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Clinical Cancer Research 13, 2115-2120, April 1, 2007. doi: 10.1158/1078-0432.CCR-06-2509
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Angiopoietin 2 Is a Potential Mediator of High-Dose Interleukin 2–Induced Vascular Leak

Diana C. Gallagher1, Rupal S. Bhatt2, Samir M. Parikh3, Parin Patel3, Virginia Seery2, David F. McDermott2, Michael B. Atkins2 and Vikas P. Sukhatme3,4

Authors' Affiliations: 1 Division of Pulmonary and Critical Care, Department of Medicine, Beth Israel Deaconess Medical Center, 2 Division of Hematology and Oncology, Renal Cancer Program, Beth Israel Deaconess Medical Center and Dana-Farber/Harvard Cancer Center, 3 Division of Nephrology and 4 Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts

Requests for reprints: Vikas P. Sukhatme, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, RW 563, Boston, MA 02215. E-mail: vsukhatm{at}bidmc.harvard.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: High-dose interleukin 2 (HDIL2) produces durable tumor regressions in 10% of patients with metastatic renal cell carcinoma and melanoma. However, a major toxicity is vascular leak syndrome (VLS). We previously reported elevated serum angiopoietin 2 (Ang2) in septic patients with vascular leak and hypothesized that Ang2 might also contribute to HDIL2 VLS.

Experimental Design: Blood was collected from 14 patients receiving HDIL2 and from 4 patients receiving HDIL2 and bevacizumab, an antibody against vascular endothelial growth factor (VEGF). The effect of Ang2 was studied in vitro by incubating high Ang2 patient serum with cultured endothelial cells.

Results: Pretreatment Ang2 levels were in the reference range (median, 3.3 ng/mL) and rose with each day of IL-2 therapy (median peak, 29.7 ng/mL). No trend was seen in free VEGF levels during therapy. Patients treated with HDIL2 and bevacizumab all developed VLS and elevated Ang2. High Ang2 patient sera induced propermeability structural changes in endothelial cells, an effect reversed by blockade with the competitive ligand angiopoietin 1 (Ang1).

Conclusions: Ang2 may be a mediator of HDIL2 VLS as evidenced by (a) an increase in Ang2 in all patients on HDIL2; (b) the effect of high Ang2 patient serum on cultured endothelial cells; (c) rescue of those structural changes by Ang1. The lack of correlation between VLS and serum VEGF levels in patients treated with HDIL2 alone or in combination with bevacizumab suggests that VEGF is not a major contributor to VLS or Ang2 release. These data suggest that the inhibition of Ang2 may mitigate VLS in patients receiving HDIL2.

High-dose interleukin 2 (HDIL2) is a Food and Drug Administration–approved treatment for patients with metastatic renal cell carcinoma (RCC) and metastatic melanoma. The mechanism of action of this cytokine-based therapy is poorly understood and is thought to depend on T cells and natural killer cell antitumor activity (1). Although only 23% of those treated will show tumor response, the duration of effect in responders can exceed 10 years. HDIL2 is the only available therapy that can offer such results (2).

As many as 65% of patients receiving HDIL2 will have interruption or discontinuation of treatment due to vascular leak syndrome (VLS; ref. 3, 4). VLS is characterized by marked vasopermeability with hypotension requiring i.v. fluids and, frequently, pharmacologic vasopressor support. Other manifestations of interleukin 2 (IL-2)–induced VLS include prerenal azotemia, metabolic acidosis, pleural effusions, and noncardiogenic pulmonary edema (5, 6). There are no proven therapies to prevent or treat VLS other than holding IL-2 doses and providing supportive care.

It is well known that IL-2 causes endothelial cell activation with loss of proper barrier function (7, 8). This may require the interaction of endothelial cells and specific circulating leukocyte populations, but specific interactions have not yet been characterized (9–12). Prior efforts to attenuate IL-2–induced VLS focused initially on coadministration with steroids, an intervention that decreased circulating tumor necrosis factor {alpha} levels (13). However, follow-up studies using tumor necrosis factor receptor blockade failed to show decreased clinical toxicity (14). More recent work with soluble IL-1 receptor also failed to show any effect on toxicity profiles (15). With little mechanistic data on pathways leading to endothelial dysfunction, further work on the biology of VLS is needed to identify key molecules and targets for novel therapies.

Our laboratory is exploring the role of the endothelial receptor Tie2 and its agonist/antagonistic ligand pair, angiopoietin 1 and 2 (Ang1 and Ang2), in VLSs. These proteins are known to be critical in postnatal vessel maturation and stabilization, yet their roles in adult vasculature are only now being explored (16). It had previously been shown that Ang1, which phosphorylates the Tie2 receptor, could attenuate leak in the adult murine vasculature (17). We have shown that Ang2, which blocks Tie2 phosphorylation, causes endothelial paracellular gap formation in vitro, and that Ang2 infusion produces pulmonary leak in murine models. Furthermore, we have shown that Ang2 is present in circulating blood of septic subjects, and that Ang2 levels correlate with the severity of pulmonary leak (18). Like IL-2–induced VLS, sepsis is a syndrome characterized by profound hypotension and end organ injury due in part to endothelial barrier derangement and unchecked vascular leak.

VLS from HDIL2, because of its clinical similarity to sepsis, is a compelling model of endothelial barrier dysfunction given the marked temporal relationship of vascular leak to IL-2 administration. The study of biomarkers before, during, and after infusion of IL-2 might provide insights into the pathogenesis of sepsis-related vascular leak as well as the mechanism of IL-2–induced toxicity.

The objective of this study was to determine whether VLS resulting from HDIL2 therapy is associated with elevations in circulating Ang2.


   Materials and Methods
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 Materials and Methods
Results
 Discussion
 References
 
Patient samples. Eligible patients included adults with metastatic renal cell or metastatic melanoma scheduled to receive HDIL2. Over a 6-month period at Beth Israel Deaconess Medical Center, we prospectively collected discarded serum and plasma samples. In our first feasibility protocol on four patients, we collected samples only on day 1 (before HDIL2) and day 6 (after HDIL2). Based on these results, we planned an observational study with a sample size of at least 10 patients on HDIL2. In this cohort, we collected serum before IL-2 infusion and with each successive day's morning labs. Whenever possible, we also obtained discarded blood from all follow-up outpatient appointments. All identifying information was removed, and data were encoded to protect patient privacy. Clinical data were collected on each patient by chart review. This study was approved by the medical center's Institutional Review Board.

Four patients received the anti-VEGF antibody bevacizumab 2 weeks before and immediately before starting HDIL2, and we also collected baseline, bevacizumab-treated, and daily HDIL2 samples on these patients.

ELISA. Ang1, Ang2, and free VEGF (isoform 165) were measured in serum samples from patients by sandwich ELISA using the respective human ELISA kits (R&D Systems, Minneapolis, MN). Preliminary experiments confirmed the stability of these proteins in serum for 6 to 12 h at room temperature as well as its stability through several freeze-thaw cycles.

Cell culture. Human microvascular endothelial cells from lung (HMVEC-L; Cambrex Bio Science Walkersville, Inc., Walkersville, MD) were cultured in EBM-2 (Cambrex) supplemented with 5% fetal bovine serum and growth factors according to the manufacturer's instructions. Passages 4 to 8 were used for all experiments.

Immunofluorescence. HMVEC-Ls were grown to confluence on glass coverslips coated with 1% gelatin. The cells were fixed for 10 min in 4% paraformaldehyde in PBS and incubated for 5 min in 0.5% Triton X-100 in PBS. After blocking, the monolayers were processed for staining with anti–VE-cadherin monoclonal antibody (BD Biosciences PharMingen, San Diego, CA) and Alexa Fluoro 488 goat anti-mouse immunoglobulin G, rhodamine phalloidin (Molecular Probes, Eugene, OR) for filamentous actin staining, and TOPRO-3-iodine (Molecular Probes) for nuclear staining. Fluorescence images were obtained using a Bio-Rad (Hercules, CA) MRC confocal fluorescence microscope. For experiments using cells treated with serum from patients, serum Ang2 concentration was first measured by ELISA. Then, patient serum was diluted to 10% with EBM-2 and filtered with low-protein-binding polyvinylidene difluoride membrane (0.22 µm, Millipore, Billerica, MA) before application on endothelial cell monolayers.

Statistical analysis. Median results are reported as median ± SE. Comparisons between groups were made with Mann-Whitney U test, and correlation was assessed by Spearman's rank correlation coefficient.


   Results
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 Materials and Methods
 Results
Discussion
 References
 
In a pilot study, we measured serum Ang2 in three patients before infusion of IL-2 and 1 day after completion of HDIL2 therapy (cycle 1, week 1). We found an average pretreatment Ang2 level of 4 ng/mL and post-treatment level of 25 ng/mL (P < 0.0008; data not shown). Based on these results, we collected daily serum samples on 14 additional subjects receiving HDIL2. Table 1 shows baseline characteristics of these patients.


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  		Table 1. Baseline characteristics

 
ELISA was used (as outlined in Materials and Methods) to measure serum Ang2. Patients received thrice daily infusions, but the final number of doses and doses per day were determined by institutional protocol designed to limit manifestations of VLS (19).

Table 2 shows detailed information on each patient's clinical course. All patients had hypotension requiring i.v. fluid boluses, and all gained weight with a median weight gain of 9.8 ± 0.5% from admission. All subjects had clinical evidence of VLS-associated end organ injury with one or more of the following: elevated creatinine, transaminitis, metabolic acidosis, and/or neurotoxicity (data not shown). Five of the 14 patients required vasopressors in addition to i.v. fluids, and four required supplemental oxygen. Neither the peak Ang2 level nor the trajectory of increase of Ang2 was correlated with a vasopressor or oxygen requirement. However, because clinicians hold doses based on cumulative VLS side effects, we questioned whether there was an association between higher early Ang2 levels and poor tolerance of the protocol. In our cohort, the median number of doses received was 12 ± 0.5, and on average, the first dose was held on day 5. We found that Ang2 levels on day 3 negatively correlated with the total number of IL-2 doses administered by the end of the treatment period (r = –0.53, P < 0.05), suggesting that higher Ang2 levels early on in therapy predict poor protocol tolerance.


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  		Table 2. Detailed IL-2 dosing, Ang2 levels, and end points

 
Figure 1A shows the increase and decrease of Ang2 for a single patient during the protocol. Figure 1B shows the cohort had a significant increase of Ang2 from baseline with a median starting Ang2 of 3.3 ± 0.5 ng/mL and a peak level of 29.7 ± 3.6 ng/mL (P < 0.005). In the 10 subjects from whom serum was available at 24 or 48 h after the final IL-2 infusion, median Ang2 level was 18.7 ±2.9 ng/mL (P = 0.03 compared with peak).


Figure 1
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  		Fig. 1. A, median serum levels of Ang2 in the 14 patients measured at admission, at peak, and off-protocol. Off-protocol refers to levels measured in the blood up to 48 h after last IL-2 infusion, and this blood was only available for 10 patients because some patients were discharged on the same day as the last infusion. bars, SE of the median. There is a statistically significant difference between baseline and peak (P < 0.005) and peak and completion (P = 0.03). B, depiction of daily serum Ang2 levels in patient 8, which shows the increase and decrease during protocol.

 
The mechanism for Ang2 inducing vasopermeability is believed to be through the blockade of normal phosphorylation of the endothelial specific Tie2 receptor. This leads to up-regulation of RhoA, a protein in the GTPase family, which, in turn, increases phosphorylation of the myosin light chain with resultant actin stress fiber formation. Such cytoskeletal changes lead to cellular contraction with disruption of endothelial junctional proteins, such as vascular endothelial cadherin (VE-cadherin), with formation of interendothelial gaps and increased permeability (18).

To determine the functional importance of elevated Ang2 in HDIL2 recipients developing VLS, we did immunostaining experiments (20–22). Cultured monolayers of human pulmonary microvascular endothelial cells (HMVEC-L) were bathed in a 1:10 dilution of patient serum, and the effect on cell structure was examined. As outlined in Materials and Methods, cells were fixed and stained for actin and for VE-cadherin. As shown in Fig. 2 , when confluent HMVEC-L monolayers were incubated for 60 min with low Ang2 (3.9 ng/mL) patient sera, the phenotype was similar to control with minimal actin stress fiber formation and circumferential VE-cadherin cell surface expression, suggesting intact cell-cell contacts. However, when the same patient's serum on HDIL2 infusion day 5 high (Ang2 = 41.8 ng/mL) was incubated with HMVEC-L monolayers, increased actin stress fibers and endothelial gaps developed. Additionally, VE-cadherin staining was markedly altered, suggesting a disruption of endothelial barrier integrity.


Figure 2
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  		Fig. 2. Confocal images using actin (left column), VE-cadherin (middle column), and merged (right column) for four conditions when patient blood is added to an endothelial confluent monolayer. A, control (10% FCS) demonstrating relatively sparse actin staining and marked junctional VE-cadherin staining. B, patient 11, day 1, Ang2 level of 3.9 ng/mL is similar to control. C, patient 11, day 5, when the Ang2 level was 41.8 ng/mL, shows increased actin stress fibers and markedly diminished VE cadherin staining, as well as the presence of intercellular gaps (white arrows). D, patient 11, day 5, Ang2 = 41.8 ng/mL with the addition of Ang1 at a level of 100 ng/mL, added 30 min after the start of incubation. The monolayer has been restored with a decrease in actin staining, an increase in junctional VE-cadherin, and the loss of gaps, reminiscent of control serum.

 
The sera from patients treated with HDIL2 contains not only Ang2, but other proteins associated with leak, including tumor necrosis factor {alpha}, IL-6, and IL-10. We did experiments with the endogenous Ang2 inhibitor, Ang1, to determine whether the endothelial barrier breakdown might be mediated by Ang2 specifically. HMVEC-L monolayers were bathed with high Ang2 patient serum for 30 min, and then Ang1 was added for the final 30 min of assay. Under these conditions, there was a significant attenuation in stress fiber formation and a reforming of contiguous VE-cadherin cell junction expression.

Having established that (a) increasing Ang2 correlates with the development of VLS; (b) decreasing Ang2 correlates with the cessation of HDIL2 and recovery from VLS; (c) Ang2 in patient sera is sufficient to induce endothelial cell disruption, we next asked whether VEGF, a canonical vascular propermeability factor, might show increased blood levels during HDIL2 therapy. The possibility that VEGF might be involved in HDIL2 VLS is supported by the fact that patients with metastatic renal cell and melanoma have higher circulating VEGF levels compared with normals (23). However, there are conflicting data on the trend of VEGF levels during treatment with IL-2 (24, 25). Using ELISA, we measured serial free VEGF levels in eight subjects on HDIL2, and there was no trend seen during HDIL2 infusion (Fig. 3 ).


Figure 3
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  		Fig. 3. A, serial measurements of free VEGF in blood of eight patients treated with high-dose IL-2 in the absence of bevacizumab. B, serial measurements of free VEGF in four patients who received HDIL2 plus bevacizumab. C, Ang2 levels in patients on HDIL2 and bevacizumab during their hospital course, showing that Ang2 levels increase during the hospital course in a manner similar to what happens in a larger set of patients when no bevacizumab is used.

 
We then studied four patients treated on a novel Cytokine Working Group protocol. Patients were pretreated with bevacizumab, a VEGF neutralizing antibody, 2 weeks and immediately before receiving HDIL2. This patient population would be expected to have near-zero levels of free VEGF at the time of initiation of HDIL2 therapy and should eliminate the question of whether VEGF is a confounding factor in HDIL2 VLS. As shown in Table 3 , all four patients treated with bevacizumab plus HDIL2 developed VLS with hypotension and weight gain. Figure 3 shows that free VEGF levels were low throughout therapy. However, similar to HDIL2-only treated patients, this subset on bevacizumab and HDIL2 had a starting Ang2 value of 3.1 ± 0.6 ng/mL with a peak of 21.0 ± 4 ng/mL.


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  		Table 3. Bevacizumab and HDIL2-treated patients

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
High-dose bolus IL-2 is a potentially curative treatment for 8% to 10% of patients with metastatic RCC and melanoma, but with considerable toxicity and cost. Over the past year, two new targeted therapies, sorafenib (Bayer-Onyx, West Haven, CT) and sunitinib (Pfizer, New York, NY) have been approved for the treatment of RCC. Although these agents expand the repertoire of options for patients with RCC, both are given indefinitely, are associated with toxicities, and lead to few complete responses. Moreover, resistance to these agents develops in about 8 to 12 months, leaving room for more treatment options, either as salvage or first line, before these newer therapies. In contrast to RCC, patients with metastatic melanoma have more limited treatment options. Thus, in this patient population, improving the toxicity profile of HDIL2 would be a valuable therapeutic advance.

VLS is a life-threatening toxicity of HDIL2 therapy for which there is little mechanistic understanding and no specific therapy, other than supportive care.

Avoidance or attenuation of VLS would allow more patients to safely receive this treatment option and potentially increase durable tumor response.

We have previously reported elevated levels of Ang2 in the serum of patients with septic shock, and because of the striking clinical similarities in sepsis and HDIL2 VLS, we hypothesized that Ang2 might mediate vascular leak in subjects on this biological therapy.

Ang2 levels correlated with VLS as they rose in 14:14 patients with HDIL2 administration and continued to increase with ongoing IL-2 exposure. Cessation of HDIL2 resulted in resolution of VLS and decrease in circulating Ang2. Patients with higher Ang2 levels by day 3 received fewer protocol doses of IL-2. The decision to withhold a dose is made by treating physician based on total side effects experienced. All patients treated with IL-2 exhibited signs and symptoms of VLS as measured by hypotension and weight gain, and all had either transaminitis, renal dysfunction, and/or neurotoxicity. A subset of patients showed more severe vascular leak as manifested by an oxygen requirement due to pulmonary edema and/or hypotension requiring vasopressor support. We did not see a correlation between the peak or the fold increase of Ang2 and the severe VLS end points of oxygen or vasopressor requirement (data not shown). It is possible that although all subjects showed a clear increase in serum Ang2, our small number of patients makes it difficult to correlate absolute Ang2 values with these end points. We did not have access to more detailed data such as continuous blood pressure monitoring, oxygen saturations, serum lactates, or PaO2 levels, which might be more sensitive in quantifying the degree of VLS. Such information might elucidate a quantitative temporal relationship between absolute Ang2 levels and manifestations of VLS in the context of each individual's own cardiopulmonary reserve. However, we did note that patients with higher Ang2 levels by day 3 had more doses of IL-2 held for overall side effect profile.

We sought a mechanistic explanation for high-serum Ang2 levels correlating with VLS. We showed that Ang2 is an important endothelial disrupting factor in HDIL2 patient serum by rescuing its effect on HMVEC-Ls with specific Ang2 antagonism via Ang1.

Because VEGF has been described as a mediator of vascular leak, we measured serial free VEGF levels in patients treated with HDIL2. Although baseline levels are elevated above normals, no trends pre-, post-, or during therapy were noted. To further explore the role of VEGF in IL-2–induced vascular leak, we studied a subset of patients treated with IL-2 and bevacizumab. Notably, despite having absent or low free VEGF levels, patients treated with IL-2 and bevacizumab still experienced the same degree of VLS and had elevated Ang2 levels similar to their IL-2 only counterparts. This finding indicates that HDIL2-induced vascular leak can occur in the absence of free circulating VEGF.

Collectively, our data suggest that inhibition of Ang2 is a logical concept and should be studied in a clinical trial setting. If an Ang2 inhibitor could attenuate VLS, it would address the major dose-limiting side effect of HDIL2 therapy. An additional reason to consider Ang2 antagonism in this population is the preclinical study by Oliner et al. (26) who showed that selective blockade of Ang2 inhibited tumor angiogenesis and decreased tumor burden in murine models of colon cancer. Blocking the effects of Ang2 effect by providing excess Ang1 would be logical, but the use of Ang1 infusions may be hampered by poor pharmacokinetics profile due to multimerization and matrix binding. Currently, a reagent which specifically neutralizes the activity of Ang2 (but not Ang1) is available and, if humanized, would be a reasonable candidate for testing in patients receiving HDIL2 (26). We speculate that Ang2 inhibition during HDIL2 therapy may not only allow for greater IL-2 dosing by attenuating VLS, but may also enhance the efficacy of IL-2 therapy.


   Acknowledgments
 
We recognize Marc Ernstoff, principal investigator of the IL-2/bevacizumab trial, and Mark Exley for access to initial patient samples. We appreciate the efforts of Kendra Bradley and all of the nurses who provide excellent care for our patients. We thank Sonia Sinha for technical support, and Steven Cannistra for helpful discussion. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.


   Footnotes
 
Grant support: Seed funds from Beth Israel Deaconess Medical Center (V.P. Sukhatme), NIH Renal Cancer Specialized Programs of Research Excellence (V.P. Sukhatme, M.B. Atkins, D.F. McDermott, R.S. Bhatt), American Society for Clinical Oncology Young Investigator Award (R.S. Bhatt), and American Association for Cancer Research-Amgen Research Fellowship (R.S. Bhatt), NIH 5 T32 HL007633 (D.C. Gallagher), NIH 5K08-DK069316-02 (S.M. Parikh).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: D.C. Gallagher and R.S. Bhatt contributed equally to this work.

Received 10/16/06; revised 1/26/07; accepted 1/31/07.


   References
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 References
 

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