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A Randomized Trial of Bevacizumab, an Anti–Vascular
Endothelial Growth Factor Antibody, for Metastatic Renal Cancer
James C. Yang, M.D., Leah Haworth, B.S.N., Richard M. Sherry, M.D., Patrick Hwu, M.D.,
Douglas J. Schwartzentruber, M.D., Suzanne L. Topalian, M.D., Seth M. Steinberg, Ph.D.,
Helen X. Chen, M.D., and Steven A. Rosenberg, M.D., Ph.D.
From the Surgery Branch (J.C.Y., L.H., R.M.S., P.H., D.J.S., S.L.T., S.A.R.), the Biostatistics and
Data Management Section (S.M.S.), and the Cancer Therapy Evaluation Program (H.X.C.), National
Cancer Institute, Bethesda, Md
Abstract
Background—Mutations in the tumor-suppressor gene VHL cause oversecretion of vascular
endothelial growth factor by clear-cell renal carcinomas. We conducted a clinical trial to evaluate
bevacizumab, a neutralizing antibody against vascular endothelial growth factor, in patients with
metastatic renal-cell carcinoma.
Methods—A randomized, double-blind, phase 2 trial was conducted comparing placebo with
bevacizumab at doses of 3 and 10 mg per kilogram of body weight, given every two weeks; the time
to progression of disease and the response rate were primary end points. Crossover from placebo to
antibody treatment was allowed, and survival was a secondary end point.
Results—Minimal toxic effects were seen, with hypertension and asymptomatic proteinuria
predominating. The trial was stopped after the interim analysis met the criteria for early stopping.
With 116 patients randomly assigned to treatment groups (40 to placebo, 37 to low-dose antibody,
and 39 to high-dose antibody), there was a significant prolongation of the time to progression of
disease in the high-dose–antibody group as compared with the placebo group (hazard ratio, 2.55;
P<0.001). There was a small difference, of borderline significance, between the time to progression
of disease in the low-dose–antibody group and that in the placebo group (hazard ratio, 1.26; P=0.053).
The probability of being progression-free for patients given high-dose antibody, low-dose–antibody,
and placebo was 64 percent, 39 percent, and 20 percent, respectively, at four months and 30 percent,
14 percent, and 5 percent at eight months. At the last analysis, there were no significant differences
in overall survival between groups (P>0.20 for all comparisons).
Conclusions—Bevacizumab can significantly prolong the time to progression of disease in
patients with metastatic renal-cell cancer.
Studies of the hereditary form of clear-cell renal carcinoma, which occurs in the von Hippel–
Lindau syndrome, led to the identification of the von Hippel–Lindau tumor suppressor gene
(VHL). The gene is mutated both in hereditary renal-cell carcinoma (where one mutation is a
germ-line mutation) and in most cases of sporadic clear-cell renal carcinoma (where both
alleles have acquired mutations or deletions).
1,2
One consequence of these mutations is the
overproduction of vascular endothelial growth factor through a mechanism involving hypoxia-
inducible factorα.
3–7
In addition, both VHL-deficient mice and vascular endothelial growth
factor–knockout mice die in utero from defective vasculogenesis.
8,9
Thus, by its regulation of
vascular endothelial growth factor, the von Hippel–Lindau protein is tightly linked to
angiogenesis. Vascular endothelial growth factor stimulates the growth of endothelial cells and
Address reprint requests to Dr. Yang at Rm. 2B-37, Bldg. 10, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892,
or at james_yang@nih.gov.
NIH Public Access
Author Manuscript
N Engl J Med. Author manuscript; available in PMC 2008 March 26.
Published in final edited form as:
N Engl J Med. 2003 July 31; 349(5): 427–434.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
appears to be a central factor in angiogenesis, particularly during embryogenesis, ovulation,
wound healing, and tumor growth.
10
Studies of human tumor xenografts in immunodeficient mice showed that neutralization of
vascular endothelial growth factor inhibited the growth of a variety of model tumors.
11,12
Presta and colleagues “humanized” the murine antibody used in these studies, A.4.6.1, by
placing its complementarity-determining (antigen-binding) regions into a human IgG1
constant-region framework and modifying further amino acid residues to optimize antigen
binding.
13
In the resulting product, bevacizumab (or rhMAb-VEGF), 7 percent of the amino
acids are from the murine antibody. In phase 1 testing, bevacizumab had a low toxicity profile
in most patients, had a terminal elimination half-life of approximately 21 days, and did not
induce antibodies to bevacizumab.
14
The severe toxic effects that occurred in the phase 1 trial
were infrequent intratumoral bleeding (including fatal hemoptysis), pulmonary emboli, and
peripheral venous thrombosis. We conducted a randomized, placebo-controlled phase 2 trial
of bevacizumab in patients with advanced renal-cell carcinoma.
METHODS
PATIENTS
Patients with histologically confirmed renal cancer of the clear-cell type, measurable metastatic
disease, and documented progression of disease were eligible for this study. Other requirements
included an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or lower
and previous therapy with interleukin-2 (or contraindications to standard interleukin-2
therapy). The exclusion criteria were a history of central nervous system involvement, any
other therapy or major surgery within the previous four weeks, a history of intratumoral
bleeding, a serum creatinine level of more than 2 mg per deciliter (17 μmol per liter), a serum
bilirubin level of more than 2 mg per deciliter (34 μmol per liter), and ischemic vascular disease.
All patients gave written informed consent. This protocol was approved by the institutional
review board of the National Cancer Institute (NCI). The study was sponsored by the Cancer
Therapy Evaluation program of the NCI, and bevacizumab was supplied by Genentech under
a cooperative research and development agreement with the NCI. Trial design, data accrual
(with the exception of assays for vascular endothelial growth factor and bevacizumab
performed by Genentech on coded patient specimens), data analysis, and manuscript
preparation were performed entirely by the authors.
The patients were evaluated by physical examination, magnetic resonance imaging of the brain,
and complete computed tomographic scanning no more than one month before randomization,
five weeks after the beginning of therapy, and then every two months for the first year of therapy
and every three months for the second year of therapy.
A complete response was defined as the absence of all evidence of disease for at least a month.
A partial response was defined as a decrease of at least 50 percent in the sum of the products
of the maximal perpendicular diameters of measured lesions, lasting for a minimum of one
month, with no progression of any lesion or appearance of new lesions. Minor and mixed
responses were not included as responses.
Annual interim evaluations were performed by an independent data safety and monitoring
board, and the method of O’Brien and Fleming was used to determine the threshold for
statistical significance at each interim evaluation that would constitute grounds to recommend
termination of the trial.
15
For the first year of the trial, this threshold was a P value of 0.0006
or less; for the second year, it was a P value of 0.015 or less; and for the third year, it was a P
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value of 0.047 or less. The estimated and actual accrual rates were similar enough that these
proposed intervals did not require revision.
RANDOMIZATION AND TREATMENT
In this phase 2 study, the patients were stratified according to whether or not they had received
inter-leukin-2 therapy and were then randomly assigned to receive either a vehicle-only
placebo, 3 mg of bevacizumab per kilogram of body weight, or 10 mg of bevacizumab per
kilogram. During all treatment and evaluations, neither the patients nor any participating health
care personnel were aware of the treatment assignment. Based on pharmacokinetic modeling,
treatment with bevacizumab began with one loading dose, in which 150 percent of the assigned
dose was administered by intravenous infusion over a 2-hour period, and then, beginning one
week later, the standard assigned dose was administered (by progressively shorter infusions
that reached a minimum of 30 minutes) every two weeks. Plasma levels of vascular endothelial
growth factor and serum levels of bevacizumab were measured. The plasma vascular
endothelial growth factor assay used the 3.5.F.8 murine antibody for both capture and detection.
This assay detects both free and bevacizumab-bound vascular endothelial growth factor
equally, with a lower limit of detection of 40 pg per milliliter.
EVALUATION
For the purposes of end-point evaluation, the criteria for declaring tumor progression were the
unequivocal appearance of new lesions; an increase of more than 25 percent in the product of
the maximal perpendicular diameters of any measured lesion, as compared with base-line
evaluation (or the smallest size subsequent to base line); or a tumor-related deterioration in
ECOG performance status to 3 or more. For a declaration of progressive disease to be made,
the lesions had to attain a minimal diameter of 1.5 cm (to ensure accurate measurement).
The indications for removing patients from the study and unblinding their treatment
assignments were as follows. To permit adequate time for the initial assessment of the therapy
while protecting patients with rapid disease progression who were assigned to placebo, the
evaluation conducted five weeks after enrollment differed from subsequent evaluations. At
five weeks, patients with increases of more than 2 cm in any lesion, a clinically significant
deterioration in performance status, or new, severe symptoms (e.g., bone pain or nerve
compression) were removed from the study. At all other evaluations, the indication for removal
from the study was progressive disease. These different indications for removal from the study
did not affect the end-point analyses, which were always based on tumor progression, as defined
above.
STATISTICAL ANALYSIS
Using NCI Surgery Branch historical data from patients with no response to interleukin-2
therapy, we used the following criteria to estimate the sample size necessary to detect a
doubling of the time to progression in patients receiving either dose of bevacizumab as
compared with those receiving placebo: a 24-month accrual period, a 12-month evaluation
period after the completion of accrual, a power of 80 percent, and an overall alpha of 0.05 to
detect a doubling of the hazard ratio for each of the two primary comparisons (high-dose
antibody vs. placebo and low-dose antibody vs. placebo). The calculation indicated that 40
patients per group would be required (50 were permitted, to allow for some patients who could
not be evaluated).
The primary evaluation was based on the time from enrollment to disease progression; a
secondary analysis examined the time to disease progres sion from the five-week assessment,
in order to determine whether the effect of treatment was delayed and to ensure that small
variations in the interval from the pretreatment evaluation to the time of randomization did not
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affect the uniform determination of the time to progression. Each P value was adjusted for the
performance of two primary comparisons on the basis of treatment groups.
The time to progression and the overall response rate were the primary end points, and the
analyses were performed on an intention-to-treat basis. Survival was declared a secondary end
point, because patients whose disease progressed while they were receiving placebo were
offered crossover either to 3 mg of bevacizumab per kilogram alone or to a combination of 3
mg of bevacizumab per kilogram and thalidomide. The time to progression of disease and
survival were assessed with use of Kaplan–Meier curves and tested for significance by the log-
rank test. Hazard ratios were determined with the Cox proportional-hazards model. All P values
are two-tailed.
RESULTS
Between October 1998 and September 2001, 116 patients were enrolled, of whom 108 had
progressive disease during the course of the study. The median follow-up time from study entry
was 27 months. Forty patients were randomly assigned to placebo, 37 to low-dose
bevacizumab, and 39 to high-dose bevacizumab. All planned doses of the study drug were
given unless grade 3 toxic effects occurred, in which case doses were withheld as specified by
the study protocol. Only one patient (who was assigned to low-dose bevacizumab) was lost to
follow-up after therapy. The three groups had similar demographic and clinical characteristics
and laboratory results (Table 1). All patients received at least one dose of the assigned drug,
and 114 of the 116 patients underwent at least one planned follow-up evaluation (evidence
concerning disease progression was available for the remaining 2 patients).
There were no life-threatening toxic effects (grade 4, major organ) or deaths possibly related
to bevacizumab (Table 2). Hypertension and asymptomatic proteinuria were associated with
bevacizumab therapy (Table 2). Of 13 patients with grade 2 or 3 hypertension, 7 (54 percent)
had grade 2 or 3 proteinuria; of 63 patients with grade 0 or 1 hypertension, 10 (16 percent) had
grade 2 or 3 proteinuria (P=0.007 by Fisher’s exact test). None of these patients, or any other
patient, had renal insufficiency. Hypertension and proteinuria uniformly decreased after the
cessation of therapy, but death from renal cancer, the slow rate of correction of hypertension
and proteinuria, and the commencement of other therapies prevented the documentation of
complete resolution of these toxic effects in all but one patient.
There were no episodes of grade 4 hypertension during randomized therapy, but in one patient
who was initially assigned to placebo, hypertension with coma developed after the patient
crossed over to low-dose bevacizumab plus thalidomide. These complications resolved
completely after therapy was stopped. Typically, hypertension during the study was treated by
the patients’ private physicians with standard regimens for essential hypertension. Among all
bevacizumab-treated patients who required therapy for newly diagnosed hypertension (for
whom the dates of onset could be most accurately determined), the median interval from the
first dose of bevacizumab to the onset of hypertension was 131 days (range, 7 to 316). Grade
1 or 2 hemoptysis developed in four patients (one receiving high-dose bevacizumab, one
receiving low-dose bevacizumab, and two receiving placebo), and one patient receiving
placebo had a pulmonary embolus.
At the second interim evaluation (which analyzed the data on 110 patients), the NCI data safety
and monitoring board recommended closure of accrual on the basis of the difference between
the placebo and high-dose bevacizumab groups in the time to progression of disease. According
to intention-to-treat analysis, progression-free survival in the group receiving 10 mg of
bevacizumab per kilogram (with a median time to progression of 4.8 months) was significantly
longer than that in the placebo group (with a median time to progression of 2.5 months, P<0.001
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by the log-rank test) (Fig. 1A). The difference between the time to progression of disease in
the group receiving 3 mg of bevacizumab per kilogram (median time, 3.0 months) and that in
the placebo group was of borderline significance (P=0.041 by the log-rank test) (Fig. 1B).
The planned analysis of progression from the five-week assessment yielded the same results.
The percentages of patients assigned to high-dose bevacizumab, low-dose bevacizumab, and
placebo who had no tumor progression were 64 percent, 39 percent, and 20 percent,
respectively, four months after randomization and 30 percent, 14 percent, and 5 percent eight
months after randomization. A Cox proportional-hazards model yielded hazard ratios for the
time to progression of disease of 2.55 among patients given high-dose bevacizumab (P<0.001)
and 1.26 among those given low-dose bevacizumab (P=0.053), as compared with those given
placebo.
Only four patients had objective responses (all of which were partial responses), and all of
these had received high-dose bevacizumab; thus, the response rate for high-dose bevacizumab
was 10 percent (95 percent confidence interval, 2.9 to 24.2 percent). One patient had a partial
response for the maximal treatment period of two years. This patient then stopped therapy, had
a relapse six months later, and is currently having a second partial response after retreatment
under a compassionate exemption (Fig. 2). Another patient treated for two years had a sustained
minor response, had a relapse after stopping therapy, and had another minor response after
being retreated.
Measurements of plasma vascular endothelial growth factor were available for 113 patients.
Of these, 76 had a base-line level below the lower limit of detection (40 pg per milliliter). There
were no significant associations between a detectable pretreatment level of vascular endothelial
growth factor and the clinical response or the time to progression in either bevacizumab group
(all P values were greater than 0.20). However, the limited sensitivity of the assay does not
permit the definitive conclusion that there is no correlation between the base-line plasma level
of vascular endothelial growth factor and the clinical response or the time to progression. After
antibody therapy was started, the plasma levels of vascular endothelial growth factor rose
steadily (the assay measures both free and antibody-bound vascular endothelial growth factor).
After 5 weeks and 13 weeks of therapy, all bevacizumab-treated patients had detectable plasma
levels of vascular endothelial growth factor. The median levels were 196 and 246 pg per
milliliter, respectively, for patients receiving high-dose bevacizumab and 155 and 170 pg per
milliliter for patients receiving low-dose bevacizumab. The percentages of patients assigned
to placebo who had undetectable plasma levels of vascular endothelial growth factor at base
line, 5 weeks, and 13 weeks were 66 percent, 67 percent, and 75 percent, respectively. Patients
receiving low-dose bevacizumab had mean (±SE) peak and trough serum levels of
bevacizumab of 101±9 and 39±3 μg per milliliter, respectively; patients receiving high-dose
bevacizumab had mean peak and trough levels of 392±24 and 157±13 μg per milliliter,
respectively. In both groups, the trough levels were above that needed to abolish detectable
free vascular endothelial growth factor in the plasma of patients in previous phase 1 studies.
14
At the most recent analysis, in February 2003, 19 of 116 patients (16 percent) were alive, and
there were no significant differences in survival between the treatment groups (all P values
were greater than 0.20) (Fig. 3). The complete radiographic records of 113 patients (3 were no
longer complete at the time of audit) were blindly audited by a team of extramural radiologists
under the supervision of the Cancer Therapy and Evaluation Program of the NCI. The
prolongation of time to progression of disease was confirmed radiologically.
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DISCUSSION
We selected vascular endothelial growth factor as a target for treatment of clear-cell kidney
cancer because mutations in the von Hippel–Lindau tumor-suppressor gene, which probably
cause most sporadic clear-cell kidney cancers, result in overproduction of this growth factor
by the tumors. In our study, the aim was to neutralize vascular endothelial growth factor with
a humanized monoclonal antibody (bevacizumab) in patients with metastatic clear-cell renal
cancer. Using a randomized, double-blind, placebo-controlled design, we found that the time
to tumor progression was prolonged by a factor of 2.55 in patients given 10 mg of bevacizumab
per kilogram every two weeks, as compared with patients in the placebo group. Survival was
not a primary end point in this trial, which allowed patients to cross over from placebo to
bevacizumab therapy at the time of disease progression. Indeed, the survival of bevacizumab-
treated patients was not significantly different from that of the patients receiving placebo.
During bevacizumab therapy, the plasma level of vascular endothelial growth factor rose. It is
important to note that the assay we used measured both free and antibody-bound vascular
endothelial growth factor. The explanation for this increase and its clinical significance are
unknown, but it might have been due to diminished clearance of bevacizumab-bound, inactive
vascular endothelial growth factor or to an antibody-mediated blockade of the binding of
vascular endothelial growth factor to its receptors.
Hypotheses about the mechanism responsible for the delay we observed in tumor progression
are based on in vitro data, the results of treatment of human tumor xenografts in
immunodeficient mice, and studies of human renal cancer. These data suggest that the
antitumor effects of the antibody against vascular endothelial growth factor are due to inhibition
of angiogenesis. Both in vitro and in tumor xenografts, vascular endothelial growth factor has
potent angiogenic activity, which is inhibited by neutralizing antibodies to vascular endothelial
growth factor; the result is a decrease in tumor blood flow and microvessel densities.
11
Human
clear-cell renal cancers have significantly higher microvessel counts than non–clear-cell renal
cancers, and these counts are correlated with the expression of vascular endothelial growth
factor.
16
Endothelial cells and hematopoietic cells (but not renal cancer cells) are the
predominant cells that express receptors for vascular endothelial growth factor, but the
inhibition of the growth of human tumor xenografts in immunodeficient mice argues against
contributions from an immunologic mechanism. For all these reasons, the inhibitory effect of
bevacizumab on the growth of clear-cell renal cancer is likely to be due to its antiangiogenic
action.
Antiangiogenic strategies for the treatment of cancer have generated widespread enthusiasm
based on promising in vitro and preclinical studies. The concepts that growing tumors require
the manufacture of new blood vessels and that very little of the rest of the normal adult body
has such a requirement have led to the belief that there is valuable therapeutic potential in this
area. Early clinical studies of antiangiogenic compounds such as endostatin, TNP-470, and
thalidomide were not designed to assess their clinical efficacy.
17,18
In retrospect, only a
randomized assessment of a time-to-progression end point could have demonstrated the activity
of bevacizumab in renal cancer. Reliance on major response rates would have resulted in the
conclusion that this drug was ineffective. Nevertheless, without a demonstration of improved
overall survival, this single-agent trial serves primarily as a proof of principle and the basis for
further investigation.
The magnitude of the clinical benefit of bevacizumab in this trial was small. The differences
in the time to the progression of disease between the high-dose bevacizumab group and the
placebo group was only a few months. Nevertheless, the likelihood is high that this difference
was due to true biologic activity. The lack of an overall survival benefit in this trial and the
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small size of the increase in the time to progression may reflect the crossover design and the
rigorous indications for declaring progression and removing a patient from the study (an
increase in diameter of any single lesion by as little as 12 percent could constitute tumor
progression). Some patients left the study with only small new lesions or mixed responses, but
often with minimal or no increase in the size of preexisting tumors. In fact, 23 patients treated
with high-dose bevacizumab showed no net increase in the size of index lesions from base line
to the time of tumor progression. Tumor progression in these patients was typically based on
the appearance of small new lesions or an increase in the size of some lesions that was offset
by regression in other lesions. It would be worthwhile to determine survival in patients allowed
to continue to receive bevacizumab despite tumor progression.
Future treatments for renal cancer that target angiogenic mechanisms should consider pathways
other than that mediated by vascular endothelial growth factor. There are other proteins in the
local microenvironment of some tumors that can promote angiogenesis. For example, fibroblast
growth factor 5, which has angiogenic activity, is secreted by most renal cancers,
19
suggesting
that combinations of bevacizumab and inhibitors of members of the fibroblast growth factor
family may have promise for treatment of this disease. It is likely that the future of
antiangiogenic therapy will require a rational combination of inhibitors, directed by a better
understanding of the biology of each individual type of cancer.
Acknowledgements
We are indebted to the Surgery Branch research nurses and immunotherapy fellows, the day hospital nursing staff,
Don White, Maria Merino, W. Marston Linehan, Richard Klausner, Gwen Fyfe, and William Novotny for their
invaluable assistance in the conduct of this study.
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Figure 1. Kaplan–Meier Analysis of Survival Free of Tumor Progression for Patients Receiving
High-Dose Bevacizumab (Panel A) or Low-Dose Bevacizumab (Panel B), as Compared with
Placebo
The high dose of bevacizumab was 10 mg per kilogram of body weight. The low dose of
bevacizumab was 3 mg per kilogram. Doses were given every two weeks. P values were
calculated by the log-rank test.
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Figure 2. Serial Radiographs of a Patient Treated with High-Dose Bevacizumab
Panel A shows the pretreatment assessment (arrows indicate lymph-node metastases). Panel
B shows a radiograph obtained two years later, when treatment was stopped during a partial
response. Panel C shows relapse of tumor six months thereafter. Panel D shows a second partial
response 3 months after therapy was restarted, which is ongoing at more than 18 months as of
this writing.
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Figure 3. Overall Survival of Patients Receiving Placebo, Low-Dose Bevaci-zumab, or High-Dose
Bevacizumab
There were no significant differences among the treatment groups.
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Table 1
Characteristics of Patients before Treatment.
*
Characteristic High-Dose Bevacizumab (N=39) Low-Dose Bevacizumab (N=37) Placebo (N=40)
Median age (yr) 53 54 53
Male sex (%) 74 84 68
ECOG performance status (no.)
†
0 30 30 31
1 or 2 9 7 9
Prior interleukin-2 therapy (no.) 37 34 37
Prior chemotherapy (no.) 10 7 8
Prior radiation therapy (no.) 8 6 12
Prior nephrectomy (no.) 35 33 38
Anemia (no.) 14 15 16
Hypercalcemia (no.) 12 18 14
Interval from diagnosis to randomization (no.)
<1 yr 14 13 12
1–2 yr 8 6 9
>2 yr 17 18 19
Liver involvement (no.) 10 10 10
Bone involvement (no.) 2 3 6
*
P>0.05 for all comparisons.
†
ECOG denotes Eastern Cooperative Oncology Group. Higher performance-status numbers indicate greater impairment.
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Yang et al. Page 13
Table 2
Toxic Effects of Treatment.
*
Effect High-Dose Bevacizumab (N=39) Low-Dose Bevacizumab (N=37) Placebo (N=40)
number
Epistaxis
8
†
5 1
Hypertension
14 (8
††
)
1 2
Fever without infection 4 1 0
Malaise 13 6 6
Hematuria
5
†
1 0
Hyponatremia 3
4
†
0
Proteinuria (≥1+ or ≥150 mg/24 hr)
25 (3)
†
15 (2) 15
Elevated alanine aminotransferase 4 2 0
Chest pain 2 (2) 0 0
*
The table lists all toxic effects of any grade that occurred in at least 10 percent of patients receiving either dose of antibody and that were more frequent
than in patients receiving placebo. The number of patients with grade 3 toxic effects is shown in parentheses (there were no grade 4 or 5 events; every
bevacizumab-associated grade 3 toxic effect occurring in more than one patient is shown). Grade 3 hypertension was defined as hypertension not completely
controlled by one standard medication. Grade 3 proteinuria was defined as urinary excretion of more than 3.5 g of protein per 24 hours. Other toxic effects
were graded according to the National Cancer Institute Common Toxicity Criteria (version 2.0).
†
Unadjusted P≤0.05 for the comparison with placebo (by chi-square test, or by Fisher’s exact test if the expected frequency was less than 5).
N Engl J Med. Author manuscript; available in PMC 2008 March 26.
