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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Molecular Pathology and
Medicine, Molecular Genetics Unit, DIBIT, San Raffaele Scientific
Institute, Milan, Italy; the Department of Infectious Diseases,
Rigshospitalet, Copenhagen; the Department of Infectious Diseases and
Clinical Research Unit, Hvidovre Hospital, Denmark; and the Royal Free
Centre for HIV Medicine and the Department of Primary Care and
Population Sciences, Royal Free and University College Medical School,
University College London, London, United Kingdom.
Human immunodeficiency virus-1 (HIV-1) infection has been shown to
result in up-regulation of the urokinase-type plasminogen activator
receptor (uPAR/CD87) on leukocytes in vitro and in vivo. The objective of this study was to investigate whether this
up-regulation is paralleled by higher serum levels of soluble uPAR
(suPAR) in patients with advanced HIV-1 disease and whether the serum
level of suPAR is predictive of clinical outcome. Using an
enzyme-linked immunosorbent assay, the level of suPAR was measured
retrospectively in serum samples from 314 patients with HIV-1
infection. By Kaplan-Meier and Cox regression analyses, the serum suPAR
levels were correlated to survival with AIDS-related death as the end
point. High levels of serum suPAR (greater than median) were associated
with poor overall survival, and Kaplan-Meier analysis on patients
stratified by suPAR level demonstrated a continuous increase in
mortality rates with higher suPAR levels. After adjustment for accepted prognostic markers The urokinase-type plasminogen activator system
consists of a proteinase (uPA), a receptor (uPAR), and inhibitors. The
system is involved in pericellular proteolysis, cell migration, and
tissue remodeling by multiple modes of action High serum levels of suPAR, the soluble form of uPAR, have
recently been associated with worse overall survival rates among cancer
patients,7,8 and it has been suggested that in cancer the
excess of suPAR in the circulation derives from tumor cells or
tumor-infiltrating macrophages,7-10 which often express
high levels of uPAR.11
The fact that enhanced serum suPAR levels are indicative for
up-regulated cellular uPAR levels, combined with the previously published data that HIV-1 infection leads to enhanced cell surface expression of uPAR on monocytes and T-lymphocytes in vitro and in
vivo,12-14 prompted us to investigate the relation between
serum suPAR levels in HIV-1-infected patients and disease progression.
Patients
Measurement of serum suPAR, CD4 count, viral load, and
Statistical analysis All statistical analyses were performed using statistical packages (SSPS version 8 and SAS version 6.12; SAS Institute, Cary, NC), and the observed differences were considered significant if P < .05. Comparisons of proportions were performed using the 2 test. Comparison of unpaired observations
for continuous variables was carried out using Kruskal-Wallis or
Mann-Whitney U tests. Correlations were calculated using the
Spearman rank correlation test. Kaplan-Meier curves were constructed
stratifying the patients by the level of serum suPAR to generate
similar numbers of patients in each stratum. Differences between
Kaplan-Meier curves were analyzed by the log-rank test and by Cox
regression. The ability of serum suPAR to predict mortality in the
context of other known prognostic markers was formally assessed using a
multivariate Cox proportional hazards model. Variables were fitted in a
continuous scale using the transformations that provided the best fit
as determined by the Wald 2 value (log-scale for viral
load, CD4 count and  2-microglobulin, and linear scale for suPAR
and age).
Levels of suPAR in serum All 314 HIV-1-infected patients had measurable levels of serum suPAR with a median value of 3.69 ng/mL (range, 1.15-15.60 ng/mL). Division of all patients into 2 equal groups by the median suPAR level revealed several significant differences (Table 1). Patients with high suPAR levels had shorter follow-up time (P < .0001) because of an increased incidence of AIDS-related death (P < .0001), lower CD4 count (P < .0001), higher viral load (P = .002), and higher2-microglobulin level
(P < .0001), and they were older (P = .007).
No significant differences were observed with respect to the gender of
the patients (P = .7) or the route of infection
(P  .2) or whether the patients received antiretroviral treatment at the time of enrollment (P = .3).
Division of the patients into 3 groups by the CDC-defined clinical
stages A, B, and C (Figure 1)
demonstrated that more advanced HIV-1 disease was associated with
higher levels of serum suPAR (P < .0001, Kruskal-Wallis
test). Within each of the clinical stages A, B, and C, the proportion
of survivors was significantly (P
Spearman rank correlation demonstrated a weak but significant negative
correlation between suPAR and CD4 count ( Kaplan-Meier analysis To investigate the association between suPAR levels and survival, we performed a Kaplan-Meier analysis (Figure 2). In the absence of any relevant cut-off value for serum suPAR, we used the 33% and 66% percentiles of suPAR level, generating 3 groups of patients of similar size (n = 104-105 patients) representing low, medium, and high serum suPAR levels. The survival curve for patients with low serum suPAR was significantly different from that of patients with medium suPAR (P = .0008;2 = 11; log-rank test), which
in turn was significantly different from that of patients with high
suPAR (P < .0001; 2 = 26;
log-rank test).
Cox regression analysis Comparison of the 3 patient groups in the Kaplan-Meier plot (Figure 2) by Cox regression analysis demonstrated a continuously increasing risk for mortality with increasing suPAR level. Compared to a low suPAR level, a medium suPAR level was associated with a hazard ratio (HR) of 2.2 (95% confidence interval [95% CI], 1.4-3.4; P < .0001) and a high suPAR level with an HR of 4.7 (95% CI, 3.1-7.2; P < .0001). Compared to a medium suPAR level, a high suPAR level was associated with an HR of 2.2 (95% CI, 1.6-3.1; P < .0001).When suPAR was fitted as a continuous variable in a Cox model including
all patients, high levels of suPAR were significantly associated with
increased mortality rates (Table 2). An
increase in serum suPAR of 1 ng/mL was associated with an HR of 1.6 (95% CI, 1.5-1.8; P < .0001). Other clinical and
serologic variables that provided significant prognostic value in
univariate Cox analysis were CD4 count, viral load,
Patients for whom data for all the serologic variables were available
(n = 231) were then fitted with suPAR in a multivariate Cox
regression model (Table 2). The predictive strength of suPAR in this
subset (n = 231) of patients was virtually identical to when the
analysis was performed on all 314 patients (not shown). In the
multivariate Cox model, suPAR, CD4 count, viral load,
In the current work we have provided evidence that serum suPAR is a strong independent prognostic variable in HIV-1 infection. In Kaplan-Meier survival analysis, higher serum levels of suPAR are associated with faster progression to death. After adjustment for all known relevant HIV-1 disease progression markers in multivariate Cox regression analysis, suPAR remains a highly significant independent predictor of survival. This is, to our knowledge, the first report demonstrating a connection between the urokinase plasminogen activator system and disease progression in HIV-1 infection. One possible explanation for the prognostic value of serum suPAR in HIV-1 infection is that it derives, at least partially, from HIV-1-infected cells. Monocytes and T-lymphocytes infected with HIV-1 have been shown to express elevated levels of cell-surface uPAR,12-14 and the serum level of suPAR may correlate with the size of the reservoir of HIV-1-infected cells in the organism. This model predicts that suPAR levels will be higher in HIV-1-infected patients than in healthy controls, a possibility we have not addressed in this study. However, in accordance with this hypothesis, our data do demonstrate that the suPAR level is higher in patients with more advanced HIV-1 infection. The striking correlation between suPAR levels and the risk of disease progression may suggest a direct relevance of uPAR in HIV-1 infection. The natural high-affinity ligand of uPAR, uPA has been shown to bind HIV-1 gp120 and to promote HIV-1 infection of macrophages in vitro.18 The interaction between uPA and gp120 involves the functionally important V3-loop of gp120 and the catalytic domain of uPA, but it leaves the ligand-binding domain of uPA free for interaction with uPAR.18 It is thus possible that uPA may form a "bridge" between gp120 on the virus and uPAR on the cell surface, which would identify uPAR as a cellular coreceptor for HIV-1. Indeed, it should be noted that the major cell types susceptible to HIV-1 infection are those in which uPAR is predominantly expressed (macrophages, activated monocytes, and T cells). Investigations addressing such an involvement now seem highly warranted. Finally, it should be emphasized that circulating suPAR might play a direct role in the pathogenesis of HIV-1 infection. In fact, suPAR fragments have chemokine-like activities, and suPAR is capable of modulating processes such as cell adhesion, migration, and proliferation in vitro.1,2 Regardless of the biologic explanation, the serum level of suPAR remains a strong and independent prognostic predictor of disease progression in HIV-1 infection. Using a single serum suPAR measurement and arbitrary cut-off points, we were able to define patient groups associated with different hazard ratios for disease progression, suggesting that suPAR measurements may be used as a prognostic tool in HIV-1 infection. The variables suPAR, CD4 count, and viral load were only poorly correlated mutually, and all 3 parameters provided highly significant independent prognostic information in multivariate Cox analysis. This implies that suPAR provides important additional prognostic information. In this respect it will be particularly important to determine whether suPAR is useful in determining whether and when to initiate highly active antiretroviral therapy (HAART). There is still considerable debate, as reflected in different national guidelines, about what combination of CD4 count and viral load constitutes an indication for therapy. Virologic and immunologic arguments for early initiation of therapy must be balanced against disadvantages of treatment, such as lifelong polypharmacy, high costs, side effects, and risk for resistance development. Because the suPAR level carries strong and independent information on the progression status, it may aid in the monitoring of patients and in the important clinical decision of when to initiate HAART. In the current study, follow-up was censored when the first patient in the cohort received a protease inhibitor, and patients therefore only received relatively light antiretroviral treatment compared to HAART. Treatment with antiretroviral drugs at the time of enrollment in CHIC was associated with poor survival in univariate Cox analysis but failed to provide significant prognostic information in multivariate Cox analysis. This most likely reflects the fact that patients who had received treatment at the time of enrollment were those with more advanced disease for whom monotherapy or dual therapy did not provide strong survival benefit. The current study is therefore likely to reflect disease development close to the natural history course of HIV-1 infection, and the possible prognostic value of suPAR in the context of HAART should be addressed in other more appropriately designed studies. Although the current study shows a strong association between suPAR levels and disease progression, it is important to realize that the amount of data from this single study does not justify suggestions of using suPAR as a standard prognostic parameter. First, the data must be confirmed in an independent cohort. Second, because this cohort consists of patients in a European country before the HAART era, its value must be confirmed in patients with access to HAART treatment or patients in resource-poor settings, or both. In this study we have used arbitrarily chosen cut-off values and statistical methods that do not require the definition of specific cut-off values. Normal values of suPAR obtained using standardized sample techniques must be determined before routine use of suPAR can be applied. We have recently shown that suPAR antigen can be readily quantified in urine and that the urinary suPAR level strongly correlates with the serum level.19 It is therefore possible that urine may provide an alternative to serum samples for suPAR-based HIV-1 prognosis, which would introduce an element of safety and convenience for patients and medical staff. The introduction of HAART in the Western world has profoundly increased the life expectancy of HIV-1-infected patients. However, because of the high cost of these drugs, only a small fraction of the HIV-1-infected patients in the world have access to effective HIV-1 therapy. The measurement of suPAR is performed using a simple and inexpensive ELISA technique. After confirmation of our results in other groups of patients, the determination of suPAR in serum (or even better, urine) from HIV-1-infected patients may provide a cost-effective supplement or alternative to the currently used decision-making tools based on CD4 counts and viral load.
We thank Gunilla Høyer-Hansen (Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark) for supplying antibodies. We also thank Terese Katzenstein, Jan Gerstoft, and Peter Skinhøj (Department of Infectious Diseases, Rigshospitalet) for their important contributions to the collection and analysis of patient samples.
Submitted June 28, 2000; accepted August 30, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nicolai Sidenius, Department of Molecular Pathology and Medicine, DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; e-mail: nicolai.sidenius{at}hsr.it.
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Top
Abstract
Introduction
including Centers for Disease Control and
Prevention-defined clinical stages, CD4 counts, viral load,
.03, 
indicates that the patient
died during follow-up. Median values of suPAR within each stage are
indicated by horizontal bars. The serum suPAR levels between the
clinical stages were significantly different (P < .0001,
Kruskal-Wallis test). Proportions of patients who survived were
significantly higher in patients with low levels of suPAR (below the
median in the respective group) in all 3 CDC stages (
= 
0.33; P < .0001) and weak but significant positive correlations
between suPAR and viral load (