VEGF Manipulation in Glioblastoma

Glioblastoma is the most common malignant primary brain tumor that is universally lethal, despite optimized treatment including surgery, radiation, and chemotherapy. Targeting angiogenesis has been and continues to be an attractive therapeutic modality in both newly diagnosed and recurrent glioblastoma patients. Vascular endothelial growth factor (VEGF) is the most abundant and important mediator of angiogenesis in glioblastoma. Multiple strategies have been developed to target VEGF/VEGF receptor (VEGFR)–mediated angiogenesis, including VEGF blockade, VEGF Trap, and suppression of VEGFR signaling via receptor tyrosine kinase inhibitors (TKIs). These strategies have been explored in a spectrum of clinical trials, yielding findings that have all contributed to furthering our overall understanding of antiangiogenic therapy. The role of these agents and how to best incorporate them into the treatment paradigm for glioblastoma continues to evolve as understanding of resistance mechanisms improves. Although the excitement surrounding antiangiogenic therapy has waned over the years due to the lack of durable responses and survival benefit, there continues to be hope that combining antiangiogenic therapies with radiation therapy, cytotoxic drugs, immunotherapy, and targeted molecular agents may greatly enhance treatment strategies for high-grade gliomas.

Glioblastoma

Glioblastoma is the most common malignant primary brain tumor in adults and invariably carries a dismal prognosis. The current standard of care is maximal safe surgical resection followed by radiation therapy with concurrent and adjuvant temozolomide. Despite this multimodality treatment approach, recent clinical trials have reported a median survival of only 14 to 16 months, with a 26% to 33% 2-year survival rate.[1,2] Recurrence or progression of disease is inevitable and salvage therapies with single or combination chemotherapy are largely ineffective, with 6-month progression-free survival (PFS) rates of 5% to 15%.[3-5] Poor treatment response is ascribed to multiple factors including intertumoral and intratumoral heterogeneity, de novo and acquired resistance, ineffective drug delivery as a result of the blood-brain barrier, and multiple redundant cellular pathways regulating cellular survival and proliferation.

Angiogenesis

Glioblastoma is one of the most highly angiogenic solid tumors. Its tumor vasculature is both structurally and functionally abnormal, characterized by a dense network of vessels that are tortuous and hyperpermeable, with increased vessel diameter and abnormally thickened basement membranes. This aberrant tumor vasculature is believed to enhance tumor hypoxia and impair the delivery of cytotoxic chemotherapy.[6,7] Angiogenesis is a pathologic hallmark of glioblastoma and is driven by both hypoxia-dependent and hypoxia-independent mechanisms. Hypoxia inactivates prolyl hydroxylases, resulting in hypoxia-inducible factor-1α (HIF-1α) accumulation that leads to activation of VEGF, one of the most important regulators of angiogenesis.[8,9] VEGF is also upregulated via hypoxia-independent mechanisms as a result of dysregulated activation of mitogenic and survival pathways including Ras/mitogen-activated protein kinase and phosphatidylinositol 3-kinase. In addition to VEGF, glioblastoma frequently expresses additional proangiogenic factors including fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), integrins, hepatocyte growth factor/scatter factor, ephrins, angiopoietins, and interleukin-8.[10-13] The critical role that VEGF plays in angiogenesis has rendered it an appealing target to exploit in cancer therapeutics.[14]

VEGF-Targeted Therapies

VEGF blockade

VEGF and its receptors can be antagonized by monoclonal antibodies to VEGF. Bevacizumab is a recombinant humanized monoclonal antibody composed of human immunoglobulin G1 (IgG1; 93%) and murine complementarity-determining regions (7%) that bind to VEGF with high affinity and specificity, preventing its interaction with VEGFRs, ultimately resulting in suppression of VEGF signaling.[15] Bevacizumab received accelerated US Food and Drug Administration (FDA) approval in May 2009 for use as a single agent in patients with glioblastoma who have progressive disease following front-line therapy consisting of surgical resection, radiotherapy, and temozolomide. The basis of this accelerated approval followed the results of two phase II studies that demonstrated an objective tumor response rate based on independent radiographic review. The first study was a single-arm evaluation of single-agent bevacizumab in 48 patients with a Karnofsky performance status (KPS) greater than 60 and any number of prior recurrences.[16] In the second study, 167 glioblastoma patients at first or second recurrence with a KPS greater than 70 were randomized to single-agent bevacizumab or bevacizumab in combination with irinotecan.[17] This study was not designed to detect superiority between the treatment arms and did allow for crossover from single-agent bevacizumab to the combination therapy. For both studies, the primary endpoint was 6-month PFS compared with historic controls. Response assessment for the single-arm study consisted of blinded independent reviewers using the Macdonald criteria,[18] while World Health Organization Response Evaluation Criteria in Solid Tumors[19] was used in the randomized study. Significantly better outcomes were reported for both of these studies when compared with historic controls.

In addition to the evaluation of bevacizumab in the recurrent glioblastoma setting, bevacizumab has also been evaluated in phase III clinical trials for newly diagnosed glioblastoma, with unfortunately no improvement in overall survival. Two recent, large, randomized phase III trials, AVAglio and Radiation Therapy Oncology Group (RTOG) 0825, demonstrated that the addition of bevacizumab to upfront treatment with radiation and temozolomide conferred no benefit in terms of overall survival in newly diagnosed glioblastoma patients when compared with the standard treatment arm.[20,21] PFS was found to be prolonged in both studies by approximately 3 to 4 months, reaching statistical significance in the AVAglio study but not in the RTOG 0825 study based on predefined criteria.[20,21] These two trials reported divergent findings regarding quality of life: The AVAglio trial found improvement in, or prolonged maintenance of, quality of life and performance status, while RTOG 0825 found a worsened quality of life and a decline in cognitive function over time. However, the latter study did independently review the radiographic images used to define progression, and thus it is not known if deterioration in quality of life was as a result of tumor progression not identified by the treating investigator. These results have spurred discussion regarding quality-of-life endpoints for each of the studies and the role that patient-reported outcomes such as quality of life could play in the approval of a new therapy for cancer, regardless of its effect on survival.[22]

The initial excitement generated by the early uncontrolled clinical studies of bevacizumab, which resulted in impressive radiographic responses and prolongation of PFS, have been tempered by the realization that VEGF pathway inhibitors result in transitory clinical and radiographic response on the order of months prior to inevitable progression. The immediate and dramatic radiographic responses that result from treatment with angiogenesis inhibitors suggest that these agents have little intrinsic antitumor activity; the main benefit is derived from the indirect effects secondary to reduction in cerebral edema, with concomitant reduction of corticosteroids, as well as the potential of angiogenesis inhibitors to enhance the efficacy of other therapies. The lack of a durable response seen with bevacizumab as a single agent has resulted in the speculation that pairing it with a cytotoxic agent may yield a more significant benefit because the combination would allow targeting of both endothelial cells and cancer cells. Bevacizumab has been evaluated in combination with lomustine in the BELOB trial, a multicenter phase II study conducted in the Netherlands.[23] Adult patients with first recurrence of glioblastoma following temozolomide-based chemoradiotherapy were randomly assigned to receive lomustine alone, bevacizumab alone, or the combination of bevacizumab and lomustine. Improvement in survival was seen in those patients receiving the combination of bevacizumab and lomustine, who had a median overall survival of approximately 12 months following treatment compared with either of the single-agent groups (lomustine median survival, 8 months; bevacizumab monotherapy median survival, 8 months). These findings are being further assessed in a phase III study from the European Organisation for Research and Treatment of Cancer (EORTC 26101), which is currently underway.

VEGF Trap

Aflibercept (VEGF Trap) is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGFRs 1 and 2 that are fused to the Fc portion of human IgG1/soluble decoy VEGFR, which acts to scavenge both VEGF and placental growth factor (PlGF). Aflibercept has a several hundred–fold greater affinity for VEGF-A than bevacizumab[24] and also demonstrates high affinity for PlGF. In preclinical studies, VEGF Trap improved survival in an orthotopic glioblastoma model[25] and was noted to enhance the activity of radiation therapy.[26] Patients with first recurrence of either glioblastoma (n = 42) or anaplastic glioma (n = 16) following concurrent radiation and temozolomide and adjuvant temozolomide were evaluated in a single-arm phase II study to assess the efficacy of aflibercept.[27] Patients received aflibercept 4 mg/kg intravenously every 2 weeks. Moderate toxicity was noted, with grade 3 hypertension, fatigue, hand-foot syndrome, thrombosis, and proteinuria as the most commonly reported adverse events. The primary endpoint for the glioblastoma patients, defined as 6-month PFS, was not met since only three patients (7.7%) were alive and free of progression at 6 months, resulting in the conclusion that single-agent aflibercept had minimal activity in the setting of recurrent glioblastoma.[27]

VEGFR-Targeted Therapies

Receptor tyrosine kinase inhibitors

Suppression of VEGFR signaling can be achieved by blocking the ligand-binding site of VEGFR with either monoclonal antibodies or genetically engineered peptides, or by blocking the tyrosine kinase activation site of VEGFR with small-molecule inhibitors (tyrosine kinase inhibitors [TKIs]). Numerous receptor TKIs have been under investigation in the setting of recurrent high-grade glioma. Cediranib, sunitinib, pazopanib, vandetanib, and sorafenib are just some of the multi-kinase VEGFR inhibitors that have been evaluated in glioblastoma. The potency of these molecules against the VEGFR family is variable and each agent inhibits multiple other potentially relevant receptors, including KIT, PDGFR, RET, RAF, and EGFR (epidermal growth factor receptor).

A number of these VEGFR TKIs have undergone evaluation in phase I/II clinical trials. Of these agents, cediranib is the only one to have moved to a phase III investigation. In the initial phase II study of single-agent cediranib (45 mg/d), 27% of patients with recurrent high-grade glioma experienced a radiographic response and a 6-month PFS of 26%. Adverse events including hypertension and fatigue were observed, and almost half of the patients required a dose reduction or interruption in therapy because of toxicity.[28] The promising results seen in the phase II REGAL (Recentin in Glioblastoma Alone and With Lomustine) study prompted a randomized, placebo-controlled, partially blinded, phase III clinical trial in patients with first recurrence of glioblastoma, which was designed to determine the efficacy of either cediranib as monotherapy (30 mg/d; n = 120), cediranib (20 mg/d; n = 120) in combination with lomustine, or lomustine alone (n = 60). The primary endpoint of PFS prolongation was not reached in the cediranib as monotherapy arm or the combination with lomustine arm compared with lomustine alone.[29]

Pazopanib, a multi-targeted TKI against VEGFR-1, -2, -3; PDGFR-alpha and -beta; and c-KIT, was evaluated in a single-arm phase II study in the setting of recurrent glioblastoma.[30] Patients with fewer than two relapses and no prior VEGF/VEGFR therapy were treated with daily pazopanib 800 mg on 4-week cycles. A total of 35 recurrent glioblastoma patients were accrued. Overall, pazopanib was reasonably well-tolerated and no unexpected adverse events associated with VEGF/VEGFR agents were observed. Radiographic responses were noted in only 6% of patients, and 6-month PFS was only 3%.[30]

Sunitinib is a small-molecule TKI of the VEGFR, PDGFR, c-KIT, FLT1, FLK1/KDR, FLT3, and RET kinases, with evidence of clinical activity in a number of HIF/VEGF-dependent, PDGFR-dependent, and KIT-dependent cancers, including renal cell carcinoma and gastrointestinal stromal tumor.[31] Unfortunately, limited activity was seen with single-agent sunitinib in a phase II study of 25 patients with recurrent high-grade glioma.[32] None of the patients on the study achieved an objective radiographic response. The median time to progression was 1.6 months and overall survival was only 3.8 months.[32]

The combination of two targeted therapies, vandetanib and sirolimus, was evaluated in a phase I study in patients with recurrent glioblastoma.[33] Vandetanib is a receptor TKI that targets both VEGF and EGF signaling, while sirolimus inhibits mammalian target of rapamycin (mTOR). Vandetanib and sirolimus were orally administered on a continuous daily dosing schedule in patients who had not previously received anti-VEGF and anti-EGF therapy or mTOR inhibitors. Ten patients were enrolled in the dose escalation phase, and 12 more patients were subsequently enrolled at the maximum tolerated dose (MTD) to explore 6-month PFS in a single-arm, single-stage phase II design. Of the 19 patients who received at least one dose at the MTD, two patients had a partial response (10.5%). The dose-limiting toxicity was elevation in aspartate aminotransferase. Lymphopenia, fatigue, rash, and hypophosphatemia were the other most commonly observed toxicities.[33]

Other VEGFR inhibitors

Suppression of VEGFR activation can also be achieved by directly blocking ligand binding. VEGF signaling via VEGFR-2 is an important mediator of angiogenesis in glioblastoma, and CT-322, a pegylated protein engineered from the 10th human fibronectin type III domain, is both a specific and potent blocker of VEGFR-2. The efficacy and safety of CT-322 was evaluated in an open-label phase II study of glioblastoma patients at either first, second, or third relapse with no prior exposure to antiangiogenic therapy.[34] The primary endpoint was 6-month PFS. The 6-month PFS rate in the patients treated at a CT-322 dose of 1 mg/kg was 18.6%, and 0.0% in those patients treated at the 2-mg/kg dose. The study was subsequently terminated prior to full planned accrual because of insufficient efficacy.[34]

Ramucirumab and icrucumab are both monoclonal antibodies that competitively bind the VEGFR ligand-binding site.[35] Ramucirumab was under investigation in a phase II trial of patients with recurrent glioblastoma and was recently completed, with study results pending (ClinicalTrials.gov identifier: NCT00895180).

Other strategies

Thalidomide is an inhibitor of angiogenesis specifically targeting basic FGF and VEGF[36] and was one of the first oral antiangiogenic agents ever evaluated in the setting of high-grade glioma.[37] Although some studies showed modest activity,[38-40] the results have largely been unimpressive, with some trials demonstrating no benefit.[41,42]

Integrins represent a class of cell surface adhesion molecules that play an important role in many cellular behaviors including proliferation, survival, and migration.[43] Integrins such as αvβ3 and αvβ5 are upregulated in several cancers including glioblastoma, with their expression seen on tumor-associated vasculature and tumor cells.[44,45] The integrins αvβ3 and αvβ5 have been implicated in playing a role in glioma angiogenesis based on their expression on activated endothelial cells within the tumor in contrast to the normal brain endothelial cells.[44] Cilengitide is a cyclicized arginine-glycine-aspartic acid (RGD)-containing pentapeptide that selectively and potently blocks the activation of αvβ3 and αvβ5 integrins.[46] Several phase I and II clinical trials of cilengitide demonstrated that the drug is well-tolerated and has evidence of antitumor activity in patients with newly diagnosed and recurrent glioblastoma.[47-49] However, a large, randomized, blinded, placebo-controlled, phase III clinical trial in patients with newly diagnosed glioblastoma did not demonstrate an improvement in median overall survival for patients randomized to receive cilengitide together with standard chemoradiation compared with patients receiving placebo.[50]

Radiographic Assessment of VEGF/VEGFR Therapy

Tumor response evaluation has become increasingly difficult in the era of antiangiogenic therapy. Inhibition of VEGF/VEGFR signaling decreases tumor vessel permeability, which can translate to radiographic responses. Historically, tumor responses are based on reduction in T1 post-gadolinium MRI, in which a reduction in permeable tumor vessels is due to tumor cell killing. The rapid (within 24 hours) and robust imaging responses seen with anti-VEGF therapy suggest that angiogenesis inhibitors have little intrinsic antitumor activity and that the main benefit is derived from a reduction in vessel permeability independent of the effect of the drug on the tumor. Figure 2 highlights the post-contrast radiographic response seen in the setting of single-agent bevacizumab in a patient with recurrent glioblastoma. Indirect benefits of antiangiogenic therapies are largely a result of a secondary reduction in cerebral edema and the ability to reduce corticosteroid use, thereby minimizing steroid-mediated toxicities. This complicates the interpretation of response in patients with high-grade gliomas as measured by contrast enhancement.[51] Despite improvement on the contrasted images, progressive non-enhancing tumor infiltration seen on T2/fluid-attenuated inversion recovery (FLAIR) sequence images is often noted. The standard Macdonald criteria do not address non-enhancing disease, rendering it a less than ideal measure to assess treatment response. The international Response Assessment in Neuro-Oncology Working Group (RANO) subsequently developed new criteria to address these challenges by incorporating measurement of T2/FLAIR signal in addition to the review of the post-contrast studies as part of the imaging response evaluation.[52] The RANO criteria are being integrated into most clinical trials, but there remain challenges, particularly in universally accepted criteria for the formal measurement of non-enhancing tumor.

Mechanisms of Resistance

Unfortunately, durable responses are not typically seen in the setting of antiangiogenic therapy, which has prompted efforts to better understand the mechanisms underlying this resistance, so that therapeutic approaches can be improved. Adaptive (evasive) resistance and intrinsic (preexisting) nonresponsiveness are the primary modes of resistance to antiangiogenic therapy.

Multiple mechanisms are believed to contribute to each type of resistance pattern observed.[53] Adaptive mechanisms described have included activation and/or upregulation of alternative proangiogenic signaling pathways within the tumor, recruitment of bone marrow–derived proangiogenic cells, increased pericyte coverage of the tumor vasculature, and activation and enhancement of invasion and metastasis. It has also been proposed that some glioblastoma-associated blood vessels are intrinsically resistant to antiangiogenic therapy based on the observation of patients on clinical trials with VEGF pathway inhibitors who had no discernible reduction in contrast enhancement on MRI after treatment with antiangiogenic therapy. A subset of patients in clinical trials with agents including bevacizumab, sorafenib, and sunitinib who did not demonstrate any transitory radiographic response or clinical benefit was identified.[54] This lack of response was characterized by no evidence of reduction in vascular permeability, no cessation of tumor growth or retardation of growth rate, no observed quality-of-life benefit, and no evidence of increased survival. It is likely that these tumors express high levels of multiple other proangiogenic growth factors in addition to VEGF, rendering tumor angiogenesis largely VEGF-independent.[53]

Clinical Implications

Unfortunately, the outcome of antiangiogenic therapy in glioblastoma patients has been largely disappointing in both the upfront and recurrent settings. Though bevacizumab originally received accelerated FDA approval in 2009 for use as a single agent in patients with progressive or recurrent glioblastoma following front-line standard therapy, collective experience over time has failed to demonstrate a survival benefit. There is no role for adding bevacizumab to the upfront treatment of patients with newly diagnosed glioblastoma based on the results of AVAglio and RTOG 0825.[20,21] Present National Comprehensive Cancer Network (NCCN) practice guidelines (Version 1.2015) continue to reflect the role of bevacizumab for use in the recurrent setting only as a result of the original FDA-approved indication. According to the NCCN guidelines, bevacizumab is recommended to be used either as a single agent or in combination with chemotherapy in the setting of recurrent anaplastic glioma or glioblastoma. Data from a recently completed phase III trial of bevacizumab alone compared with bevacizumab plus lomustine are anxiously anticipated.

The lack of a durable response and survival benefit has underscored the need to further our understanding of the mechanisms underlying resistance to antiangiogenic therapy. VEGF pathway inhibitors typically result in only transitory clinical and radiographic responses, which are largely secondary to the benefit derived in the reduction of cerebral edema. Although this benefit is only transitory, it is still frequently of paramount importance in reducing neurologic symptoms, which can be functionally disabling. The dramatic and rapid reduction in cerebral edema makes antiangiogenic therapy very relevant clinically in the effort to preserve neurologic function and, ultimately, quality of life. For those patients who are steroid-dependent, the initiation of antiangiogenic therapy can be a mechanism to wean them off steroids, which is an important step in improving quality of life, given the large number of adverse side effects associated with long-term steroid use.

Antiangiogenic therapy harbors the potential to enhance the efficacy of other therapies when combined with cytotoxic therapies, and even immunotherapeutic strategies, which has renewed a sense of optimism that there are still potentially important roles for antiangiogenic therapies in the treatment of glioblastoma. As previously mentioned, the recently published BELOB study demonstrated a significant improvement in overall survival with the combination of bevacizumab and lomustine compared with either agent alone.[23] A confirmatory phase III trial is still accruing data. Anti-VEGF therapies have also been noted in preclinical studies to result in increased tumor permeability to activated T cells, which has the overall result of making tumor cells more susceptible to immune attack.[55] Efforts are currently underway to exploit the role of anti-VEGF therapy in overcoming immunosuppression via the exploration of treatment approaches using combinations of bevacizumab with immunotherapy. Immunotherapeutic strategies under investigation in combination with bevacizumab include vaccines and immune checkpoint inhibitors. The ReACT trial is one example of a combinatorial treatment strategy evaluating bevacizumab with the rindopepimut vaccine in patients with recurrent EGFRvIII-positive glioblastoma (ClinicalTrials.gov identifier: NCT01498328).

The role of antiangiogenic therapy remains to be fully defined and timing of its use has evolved into a challenge routinely encountered by practitioners. There are an increasing number of clinical trials excluding patients with prior bevacizumab exposure in part due to the confounding imaging results seen following tumor progression on antiangiogenic therapy. Clinical trial accessibility has now created a culture that reserves bevacizumab for cases in which other lines of treatment-including clinical trials-have been fully exhausted. In our practice, we typically advocate for deferring the use of bevacizumab for patients who have other treatment alternatives, including enrollment onto clinical trials, given the potential limitation that bevacizumab exposure can have on clinical trial eligibility. In the absence of clinical trial options, we favor the use of bevacizumab in combination with cytotoxic chemotherapy over bevacizumab alone for those patients with good functional status.

Conclusion

Angiogenesis is a pathologic hallmark of glioblastoma and continues to be an appealing therapeutic target in cancer, including high-grade gliomas. Among other proangiogenic cytokines, VEGF is the most important regulator of angiogenesis in glioblastoma. Multiple approaches to targeting the VEGF/VEGFR pathway, including VEGF sequestration, vascular disruption, and suppression of VEGFR signaling using TKIs, have been explored in a spectrum of clinical trials. Many of these strategies remain under active investigation. As a result of the collective investigative effort in evaluating antiangiogenic therapy in clinical trials, tumor response evaluation has evolved due to the increased understanding of the radiographic changes antiangiogenic therapy can produce. Bevacizumab continues to be the most studied antiangiogenic agent in high-grade gliomas. Although bevacizumab has yet to conclusively demonstrate improved survival in any high-grade glioma setting to date, it is clearly useful in patients with peritumoral edema. However, the exact timing of administration remains undefined.

Angiogenesis is highly complex, which underscores the need for an improved understanding of the mechanisms of action and resistance to VEGF/VEGFR therapies in order to determine how to optimally incorporate the use of antiangiogenesis inhibitors into the treatment paradigm for high-grade gliomas. Recent data implicating VEGF as a potent mediator of immunosuppression suggest a rational combination of anti-VEGF therapies with agents designed to activate antitumor immune responses such as vaccines, adoptive T-cell therapies, and checkpoint inhibitors (programmed cell death 1 and programmed death ligand 1 antibodies). Combining antiangiogenic therapies with radiation therapy, cytotoxic drugs, immunotherapy, and targeted molecular agents offers the hope of improving existing treatment strategies.

Financial Disclosure: Dr. de Groot serves on advisory boards for Genentech, Celldex, Novartis, and Foundation Medicine; serves as a consultant for Celldex and Deciphera Pharmaceuticals; is on the Data Safety Monitoring Board for VBL Therapeutics; and receives research support from Sanofi-Aventis, AstraZeneca, EMD-Serono, Eli Lilly, Novartis, and Deciphera Pharmaceuticals. Dr. Weathers has no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

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