J Neurol (2000) 247 : 597–608 © Steinkopff Verlag 2000
Jaap C. Reijneveld Emile E. Voest Martin J. B. Taphoorn
Received: 25 May 1999 Accepted: 7 January 2000
J. C. Reijneveld () · M. J. B. Taphoorn Department of Neurology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands e-mail:
[email protected] Tel.: +31-30-2507939 Fax: +31-30-2542100 J. C. Reijneveld · E. E. Voest Laboratory of Medical Oncology, Department of Internal Medicine, University Medical Center, Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands
OCCASIONAL REVIEW
Angiogenesis in malignant primary and metastatic brain tumors
Abstract Patients with malignant primary and metastatic brain tumors have a poor prognosis, despite developments in diagnostic and therapeutic modalities. Therefore in the past decade a search for new therapeutic possibilities has started. The inhibition of angiogenesis, the sprouting of new capillaries from preexisting vasculature, which is an absolute requirement for the growth of tumors beyond a size of a few cubic millimeters, is one of the most promising approaches with which to influence tumor growth. This review focuses on the critical role of angiogenesis in the development of normal brain and the blood-brain bar-
Introduction Approximately 70% of brain tumor cases are malignant primary tumors, predominantly high-grade gliomas, and metastatic brain tumors, both intracerebral and leptomeningeal [107]. Despite developments of both diagnostic modalities and new therapeutic strategies, the majority of patients with these tumors cannot be cured and has a poor outlook. Median survival is 12–18 months in patients with high-grade glioma and less than 6 months in patients with cerebral metastases. These figures have not substantially changed during the past two decades [20, 23, 122, 128]. It is now known that tumor growth requires the formation of a new blood vessel network, a process called angiogenesis [36]. An adequate supply of oxygen and nutrients through newly formed microvessels is necessary for tumor growth beyond a few cubic millimeters. Several an-
rier. We discuss the importance of angiogenesis in the formation of malignant brain tumors and in bloodbrain barrier function in these tumors and possible consequences of altered blood-brain barrier properties for antiangiogenic therapy. Furthermore, results of current clinical trials with antiangiogenic drugs are reviewed, and clinical perspectives of antiangiogenic therapy in malignant brain tumors are outlined. Key words High-grade glioma · Cerebral metastasis · Leptomeningeal metastasis · Angiogenesis · Blood-brain barrier
tiangiogenic agents have been shown to retard the growth of various tumors in experimental models [9, 97]. Highgrade gliomas and cerebral metastases are among those characterized by endothelial cell proliferation and a high degree of vascularity. In fact these tumors are among the most angiogenic of all human tumors, and the amount of neovasculature in high-grade glioma is closely correlated with the degree of malignancy and the prognosis of patients [73]. The development of antiangiogenic strategies may open new ways for more effective treatment in patients with malignant brain tumors. This review describes the process of angiogenesis and its role in the development of the normal brain and in brain tumor formation. We focus on the effect of neovascularization and antiangiogenic therapy on the blood-brain barrier function in malignant brain neoplasms. Finally, clinical perspectives in antiangiogenic therapy in these tumors are discussed.
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The process of angiogenesis The angiogenic switch Only few tumors are angiogenic at the beginning of their development [34, 51]. Microscopic tumors may remain dormant for months or years, as tumor cell proliferation is balanced by an equivalent rate of apoptosis [54]. At some point, however, tumor cells switch to the so-called angiogenic phenotype. The angiogenic switch is activated by a local imbalance between positive and negative angiogenic factors, probably through environmental and genetic alterations [10, 11, 51, 79, 112, 119]. The subsequent neovascularization results in a better supply of nutrients, leading to exponential tumor growth, with or without metastasis [54, 100]. Interaction between endothelial cells and the extracellular matrix and basement membrane Angiogenesis requires a number of sequential steps: (a) induction of vascular discontinuity, (b) endothelial cell proliferation, (c) endothelial cell migration, and (d) structural reorganization of the new vasculature (Fig. 1). The process depends on a complex interaction between endothelial cells and the extracellular matrix [10, 11, 51, 72, 78, 112]. Angiogenesis requires migration of endothelial cells. Cell migration requires attachment and deattachment of the cell as it moves forward. The formation and subsequent breakdown of an angiogenic matrix plays a crucial role in the ability of endothelial cells to migrate [102]. Angiogenic factors induce hyperpermeability, which results in the extravasation and deposition of plasma proteins and hence the formation of a temporary matrix Fig. 1 The process of angiogenesis developed by sequential morphological steps: 1 induction of vascular discontinuity; 2 endothelial cell proliferation; 3 endothelial cell migration; 4 structural reorganization of the new vasculature
[26]. Migration of endothelial cells through this matrix depends on the ability of these cells to move along their basement membrane. Several extracellular matrix proteins, such as fibronectin and vitronectin, bind to the integrin receptors αvβ3 and αvβ5, which are upregulated on the surface of activated endothelial cells [28, 45, 145]. This suggests an essential role for these integrins in angiogenesis. Two types of serine proteases, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), play an important role in the breakdown of the angiogenic matrix and basement membrane [2, 83, 86]. These proteases are produced by tumor cells and stimulated endothelial cells and cleave plasminogen into active plasmin. Plasmin in turn cleaves extracellular matrix proteins, such as fibrin and vitronectin, and activates procollagenases [16, 45]. Furthermore, matrix metalloproteinases (MMPs), produced by tumor and probably also stromal cells, contribute to the degradation of extracellular matrix components, such as gelatin and collagen [63, 90, 152]. Inhibitors of both types of proteinases, plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2) and tissue inhibitors of metalloproteinases (TIMPs), are also upregulated. This activation of antiproteolytic action is probably meant to prevent excessive destruction of the extracellular matrix that would result in dysfunctional vasculature [78, 96]. Evidence is accumulating that the process of matrix proteolysis by the activation of plasmin and the process of endothelial cell migration through the integrin receptors do not operate independently but are closely correlated with each other [45, 99].
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Angiogenic factors The first angiogenesis inducers to be characterized were basic (bFGF) and acidic fibroblast growth factor (aFGF). Both FGFs are mitogenic for a spectrum of cells, including endothelial and tumor cells [12]. At about the same time a protein that elicits vascular permeability was detected. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a specific mitogen for endothelial cells [126]. Hypoxia, which is the result of a lack of perfusion and vascular compression in tumor tissue, is one of the strongest inducers of VEGF expression [58, 133]. Both FGF and VEGF bind to tyrosine kinase receptors. The three VEGF receptors [VEGFR-1 (flt-1), VEGFR-2 (flt-1), and VEGFR-3 (flt-4)]) are selectively expressed on endothelial cells, although expression has also been reported in nonendothelial cells [4, 18]. The four FGF receptors (FGFR-1–FGRF-4) are more widely expressed [23]. Evidence is growing that VEGF and bFGF act synergistically in stimulating angiogenesis [3, 49, 101]. Another class of tyrosine kinase receptors expressed on endothelial cells, the Tie receptors, is also essential for angiogenesis [76]. Angiopoietin-1, the natural ligand for the Tie-2 receptor, plays a role in the end stage of tumor angiogenesis, remodeling the new vasculature [77, 137]. However, the expression of angiopoietin-2, the natural antagonist of angiopoietin-1 for the Tie-2 receptor, is also upregulated in tumor vasculature. It has been hypothesized that angiopoietin-1 is important for stabilizing the vessel wall, while angiopoietin-2 has a role in keeping endothelial cells accessible to angiogenic inducers, such as VEGF, by loosening contacts between endothelial and periendothelial cells [52, 134]. Exogenous and endogenous antiangiogenic factors In 1982 the first endogenous angiogenesis inhibitor, platelet factor 4 (PF-4), a protein that is normally found in the αgranules of platelets, was identified [81, 142]. Seven years later thrombospondin-1 was found to be an inhibitor of endothelial cell proliferation in vitro and of tumor growth and angiogenesis in vivo [47, 114]. Thrombospondin-1 is expressed at high levels in several normal cell types and at lower levels in many tumor cell lines. Expression of this glycoprotein is under regulatory control of the tumor-suppressor gene p53 in fibroblasts [22]. A very potent angiostatic factor was identified in 1994 [97]. Angiostatin, a 38-kDa proteolytic internal fragment of plasminogen, is probably generated by cancer-mediated proteolysis of this proenzyme by uPA and other proteolytic enzymes, suggesting a link between degradation of extracellular matrix and angiogenesis inhibition [44]. Systemic administration of angiostatin blocks angiogenesis and growth in a variety of tumor types [97]. More re-
cently another endogenous angiogenesis inhibitor was discovered. The protein endostatin, a fragment of collagen XVIII, is present in the basement membrane of blood vessels. Systemic administration leads to rapid regression of several experimentally induced tumors [9, 98]. TNP-470, also called AGM-1470, is a potent exogenous inhibitor of angiogenesis. TNP-470 is a synthetic analogue of the fungal-produced agent fumagillin and has shown to be antiangiogenic in vitro and in vivo, possibly through interaction with bFGF-stimulated DNA synthesis [55, 59]. Both endogenous and synthetic inhibitors of MMPs have been considered as potential therapies for the treatment of cancer. Recombinant TIMP-1 and TIMP-2 and the synthetic MMP inhibitors batimastat and marimastat have been shown to be antiangiogenic and to retard tumor growth in several experimental tumor models [1, 29, 63, 130]. It appears that TIMPs mainly act by inhibiting endothelial cell proliferation, while the synthetic MMP inhibitors block endothelial cell invasion [91]. Synthetic uPA receptor (uPAR) antagonists and uPA inhibitors have been demonstrated to retard the growth and metastasis of several types of experimental tumors [68, 74, 90, 111, 125]. Furthermore, well known agents such as thalidomide and suramin have shown antiangiogenic properties. The underlying mechanism of antiangiogenic activity in these drugs, however, remains to be elucidated [21, 32]. Angiogenesis in the normal brain Although angiogenesis is frequently noted as an absolute requirement for pathophysiological processes such as the growth of solid tumors, it also plays an indispensable role in embryogenesis. The study of neovascularization in the brain during the embryonic stages and in the normal adult brain has led to a better understanding of its role in brain tumor formation and in maintenance of blood-brain barrier properties in adult life. Angiogenesis during embryonic brain development The development of the brain vascular system starts with the formation of the perineural vascular plexus out of the putative common progenitors of hematopoietic and endothelial cells, the hemangioblasts [88]. This plexus covers the entire surface of the neural tube. Vascular sprouts from this plexus invade the proliferating neuroectoderm and form the vasculature within the central nervous system [12]. Cumulative evidence has been gathered that these processes of subsequent vasculogenesis (the appearance of new vessels) and angiogenesis (the sprouting of vessels from preexisting vasculature) are regulated by factors that are
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secreted by the developing brain [12, 40, 116, 118]. VEGF is released by cells of the ventricular layer of the developing neuroectoderm, inducing migratory and mitogenic responses by endothelial cells from the perineural vascular plexus, and this results in the radial ingrowth of capillaries from the convexity into the brain parenchyma [13]. Haploid VEGF-deficient mice die early in embryonic development and do not show a vascular system within the brain [15]. VEGFR-2 (Flk-1) expression is upregulated during embryogenesis in mice compared to the adult brain [13]. Disruption of the VEGFR-1 (Flt-1) gene results in abnormal and disorganized blood vessels. Interference with the VEGFR-2 (Flk-1) gene inhibits endothelial cell differentiation, while inactivation of the VEGFR-3 (Flt-4) leads to defective remodeling of vascular networks. All three mutations lead to embryonic death in mice [27, 41, 127]. The Tie receptors are also involved in embryonic vascular development [76]. Angiopoietin-1 probably plays a role in vascular sprouting and remodeling in the embryonic stage [136]. The mRNA expression of Tie-1 and Tie-2 receptors in the developing brain is upregulated in the same pattern as the VEGF-R2. Overexpression of angiopoietin2, the natural antagonist of angiopoietin-1, leads to malformation of the vascular network [82, 123]. Tie-1 knockout mice show edema and localized hemorrhage, while Tie-2 knock-out mice lack sprouting capillaries in the brain, although a perineural plexus is formed normally. Both genetic defects result in embryonic or early postnatal death [110, 123]. Angiogenesis in the normal adult brain As there is hardly any neovascularization in the adult brain, one would expect the complete absence of angiogenic factors. However, VEGF mRNA is observed in the normal adult mouse and rat brain, especially in epithelial cells that are in close relation to fenestrated endothelium in the choroid plexus. It has been hypothesized that VEGF plays a role in the establishment or maintenance of fenestrated endothelium in the choroid plexus, facilitating the exchange of low molecular weight substances [12, 105]. Furthermore, astroglial or microglial cells throughout the adult brain express VEGF mRNA, suggesting that VEGF is important in maintenance of normal vascular function [103]. Angiogenesis and the blood-brain barrier The blood-brain barrier provides precise control over substances that enter or leave the brain. Brain endothelium is characterized by tight junctions between the endothelial cells that restrict the passage of macromolecules. This unique phenotype is believed to result from interactions with astrocytic processes that form endfeet and surround
brain microvessels [24, 62, 117]. Tumor growth leads to disruption of the blood-brain barrier, resulting in cerebral edema. It has been suggested that the continuous maintenance of the barrier, provided by the tissue microenvironment, is interrupted [118]. Experimental studies have demonstrated that continuous VEGF administration into the cortex of rats results in leaking vessels and local neovascularization, with the majority of the new vasculature lacking the specific bloodbrain barrier phenotype marker GLUT-I [11, 26]. Dexamethasone, which is known for its ability to decrease cerebral vasogenic edema in brain tumor patients, has been shown to inhibit expression of VEGF in an experimental brain tumor model [42, 53]. From these data it is hypothesized that upregulation of VEGF is responsible not only for neovascularization in brain tumors but also for the increased vascular permeability and disruption of blood-brain barrier function [103].
Angiogenesis in malignant primary brain tumor formation Angiogenesis plays a crucial role in malignant primary brain tumor formation Angiogenesis has always been thought to play an important role in malignant primary brain tumors. The frequently encountered microvascular proliferations suggest that these tumors are excellent candidates for antiangiogenic therapy [35]. According to some authors, however, the diffuse infiltrative growth pattern of malignant gliomas may make angiogenesis totally redundant for outgrowth. They argue that glioma cells may encounter nutrient supplies at frequent intervals as they advance and therefore would escape the need of neovascularization [150]. In recent years, however, proof has accumulated that angiogenesis is essential in primary brain tumor formation (Fig. 2). Several studies have confirmed the important role of VEGF and bFGF in glioma growth. VEGF is expressed in human high-grade glioma but is not found in normal brain [104, 135]. bFGF is detected in the cerebrospinal fluid of approximately 60% of patients with primary brain tumors and is correlated with microvessel density in histological sections of these tumors [75]. Increased expression of bFGF, VEGF, and VEGF receptors is correlated with tumor vascularity and malignancy grade in human gliomas [14, 95, 104, 121]. In addition, there is a striking association between upregulation of VEGF and areas of necrosis in glioma [104]. The high proliferation rate of malignant glioma may lead to areas of necrosis and hypoxia, resulting in increased expression of VEGF [58]. Less clear is the role of the Tie receptor family in primary brain tumor angiogenesis. It has been shown that these receptors are upregulated in the endothelium of hu-
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expect the opposite, accompanied by overexpression of TIMP-1 [92, 93, 113]. Furthermore, it has been shown that the endogenous angiogenesis inhibitor thrombospondin-1 is produced by normal brain and low-grade gliomas but is completely absent in glioblastomas [57]. Antiangiogenic treatment inhibits the growth of experimental brain tumors Inhibition of angiogenic factors Treatment of mice bearing subcutaneous glioma with a monoclonal antibody against VEGF results in inhibition of tumor growth and a decrease in vessel density [66]. Subcutaneous or intracerebral implantation of C6 glioma cells which are ex vivo transfected with antisense VEGF DNA results in retarded tumor growth and decreased vascularization [17, 120]. Local treatment with bFGF antibodies of mice with intracranial human glioma xenografts results in decreased tumor growth and vascularization [132]. Inhibition of plasminogen activation through ε-amincaproic acid leads to a decreased rate of tumor growth and tumor volume at the time of death and to prolonged survival in rats bearing subcutaneous glioma [124]. Neither synthetic uPAR antagonists and uPA inhibitors nor recombinant TIMPs and synthetic MMP inhibitors have been tested in experimental glioma models so far. Administration of endogenous and exogenous angiogenesis inhibitors
Fig. 2 Immunohistochemical staining against factor VIII related antigen shows increased microvascular density in experimentally induced rat C6-glioma (above) compared to normal murine brain parenchyma (below). Arrowheads Individual microvessels. × 50
man brain tumors, while angiopoietin-1 and angiopoietin2 are preferentially expressed in glioma cells and endothelial cells, respectively [134]. Other investigators have focused on the role of serine proteases and MMPs. Increased expression of uPA and uPAR in human gliomas is correlated significantly with a higher tumor grade and worse prognosis [46, 56, 153, 154]. In experimental glioblastoma uPA/uPAR coexpression is predominantly found at the tumor edges, indicating a role in glioma invasion [46]. Overexpression of MMP-2 (gelatinase A), MMP-7 (matrilysin), and MMP-9 (gelatinase B) is also associated with the malignancy grade of gliomas and probably determines the invasive capacity of these tumors. This overexpression is, although one might
Systemic administration of angiostatin in mice bearing intracerebral glioma leads to inhibition of tumor growth and vascularization. An important conclusion that has been drawn from this finding is the fact that angiostatin acts as an endothelial cell inhibitor independently of the bloodbrain barrier [67]. Preliminary results of experiments with systemic administration of endostatin in mice with subcutaneous or intracranial gliomas indicate that this leads to reduced tumor growth and prolonged survival [131]. Systemic TNP-470 administration in murine models with intracranial gliomas shows tumor growth inhibition in many but not all models, possibly depending on the type of tumor xenograft and the time between tumor inoculation and start of treatment [8, 60, 138, 139, 151]. Antiangiogenic gene therapy Assuming that effective antiangiogenic treatment requires continuous and prolonged suppression of neovascularization, gene therapy approaches have been used [39]. In vivo gene transfer offers a means of long-term treat-
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ment without the disadvantages of daily drug administration. Ideally a single and preferably local administration of a vector leads to long-term inhibition of angiogenesis [115]. Brain tumors are particularly attractive for gene therapy as the presence of a blood-brain barrier and the lack of a lymphatic system offer an immunological barrier, leading to an immunologically privileged site [122]. This new approach has been used successfully in several experimental models of brain tumors. Treatment of mice bearing subcutaneous glioma with a retrovirus, encoding a dominant-negative mutant of the VEGF receptor Flk-1, resulted in inhibition of tumor growth and a decrease in vessel density [89]. Local gene therapy, using a recombinant adenoviral vector with the cDNA encoding for angiostatin, in preestablished subcutaneous and intracerebral glioma in nude mice led to prolonged survival of treated animals, with lower intratumoral vascular densities and higher apoptotic indices [50, 141]. Intratumoral administration of an adenoviral vector containing cDNA encoding for a soluble form of PF-4 in mice with intracerebral glioma resulted in prolonged survival combined with a lower vascular density [140]. PF-4 has been tested in a phase I clinical trial, but the short half-life of this protein caused insurmountable problems [6, 64].
Angiogenesis in metastatic brain tumors
Leptomeningeal metastases Cancer cells that reach the cerebrospinal fluid may float to other areas of the nervous system, where they may settle and grow. Subsequently these tumor cells infiltrate the leptomeninges of the brain and spinal cord, and clinically relevant leptomeningeal metastases (LM) develop [107]. The initial growth pattern of these metastases, as monoor multilayers fed by diffusion from the cerebrospinal fluid and the subarachnoid vasculature, does not seem to require neovascularization. Later in the metastatic process, however, larger tumor nodules develop, which, given their size, depend on the growth of new microvessels. Until now, however, there is only circumstantial evidence to support this hypothesis. It was suggested even more than two decades ago that neovascularization plays a role in LM. This was demonstrated by alteration of the blood-brain barrier properties in a rat tumor model [147]. Systemic chemotherapy in rats with LM was more effective when started a longer time after tumor establishment, suggesting the formation of new blood vessels that lack the typical blood-brain barrier properties [107, 148]. Indeed, study of experimentally induced LM has revealed microvessels with fenestrated endothelium, lacking the usual blood-brain barrier properties and suggesting newly formed vasculature [129]. Further support is the finding that activated MMP-2, MMP-9 and VEGF are upregulated in the cerebrospinal fluid of patients with LM [43].
Intraparenchymal brain metastases Relatively little is known about the role of angiogenesis in the formation of intracerebral metastases, as research efforts have focused mainly on primary brain tumors. Histological evaluation of brain metastases of various types of lung cancer, however, has demonstrated increased vascular proliferation [61]. Two studies of human metastatic brain tumors have found a high correlation between production or expression of VEGF and tumor vascularity and peritumoral edema [7, 135]. Cerebral melanoma metastases show an upregulation of the Flk-1, Flt-1, and Tie receptors [52, 65]. Significant increases in the levels of several MMPs have been observed in brain metastases originating from lung, colon, and breast carcinoma [93]. PAI-1 immunoreactivity is prominent in brain metastases from lung carcinoma, breast cancer and melanoma, and is confined to glomeruloid-shaped proliferative blood vessels and areas within or adjacent to necrosis [69]. Furthermore, the cerebrospinal fluid of patients with brain metastases contains precursor MMP-9, in contrast with samples of healthy controls [43]. No intervention studies with antiangiogenic agents have as yet been reported in experimental metastatic brain tumor models.
Effect of antiangiogenic therapy on blood-brain barrier properties In evaluating the possibilities of antiangiogenic therapy of malignant brain tumors it is important to consider the effects of antiangiogenic drugs on blood-brain barrier properties. The necessity for antiangiogenic agents to cross the blood-brain barrier It is unclear whether antiangiogenic drugs must cross the blood-brain barrier to be effective [37, 39]. Antiangiogenic drugs that inhibit endothelial cell function probably do not have to pass this barrier. Systemic administration of angiostatin leads to a decrease in brain tumor growth and vascularization [67]. It is unlikely that this hydrophilic 38-kDa peptide easily crosses the blood-brain barrier [33]. On the other hand, antiangiogenic agents that seize the proteolytic activity of proteases probably must pass the barrier to reach the extracellular matrix.
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Table 1 Current clinical trials of antiangiogenic therapy in brain tumor patients (from [155])
Drug type
Phase
Mechanism of action
Patients
Marimastat TNP-470 Thalidomide Suramin
III II II II
Synthetic MMP inhibitor Inhibits endothelial cell growth Unknown Unknown
SU101
II/III
Inhibits signaling of PDGF receptor
PTK787/ZK 22584
I/II
Blocks VEGF receptor signaling
Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Astrocytoma III/IV and oligodendroglioma Anaplastic astrocytoma/ oligoastrocytoma and glioblastoma multiforme Glioblastoma multiforme
The effect of antiangiogenic agents on tumor uptake of cytotoxic drugs Angiogenesis is commonly associated with increased vascular permeability [11, 103]. Disruption of blood-brain barrier properties facilitates tumor uptake of drugs. Antiangiogenic treatment may result in restoration of bloodbrain barrier properties which could hamper local delivery of concomitantly administered cytotoxic drugs [25]. Antiangiogenic therapy, however, also leads to a decrease in interstitial pressure within the tumor. This results in decreased intratumoral hypoxia, which makes tumor cells more vulnerable to the cytotoxic actions of many chemotherapeutic agents and ionizing radiation [37]. Coadministration of antiangiogenic agents and cytotoxic drugs potentiates the efficacy of cytotoxic agents in subcutaneous Lewis lung carcinoma and intracerebral glioma models [143, 144]. The combination of angiostatin and irradiation in subcutaneous tumors enhances tumor eradication [48, 84]. The synergistic effect of combined antiangiogenic and cytotoxic treatment apparently completely outweighs possible negative effects of antiangiogenic agents on bloodbrain barrier permeability and local drug delivery.
Clinical prospects The promising results obtained with antiangiogenic treatment in animal studies have raised high hopes for this approach in patients. Antiangiogenic agents have reached their ultimate test: over 20 angiogenesis inhibitors are currently being tested in clinical trials [5, 6, 19, 30, 37, 38, 64, 80, 85, 87, 106, 108, 109, 146]. At least six of these trials include patients with brain tumors [70, 71] (Table 1). Most of the compounds are in phase I trials, but several antiangiogenic agents have already reached phase III. No data are yet available, and the first phase III trial results of treatment of glioblastoma patients with marimastat are expected some time in 1999 [94]. The design of these trials, however, deserves careful evaluation. The conceptual basis of antiangiogenic therapy is that it is a cytostatic rather than a cytotoxic treat-
ment. This implies that prolonged, and preferably continuous, treatment is required to obtain optimal antitumor effects. The goal of a conventional phase I study is to find the maximal tolerated dose (MTD) and dose-limiting toxicity. Because the ideal antiangiogenic drug is specific to the tumor vasculature and must be taken for the rest of the patient’s life, toxicity should be limited. To determine the MTD may therefore be difficult and not essential for further treatment design. Information on biological endpoints which indicate that angiogenesis is actually inhibited is crucial and will determine optimal treatment schedules. In addition, phase II trials may fail to demonstrate efficacy of the antiangiogenic drug because it is unlikely that regression will be obtained in patients with bulky tumors, and in a time span of 6–8 weeks. Bulky tumors may produce large amounts of growth factors which are difficult to counterbalance by the inhibitor. The result could be that effective agents, unjustly, will not reach phase III evaluation [149]. The current design of early clinical trials does not provide an optimal framework to determine the true clinical value of these novel drugs. Surrogate endpoints, other than tumor regression, are highly needed. Furthermore, several preclinical studies have already demonstrated the added value of combining chemotherapy or radiotherapy with inhibitors of angiogenesis [48, 84, 143, 144]. Antiangiogenic agents should therefore be tested as adjuvant treatment in patients who undergo successful conventional treatment modalities, resulting in small residual tumor burden [31, 37]. In conclusion, antiangiogenic therapy may offer new opportunities in the treatment of malignant primary brain tumors. Further research must focus on the interaction of these drugs and the blood-brain barrier, the role of angiogenesis in brain metastases, and the development of longterm treatment strategies. Furthermore, the design of clinical trials may require adjustments to adequately evaluate antiangiogenic therapy. Acknowledgements J.C.R. is supported by grants from the Dutch Cancer Society (N.K.B.) and the Dutch Organization for Scientific Research (NWO, reg. no. 920-03-075).
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