Bevacizumab: Review of Development, Pharmacology, and Application to Brain Tumors

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1 Clinical Medicine: Therapeutics R e v i e w Open Access Full open access to this and thousands of other papers at Bevacizumab: Review of Development, Pharmacology, and Application to Brain Tumors Herbert B. Newton Dardinger Neuro-Oncology Center and Division of Neuro-Oncology, Departments of Neurology and Neurosurgery, The Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute Columbus, Ohio USA. newton.12@osu.edu Abstract: Bevacizumab is a humanised monoclonal antibody targeted to the vascular endothelial growth factor (VEGF). VEGF is the ligand for VEGF receptors (VEGFR), which are important for the development and maintenance of the angiogenic phenotype in highgrade solid tumors, including malignant gliomas. An overview of angiogenesis, VEGF, VEGFR, and the pharmacology of bevacizumab will be presented. Bevacizumab is active in pre-clinical testing against glioma tissue cultures and xenograft models. In the clinical setting, in combination with irinotecan and other chemotherapy agents, it has shown significant activity in patients with glioblastoma multiforme (GBM) and other brain tumors. Objective responses on neuro-imaging have been noted in 30% 60% of reported cases. Prolongation of progression-free survival and overall survival have also been suggested in many reports. Treatment of bevacizumab is associated with potential side effects, including thromboembolic disorders, fatigue, intracranial hemorrhage, proteinuria, hypertension, and bowel perforation. Keywords: bevacizumab, chemotherapy, vascular endothelial growth factor, angiogenesis, brain tumor, glioblastoma multiforme, glioma Clinical Medicine: Therapeutics 2009: This article is available from Libertas Academica Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution and reproduction provided the original work is properly cited. The authors grant exclusive rights to all commercial reproduction and distribution to Libertas Academica. Commercial reproduction and distribution rights are reserved by Libertas Academica. No unauthorised commercial use permitted without express consent of Libertas Academica. Contact tom.hill@la-press.com for further information. Clinical Medicine: Therapeutics 2009:1 1577

2 Newton Primary brain tumors (PBT) remain a significant health problem in the USA and worldwide. They comprise some of the most malignant tumors known to affect humans and are generally refractory to all modalities of treatment. 1 Between 30,000 and 35,000 new cases of PBT will be diagnosed in the upcoming year. 1 3 The majority of these tumors will be malignant gliomas of neuroectodermal origin. 4 Most studies suggest that approximately 14 per 100,000 people in the USA will be diagnosed with a PBT each year. Among this cohort, 6 to 8 per 100,000 will have a high-grade glioma. The prognosis and survival of patients with malignant gliomas remains poor. 1 3 Although an uncommon neoplasm, PBT are among the top 10 causes of cancer-related deaths in the USA and account for 2.4% of all yearly cancer-related deaths. 5 The median survival for a patient with glioblastoma multiforme (GBM) is approximately 12 to 15 months. For patients with a lowgrade astrocytoma or oligodendroglioma, the median survival is still significantly curtailed and is about 6 to 10 years. It is obvious from these survival data that the current treatment of brain tumor patients (i.e. surgical resection, irradiation, chemotherapy) is inadequate and unable to overcome the malignant biology of these neoplasms. Although cytotoxic chemotherapy has been demonstrated to provide a survival benefit to highgrade glioma patients in several recent meta-analyses, the results have been modest. 6,7 One prominent explanation for the poor results of conventional chemotherapy is the non-specific, non-targeted nature of most of the currently used drugs (e.g. alkylating agents, DNA topoisomerase inhibitors). 8,9 As mentioned above, conventional forms of treatment for malignant glioma are not predicated on the biology of the malignant phenotype. It has become apparent that the transformed phenotype of brain tumor cells is highly complex and results from the dysfunction of a variety of inter-related regulatory pathways The transformation process involves amplification or over-expression of oncogenes in combination with loss or lack of expression of tumor suppressor genes. Oncogenes that have been demonstrated to be important for gliomagenesis include platelet-derived growth factor and its receptor (PDGF, PDGFR), epidermal growth factor and its receptor (EGF, EGFR), CDK4, mdm-2, Ras, Akt, and mtor (mammalian target of rapamycin) (see Figs. 1 3). Tumor suppressor GF PDGF/EGF/FGF/IGF-IR receptor cell membrane Grb2 Sos-1 Ras GDP Gap Ras GTP PL-C Raf-1 PI3K DAG MEK Akt PKC MAPK Proliferation Gene transcription & survival Figure 1. Ligand (e.g. PDGF, EGF, FGDF, IGF) binds to the extracellular portion of a protein tyrosine kinase receptor, inducing dimerization of two receptors and autophosphorylation of the catalytic domain tyrosine kinase residues. Receptor activity leads to attachment of molecules with SH2 domains, such as Grb2, PL-C, and PI3K, activating downstream signaling pathways that promote proliferation, gene transcription, and cellular survival. Adapted from reference 15 and used with permission from Future Drugs, Ltd Clinical Medicine: Therapeutics 2009:1

3 Bevacizumab and brain tumors GF PDGF/EGF/FGF/IGF-IR receptor cell membrane PI3K PDK1 Akt PDK2 PIP2 PIP3 Akt E2F MDM2 p21/p27 BAD FKHR NF-κ B caspase-9 GSK3β mtor proliferation & survival anti-apoptosis & survival glycogen synthesis Figure 2. PI3K is activated by growth factor receptors after stimulation by the various ligands (e.g. PDGF, EGF, IGF, FGF), as well as by the Ras pathway. Activity of PI3K leads to the production of PtdIns(3,4,5)P (PIP3), after phosphorylation of PtdIns(4,5)P (PIP2). PIP3 binds Akt and translocates it near the cell membrane, where it can be phosphorylated and activated by PDK1 and PDK2. After the activation of Akt, it interacts with and phosphorylates several downstream effectors that promote proliferation and survival, including MDM2, p21/p27, BAD, FKHR, NF-κB, caspase-9, GSK3β, and mtor. Adapted from reference 16 and used with permission from Future Drugs, Ltd. GF PDGF/EGF/FGF/IGF-IR receptor cell membrane PI3K PDK1 Akt PDK2 PIP2 PIP3 mtor Tuberin Hamartin prb P70 S6K 4E-BP1 eif-4e eif-4g S6 Translation Figure 3. mtor is activated directly by Akt, and indirectly via Akt-mediated inhibition of the tuberin/hamartin complex. Activated mtor then phosphorylates several key regulators of cellular protein translation, including p70s6k and 4E-BP1. mtor also interacts with other proteins, such as prb, that are involved in cell growth and metabolism. Adapted from reference 16 and used with permission from Future Drugs, Ltd. Clinical Medicine: Therapeutics 2009:1 1579

4 Newton genes of importance in glial transformation include p53, retinoblastoma (Rb), p16 and p15 (i.e. INK4a, INK4b), DMBT1, and PTEN. Most of these tumor suppressor genes function as negative regulators of the cell cycle, while others are inhibitors of important internal signal transduction pathways. The net effect of these acquired abnormalities is dysregulation of, and an imbalance between, the activity of the cell cycle and apoptotic pathways. Due to the inadequacy of conventional therapeutic approaches, it is obvious that new treatment modalities must be developed that have a more molecular, targeted mechanism of action, with the ability to overcome the transformed phenotype of malignant gliomas Recent advances in the biology of growth factor receptors, internal signal transduction, and angiogenesis are now providing the background for development of molecular therapeutics, a new class of drugs that manipulate and exploit these pathways. Many investigators have begun to focus on agents that target the ligands and receptors that mediate neoplastic angiogenesis. The vascular endothelial growth factor (VEGF) pathway appears to be the dominant angiogenic pathway in solid tumors, including malignant gliomas. 16,19,20 Intense efforts are underway to develop targeted therapeutics to VEGF and its receptors (VEGFR). The following sections will review angiogenesis, the VEGF signaling pathway, and the pharmacology and clinical applications of bevacizumab, a monoclonal antibody targeted against VEGF. Overview of Angiogenesis Angiogenesis is a tightly controlled process that involves growth and maintenance of blood vessels within tissues and organs. 21 A delicate equilibrium exists between positive angiogenic factors (e.g. basic fibroblast growth factor [bfgf], VEGF, transforming growth factor-β [TGF-β], platelet-derived endothelial cell growth factor), and inhibitory factors (e.g. thrombospondin-1 [TSP-1], glioma-derived angiogenesis inhibitor factor, tissue inhibitor of matrix metalloproteinases-1, angiostatin, endostatin) These factors interact with specific receptors on endothelial cells and the extracellular matrix (ECM), such as VEGF receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, TIE1, and TIE2. 26 The surrounding ECM, associated adhesion molecules (i.e. integrins), and matrix metalloproteinases (MMP s) are also important contributors to normal and neoplastic angiogenesis In the normal adult brain, angiogenesis is tightly downregulated, except for low-level activity within vascular endothelial cells. 26 After initial clonal expansion, a PBT must acquire the angiogenic phenotype and induce neovascularization to enlarge beyond 2 to 3 mm 3 in size (approximately one million cells). The switch to the angiogenic phenotype involves up-regulation of selected angiogenic factors (e.g. 50-fold increase in expression of VEGF in GBM cells) and their receptors, and down-regulation of angiogenesis inhibitors. There are several important stimuli for conversion to the angiogenic phenotype in GBM. The presence of hypoxia induces up-regulation of secretion of VEGF and expression of VEGFR s in tumor endothelial cells and surrounding regional vasculature. 26 Another critical element for the switch to the angiogenic phenotype is over-activity of the major growth factor signaling pathways and loss of certain tumor suppressor genes. 12,13 Overexpression and excessive activity of PDGF, EGF, insulinlike growth factor (IGF), FGF, Ras, and TGF-α and TGF-β, are critical to the development of the angiogenic phenotype through several mechanisms. 12,31 34 Increased levels of these tumor-derived growth factors and/or their receptors can directly initiate endothelial activity and blood vessel tube formation, stimulate the increased expression and/or secretion of VEGF, and upregulate critical proteases from endothelial cells to remodel surrounding ECM and permit endothelial cell migration. In addition to growth factor activity, internal signal transduction mediators also appear to play a role in the angiogenic phenotype. The phosphatidylinositol-3 kinase (PI3K)/Akt/PTEN signaling pathway is involved in the regulation of angiogenesis through the control of expression of VEGF, hypoxia-inducible factor-1 (HIF-1), and TSP Over-activity of the PI3K/Akt signaling pathway, either by excessive stimulation by upstream growth factors or by loss of PTEN function, leads to upregulation of VEGF expression directly and secondarily, through increased expression of HIF-1 or HIF-1α. HIF-1 is a heterodimeric transcriptional activator composed of HIF-1α and HIF-1β subunits that are highly expressed in endothelial cells of malignant gliomas, especially under hypoxic conditions, and can induce expression of VEGF. Activation of PI3K/Akt leads to an increase in HIF-1α protein levels without 1580 Clinical Medicine: Therapeutics 2009:1

5 Bevacizumab and brain tumors an increase in the levels of HIF-1α mrna, suggesting regulation at the post-transcriptional level. 37,39 The levels of HIF-1α and VEGF can be subsequently reduced by treatment with wortmannin or LY294002, inhibitors of the PI3K/Akt pathway, or by replacement of wild-type PTEN. TSP-1 is a negative regulator of angiogenesis that is downregulated in cells with loss of PTEN function and overactivity of the PI3K/Akt signaling pathway. Recent studies suggest that angiogenesis is essential for the growth of GBM and other PBT, similar to systemic solid neoplasms. 26,40,41 There is a significant inverse correlation between density of vessels in astrocytic gliomas and postoperative survival. 42 Patients with the highest microvessel density had the most malignant tumors and the shortest survival times. Vascular density is most prolific in GBM, which demonstrates intense vascular and microvascular hyperplasia, including the presence of glomeruloid bodies. As mentioned above, the exaggerated vascularity and necrosis in GBM appears to result from a combination of a high rate of proliferation and the presence of widespread hypoxia throughout the tumor. Necrosis is present in areas of severe hypoxia, often surrounded by pseudopalisading tumor cells. 26,41 Levels of HIF-1α are very high in these hypoxic pseudopalisading cells, as well as in cells at the leading edge of the infiltrative, growing tumor. The high levels of HIF-1α are due to increased protein stability, mediated via a hypoxia-induced reduction in interaction with the Von Hippel-Lindau (VHL) tumor suppressor protein. 43 Under non-hypoxic conditions, HIF-1α is hydroxylated and acetylated, which promotes interaction with the VHL protein, an E3 ubiquitin ligase that induces the rapid ubiquitination and proteosomal degradation of HIF-1α. In addition, expression of VEGF is also very high in hypoxic pseudopalisading tumor cells, on the order of 200 to 300-fold higher than concentrations in serum. 44 Molecular Biology of VEGF, VEGFR, and VEGF/VEGFR Signaling VEGF was first discovered in 1983 by Senger et al, who described the partial purification of a protein with the ability to induce vascular leakage when injected into guinea pig skin. 19,45 The protein was named vascular permeability factor (VPF), and was thought to function as a regulator of the permeability of tumor vasculature. In 1989, Ferrara and Henzel reported the purification and partial sequencing of an endothelial cell-specific mitogen, which they named VEGF. 19,46 Subsequent cloning and expression of VPF and VEGF revealed that they were the same molecule. 19 The VEGF gene family is now known to include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF) The human VEGF-A gene is located on chromosome 6p21.3, and is organized into eight exons, separated by seven introns. 50,51 The structure and organization of the other VEGF genes are similar to VEGF-A, with different chromosomal locations (e.g. VEGF-B gene is on 11q13; VEGF-C gene is on 4q34). The VEGF genes are under regulatory control from several mechanisms, including hypoxia, cell surface receptors, transcription factors, inflammatory mediators, mechanical forces, and oncogenes The predominant mechanism involves ambient oxygen tension, with an increase in expression of VEGF in conditions of hypoxia. During hypoxia, HIF-1α dimerizes with the constitutively active HIF-1β, to form a transcription factor that binds to the hypoxia responsive elements (HRE) in the promoter region of the VEGF gene, thereby increasing VEGF transcription. Alterations in the expression or activity of the VHL tumor suppressor protein can also effect VEGF expression, since VHL is important for regulating the available concentrations of HIF-1α (see above). Other regulatory mechanisms capable of increasing VEGF expression include growth factors and receptors (e.g. EGF/EGFR, PDGF/ PDGFR, IGF, FGF, TGF-α, TGF-β), internal signal transduction pathways (e.g. Ras, PI3K/Akt), and inflammatory cytokines (e.g. interleukin-1α and -1β, interleukin-6, tumor necrosis factor-α). In addition, mechanical forces such as stretch and shear stress can also induce VEGF expression. In general, functional VEGF exists as a basic, heparin-binding homodimeric glycoprotein comprised of two identical 23 kda subunits, assembled in an antiparallel fashion. 46,47 Each VEGF monomer is similar in structure to PDGF, with a more helical N-terminal segment. Because of alternative splicing of the VEGF genes, various isoforms of VEGF protein are produced For the VEGF-A gene, there are six known isoforms of VEGF-A polypeptides, each with a different amino acid length beyond the signal sequence (VEGF-A 121, VEGF-A 145, VEGF-A 165, Clinical Medicine: Therapeutics 2009:1 1581

6 Newton VEGF-A 183, VEGF-A 189, VEGF-A 206 ). 48 VEGF-A 165 is the predominant species produced by numerous normal and neoplastic cells, including GBM. 50 VEGF-A 121 and VEGF-A 189 are also frequently present in the majority of cells and tissues that express the VEGF-A gene. VEGF-A 165 lacks exon 6 and has moderate heparin binding ability. It exists as both bound and freely diffusible protein, with the bound fraction undergoing proteolysis via plasmin and MMP s. The longer isoforms of VEGF-A, VEGF-A 189 and VEGF-A 206, bind strongly to heparin and heparin-like moieties in the ECM, and remain sequestered in the ECM and at the cell surface. In contrast, VEGF-A 121 lacks exons 6 and 7, and associated basic residues, thereby not allowing any binding to heparin or the ECM. The fraction of VEGF sequestered in the ECM is thought to constitute a reserve of available growth factor that can be released by plasmin or MMP s, in the form of truncated and freely soluble bioactive peptides. Once secreted or released from sequestration, activated VEGF is then free to bind to several different receptor types, including the VEGF receptor (VEGFR-1; Flt-1), VEGFR-2 (Flk-2; KDR), VEGFR-3, Neuropilin-1 (NRP-1), and NRP-2. VEGF-A 165 is the main isoform that interacts with NRP-1 and NRP-2. Experiments in animal models have suggested the importance of VEGF activity in gliomas For example, if VEGF is neutralized by antibodies, vascularization and growth in glioma xenografts is inhibited. Several investigators have demonstrated extensive expression of VEGF and its receptors in gliomas and speculate that VEGF may be the major endothelial mitogen for these tumors. 26,40,41,52 55 In GBM, VEGF 121, VEGF 165, and VEGF 189 can be detected, with VEGF 165 being the most common form. There are three known VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (flk-1/kdr), and VEGFR-3 (Flt-4) All three receptors have a similar structure, with seven immunoglobulin-like domains that form the extracellular portion of the molecule, a single transmembrane region, and an intracellular domain composed of a consensus tyrosine kinase sequence (see Fig. 4). A soluble form of VEGFR-1 (i.e. svegfr-1) has also been identified, that consists of only the extracellular domain of the receptor. The binding site of VEGF has been mapped to the second immunoglobulin-like domain of both VEGFR-1 and VEGFR-2; deletion of this domain will completely abolish VEGF binding. Crystal structure studies suggest that the binding is mediated via hydrophobic interactions with the poles of the VEGF dimer. VEGFR-1 binds VEGF-A, VEGF-B, and PlGF, and is expressed in endothelial cells, as well as pericytes, monocytes/ macrophages, osteoblasts, and some hematopoietic VEGFR-1 (Flt-1) VEGFR-2 (Flk-1/KDR) VEGFR-3 (Flt-4) Neuropilin-1 PLGF-1 PLGF-2 VEGF 165 VEGF 121 VEGF-B VEGF-C VEGF-D VEGF 165 PLGF-2 endothelial cell membrane Figure 4. VEGF and Neuropilin receptors. All three receptors have seven immunoglobulin-like domains that form the extracellular portion of the molecule, a single transmembrane region, and an intracellular domain composed of a consensus tyrosine kinase sequence All VEGF-A isoforms interact with VEGFR-1 and VEGFR-2. VEGF-B and PlGF can bind with VEGFR-1 but not VEGFR-2. VEGF-C and VEGF-D can bind VEGFR-2 and VEGFR-3, but not VEGFR-1. Neuropilin-1 is a receptor for the heparin-binding isoforms of VEGF and PlGF. Adapted from references 46,52 and used with permission from Springer-Verlag Clinical Medicine: Therapeutics 2009:1

7 Bevacizumab and brain tumors stem cells; it has the highest affinity for VEGF, with a K d of approximately pm. Homodimeric VEGFR-1 transmits weak mitogenic signals to endothelial cells, but can relay a stronger signal when it forms a heterodimer with VEGR-2. VEGFR-1 expression is upregulated during angiogenesis, hypoxia, and via transcriptional regulation by VEGF. svegfr- 1 binds VEGF with high affinity (K d of pm) and is inhibitory to VEGF-induced mitogenic signaling. VEGFR-2 binds VEGF-A, VEGF-C, and VEGF-D, with a lower affinity (K d of approximately pm) than VEGFR-1, and is the primary receptor for transmission of VEGF signaling in endothelial cells. The VEGFR-2 pathway is responsible for mediating VEGF effects such as vasodilatation, endothelial cell migration, and endothelial cell proliferation. VEGFR-3 binds to VEGF-C and VEGF-D, and is present on all endothelial cells during development. In adulthood, VEGFR-3 becomes restricted to endothelial cells of lymphatic vessels and selected fenestrated vasculature. As mentioned above, VEGF also interacts with the Neuropilin receptors (NRP-1, NRP-2), which have important roles in immunology and neuronal development, as well as angiogenesis. NRP-1 binds with VEGF-A 165, VEGF-B, and PlGF. NRP-2 binds to VEGF-A 165, VEGF-C, and PlGF. NRP-1 appears to act as a co-receptor to enhance VEGF-VEGFR-2 interactions, forming complexes with VEGFR-1. After binding with VEGF, the VEGFR s undergo dimerization and tyrosine kinase autophosphorylation, similar to other tyrosine kinase receptors (e.g. PDGF, EGF) Dimerization can result in the formation of homodimers (e.g. PDGFR-1/PDGFR-1) and heterodimers (e.g. PDGFR-1/PDGFR-2). The profile of subsequent signaling cascades are variable, depending on the pairing of receptor subtypes. Once activated, VEGFR s induce a cascade of internal signal transduction mediators, including the PI3K/ Akt and MAPK pathways, Ras/Raf/MEK/ERK, focal adhesion kinase (FAK), Paxillin, and protein kinase C (see Fig. 5). The different signal transduction cascades mediate different functions within endothelial cells. Cell survival is enhanced through the PI3K and Akt pathway, while DNA replication and cell proliferation are mediated via the Ras/Raf/MEK/ERK and MAPK pathways. Cell migration is also enhanced, Bevacizumab VEGF Grb2 VEGFR-2 Sos-1 Ras GDP Gap Ras GTP endothelial cell membrane Raf Akt PI3K PL-C PTEN DAG MEK Nucleus PKC ERK Nucleus Angiogenesis AP-1 Figure 5. Within the vascular endothelial cells, VEGF binding to VEGFR-2 results in activation of several signal transduction pathways, including Ras-GTP, Raf/MEK/ERK, protein kinase C, and PI3K/Akt. The activation of these various pathways results in a net increase in angiogenic activity. Molecular targeted therapy of the VEGF pathway is depicted, using bevacizumab (Avastin ). Adapted from References. 16,47 49,52 Clinical Medicine: Therapeutics 2009:1 1583

8 Newton mainly through activity of FAK and Paxillin, but also through signaling from the PI3K/Akt and MAPK pathways. Molecular Biology, Pharmacology, and Clinical Application of Bevacizumab Strategies to disrupt tumor angiogenesis have been under study for many years, and have tested numerous molecules and treatment approaches, including angiostatin, endostatin, thalidomide, combrestatin, thrombospondin, receptor tyrosine kinase inhibitors (e.g. SU5416, PTK 787, gefitinib, erlotinib, imatinib), and antiangiogenic monoclonal antibodies. 21,49,56 Once VEGF was demonstrated to be the predominant proangiogenic mitogen in the majority of solid tumors, such as colorectal carcinoma, breast cancer, nonsmall cell lung cancer, renal cell cancer, head and neck cancer, pancreatic cancer, and gynecological malignancies, the search began for the best strategy to inhibit the VEGF/VEGFR signaling pathway. 57,58 Kim and co-workers were the first to generate a series of four murine monoclonal antibodies (mabs; A3.13.1, A4.6.1, B4.3.1, B2.6.2) against recombinant V H C H V L S S C L Figure 6. Structure of Bevacizumab. The humanised Bevacizumab antibody consists of six murine specificity sequences (green dots) grafted onto a backbone of disulfide linked heavy and light chains containing variable (V H and V L ) and constant (C H and C L ) regions. Adapted from references 46,57,60,61 and used with permission from Bentham Science Publishers Ltd. S S S S S S human VEGF-A. 59 The mabs belong to the IgG 1 isotype and have high affinity for two different epitopes of VEGF. mab A4.6.1 was able to recognize VEGF-A 121, VEGF-A 165, and VEGF-A 189, and was noted to be a potent neutralizer of VEGF activity in in vitro and in vivo assays. However, since using a murine mab for human applications can be limited by immunological responses and toxicity, mab A4.6.1 was humanised by Presta et al. 60 The humanized mab contains the amino acid sequences from the six complementarity-determining regions (CDRs) of mab A4.6.1, grafted onto a consensus human framework of disulfide linked heavy and light chains containing variable and constant regions (see Fig. 6). Unfortunately, the initial humanised mab had an affinity for VEGF that was significantly weaker than murine mab A To restore binding equivalency to A4.6.1, the humanised mab was further engineered by changing seven framework residues in the variable heavy (VH) domain and one framework residue in the variable light (VL) domain from human to murine. The final version of the recombinant humanised mab (RhumAb) against VEGF is known as bevacizumab (Avastin ; Genentech, Inc.; South San Francisco, CA), and is composed of the consensus human IgG 1 framework and antigen-binding regions (93%) and the complement determining regions from the murine mab A4.6.1 (7%). 60,61 Bevacizumab neutralizes all isoforms of VEGF-A in a similar manner to A4.6.1, with dissociation constants (K d ) of 1.1 and 0.8 nmol/l, respectively. In addition, it inhibits VEGF-induced proliferation of endothelial cells in vitro with an ED 50 of 50 ± 5 ng/ml, also equivalent to A4.6.1 (48 ± 8 ng/ml). 61 In xenograft studies in nude mice, bevacizumab and A4.6.1 have identical potency and efficacy against a rhabdomyosarcoma cell line, with percentage inhibition of growth of 95% and 93%, respectively. Distribution studies in rabbits have demonstrated that the majority of bevacizumab remains in the plasma, with a significant proportion of drug distributed to the heart, testes, bladder, and kidney in comparison to other systemic organs. 61 Bevacizumab appears to be cleared from the circulation similar to endogenous antibodies, with a terminal elimination half-life of 1 2 weeks and a linear pharmacokinetic profile. Pre-clinical studies in mice, rats, and rabbits have shown that plasma concentrations of bevacizumab would need to be in the range 1584 Clinical Medicine: Therapeutics 2009:1

9 Bevacizumab and brain tumors of µg/ml, to achieve maximal tumor growth inhibition. Systemic doses of bevacizumab 0.3 mg/kg were able to produce complete suppression of free serum VEGF, while doses of 1 mg/kg resulted in serum levels in the target range of 10 µg/ml for at least 14 days. In further pre-clinical studies, mab A4.6.1 and/or bevacizumab have shown significant activity against a broad range of tumor types, in in vitro testing and in xenograft models. 57,61 Overall, these studies demonstrate that anti-vegf treatment results in a 25% to 95% inhibition of tumor growth in comparison to controls. Dose-dependent tumor inhibition was noted regardless of the route of administration or tumor location. The degree of inhibition was somewhat dependent on the timing of treatment, with more robust effects when drug was instituted early on, before tumors could attain a significant size and begin to secrete VEGF. Anti-VEGF therapy showed activity against many tumor types, including rhabdomyosarcoma, leiomyosarcoma, ovarian carcinoma, prostate carcinoma, colon adenocarcinoma, Wilms tumor, hepatoblastoma, neuroblastoma, breast carcinoma, and GBM. 61 In the GBM study, Rubenstein and colleagues assessed survival in xenografted rats after treatment with A4.6.1, and noted significant prolongation of survival, reduction in tumor vascularity, and increased tumor cell apoptosis. 62 Studies on tumor cell lines by Kim et al have shown that the use of bevacizumab or A4.6.1 had no effect on proliferation rates, further emphasizing the fact that anti- VEGF therapy is more directed towards endothelial cells and tumor vascularity. 63 In addition, xenograft models demonstrate that anti-vegf treatment can be effective in the suppression of metastases to the lungs, liver, and peritoneal cavity. 61 These results are not surprising, since the growth of systemic metastases are very dependent on neoangiogenesis and the development of their own vascular supply. Furthermore, the use of bevacizumab or A4.6.1 in combination with chemotherapy (e.g. doxorubicin, topotecan, paclitaxel, docetaxel) or radiotherapy appeared to have an additive antitumoral effect. 57,61 Combination therapy was noted to be more effective in xenografts from colon adenocarcinoma, GBM, breast cancer, Wilms tumor, prostate cancer, ovarian cancer, and pancreatic cancer. One explanation for the additive effect of anti-vegf therapy and chemotherapy, as espoused by Jain, is that anti-vegf treatment may result in the normalization of tumoral vasculature, with better delivery of drug to tumor cells. 64 The normalization effect may result through several mechanisms, including apoptosis of tumor endothelial cells; a decrease in tumor vessel diameter, density, and permeability; a subsequent decrease in tumor interstitial fluid pressure; and improved intratumoral oxygenation. This hypothesis is somewhat born out by other pre-clinical studies that examined the effect of anti-vegf therapy and vascular permeability. In a study by Yuan et al., the use of A4.6.1 against mouse xenografts from human GBM, colon adenocarcinoma, and melanoma caused a reduction in tumor vascular permeability and vessel diameter, eventually leading to vascular regression. 65 Brasch and co-workers used MRI techniques to study the vascular effects of A4.6.1 on breast carcinoma xenografts in athymic rats. 66 They noted a significant decrease in microvascular permeability, with a 98% reduction in fractional leak rate and fractional reflux rate, along with a 97% decrease in surface area product, in comparison to control animals. Similar findings have been documented using dynamic contrast-enhanced MRI in GBM mouse xenografts. 67 Numerous phase I, II, and III clinical trials have been performed over the past decade, confirming the efficacy and relative safety of bevacizumab for the treatment of solid tumors and other malignancies. 49,57,58,68,69 In a phase I trial, as well as in a summary of data compiled from a series of 8 clinical trials (N = 491 patients) and submitted to the Federal Drug Agency (FDA), there was a linear pharmacokinetic relationship at doses ranging from 0.3 to 10 mg/kg, with a C max ranging from 2.80 to 284 µg/ml. 68,70 The mean clearance of bevacizumab over a dose range of 0.3 to 10 mg/kg was 2.75 to 5.07 ml/kg/day after the first dose. Bevacizumab exhibited a 2-compartmental model with first-order elimination; the estimated elimination t 1/2 was approximately 20 days. Although the exact route of its metabolism and elimination remains uncertain, it is likely to be cleared via the reticuloendothelial system. When used in combination with chemotherapy (e.g. doxorubicin, carboplatin), there did not appear to be any significant alteration of the pharmacokinetic profile of bevacizumab. Phase II and III clinical trials are either completed or ongoing for several solid tumors, including Clinical Medicine: Therapeutics 2009:1 1585

10 Newton colorectal carcinoma, breast cancer, non-small lung carcinoma, renal cell carcinoma, pancreatic adenocarcinoma, prostate cancer, hepatocellular carcinoma, head and neck cancer, and soft tissue sarcomas, as well as selected hematological malignancies. 57,58,68,69,71,72 Overall, when used in combination with cytotoxic chemotherapy, bevacizumab has been shown to prolong survival in patients with colorectal, lung, and breast cancers, and may have activity as a single agent in the treatment of ovarian and renal cancer. It is the first angiogenesis inhibitor drug to be approved for use against cancer, and is now FDA approved for treatment of metastatic colon and lung cancer, in combination with chemotherapy. Although bevacizumab has been well tolerated by the majority of cancer patients, severe toxicity can occur, including hypertension, proteinuria, thromboembolic events, hemorrhage, gastrointestinal perforation, and impaired wound healing Hypertension and proteinuria have been reported most often, ranging from 22% to 38% of patients with renal cell carcinoma followed in clinical trials. 74,75,78 80 Most patients that develop hypertension while receiving bevacizumab will respond well to oral antihypertensive agents. If a given patient does not respond to oral antihypertensive therapy, bevacizumab should be discontinued. Bevacizumab-induced proteinuria is usually an asymptomatic event noted on laboratory analysis; however, full-blown nephrotic syndrome can occur. 75,79,80 Patients receiving bevacizumab should be closely monitored for proteinuria and renal injury via serum BUN and creatinine, and urinalysis (including urine creatinine, protein, and UPC ratio). Bevacizumab should be dose-reduced if there is evidence for severe proteinuria or renal insufficiency. The use of bevacizumab does not appear to increase the overall risk of thromboembolism or the incidence of venous thromboembolic events. 74,76,79 However, it does increase the incidence of arterial thromboembolic events (3.8% versus 1.7%). Bevacizumab should be discontinued immediately if there is evidence for an arterial thromboembolism. Hemorrhagic complications, including pulmonary hemorrhage, hemoptysis, epistaxis, and intracranial bleeding have been reported in patients receiving bevacizumab. 76,79 Bowel perforation is an infrequent (1.5%) complication of therapy with bevacizumab, and can be life-threatening. 74,77,79 Patients should be monitored for persistent abdominal pain associated with constipation and/or vomiting. Application of Bevacizumab to Recurrent High-Grade Gliomas The first report of the use of bevacizumab for brain tumor patients was by Stark-Vance in 2005, who treated 21 patients with malignant gliomas, in combination with irinotecan. 81 The response rate was an impressive 43%, although there were two treatmentrelated deaths (intracranial hemorrhage, intestinal perforation). Based on this promising preliminary work, Vredenburgh and his colleagues at Duke organized a prospective phase II trial of bevacizumab and irinotecan for patients with recurrent malignant gliomas (GBM, anaplastic astrocytoma [AA], anaplastic oligodendroglioma [AO]). 82 Patients with evidence of intracranial hemorrhage on screening MRI scans were excluded. Thirty-two patients were enrolled (GBM 23, AA/AO 9) and received intravenous bevacizumab (10 mg/kg) and irinotecan (340 mg/m 2 on enzyme-inducing antiepileptic drugs [EIAED], 125 mg/m 2 on non-enzyme-inducing antiepileptic drugs [NEIAED]) every two weeks. Radiographic responses were noted in 20 of 32 patients (63%; complete response [CR] 1, partial response [PR] 19), including 14 of 23 in the GBM cohort and 6 of 9 in the anaplastic glioma sub-group. The median overall progression-free survival (PFS) was 23 weeks; 20 weeks in GBM patients and 30 weeks in patients with AA or AO. The 6-month PFS and overall survival rates were 38% and 72%, respectively. The regimen was tolerated relatively well; no CNS hemorrhages were noted. However, three patients developed deep venous thromboses or pulmonary emboli, and one patient had an arterial ischemic stroke. A recent update of this series by Goli and co-workers, reports the data from 68 patients (GBM 35, AA/ AO 33). 83 The MRI response rate was similar (59%), with 2 CR and 38 PR. For the GBM cohort, the 6-month PFS rate was 43%, with a median PFS of 23 weeks. For the anaplastic glioma sub-group, the 6-month PFS rate was 61%, with a median PFS of 42 weeks. Eight patients were taken off study for thrombotic complications, including pulmonary emboli (4), deep venous thrombosis (2), thrombotic thrombocytopenic purpura (1), and thrombotic stroke (1). In addition, two patients stopped treatment secondary to grade II 1586 Clinical Medicine: Therapeutics 2009:1

11 Bevacizumab and brain tumors proteinuria. The preliminary use of bevacizumab and irinotecan at other institutions has resulted in similar MRI response rates, 6-month PFS rates, median PFS, and toxicity Kang et al. reported a series from the Cleveland Clinic of 27 patients with recurrent highgrade gliomas that received standard bevacizumab and irinotecan. 84 The 6-month and median PFS was 46% and 5.1 months, respectively. The overall survival at 6 months was 84%, with a median survival of 12.6 months. Treatment was tolerated well except for one case of intracranial hemorrhage. Gilbert and colleagues from the RTOG used bevacizumab (10 mg/kg) and irinotecan (200 mg/m 2 ) every two weeks in a series of 57 patients with recurrent GBM that were all on NEIAED; all patients had failed chemoradiation and adjuvant therapy with temozolomide. 85 The median age and KPS of the cohort were 57 years and 80, respectively. The 6-month PFS rate was 37%, with 21 of the 57 patients still progression-free. Moderate toxicity was noted in 21 patients (37% grade III, 12% grade IV), including one fatal intracranial hemorrhage. Other complications included DVT (6), grade 3 or 4 hematological toxicity (14), fatigue (9), and hypertension (2). A report from Denmark describes a series of 52 consecutively treated patients with recurrent malignant brain tumors (27 GBM, 13 AA, 5 AO) who received bevacizumab and irinotecan using the standard Duke regimen. 88 Objective responses (CR + PR) were noted on MRI in 25% of all cases (30% in grade IV glioma). The median PFS for grade IV and III tumors was 22 weeks, with an overall 6-month PFS of 32%. Median overall survival was 30 weeks; 28 weeks in grade IV gliomas and 32 weeks in grade III gliomas. Four patients discontinued treatment due to severe toxicity (cerebral hemorrhage, cardiac arrhythmia, intestinal perforation and diarrhea). An Israeli group reports the use of standard bevacizumab and irinotecan in a series of 20 patients with recurrent high-grade gliomas (20 GBM, 2 AO, 1 AOA). 87 In 19 assessable patients, there were 9 objective responses (47.3%; 2 CR, 7 PR). Median overall TTP was 4.7 months, with a 6-month PFS and median survival of 25% and 55%, respectively. Treatment was well tolerated, without any thrombotic complications or grade III/IV toxicity. A similar series by Ali et al. treated 13 patients with heavily pre-treated recurrent GBM using bevacizumab (5 10 mg/kg every 2 weeks) and irinotecan (125 mg/m 2 every 3 weeks). 86 Ten patients (77%) had PR on follow-up MRI scans. The median TTP was 24 weeks, with a median overall survival of 27 weeks. Two patients had to discontinue bevacizumab due to non-fatal intracranial hemorrhages. Another follow-up report from Vredenburgh and co-workers from Duke describes a phase II trial in a series of patients with two cohorts of recurrent GBM. 89 One cohort of 23 patients received bevacizumab and irinotecan using the standard dosing approach every two weeks. The other cohort of 12 patients received bevacizumab at 15 mg/kg every 21 days and irinotecan on days 1, 8, 22, and 29, with dosing stratified by EIAED versus NEIAED; each cycle was 6 weeks long. Twenty of the 35 patients (57%) had at least a PR on MRI follow-up. The 6-month PFS for the entire group was 46%, with a 6-month overall survival of 77%. Moderate toxicity was noted, including one CNS hemorrhage and four patients with thromboembolic complications (DVT and/or pulmonary embolus). In a molecular study performed in parallel with the above clinical trial, VEGF, VEGFR-2, CD31, hypoxia-inducible carbonic anhydrase 9 (CA9), and HIF-2α were assessed by semiquantitative immunohistochemistry in a cohort of 45 patients (27 GBM, 18 AA) and correlated with radiographic response and survival. 90 High VEGF expression was associated with a higher likelihood of radiographic response (p = 0.024), but did not effect survival. The only factor with a significant effect on survival was high CA9 expression, which was correlated with a poor survival outcome (p = 0.016). The Duke group has also performed a phase II study of bevazicumab and irinotecan that was restricted to only patients with recurrent grade III gliomas (25 AA, 8 AO). 91 There were two cohorts of patients: the first group of 9 patients received the standard combination regimen every two weeks, while the second group received bevacizumab (15 mg/kg every 3 weeks) and irinotecan (days 1, 8, 22, 29 of each cycle; 340 mg/m 2 on EIAED, 125 mg/m 2 on NEIAED) on a 6 week cycle. Objective responses were noted in 29 patients (61% with at least a PR). The 6-month PFS and overall survival rates were 55% and 79%, respectively. There was no difference in PFS or survival rates between the two treatment cohorts. Treatment was well tolerated, with only one patient with CNS hemorrhage and another with thrombotic thrombocytopenic purpura. Clinical Medicine: Therapeutics 2009:1 1587

12 Newton The results of the BRAIN trial have just recently been reported by Friedman and colleagues. 92 This study was a phase II, multicenter, open-label, non-comparative trial evaluating the efficacy of bevacizumab, alone or in combination with irinotecan, in a group of 167 patients with GBM in first or second relapse. Patients were randomly assigned to receive bevacizumab (10 mg/kg) as a single agent, or in combination with irinotecan (340 mg/m 2 or 125 mg/m 2, depending on EIAED or NEIAED), on an every 2-week schedule. The primary endpoints for the study were 6-month PFS and objective response rate on follow-up MRI, with secondary endpoints of safety and overall survival. The 6-month PFS rates in the bevacizumab alone and bevacizumab plus irinotecan groups were 42.6% and 50.3%, respectively. Objective response rates on followup MR imaging were 28.2% for the bevacizumab alone cohort and 37.8% for the bevacizumab plus irinotecan group. Median PFS times for the bevacizumab alone and bevacizumab plus irinotecan cohorts were 4.2 months and 5.6 months, respectively. The overall survival times were also similar between the single agent bevacizumab and bevacizumab plus irinotecan groups (9.2 months versus 8.7 months). There was a trend for patients on corticosteroids at the beginning of the trial to remain on stable or decreasing doses over time. Grade 3 or 4 toxicity was more frequent in the bevacizumab plus irinotecan group (65.8%) in comparison to those receiving bevacizumab alone (46.4%). The most common adverse events were hypertension, fatigue, neutropenia, and seizures. Intracranial hemorrhage was uncommon in both groups (2.4% bevacizumab alone, 3.8% bevacizumab plus irintotecan; 5 total patients, only 1 was grade 4). The Authors concluded that bevacizumab, alone or in combination with irinotecan, was active against recurrent GBM, with 6-month PFS rates that were far superior to the expected 15% rate for salvage chemotherapy and irinotecan alone. In a report by Raizer and colleagues of a phase II study of 61 patients with recurrent malignant gliomas (50 GBM, 5 AA, 6 AO/AOA), single agent bevacizumab (15 mg/kg every 3 weeks) did not appear to be quite as active as the combination regimen. 93 There were objective responses by MRI in 25% of the cohort (0% CR, 25% PR), with a 50% rate of disease stabilization. The 6-month PFS rate was 32%, with a median PFS of 3.9 months. The median overall survival for the entire cohort was 6.6 months. Grade 3 4 toxicities included non-fatal intracranial hemorrhage (1), fatal GI perforation (1), DVT (1), and rectal bleeding (1). In a similar study by Kreisl et al, single-agent bevacizumab (10 mg/kg every 2 weeks) was used for 48 patients with recurrent GBM, followed by standard bevacizumab plus irinotecan at the time of tumor progression. 94 Objective responses were noted on MRI follow-up in 17 patients (35%; 1 CR, 16 PR). The 6-month PFS rate was 29%, with a median PFS of 16 weeks. The 6-month and median overall survival were 57% and 31 weeks, respectively. Early responses by MRI (96 hours and 4 weeks) were predictive of long-term PFS. In the cohort of 19 patients treated with bevacizumab and irinotecan at the time of disease progression, there were no objective radiographic responses and the median PFS was only 30 days. Bevacizumab has also been used in combination with other drugs for treatment of patients with recurrent malignant gliomas. Norden and colleagues from the Dana-Farber Cancer Institute describe their experience with bevacizumab in combination with irinotecan, carboplatin, and carmustine. 95 In a cohort of 55 consecutive patients with recurrent malignant gliomas (33 GBM, 21 AA/AO, 1 high-grade glioma), the regimen consisted of bevacizumab (10 mg/kg every 2 weeks, except for one patient 5 mg/kg) in combination with irinotecan (47 patients; 125 or 340 mg/m 2, depending on AED status; every 2 weeks), carboplatin (6 patients; AUC of 5 6), carmustine (1 patient; 200 mg/m 2 every 6 weeks), or temozolomide (1 patient; mg/m 2 for 5 days every 4 weeks). Objective responses were noted in 63.6% of the cohort (CR 2.3%, PR 31.8%, 29.5% MR). Median TTP was 19.3 weeks, with a 6-month PFS of 42% for GBM and 32% for anaplastic gliomas. In 23 patients who progressed on their initial therapy with bevacizumab and chemotherapy, the concurrent drug was changed and then therapy was continued. No objective MRI responses were noted in this group, although two patients had extended PFS (20 and 31 weeks). In patients with tumor progression, a high percentage of patients were noted to have extensive infiltrative disease, even with continued inhibition of the tumor vasculature. A similar study was reported by the UCLA group, in a cohort of 44 patients with recurrent GBM. 96 Each patient received bevacizumab (5 mg/kg every 2 weeks) in combination with concurrent irinotecan (31 patients), 1588 Clinical Medicine: Therapeutics 2009:1

13 Bevacizumab and brain tumors carboplatin (8 patients), lomustine (3 patients), or etoposide (2 patients). In comparison to a group of control GBM patients that had not received bevacizumab-based chemotherapy, the bevacizumab group had statistically significant improvements in median PFS (4.25 versus 1.82 months; p = 0.01) and median overall survival (9.01 versus 6.11 months; p = 0.04). In addition, the 6-month PFS rate was also superior in the bevacizumab group (41% versus 18%). Further analysis demonstrated that patients of older age ( 55 years) and poor KPS ( 80) had significantly better PFS when treated with bevacizumab. In addition, the older patients also had significantly longer overall survival after bevacizumab treatment. Microarray expression analysis of VEGF was significantly higher in GBM patients 55 years of age. The bevacizumab cohort also required less dexamethasone and maintained their functional status longer than the control patients. In a study by Narayana et al, 61 consecutive patients with recurrent high-grade glioma (37 GBM, 24 AA/AO) received bevacizumab (10 mg/kg every 2 weeks) in combination with either irinotecan (54 patients; 125 mg/m 2 every 2 weeks) or carboplatin (7 patients; AUC 6 every 4 weeks). 97 The median PFS for the entire cohort was 5 months, with a median overall survival of 9 months. Of 53 evaluable patients, there were 39 objective responses (73.6%; 7 CR, 32 PR). The extent of resection prior to initiation of bevacizumab or choice of chemotherapy drug in combination with bevacizumab did not affect PFS or length of survival. At the time of progressive disease, 70% (35 of 50) occurred locally around the original site. In the remaining patients, the disease was more diffuse, similar to gliomatosis, with a minor component of local recurrence in all but one case. Biopsies were performed in 6 of these cases, and the pathology was suggestive of a more invasive mesenchymal phenotype (i.e. increased expression of D2-40, CD34, fascin). Significant toxicity consisted of DVT/PE (6), severe fatigue (10), intracerebral hemorrhage (6; 3 with symptoms), and colonic perforation (1). Recent phase II studies of bevacizumab, in combination with etoposide and erlotinib for recurrent malignant gliomas, also suggest encouraging anti-tumor activity. 98,99 In one study by Reardon et al, 59 patients with recurrent malignant gliomas (27 GBM, 32 AA/AO) were treated with bevacizumab (10 mg/kg every 2 weeks) and oral etoposide (50 mg/m 2 on days 1 21 of each 28 day cycle). 98 The 6-month PFS rate was 44% in GBM patients and 40.6% in anaplastic gliomas. The overall survival for GBM and anaplastic gliomas was 46 and 47 weeks, respectively. Complications included neutropenia (41%), fatigue (22%), infection (20%), mild intracranial hemorrhage (1 patient), and grade 4 GI perforation (1 patient). A similar study from the same group evaluated 56 patients with recurrent malignant gliomas (24 GBM, 32 AA/AO), using a regimen of standard bevacizumab in combination with erlotinib (500 mg/day on EIAED, 200 mg/day on NEIAED). 99 The 6-month PFS rates were 25% for GBM and 50% for anaplastic gliomas. No survival difference was noted for patients on EIAED versus those on NEIAED. The most common toxicity was rash, noted in 92% of patients (54% grade 1 2, 38% grade 3). Mild grade 1 2 nausea, diarrhea, and fatigue were also frequent. Severe complications were uncommon, and included two patients with PE, and one each with intestinal perforation, ischemic stroke, gastric bleeding, and nasal septal perforation. A neuro-imaging study of patients treated with bevacizumab in combination with irinotecan, carboplatin, or etoposide noted a response rate of 50%. 100 The responses could manifest very quickly in some cases, with a reduction in tumor enhancement and edema within 18 days of treatment. In addition, the reduction of peri-tumoral edema could be striking in some patients, even when the tumor remained stable in size. A more recent PET study from the same group used [ 18 F]fluorothymidine (FLT) as an imaging biomarker of metabolic response to bevaciumab and irinotecan in a cohort of 19 assessable patients. 101 Patients were studied at baseline, after 1 2 weeks, and after 6 weeks from the onset of treatment. A reduction in tumor FLT uptake of more than 25% was defined as a metabolic response; FLT responses were correlated with MRI results and patient survival. Metabolic responders were noted to survive three times longer than non-responders (10.8 versus 3.4 months; p = 0.003), and had a strong trend towards a prolonged PFS (p = 0.061). In addition, early and late FLT-PET responses were more significant predictors of overall survival (1 2 weeks, p = 0.006; 6 weeks, p = 0.002) then the comparable MRI responses. A more recent MRI study from the UCLA group evaluated a series of 82 patients with recurrent GBM, using apparent diffusion coefficient (ADC) histogram analysis, in an Clinical Medicine: Therapeutics 2009:1 1589

14 Newton attempt to stratify patients prior to treatment with bevacizumab. 102 The patients were separated into two cohorts of 41 patients: bevacizumab-treated and matched controls. Pre-treatment ADC histograms were generated using a two-mixture normal distribution for optimal fitting (including overall mean ADC and ADC from the lower curve, ADC L ), and then correlated with PFS at 6 months and overall survival. It was hypothesized that tumors with more necrosis and edema would have higher ADC values, and be more responsive to bevacizumab treatment. In the bevacizumab treated cohort, mean ADC, ADC L, and the lower curve proportion (LCP) were all significantly predictive of 6-month PFS (p = 0.016, p = 0.001, p = 0.001, respectively). Using a univariate Cox model, ADC L was the most accurate predictor of 6-month PFS. Pre-treatment ADC values were noted to be more accurate for predicting 6-month PFS than response at first follow-up MRI (73% versus 58% accuracy). In the multivariate Cox model, ADC L, LCP, and change in enhancing tumor volume were all significant predictors of 6-month PFS (p = 0.004, p = 0.014, p = 0.004, respectively). ADC L values were significantly correlated with overall PFS in the bevacizumab cohort, but not in the control patients. In the combined group of patients with an ADC L value of 1201 or greater, not receiving bevacizumab treatment resulted in a hazard ratio for progression by 6 months of In this same group of patients, treatment with bevacizumab resulted in an increase in median survival of 6.6-fold. Based on the studies and data outlined above, it is now apparent that bevacizumab, in combination with irinotecan and other forms of chemotherapy, is an active treatment for patients with recurrent malignant gliomas, and appears to improve PFS and overall survival in comparison to other forms of treatment. Several authors have now suggested that bevacizumab, in combination with irinotecan, should be considered for all patients with recurrent GBM The Federal Drug Administration (FDA) is in agreement with this assessment, and approved the use of bevacizumab for the treatment of recurrent GBM in May of The author also has extensive experience using bevacizumab and irinotecan for treatment of patients with recurrent GBM, including significant objective responses by MRI (see Figs. 7 and 8). Similar to the reports of other investigators, however, improvements in MRI enhancement and edema may be followed rapidly by dramatic progression of infiltrative disease (see Fig. 9). Application of Bevacizumab to Newly Diagnosed High-Grade Gliomas Clinicians have begun to evaluate the safety and feasibility of using bevacizumab in combination with temozolomide during chemoradiation in newly diagnosed patients with high-grade gliomas. 106,107 An early pilot study of bevacizumab (10 mg/kg every 2 weeks) in combination with temozolomide (75 mg/m 2 during irradiation, followed by mg/m 2 5 days every 4 weeks) was performed by Lai and colleagues in 10 patients with newly diagnosed GBM. 106 The toxicity was considered to be acceptable, and consisted of fatigue, myelotoxicity, wound breakdown, DVT/PE, and one case of radiation-induced optic neuropathy. Preliminary efficacy analysis suggested an encouraging mean PFS (range weeks). A recent update of this study with the completed cohort of 70 patients demonstrates acceptable overall toxicity and a promising TTP in comparison to control patients. 108 A similar feasibility study from Narayana et al. evaluated 15 patients with high-grade glioma, with the adjuvant therapy phase planned over one year (temozolomide 150 mg/m 2 /day). 107 Thirteen patients (86.6%) completed the entire year of adjuvant treatment; radiographic responses were noted in 13 of 14 assessable patients (92.8%). The 1-year PFS and overall survival rates were 59.3% and 86.7%, respectively. Grade III/ IV non-hematologic toxicity consisted of thromboembolism (2), non-healing skin ulcers and cellulitis of the lower extremities (1), and malignant hypertension (1). Grade III/IV hematologic toxicity consisted of thrombocytopenia (3) and neutropenia (1). Nicholas and co-workers reported a cohort of 42 patients with newly diagnosed GBM, in which patients received standard chemoradiation with temozolomide, followed by adjuvant treatment with the combination of temozolomide ( mg/m 2 5 days every 28 days) and bevacizumab (10 mg/kg every 2 weeks). 109 Objective radiographic responses have been noted in 14 patients (5 CR, 9 PR). Toxicity has been acceptable, with only one unexpected event (GI bleed). Using a more aggressive approach, Vredenburgh et al. treated 75 patients with newly diagnosed GBM with standard chemoradiation plus bevaciumab (10 mg/kg every 2 weeks), followed 1590 Clinical Medicine: Therapeutics 2009:1

15 Bevacizumab and brain tumors Figure year-old male with multifocal GBM. A) After chemoradiation and 4 cycles of adjuvant TMZ, the original focus of tumor in the right posterior temporal region is stable, with progression on the left side near the ventricular trigone. B) Following 9 months of treatment with bevacizumab and irinotecan (every 2 week schedule), there is almost complete resolution of the enhancing lesion on the left side. Figure year-old male with GBM. A) After chemoradiation and 4 cycles of adjuvant temodar, there was mild progression of disease within the right thalamus. B) Following 2 additional months of adjuvant temodar in combination with bevacizumab (every 2 week schedule), there is almost complete resolution of enhancement within the thalamus. Clinical Medicine: Therapeutics 2009:1 1591

16 Newton Figure year-old male with progressive GBM. (A and B) After chemoradiation and 14 cycles of adjuvant temodar, there was progression of disease within the periventricular white matter, corpus callosum, and septum pellucidum, with edema and new enhancement. (C and D) Following 2 cycles of standard bevacizumab and irinotecan, there was dramatic progression of T2 and FLAIR signal into the left frontal lobe, while the corresponding region of enhancement had been significantly reduced in size Clinical Medicine: Therapeutics 2009:1