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Interleukin-2 cycling causes transient increases in high-sensitivity C-reactive protein and D-dimer that are not associated with plasma HIV-RNA levels
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AIDS:
24 September 2009
Porter, Brian O; Shen, Jean; Kovacs, Joseph A; Davey, Richard T; Rehm, Catherine; Lozier, Jay; Csako, Gyorgy; Nghiem, Khanh; Costello, Rene; Lane, Henry Clifford; Sereti, Irini
aNational Institutes of Health, National Institute of Allergy and Infectious Diseases, USA
bNational Institutes of Health, Critical Care Medicine Department, US
cNational Institutes of Health, Clinical Center (CC), USA
dNational Institutes of Health, CC, Department of Laboratory Medicine (DLM), Bethesda, Maryland, USA.
"With outcome data from the SILCAAT and ESPRIT trials indicating that IL-2 provides no clinical benefit in chronic HIV infection treated with HAART [5,6], the present findings suggest a possible mechanism to explain some of the adverse clinical events associated with IL-2 treatment.....This study demonstrates that IL-2 cycles cause marked transient increases in plasma levels of the acute phase reactant CRP and the thrombotic marker D-dimer, both in the setting of inadequate HIV-RNA suppression (i.e. mono or dual nucleoside analogue therapy) and with viral suppression to less than 50 copies/ml on HAART......Of note, however, a significantly greater number of serious cardiovascular events were observed in the IL-2 treatment arm compared with the placebo arm (40 versus 14 events, respectively) of the multicenter ESPRIT trial, the most common being peripheral emboli and thromboses (13 versus two events, respectively) [6]. Given the randomized design of the ESPRIT trial, this finding supports a causal relationship for these adverse events to IL-2. However, the timing of their clinical presentation raises questions as to the underlying pathologic mechanism."
Abstract
Objective: To determine the effects of interleukin (IL)-2 treatment on inflammatory and thrombotic biomarkers in chronically HIV-infected adults receiving antiretroviral therapy.
Methods: Cryopreserved plasma was evaluated retrospectively for C-reactive protein (CRP) and D-dimer at baseline, end of an IL-2 cycle, and long-term follow up from two randomized, controlled trials: 57 IL-2-naive adults receiving either three to six cycles of IL-2 as well as antiretroviral therapy (nucleoside analogues) or antiretroviral therapy alone for 12 months, and 40 IL-2-experienced adults on highly active antiretroviral therapy who either interrupted or continued therapy for 6 months after a baseline IL-2 cycle. High-sensitivity CRP (hsCRP) was measured by immunonephelometry (detection limit 0.175 mg/l) and D-dimer by latex agglutination (detection limit 0.20 mg/l). Median within-group differences and pre and post-IL-2 changes between groups were assessed via nonparametric Wilcoxon signed-rank and Mann-Whitney U-tests. Spearman's rank test was used to assess correlations between changes in hsCRP, D-dimer, and HIV-RNA viral load.
Results: Significant increases in hsCRP (study 1: 138.6 mg/l; study 2: 58.9 mg/l) and D-dimer (study 1: 3.1 mg/l; study 2: 0.4 mg/l, all P < 0.0001) occurred by the end of the initial IL-2 cycle, returning to baseline by the end of study. No correlations were seen between changes in hsCRP or D-dimer and HIV-RNA, CD4 T-cell count, or proliferation (Ki67 expression). No thrombotic or cardiovascular serious adverse events occurred during these study periods.
Conclusion: IL-2 dosing caused transient increases in plasma hsCRP and D-dimer levels, regardless of HIV-RNA viral load, suggesting the possibility of increased risk for thrombotic events.
Introduction
Several phase I/II studies [1,2] have demonstrated that intermittent cycling of interleukin (IL)-2 leads to long-term CD4 T-cell increases in chronic HIV infection. Recently, data from large-scale multicenter phase III trials [Evaluation of Subcutaneous Proleukin (IL-2) in a Randomized International Trial (ESPRIT) and Study of IL-2 in people with low CD4+ T-cell counts on Active Anti-HIV Therapy (SILCAAT)] [3,4] showed that these CD4 T-cell increases are not associated with clinical benefit and may be associated with more grade 4 clinical events [5,6].
IL-2 causes the release of proinflammatory cytokines, including tumor necrosis factor-α, IL-1ß, and IL-6 [7-10], which are known to increase acute phase reactants such as C-reactive protein (CRP) and activate prothrombotic pathways [11,12]. Multiple studies [10,13-15] have established associations between levels of CRP and the fibrinogen breakdown product D-dimer with both cardiovascular and thrombotic disease. These biomarkers have also been shown to be elevated in HIV-infected patients [16,17]. Recent findings from the Strategies for the Management of Antiretroviral Therapy (SMART) trial demonstrated that baseline levels of CRP and D-dimer were predictors of all-cause mortality in a chronically HIV-infected sample of patients who were undergoing highly active antiretroviral therapy (HAART) interruption [18]. Understanding the acute and chronic effects of IL-2 on these markers may thus provide insights into potential IL-2-related complications.
In this study, plasma CRP and D-dimer levels were analyzed longitudinally in two randomized clinical trials that evaluated IL-2 as an adjuvant therapy in HIV infection. The first study [19,20] evaluated the effect of three to six IL-2 cycles added to single or dual nucleoside analogue therapy administered over 1 year compared with antiretroviral therapy (ART) alone. The second study [21,22] evaluated the effect of a baseline IL-2 cycle in patients who either subsequently interrupted or continued HAART for 6 months. Biomarkers and immune parameters were analyzed pre and post-IL-2 administration, as well as long term at the end of each trial, in order to characterize the duration of any IL-2 effect, its relationship to HIV viremia, and changes in CD4 T-cell proliferation.
Discussion
This study demonstrates that IL-2 cycles cause marked transient increases in plasma levels of the acute phase reactant CRP and the thrombotic marker D-dimer, both in the setting of inadequate HIV-RNA suppression (i.e. mono or dual nucleoside analogue therapy) and with viral suppression to less than 50 copies/ml on HAART.
Significant associations between inflammatory biomarkers and endothelial activation markers have been demonstrated in HIV-infected patients [17], suggesting a potential mechanism by which such markers may portend cardiovascular or thrombotic disease. In addition, data from the SMART study-suggested increases in biomarker levels observed following HAART interruption were associated with increased rates of non-HIV-related adverse events [18]. However, the clinical significance of the hsCRP and D-dimer elevations observed in these two trials is unclear because these levels returned to baseline by 1 month post-IL-2 and remained at baseline when measured up to 12 months after an initial IL-2 cycle. This finding is consistent with studies in malignant melanoma patients, which demonstrated that IL-2-induced increases in several procoagulant markers, including thrombin-antithrombin and plasmin-antiplasmin complexes, returned to baseline within 24 h post-IL-2 administration [11]. Moreover, no serious clinical events related to thromboses, emboli, or cardiovascular events were reported during the primary study periods in either of these trials. In contrast to findings from the SMART study [18], increased adverse clinical events were not observed among participants in study 2 who interrupted HAART, possibly due to the small sample size, shorter duration of follow up (6 months), and better immune status (median CD4 T-cell count, 947 cells/µl) compared with participants in the SMART study.
Of note, however, a significantly greater number of serious cardiovascular events were observed in the IL-2 treatment arm compared with the placebo arm (40 versus 14 events, respectively) of the multicenter ESPRIT trial, the most common being peripheral emboli and thromboses (13 versus two events, respectively) [6]. Given the randomized design of the ESPRIT trial, this finding supports a causal relationship for these adverse events to IL-2. However, the timing of their clinical presentation raises questions as to the underlying pathologic mechanism.
These data offer the first randomized and controlled comparative analysis of inflammatory and thrombotic biomarkers in HIV-infected IL-2 recipients versus nonrecipients, both in the presence and absence of HAART. The striking impact of IL-2 treatment on these biomarkers should warrant evaluating thrombotic and cardiovascular risk factors prior to IL-2 treatment, regardless of HIV status. With outcome data from the SILCAAT and ESPRIT trials indicating that IL-2 provides no clinical benefit in chronic HIV infection treated with HAART [5,6], the present findings suggest a possible mechanism to explain some of the adverse clinical events associated with IL-2 treatment.
Results
Participant characteristics
Demographic characteristics and median values for baseline immune parameters and biomarkers for both treatment groups in each trial are summarized in Table 1. No thrombotic or cardiovascular serious adverse events occurred in either trial during these study periods.
Effects of interleukin-2 on high-sensitivity C-reactive protein and D-dimer
By the end of the initial IL-2 cycle, IL-2 recipients from both trials demonstrated significant increases (all P < 0.0001) in median plasma levels of hsCRP [study 1: 138.6 mg/l, interquartile range (IQR) = 87.9-173 mg/l; study 2: 58.9 mg/l, IQR = 35.9-84.5 mg/l] and D-dimer (study 1: 3.1 mg/l, IQR = 2.3-4.7 mg/l; study 2: 0.4 mg/l, IQR = 0.2-0.8 mg/l). However, as shown in Fig. 1, these increases were transient, as hsCRP (a and b) and D-dimer (c and d) levels returned to baseline in IL-2 recipients from each trial by the end of their respective study periods (Fig. 1). In the second trial, both hsCRP and D-dimer had returned to baseline as early as 1-month post-IL-2 administration (data not shown).
Association between changes in high-sensitivity C-reactive protein, D-dimer, and immune parameters
Peri-IL-2 cycle increases in hsCRP and D-dimer positively correlated among IL-2 recipients in study 1 (r = 0.43; P = 0.036) and study 2 (r = 0.57; P = 0.0002); however, levels of hsCRP and D-dimer were not associated at baseline in either trial. These end of IL-2 cycle increases in hsCRP and D-dimer were not correlated with baseline HIV-RNA viral load or CD4 T-cell count in either study. Long-term changes (baseline to either month 12 in study 1 or month 6 in study 2) in hsCRP or D-dimer with either HIV-RNA viral load or CD4 T-cell count were also not associated (data not shown). In addition, no correlations were observed between changes in CD4 T-cell proliferation (Ki67 expression), hsCRP, or D-dimer levels over the same time periods in either trial (data not shown).
Methods
Clinical trials
The methodology and primary outcomes of both these trials have been published previously [19-22]. In the first study [19], 57 chronically HIV-infected IL-2-naive adults on nucleoside reverse transcriptase inhibitor monotherapy or combination therapy were randomized in the ratio of 1: 1 to receive either three to six intermittent cycles of IL-2 as well as ART or ART alone and were followed for 1 year. Cycles of human recombinant IL-2 (Novartis, Emeryville, California, USA) were administered no more frequently than every 8 weeks for 5 consecutive days as continuous infusions of 6-18 million international units per day.
The second study [21,22] consisted of 40 chronically HIV-infected IL-2-experienced adults who were on HAART prior to receiving a baseline cycle of IL-2 (subcutaneous injections of 7.5 million international units twice daily). Patients were then randomized in the ratio of 2: 1 either to interrupt or continue HAART for 6 months, during which time additional IL-2 cycles were administered with 10 days of pericycle HAART, if CD4 T-cell counts fell below 90% of baseline.
Participants in both trials were evaluated monthly throughout their study periods. Intracellular Ki67 expression in CD4 T cells was determined via flow cytometry at baseline in both studies, at 12 months follow up in study 1 and at 1-month post-HAART interruption in study 2, as described previously [20].
C-reactive protein and D-dimer measurements
Concentrations of CRP and D-dimer were determined from cryopreserved plasma collected at baseline on completion of the first IL-2 cycle (day 6 in the first study and day 5 in the second study) and at the end of the study period (month 12 in study 1 and month 6 in study 2). For participants in study 2 who interrupted HAART, levels were also measured 1 month after the baseline IL-2 cycle.
Plasma CRP levels were determined using high-sensitivity CRP (hsCRP) immunonephelometry (BN-II; Siemens, Deerfield, Illinois, USA) with a detection limit of 0.175 mg/l. D-dimer concentration was determined via latex agglutination (Liatest; Diagnostica Stago, Asnieres, France) with a detection limit of 0.20 mg/l.
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