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Functional Evaluation of Plasmin Formation in Primary Breast Cancer

From the Division of Oncology, Department of Medicine, and Department of Gynaecology and Obstetrics, Hôpitaux Universitaires, and Department of Pathology, Medical University Centre, Geneva, Switzerland; and Departments of Medicine and Human Genetics, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada.

Address reprint requests to Pierre O. Chappuis, MD, Division of Medical Genetics, Montreal General Hospital/Rm L10-120, 1650 Ave Cedar, Montreal, QC H3G 1A4, Canada; email: pierre. chappuis{at}muhc.mcgill.ca

	ABSTRACT

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PURPOSE: Plasmin generation is controlled by the plasminogen activators (PA)/plasmin system, which comprises proteases (urokinase-type PA [uPA] and tissue-type PA [tPA]) and antiproteases (PA inhibitors, PAI-1 and PAI-2). The tumoral content of uPA and PAI-1 has been shown to carry prognostic value in breast cancer; however, because most assays used so far have relied on immunometric determinations, we have explored the enzymatic activities governing plasmin formation in breast cancer specimens.

PATIENTS AND METHODS: We applied semiquantitative histochemical zymography to 201 primary breast cancer tissue sections. Enzymatic activities were correlated with histopathologic parameters and clinical outcome. The median follow-up was 91 months.

RESULTS: A wide range of PA-mediated catalytic activities was detected. The overall survival was significantly worse for patients with tumors showing tPA in the lowest quartile of activity (P = .003). The 5-year overall survival of patients with tPA activity in the lowest quartile was 58% compared with 81% for patients with tPA value in the other three quartiles. Tumor size, axillary lymph node metastasis, histologic grade, lymphovascular infiltration, TP53 mutation, and tPA activity were all major risk factors in univariate analysis. tPA activity was an independent prognostic factor in a multivariate Cox regression model, both in the whole population (relative risk = 0.5, 95% confidence interval, 0.3 to 0.9; P = .02) and in the node-negative subgroup (relative risk = 0.2, 95% confidence interval, 0.08 to 0.6; P = .004).

CONCLUSION: By using a zymographic assay performed directly on primary tumor tissue sections, we demonstrate that reduced tPA-mediated plasmin production is an independent adverse prognostic factor in breast cancer.

	INTRODUCTION

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METASTASIS FORMATION is considered to result from the concerted activation of multiple molecular pathways imparting malignant cells with the capacities to modulate their interactions with the extracellular environment. In particular, invasive and disseminative processes appear to require the production of extracellular proteolytic enzymes by tumoral tissues.¹ Among the diverse proteolytic systems known to be produced by human cancers, the plasminogen activators (PA)/plasmin system is postulated to contribute to the invasive and metastatic phenotype by upregulating plasminogen conversion into plasmin, a protease capable of degrading most components of the extracellular matrix.² The PA/plasmin system is a multicomponent, proteolytic cascade that comprises the serine proteases urokinase-type PA (uPA) and tissue-type PA (tPA), the serine protease inhibitors PAI-1 and PAI-2, and the cell surface receptor for urokinase uPAR.³ In physiologic conditions, the coordinated expression of PA, PAI, and uPAR in a defined location provides a means to tightly control plasmin production in the extracellular compartment. uPA is primarily involved in cellular migration and in tissue remodeling.⁴ tPA is essentially produced by endothelial cells, and its main function is thought to be fibrinolysis,⁵ but its contribution in tumor progression has been reported.⁶ The PA/plasmin system also appears to be important in controlling the action of several growth factors⁷ or other proteolytic systems.⁸ In pathologic conditions such as neoplasia, alterations in the protease-antiprotease balance lead to increased plasmin formation and are thought to account for excessive tissue degradation.⁹ Experimental and clinical evidence supports a critical role for uPA in the biology of tumor invasion and metastasis formation. In primary human tumors, including breast cancer, elevated levels of uPA have been consistently associated with a poor outcome.^10-13 Although tPA is also known to be produced by breast cancer, its contribution has not been conclusively defined.^14-20 Paradoxically, elevated amounts of PAI-1 have been correlated to unfavorable prognosis,^21,22 corroborating experimental data that attributes to PAI-1 a dual role in invasion and adhesion.²³ Finally, recent studies indicate that integrated evaluation of uPA, tPA, PAI-1, and PAI-2 levels may help to better define the prognosis of node-negative breast cancer.²⁴ However, most available data remain based on immunometric determinations in tissue extracts rather than on evaluations of enzymatic activities.²⁵ The most widely used techniques to evaluate the amount of the different components of the PA/plasmin system are enzyme-linked immunoabsorbent assays, which measure the total amount of a given component. Pro-forms, active and inactive forms, and complexes between uPA (or tPA) with its inhibitors and the receptor are measured together.²⁶ Thus, individual analysis of each component of the PA/plasmin system fails to reflect their complex interactions. Moreover, different sets of tumor tissue extraction buffers, antibodies, and standards preclude any comparison between results of studies based on enzyme-linked immunoabsorbent assays.

To better delineate the prognostic relevance of plasmin formation in breast cancer, we applied a semiquantitative histochemical method to assess plasminogen activation directly on breast cancer tissue sections. By using in situ zymography, which provides a means to detect the respective expression of uPA and tPA-mediated catalytic activities at a tissular level, we confirmed the prognostic importance of PA production in breast cancer. Notably, our functional approach revealed the opposite prognostic significance of the tPA and uPA catalytic activities. A decreased enzymatic activity mediated by tPA and an increased uPA activity were associated with adverse outcome.

	PATIENTS AND METHODS

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Study Population
The study included 214 female patients with a primary invasive breast cancer, diagnosed between 1987 and 1989, for whom frozen tissues were available at the Department of Pathology of the University Hospital of Geneva, Switzerland. Tumor specimens were drawn from a pool of frozen specimens originally submitted for steroid-hormone receptor analysis. Twelve cases were excluded because of evidence of metastatic dissemination after review of medical records,⁵ second primary breast cancer,⁶ or the presence of in situ lesions only on the archived tumor block.² The study included 68% of all primary breast cancers diagnosed during the same period of time at the University Hospital of Geneva. No statistical difference regarding age, size, lymph node status, histologic type, estrogen receptor (ER) content, treatment, and follow-up was found between the studied population and the patients not included in the study (frozen samples unavailable or excluded cases). The routine baseline evaluation of the patients included laboratory analyses, chest radiography, mammography, liver ultrasonography, and bone scanning. All patients were treated surgically by mastectomy or lumpectomy and axillary dissection. Postoperative radiotherapy was given to 95 (48%) patients. Various regimens of adjuvant chemotherapy were administered to 71 (36%) patients. Adjuvant hormonotherapy was prescribed to 73 (37%) postmenopausal women with ER-positive tumors. The median age of patients at the time of diagnosis was 55.8 years (range, 23.8 to 86.7 years). On average, 19 axillary lymph nodes were examined (range, three to 44 nodes). The median size of collected tumors was 2.0 cm (range, 0.1 to 7.2 cm). Histopathologic type and grade were classified according to World Health Organization criteria and Bloom-Richardson grading, respectively. Tumors were pure histologic variants of invasive breast carcinomas consisting of 93% ductal and 7% other types. Presence or absence of peritumoral vascular invasion was recorded. The ER and progesterone receptor (PR) status was determined by immunocytochemistry in 97% of cases and by the dextran-coated charcoal method in the remaining cases. p53 functional evaluation was determined as described elsewhere.²⁷ After treatment completion, patients were seen on a regular basis at least every 6 months for 5 years, then once a year. The follow-up evaluation included a physical examination, a chest radiogram, and serum biochemistry. The median follow-up duration was 91 months (range, 5 to 113 months). Follow-up status and causes of death were determined from hospital charts, physicians’ reports, and the Geneva Cancer Registry files. Three patients were lost to follow-up. Overall survival (OS) rate was calculated as the number of months from the date of primary surgery to the date of death. Sixty-nine patients died during the follow-up period, 47 from breast-cancer–related causes.

Semiquantitative In Situ Zymography
The enzymatic assay was performed on 10-µm cryostat tissue section as described elsewhere.²⁸ For each cryopreserved tumor block, four sections were used to evaluate the global PA-mediated (uPA + tPA) enzymatic activities, four sections were used to evaluate tPA-mediated activity, and one section was used as a control. Briefly, sections were overlaid with a mixture containing 0.75 mL of phosphate-buffered saline (with 0.9 mmol/L Ca²⁺ and 1 mmol/L Mg²⁺), 2% nonfat dry milk, 0.9% agar, and 40 µg/mL of purified human plasminogen in phosphate-buffered saline. The slides were then incubated in a humid chamber at 37°C for 2.5 hours. We previously determined that 2.5 hours was the incubation period allowing an optimal in situ caseinolytic reaction (data not shown). Control experiments were carried out with an overlay mixture from which plasminogen was omitted. No proteolytic zones were observed in the absence of plasminogen, demonstrating that the lytic activity was caused by PA. To discriminate uPA-mediated from tPA-mediated catalytic activities, amiloride—a specific inhibitor of uPA-mediated proteolytic activity²⁹—was added to the overlay mixture at 1 mmol/L final concentration. Photographs were taken using dark ground illumination with a video camera (Ikegami FCD-12, Ikegami Tsushinki Co., Tokyo, Japan) coupled to a macroscope (Leica M420, Leica Microsystems, Cambridge, UK), and the photographs were stored on a computer. The pictures were then analyzed using a computer-assisted image analyzer (Qwin software, Leica Microsystems, Cambridge, UK). All image analyses were performed by one observer, blinded to the clinical data. Evaluation of the surface of caseinolysis visualized as black areas over the tissue section and of the total surface of the section was performed for global PA and tPA-specific activities. The areas were defined as pixel counts, and the ratio between lytic/total section surface was expressed as a percentage. uPA activity was determined by subtraction of tPA-specific activity to the global PA/plasmin system activity.

Statistical Analysis
A mean of four measures of global PA and tPA activities was calculated. An association between clinicopathologic characteristics and PA/plasmin proteolytic activity was established by ² test and the analysis of variance. The association between tPA and uPA activities was measured by Spearman rank correlation coefficient. Five-year survival rates were estimated using the actuarial method (Kaplan-Meier), and survival rates are also presented for the median follow-up time of 91 months. Survival curves were compared using the Lee-Desu test (SPSS, Allegiant Technologies, Chicago, IL). Multivariate survival analysis was performed by the Cox regression model (Egret, SERC, Seattle, WA).

	RESULTS

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Clinicopathologic characteristics of the study population are listed in Table 1. A wide range of caseinolytic activities was found. Representative examples of tPA-mediated and uPA-mediated caseinolytic activities in breast cancer specimens evaluated by in situ zymography are shown in Fig 1. The respective distribution of the tPA and uPA caseinolytic activities after 2.5 hours of incubation is presented in Fig 2. tPA and uPA enzymatic activities were significantly inversely associated (r = -0.65; P < .001). uPA-related catalytic activity, considered as a continuous variable, was significantly associated with tumor size (P = .048) and inversely associated with ER status (P = .038). Considered as a continuous variable, tPA activity was associated with ER (P < .001) and PR (P < .001) status and inversely associated with tumor size (P = .034), histologic grade (P = .007), and TP53 mutation (P < .001) (Table 1). A cutoff at the first quartile of the tPA activity range was chosen, as higher levels of activity were significantly associated with a better prognosis at all incubation times (data not shown). The tPA distribution within the subcategories of other dichotomous variables is listed in Table 1. Neither tPA nor uPA activity was associated with age, axillary node status, or histologic type. No significant differences were noted between median values for the tPA and uPA activities in node-negative and node-positive subgroups.

View larger version (108K): [in this window] [in a new window]   Fig 1. In situ zymography of three breast tumor specimens with various caseinolytic activities (2.5 hours of incubation at 37°C). The global PA-dependent caseinolysis (ie, uPA + tPA activities) is shown, resulting predominantly from (A) uPA-mediated activity, (B) uPA and tPA combined activity, (C) and mainly from tPA-mediated activity.

View larger version (30K): [in this window] [in a new window]   Figure 2. Box and whisker plots of the distribution of uPA and tPA caseinolytic activities in 201 primary breast cancers after 2.5 hours of incubation. The bold line in the box shows the median value.

View larger version (14K): [in this window] [in a new window]   Figure 3. Overall survival curves according to tPA activity (cutoff at first quartile) for the whole population. At 60 months, there were 31 patients at risk with tPA less than 4% and 111 patients at risk with tPA 4%.

View larger version (14K): [in this window] [in a new window]   Figure 4. Overall survival curves according to tPA activity (cutoff at first quartile) for the axillary node-negative patients. At 60 months, there were 20 patients at risk with tPA less than 4% and 65 patients at risk with tPA 4%.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
By using a functional assay, we demonstrate here that breast cancers express active enzymes capable of catalyzing plasmin formation. We confirm that uPA and tPA are the proteases responsible for plasmin generation and that tumors display wide heterogeneity in the amounts of enzymes produced. Semiquantitative evaluations indicate that a majority of tumors express moderate to high levels of uPA-mediated enzymatic activity while a minority of tumors are associated with high tPA production. In previous studies, the median fraction of total PA/plasmin activity accounted for by tPA was reported to range from 13% to 86%.^30,31 Our observation (median 17%) is close to the data reported by Mira-y-Lopez et al (13%).³⁰ Noteworthy, the assay used by Yamashita et al was biased in favor of an excess amount of tPA.³¹ High tPA levels were previously associated with ER-positive/PR-positive and low-grade tumors.^16-18,32 The absence of inverse correlation between tPA levels of activity and nodal status is somewhat surprising, but similar results have been shown when uPA or PAI-1 were reported to be independent prognostic factors of survival.^12,18,33,34 High levels of uPA-mediated activity in primary tumors failed to show an independent prognostic value. In contrast, we show that reduced tPA-mediated catalytic activity is associated with adverse OS and that downregulation of tPA enzymatic activity constitutes an independent prognostic factor, in particular for node-negative patients.

Numerous investigations have documented the prognostic impact of the PA/plasmin system in breast cancer. Raised intratumoral contents of uPA and of uPAR have been shown to correlate with poor clinical outcome,^10-13,35 while high tPA levels have been associated with more favorable evolution.^14,15,17-20 Paradoxically, increased PAI-1 levels have also been found to carry prognostic value and in multivariate analysis to be a stronger prognostic indicator of unfavorable outcome than uPA itself.^13,20-22,36 Interestingly, some studies did not show an independent impact on survival of uPA or PAI-1 in premenopausal or node-negative women.^36,37 By minimizing the prognostic impact of uPA-mediated activity and by emphasizing the contribution of reduced tPA-mediated activity as an independent prognostic factor, our observations diverge from data obtained by immunometric determinations. These apparent discrepancies are most likely attributable to differences in the methodological approaches: estimations based on antigen recognition fail to assess enzymatic activities, while histologic zymography evaluates the net proteolytic activities available to tumoral tissues by taking into account the interactions of both enzymes with their inhibitors PAI-1 and PAI-2.

The biologic plausibility of an association between elevated uPA production and unfavorable clinical outcome rests on the consideration that the enzyme promotes normal tissue invasion and metastasis formation via plasmin generation, a broad-spectrum proteolytic enzyme capable of degrading most extracellular proteins. However, localization studies in breast cancer have shown that uPA is mainly produced by stromal cells and not by tumor cells,^9,38 suggesting that the functional consequences of upregulated uPA production may not be restricted to the mediation of tissue invasiveness. In that context, the lack of a significant association between the amount of uPA-mediated catalytic activity and prognosis raises further doubts about the exact contribution of uPA in breast cancer biology. By integrating the respective actions of PA and PAI, our results provide information susceptible to clarify the apparent paradox of the reported association between elevated PAI-1 levels and unfavorable outcome in breast cancer. Localization studies have shown that tPA and PAI-1 are predominantly produced by endothelial cells in diverse carcinomas;²⁸ as previously demonstrated in colorectal neoplasia, downregulation of tPA-mediated plasmin formation appears to result from the concomitant production of tPA and PAI-1 in the neovascular compartment of tumoral tissues.²⁸ As for uPA, the functional repercussion of tPA production on tumor behavior remains speculative. Several hypotheses have been raised to explain the favorable prognostic impact of high tPA content in breast cancer. The presence of ER in breast carcinomas is generally thought to be associated with good prognosis. Because tPA is an estrogen-induced protein, higher tPA amounts have been thought to reflect a functional ER pathway.^30,32 However, tPA has also been found to be a factor independent of ER in predicting survival,^15,17,18 and in our series, tumors of patients who relapsed or died had lower tPA activity regardless of the type of adjuvant therapy. Furthermore, tPA has been shown to participate in the control of apoptosis³⁹ and angiogenin formation,⁴⁰ both processes susceptible to influence more directly tumor progression. Our findings in breast cancer are similar to previous observations obtained in other epithelial tumors; they indicate that reduced tPA catalytic activity is occurring in the vascular compartment and that downregulation of tPA activity results mainly from the concomitant production of PAI-1. This spatial distribution may challenge interpretations conferring a role for PAI-1 in cellular adhesion control and neoangiogenesis to explain the adverse impact of PAI-1 expression in breast cancer;²³ for instance, inadequate fibrinolysis because of reduced tPA enzymatic activity within tumoral vessels may promote the formation of tumor emboli.

We show here that marked and contrasting alterations in the proteolytic balance governing plasmin formation take place in breast cancer. By demonstrating that downregulation of tPA-mediated catalytic activity is an adverse prognostic factor, we add evidence arguing against an exclusive role of plasmin in tissue invasion. Although the clinical utility of our functional assay remains to be prospectively validated, it offers a novel means to identify high-risk, node-negative patients. Moreover, these data give some new biologic insights for the ongoing prospective trials that evaluate the impact on survival of antithrombotic drugs combined with chemotherapy in breast cancer treatment.⁴¹

	ACKNOWLEDGMENTS

 
P.O.C. is supported by grants from la Ligue Genevoise Contre le Cancer and Cancer and Solidarité Fondation, Geneva, Switzerland.

We are indebted to the family of a patient who helped us accomplish this study. We particularly wish to thank Bernadette Mermillod and Dr William D. Foulkes for helpful comments and Gabriel Andrey for technical assistance.

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Submitted September 14, 2000; accepted February 16, 2001.

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