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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

From the Clinical Research Group, Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany.

Address reprint requests to Nadia Harbeck, MD, Frauenklinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 22, D-81675 Munich, Germany; email: nadia.harbeck@ lrz.tum.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: A strong prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor and plasminogen activator inhibitor type 1 (PAI-1) as individual factors is well established in breast cancer. The improvement in clinical risk assessment gained by combining these factors is evaluated here.

PATIENTS AND METHODS: uPA and PAI-1 levels were prospectively measured by enzyme-linked immunosorbent assay in tumor tissue extracts of 761 patients with primary breast cancer.

RESULTS: In the clinically important subgroup of node-negative patients without adjuvant systemic therapy (n = 269; median follow-up, 60 months), the clinical value of testing both uPA and PAI-1 is demonstrated. The criterion either or both high identifies with high sensitivity the patients at high relapse risk while keeping more than half in the low-risk group. uPA/PAI-1 is the strongest predictor of disease-free survival and overall survival; patients with high uPA/PAI-1 have an increased relapse risk (P < .001; relative risk, 4.8; 95% confidence interval [CI], 2.5 to 9.1), in particular for early relapse. Even within risk groups stratified by established criteria (nodal or menopausal status, tumor size, grade, or steroid hormone receptors), uPA/PAI-1 provides significant risk group discrimination. In the whole collective, the significant interaction between uPA/PAI-1 and adjuvant systemic therapy suggests a benefit from adjuvant therapy in high-risk patients as defined by uPA/PAI-1.

CONCLUSION: The clinical relevance of the two tumor-invasion factors uPA and PAI-1 is greatest when they are used in combination. The particular combination of uPA and PAI-1 (both low v either or both high) is superior to either factor alone and supports risk-adapted individualized therapy decisions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ADEQUATE RISK-GROUP assessment for subsequent decisions on adjuvant systemic therapy is a prerequisite for individualized therapy concepts in primary and, particularly, in node-negative breast cancer. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) are key factors in efficient focal proteolysis, adhesion, and migration of tumor cells and, hence, in subsequent tumor invasion and metastasis.^1,2 Increased levels of uPA and PAI-1 are present in breast carcinoma tissue compared with benign lesions or normal breast tissue.¹ A strong prognostic impact of uPA and PAI-1 in primary breast cancer has been reported by several investigators using biochemical assays.^3-12 Patients with high antigen levels of either factor in their tumors have a significantly worse survival rate than patients with low levels.

So far, most researchers have looked at the prognostic impact of each factor separately. It is known that PAI-1 or uPA alone already defines a low-risk group; hence, it is of immediate clinical interest to investigate whether this low-risk group can be additionally stratified by combining the two factors. This study addresses the clinical relevance of the particular combination of both factors, uPA/PAI-1 (both factors low v either or both high), in a cohort of 761 patients with primary breast cancer. To evaluate the impact of uPA/PAI-1 on the natural course of the disease, we focus on the clinically important subset of node-negative patients without adjuvant systemic therapy.

Concerning the response to systemic therapy in patients stratified by uPA or PAI-1, few data were available until now. Neither data from neoadjuvant therapy looking at local response¹³ nor analysis of a relationship between uPA and PAI-1 levels in primary tumor tissue and later response to palliative systemic therapy data^14,15 can be readily transferred to the adjuvant setting. Recently, first results of a prospective randomized multicenter therapy trial (Chemo N₀) indicated that high-risk node-negative patients, as defined by high uPA or PAI-1, seem to benefit from adjuvant cyclophosphamide, methotrexate, fluorouracil chemotherapy¹⁶; yet the survival difference did not reach significance in the intention-to-treat analysis (median follow-up, 32 months). Our analysis will now shed light on possible benefit of adjuvant systemic therapy in the risk groups defined according to uPA/PAI-1 by considering the relative impact of uPA/PAI-1 in patients receiving adjuvant systemic therapy versus those not receiving such therapy.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
In 761 consecutive patients with primary breast cancer (Table 1), the clinical relevance of the combination of uPA and PAI-1 was evaluated. Of these, less than half (n = 316) have been reported previously,¹⁷ and follow-up has been updated on all patients. Patients either underwent a modified radical mastectomy (n = 389) or breast-conserving surgery with subsequent breast irradiation (n = 372) at the Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany, between 1987 and 1998. Informed consent for analysis of tumor biologic factors was obtained at primary surgery. Therapy decisions were based solely on consensus recommendations at the time but not on uPA and PAI-1. For 745 patients, information on adjuvant systemic therapy was available (Table 1). Median age of the patients at time of primary surgery was 56 years (range, 28 to 92 years). At time of primary therapy, no patient had any clinical or x-ray evidence of distant metastases. Median follow-up time of all patients still alive at time of analysis was 48 months (range, 1 to 142 months). Within the follow-up period, 194 (25%) patients experienced disease recurrence, and 164 (22%) patients died. In addition to the collective as a whole, the subset of node-negative patients without adjuvant systemic therapy (n = 269; median follow-up, 60 months) was analyzed separately (Table 1).

Tumor grade was determined using the well-established Bloom-Richardson criteria. Steroid hormone receptors (estrogen and progesterone receptors) were initially determined biochemically in cytosol fractions and considered positive if they contained at least 20 fmol/mg protein. Starting in 1991, immunohistochemical staining on paraffin-embedded tissue sections was performed; positive staining denoted receptor positivity. Steroid hormone receptor status was considered positive if either or both receptors were positive.

Statistical Analyses
The continuous variables uPA and PAI-1 were first coded as binary variables using the previously optimized and re-evaluated cutoffs of 3 ng uPA/mg protein and 14 ng PAI-1/mg protein to distinguish between high and low antigen levels of the analytes in primary tumor tissue extracts.¹⁷ A new binary variable, uPA/PAI-1, representing the combination of these two factors, was then defined as both factors low versus either or both factors high. Established prognostic factors were dichotomized as described elsewhere.¹⁹ For univariate analysis of disease-free survival (DFS) and overall survival (OS), Kaplan-Meier curves were plotted and then compared using log-rank statistics. Multivariate analyses were performed in a stepwise forward fashion by applying the Cox proportional hazards model and Cox models with time-varying covariates using the SPSS software package (SPSS Inc, Chicago, IL). Interactions were included within the Cox models in the second stage of the model using forward selection. All tests were performed at a significance level of alpha = .05. Confidence intervals (Cis) refer to the 95% level.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Combination of uPA and PAI-1 Identifies Low-Risk Patients Better Than Either Factor Alone
To address the question of improved risk group discrimination by the combination of uPA and PAI-1, we focused on node-negative patients without adjuvant systemic therapy, because in this subset, the prognostic impact reflects the natural course of the disease.

Both uPA (P = .022; relative risk [RR], 2.3; 95% CI, 1.1 to 4.1) and PAI-1 (P = .049; RR, 2.0; 95% CI, 1.0 to 4.0) separately as well as grade (P = .026; RR, 2.1; 95% CI, 1.1 to 4.0) are significant in multivariate Cox regression for DFS (including established factors tumor size, grade, hormone receptor, and menopausal status). However, if the dichotomized combination of uPA and PAI-1 (low-low v either or both high) is entered into Cox regression on this cohort, then uPA and PAI-1 both drop out of the model. To understand the special role of the combination of uPA and PAI-1, we stratified by each of the two factors and performed a Cox regression on the remaining factors in the respective low-risk subgroup. In patients with low uPA, only PAI-1, but none of the established factors, provides additional risk discrimination (P < .001; RR, 5.9; 95% CI, 2.2 to 16.0). Similarly, in patients with low PAI-1, uPA provides additional prognostic information (P = .001; RR, 4.2; 95% CI, 1.9 to 9.6), even in multivariate analysis. This behavior is illustrated by the respective Kaplan-Meier curves in Fig 1. As shown in the top panels, PAI-1 provides statistically significant risk group separation (PAI-1 low: n = 171, 13 events; PAI-1 high: n = 23, six events) in patients considered low-risk by uPA levels in their primary tumor tissue (uPA low: n = 194, 19 events; uPA high: n = 75, 25 events). In the bottom panels, it is seen that uPA provides statistically significant risk group separation (uPA low: 171 patients, 13 events; uPA high: 40 patients, 12 events) in patients considered low-risk according to PAI-1 (PAI-1 low: n = 211, 25 events; PAI-1 high: n = 58, 19 events). Interestingly, the influence of either of these two factors is not uniform with respect to the other but is significant only in the low-risk subgroup of the other.

View larger version (32K): [in this window] [in a new window]   Fig 1. Enhanced risk group separation achieved by PAI-1 in low-uPA patients (top panels) and by uPA in low-PAI-1 patients (bottom panels). Impact on DFS in node-negative breast cancer (no adjuvant systemic therapy).

View larger version (24K): [in this window] [in a new window]   Fig 2. Prognostic impact of the four different combinations of uPA and PAI-1 on DFS in node-negative breast cancer (no adjuvant systemic therapy).

is used, where T is the time in months. This functional form allows the contribution of uPA/PAI-1 to remain strong through approximately 3 years and then rapidly diminish toward zero with longer follow-up; the precise form of F(T) affects the fit but otherwise makes little qualitative difference in understanding the interaction. Models were constructed including all dichotomized established factors and dichotomized uPA*F(T), PAI-1*F(T), and the interaction term uPA*(PAI-1)*F(T) as well as these factors without the time dependence. The model with the best fit includes only grade, uPA*F(T), PAI-1 *F(T), and uPA*(PAI-1)*F(T). The beta coefficients of all three terms are the same in magnitude, approximately 2.7 (corresponding to a relative risk of approximately 15), but the coefficient of the interaction is negative. This result means that the log RR associated with the combination of high uPA and high PAI-1 is not twice the log RR, as would be expected from a linear model, but is close to that for either factor alone. These relationships are reflected in Fig 2. These results support the statement that the particular combination of uPA and PAI-1 as used here (either or both high v both low) correctly characterizes the risk associated with uPA and PAI-1.

Prognostic Impact of uPA/PAI-1 in Node-Negative Patients Without Adjuvant Therapy
The combination uPA/PAI-1 is a highly significant discriminator between patients at low and those at high risk not only for relapse but also for death in univariate analysis in this clinically relevant subgroup. In multivariate analysis, uPA/PAI-1 is the strongest prognostic factor not only for DFS but also for OS (Table 2). Moreover, uPA/PAI-1 enables identification of high-risk patients even within established risk groups defined by tumor size, grade, steroid hormone receptor, or menopausal status. In Fig 3, RR of recurrence is given as a function of high antigen levels of either or both factors versus low levels of both uPA and PAI-1 as determined in primary tumor tissue extracts.

View larger version (46K): [in this window] [in a new window]   Fig 3. RR of recurrence associated with high uPA/PAI-1 in clinically relevant subgroups of node-negative breast cancer patients (without adjuvant systemic therapy).

View larger version (25K): [in this window] [in a new window]   Fig 4. Impact of uPA/PAI-1 on DFS reflects effect of adjuvant systemic therapy in primary breast cancer patients.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

The ELISAs for uPA and PAI-1 are robust enough for clinical routine use, and international quality assurance is guaranteed.²⁰ For testing, a minimum of 100 µg tumor tissue (corresponding to approximately 1 µg protein extract) is sufficient. Hence, the ELISAs can also be applied to extracts prepared from core biopsy specimens or cryostat sections. The optimized cutoffs for the assays used here are stable over time, correspond well to those found by other researchers using the same biochemical assays,⁶ and have recently been validated in a multicenter prospective trial.¹⁶ In contrast, no consistent clinically relevant data have been generated applying immunohistochemistry (IHC) or other techniques for determination of uPA and PAI-1 protein expression in breast carcinoma tissue. A recent IHC study showed that expression of uPA and PAI-1 in stromal fibroblasts is of more clinical relevance than expression in the tumor cells themselves, at least for the antibodies used.²¹ Such tissue heterogeneity with regard to expression of uPA and PAI-1 in different cell types is well accounted for using the ELISA procedure.

The fact that there is no contradictory evidence on the prognostic impact of uPA and PAI-1 in breast cancer is unique for any tumor biologic factor, in particular given the fact that the data have been generated under a variety of demographic conditions (in Europe, United States, and Japan). A pooled analysis comprising more than 8,000 patients has recently validated the unicenter data.²² uPA and PAI-1 are hardly or not at all correlated with any of the established prognostic factors.¹⁷ In a small node–negative collective without adjuvant systemic therapy, we previously found by classification and regression trees analysis that the combination of uPA and PAI-1 is superior to established prognostic factors with regard to selection of low-risk patients. It also outperforms other tumor biologic factors, such as HER2 protein overexpression, cathepsin D, p53, S phase, MIB1, or DNA ploidy.¹⁹ Even HER2 gene amplification, as determined by fluorescence in situ hybridization, which is also a strong prognostic factor in node-negative breast cancer,²³ is complementary, although weaker than uPA/PAI-1 for risk-group selection in node-negative breast cancer.²⁴

The present study illustrates the potential clinical value of testing both uPA and PAI-1 levels. In a collective of 269 node-negative patients without adjuvant systemic therapy, the condition either or both high identifies with high sensitivity those patients who are at high risk of relapse while still preserving a substantial, clinically relevant low-risk group. Thus, this combination captures and effectively dichotomizes the essential information obtained by the two factors. The significant improvement in risk discrimination (compared with either factor taken separately) is all the more remarkable in view of the biologic relationship and the significant correlation (r = .4)¹⁷ between these two factors.

The presence of time variation was previously discussed by Schmitt et al²⁵ for uPA and PAI-1 as single factors. In the present study, an interaction of uPA and PAI-1 was detected both using the proportional hazards assumption and using a time-varying model. The impact of uPA and PAI-1 can be described parsimoniously by the particular combination of uPA and PAI-1. The time variation implies that the relapses in the high-risk group will tend to occur within the first 3 to 4 years. Thus, not measuring one of these two factors might mean missing patients at high risk for subsequent systemic disease and, in particular, those at risk for early relapse. Even within risk groups defined by established prognostic factors, the combination of uPA and PAI-1 enables significant risk-group assessment (Fig 3). Patients with node-negative breast cancer with low levels of both PAI-1 and uPA in their primary tumor, comprising more than half of node-negative patients, have an excellent 5-year DFS rate of higher than 90% (Fig 2). In contrast, those patients with high levels of either or both factors have a 5-year DFS rate comparable with that of patients with several involved lymph nodes (Fig 2). These results are in concordance with data from the Chemo N₀ trial, which validated the independent prognostic impact of uPA/PAI-1 in node-negative breast cancer.¹⁶

Testing of both uPA and PAI-1 provides a valuable basis for patient counseling in node-negative breast cancer both for low- and high-risk patients. Some of these patients are willing to undergo systemic treatment for even a small benefit probability.²⁶ However, other patients or their physicians will express a preference for a no-treatment option, in particular with regard to chemotherapy. The low-risk group identified by uPA/PAI-1 is substantially larger than that characterized by the St Gallen criteria²⁷ and thus much closer to the actual 70% of node-negative patients cured by locoregional treatment alone;²⁸ they are candidates for being spared the burden of adjuvant chemotherapy. The results of this study support the potential value of uPA and PAI-1 measurements to define those node-negative patients who are clearly at high risk and for whom adjuvant systemic treatment would be strongly recommended.

Our data suggest a benefit from adjuvant chemotherapy or endocrine therapy in patients with high uPA/PAI-1. In the patient collective as a whole, the univariate prognostic impact of uPA/PAI-1 on DFS was substantial in patients without adjuvant systemic therapy (Fig 4), underlining the strong association of uPA and PAI-1 with an aggressive tumor phenotype leading to invasion and metastasis. However, this prognostic strength is diminished in patients who received adjuvant systemic therapy, suggesting a benefit from adjuvant systemic therapy in this high-risk group, at least for DFS (Fig 4). Because most untreated patients are node-negative, the interaction of adjuvant treatment and uPA/PAI-1 is difficult to distinguish from a hypothetical interaction of nodal status with uPA/PAI-1 because of the confounding. However, our results and interpretation are supported by the finding of Foekens et al⁶ that both uPA and PAI-1 are strong and significant, even within the subgroup of node-positive patients, most of whom did not receive adjuvant systemic therapy. Moreover, for adjuvant chemotherapy, our findings agree well with the observed benefit from adjuvant cyclophosphamide, methotrexate, fluorouracil in node-negative patients with high uPA/PAI-1 data in the Chemo N₀ trial.¹⁶ A European follow-up therapy trial will now compare different chemotherapy regimens for these high-risk patients. With regard to adjuvant endocrine therapy, no data are available looking at the predictive impact of uPA and PAI-1. Retrospective evidence from studies in metastatic breast cancer, implying that high uPA or PAI-1 tumor levels at primary therapy are associated with poor response to later palliative endocrine therapy,^14,15 cannot be directly translated to the adjuvant setting. Indeed, the two findings are consistent with the tumor biology of uPA and PAI-1.¹ High levels do reflect an aggressive phenotype that may be overcome or suppressed by early systemic therapy in the adjuvant setting but may be far too advanced for response to palliative therapy at a later stage. Concerning overall survival, definite statements concerning the interaction of uPA/PAI-1 with adjuvant systemic treatment cannot be based on retrospective evidence, due to the heterogeneity of palliative systemic treatment after first relapse.

Strict criteria to be satisfied before any new prognostic marker can be transferred into clinical routine have been put forward.^29,30 uPA and PAI-1 are the only novel tumor biologic factors so far for which all of these criteria have been fulfilled. As stated above, assessment of tumor expression of uPA and PAI-1 by IHC has not been sufficiently prognostic, because the proteins are synthesized and expressed in varying proportions by both tumor and stroma cells.¹ Such heterogeneity is difficult to quantify reproducibly using IHC. Nonetheless, there is undisputed evidence for the prognostic value of uPA and PAI-1 as single factors measured by robust and quality-assured ELISAs. Our data now show that the combination of both factors is superior to either factor taken alone and outperforms established prognostic factors with regard to risk group stratification, in particular in node-negative breast cancer. Moreover, our data suggest that high-risk patients, according to uPA/PAI-1, benefit from adjuvant systemic therapy, which is consistent with the Chemo N₀ trial.¹⁶

On the basis of the Chemo N₀ trial¹⁶ and the pooled analysis,²² uPA and PAI-1 have reached the highest level of evidence (LOE I) according to the Tumor Marker Utility Grading System.³¹ This study shows that the clinical relevance of these two factors is greatest when used in combination and that the combination uPA/PAI-1 supports risk-adapted individualized therapeutic strategies in the adjuvant setting. No change in treatment strategy can be recommended solely on the basis of the retrospective data presented here. However, our results strongly indicate that there is a potential for improved treatment strategies that should be additionally pursued in prospective randomized trials.

	ACKNOWLEDGMENTS

 
Supported in part by a grant from the State of Bavaria (KKF project no. 8756159) to N.H.

The authors thank Erika Sedlaczek and Christel Schnelldorfer for their expertise in performing the ELISA assays. The continuous technical support of American Diagnostica Inc, Greenwich, CT, is acknowledged.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted March 13, 2001; accepted October 24, 2001.

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