multivariate regression analysis. In Table 4Citation the results of the Cox multivariate regression analysis for RFS in all of the patients are listed. In the final multivariate model, younger age, larger tumor size, more lymph nodes affected, negative PgR status, no adjuvant endocrine therapy or chemotherapy, and increasing levels of uPA were significantly associated with a poor RFS. High levels of uPA:PAI-1 complex were also independently associated with a worse RFS [Q4, hazard ratio (HR) = 1.57; 95% confidence interval, 1.13–2.20; P = 0.033]. Adjuvant radiotherapy was not associated significantly with RFS.


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  		Table 4 Final model of the Cox multivariate regression analysis of relapse-free survival in all patients (n = 1119)

 
The additive value to this basic model of interaction terms (endocrine therapy or chemotherapy with uPA, PAI-1, or uPA:PAI-1 complex; endocrine therapy with chemotherapy; and lymph node status with uPA, PAI-1, or uPA:PAI-1 complex levels) was assessed by an additional round of reverse stepwise regression analysis to determine the predictive impact of uPA:PAI-1 complex (and other parameters) with regard to effectiveness of adjuvant systemic treatment.

There was a statistically significant interaction between adjuvant chemotherapy and uPA:PAI-1 complex level (P = 0.033). This interaction implies that the higher HR of recurrence (1.57) for patients with high tumor levels of uPA:PAI-1 complex (Q4) is significantly more reduced (0.49 *1 0.57 = 0.77) in patients who were treated with adjuvant chemotherapy compared with the patients with lower tumor levels of uPA:PAI-1 (Q1, 0.85 *1 0.02 = 0.87). This benefit occurs in addition to the independent overall reduction in recurrence rate of ~50% due to treatment with adjuvant chemotherapy (HR = 0.47; 95% confidence interval, 0.32–0.70; P < 0.001). Furthermore, there was a significant interaction between adjuvant endocrine therapy and uPA (P = 0.031). This interaction means a significant reduction in recurrence for patients with high uPA levels (Q4, HR = 1.49) who were treated with adjuvant endocrine therapy (0.45 *1 0.49 = 0.67) compared with patients with lower uPA levels (Q1, 0.89 *0.90 = 0.80). This reduction in recurrences occurs in addition to the independent overall reduction of ~75% due to adjuvant endocrine therapy (HR = 0.77; 95% confidence interval, 0.56–0.89; P < 0.001). No other interactions were found to be significant. Similar findings were seen when uPA, PAI-1, or uPA:PAI-1 complex were tested as log-transformed continuous variables instead of categorized variables (data not shown).

The uPA:PAI-1 complex by chemotherapy interaction was subsequently characterized by exploratory subset analyses. These analyses focused on treatment effects within subsets, i.e., the effect of adjuvant endocrine therapy and chemotherapy in patients with low levels of uPA:PAI-1 complex (Q1) and in patients with high levels of uPA:PAI-1 complex (Q4). In the low subset, the HR was 0.80 for endocrine therapy and 0.62 for chemotherapy. In the multivariate model for the high subset, the HR for endocrine therapy was 0.74 and for chemotherapy 0.37. These findings were according to the idea that patients with higher levels of uPA:PAI-1 complex benefited more from adjuvant chemotherapy than patients with lower uPA:PAI-1 complex levels, after correcting for other clinicopathological parameters. Furthermore, there was hardly a difference in HR for endocrine therapy between patients with low levels of uPA:PAI-1 complex (0.80) and patients with high levels (0.74).

A Cox multivariate model was also prepared separately for uPA and PAI-1 without inclusion of uPA:PAI-1 complex. In the basic multivariate model, younger age, larger tumor size, more lymph nodes affected, negative PgR status, no adjuvant endocrine therapy or chemotherapy and increasing levels of uPA and PAI-1 were associated significantly with a poor RFS (P = 0.007 and P < 0.001, respectively). In this analysis, the interaction between tumor levels of PAI-1 and adjuvant chemotherapy but not endocrine therapy reached statistical significance (P = 0.012) when added to the basic multivariate model (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to show that there is a difference in benefit from adjuvant chemotherapy for patients with higher versus lower tumor levels of uPA:PAI-1 complex. We find that the expression of uPA:PAI-1 complex independently predicts the efficacy of adjuvant chemotherapy in patients with primary breast cancer. In addition, uPA had predictive value for endocrine therapy success. When complex levels were not entered in the analysis, PAI-1 could significantly predict chemotherapy success.

For the individual high-risk patient predictive factors may be useful to support the choice of the most optimal adjuvant treatment regimen. Currently, the steroid hormone receptor status is the only established factor predicting the responsiveness to adjuvant endocrine therapy in breast cancer (26) . The best way to determine the predictive value of a parameter is to conduct a prospective study in which the patient cohorts are stratified by the presence or absence of the parameter under investigation and randomized between adjuvant versus no adjuvant systemic therapy. However, it is considered not ethical anymore to withhold adjuvant systemic therapy from such high-risk patients. Therefore, at present the predictive value of a particular marker is generally assessed from large retrospective data sets containing substantial patient numbers with and without therapy. A multivariate analysis with a term for interaction is a well-accepted way to ascertain the predictive value of a factor from such retrospective studies (15 , 25) .

There are two prior studies in breast cancer patients that relate the levels of components of the plasminogen activation system to the efficacy of adjuvant systemic therapy (14 , 15) . Both studies of Harbeck et al. (14 , 15) focused on the predictive value of uPA and PAI levels in tumor tissue. In the first study, these authors showed that there was a significant interaction between the combined variable of uPA and PAI-1, and adjuvant systemic therapy in 761 patients with primary breast cancer, with a larger benefit from adjuvant systemic therapy for patients with high risk, as defined by uPA and PAI-1 (14) . In the other study, comprising 3424 patients with primary breast cancer, Harbeck et al. (15) showed that high-risk patients, i.e., patients with tumor levels of uPA and PAI-1, either or both, above their respective cutoff values, benefited more from conventional adjuvant systemic therapy, particularly from chemotherapy, than low-risk patients. This effect was seen as a statistically significant interaction between chemotherapy and the combined variable of uPA and PAI-1, with a HR of 0.68 (95% confidence interval, 0.53–0.88; P < 0.003).

The components of the plasminogen activation system have different biological properties that might be responsible for their predictive impact. Higher levels of these components are associated with a more aggressive tumor behavior. From the Early Breast Cancer Trialists’ Collaborative Group meta-analyses, an increase in absolute efficacy of adjuvant systemic therapy may be expected in patients at increased risk for relapse. However, in the present study we demonstrated that also the relative difference in benefit was increased. At the moment, there are no studies available that investigate how these biological differences translate into the responsiveness to adjuvant systemic therapy. Additional in vitro and in vivo models are necessary to explain the findings from the current study on the predictive value of uPA:PAI-1 complex and from the previous studies (14 , 15) .

uPA exists in chemically different forms, some of them active and some inactive, and the assays used for measuring uPA are generally not able to distinguish between the different forms of uPA, so that the overall assay signal comes from a mixture of substances (27) . The ELISA for uPA:PAI-1 complex can be considered as specific, and the signal represents the small fraction of uPA that has been activated from proenzyme and had become inactive by forming an inactive complex with active PAI-1 (28 , 29) . Because complex formation between uPA and PAI-1 occurs only when both molecules are in their active form, one might assume that the level of uPA:PAI-1 complex represents previous active uPA and PAI-1. Thereby, selective assessment of uPA:PAI-1 complex levels in tumor tissue would therefore yield more accurate prognostic information than analysis of total uPA levels.

We measured soluble, nonmembrane bound uPA:PAI-1 complex. The complex selectively reflects the amount of uPA that has been activated from proenzyme uPA and subsequently inactivated by PAI-1. We have earlier shown that the complex of tissue-type plasminogen activator:PAI-1 as measured in the soluble, nonmembrane bound fraction has significant clinical relevance in terms of RFS and OS, whereas levels in pelleted (membrane) fractions were substantially less relevant (17) . Furthermore, the levels of uPA and PAI-1 in the pellet extracts provided less prognostic information as compared with those in the soluble, nonmembrane bound extracts (30) . We assumed the same would hold true for the uPA:PAI-1 complex, and, therefore, measured this complex in the soluble, nonmembrane bound fraction of our tissue homogenates. The clinical relevance found for the tissue-type plasminogen activator:PAI-1 and uPA:PAI-1 complexes confirm that this fraction has particular relevance in breast cancer pathogenesis.

This present retrospective study is the first study in which the predictive value of uPA:PAI-1 complex was evaluated in patients with primary breast cancer. Four hundred and thirty-two of these patients (39%) were treated with adjuvant systemic therapy, thus enabling for the determination of whether uPA:PAI-1 is an independent feature predictive of clinical outcome with regard to adjuvant systemic therapy. In this cohort, results of the univariate analysis showed that increasing levels of uPA, PAI-1, and uPA:PAI-1 complex were associated with a reduced RFS. In the multivariate analysis, uPA and uPA:PAI-1 complex were still associated significantly with poor RFS, but, of note, this analysis also included patients who actually had received adjuvant systemic therapies. Thus, such an association incorporates in part a prognostic and a predictive characteristic of the specific parameter. To investigate a strict predictive impact, tumor levels of the complex and individual uPA and PAI-1 parameters were additionally tested by a term of interaction (15 , 25) . Tumor levels of uPA were found to independently predict the efficacy of adjuvant endocrine therapy, whereas the levels of uPA:PAI-1 complex predict responsiveness to adjuvant chemotherapy, where patients with high tumor levels of uPA:PAI-1 complex appear to benefit more strongly from adjuvant chemotherapy than those with low levels. Similar results were found regarding OS (data not shown). However, RFS, rather than OS, should be considered the primary end point of the study, because after recurrence patients are treated with additional therapy for advanced disease, obscuring pure adjuvant treatment effects. When uPA:PAI-1 complex levels were not entered in the analysis, PAI-1 could significantly predict chemotherapy success. Earlier, Harbeck et al. (14 , 15) also found associations between components of the plasminogen activation system and adjuvant systemic therapy. Comparison with our results is difficult, because Harbeck et al. (14 , 15) used a combined variable of uPA and PAI-1, and different cutoff values were applied. In addition, Harbeck et al. (14 , 15) did not assess uPA:PAI-1 complex levels. However, both these studies (14 , 15) and our results indicate a significant role of the plasminogen activation system in chemotherapy resistance. Additional studies are mandatory to confirm the results of the current study before any definitive treatment decisions can be drawn and uPA:PAI-1 complex can be considered ready for clinical use.


   ACKNOWLEDGMENTS
 
We thank the contributors, especially the surgeons and internists, of the University Medical Centre Nijmegen and of the community hospitals in the region: Ziekenhuiscentrum Apeldoorn, Apeldoorn, Rijnstate Ziekenhuis, Arnhem, Maasziekenhuis, Boxmeer, Deventer Ziekenhuis, Deventer, Gelderse Vallei, Ede, Canisius-Wilhemina Ziekenhuis, Nijmegen, Streekziekenhuis Zevenaar, Zevenaar, and Nieuw Spitaal, Zutphen, for their assistance in collecting the patient clinical follow-up data. The excellent technical assistance of Anneke Geurts-Moespot and Doorlène van Tienoven of the Department of Chemical Endocrinology of the University Medical Centre Nijmegen is gratefully acknowledged.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Peggy Manders, Department of Chemical Endocrinology–530, University Medical Centre Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, the Netherlands. Phone: 31-24-3614013; Fax: 31-23-3541484; E-mail: p.manders{at}ace.umcn.nl

Received 6/20/03. Revised 10/28/03. Accepted 11/12/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv. Cancer Res., 44: 139-266, 1985.[Medline]
  2. Schmitt M., Harbeck N., Thomssen C., Wilhelm O., Magdolen V., Reuning U., Ulm K., Höfler H., Jänicke F., Graeff H. Clinical impact of the plasminogen activation system I tumor invasion and metastasis: prognostic relevance and target for therapy. Thromb. Haemost., 78: 285-296, 1997.[Medline]
  3. Duffy M. J., Maguire T. M., McDermott E. W., O’Higgins N. Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J. Surg. Oncol., 71: 130-135, 1999.[CrossRef][Medline]
  4. Vassalli J. D., Baccino D., Belin D. A cellular binding site for the Mr 55, 000 form of the plasminogen activator, urokinase. J. Cell Biol., 100: 86-92, 1983.
  5. Cubellis M. V., Nolli M. L., Cassani G., Blasi F. Binding of single-chain prourokinase receptor to human U937 cells. J. Biol. Chem., 261: 15819-22, 1986.[Abstract/Free Full Text]
  6. Conese M., Blasi F. Urokinase/urokinase receptor system: internalization/degradation of urokinase-serpin complexes: mechanisms and regulation. Biol. Chem. Hoppe-Seyler, 376: 143-155, 1995.[Medline]
  7. Rijken D. C. Plasminogen activators and plasminogen activator inhibitors: biochemical aspects. Baillière’s Clin. Haematol., 8: 291-312, 1995.
  8. Andreasen P. A., Kjoller L., Christensen L., Duffy M. J. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer, 72: 1-22, 1997.[CrossRef][Medline]
  9. Foekens J. A., Peters H. A., Look M. P., Portengen H., Schmitt M., Kramer M. D., Brünner N., Jänicke F., Van Meijer-Gelder M. E., Henzen-Logmans S. C., Van Putten W. L., Klijn J. G. The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res., 60: 636-643, 2000.[Abstract/Free Full Text]
  10. Look M. P., Van Putten W. L. J., Duffy M. J., Harbeck N., Christensen I. J., Thomssen C., Kates R., Spyratos F., Fernö M., Eppenberger-Castori S., Sweep C. G. J., Ulm K., Peyrat J. P., Martin P. M., Magdelenat H., Brünner N., Duggan C., Lisboa B. W., Bendahl P. O., Quillien V., Daver A., Ricolleau G., Van Meijer-Gelder M. E., Manders P., Fiets W. E., Blankenstein M. A., Broë P., Romain S., Daxenbichler G., Windbichler G., Cufer T., Borstnar S., Kueng W., Beex L. V. A. M., Klijn J. G. M., O’Higgins N., Eppenberger U., Jänicke F., Schmitt M., Foekens J. A. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J. Natl. Cancer Inst., 94: 116-128, 2002.[Abstract/Free Full Text]
  11. Jänicke F., Prechtl A., Thomssen C., Harbeck N., Meisner C., Untch M., Sweep C. G. J., Selbmann H. K., Graeff H., Schmitt M. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J. Natl. Cancer Inst., 93: 913-920, 2001.[Abstract/Free Full Text]
  12. Pierga J. Y., Laine-Bidrom C., Beuzeboc P., DeCremoux P., Pouillart P., Magdelenat H. Plasminogen-activator inhibitor-1 (PAI-1) is not related to response to neoadjuvant CT in breast cancer. Br. J. Cancer, 76: 537-540, 1997.[Medline]
  13. Foekens J. A., Look M. P., Peters H. A., van Putten W. L. J., Portengen H., Klijn J. G. M. Urokinase-type plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer. J. Natl. Cancer Inst., 87: 751-756, 1995.[Abstract/Free Full Text]
  14. Harbeck N., Kates R. E., Schmitt M. Clinical relevance of invasion factors urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 for individualized therapy decisions in primary breast cancer is greatest when used in combination. J. Clin. Oncol., 20: 1000-1007, 2002.[Abstract/Free Full Text]
  15. Harbeck N., Kates R. E., Look M. P., Meijer-van Gelder M. E., Klijn J. G., Kruger A., Kiechle M., Janicke F., Schmitt M., Foekens J. A. Enhanced benefit from adjuvant chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (n=3424). Cancer Res., 62: 4617-4622, 2002.[Abstract/Free Full Text]
  16. Hekman C. M., Loskutoff D. J. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J. Biol. Chem., 260: 11581-7, 1985.[Abstract/Free Full Text]
  17. Witte J. H. de, Sweep C. G. J., Klijn J. G., Grebenschikov N., Peters H. A., Look M. P., Tienoven T. H. van, Heuvel J. J., Bolt-Vries J. de, Benraad T. J., Foekens J. A. Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer. Br. J. Cancer, 80: 286-294, 1999.[CrossRef][Medline]
  18. Manders P., Beex L. V. A. M., Tjan-Heijnen V. C. G., Geurts-Moespot J., van Tienoven T. H., Foekens J. A., Sweep C. G. J. The prognostic value of vascular endothelial growth factor in 574 node-negative breast cancer patients who did not receive adjuvant systemic therapy. Br. J. Cancer, 87: 772-778, 2002.[CrossRef][Medline]
  19. Koenders A. J. M., Geurts-Moespot A., Zollingen S., Benraad Th. Progesterone and estradiol receptors in DMBA-induced mammary tumors before and after ovariectomy and after subsequent estradiol administration McGuire W. Raynaud J. Baulieu E. eds. . Progesterone Receptors in Normal and Neoplastic Tissues (Progress in Cancer and Therapy), 71-84, Raven Press New York, NY 1977.
  20. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurements with the folin reagent. J. Biol. Chem., 193: 265-275, 1951.[Free Full Text]
  21. Grebenschikov N., Geurts-Moespot A., de Witte H., Heuvel J., Leake R., Sweep F., Benraad T. A sensitive and robust assay for urokinase and tissue-type plasminogen activators (uPA and tPA) and their inhibitor type I (PAI-1) in breast tumor cytosols. Int. J. Biol. Markers, 12: 6-14, 1997.[Medline]
  22. Grebenschikov N., Sweep F., Geurts A., Andreasen P., de Witte H., Schousboe S., Heuvel J., Benraad T. ELISA for complexes of urokinase-type and tissue-type plasminogen activators with their type-1 inhibitor (uPA-PAI-1 and tPA-PAI-1). Int. J. Cancer, 81: 598-606, 1999.[CrossRef][Medline]
  23. Sweep C. G. J., Geurts-Moespot J., Grebenschikov N., de Witte J. H., Heuvel J. J., Schmitt M., Duffy M. J., Jänicke F., Kramer M. D., Foekens J. A., Brunner N., Brugal G., Pedersen A. N., Benraad T. J. External quality assessment of trans-European multicenter antigen determination (enzyme-linked immunosorbent assay) of urokinase plasminogen activator (uPA) and its type-1 inhibitor (PAI-1)in human breast cancer extracts. Br. J. Cancer, 78: 1434-1441, 1998.[Medline]
  24. Cox D. R. Regression models and life-tables. J. R. Statist. Soc. B., 34: 187-220, 1972.
  25. Simon R., Altman D. G. Statistical aspects of prognostic factor studies in oncology. Br. J. Cancer, 69: 979-985, 1994.[Medline]
  26. Goldhirsch A., Glick J. H., Gelber R. D., Coates A. S., Senn H.-J. Meeting highlights: International consensus panel on the treatment of primary breast cancer. J. Clin. Oncol., 19: 3817-3827, 2001.[Free Full Text]
  27. Benraad T. J., Geurts-Moespot J., Grøndahl-Hansen J., Schmitt M., Heuvel J. J., de Witte J. H., Foekens J. A., Leake R. E., Brünner N., Sweep C. G. J. Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop. Eur. J. Cancer, 32A: 1371-1381, 1996.
  28. Skriver L., Larsson L. I., Kielberg V., Nielsen L. S., Andresen P. B., Kristensen P., Danø K. Immunocytochemical localization of urokinase-type plasminogen activator in Lewis lung carcinoma. J. Cell Biol., 99: 752-757, 1984.
  29. Kielberg V., Andreasen P. A., Grondahl-Hansen J., Nielsen L. S., Skriver L., Danø K. Proenzyme to urokinase-type plasminogen activator in the mouse in vivo. FEBS Lett., 182: 441-445, 1985.[CrossRef][Medline]
  30. Witte J. H. de, Sweep C. G. J., Klijn J. G., Grebenschikov N., Peters H. A., Look M. P., Tienoven T. H. van, Heuvel J. J., Putten W. L. van, Benraad T. J., Foekens J. A. Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients. Br. J. Cancer, 79: 1190-1198, 1999.[CrossRef][Medline]



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