Affiliations of authors: R. W. Stephens, I. J.Christensen, K. Danø, N. Brünner, The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark; H. J. Nielsen, Department of Surgical Gastroenterology, Hvidovre Hospital, Copenhagen; O. Thorlacius-Ussing, Department of Surgical Gastroenterology, Aalborg Hospital, Denmark; S. Sørensen, Department of Clinical Biochemistry, Hvidovre Hospital, Copenhagen.
Correspondence to: Ross W. Stephens, Ph.D., The Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark (e-mail: stephens.r{at}finsenlab.dk).
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ABSTRACT |
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INTRODUCTION |
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It is now well established that proteolytic enzymes produced by cancer cells and/or cells in the tumor stroma are involved in the intensive tissue remodeling that accompanies cancer cell invasion and metastasis (1-3). Among several enzyme systems expressed in cancer tissue, plasmin generated by urokinase plasminogen activator (uPA) is thought to play a key role in tissue degradation (4,5), activation of pro-metalloproteases (6), activation of cytokines (7), and angiogenesis (8), all of which could lead to an increase in the metastatic potential of the cancer cells. uPA is secreted as an inactive proenzyme (9), which localizes on cell surfaces (10) by binding through its epidermal growth factor-like domain to a specific high-affinity cell surface receptor (urokinase plasminogen activator receptor [uPAR]) (11). uPAR is a cell surface glycoprotein with a molecular mass of 55-60 kd and consists of three homologous protein domains, all of which are required for high-affinity binding of uPA. At the carboxyl terminal of domain 3, uPAR is anchored to the cell membrane by a glycosylphosphatidylinositol moiety (11). Compared with activation of uPA proenzyme in free solution, activation of uPAR-bound proenzyme is strongly enhanced as a result of the proximity of cell surface-bound plasminogen and plasmin (12-14). There is considerable experimental evidence that uPAR is functionally involved in cancer invasion (15-18), consistent with its ability to concentrate and enhance uPA activity on cell surfaces (10).
We and others have previously reported the association of uPAR levels in tumor tissues with prognosis for patients with squamous cell lung cancer (19), colon cancer (20), and breast cancer (21). In the latter study, it was found that a fraction representing soluble uPAR (suPAR), i.e., uPAR protein without the glycolipid anchor, was inversely related to survival. We have previously found that suPAR is normally present at low levels in the blood (22) but that the levels are increased in patients with non-small-cell lung cancer (23), metastatic breast cancer (24), Dukes' stage D colorectal cancer (24), and ovarian cancer (25), probably as a result of the release of suPAR into the circulation from tumors.
We have now undertaken a retrospective study of suPAR levels in preoperative plasma from 591 patients who had surgery for colorectal cancer. We tested for an association between the levels of suPAR and patient survival.
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SUBJECTS AND METHODS |
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Sampling of blood. Blood samples (5 mL) for suPAR analysis were taken preoperatively from patients on the day of their surgery and were collected in ethylenediamine tetraacetate (EDTA)-containing anticoagulant tubes (Becton Dickinson, Mountain View, CA). Plasma was separated within 1.5 hours and stored frozen at -80 °C until analyzed. Immediately before suPAR assay, the plasma samples were thawed rapidly at 37 °C and diluted 1 : 10 as previously described (24). Blood (5 mL) was also collected preoperatively for serum CEA measurement, by use of the ImmuliteTM CEA assay kit (Diagnostics Products Corporation, Los Angeles, CA).
suPAR analysis. The plasma concentration of suPAR was determined by use of a modification of a new kinetic enzyme-linked immunosorbent assay (ELISA) that meets strict criteria of specificity and sensitivity (24). Briefly, the ELISA consisted of a catching layer of the monoclonal antibody R2 and a detection layer of rabbit polyclonal antibodies to human uPAR, i.e., an inversion of the two layers in the described ELISA (24). This modification eliminated a background signal found in approximately 4% of individuals, independent of cancer diagnosis. The R2 monoclonal antibody has high affinity for domain 3 of the human uPAR molecule, so that both the full-length (domains 1 + 2 + 3) and proteolytically cleaved (domains 2 + 3) forms of suPAR (11) were measured by this assay. A monoclonal anti-rabbit immunoglobulin/alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, MO) was used in the final step, so that rate measurements for phosphatase enzyme activity could be automatically collected over a 1-hour incubation period in a Ceres 900TM plate reader (Bio-Tek Instruments, Winooski, VT) (24). KinetiCalc software (version 2.16; Bio-Tek Instruments) was used to manage the data and to calculate the rate of color change for each well by linear regression analysis. The suPAR concentration of each plasma sample was calculated by use of a four-parameter fitted standard curve computed from the rates for the recombinant suPAR standards. The absolute concentration of the recombinant suPAR standard was previously determined by amino acid analysis (27). The limit of detection for the assay was 3 pg/mL or approximately 0.3% of the median concentration found in donor plasma. The intra-assay variation for a plasma pool was 4.8% (n = 21), and the inter-assay variation for 30 successive assays of aliquots of the same plasma pool (on different days) was 7.6%. suPAR was evidently stable in frozen plasma for at least several months. When recombinant suPAR was added to plasma as an internal control, 97% of the standard could be detected by ELISA. Specificity was rigorously controlled by plasma immunoabsorption experiments as described previously (24).
Statistical analyses. The SAS® software package (version 6.12; SAS Institute, Cary, NC) was used to manage the patient data and to perform all statistical analyses. The plasma suPAR measurements were log transformed (i.e., ln suPAR) for survival analysis; for graphical representation, the patients were stratified into four groups based on the suPAR value, such that each stratum yielded an equal number of events (deaths of patients). For each Dukes' stage, the median value of plasma suPAR determined for all patients was used for arbitrary dichotomization. The Kaplan-Meier method was used to estimate survival probabilities, and the logrank test was used to test for equality of strata. The Cox proportional hazards model was used for analysis of continuous covariates as well as for multivariate analysis. The assumption of proportional hazards was verified graphically. Rank statistics were used to calculate correlation coefficients and to test hypotheses on location. Tests of independence were done with the use of the chi-squared test. The significance level was set to 5%. The expected survival for patients in each Dukes' stage was calculated for age- and sex-matched cohorts (28) by use of official vital statistics recorded and tabulated for the Danish population (29). All P values reported are two-sided.
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RESULTS |
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suPAR was measured by a modified kinetic ELISA method in
EDTA-anticoagulated plasma obtained preoperatively from each patient
with colorectal cancer. All the plasma samples had measurable levels of
suPAR, with a median value of 1.37 ng/mL (range, 0.46-8.0 ng/mL). When
the patient data were broken down according to Dukes' stage, there
were statistically significant differences in plasma suPAR levels, with
Dukes' stage A being the lowest and Dukes' stage D being the highest
(Kruskal-Wallis test, P = .001). Nevertheless, it was clear
that higher levels of plasma suPAR were not restricted to advanced
disease. The means, standard deviations, medians, and interquartile
ranges for plasma suPAR are summarized in Table 1.
There was a significant but relatively weak correlation between the
suPAR levels and age of patients with cancer (Spearman's rho = .28;
P<.0001), but no significant association was found between
sex and level of suPAR (Wilcoxon rank sum test, P = .11). The
median CEA level was 3.8 ng/mL (range, 0.34-9800 ng/mL), and there was
a significant but relatively weak correlation between the level of CEA
and the level of suPAR (Spearman's rho = 0.31; P<.0001).
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Treated as a continuous variable, the log-transformed plasma level
of suPAR (i.e., ln suPAR) was statistically significantly associated
with survival (Cox regression model, P<.0001; Table
2); higher levels of suPAR were found in plasma from
patients who had a shorter survival. For survival analysis in which the
Kaplan-Meier method was used, the patients were divided into four
strata based on the plasma suPAR value, such that each stratum yielded
an equal number of events (deaths of patients; see Fig.
1).
This procedure produced strata with different
hazard ratios (HRs) (see legend to Fig. 1
), and the analysis
indicated a continuously increasing risk of mortality with increasing suPAR level.
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Patients with Dukes' stage C disease who had plasma suPAR levels above the median
level (72 of 170 patients, 42%) had an HR of 1.8 (95% CI = 1.2-2.6; P = .005) relative to those who had levels below the median level (Fig. 2). Thus, the
median plasma suPAR level could also divide patients with more advanced, less surgically
curable disease into groups with differing survival. However, virtually all patients with
Dukes' stage C disease had a significantly higher death rate than an age- and sex-matched
cohort drawn from the general population.
Division by the median in patients with Dukes' stage D disease also showed a
relationship between suPAR level and prognosis (Fig. 2), but all patients
with this disease stage
had relatively poor survival due to disseminated disease. In summary, the results that we
obtained using an arbitrary cut point for analyses of prognosis in each Dukes' stage show
that the suPAR level is a statistically significant prognostic factor in Dukes' stages B, C,
and D, but its greatest potential value is as a clinical marker in early stage disease.
Multivariate Analysis
Multivariate Cox analysis of the survival data was performed and
included the clinical parameters Dukes' stage, sex, and age, as
well as CEA dichotomized by its median level (3.8 ng/mL) and suPAR
analyzed continuously as the log of suPAR concentration (i.e., ln
suPAR). The results are summarized in Table 2. Dukes' stage was
statistically significantly associated with survival; patients with
Dukes' stage D disease had an HR of 7.5 (95% CI = 5.6-10.2)
compared with those with Dukes' stage B disease, and patients with
Dukes' stage C disease had an HR of 2.2 (95% CI = 1.6-3.0)
compared with those with Dukes' stage B disease. Age scored in years
at entry was statistically significantly associated with survival
(P = .02), whereas serum CEA (P = .18) and sex
(P = .19) were not. However, it was also notable that in the
multivariate analysis high levels of plasma suPAR were found to be an
independent prognostic indication for shorter overall survival, with an
HR of 1.9 (95% CI = 1.4-2.5; P<.0001) for an increase
in ln suPAR of 1.0 or a 2.7-fold increase in suPAR concentration.
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DISCUSSION |
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In the group with Dukes' stage C disease, and even more so in the group with Dukes' stage D disease, almost all patients (and thus all suPAR levels) were found to have a greater risk than their respective matched population cohorts. The difference in this regard between patients with Dukes' stage B disease and patients diagnosed with more advanced disease implies that the preoperative plasma level of this functional marker can be a useful measure of the invasive potential of the tumor (and thus survival) in only early stage disease, since survival at later stages of tumor dissemination is predominantly determined by the difficulty experienced in complete surgical removal of the patient's tumor. The potential value of suPAR as a clinical marker is therefore likely to be greatest in managing the disease in patients with Dukes' stage B, where the cancer is still potentially curable by surgery but the expected mortality is statistically significant. Plasma suPAR levels could be used in identifying those patients with Dukes' stage B disease who are at high risk and who, therefore, can potentially benefit most from adjuvant treatment. Conversely, plasma suPAR levels could be used to identify patients with Dukes' stage B disease who should not be subjected to adjuvant treatment because they do not have a significantly increased risk from their disease. Note that none of the patients in this study received adjuvant therapy.
This study was based on a newly developed suPAR kinetic ELISA method that has a high level of sensitivity and specificity in the measurement of suPAR levels in plasma and serum (24). The results are consistent with those of previous studies performed on extracts of resected tumor tissue from colorectal cancer (20), lung cancer (19), and breast cancer (21); those studies demonstrated that high levels of uPAR in the tumor tissue were related to poor patient survival. Furthermore, we found earlier that, in patients with non-small-cell lung cancer (23) and in patients with advanced breast cancer (24), colorectal cancer (24), and ovarian cancer (25), plasma concentrations of suPAR are statistically significantly increased compared with those in healthy individuals. This increase is most likely the result of enzymatic cleavage of uPAR from the surface of tumor cells and/or stromal cells. In colon cancer, uPAR messenger RNA expression and immunoreactivity are enhanced in both tumor cells and tumor-infiltrating macrophages (33).
In conclusion, invasion and metastasis are dependent on proteolytic activity in tumors; accordingly, a large number of studies involving different cancer types, including colorectal cancer, have shown that tumor tissue levels of uPA, uPAR, and type 1 plasminogen activator inhibitor are associated with shorter survival (19-21). Our present findings on plasma suPAR levels in patients with colorectal cancer add new data to these studies and strengthen the view (25,32,34) that measurement of functionally relevant blood parameters before resection of a primary tumor may provide valuable prognostic information for patients with cancer. The potential for application of suPAR as a useful clinical marker remains to be determined, but this should be sought in the setting of early stage disease.
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NOTES |
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We thank the RANX05 colorectal cancer study group and the blood bank at the Hvidovre Hospital for the collection of the plasma and serum samples. We also thank Maria Hamers for her excellent technical assistance in analyses of samples.
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Manuscript received July 10, 1998; revised March 12, 1999; accepted March 20, 1999.
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