Urokinase plasminogen activator: a prognostic marker in breast cancer including patients with axillary node-negative disease

Departments of
¹ Nuclear Medicine and
² Surgery, St. Vincent's Hospital, Dublin 4, Ireland.
³ Department of Gastroenterology, St. Bartholomew's Hospital, London EC1 M 6BQ, UK.
^a Address correspondence to this author at: Department of Nuclear Medicine, St. Vincent's Hospital, Elm Park, Dublin 4, Ireland. Fax 353-1-269 6018; e-mail mjduffy{at}SVHERC.ucd.ie.

	Abstract

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Urokinase plasminogen activator (uPA) is a serine protease causally involved in cancer invasion and metastasis. In this study, high concentrations of uPA in primary breast cancers were independently associated with both a shortened disease-free interval and overall survival. For the disease-free interval as endpoint, uPA was a stronger indicator of outcome than lymph node status, whereas for overall survival, nodal status was stronger than uPA. In patients without metastasis to axillary nodes, uPA was also an independent prognostic marker, using both the disease-free interval and overall survival as end points. In contrast to uPA, neither tumor size nor estrogen receptor status was prognostic in the node-negative patients. Measurement of uPA concentrations might thus be of value in selecting the more aggressive subpopulation of node-negative breast cancer patients that could benefit from adjuvant therapy.

	Introduction

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Breast cancer is the most common fatal malignancy in women in the Western world (1). Recently, randomized clinical trials have shown that adjuvant treatment with either combination chemotherapy or hormonal therapy improved the outcome of patients with this malignancy (2). Because of these findings, it has been argued that adjuvant therapy should be given to all breast cancer patients. However, for many, this approach would be overtreatment (3). Thus, ~70% of axillary node-negative patients are cured of their disease by local surgery, radiotherapy, or a combination of these two treatments, without any recourse to adjuvant therapy. A large number of women without disease would therefore be subjected to both the toxic side effects and costs of this therapy. In addition, because the clinical benefits of presently available therapies are relatively small, only a minority of these node-negative patients would benefit. A practical solution to this dilemma is to use prognostic markers to separate patients into low- and high-risk groups, based on metastatic potential, and focus treatment on patients with high risk of relapse (3).

Urokinase plasminogen activator (uPA)¹ is a serine protease with multiple functions, enabling it to play a role in cancer progression [reviewed in (4)(5)]. In particular, it can both catalyze the degradation of the extracellular matrix and stimulate cellular migration (4)(5). These two events are critical for malignant cell invasion and metastasis. Results from model systems have shown that uPA is causally involved in cancer spread (4)(5). Consistent with these findings from experimental systems, uPA has also been shown to be a marker of metastatic potential or prognosis in several human cancers, including breast, colorectal, and gastric malignancies [reviewed in (6)]. In this study, we show, using a commercially available kit, that high concentrations of uPA correlate with both shortened disease-free interval and overall survival in breast cancer patients, including women with node-negative disease.

	Materials and Methods

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methods
After histological examination, breast cancers were snap frozen in liquid nitrogen and then transferred to a -70 °C freezer. Tumors were homogenized in 50 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L monothioglycerol, as described previously (7). The homogenate was centrifuged at 2000g for 10 min, and the supernatant was extracted using 10 mL/L Triton X-100. Centrifugation was then carried out at 10 000g for 20 min. uPA was assayed on the supernatant using ELISA (American Diagnostica, Greenwich CT). This ELISA kit detects precursor uPA, active uPA, and uPA inhibitor complexes and has a stated detection limit of 10 ng/L. Total protein was measured by using kits obtained from Bio-Rad. uPA concentrations were expressed as ng uPA/mg protein.

Estrogen receptors (ERs) were assayed as described previously (8). The cutoff point used was 200 fmol/g wet weight of tissue.

patient cohort
We studied 184 consecutive patients on whom ER status, axillary node status, and tumor size were known. All of the carcinomas used were submitted for routine steroid receptor determination. Detailed characteristics of the tumors, as well as patients' ages and adjuvant therapies administered, are listed in Table 1 . Median follow-up of patients alive at the end of the study was 6.9 years (range, 0–10.4 years). Fifty-six patients died during the follow-up period.

The cutoff point used for discriminating between patients with high and low uPA concentrations was 0.81 ng/mg total protein. This was determined by using the maximal log-rank test and was identical to that found previously by us (7). Cox regression analysis was performed to determine that uPA was an independent prognostic marker. A backwards regression model was used to achieve the most powerful set of predictor variables. Two-sided P values of <0.05 were considered significant.

	Results

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preliminary experiments
The concentration of uPA in the breast cytosols varied widely, i.e., from 0–63.3 µg/L (median value, 0.66 µg/L) or from 0–10.2 µg/g protein (median value, 0.34 µg/g protein). These values are shown in the form of a frequency distribution graph in Fig. 1 . Because the concentration of calibrators in the ELISA ranged from only 0–1 µg/L, many cytosol samples had to be diluted. It was, therefore, essential to verify that the values obtained after dilution displayed parallelism. Values corrected for dilution should give the same result, irrespective of the extent of dilution. Fig. 2 shows the linearity of values after dilution of four different breast cancer cytosols. Clearly, good parallelism was obtained over the dilutions used, i.e., 1:2, 1:4, 1:8, 1:16, and 1:32. We also checked the between-assay variation for the uPA ELISA kit. For this purpose, pooled sera from patients with metastatic breast cancers was used. This control sample was assayed on 17 different days over a period of 15 months, using kits with six different lot numbers. The mean uPA value of the 17 determinations was 0.303 µg/L, with an overall CV of 11.43%.

View larger version (17K): [in this window] [in a new window]   Figure 1. Frequency distribution of uPA in breast cytosols.

View larger version (13K): [in this window] [in a new window]   Figure 2. Linearity of uPA values for four different breast cytosols after dilution. Dilutions were as follows: (1) 1:2; (2) 1:4; (3) 1:8; (4) 1:16; and (5) 1:32.

correlation of uPA with patient outcome
All patients.
As shown in Fig. 3 , patients with high concentrations of uPA had both a shorter disease-free interval (P = 0.0008) and shorter overall survival (P = 0.0004) than patients with low concentrations of the protease. As a continuous variable, uPA concentrations were also significantly related to both the disease-free interval (P <0.0001) and overall survival (P = 0.0018). Table 2 compares the relative strength of uPA with established prognostic markers for breast cancer, using both univariate and multivariate analyses. With the exception of tumor size for the disease-free interval, all of the investigated parameters, (i.e., nodal status, ER status, tumor size, and uPA) correlated significantly with patient outcome in univariate analysis. By using multivariate analysis, however, only uPA and lymph node status predicted the disease-free interval, whereas uPA, lymph node status, and size correlated with overall survival. With disease-free interval as end point, uPA was a stronger prognostic marker than either nodal status, ER status, or tumor size. On the other hand, for overall survival, uPA was weaker than lymph node status but more powerful than either tumor size or ER status in predicting outcome.

View larger version (17K): [in this window] [in a new window]   Figure 3. Disease-free survival and overall survival of 184 patients with breast cancer, according to tumor uPA expression (u-PA low <0.81 µg/g protein; uPA high >0.81 µg/g protein).

Axillary node-negative patients.
In the axillary node-negative patients, high concentrations of uPA were also associated with both a shortened disease-free interval (P = 0.0001) and overall survival (P = 0.0002; Fig. 4 ). In this subgroup of patients, uPA was the only significant predictor of outcome and was significant when using both univariate and multivariate analyses (Table 3 ).

View larger version (17K): [in this window] [in a new window]   Figure 4. Disease-free survival and overall survival of 87 patients with node-negative breast cancer, according to tumor uPA expression (uPA low <0.81 µg/g protein; uPA high >0.81 µg/g protein).

Patients receiving different adjuvant therapies.
uPA was also investigated for possible prognostic value in patients receiving different types of adjuvant treatment. For the group of patients treated with adjuvant tamoxifen, uPA concentrations correlated significantly with both disease-free interval (relative risk, 3.27; P = 0.0002; , 13.9) and overall survival (relative risk, 4.7; P <0.0001; = 22.5). uPA was not prognostic in those groups of patients who either received no adjuvant therapy or who were given adjuvant chemotherapy. It should, however, be pointed out that the numbers of patients in both of these subgroups were relatively small, i.e., 44 and 21, respectively.

	Discussion

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To our knowledge, this is the first report to show that high concentrations of uPA in node-negative breast cancers are significantly associated with both a shortened disease-free interval and overall survival. Previously, uPA was shown to be a prognostic marker for total populations of breast cancer [i.e., both node-negative and node-positive patients; reviewed in (6)]. By using a different group of patients and a different ELISA for uPA, we demonstrated previously that high concentrations of the protease predicted a shortened disease-free interval in axillary node-negative breast cancer patients (8). However, in that study, uPA was not shown to be an independent prognostic marker for node-negative disease. Furthermore, in our previous study (9), uPA was not related significantly to overall survival, using P <0.05 as a criteria of significance. Others have also reported uPA to be a prognostic marker in node-negative breast cancer by using the disease-free interval as the end point (10)(11)(12).

Although multiple groups have now reported uPA to be prognostic in breast cancer patients, it should be pointed out that the cutoff point used by the different investigators varied widely, i.e., from 0.62 µg/g protein to 10 µg/g protein (9)(10)(11)(12). The reasons for these widely different cutoff points are likely to include: (a) type of tumor extract used for assaying uPA, e.g., whether whole homogenate, low speed, or high speed supernatant was used; (b) whether a detergent such as Triton X-100 was used to extract the protease; (c) different calibrators used in the various assays; and (d) specificity of the antibodies used in the different ELISAs. This last point may be particularly difficult to control because uPA can exist in multiple forms in vivo. Thus, in addition to its precursor and mature and low molecular weight forms, uPA can form complexes with its two inhibitors, PAI-1 and PAI-2, and with its receptor uPAR. Ternary complexes such as uPA-PAI-1-uPAR and uPA-PAI-2-uPAR, as well as quaternary complexes, involving associations between these ternary complexes and the ₂-macroglobulin receptor, may also exist (6).

Another protease implicated in cancer progression, i.e., cathepsin D (13), has also been shown in some studies to be a prognostic marker in node-negative breast cancer patients (14)(15). However, these results with cathepsin D in node-negative patients have not been confirmed [reviewed in (6)(16)]. Similarly, other biochemical prognostic factors such as ER, progesterone receptor, c-erbB-2 and p53, although prognostic in the total population of breast cancer patients, are not consistently found to correlate with outcome in the node-negative subgroup [reviewed in (17)(18)(19)]. Interestingly, PAI-1, a naturally occurring inhibitor of uPA, has also been found to be associated with patient outcome in node-negative breast cancer patients (11)(20).

According to Clark (21), a prognostic marker in breast cancer should not only predict outcome but should also be able to identify patients likely to be responsive or resistant to particular forms of treatment. uPA may also be of value in this latter area of breast cancer management. Recently, Foekens et al. (22) showed that uPA concentrations in primary breast cancers predicted response of metastatic disease to tamoxifen therapy. This effect of uPA appeared to be independent of both ER and progesterone receptor status. (22).

Because uPA is an independent prognostic marker in breast cancer [reviewed in (6)], prognostic in the node-negative subgroup of patients (6), and a possible predictor of response to hormonal therapy (22), it is a potential candidate for routine use in the management of patients with malignancy of the breast. However, before any new biological marker enters routine use, it should be evaluated properly (23). In particular, it should be evaluated with regard to both analytical performance and clinical value (24).

Unlike most new biological prognostic markers described for breast cancer in recent years, these studies have either been carried out or are currently in progress for uPA. For example, in 1996, Benraad et al. (25) reported on the evaluation of six different ELISA kits. Some of the conclusions to emerge from this study were as follows:

(a) Although the absolute uPA values measured with the various kits differed, good correlations were obtained between any two of the ELISAs tested.

(b) In the two kits evaluated for the effects of cytosol dilution on the measured value, no systematic increase or decrease in uPA concentrations were found. With xenograft cytosols, dilutions as low as 1:400 yielded CVs <5%.

(c) Human breast cancer cytosols with added recombinant pro-uPA and lyophilized gave a mean CV of 11.5% when assayed on 26 separate days over a period of 18 months. This between-assay variation of Benraad et al. (25) is almost identical to what we found in the present study, i.e., 11.43%. In the present report, we also found an acceptable degree of parallelism for uPA after cytosol dilution. Additional work in the area of uPA methodology will require assay standardization and evaluation in External Quality Assurance trials. Regarding the latter, it should be pointed out that such studies have recently been carried out in Europe (Sweep et al., manuscript in preparation).

Finally, before routine application of a new marker, randomized trials should show that determination of marker concentrations leads to either enhanced disease-free interval, extended overall survival, or increased quality of life. To address whether assay of uPA can positively contribute to the management of breast cancer patients, a prospective randomized multicenter study was recently initiated in Germany. In this trial, axillary node-negative breast cancer patients with high concentrations of uPA (or of its inhibitor, PAI-1) were randomized to receive either six cycles of cyclophosphamide or to be observed. Patients with low concentrations of uPA/PAI-1 were not treated. With this trial, it is hoped that the subgroup of patients that are at high risk of early disease relapse and those who will benefit from adjuvant chemotherapy can be identified. The results of this study may thus lead to a test that will prevent the administration of unnecessary and potentially toxic therapy to the majority of node-negative patients.

	Acknowledgments

 
This work was supported by The Irish Cancer Society, The Health Research Board of Ireland, The International Association for Cancer Research, and the BIOMED 1 Program of the European Union (Project: Clinical Relevance of Proteases in Tumor Invasion and Metastasis; contract no. CT931346). Some of the uPA kits used were supplied free of charge by American Diagnostica, Inc.

	Footnotes

 
¹ Nonstandard abbreviations: uPA, urokinase plasminogen activator; ER, estrogen receptor; uPAR, uPA receptor; and PAI, plasminogen activator inhibitor.

	References

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