Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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Clinical Cancer Research Vol. 10, 5732-5740, September 1, 2004
© 2004 American Association for Cancer Research

Clinical Trials

Phase II Study of the Antiangiogenic Agent SU5416 in Patients with Advanced Soft Tissue Sarcomas

John V. Heymach1, Jayesh Desai1, Judith Manola1,2, Darren W. Davis8, David J. McConkey8, David Harmon4, David P. Ryan4, Geraldine Goss1, Travis Quigley1, Annick D. Van den Abbeele3, Stuart G. Silverman7, Susan Connors5, Judah Folkman5, Christopher D. M. Fletcher6 and George D. Demetri1

Departments of 1 Medical Oncology, 2 Biostatistical Science, and 3 Nuclear Medicine, Dana-Farber Cancer Institute, Boston, Massachusetts; 4 Division of Hematology-Oncology, Massachusetts General Hospital, Boston, Massachusetts; 5 Department of Surgery, Children’s Hospital, Boston, Massachusetts; Departments of 6 Pathology and 7 Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; and 8 The University of Texas M. D. Anderson Cancer Center, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: SU5416 (semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor-2 and KIT receptor tyrosine kinases. This Phase II study was conducted to investigate the safety and efficacy of SU5416 for patients with soft tissue sarcomas.

Experimental Design: Thirteen patients with locally advanced or metastatic soft tissue sarcomas were treated with SU5416 via intravenous infusion at a dose of 145 mg/m2 twice weekly. In selected cases tumor biopsies were taken before and after 2 months of treatment.

Results: The median progression-free survival was 1.8 months. Median overall survival was 22.8 months. No objective tumor responses were observed. There was evidence of shorter survival among patients with high baseline urine VEGF levels (P = 0.04). No grade 4 toxicities were observed. The most common grade 3 toxicities were headache and thrombosis. Other less serious toxicities included fatigue, nausea, and abdominal pain. The median systolic blood pressure increased from 118 mmHg at baseline to 133 after 1 month of treatment (P = 0.01). Post-treatment tumor biopsies showed no significant decreases in VEGF receptor phosphorylation compared with baseline in 3 evaluable patients. One patient with gastrointestinal stromal tumor who had rapid progression during SU5416 treatment was subsequently treated with another KIT inhibitor, imatinib mesylate, and had a partial response lasting >36 months.

Conclusions: SU5416 was relatively well tolerated but did not demonstrate significant antitumor activity against advanced soft tissue sarcoma. Correlative studies suggest that VEGF receptor or KIT inhibition was incomplete in at least some cases, providing a possible explanation for the observed lack of activity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growth and metastatic spread of tumors are dependent on angiogenesis, a multistep process regulated by the local balance of endogenous pro- and antiangiogenic factors (1) . Among the known stimulators of tumor angiogenesis, vascular endothelial growth factor (VEGF) is thought to play a pivotal role. The activities of VEGF are primarily mediated by two receptors, VEGF receptor-1 (VEGFR-1 and Flt-1) and VEGFR-2 (KDR and Flk-1; ref. 2 ). VEGF-induced endothelial migration, proliferation, and vascular permeability appear to be dependent on VEGFR-2 signaling via its tyrosine kinase domain. In preclinical models, blockade of the VEGF pathway inhibits tumor growth as demonstrated by investigators using monoclonal antibodies that bind VEGF or VEGFR-2 or small molecule inhibitors of the VEGFR-2 tyrosine kinase domain (3, 4, 5, 6) . These approaches are currently being evaluated in human clinical trials.

SU5416 (semaxanib) is a small molecule inhibitor of VEGFR-2 tyrosine kinase (3) . In murine models, SU5416 reduced tumor angiogenesis and inhibited the growth of a broad range of tumor types (3 , 7) . Phase I and II clinical trials have been conducted using a twice-weekly dosing schedule, with a maximum tolerated dose of 145 mg/m2. Dose-limiting toxicities were observed including nausea, vomiting, and headache (8 , 9) . A once-weekly schedule has also been tested, which also showed that doses up to 145 mg/m2 were well tolerated (10) . Evidence of antitumor activity has been observed in several studies (10, 11, 12) . When SU5416 was given in combination with gemcitabine and cisplatin, a high incidence of thromboembolic complications was observed and was accompanied by laboratory evidence of endothelial cell perturbation, discouraging additional use of this combination (13) . After initiation of this trial, it was also reported that SU5416 also inhibits the KIT receptor tyrosine kinase (14 , 15) . Mutations causing constitutive activation of the KIT receptor appear to play a critical role in the pathogenesis of gastrointestinal stromal tumors and a KIT inhibitor, imatinib, is effective therapy for the treatment of gastrointestinal stromal tumor (16, 17, 18, 19) .

Soft tissue sarcomas are a diverse group of tumors of mesenchymal origin. For most histologic subsets of advanced or metastatic soft tissue sarcomas, chemotherapy is currently the treatment of choice. Unfortunately, these drugs carry substantial toxicity, and resistance tends to arise quickly. The vast majority of soft tissue sarcomas were found to express VEGF, and elevated serum VEGF levels were found to correlate with higher histologic tumor grade (20 , 21) . VEGF receptor inhibitors showed antitumor activity in preclinical models of sarcoma (3 , 7) . Therefore, soft tissue sarcomas represent a rational target in which to perform therapeutic testing of VEGF pathway inhibitors. For this reason, we have conducted a Phase II study of SU5416 in patients with advanced or metastatic soft tissue sarcoma. We have also explored potential markers of antiangiogenic activity including urine VEGF and basic fibroblast growth factor (bFGF) levels and tumor endothelial cell VEGFR phosphorylation assessed by laser scanning cytometry (22) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Eligibility.
All of the patients were required to have histologically documented soft tissue sarcoma with measurable disease that was metastatic or inoperable. This study was designed to include patients who were treated previously with chemotherapy and who had either not responded or had progression of disease after an initial response. All of the patients had to have had prior chemotherapy. All of the patients were ≥18 years of age, signed a written informed consent, and had an Eastern Cooperative Oncology Group performance status of 0–1, with a life expectancy ≥12 weeks. Other inclusion criteria included adequate hematologic function (white blood cell count >2,000 cells/µL and platelet count >100,000 cells/µL), renal function (serum creatinine ≤1.5 mg/dL), hepatic function (serum bilirubin ≤ the upper limit of normal and transaminases <1.5 times the upper limit of normal) and coagulation function (prothrombin and partial thromboplastin time <1.25 times the upper limit of normal, fibrin split products ≤1.0 µg/mL, and fibrinogen >200 mg/dL). At least 3 weeks had to have elapsed from any prior radiotherapy or chemotherapy (6 weeks from any prior mitomycin C or nitrosurea therapy) and 4 weeks from any major surgery. Exclusion criteria included patients with brain metastases, a bleeding diathesis, a history of venous or arterial thrombosis (including pulmonary embolism) in the previous 3 months, a history of allergic reaction to poloxyl 35 castor oil (Cremophor) or paclitaxel, patients with uncompensated cardiac disease, diabetes mellitus, severe peripheral vascular disease, and patients who were pregnant or breastfeeding. Women of childbearing potential were required to have a negative pregnancy test before enrollment, and both partners were required to practice suitable contraception during the study. All of the patients were required to sign a written informed consent approved by the Dana-Farber/Partners Cancer Care Institutional Review Board before treatment could be started.

Treatment Plan.
SU5416 (produced by Sugen, Inc., South San Francisco, CA) was obtained from the Cancer Therapy Evaluation Program of the National Cancer Institute. The drug was administered via intravenous infusion in an outpatient setting at a dose of 145 mg/m2 twice weekly. The first dose of SU5416 was administered at 100 mL/hour for the first 15 minutes before increasing to full speed (200 mL/hour). If the patient had any evidence of venous irritation, the infusion rate was slowed to 100 mL/hour. Patients were observed for a minimum of 3 hours after their first three doses. Components in the formulation included Cremophor (poloxyl 35 castor oil), polyethylene glycol 400, benzyl alcohol, and dehydrated alcohol (supplied by Sugen, Redwood City, CA). To prevent allergic reaction to Cremophor, premedication with dexamethasone and an H1-receptor blocker were used. Patients also received prophylaxis against thromboembolic disease with coumadin 1 mg/day, Dalteparin 2,500 IU subcutaneously/daily, or Lovenox 30 mg subcutaneously/daily.

Evaluations During Treatment.
Pretreatment evaluation was carried out within 14 days before treatment and included informed consent, a complete history, and physical examination. Baseline laboratory studies included complete blood count with differential, comprehensive coagulation profile, liver function tests, renal function, serum chemistries, urinalysis, and pregnancy test. Baseline electrocardiogram; chest X-ray; computed tomography scans of the chest, abdomen, and pelvis with quantification of measurable disease; and bone scan (if relevant) were also performed. 2-Deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18FDG-PET) scans were also performed to complete the baseline assessment. During treatment, patients were seen by a physician weekly and had a complete physical examination, assessment of performance status, complete blood count, serum chemistries, coagulation profile, and urine and serum assays for angiogenic factors performed. At each visit, patients were also evaluated for toxicity, which was graded according to the National Cancer Institute Common Toxicity Criteria. Patients had a 60-minute corticotrophin stimulation test performed every 4 weeks while on study to monitor for adrenal insufficiency. Disease response was assessed every 8 weeks with tumor imaging via computed tomography scans and PET scans. Response to therapy was evaluated according to Response Evaluation Criteria in Solid Tumors criteria (23) . Where possible, accessible tumor was rebiopsied after 8 weeks of treatment for evaluation of tumor vasculature. Urine was collected before therapy on days 1, 8, 15, and 22 of each cycle, centrifuged to remove debris, stored at –70°C, and batched for enzyme-linked immunosorbent assay (ELISA) analysis. VEGF and bFGF levels were measured using the Quantikine human VEGF and Quantikine High Sensitivity Human bFGF ELISA kits (R&D Systems, Inc., Minneapolis, MN) as per the manufacturer’s instructions. The limits of detection were 5 pg/mL and 0.2 pg/mL for VEGF and bFGF ELISAs, respectively.

Dose Adjustments.
Dose adjustments were based on the worst toxicity occurring in the previous week of treatment. For any grade 3 or 4 hematologic toxicity, drug was withheld until recovery to baseline and was then recommenced at 110 mg/m2. Patients with ≥grade 3 nausea and vomiting, despite treatment, had the dose of drug reduced to 110 mg/m2. Patients with ≥ grade 2 neurologic toxicity were restarted at 110 mg/m2 once recovery to ≤grade 1 was reached. Patients with all other ≥grade 3 nonhematologic toxicities had drug withheld until recovery to ≤grade 1, with recommencement at 110 mg/m2. In patients with recurrence of any ≥grade 3 toxicity, dose was additionally reduced to 85 mg/m2, followed by reduction to 65 mg/m2. Patients who did not recover from any ≥grade 3 toxicity to ≤grade 1 were removed from study. Patients experiencing grade 3 or 4 anaphylactic reactions, despite full-dose dexamethasone premedication, were removed from study.

Statistical Considerations.
The primary objective of this study was to determine the progression-free rate at 6 months. The secondary objective was to determine the overall response rate (partial and complete response). Historically, survival for patients with metastatic soft tissue sarcomas is very poor. Long-term disease-free survival is observed in <5% of patients, with objective complete response rates being ~15%. This study was designed to accrue 60 patients in two stages to distinguish a 15% response rate from a null rate of 5% but terminated before reaching the first-stage accrual goal due to a lack of antitumor activity observed.

Descriptive statistics were used to characterize patients at baseline and laboratory correlates. The method of Kaplan and Meier was used to estimate overall and progression-free survival. We used 95% exact binomial confidence intervals to estimate the response rate and the proportion progression-free at 6 months. ANOVA was used to test for a change over time in blood pressure.

Correlative Studies of Tumor Biopsies.
Laser scanning cytometry was performed using a laser scanning cytometry instrument (CompuCyte, Corporation, Cambridge, MA) consisting of a base unit containing an Olympus BX50 fluorescent microscope and an optics unit coupled to an Argon and HeNe laser. The laser scanning cytometry analysis and identification of endothelial cells was performed as described previously (22 , 24) . Briefly, endothelial cells were stained using a monoclonal antihuman CD31 (clone JC/70A, Dako Corporation, Carpinteria, CA) followed by Cy5-conjugated goat antimouse secondary (Jackson ImmunoResearch Laboratories, West Grove, PA). For detection of total VEGFR-2, formalin-fixed tissues were incubated with rabbit anti-Flk-1 antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) followed by a secondary goat antirabbit IgG conjugated to Cy5 (Jackson ImmunoResearch Laboratories). For detection of phospho-VEGF, a rabbit antiphospho-VEGFR-2 antibody (Ab-1, Oncogene, La Jolla, CA) was used followed by biotinylated goat antirabbit IgG (Biocare Medical, Walnut Creek, CA) and Avidin- fluorescein isothiocyanate (PharMingen, San Diego, CA). Cells were counterstained with propidium iodide (1 µg/mL) for identification of cell nuclei. The relative levels of fluorescence for each cell were plotted on a scattergram. The analytical gates define four quadrants that determine the total number of cells within each population (i.e., CD31–/pVEGFR–, CD31+/pVEGFR+, and so forth). Staining with negative controls was performed in parallel as controls and were used to set analytical gates. Each slide was placed on a computer-controlled motorized stage, and the area to be analyzed was visually identified using the epifluorescent microscope of the instrument, excluding normal tissue and necrotic regions. The sections were then subjected to quantitative analysis. Three to five randomly selected areas were quantitatively assessed on each slide, and levels were standardized relative to total VEGFR-2. At baseline, phosphorylated VEGFR was detected in 30–90% of CD31+ endothelial cells.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics.
A total of 15 patients were enrolled from May 2000 to October 2000. Two patients were registered to study but never treated with SU5416: 1 patient died unexpectedly before receiving any protocol therapy, and the second patient was excluded because of a recent history of deep venous thrombosis. Thus, there were 13 treated patients evaluable for this study.

Table 1Citation shows patient characteristics at study entry. The median age was 49 years (range, 22–72), and the majority (62%) were rated as Eastern Cooperative Oncology Group performance status 1. The patients were heavily pretreated, with 54% having received three or more prior chemotherapy regimens and 85% treated previously with radiotherapy. Synovial sarcoma was the most common histologic subtype (31%).


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  		Table 1 Patient characteristics

 
Toxicities and Physiologic Changes Associated with SU5416 Administration.
Table 2Citation summarizes all of the reported toxicities of grade 2 or higher. Toxicities were rated as whether they were attributable to SU5416 treatment, but this table shows all of the toxicities regardless of attribution. There were no grade 4 toxicities observed. No patients developed life-threatening or lethal toxicities. The most frequent toxicity was headache, which was grade 3 in 4 cases (31%). Headache typically started within several hours after receiving SU5416, was frequently associated with nausea, and usually resolved within 24 hours. Grade 2 rash occurred in 2 patients (15%) and erythema multiforme in 1 patient (8%), which could be attributable to SU5416 or the Cremophor vehicle used in the drug formulation.


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  		Table 2 National Cancer Institute Common Toxicity Criteria grade 2 and 3 toxicities

 
Patients received prophylaxis against thromboembolic disease with either coumadin or low molecular weight heparin during the study because of an increased incidence of thromboembolic events, particularly catheter-associated clots, noted in the Phase I studies (13 , 25, 26, 27) . One patient developed an internal jugular vein thrombosis on day 10 of the first treatment cycle in the context of having an internal jugular Port-a-Cath in place. This occurred despite prophylactic anticoagulation with low-dose warfarin. Study drug was withheld, and the patient was fully anticoagulated with enoxaparin. The port was left in place after radiologic studies revealed it to be fully patent. SU5416 was recommenced on resolution of symptoms, 3 weeks later. Ten days after recommencing SU5416, despite being fully anticoagulated, he went on to develop a recurrence of the thrombosis. Study drug was promptly stopped, and the patient was removed from protocol. There were no other serious hematologic toxicities. Two patients (15%) experienced grade 2 anemia, and 1 patient (8%) developed grade 2 leukopenia.

Hypertension has been reported in clinical trials for several VEGF pathway antagonists (28 , 29) . No patients in this study experienced grade 2 or higher hypertension. There was, however, a statistically significant increase in the median systolic blood pressure from 118 mmHg at baseline to 133 mmHg after 1 month of treatment (P = 0.01; Table 3Citation ). This difference had disappeared by the end of cycle 2. The diastolic blood pressure decreased slightly during treatment, whereas the pulse pressure (systolic minus diastolic blood pressure) increased from baseline to cycle 1 (P = 0.007).


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  		Table 3 Median blood pressure (mmHg) and pulse pressure (systolic minus diastolic pressure) for patients treated with SU5416

 
SU5416 was also reported recently to cause secondary polycythemia vera in 3 patients with Von Hippel-Lindau disease (30) . No significant change in hematocrit was observed during treatment in this study (data not shown), suggesting that this effect may be specific to patients with von Hippel-Lindau disease.

Treatment Duration.
The median duration of treatment was two cycles. One patient with leiomyosarcoma received four cycles before progressing. Treatment was discontinued in 12 patients for disease progression and in 1 patient for toxicity (deep vein thrombosis as discussed above).

Response.
Using formal Response Evaluation Criteria in Solid Tumors criteria to assess response to treatment, 3 of 13 treated patients were inevaluable for response. Two of these patients demonstrated clinical evidence of progression, whereas the third patient was removed from treatment due to toxicity before completing two cycles but had no evidence of progression. One patient had a follow-up scan before completion of two cycles. At the time of the scan, tumor measurements indicated stable disease. This patient demonstrated clinical progression by the end of cycle 2 though, and is, thus, considered to have progressed. There were no responses observed in 13 evaluable patients (0%, 95% one-sided exact binomial confidence interval, 0–21%).

Fig. 1ACitation shows the progression-free survival. The median time to progression was 1.8 months. The study was originally designed to estimate the proportion of patients free of progression at 6 months, because that was considered to be potentially reflective of clinical activity and stabilization of disease. We observed a 3-month progression-free rate of 8% (95% confidence interval, 1–54%) and the 6-month progression-free rate of 0% (95% confidence interval, 0–22%).



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  		Fig. 1. Kaplan-Meier curves for progression-free survival (PFS; A) and overall survival (OS; B). Median PFS was 1.8 months, and the median OS was at the time of this analysis was 22.8 months. Dashed lines, 95% confidence interval.

 
The overall survival is shown in Fig. 1BCitation . At the time of this analysis, 7 patients have died and 6 patients remain alive. Median overall survival is 22.8 months.

One informative case study requires expansion. A 57-year-old man diagnosed in 1998 with metastatic gastrointestinal stromal tumor was treated previously with four chemotherapy regimens and began treatment with SU5416 on August 1, 2000. After two cycles of therapy, he had evidence of disease progression by computed tomography and 18FDG-PET scans (Fig. 2, A and B)Citation and discontinued SU5416 in early October, 2000. In November 2000 he initiated therapy with imatinib mesylate (Gleevec, STI571), and by the end of the first cycle a dramatic reduction in tumor FDG uptake was observed (Fig. 2, C and D)Citation , which was accompanied by tumor shrinkage by computed tomography. The patient has had a partial response to imatinib, which has lasted >3 years at the time of this analysis.



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  		Fig. 2. 18FDG-PET of patient with gastrointestinal stromal tumors treated initially with SU5416 who subsequently had a durable partial response to imatinib. A, baseline scan before initiating therapy. Arrow shows location of large hepatic metastases. B, after ~2 months of treatment with SU5416. Patient was discontinued from protocol for tumor progression as judged by computed tomography scan. C, scan 1 month later, before initiating therapy with imatinib. D, dramatic reduction in FDG-avid disease after 1 month of therapy with imatinib. Patient has been treated with imatinib for >3 years at the time of this analysis without evidence of progression.

 
18FDG-PET Scans.
To assess changes in tumor glucose uptake and metabolism during therapy, 18FDG-PET scans were performed. In 7 of 7 patients assessed at baseline and at the end of cycle 2, an increase in tumor 18FDG uptake was observed. Computed tomography scans performed for all 7 of the patients at the end of cycle 2 showed concordant tumor progression.

Angiogenic Markers.
Table 4Citation shows urinary bFGF and VEGF levels for patients at baseline and after cycle 1 of treatment. The median urinary VEGF and bFGF levels increased by 21% and 14%, respectively over this interval. For 11 patients with both evaluable progression status at 6 months and known laboratory levels, differences in time to progression by baseline VEGF levels were examined. There was a trend toward shorter time to progression among patients with high (greater than median) baseline VEGF levels, but power is limited by a small sample size (Fig. 3A)Citation . As seen in Fig. 3BCitation , there was evidence of shorter survival time among patients with high baseline VEGF levels (P = 0.04). There was no difference in survival or time to progression associated with baseline bFGF levels (data not shown).


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  		Table 4 VEGF and bFGF levels (pg/ml) at baseline and end of cycle 1

 


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  		Fig. 3. Relationship between baseline VEGF levels and clinical outcome. A, progression-free survival and (B) overall survival in patients with high (≥median level of 101 pg/ml) or low (<101 pg/ml) baseline levels of urinary VEGF. A shorter overall survival time was observed for patients with high baseline VEGF levels (P = 0.04).

 
Tumor Biopsies.
To directly assess the effects of SU5416 on the tumor, we used laser scanning cytometry to measure VEGFR phosphorylation status from biopsies taken at baseline and after two cycles of therapy. Of the 13 patients treated in the study, 5 underwent repeat biopsies, yielding three pairs of evaluable biopsies (in 1 case, the second biopsy yielded necrotic tissue that could not be evaluated; in another, the amount of tissue was not adequate for a full analysis). Treatment with SU5416 did not lead to significant decreases in VEGFR phosphorylation in any of these three pairs (Table 5)Citation . In 2 cases, VEGFR phosphorylation decreased slightly after treatment, whereas in 1 case it increased. Interestingly, 1 of the cases in which partial inhibition was observed was the aforementioned patient with gastrointestinal stromal tumor who subsequently had a prolonged clinical response to imatinib, providing additional evidence for incomplete target inhibition by the drug. An analysis of additional angiogenic markers using tumor biopsies from this study and two other clinical trials using SU5416 or a related molecule, SU6668, is detailed in a separate study.9


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  		Table 5 Quantitative analysis of VEGFR-2 phosphorylation by laser scanning cytometry

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For most advanced or metastatic soft tissue sarcoma, conventional cytotoxic chemotherapy remains standard for palliative management of patients. Unfortunately these tumors are relatively chemoresistant, and responses to these chemotherapy agents are usually suboptimal and short-lived. The vast majority of soft tissue sarcomas express VEGF (20 , 21) . Preclinical studies indicate that VEGF pathway blockers inhibit tumor growth in animal models of sarcoma and other malignancies (4 , 7 , 31) , suggesting that VEGFR-2 may be a viable target. To test this hypothesis, we conducted a Phase II trial of the small molecule VEGFR-2 inhibitor SU5416.

In Phase I testing, 145 mg/m2 twice weekly was determined to be a dose that was acceptable for chronic dosing and resulted in a systemic exposure comparable with what was required in animals for effective tumor inhibition (8 , 32) . This dose has subsequently been used for Phase II testing (12 , 25, 26, 27 , 33, 34, 35) . At higher doses, dose-limiting toxicities were observed including headache, nausea, and vomiting. We found that SU5416 could be safely administered chronically at this dose. There were no grade 4 toxicities observed. Consistent with previous studies, headache was the most frequent grade 3 toxicity in this study and was typically reversible within 24 h. Certain toxicities, such as hyperglycemia and insomnia, were likely attributable to the steroid premedication.

It is interesting to note that systolic blood pressure and pulse pressure increased slightly after one cycle, although this difference had disappeared by the end of cycle 2 (Table 3)Citation . Hypertension has been reported for bevacizumab (29 , 36) and VEGFR-2 antagonists, raising the possibility that this effect may be related to blockade of the VEGF/VEGFR-2 pathway. The mechanism by which VEGFR-2 blockade might cause an increase in blood pressure has not been established. VEGF is known to be a regulator of vascular tone and permeability, in part through modulation of endothelial nitric oxide synthase (37 , 38) . By inhibiting VEGFR-2, SU5416 may interfere with these processes and inhibit nitric oxide-mediated vasodilation, causing a change in vessel compliance.

On the basis of preclinical studies, VEGF pathway inhibitors, if active, would be expected to result in disease stabilization as opposed to tumor shrinkage. Conventional radiologic means of assessing activity based on response rate may, therefore, not be an optimal indicator of the biological activity of these agents. In view of this, it has been proposed that progression-free survival or progression-free rate may be a more appropriate end point for Phase II studies of agents expected to be cytostatic (39 , 40) .

In this study we observed no objective tumor responses out of 13 evaluable patients. The median progression-free survival was 1.8 months, whereas the 3- and 6-month progression-free rates were 8% (95% confidence interval,1–54%) and 0% (95% confidence interval, 0–22%), respectively. The results were not considered sufficiently promising to continue accrual to study, and the production and testing of SU5416 was ceased by the biopharmaceutical firm that manufactured it. Due to the small sample size, the latter confidence interval includes both 5% (the rate signifying minimal activity) and 15% (the rate indicating SU5416 was worthy of additional study). Given this broad confidence interval, we cannot exclude the possibility that SU5416 might be active in slowing tumor progression in some cases. The available data suggests, however, that the magnitude of any such activity would be small. In a review of European Organization for Research and Treatment of Cancer studies of patients with advanced soft tissue sarcoma, 380 patients treated previously with chemotherapy had a 3-month progression-free rate of 28% and a 6-month progression-free rate of 10% (39) . The subgroup of patients treated with inactive agents had 3-month and 6-month progression-free rates of 21% and 8%, respectively. On the basis of these studies, the authors suggest that a 3-month progression-free rate of ≤20% suggests inactivity, whereas a progression-free rate ≥40% suggests activity. The 3-month progression-free rate of 8% observed in our study and the absence of objective tumor responses suggest that the agent does not have significant activity in advanced soft tissue sarcoma. In other studies of SU5416 a limited degree of antitumor activity has been observed. In a recently published study, which included 26 patients with soft tissue sarcoma treated with SU5416 at the same dose and schedule as this study, 1 patient had a partial response (4%) and 5 patients had stable disease for ≥3 months for a 3-month progression-free rate of 23% (12) . Evidence of antitumor activity has also been observed in patients with other malignancies treated with SU5416 (10 , 11 , 33 , 34) .

Although SU5416 did not demonstrate significant antitumor activity in this study, the median survival of 22.8 months was longer than would be expected for patients with metastatic soft-tissue sarcoma, which was ~12 months in a large analysis of patients treated in first-line chemotherapy studies (41) . This is likely explained by the heterogeneous population group in this small study and, in particular, the large percentage of patients with synovial cell sarcoma (31%), a tumor subtype generally associated with longer survival (41) .

There are a number of possible explanations for the lack of antitumor activity observed. One possibility is that the inhibition of the VEGFR-2 by SU5416 may not have been complete or consistent. To assess the interaction between SU5416 and its targets, biopsies were performed on a subset of patients at baseline and after 2 months of therapy and assessed for VEGFR phosphorylation by laser scanning cytometry. In 2 patients VEGFR phosphorylation decreased slightly after therapy, whereas in the third patient, an increase was observed (Table 5)Citation . Because of the small number of paired specimens analyzed and the absence of clinical responses to SU5416, it is not possible to determine whether the degree of inhibition correlates with clinical response. For comparison, however, in a murine orthotopic bladder tumor model, a monoclonal antibody inhibitor of murine VEGFR-2 effectively inhibited tumor growth and led to a 50% inhibition of VEGFR phosphorylation as assessed by Laser scanning cytometry.9 These results are consistent with the hypothesis that SU5416 incompletely inhibited its intended targets in this study. Similar findings were observed in a set of 8 additional patients treated with SU5416 or SU6668 in other studies.9

Incomplete receptor inhibition may be a result of inadequate systemic exposure. In Phase I testing an elimination half-life of <1 hour was observed (8) . Furthermore, using the twice weekly schedule, a 50–60% induction of SU5416 clearance was observed with chronic dosing (10 , 32 , 42) resulting in lower systemic exposures. A schedule using a 5-day loading dose followed by once a week dosing at 145 mg/m2 prevented this induction, leading to consistently higher serum levels than twice weekly dosing (10) . Given the short half-life, dose-limiting toxicities, and induction of SU5416 clearance, sustained levels needed for therapeutic efficacy may not have been achieved or sustained using a twice-weekly dosing schedule. The lack of antitumor response in a patient with gastrointestinal stromal tumor, who subsequently responded to imatinib, additionally supports this possibility (discussed below). Therefore, based on this study, we cannot rule out the possibility that agents achieving a higher degree or more prolonged VEGFR inhibition may be useful for the treatment of soft tissue sarcoma.

It is noteworthy that a patient with gastrointestinal stromal tumor who had progressive disease after 2 months of treatment with SU5416 subsequently has had a partial response lasting >3 years to imatinib mesylate. Imatinib is thought to exert its antitumor effect through inhibition of the mutated KIT receptor kinase, which is constitutively active in most cases of gastrointestinal stromal tumor (16, 17, 18 , 43 , 44) . Interestingly, SU5416 was reported to inhibit KIT receptor tyrosine phosphorylation induced by stem cell factor with an IC50 of 0.01 µmol/L in H526 small cell carcinoma cells (15) , and 0.1–1.0 µmol/L in M07e cells (14) , similar to the IC50 of 0.1 µmol/L observed for imatinib in M07e cells (46) . Therefore, SU5416 showed no antitumor activity in this patient despite its ability to inhibit KIT tyrosine kinase activity at concentrations similar to or lower than imatinib (14 , 15 , 44 , 45 , 46) . The dramatic and sustained antitumor activity observed for imatinib, but not SU5416, may be due to differences in the dosing schedule (daily for imatinib versus twice weekly for SU5416), drug pharmacokinetics (the elimination half-life of SU5416 is <1 hour versus 13–16 hours for imatinib; Ref. 47 ), or a difference in sensitivity of the specific activating KIT mutation to either drug.

Another possible explanation for the overall observed lack of antitumor activity is that for advanced tumors with established vasculature, targeting one receptor for a single angiogenic factor may not be sufficient. Sarcomas are known to produce a variety of proangiogenic factors in addition to VEGF, including bFGF, acidic fibroblast growth factor, VEGF-B, VEGF-C, interleukin 8, transforming growth factor {alpha}, and matrix metalloproteinases (20 , 48, 49, 50, 51) , which could presumably help sustain tumor vasculature even in the presence of VEGFR-2 inhibition. VEGFR-1 and other VEGFRs may play an important role in tumor vasculature as well (52) . For this reason, combinations of agents or inhibitors with broader specificity may prove to be more effective.

Elevated baseline levels of urinary VEGF but not bFGF appeared to be associated with shorter survival (Fig. 3B)Citation , although this finding was of borderline significance (P = 0.04). Furthermore, there was a trend toward shorter time to progression in patients with high baseline VEGF (Fig. 3A)Citation . Interestingly, in a Phase I study of SU5416, 4 patients who obtained clinical benefit (defined by tumor regression or diseases stabilization for at least 12 weeks) had higher baseline 24-hour urinary levels of VEGF than the 18 patients who did not have clinical benefit (323.4 versus 182.7, P < 0.05; Ref. 10 ). Because no patients in this study obtained clinical benefit by these criteria, such a comparison is not possible in our study. Nevertheless, the trend toward shorter time to progression in patients with high baseline VEGF in this study suggests that elevated baseline VEGF would most likely not be useful as a marker of responsiveness to a VEGFR-2 antagonist.

The mean urinary VEGF and bFGF levels were 21% and 14% higher, respectively, at the end of the first cycle of therapy as compared with baseline. There was considerable variability in these levels for a given patient during therapy, and no clear pattern of change could be discerned. Furthermore, it is not known whether changes observed were due to treatment with SU5416, disease progression, or some other cause. Our findings suggest that measurement of levels of angiogenic factors may provide prognostic information. This is consistent with earlier studies in sarcoma and other diseases in which elevated VEGF correlated with higher stage and/or poor outcome (20 , 51) , although in one study of synovial sarcoma a correlation was not observed (53) . It remains to be seen whether urinary VEGF levels will useful for predicting or monitoring responses to therapy (22) .

In summary, SU5416, an inhibitor of VEGFR-2 and KIT tyrosine kinases, was relatively well tolerated but did not appear to have significant antitumor activity in patients with soft tissue sarcoma. In a limited analysis of 3 patients, significant decreases in VEGFR photophosphorylation were not observed, suggesting that target inhibition was incomplete. This possibility was additionally supported by a patient with gastrointestinal stromal tumor who had tumor progression after two cycles of SU5416 but subsequently had a prolonged response to imatinib, another KIT inhibitor. This case highlights the importance of not only identifying the critical molecular target(s) for a given tumor type, but also determining the duration and degree of target inhibition necessary for antitumor activity.


   ACKNOWLEDGMENTS
 
The authors would like to thank Amy Potter for her assistance in tabulating clinical trial data and Beth Staron for data management in the DFCI Quality Assurance Center for Clinical Trials.


   FOOTNOTES
 
Grant support: Biostatistics support provided by Grant CA-06516 from the National Cancer Institute. J. Heymach is supported in part by American Society for Clinical Oncology Young Investigator Award and American Association for Cancer Research-Amgen Research Fellowship. G. Demetri and J. Desai are supported in part by the Richard I. Kaner Ewing’s Sarcoma Research Fund, the Leslie’s Links Foundation, the Abraham J. and Phyllis Katz Foundation, the Quick Family Sarcoma Research Fund, and the Ludwig Trust for Cancer Research at Harvard Medical School. D. Davis is supported in part by the Commonwealth Foundation for Cancer Research.

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.

Requests for reprints: George Demetri, Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, SW530, 44 Binney Street, Boston, MA 02115. E-mail: gdemetri{at}partners.org

9 Darren W. Davis, Henry Q. Xiong, Roy S. Herbst, Walter M. Stadler, John V. Heymach, George D. Demetri, Asif Rashid, Yu Shen, Sijen Wen, Steven Canfield, James L. Abbruzzese, and David J. McConkey. Pharmacodynamic analysis of target inhibition and tumor cell death in biopsies obtained from patients treated with the VEGF receptor antagonists SU-5416 or SU-6668. Submitted for publication. Back

Received 1/26/04; revised 5/ 5/04; accepted 5/11/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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