Plasma Plasminogen Activator Inhibitor-1 (PAI-1) Levels in Breast Cancer – Relationship with Clinical Outcome

Discussion

The coagulation/fibrinolytic systems include a large spectrum of proteolytic enzymes with pathophysiological functions in a variety of processes, such as hemostasis, wound repair and tissue remodeling, tumor invasion and angiogenesis. Although only poorly-understood, up-regulation of coagulation or fibrinolytic factors has been associated with an unfavorable prognosis in breast cancer (1, 2, 21). Tumor PAI-1 expression, for example, was shown to have a strong and independent prognostic value in both node-negative and node-positive primary breast cancer (6). Moreover, in patients with node-negative breast cancer, those with low levels of uPA/PAI-I in their primary tumor were shown to have a very good prognosis, and may, thus, be candidates for being spared the burden of adjuvant chemotherapy (5, 6, 10). Based on these observation, the American Society of Clinical Oncology (ASCO) stated in the 2007 guideline update that “uPA/PAI-1 measured by ELISAs on a minimum of 300 mg of fresh or frozen breast cancer tissue may be used for the determination of prognosis in patients with newly-diagnosed, node-negative breast cancer” (11). Further support for these recommendations was recently provided by the final analysis of the prospective multicentre Chemo-N0 trial (10) and by two cost-effectiveness studies demonstrating that uPA/PAI-1 testing is beneficial for patients with breast cancer with no lymph node involvement (22, 23), with a return-on-investment ratio of 8.4:1, which was further increased by indirect cost savings (23).

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Figure 3.

Kaplan–Meier curves of overall survival (OS) of patients with breast cancer. Comparison between patients with low (dotted line) and high (solid line) plasma PAI-1 levels (cut-off ≤30 ng/ml). A: Kaplan–Meier curves for the entire population (n=152, 10 year OS rates 45% vs. 67%). B: Kaplan–Meier curves for non-metastatic breast cancer (n=134, 10-year OS rates 58% vs. 84%).

These recommendations, however, are far from being introduced into clinical practice since, despite the introduction of novel assays needing very little amounts of tissue (24), tumor uPA/PAI-1 expression still necessitates fresh/frozen tumor samples in order to perform the analysis, limiting its determination to particular research settings (12). Thus, the possibility of using a plasma matrix to determine the prognostic value of PAI-1 is appealing, as it might allow a more widespread use of this marker. Unfortunately, available data are scarce and controversial (13-16), probably because of incorrect sample choice and processing (choice of anticoagulant, temperature conditions and elapsed time from sampling to centrifugation), ultimately leading to in vitro platelet activation and uncontrolled release of PAI-1. The latter, in fact, is mainly produced by endothelial cells and megakaryocytes, as well as smooth muscle cells, and is then stored in platelets (3). Most studies on plasma PAI-1 distribution in breast cancer were performed on samples using EDTA or heparin as anticoagulants, which may induce platelet changes over time (24). For example, the independent prognostic role of plasma PAI-1 in breast cancer was rejected by some authors on the basis of a lack of correlation between plasma and tumor tissue levels of PAI-1, suggesting that determination of this factor in plasma does not reflect its concentration in tumor tissue (15). The use of EDTA-anticoagulated samples in this study might have affected plasma PAI-1 levels, also dependent on the elapsed time between blood withdrawal and processing (25). This effect appears to be unpredictable and must be controlled, either with the use of different anticoagulants or by standardizing the duration between sampling and analysis (26).

In our study, samples were collected as part of a biobank project, the BioBIM institutional project, using standard operating procedures and information and communications technology (ICT) tools to monitor temperature conditions (27) and in which all preanalytical variables, including elapsed time between blood withdrawal and processing, are encapsulated in a standard code calculated by means of a dedicated software tool (28). This ensures relative homogeneity among the samples used in a given study and minimizes the differences in sample analyses. The results obtained here using such a controlled set of samples support the idea that plasma PAI-1 levels might be used as a prognostic indicator in women with breast cancer. In our analysis, in fact, elevated plasma PAI-1 levels were associated with increasing tumor stage and disease relapse, and were predictive of shorter RFS and OS. This prognostic value was fully retained in node-positive cases, in agreement with the results obtained in the EORTC pooled analysis showing that tumor PAI 1 expression had a strong and independent prognostic value in node-positive primary breast cancer and reporting HRs similar to those found in our study (6). Moreover, the prognostic value of plasma PAI-1 was confirmed in the subgroup of women treated with adjuvant chemotherapy, with a 5-year RFS rate of 59% in women with PAI-1 levels above 30 ng/ml vs. 84% in those with PAI-1 levels below this cut-off, suggesting a benefit from adjuvant therapy in patients with high plasma PAI-1 levels, as previously shown for tumor PAI-1 expression (7).

Tumor heterogeneity, however, complicates this picture and PAI-1 production can be increased by a wide array of soluble factors released into the tumor microenvironment (e.g., vascular endothelial growth factor and inflammatory cytokines). In this respect, we must also take into consideration the fact that an elevated plasma PAI-1 level might be the result of a hypercoagulable state (17), which is a common finding in patients with solid cancer. The possibility of an involvement of coagulation activation in determining PAI-1 plasma levels could, at least partially, explain the lack of correlation between plasma and tumor tissue levels of PAI-1 discussed above (15). In this study, APC-dependent thrombin generation and HS D-dimer determination were used as markers of abnormal coagulation/fibrinolysis in order to discriminate the relative contribution of coagulation activation to the prognostic value of elevated plasma PAI-1. Of interest, plasma PAI-1 correlated with HS D-dimer level, but no association was found with APC-dependent thrombin generation, although a hypercoagulable state was present in patients as evidenced by impaired ThromboPath values. Moreover, neither HS D-dimer nor ThromboPath were associated with survival outcomes, further suggesting that the tumor-associated plasminogen activator pathway, not the coagulation pathway, is a key distinguishing feature of more aggressive breast cancer phenotypes, as recently demonstrated in an in vitro model (29). This finding is of importance given that both coagulation and fibrinolytic pathways have been explored for therapeutic purposes (30, 31) and that blockade of PAI-1 has recently been reported to exert anticancer effects by modulating the function of uPA receptor (32). Of particular interest is the recent demonstration that SK-216, a specific PAI-1 inhibitor, was capable of reducing tumor size and extent of metastases in an animal model (33). Given that PAI-1 can be produced by host and tumor cells in the tumor microenvironment, a PAI-1-deficient mouse model was employed in that study to determine whether host or tumor PAI-1 was more crucial in tumor progression and angiogenesis. The results showed that host, not tumor, PAI-1 levels were able to control both processes, further suggesting that administration of SK-216 PAI-1 inhibitor exerts its antitumor actions through interaction with host PAI-1, regardless of tumor PAI-1 level (33).

On this context, the possibility of gaining prognostic information from plasma PAI-1 determination is of clinical relevance as it might provide the basis for a tailored therapeutic approach. There are, of course, some limitations to our study that need to be acknowledged. The most obvious resides in the small sample size that might have weakened the statistical power. Moreover, the study was a retrospective analysis, although all eligible consecutive patients within the designated timeframe were included and all measurements were performed while blinded to the patient outcome. On the other hand, the strength of our analysis is represented by the use of samples collected and processed using standard operating procedures, which ensures relative homogeneity among the samples used and minimizes the difference in sample analyses.

In conclusion, enhanced PAI-1 expression contributes to outcome measures in breast cancer patients and its determination might provide important information in risk stratification of patients with breast cancer. The potential limitations described above suggest that these results should be regarded with caution and that detailed experimental evaluation is needed before the ultimate prognostic significance of PAI-1 in breast cancer can be established. Additional studies are required to prospectively evaluate the clinical value of plasma PAI-1 in breast cancer. Nevertheless, we believe that its determination might provide important information in risk stratification, and encourage future investigations addressing the role of plasma PAI-1 levels in the management of patients with breast cancer, and in providing the rationale for new therapeutic strategies.