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Clinical Investigations |
The Finsen Laboratory, Rigshospitalet [A. N. P., I. J. C., R. W. S., K. D., N. B.]; Department of Tumor Endocrinology, the Danish Cancer Society [P. B.]; and the Danish Breast Cancer Cooperative Group [H. T. M.], DK-2100 Copenhagen, Denmark
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.002). The tumor levels of complex,
uPA, and PAI-1 were all associated with survival; high complex levels
predicted longer recurrence-free survival (P = 0.03) and overall survival [OS (P = 0.005)], whereas high uPA or PAI-1 levels significantly
predicted shorter survival. In multivariate Cox analysis, the only
parameters that independently predicted survival were total PAI-1 level
and lymph node status for recurrence-free survival and OS and,
additionally, steroid hormone receptor status and grade for OS. This is
the first demonstration of a relationship between uPA·PAI-1 complex
tumor level and patient survival. However, total PAI-1 level showed
superior prognostic power. Additional studies are needed to understand
the relationship of these parameters to cancer biology and to assess
the clinical utility of the uPA·PAI-1 complex. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A central role of
uPA2
in cancer
invasion and metastasis is well established (4
, 5)
. This
Mr 52,000 serine proteinase is
secreted as an inactive precursor, pro-uPA, that binds to a specific
cell surface receptor, uPAR, where formation of active uPA leads to
localized potentiation of plasminogen activation (6)
. The
plasmin thus generated mediates broad spectrum proteolysis,
facilitating cell migration, proliferation, and invasion
(4)
. uPA activity, whether in solution (7)
or
receptor-bound (8
, 9)
, is rapidly neutralized by its
specific high-affinity inhibitors, PAI-1 and PAI-2. PAI-1, the
principal physiological uPA inhibitor, is a
Mr 52,000 protein secreted in an
active but conformationally unstable form that gradually loses activity
unless it is stabilized by binding to the extracellular matrix protein
vitronectin (10
, 11) . Active PAI-1 forms an
equimolar, SDS-stable, Mr 100,000,
enzymatically inactive complex with active uPA, whereas the inactive
forms of these two components cannot form a complex (12
, 13)
. By a process dependent on uPAR and the
low-density lipoprotein receptor-related protein
(
2-macroglobulin receptor), cell surface
uPA·PAI-1 complex is internalized and ultimately degraded in
lysosomes (5
, 14)
.
ELISA measurements of levels of uPA, PAI-1, PAI-2, and uPAR in tumor tissue extracts have enabled several studies of their relationship to cancer patient survival (5 , 15) . High levels of uPA and uPAR were found to predict short survival, consistent with the proposed role of the protease in facilitating matrix degradation and the proposed role of the receptor in promoting uPA activity on cell surfaces. Surprisingly, high levels of PAI-1 have also been reproducibly found to be predictive of short survival, despite the known ability of PAI-1 to inhibit uPA activity. On the other hand, high levels of PAI-2 seem to predict longer survival in breast cancer patients.
ELISAs used in the prognostic studies were developed to measure the total amount of each molecule, including proforms, active, inactive, and complex-bound forms. However, to further investigate the relationship of tumor tissue uPA activity and its PAI-1 inhibition to patient survival, we decided to measure the level of uPA·PAI-1 complex because this parameter selectively represents that minute fraction of uPA (16 , 17) that has been activated from proenzyme and subsequently inactivated by active PAI-1. For this purpose, we have recently developed a kinetic uPA·PAI-1 complex ELISA for extracts of patient tumor tissue (18) . We now report the results of an exploratory retrospective study of the complex levels in 342 primary breast tumors to test for associations with clinicopathological parameters, RFS, and OS of the patients, including the important subgroup of axillary lymph node-negative patients.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, of whom 164 were free of axillary
lymph node metastases, and 178 were lymph node positive at the time of
primary surgery. Lymph node-negative patients with a tumor larger than
5 cm in diameter (pathologists’ estimate), patients in the DBCG-82
protocol with skin or thoracic wall invasion, premenopausal patients in
the DBCG-89 protocol with a ductal carcinoma showing grade II or III
cell anaplasia (20)
, and all lymph node-positive patients
received adjuvant therapy. The median patient age at time of surgery
was 56 years (range, 29–75 years), and the median follow-up time at
the time of data analysis was 67 months (range, 53–101 months). The
anticipated follow-up period was intended to be 10 years, as described
previously (19
, 21)
, and the information was updated
annually. During the observation period, there were 130 recurrences and
113 deaths. Recurrence was confirmed by biopsy and/or other relevant
diagnostic procedures and defined as the appearance of new breast
cancer lesions after primary surgery. The actual cause of death was not
available for the patients, therefore recording of survival was based
on death from all causes. Tumor tissue samples were obtained in
accordance with the Helsinki Declaration.
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Assays.
All assays were performed without knowledge of the clinicopathological
data and patient outcome.
Total protein concentrations in the extracts were measured by the Bradford method for protein analysis (26) , using a protein assay kit using BSA as a standard (Bio-Rad, Hercules, CA). For reference purposes, a pool of tumor tissue extracts was made, comprising equal volumes from 17 randomly chosen tumor tissues. The pool had a protein content close to the mean value found for extracts from all of the patients, and the interassay variation for protein measurement of this pool was 8.3% (n = 9 assays).
Total uPA concentrations and total PAI-1 concentrations in extracts were determined using ELISA kits (Oncogene Science Diagnostics, Cambridge, MA) as described previously (27 , 28) . The uPA assay has a detection limit of 25 pg/ml and detects the uPA antigen with equal efficiency, regardless of whether it is present as pro-uPA, uPA, uPA·PAI-1, or uPA·uPAR complexes with closely similar efficiency. The interassay variation for the reference pool was 9% (n = 9 assays). The PAI-1 ELISA has a detection limit of 100 pg/ml and measures the PAI-1 antigen with equal efficiency whether present as the latent or the active form. However, the efficiency of measurement of PAI-1 in complex with plasminogen activators is approximately 50%.3 The interassay variation was 3.9% (n = 9 assays).
The concentration of uPA·PAI-1 complex in extracts was determined using a new kinetic ELISA that meets strict criteria of specificity and sensitivity (18) . In short, this sandwich ELISA consists of a coating with two murine monoclonal antihuman PAI-1 capture antibodies. After blocking the remaining protein binding sites, standard dilutions of purified, stable uPA·PAI-1 complex and diluted extracts were incubated. The complex standard was prepared from purified high-molecular weight active human uPA (Serono, Aubonne, Switzerland) and activated human PAI-1, purified from conditioned media of dexamethasone-treated HT-1080 fibrosarcoma cells (18) . The standard was measured with a detection limit of 8 pg/ml, and neither free uPA nor free PAI-1 was detected by the assay. To block ex vivo formation of the uPA·PAI-1 complex from free uPA and PAI-1 in the diluted extracts during incubation of the assay plates, p-nitrophenyl guanidinobenzoate (an inactivator of uPA; Sigma, St. Louis, MO) was added to the dilution buffer (18) . A combination of three biotinylated monoclonal antihuman uPA antibodies was used as the detection reagent, and the data were collected as kinetic measurements of bound streptavidin alkaline phosphatase activity (Dako, Glostrup, Denmark). The interassay variation of the extract pool for this study was 13% (n = 9 assays).
Western blotting of the uPA·PAI-1 complex was carried out as described previously (29) . Briefly, 35 µl of pooled crude tissue extract were diluted 3:1 in Laemmli sample buffer, the sample was electrophoresed on a 10% SDS-polyacrylamide minigel, and the separated proteins were blotted electrophoretically onto a polyvinylidene difluoride membrane. After blocking, the membrane was sequentially incubated with rabbit polyclonal antihuman PAI-1 antibodies and alkaline phosphatase-conjugated monoclonal antirabbit antibody (Dako). Finally, color was developed with the phosphatase substrate solution nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate.
Statistical Analyses.
Spearman rank correlation coefficients (rs) were
calculated for matched pairs of sample measurements of total uPA, total
PAI-1, and uPA·PAI-1 complex. Tests of hypotheses on the location
parameter for these variables were done using Wilcoxon’s rank-sum test
(the Kruskal-Wallis test was used for comparisons of more than two
groups). Survival curves for the time to recurrence and the time to
death were estimated by the Kaplan-Meier method. It was prespecified
that total uPA, total PAI-1, and uPA·PAI-1 complex were to be scored
first as the actual value (log transformed) and then as indicator
variables for each quartile. The ratios of uPA·PAI-1 complex to total
uPA and PAI-1, respectively, were considered in a similar manner. The
log-rank test was used to test for equality of strata. The Cox
proportional hazards model was used for analysis of survival. The
assumption of proportional hazards was verified graphically. Reduced
models were obtained by backward selection in a monotonic fashion; the
exclusion level was 0.05. P < 0.05 was
considered significant, and the 95% CI for each RR was calculated.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, primary tumor tissue
detergent extracts were analyzed for total protein concentration, total
uPA, total PAI-1, and uPA·PAI-1 complex level. For comparison, Table 1
also shows the clinicopathological data for the other breast cancer
patients (n = 10,576) who were operated on
during the study period. It appears that the study patients in general
presented characteristics that were representative of all of the
patients operated on during the study period. Minor exceptions were
related to patient age (no patients >75 years old were included in any
protocol) and tumor size (the availability of frozen tissue was a
prerequisite for this study). The characteristics of the study group
and other breast cancer patients appeared comparable with regard to
histological type, tumor grade, and steroid hormone receptor or lymph
node status (Table 1)
.
Levels of uPA·PAI-1 Complex, Total uPA, and Total PAI-1.
One tissue specimen was lost during handling, and thus only 341
extracts were analyzed by ELISA. All of the extracts analyzed had
measurable levels of uPA·PAI-1 complex, total uPA, and PAI-1. Each
ELISA determination was referred to the protein concentration in the
same extract. Because the frozen tumor tissue had been stored for
several years before the extraction procedure, we first investigated
whether the storage time had any influence on the measured antigen
levels. Tests for any association between tissue storage time and the
measured parameters did not reveal any significant relationship. Also,
the presence of intact tumor tissue uPA·PAI-1 complex in extracts
stored for several months was demonstrated by Western blots (Fig. 1
, inset).
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shows the
percentiles plot for the level of complex found in the individual
extracts, and it is evident that the distribution is skewed toward the
higher values. For total uPA, the median value was 5.2 ng/mg protein
(range, 0.17–61 ng/mg protein), and for total PAI-1, the median value
was 2.4 ng/mg protein (range, 0.19–80 ng/mg protein). The
distributions of uPA and PAI-1 levels were also skewed toward the
higher values. uPA·PAI-1 complex was found to represent a minor,
variable fraction of total uPA (rs = -0.24; P < 0.0001; Fig. 2A
) as well as total PAI-1
(rs = -0.21; P < 0.0001; Fig. 2B
). In contrast, there was a close and
positive association between total uPA and PAI-1
(rs = 0.78; P < 0.0001; data not shown). We then determined the ratio of uPA·PAI-1
complex to total uPA and total PAI-1, respectively, for each extract by
dividing the complex value by the total value. The median value for the
complex:total uPA ratios was 0.13, and the median value for the
complex:total PAI-1 ratios was 0.27. Because the complex was only a
minor fraction of the total antigen levels and represented a
narrow range as compared with the total antigen levels, there was a
very close association between the ratios and the corresponding total
antigen levels (both, rs = 0.9;
P < 0.0001).
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shows the comparison of the
measured biochemical and clinicopathological parameters. It is
noteworthy that for uPA·PAI-1 complex, the levels were significantly
higher in lymph node-negative tumors than in lymph
node-positive tumors (P < 0.0001), and
complex levels were also higher in small tumors (P < 0.0001) and in those with a low grade of anaplasia
(P = 0.002). Conversely, total uPA or PAI-1
levels were higher in large tumors and those with a high grade of
anaplasia, whereas no significant association was found between nodal
status and uPA or PAI-1 levels (Table 2)
. To further illustrate the
association between complex and nodal status, Table 3
shows the frequencies of lymph node
involvement with the complex values divided into quartile groups.
Although a significant relationship (
2
test
for trend, P < 0.0001) between the
parameters could be demonstrated at the extremes, the relationship
broke down in the intermediate complex concentration range.
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]; increasing uPA levels were
associated with shorter survival. Comparing patients at the 75th
percentile value with those at the 25th percentile value, the RR was
1.2 (CI, 1.1–1.3) for RFS and 1.2 (CI, 1.1–1.4) for OS. Increasing
total PAI-1 levels also predicted shorter RFS and OS
(P < 0.0001 for both). When comparing
patients at the 75th and 25th percentile values, the RR for RFS was 1.4
(CI, 1.3–1.6), and the RR for OS was 1.6 (CI, 1.4–1.8).
Conversely, increasing levels of uPA·PAI-1 complex predicted longer
RFS and OS. However, the scoring of the complex by its log-transformed
value did not adequately fit a linear model in the analysis;
consequently, this approach was not pursued further. However, scoring
of the ratio of complex to total uPA or PAI-1 fitted the data well;
both ratios highly significantly predicted survival
(P < 0.0001 for both), with increasing
ratios associated with longer survival. For example, for the
complex:total PAI-1 ratio, the RR was 0.5 (CI, 0.4–0.6) for RFS and
0.4 (CI, 0.3–0.5) for OS when comparing patients at the 75th and 25th
percentile values. Clinicopathological parameters of prognostic
significance in the univariate Cox model were large tumor size, high
tumor grade of anaplasia, and positive lymph node status; each of which
predicted shorter survival, whereas positive steroid hormone receptor
status predicted longer survival (Table 4)
.
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) and OS
(P = 0.005) than patients with lower values.
A comparison of patients in the fourth and first quartiles gave a RR of
0.5 (CI, 0.3–0.8) for RFS and a RR of 0.4 (CI, 0.2–0.8) for OS. For
total uPA, high values were associated with a shorter survival [RFS,
P = 0.007 (Fig. 3B)
; OS,
P = 0.003, which was also the case for total
PAI-1 [for both RFS (Fig. 3C)
and OS,
P < 0.0001]. Moreover, divided by the
quartiles, the complex:total uPA and complex:total PAI-1 ratios clearly
separated the four patient groups with regard to prognosis. For
example, for the ratio to uPA, the RR was 0.4 for RFS (Fig. 4A)
for patients in the fourth
quartile versus those in the first quartile, and
for the ratio to PAI-1, the RR was 0.3 for RFS (Fig. 4B)
.
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, inset):
(a) those with long-term RFS; and (b) those with
short-term RFS (Fig. 5)
. A comparison of patients with a tumor tissue
uPA·PAI-1 complex value above the optimal cutoff value with those
with a value below the optimal cutoff value gave a RR of 0.4 for RFS.
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Firstly, all studied parameters were included in the model. In this
analysis, the only parameters that independently and significantly
predicted survival were total PAI-1 and lymph node status for RFS and
OS (P < 0.0001 for all) and additionally
steroid receptor status for OS (Table 5)
.
High PAI-1 was strongly associated with both short RFS with a RR of 3.8
(CI, 2.2–6.4) and short OS with a RR of 3.5 (CI, 1.9–6.3) when
comparing patients in the fourth quartile with those in the first
quartile. If tumor grade of anaplasia, which was only available for the
ductal carcinomas, was included in the model, grade II or III
versus grade I gave a RR of 2.4 (CI, 1.3–4.7) for OS
(P = 0.007), and the level of significance
for independent prediction of RFS was 0.06. The results for the other,
previously included parameters did not change appreciably (data not
shown).
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Subgroup Analysis.
A separate exploratory analysis of the clinically important
node-negative patient subgroup was performed, with the understanding
that the uPA·PAI-1 complex levels were significantly higher in lymph
node-negative tumors than in node-positive tumors. In the node-negative
group of 164 patients, there were only 39 recurrences and 25 deaths
during the observation period. Thus, in univariate analysis using the
uPA·PAI-1 complex, total uPA, and PAI-1 quartiles to divide this
patient material, only PAI-1 showed a significant association with
survival (data not shown). For PAI-1, a comparison of patients in the
fourth and first quartiles gave a RR of 6.1 (CI, 2.2–17) for RFS
(P = 0.0002) and a RR of 8.4 (CI, 2.5–29)
for OS (P < 0.0001). In the fourth quartile,
there were 41 patients among whom 17 recurrences and 17 deaths occurred
(data not shown). None of the clinicopathological parameters were of
prognostic significance. Multivariate analysis of the node-negative
patient group confirmed PAI-1 to be the only parameter that
independently and significantly predicted survival (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study was based on a newly developed kinetic uPA·PAI-1 complex ELISA that allowed a high level of sensitivity and specificity in the quantitation of this complex in tissue extracts (18) . Using a sandwich format of two monoclonal anti-PAI-1 capture antibodies and three anti-uPA detector antibodies, this assay selectively detects the complex but not the free forms of the individual components. Furthermore, free uPA and PAI-1 in relevant pathophysiological concentrations do not interfere with the detection of complex. Thus, in the present study of 341 breast cancer extracts, uPA·PAI-1 complex could be readily detected in all of the samples. Importantly, the assay also assures that only preformed complex is quantified. This is achieved in two steps: (a) extraction of the tumor tissue at a low pH prevents ex vivo formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed complex; and (b) during incubation of the assay plate at neutral pH, uPA·PAI-1 complex formation from free components in the diluted extracts was blocked by p-nitrophenyl guanidinobenzoate, which rapidly inactivates uPA. Moreover, there was no evidence of complex instability during tissue and extract storage because the complex levels did not change with tissue storage time, and complex was preserved as shown by Western blots of tissue extracts. Thus, the data obtained by the uPA·PAI-1 complex ELISA were suitable for further analyses of the prognostic implications of the complex in breast cancer extracts.
The uPA·PAI-1 complex level among the 341 extracts varied moderately
(
20-fold) as compared with the concentration variation for total uPA
and PAI-1 (>100-fold). Furthermore, the complex was found to represent
a minor, variable fraction of total uPA or PAI-1, whereas a close
association between uPA and PAI-1 was found. Thus, the complex levels
appeared to be largely independent of the total uPA and PAI-1 levels,
and the complex levels measured could therefore not merely be equated
with consumption of uPA or PAI-1. This finding may reflect separate
cellular locations of the components, limited activation of uPA, and a
preponderance of latent PAI-1. With regard to tumor dissemination, it
is noteworthy that in contrast to total uPA and PAI-1, the complex
levels were related to lymph node status; patients in the node-negative
group had higher tumor levels of complex than did node-positive
patients. However, a considerable overlap in the intermediate complex
concentration range suggested that the primary tumor complex level
could not be used as a surrogate measure of disseminated disease.
The prognostic value of uPA·PAI-1 complex was assessed by comparing survival for patient groups stratified into quartiles of the corresponding complex levels because scoring of the complex as a continuous variable did not adequately fit a linear model in the analysis. High levels of uPA·PAI-1 complex were associated with a lower probability of developing recurrences and of experiencing an early death. We further observed that the patient group with the highest complex values was most clearly separated, indicating that we could search for an optimal cutoff point between good and poor prognosis. Thus, using uPA·PAI-1 complex as an optimally dichotomized variable, a group with the highest complex levels and comprising 20% of the patients could be identified as having a particularly favorable prognosis. We further analyzed the prognostic value of uPA·PAI-1 complex compared with the value of the total uPA or PAI-1 levels. Interestingly, the uPA·PAI-1 complex levels in the extracts were by themselves of less prognostic value than total uPA or PAI-1. Moreover, in multivariate analyses, which also included classical prognostic parameters, neither uPA·PAI-1 complex nor total uPA appeared to be independent predictors of recurrence and death. Total PAI-1, however, was a highly significant and strong independent prognostic marker, consistent with the many previous reports (30 , 31) . The strong prognostic power of PAI-1 was also confirmed in the clinically important subgroup of lymph node-negative patients, although only a limited number of events were registered during the observation period.
These observations are consistent with the hypothesis that effective
local regulation of active uPA by active PAI-1, i.e.,
formation of complex, in the tumor may limit tumor invasion and
metastasis. However, in the absence of a simple inverse relationship
between complex and either uPA or PAI-1, these observations do
not offer an explanation why high levels of PAI-1 are so strongly
associated with short survival. An alternative explanation of why high
levels of uPA·PAI-1 complex are related to longer survival takes into
account the published data showing that high levels of uPAR predict
poor outcome for breast cancer patients (32
, 33)
: because
one pathway of uPA·PAI-1 complex removal is mediated by the
internalization of cell surface uPAR and the low-density lipoprotein
receptor-related protein [2-macroglobulin
receptor (5
, 14)
], it can be speculated that high complex
levels reflect low uPAR or low-density lipoprotein receptor-related
protein expression, leading to impaired removal of uPA·PAI-1 complex.
Thus, lower levels of uPAR expression may be the basis for the
relationship between high levels of complex and better prognosis.
However, this hypothesis is inconsistent with the proposed role of uPAR
in facilitating activation of uPA, which is necessary for the
formation of uPA·PAI-1 complex, and also with the fact that high
levels of total PAI-1 predict poor outcome.
To reconcile the new finding that high complex levels are related to good patient outcome with the established finding that high PAI-1 levels predict poor outcome, our previous proposal of the twocompartment model (34) may be invoked: in one compartment, uPA promotion of local tumor invasion is attenuated by PAI-1 complexation; whereas in another compartment, high levels of PAI-1 protect the provisional pericellular matrix of nascent capillaries forming behind uPA promoted extensions. This interpretation is consistent with the immunohistochemical localization of PAI-1 in endothelial cells in breast cancer tissue (15) and the absence of tumor-inducible angiogenesis in PAI-1-deficient mice (35) .
Because the complex comprised a minor, variable fraction of total uPA and PAI-1, the biological significance of the complex could possibly relate more to the ratio of the complex to total antigen level than to the complex level alone, i.e., a high ratio may indicate an effective role of the PAI-1 in the first compartment of the model above. Therefore, we additionally investigated whether the ratios represented an enhancement of prognostic power. Indeed, a high complex:total uPA or complex:total PAI-1 ratio conferred a strong association with longer breast cancer survival as evaluated by univariate analysis. However, multivariate analysis simultaneously including the ratios and the total antigen levels still showed the total (PAI-1) antigen level to be of superior prognostic power. This could suggest that the role of PAI-1 in the second compartment above, i.e., protection of new capillary growth, is the predominant factor in determining tumor expansion. The above-mentioned findings of the relationship between uPA·PAI-1 complex and survival may be compared with reports showing that the complex between metalloproteinase-9 and tissue inhibitor of metalloproteinases in combination with total plasma metalloproteinase-9 is a prognostic marker in gastrointestinal cancer (36) and that the ratio between complex-bound:free serum prostate-specific antigen and the total antigen level is actually a better diagnostic marker for prostate cancer than total prostate-specific antigen itself (37 , 38) .
In conclusion, our present findings on uPA·PAI-1 complex add new data to the emerging picture we have of the relationship between the tumor tissue levels of components of the uPA system and patient survival. This report demonstrates for the first time that high levels of uPA·PAI complex in tumor tissue are associated with longer survival of breast cancer patients. The results justify further exploratory studies of the utility of tumor uPA·PAI-1 complex levels, including identification of clinically relevant cutoff points for identification of low- and high-risk patients with breast cancer or other malignancies in focused adjuvant therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Oncology, Rigshospitalet, Blegdamsvej 9,
DK-2100 Copenhagen, Denmark. Phone: 45-35454676; Fax: 45-35456966. 
2 The abbreviations used are: uPA, urokinase; PAI,
plasminogen activator inhibitor; uPAR, urokinase receptor; RFS,
recurrence-free survival; OS, overall survival; CI, 95% confidence
interval; RR, relative risk; DBCG, Danish Breast Cancer
Cooperative Group.
3 A. N. Pedersen, unpublished results.
Received 4/10/00. Accepted 10/ 9/00.
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REFERENCES |
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