SledgeJr 2002 Current Problems in Cancer

Angiogenesis and Antiangiogenic Therapy
hat makes a cancer a cancer? What allows a cancer to grow? What allows a cancer to spread outside its organ of origin? What allows it to colonize the hostile environment of a distant site? These are, as all will recognize, simple questions with inordinately complex answers. The malignant phenotype has been the subject of decades of study, and we have glimpsed its broad outlines only in recent years. Growth of solid tumors in both the primary and metastatic sites depends on angiogenesis, the formation of new blood vessels, to nourish the tumor. In pioneering work by Folkman,1 cancer cells implanted in vascular sites in animals grew rapidly and formed large tumors. In contrast, cells implanted in avascular sites were unable to form tumor masses 1 to 2 mm in size. This work led Folkman1 to hypothesize that angiogenesis was obligatory for tumor growth. It has become increasingly certain that angiogenesis, or new blood vessel formation, plays a central role in the malignant phenotype. As we will discuss, angiogenesis is activated early in carcinogenesis. The presence of new blood vessels allows for the growth of local tumors, offers them a royal road to other parts of the body, and feeds their growth in distant sites. Unsurprisingly, then, new blood vessel formation has been shown to be an important prognostic factor in many tumors in human beings. This realization has led to the novel and important concept of angiogenesis as a potential therapeutic target (indeed, targets, as we shall discuss below). This therapeutic potential is now approaching practical realization in the clinic, with the reasonable expectation of commercially available products in coming years. Therapeutic targeting is based to a signicant extent on our growing understanding of the biologic factors relating to angiogenesis. Although the excitement surrounding this approach is certainly justied by the ubiquity and centrality of angiogenesis to the metastatic phenotype, the development of these novel agents is certainly not lacking in potential concerns regarding drug resistance.
Curr Probl Cancer 2002;26:1-60. 0147-0272/2002/$35.00 0 56/1/122105

doi:10.1067/mcn.2002.122105
Curr Probl Cancer, January/February 2002
The Process of Angiogenesis

Blood vessel formation consists of two related but separate processes, vasculogenesis and angiogenesis. Vasculogenesis, which occurs during embryogenesis, involves the differentiation of endothelial cells from precursor angioblasts and the subsequent formation of a capillary plexus. Angiogenesis consists of the formation of new capillaries through sprouting or splitting from preexisting vessels (both embryonic and adult). Under physiological conditions, angiogenesis involves initial activation, including sequential basement membrane degradation, cell migration, extracellular matrix invasion, endothelial cell proliferation, and capillary lumen formation. The newly formed microvasculature matures through the process of resolution, which includes inhibition of proliferation, basement membrane reconstitution, and junctional complex formation.2 In addition, newly formed vasculature recruits periendothelial cells (pericytes for small capillaries and smooth muscle cells for larger vessels) to provide support. These periendothelial support cells are known to act as a survival factor for endothelial cells.3,4 Physiological vascularization and angiogenesis are tightly regulated processes, governed by specic paracrine signals. Although many regulators of vascularization and angiogenesis have been identied (Table 1), gene knockout studies in mice have identied a few receptor tyrosine kinase complexes as being crucial for normal vascular development. These include the vascular endothelial growth factor receptors (VEGF-R1 and R-2, also known as Flt-1 and Flk-1/KDR), and Tie-1 and Tie-2. Each of these knockouts results in embryonic lethality with vascular defects (reviewed in Hanahan5). VEGF-R2 knockouts lack endothelial cells and a developing hematopoietic system, suggesting that VEGF-R2 plays an important role in conversion of angioblasts to endothelial cells. VEGF-R1 appears crucial to the formation of functional vascular tubules but knocking it out does not affect the abundance of endothelial cells. Tie-2 knockouts lack mature vessels, branching networks, and proper organization into larger and smaller vessels. Tie-1 signaling is thought to be involved in control of uid exchange across capillaries and in hemodynamic stress resistance. Unsurprisingly, as discussed below, these physiologically important receptor tyrosine kinase complexes, in conjunction with their ligands, often play an important role in pathologic angiogenesis. In contrast to what is seen with physiological angiogenesis, tumor microvessels frequently lack complete endothelial linings and basement
Curr Probl Cancer, January/February 2002 7
TABLE 1. Natural positive and negative regulators of angiogenesis Positive regulators VEGF (vascular endothelial growth factor) Basic and acidic FGF (broblast growth factor) Transforming growth factor Transforming growth factor- 1 Platelet-derived growth factor Insulin-like growth factor Angiogenin Thymidine phosphorylase Angiopoietin-1 Epidermal growth factor HER-2/neu Interleukin-8 Leptin Hepatocyte growth factor/scatter factor Hypoxia-induced factor 1 Cyclooxygenase-2 Negative regulators Angiostatin Endostatin 2-Methoxyestradiol Thrombospondin-1 Platelet factor IV Tissue inhibitors of metalloproteinases InterferonAngiopoietin-2 Troponin-1 Retinoic Acid Interleukin-12 Vasostatin Prolactin Prostate specic antigen
membranes. They tend to be highly irregular and tortuous, with arteriovenous shunts and blind ends being common. Blood ow through tumors tends to be sluggish, and the tumor associated vessels leakier than normal.6 Many (although by no means all) tumor vessels lack a supporting pericyte structure.3
Regulators of Angiogenesis
The presence of an angiogenic process implies the existence of regulators of the process. These regulators, both positive and negative, have been increasingly well characterized in recent years, and occur in astonishing numbers (Table 1). In malignancy in human beings, the balance between positive and negative regulators may play a crucial role
8 Curr Probl Cancer, January/February 2002
TABLE 2. Negative prognostic value of VEGF Investigator Toi 1994325 Toi 1995326 Obermair 199789 Relf 1997237 Linderholm 200113 Eppenberger 199847 Gasparini 1999328 Gown 2001329 Gasparini330 Eppenberger 199847 Patients 103 328 89 64 1307 305 301 123 260 190 Nodal Status N N N N N N /N /N /N /N /N /N N N 4 N N Method IHC IHC IMA RNase ELISA ICMA IMA IHC IMA ICMA High vs low VEGF (P value) RFS .039 .01 NS .03 .034 .001 .05 NR .001 .04 OS NR NR NR NR .0017 NR .05 .006 .001 NR
RFS, Relapse free survival; OS, overall survival; N , lymph node positive; N , lymph node negative; IHC, immunohistochemistry; ICMA, chemiluminescence immunosorbent assay; IMA, chemiluminescence immunoassay; ELISA, enzyme-linked immunosorbent assay, RNase, ribonucleotide protection assay, NS, not signicant; NR, not reported.
in angiogenesis and tumor progression.7 Although it is impossible to discuss all of these regulators in detail, we will focus on several in each category that have proved especially important from a biologic or therapeutic standpoint.
Positive Regulators of Angiogenesis

Vascular Endothelial Growth Factor. VEGF plays an important role in cancers in human beings. In breast cancer, VEGF is detectable at the transition from atypical hyperplasia to ductal carcinoma in situ, suggesting that its occurrence precedes tumor invasion and metastasis.8-10 Similarly, VEGF expression is found in preneoplastic Barretts esophagus.11 In contrast, VEGF expression does not occur in dysplastic adenomas of the colon and only becomes apparent with the advent of mucosal involvement of colorectal cancers.12 Numerous studies have examined the relationship of VEGF to clinical outcome in cancers in human beings, particularly breast cancer (Table 2). In general, these trials have suggested that VEGF is associated with impaired outcome in patients with early-stage cancers, being associated with worsened relapse-free and overall survival. The largest of these trials, recently reported by Linderholm et al13 at the 2001 American Society of Clinical Oncology Meetings, suggested that VEGF is an independent prognostic factor in breast cancer for both relapse-free and overall survival in multivariate analysis. Its presence is correlated with larger tumor size, steroid receptor negativity, the presence of mutant p53,
and poor tumor differentiation. In addition, Linderholms data suggest that VEGF production is associated with an increased risk of brain and visceral metastasis, a nding conrmed by Foekens et al.14 Although perhaps best studied in breast cancer, the number of cancers in which VEGF tissue expression has been correlated with impaired prognosis is truly impressive and includes (but probably is not limited to) bladder cancer,15 esophageal cancer,16 gastric cancer,17 prostate cancer,18 acute myelogenous leukemia,19 head and neck cancer,20,21 soft tissue sarcoma,22 osteosarcoma,23 non-small cell lung cancer,24-27 papillary thyroid cancer,28 cervical cancer,29,30 ovarian cancer,31 pancreatic cancer,32 and glioma.33 The literature surrounding this association is as vast as it is confusing, with great variation in technique, reagents, and scoring. A signicant number of these trials are characterized by small numbers of patients and varying disease stage and histopathologic type. Taken together, they represent impressive support for the relationship between VEGF expression and patient outcome, yet are of uncertain value for the practicing clinician. Although most studies of the prognostic effects of the VEGFs have focused on the parent (VEGF) ligand, emerging evidence suggests that VEGF-B and VEGF-C may play a role in breast cancer (Fig 1). VEGF-B, like VEGF, acts as a ligand for VEGFR-1, although not (unlike VEGF) for VEGFR-2. In vitro data suggest that activation of VEGFR-1 by VEGF-B is only weakly mitogenic for endothelial cells. A recent publication by Gunningham et al34 suggests that VEGF-B over-expression is associated with lymph node metastasis but not angiogenesis. This trial was not powered to determine the effect of VEGF-B expression on overall survival. VEGF-C is the ligand for VEGFR-3. This ligand-receptor complex is believed to play a role in lymphangiogenesis. Karpanen et al35 have recently demonstrated that VEGF-C transfection of MCF-7 breast cancer cells promoted the growth of tumor-associated lymphatic vessels, which in the tumor periphery were commonly inltrated with the tumor cells. Related experiments in an orthotopic model demonstrated that VEGF-C overexpression resulted in signicantly enhanced metastasis to regional lymph nodes and to lungs; the degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases.36 It is unsurprising that VEGF-C is expressed in many invasive breast cancers37 and that its overexpression is associated with lymphatic vessel invasion and (in one small study) disease-free survival.38 Angiopoietin/Tie-2. The angiopoietin/Tie-2 ligand-receptor complex also plays an important role in physiologic and pathologic angiogenesis,
Fig 1. VEGF and VEGF receptors. In addition to the parent VEGF ligand, several other related ligands
have been identied. In turn, these ligands are recognized by several transmembrane receptor tyrosine kinases shown above.
as discussed above. The regulation of the angiopoietins and Tie-2 are still being elucidated. HER-2 signaling up-regulates angiopoietin-2, which disrupts endothelial cell adherens junctions.39 Angiopoietin-1 is inversely related to thymidine phosphorylase expression in human breast cancer.40 Angiopoietins and their receptor Tie-2 are expressed in numerous human cancers, including prostate cancer,41 glioma,42 gastric cancer,43 non-small cell lung cancer,44 and breast cancer,45,46 and have been reported to have prognostic signicance in some of these cases,46 although they have not yet been well characterized. Fibroblast Growth Factor. Fibroblast growth factor (FGF), although commonly expressed in many cancers (especially basic FGF), has either no prognostic effect whatsoever, or may be associated with slightly better overall prognosis.13,47-51 This lack of negative prognostic effect may be due in part to direct effects of FGF on the cancer cells; bFGF has been
shown to induce apoptosis (by down-regulating Bcl-2) and increase the chemosensitivity of breast cancer cells in vitro.52,53 Integrins. Integrins are cell surface adhesion receptors. The v 3 integrin is the most promiscuous member of the integrin family, allowing endothelial cells to interact with a wide variety of extracellular matrix components. In the mid 1990s Brooks et al54 established that v 3 integrin is up-regulated in proliferating endothelial cells, and antibodies to this integrin affect angiogenesis without affecting normal pre-existing blood vessels. From a mechanistic standpoint, v 3 integrin is required for sustained mitogen-activated protein kinase activity during angiogenesis.55 Integrins may function as endothelial cell survival factors by preventing anoikis by enhancing binding to the extracellular matrix. In addition, integrins may function in concert with VEGF to promote endothelial cell survival.4 Antagonists to v 3 integrin promote tumor regression in preclinical models by inducing apoptosis of tumor blood vessels.56,57 In spite of these interesting observations, v 3 integrin has not been well studied in human cancers, primarily because of the lack of a readily available reagent for use with formalin-xed, parafn-embedded tissue. Gasparini et al have demonstrated that increased vascular expression of v 3 integrin is associated with a greater relapse rate in early stage breast cancer.82,311,317,328
Negative Regulators of Angiogenesis

Perhaps unsurprisingly, the rst regulators of angiogenesis to be discovered were positive regulators. More recently, however, several negative regulators of angiogenesis have been discovered. The importance of these negative regulators for normal physiology is self-evident; negative feedback loops are necessary to prevent uncontrolled proliferation of blood vessels. The importance of negative regulators in cancer is perhaps less obvious for existing macroscopic tumors, which by denition have already undergone an angiogenic switch. The presence of uncontrolled growth and the development of progressive metastasis would seem to imply a failure of negative regulation. As we will describe below, the story is somewhat more complicated. Angiostatin. Angiostatin is a 38-kDa plasminogen fragment generated by several proteases. Originally described by OReilly et al58 as an inhibitor of angiogenesis in Lewis lung carcinoma, it inhibits capillary endothelial cell proliferation and induces endothelial cell apoptosis in vitro and tumor growth in vivo (in multiple preclinical models). In addition, it binds ATP synthase on the surface of endothelial cells, a
mechanism that has been tied to its other actions.59 Recent data suggest that the cytotoxic effects are limited to proliferating endothelial cells.60 Its role in human cancers is poorly documented. A recent study of patients with non small cell lung cancer suggests that tumor expression of angiostatin is (as might be expected) associated with improved clinical outcome.61 Prostatic cancers generate angiostatin-like fragments, and their generation is associated with tumor grade (by Gleason scoring).62 Elevated levels of angiostatin are detectable in the urine of patients with cancer, although their prognostic import is unknown.63 Endostatin. Endostatin is a 20-kDa C-terminal fragment of collagen XVIII that potently inhibits angiogenesis in vitro and in vivo.64 It is generated by proteolytic cleavage of the parent collagen by multiple proteases.65 It affects multiple endothelial cell functions, including modulation of plasminogen activation, disassociation of focal adhesions, and disassembly of actin stress ber networks66; it alters the intracellular calcium signaling response to VEGF and bFGF.67 Endostatin binds to tropomyosin, and a peptide blocker of this binding inhibits endostatins growth-inhibitory activity in vivo.68 Endostatin rapidly down-regulates many genes in exponentially growing endothelial cells, including immediate early response genes, cell cycle-related genes, and genes regulating apoptosis inhibitors, mitogen-activated protein kinases, focal adhesion kinase, G-protein-coupled receptors mediating endothelial growth, a mitogenic factor, adhesion molecules, and cell structure components.69 Endostatins role in human cancer is not well examined. In the ovary, endostatin gene expression is higher in ovarian cancer than in normal ovaries, and high endostatin expression is associated with impaired prognosis.70 In contrast, in hepatocellular carcinoma increased expression of collagen XVIII (endostatins precursor) is associated with smaller tumor size and improved prognosis.71 Serum levels of endostatin have been measured in breast cancer, clear cell renal carcinoma, and soft tissue sarcoma, with variable results: increased plasma endostatin levels are associated with improved prognosis in premenopausal breast cancer and impaired prognosis in sarcoma.72-74 Thrombospondin. Thrombospondins are a multigene family of 5 secreted glycoproteins involved in the regulation of cell proliferation, adhesion, and migration. They are widely distributed in the extracellular matrix of numerous tissues. Two members of the thrombospondin family, namely TSP-1 and TSP-2, are naturally occurring inhibitors of angiogenesis.75 Preclinical evidence suggests that stromal TSP-2 may act as a natural inhibitor of chemically induced carcinogenesis and early tumor formation.76
The role of the thrombospondins as prognostic factors is currently being dened. In several tumors, low thrombospondin levels have been associated with either increased measures of angiogenesis or progressive disease stage and impaired prognosis. These include cervical cancer,77 colorectal cancer,78,79 bladder cancer,80 and glioma.81 In contrast, elevated thrombospondin levels have been reported to have either no effect on prognosis in some tumors (for example, in breast cancer82) or even to be associated with increased angiogenesis and impaired prognosis (for example, in endometrial cancer83).
Angiogenesis in Carcinogenesis and Tumor Progression

The role of angiogenesis in the malignant phenotype has been studied both at a preclinical and a clinical level. It is clear from these studies that angiogenesis is crucial to the early development of many cancers. Preclinical transgenic mouse models (reviewed by Hanahan and Folkman84) suggest that new blood vessel formation occurs after initial tumor initiation but before the development of invasive tumors. Studies from the clinic have conrmed this observation: premalignant lesions of breast, cervix, and melanocytes all demonstrate increased microvessel density.84 Breast cancer provides a representative clinical example of what has been termed the angiogenic switch in cancer. Stromal microvessel density increases signicantly during the transition from atypical hyperplasia to ductal carcinoma in situ of the breast,85,86 and this transition is associated with increased epithelial cell production of the proangiogenic factors VEGF, VEGF-C, hypoxia-inducible factor-1 (which regulates VEGF production), and placental-derived endothelial cell growth factor (PD-ECGF).8,9,37,86 After the transition from atypical hyperplasia to ductal carcinoma in situ, increasing angiogenesis appears to be associated with virtually every step of tumor progression. Comedo ductal carcinomas in situ, for instance, have higher microvessel density measurements than noncomedo ductal carcinomas in situ.87,88 Similarly, microinvasive cancers have higher microvessel densities than noninvasive cancers.85 The angiogenic ability of a primary invasive tumor and of its lymphatic metastases (as assessed by microvessel density) is correlated.89
Angiogenesis and Metastasis

From a teleologic standpoint, angiogenesis is important not only because it offers the growing cancer nutrients, but also because it offers cancer cells a route to distant sites and a means of growing in those sites.
The relationship of angiogenesis to distant metastasis in patients with breast cancer entered the modern era in 1991 with the seminal study by Weidner et al90 in the New England Journal of Medicine. Previous investigators had examined the invasion of tumor blood vessels by cancer cells and correlated such vascular or lymphatic invasion with outcome, with widely varying results. Weidner et al,90 in contrast, measured the blood vessel content of human breast cancers and argued that microvessel density (MVD) was directly correlated with clinical outcome. Numerous other authors have followed their lead in breast cancer (Table 2) and in other malignancies. In general, the results of MVD analyses have suggested that increasing MVD is associated with impaired prognosis for patients with metastatic breast cancer, both with regards to relapse-free and overall survival (Table 3). Not all studies concur with this conclusion. The conicting results obtained may be due to differences in study sample size (which vary widely), differences in technique, or (although this seems unlikely) the inadequacy of the underlying hypothesis. Technique differences abound. First, the means of identifying tumor blood vessels is based on the type of antibody used in the assay. Numerous endothelial cell markers (eg, CD31, CD34, factor VIII-related antigen, CD101, Tie-2) have been studied, and these markers vary with regard to which endothelial cells are delineated. Second, pathologists vary tremendously in their counting measures for tumor endothelial cells. Weidner et al90 originally suggested visual counts of tumor microvessel hot spots, the portion of a given tumor with the (subjectively determined) greatest number of tumor microvessels. Hot spot analysis has the biologic rationale of examining the portion of the tumor with the greatest concentration of new blood vessels and therefore presumably detecting the clonal populations with the greatest potential for metastasis. However, hot spot analysis is inherently subjective and obviously subject to selection bias. In addition, de Jong et al91 have demonstrated that it will affect outcome of hot spot analysis, depending on which tumor block is used and which portion of a given tumor block is examined.91 Because of these concerns, investigators have used several other variations of measuring microvessel density, including counts with a grid (so-called Chalkley counts)92-95 and computer-assisted image analysis.92,96 Comparison of these techniques has suggested that they may be more reproducible than standard hot spot analysis. Third, MVD counting is subject to all of the usual technical problems associated with immunohistochemical technique, such as antigen preserCurr Probl Cancer, January/February 2002 15
TABLE 3. Negative prognostic value of tumor microvessel density in breast cancer Nodal status N N N N N N N N N N N /N /N /N /N /N /N /N /N /N /N /N Counting method Hot spot Hot spot Hot spot Hot spot Chalkley Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot Chalkley Hot spot Hot spot Hot spot Chalkley Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot Hot spot High vs low MVD (P value) RFS Weidner 1991 Weidner 1992304 Hall 1992305 Obermair 1994306 Fox 1995307 Toi 1995308 Ogawa 1995309 Axelsson 199597 Morphopoulas 1996310 Gasparini 1998311 Kumar 1999269 Tynninen 1999312 Hansen 200094 Vincent-Salomon313 Bosari 1992314 Van Hoef 1993315 Fox 1994316 Gasparini 1994317 Bevilacqua 1995318 Inada 1995319 Obermair 1995306 Costello 1995320 Karaiossidi 1996320 Heimann 1996322 Ozer 1997323 Medri 2000324
90
Investigator
Patients
Antibody
OS NR .001 NS NR .05 NR .01 NS NS .05 .0029 NS NR NR NS NF NS NS .028 .047 .044 .05 NR NS NR NR NR NS
49 165 87 106 211 328 155 220 160 531 106 84 836 685 88 32 93 109 254 211 110 230 87 52 167 35 378
N /N N /N N /N N N N N N N N N N N N N N
FVIII FVIII FVIII FVIII CD31 FVIII FVIII FVIII FVIII CD31 CD105 CD34 FVIII CD34 FVIII FV FVIII CD31 CD31 CD31 FVIII FVIII FVIII FVIII CD34 FVIII FVIII
.003 .001 NS .0002 NR .00001 .025 NS NS .05 .0362 NS NS .05 NS .01 NS NS .01 .0004 .0001 .05 .05 NS .05 .018 .034 NS
RFS, relapse free survival, OS, overall survival, N , lymph node positive, N , lymph node negative; NS, not signicant; NR, not reported.
vation, investigator experience, and intraobserver and interobserver variability. Interobserver reproducibility has varied signicantly in various publications where it has been examined.97-99 Although MVD studies support a prognostic effect for increasing microvessel density in breast cancer, the very real technical concerns limit the usefulness of MVD as a routine prognostic indicator. The 1999 review by the College of American Pathologists of prognostic factors considered MVD to fall into category III (factors not sufciently studied to demonstrate their prognostic value) as a prognostic factor.100 In the absence of larger studies that use standardized methods, this seems a reasonable judgment.
Angiogenesis and Tumor Dormancy

Patients may harbor micrometastatic cancers for prolonged periods before the emergence of overt metastatic disease. The following possible explanations for such tumor dormancy have been suggested: (1) tumor dormancy simply may represent the natural history of slowly-dividing tumors, in which overt metastatic disease is the temporal tip of the iceberg (in essence, pseudodormancy); (2) tumor dormancy may represent a cancer composed of nondividing tumor cells that eventually begin dividing as a result of a mutation event favoring progressive growth; (3) tumor dormancy may represent the presence of tumors composed of normally dividing cells whose progressive growth (as opposed to cell division) is inhibited by some external force (eg, immune surveillance). Recent clinical and laboratory data have suggested that angiogenesis may play a role in tumor dormancy. Examinations of scar line recurrences in patients with breast cancer have suggested that true tumor dormancy is a real event; that is, that the tip of the iceberg theory cannot explain the clinical behavior of micrometastatic disease.101 Second, laboratory data from the study of angiogenesis have suggested a synthesis of the second and third explanations offered above. Micrometastatic cancer cells continue dividing at a normal pace during their dormant phase, but such cell division is balanced by cell death.102 Tumor growth, as opposed to cell division, is held in check by the absence of new blood vessel formation sufcient to support the progressive growth of the cancer. It has recently been suggested that tumor dormancy is an active rather than a passive event, in that its maintenance results from tumor production of the natural inhibitor angiostatin, which prevents development of new blood vessel formation.103 Tumor dormancy presumably ends as the result of mutational events that allow new blood vessel formation. The active maintenance of tumor dormancy by such mechanisms implies the strategy of inducing tumor dormancy as a means of treating micrometastatic cancers. Of note, this view of tumor dormancy has been challenged (or at least qualied) by Guba et al,104 who demonstrated the presence of single dormant microscopic metastatic cells independent of angiogenesis inhibition (although such inhibition was also noted in their model system).
Angiogenesis in Hematologic Malignancies

One of the more fascinating outcomes of research involving angiogenesis involves its role in hematologic malignancies. Such a role is not readily intuitive, given the fact that hematologic malignancies are literally bathed in blood, either in the marrow or in the peripheral circulation. In
recent years, however, investigators have demonstrated that angiogenesis plays an important role in the development and progression of these cancers. As with solid tumors, the evidence supporting a role for angiogenesis (reviewed by List105) comes both from studies of microvessel density and from studies of proangiogenic growth factors, particularly VEGF. Increased microvessel density is seen in the marrow of patients with acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, small lymphocytic lymphoma, multiple myeloma, idiopathic myelobrosis, and myelodysplatic syndromes.19,106110 Increased microvessel density has been correlated with impaired clinical outcome in multiple myeloma107,111 and idiopathic myelobrosis.110,112 Studies of VEGF have provided a particularly interesting view into the role of angiogenesis in hematologic malignancies. Several hematologic malignancies have been demonstrated to produce VEGF, which may be measured either in the minor cells themselves or in the serum of patients with these malignancies. In some of these malignancies VEGF expression has been correlated with impaired clinical outcome. These include acute myelogenous leukemia19 and non-Hodgkins lymphoma.113 In addition, there is now growing evidence that several hematologic malignancies express receptors for VEGF (both VEGF receptor-1 [R-1] and receptor-2 [R-2]), raising the possibility that the VEGF/VEGF receptor axis may serve as an autocrine pathway in some tumors.114,115 Dias et al116 have recently studied this role. The HL-60 promyelomonocytic leukemia cell line (expressing both VEGF and VEGF R-2) was inoculated into sublethally irradiated NOD-SCID mice, and then treated with monoclonal antibodies directed against either human or murine VEGF R-2. This approach allowed the investigators to examine the respective roles of the stromal (murine) and leukemic (human) VEGF/VEGF R-2 pathways. Of note, although both antibodies retarded progression of the leukemia in this model, neither was capable of curing the treated animals. In contrast, the combination of both antibodies resulted in cure of the animals. These data suggest that both paracrine and autocrine VEGF R-2 pathways (and, perhaps, both stromal-mediated angiogenesis and VEGF-stimulated tumor growth) play a role in the progression of this tumor. To date there are no published trials of agents targeting either VEGF or VEGF R-2 in patients with leukemia, although such trials are in development for a number of such agents.
Therapeutic Implications of Angiogenesis

Chemotherapeutics as Antiangiogenics
Oncologists may have been unknowingly administering antiangiogenic therapy for years.117 Tamoxifen, initially believed to be merely a competitive inhibitor of estradiol, may have estrogen-independent mechanisms of action.118 Tamoxifen inhibits VEGF-stimulated and FGFstimulated embryonic angiogenesis in the chick chorioallantoic membrane model. This effect was not reversed by excess estradiol, suggesting that the antiangiogenic mechanism is not dependent on estradiol concentration or estrogen receptor content.119,120 Treatment with tamoxifen resulted in a more than 50% decrease in the endothelial density of viable tumor and an increase in the extent of necrosis in MCF-7 tumors growing in nude mice.121 The inhibition of angiogenesis was detected before measurable effects on tumor volume.122 Using differential display technology to assess gene expression in tumor and normal breast tissue from two patients, Silva et al123 reported that brief treatment with tamoxifen resulted in down-regulation of CD36, a glycoprotein receptor for matrix proteins thrombospondin-1 and collagen types I and IV. Thrombospondin-1 is involved in hematogenous tumor dissemination, invasion, and angiogenesis; thus down-regulation of CD36 represents a potential mechanism for the observed antiangiogenic effect. Several chemotherapeutic agents used routinely in breast cancer treatment have known antiangiogenic activity.124-130 Maximal antiangiogenic therapy typically requires prolonged exposure to low drug concentrations, exactly counter to the maximum tolerated doses administered when optimal tumor cell kill is the goal.131 Three recent reports conrm the importance of dose and schedule. In all 3 the combination of low, frequent-dose chemotherapy plus an agent that specically targets the endothelial cell compartment controlled tumor growth much more effectively than the cytotoxic agent alone. An antiangiogenic schedule (170 mg/kg every 6 days) of cyclophosphamide was more effective than the conventional maximum tolerated dose (150 mg/kg every other day for 3 doses every 21 days) in Lewis lung carcinoma and L1210 leukemia models; the antiangiogenic schedule was 3 times more effective in controlling growth of chemotherapy-resistant Lewis lung carcinoma and EMT-6 breast cancer cell lines.132 The addition of the angiogenesis inhibitor, TNP-470, to the antiangiogenic cyclophosphamide schedule induced endothelial cell apoptosis within tumors, an effect that preceded apoptosis of drug-resistant Lewis lung carcinomas. Low-dose vinblastine (0.75 mg/m2 intraperitoneal with 1 mg/m2/d continuous infusion for 3
weeks followed by 1.5 mg/m2 intraperitoneal twice per week) plus an antibody against the VEGF-receptor-2 controlled growth of neuroblastoma xenografts during 210 days of therapy.133 Similar ndings have been reported with carboplatin plus a VEGF-neutralizing antibody.134 Thus far only few clinical trials have tested antiangiogenic schedules of chemotherapy, so-called metronomic therapy.135 Nonetheless, the limited clinical evidence available is intriguing. Remissions can be induced, albeit infrequently, in patients resistant to taxane therapy administered on an every 3-week basis by altering the drug schedule. Specically, Fennelly et al136 reported that 4 of 13 patients with ovarian cancer, who had received and subsequently relapsed from prior paclitaxel therapy, responded when treated with increasing doses of paclitaxel administered weekly. Moreover, 2 of these patients had progressed while receiving paclitaxel. Prolonged infusion paclitaxel (140 mg/m2 over 96 hours) induced responses in 7 of 26 patients who had relapsed within a median of 1 month from prior short taxane infusions.137 The European Organization for Research and Treatment of Cancer studied 2 cyclophosphamide, methotrexate, and 5-uorouracil (CMF) regimens: a classic 28-day regimen incorporating daily oral cyclophosphamide for 14 days and a modied intravenous schedule with bolus cyclophosphamide every 3 weeks. Overall response rate and survival clearly favored the classic regimen.138 Although generally viewed as a test of dose intensity (the classic regimen delivered higher total doses of both cyclophosphamide and 5-uorouracil), this study may also be considered as a test of an antiangiogenic versus bolus schedule. A phase II study of low-dose methotrexate (2.5 mg twice daily for 2 days each week) and cyclophosphamide (50 mg daily) in patients with previously treated metastatic breast cancer found an overall response rate of 19% (the conditions of an additional 13% of patients were stable for 6 months). Serum VEGF levels decreased in all patients continuing to receive therapy for at least 2 months but did not correlate with response.139
Angiogenesis as Predictive Factor

Angiogenesis appears well established as a prognostic factor. More recent studies suggest that it may also function as a predictive factor. Toi et al140 have provided evidence that VEGF expression is associated with a phenotype of hormone resistance. Similarly, Foekens et al14 have suggested that tumor levels of VEGF represent a predictive, as well as a prognostic, factor both for hormonal therapy and chemotherapy. Examining a group of 845 patients in whom VEGF had been measured in the
primary tumor cytosol and who then had development of a recurrence during follow-up, they demonstrated that high tumor VEGF levels were associated with impaired response to hormonal therapy with tamoxifen and to chemotherapy with 5-uorouracil, adriamycin, and cyclophosphamide (FAC) or CMF. In addition, high VEGF levels were associated with short progression-free and overall survival. Why might this be the case? Recent data from Pidgeon et al141 suggest that VEGF may inhibit apoptosis not only in tumor endothelial cells, but in breast cancer cells as well. This protection against apoptosis in breast cancer cells has recently been shown to be mediated at least in part through the neuropilin receptor, for which VEGF acts as a ligand.142 The belief that VEGFs effects, mediated through the classic receptors Flt-1 and KDR, are limited to endothelial cells is no longer tenable.
Targeting Angiogenesis
Our burgeoning understanding of angiogenesis has fostered the development of agents targeting specic steps in the angiogenic cascade, many of which have entered the clinic (Table 4). Part of the very real enthusiasm surrounding these reagents is due to the large variety of potential targets, in contrast to the relatively small number of targets of standard chemotherapy (Table 5). A detailed list of agents in clinical development can be obtained from the Angiogenesis Foundation (http://www.angiogenesis.org) or from the National Cancer Institute (http://cancernet.nci.nih.gov). Although the number of ongoing phase I and II trials has grown rapidly, few have been reported in the peerreviewed literature; no phase III trials (with the exception of matrix metalloproteinase inhibitors) have been completed. Rather than an exhaustive review of all agents currently in clinical testing, we have conceptually grouped agents into several categories: protease inhibitors, which either directly inhibit or otherwise interfere with the action of proteases critical for invasion, growth factor/receptor antagonists, which thwart signaling of proangiogenic growth factors, endothelial toxins, which specically target endothelial antigens, and natural inhibitors, which stimulate or mimic substances known to naturally inhibit angiogenesis. The clinical experience with representative agents in each category will be reviewed. Protease Inhibitors. Degradation of the basement membrane and surrounding stroma by the matrix metalloproteinases (MMPs) is crucial for direct tissue invasion and angiogenesis. Inhibition of the MMPs decreases angiogenesis in preclinical systems and mouse xenografts.143-149 Marimastat is a low molecular weight peptide mimetic
TABLE 4. Representative anti-angiogenic agents currently in clinical trials Agent -941 (Neovastat) Phase II III III I I I II II II II II II II II II/III II/III II/III III III Patient population Myeloma Renal cell IIIa/IIIb nonsmall cell All solid tumors All solid tumors Advanced head and neck Relapsed myeloma Colorectal Renal cell, cervical, ovarian, lymphoma, Hormone-refractory prostate Hematologic malignancies Breast Ib-IIIa resectable nonsmall cell Inammatory breast Colorectal Nonsmall cell Breast Breast Advanced colorectal Combination therapy
Platinum-based chemotherapy RT 5-uorouracil hydroxyurea Thalidomide 5-uorouracil leucovorin
RT
Angiostatin Avastin (rhuMab VEGF)
RT
Docetaxel, estramustine Cytarabine, mitoxantrone vinorelbine Neoadjuvant paclitaxel carboplatin Neoadjuvant docetaxel doxorubicin 5-uorouracil leucovorin CPT-ll Paclitaxel carboplatin Paclitaxel Capectabine Oxaliplatin 5uorouracil leucovorin
BMS-275291
I/II II II/III I II I/II II
HIV-related Kaposis sarcoma Ia-IIIa breast Nonsmall cell All solid tumor and lymphoma Renal cell, ovarian Advanced solid tumors, glioma HIV-related Kaposis sarcoma Cervical Colorectal All solid tumors Glioma All solid tumors Ovarian Colorectal
Carboxyamodotriazole
Chemotherapy or tamoxifen Paclitaxel carboplatin Paclitaxel
COL-3
Celecoxib
I/II II
Cisplatin 5-uorouracil RT 5-uorouracil leucovorin CPT-11
EMD-121974 Endostatin IM-862
I I/II I/II II II
Paclitaxel carboplatin 5-uorouracil
22
TABLE 4. (Continued) Agent Interleukin-12 Marimastat 2-methoxyestradiol Phase I/III III I I II II II II II I/II I I/II I/II II II III Suramin Thalidomide II I I/II II Patient population HIV-related Kaposis sarcoma Small cell lung Breast, all solid tumors Breast Hormone-refractory prostate, myeloma Renal cell Breast Ovarian Glioma All solid tumors, glioma, hematologic malignancies All solid tumors Soft tissue sarcoma Colorectal Renal cell Hormone-refractory prostate Colorectal Myeloma Glioma Melanoma Myelodysplastic syndromes, colorectal, ovarian, uterine sarcoma, endometrial, chronic lymphocytic leukemia, low grade lymphoma, hepatocellular Ovarian Hormone-refractory prostate Myeloma Chronic lymphocytic leukemia Nonsmall cell Low grade lymphoma, hepatocellular Hepatocellular Nonsmall cell Myeloma Combination therapy Liposomal doxorubicin
Docetaxel
RPI-4610 Soy isoavone Squalamine SU-5416
Carboplatin RT
Paclitaxel Neoadjuvant RT CPT-11 Interferon alfa-2b Dexamethasone 5-uorouracil leucovorin CPT-11 Topotecan Temozolomide
II II II II II II III III
Carboplatin Docetaxel Prednisone Fludarabine Carboplatin CPT-11 Interferon-alfa Doxorubicin chemoembolization Carboplatin paclitaxel RT Dexamethasone cyclophosphamide etoposide cisplatin Interferon alfa-2b Hormone ablation
III III
Renal cell Prostate
RT, Radiation therapy. Source: http://cancernet.nci.nih.gov
23
TABLE 5. Chemotherapy versus antiangiogenic therapy: targets of opportunity Chemotherapy targets DNA Topoisomerases Microtubules Antiangiogenesis targets Circulating Ligands Receptor Tyrosine Kinases Integrins Cell adhesion markers Proteases Membrane glycoproteins Enzymes DNA Microtubules Adapted from: Kerbel RS. Clinical trials of antiangiogenic drugs: opportunities, problems, and assessment of initial results. J Clin Oncol 2001;18(Suppl.):45s-51s.
containing a hydroxyamate group that chelates the zinc atom of the active site if the MMPs. Marimastat inhibits a broad spectrum of the MMPs and has activity in multiple human tumor xenograft models.150 Phase I trials identied musculoskeletal syndromes including arthralgia/arthritis, tendonitis, and bursitis as the dose-limiting toxicity with biologically active levels achieved in patients with advanced malignancy with doses ranging from 5 to 10 mg administered 2 times daily.151 Although not predicted to induce substantial clinical response in patients with well-established bulky tumors, long-term therapy with an MMP inhibitor may delay or eliminate tumor regrowth after initial surgical excision or systemic chemotherapy. Two recently completed trials evaluated marimastat in breast cancer. ECOG-2196 measured time to progression in patients with metastatic cancers whose conditions were responding or stable after initial chemotherapy randomized to treatment with either marimastat or placebo. ECOG-2196 recently closed to accrual; preliminary results were expected in late 2001. In a limited institution phase II pilot study, 63 patients with early stage breast cancer received marimastat at 1 of 2 dose levels either after doxirubicin-based chemotherapy or concomitantly with tamoxifen. Musculoskeletal toxicity resulted in signicant dose reductions and limited chronic administration to doses yielding plasma levels below the target range.152 BMS-275291 is a peptidomimetic MMP inhibitor that contains a chemically novel mercaptoacyl zinc-binding group. BMS-275291 inhibits a broad spectrum of MMPs without inhibiting the sheddases, related metalloproteinases which regulate proinammatory cytokine and cyto24 Curr Probl Cancer, January/February 2002
kine receptor shedding from the cell surface. BMS-275291 preserves the homeostasis of tumor necrosis factor- and other proinammatory cytokine and cytokine receptors hypothesized to play a role in the dose limiting arthritis/arthralgia seen with other MMP inhibitors. A similar adjuvant pilot trial is ongoing with this agent. Proangiogenic Growth Factor/Receptor Antagonists. Angiogenesis requires stimulation of vascular endothelial cells through the release of angiogenic peptides including VEGF. An antibody directed against VEGF inhibited the growth of several human tumors in animal models153,154; a humanized recombinant version of this antibody has entered clinical trials. RhuMAb-VEGF was well tolerated and produced the expected decrease in free plasma VEGF levels in a multicenter phase I trial of 25 patients. Three patients had tumor-related bleeding episodes, including an intracranial hemorrhage into an unrecognized cerebral metastasis in a patient with hepatocellular carcinoma. Although no objective responses were seen, 14 patients had stable disease at evaluation on day 72.155 A dual-institution phase II study of rhuMAb-VEGF in patients with previously treated metastatic breast cancer has recently been completed with 75 patients in 3 successive dose cohorts, 3 mg/kg (n 18), 10 mg/kg (n 41), and 20 mg/kg (n 16) every other week. Overall the conditions of 17% of patients were responding or stable at 22 weeks; 3 patients continued to receive therapy without progression for 12 months. Therapy was generally well tolerated with mild hypertension and proteinuria in several patients; no signicant bleeding episodes were noted.156 Inhibition of the VEGF receptor tyrosine kinase domain (Flk-1 and Flt-1) also represents a fruitful therapeutic target. The critical role of Flk-1 in tumor angiogenesis was demonstrated by use of a dominantnegative Flk-1 transfectant. Eight of 9 tumor cell lines with dominant negative Flk-1 showed growth inhibition and reduced MVD in athymic mice.157 A synthetic inhibitor of the Flk-1 kinase has been developed (SU-5416) that inhibits VEGF-dependent growth of endothelial cells without altering tumor growth in vitro. Systemic administration of SU-5416 inhibited the growth of human tumors in mice without apparent toxicity.158,159 SU-5416 was well tolerated in a phase I clinical trial with dose-limiting toxicity being a severe migraine syndrome with headache and projectile vomiting.160,161 Coadministration of SU-5416 did not alter pharmacokinetics of either agent and produced no unexpected toxicity.162 A phase III trial in colorectal cancer, as well as multiple disease-specic phase II trials are ongoing. ZD6474, an oral inhibitor of the Flk-1 tyrosine
kinase that also inhibits the epidermal growth factor receptor tyrosine kinase, has also entered clinical development.163 Ribozymes are small RNA elements with specic catalytic activity. Angiozyme is a synthetic ribozyme designed to cleave the messenger RNA for the VEGF Flt-1 receptor. Preclinical studies conrmed inhibition of both primary tumor growth and metastasis.164-167 A phase I study in patients with refractory solid tumors found limited toxicity and no evidence of drug accumulation. Immunohistochemical staining of accessible tumor samples found variable VEGF Flk-1 and Flt-1 expression with Angiozyme localized to tumor endothelial cells.167 Multiple diseasespecic phase II studies are underway. PNU-145156E (formerly FCE26644), a sulfonated distamycine A derivative, is a noncytotoxic molecule whose antitumor activity is exerted through the formation of a reversible complex with growth/angiogenic factors.168,169 In vitro PNU-145156E did not modify the cytotoxicity induced by several chemotherapeutic agents. However, in vivo, at the optimal dose of each compound, the antitumor activity was signicantly increased in all combinations, with no associated increase in general toxicity. In healthy mice treated with cyclophosphamide or doxorubicin, the addition of PNU-145156E did not enhance the myelotoxic effect.170,171 In phase I testing PNU-145156E induced an unpredictable and short-lasting decrease in antithrombin III levels without effects on serum FGF or VEGF concentrations.172 Endothelial Toxins. The antiangiogenic antibiotic TNP-470 (AGM1470) inhibits endothelial cell proliferation and entered clinical trials nearly a decade ago.173-175 Phase I studies found reversible dosedependent neurologic toxicity; only one transient objective response was reported, although several patients had stabilization of disease.176,177 The half-life of TNP470 was extremely short with practically no drug detectable an hour after treatment suggesting alternate treatment schedules might be required for maximal activity.178,179 Disruption of endothelial cell chemotaxis and migration interferes with angiogenesis. The integrins, particularly v 3 integrin, provide critical attachment between the migrating endothelial cell and the extracellular matrix180; v 3 integrin also localizes MMP-2 to the membrane of endothelial cells in the leading podosomes of new vessels providing carefully targeted matrix destruction.181 Moreover, immunohistochemical studies of clinical specimens from ocular pathologies suggest that both v 3 and v 5 integrins are of importance for endothelial cell function in angiogenic neovascular disease.182 Antibodies that block v 3 integrin inhibit angiogenesis and tumor growth in vitro56 and in vivo.57 A
humanized monoclonal antibody against v 3 integrin, vitaxin was well tolerated and showed some activity in a phase I trial.183 Phase II trials are ongoing. Specic antibodies used to characterize the vitronectin receptors v 3 integrin and v 5 integrin in vitro were used to study the function of these integrins in vivo.184 RGD (arg-gly-asp) epitope is critical for the function of many 1 integrins and is the same epitope that v 3 integrin recognizes in its extracellular matrix ligands. This has led to the development of a family of RGD-containing peptides that can serve as potent and selective inhibitors of the vitronectin receptors. EMD 121974 is the inner salt of a cyclized pentapeptide c-[Arg-Asp-DPhe-(NMeVal)] with signicant antiangiogenic activity in a variety of preclinical in vitro and in vivo models. Phase I trials of EMD 121974 are ongoing. Resting endothelial cells are normally quiescent; proliferation increases dramatically in the leading podosomes of new capillaries. The protein endoglin is expressed much more strongly in growing tumor microvasculature than in the vasculature of surrounding normal tissues. Antibodies directed against endoglin decreased tumor growth in mice xenografts.185,186 Antiendoglin antibodies complexed to deglycosylated ricin A chain produced long-lasting complete remission of preformed tumors in immunocompromised mice.187,188 Natural Inhibitors. The exact mechanism of action of angiostatin remains unclear; however, angiostatin binds ATP-synthase on the surface of endothelial cells inducing endothelial cell apoptosis.59,189,190 A phase I study of recombinant human angiostatin treated patients with refractory solid tumors with doses ranging from 15 to 240 mg/m2 as a daily 10-minute intravenous infusion. No dose-limiting toxicity or changes in coagulation factors were identied. Pharmacokinetics were linear, with dose-proportionate increases in both peak concentration and total exposure. Antibodies to rhu-angiostatin were identied in 2 of 15 patients and did not appear clinically signicant. No objective responses were reported, although some patients had measurable decreases in urine bFGF and VEGF levels.191 Treatment with angiostatin increases sensitivity to radiation in preclinical models,192,193 providing support for an ongoing phase I trial of angiostatin with radiation. Endostatin, a 20-kD proteolytic fragment of collagen XVIII, has antiangiogenic activity similar to angiostatin.194 Endostatin has a highly basic region with signicant afnity for heparin, suggesting that binding to heparin sulphate proteoglycans involved in growth factor signaling may be partly responsible for its activity.195 In addition endostatin binds to tropomyosin in vitro and to tropomyosin-associated microlaments in
endothelial cells; a peptide that mimics the endostatin-binding epitope of tropomyosin blocks endostatin antitumor activity. Thus disruption of microlament integrity seems central to endostatin activity.68 Phase I studies of endostatin have recently been reported.196-198 Patients with refractory solid tumors received daily bolus infusions ranging from 15 to 300 mg/m2 with no apparent toxicity. Pharmacokinetics were linear199; endostatin treatment had no effect on physiological wound healing.200 Correlative studies found dose-dependent decreases in tumor associated blood ow with oxygen 15-labeled positron emission tomography (PET) scanning and dynamic computed tomography imaging, increased tumor apoptosis and decrease in peripheral blood endothelial cell colony-forming precursors. A naturally occurring metabolite of estradiol, 2-methoxyestradiol (2ME2), has a dual mechanism of action: (1) as an antiproliferative drug acting directly on the tumor cell compartment and (2) as an antiangiogenic drug acting on tumor vasculature. In vitro, 2ME2 exhibits antiproliferative activity in tumor cell lines with IC50 values generally in the submicromolar to low micromolar range independent of the estrogen responsiveness of the cell line. In vivo, 2ME2 is effective in xenograft and metastatic disease models.201-204 The antiangiogenic activity of 2ME2 has been demonstrated in vivo in corneal micropocket202 and chorioallentoic membrane systems,205 as well as by the observation of reduced tumor vasculature in 2ME2-treated mice. In vitro, 2ME2 inhibits tubule formation in bovine microvascular endothelial cells stimulated by basic broblast growth factor201 and the proliferation of human umbilical vein endothelial cells.206 Several mechanisms of action have been suggested, including induction of apoptosis, possibly through the activation of p53,207 and slowing the rate, but not the degree, of tubulin (de)polymerization inhibited by 2ME2 treatment in certain cell lines208 and inhibition of superoxide dismutase resulting in increased oxidative stress.209 Preliminary results of an ongoing phase I study of 2ME2 in patients with previously treated metastatic breast cancer have been reported.210 No dose-limiting toxicity was identied with doses ranging from 200 to 1000 mg once daily. Metabolism was variable with a half-life of approximately 10 to 12 hours. Conversion to 2-methoxyestrone, an inactive metabolite, was signicant, with 2ME1 concentrations generally 10-fold higher than 2ME2 levels. No objective responses were produced, although prolonged disease stabilization was achieved in several patients. Changes in VEGF and basic broblast growth factor levels were inconsistent. Accrual continues with a twice-daily dosing schedule. A
phase I study of 2ME2 in combination with docetaxel in patients with newly diagnosed metastatic breast cancer completed accrual in late 2001 but has not yet been reported.
Conceptual Approaches to Antiangiogenic Therapy

Antiangiogenic agents may be conceptually divided into 2 general categories. What might be called vasculotoxins have a direct toxic effect on proliferating endothelium, inducing endothelial apoptosis and cell death. Assuming that a given number of endothelial cells are required to support a population of tumor cells and that some more-or-less xed ratio between the two exists, the vasculotoxins might be expected to have a multiplier effect in tumors in human beings. As such they may produce clinical responses similar to traditional antineoplastics; standard drug development and clinical trials may be appropriate. Conversely, we might term vasculostatins agents that merely prevent further new blood vessel formation without directly damaging the existing microvasculature. Such vasculostatins may require prolonged administration to induce and maintain tumor dormancy. Classic phase II trials of the vasculostatins in patients with well-established tumors may result in few (if any) objective responses without refuting the theoretical basis for their use. As such, delayed responses would be expected; prolonged stable disease might well be considered a win for such agents. Successful development of antiangiogenic therapy, particularly the vasculostatins, will require a new conceptual approach to clinical research: biologically active rather than maximal tolerated dose, long-term rather than intermittent therapy, and induction of minor dormancy rather than tumor cell kill. Current testing of new clinical agents in the phase II setting regularly focuses on overall response rate; agents failing to pass some level of response determined a priori are frequently discarded. This may represent a strategic error for agents that only prevent further tumor growth; progression-free survival may represent the preferred end point for such agents. Alternatively, it may be necessary and appropriate for some agents to move quickly into a randomized trial once appropriate safety concerns have been met.211 Hahnfeldt et al212 have explored a model of tumor growth under angiogenic signaling. This model considers growth of the tumor vasculature to be explicitly time dependent (rather than dependent on tumor volume) and to be under the control of distinct positive and negative signals arising from the tumor. Overall the model parallels Gompertzian kinetics with tumor growth slowing as tumor size increases. Tumor
growth eventually reaches a plateau as the action of stimulators of vascular growth is offset by the increasing production of vascular inhibitors by the primary tumor. Antiangiogenic therapies act to lower this plateau tumor size, it is hoped to a level compatible with asymptomatic host survival. Importantly, the nal tumor size is dependent only on the balance of positive and negative angiogenic factors and is independent of tumor size at the start of treatment. The model also predicts initial tumor growth with some inhibitors of angiogenesis (particularly angiostatin) before stabilization at the plateau size is achieved. This early growth could easily be interpreted (perhaps misinterpreted) as treatment failure unless surrogate markers of angiogenesis are used to guide therapy. If such a model is a correct approximation of clinical reality, then clinical trialists (and their patients) will need to learn to tolerate the prospect of initial disease progression. This will not be a comfortable prospect for many.
Potential Strategies for Thwarting Resistance to Antiangiogenic Therapy

Initial Assumptions and Clinical Realities
Antiangiogenic therapy was initially proposed as being a therapy resistant to resistance.213 This concept was based on both theoretical expectations and preclinical observations. From a theoretical standpoint, cancer cell mutational events form the basis of resistance to cancer chemotherapy; endothelial cells, lacking the genetic plasticity of malignant tissues, should be unable to develop resistance to antiangiogenic therapy. Observations in animal models of antiangiogenic therapy seem to support this hypothesis; treatment of mice bearing a variety of tumors (Lewis lung carcinoma, T241 brosarcoma, or B16F10 melanoma) with endostatin until tumor regression is followed by regrowth cessation of therapy. Retreatment with endostatin following regrowth results in a repeat of tumor regression (and eventual tumor disappearance), suggesting that drug resistance does not develop.214 The clinical reality of human tumors is something different. Although occasional trials have demonstrated some modest evidence of clinical efcacy (eg, VEGF-targeting therapies in breast, lung, and colorectal cancers and thalidomide in myeloma), there is currently no suggestion in the clinical literature that antiangiogenic therapies represent the therapeutic home run initially hoped for. The problem of resistance continues. The discussion above is not meant to imply that antiangiogenic therapy will prove fruitless, nor that resistance will occur to all patients in all
settings. Rather it is meant to suggest that the justied enthusiasm over a novel therapeutic modality should not blind us to the very real challenges facing this modality. Resistance is not a new problem, but rather one faced by physicians for multiple diseases, both oncologic and infectious. Antiangiogenic resistance is one thread of the larger fabric of drug resistance in cancer therapy. The lessons learned in combating other types of drug resistance, offer hope that we might be able to overcome resistance to antiangiogenic therapies. In addition, an understanding of issues specic to antiangiogenesis resistance suggests possibilities for ameliorating or bypassing resistance.
Means of Thwarting Resistance to Antiangiogenic Therapy

Use Standard Therapies with Antiangiogenic Intent. Chemotherapeutic agents have long been developed on the basis of the concept of maximum tolerated dose, and with the assumption that the cancer cells are the sole or at least primarytarget. Recent preclinical studies call these assumptions into question. Numerous chemotherapeutic agents have antiangiogenic activity, and this activity may occur at levels far lower than those required to kill cancer cells.117 Recent data have suggested that the use of long-term low-dose chemotherapy (so-called metronomic therapy) may be potently antiangiogenic, although this effect seems most pronounced when the chemotherapeutic agent is combined with an antiangiogenic agent.132,215 Use of chemotherapeutics to target the tumor vasculature has the advantage of using agents that are already commercially available. The disadvantages of this approach are equally real. For virtually all of the chemotherapeutic agents said to have antiangiogenic activity, we have no idea of the drug doses and schedules that will optimize an antiangiogenic effect. Similarly, if the concentration X time aspects of antiangiogenic chemotherapy differs signicantly from those of antitumor chemotherapy, we may be faced with the dilemma of not knowing which approach to forego. Combine Antiangiogenic Agents with Standard Chemotherapy Regimens. An iteration on the use of chemotherapeutic agents as antiangiogenic therapy involves the combination of chemotherapeutics with antiangiogenic agents. This approach has an extensive preclinical basis for support, with multiple antiangiogenic agents and multiple chemotherapeutic agents showing evidence of combinatorial activity.124,132,215-217 The mechanistic rationale for many of these combinations is poorly
understood and not intuitive. Both radiotherapy and chemotherapy depend on an effective blood supply for therapeutic efcacy. One angiogenic factor that has been extensively examined with regard to its potential interactions with chemotherapeutic agents is VEGF. VEGF is antiapoptotic for endothelial cells via several pathways, including induction of expression of the antiapoptotic proteins Bcl-2 and A1, activation of the PI 3-kinase/Akt signaling pathway, stimulation of NO and PGI2, and increased FAK tyrosine phosphorylation.218 This survival function may play a role in the protection of tumor endothelial cells against the antiangiogenic effects of commonly used chemotherapeutic agents. Sweeney et al124 have recently demonstrated that VEGF protects endothelial cells against docetaxel, an effect reversed by anti-VEGF monoclonal antibody. Recent data suggest that the antiapoptotic effects of VEGF may not be limited to endothelial cells. Neuropilin-1, a receptor important in neuronal guidance, has recently been found to act as a receptor for VEGF219 and is highly expressed by some tumor cells.220 In these, VEGF acts as an antiapoptotic factor, potentially protecting tumor cells against chemotherapeutic agents.142 It is reasonable to expect that, both from the aspect of antineoplastic and antiangiogenic activities, the combination of chemotherapeutic agents with agents targeting VEGF will allow one to increase therapeutic efcacy. Combine Antiangiogenic Agents with Each Other. If, as has been suggested above, tumor progression is associated with tumor acquisition of increasing numbers of proangiogenic factors, then the use of multiple antiangiogenic agents targeting this multiply redundant process simultaneously seems a reasonable means of thwarting resistance to individual agents. This approach is, of course, not unique to antiangiogenic therapy, having previously been used for chemotherapy, antimicrobial therapy, and antiviral therapy. The combination of antiangiogenic agents has been used in preclinical models with success, such as interferon and TNP1470,221 and angiostatin with endostatin.222 The major barriers to the use of such combinations in clinical practice will likely be regulatory and commercial. Combine Antiangiogenic Agents with Other Biologics. Many of the substances regulating angiogenesis are not solely proangiogenic. For instance, epidermal growth factor receptors and HER-2 both regulate VEGF in human tumors, and their blockade reduces VEGF production and angiogenesis.223-231 Given this plethora of indirect inuences on angiogenesis, might we be able to use the combination of biologic agents targeting growth factor receptors such as EGFR and HER-2 as a means of
inhibiting angiogenesis? More specically, might we be able to combine antiangiogenic agents with anti-growth factor receptor agents as a means of overcoming resistance? This strategy has been shown in preclinical models to be an effective antitumor strategy232 and is currently under examination by use of combinations of antiangiogenic agents and trastuzumab in patients with HER-2-positive breast cancer. Conversely, antiangiogenic agents might offer a means of overcoming resistance to growth factor-targeting agents. Recent data from VitoriaPetit et al233 suggest that increased production of VEGF represents one mechanism by which tumor cells escape control with an anti-EGFR monoclonal antibody therapy. The combination of a VEGF-targeting agent with an anti-EGFR agent might prevent this mechanism of resistance to growth factor receptor therapy. Use Antiangiogenic Therapy as Adjuvant Therapy. It is a rare therapy that is more effective for large tumors than for small. Tumor progression, as we have argued above, implies drug resistance for antiangiogenics as for other anticancer agents. One means of thwarting the development of drug resistance associated with tumor progression is to treat cancers when they are small rather than large in volume. The adjuvant setting (or, similarly, a minimal residual disease setting) is the logical place to accomplish this goal. The use of antiangiogenics as adjuvant therapy has its own potential barriers. Physicians are frequently loath to use agents in the adjuvant setting until there is evidence of activity in an advanced disease setting. The toxicity of long-term antiangiogenic therapy remains largely unexplored, as is the toxicity of combinations of chemotherapy with antiangiogenic therapy. At a minimum, it would seem reasonable to require of an antiangiogenic therapy being considered as adjuvant therapy that it can be administered on a long-term basis with established safety and acceptable pharmacokinetics. For instance, we might require that a matrix metalloproteinase inhibitor could be given safely for a year at levels above the range required for successful inhibition of trough serum metalloproteinase enzyme activity. Such a trial has already been conducted for the matrix metalloproteinase inhibitor marimistat in the adjuvant breast cancer setting.152 Use Antiangiogenic Therapy as Targeted Therapy. Antiangiogenic therapy has been viewed to date essentially as another form of chemotherapy: a general therapy given on a population basis, rather than as a targeted therapy given to patients with specic molecular targets. It is reasonable to ask whether we can call failure to respond to a therapy drug resistance if the target at which the therapy is aimed is not present
in the tumor. For instance, although VEGF is an important, perhaps obligatory, part of new blood vessel formation for many cancers, it certainly is not the only proangiogenic factor. Indeed, it is likely to be of little importance to the growth of some individual tumors or even entire classes of tumors. If a patients tumor fails to respond to an anti-VEGF approach, is the tumor resistant or is the therapy merely misguided? This is a practical, as well as, a semantic issue. If insensitivity due to lack of therapeutic target equates with resistance at the patient level, than proper targeting is the means of overcoming such resistance. Targeted therapy requires specic molecular targets. Ideally these targets should be biologically relevant (in the sense of being crucial to the tumors malignant phenotype), clinically measurable, and denably correlated with clinical benet. Examples that t into such criteria include estrogen receptor or HER-2 for breast cancer, c-kit for GIST (gastrointestinal stromal tumors), or bcr-abl for chronic myelogenous leukemia. At present we are unable to point to any targeted antiangiogenic therapy. But it is clear that such targeting is at least potentially realizable. Proangiogenic factors, such as VEGF, and endothelial markers such as v 3 integrin, are clinically measurable. It is reasonable, as part of the development of phase III proof-of-concept trials for such agents, to require mandatory tissue collection for such testing. We lack validated assays for most of the therapeutic targets, but the availability of tissues for testing has a way of stimulating the development of such assays.
Soluble Measures of Angiogenesis

Correlative laboratory studies assessing biologically meaningful intermediate endpoints of angiogenesis are a necessity. Unfortunately, the correlation between intermediate endpoints, angiogenesis and biological activity remains unproven. In spite of the wealth of laboratory data, direct clinical evidence linking antiangiogenic activity, changes in tumor microvessel density and objective tumor response is lacking. In addition to the need to establish the clinical relevance of antiangiogenic activity, the development of reliable surrogate markers of angiogenesis that could guide therapy without repeated tissue samples is urgently needed. Although as yet no clear standard has emerged, the search for reliable surrogates of antiangiogenic activity has focused on two main areas: soluble factors and imaging of the tumor vasculature. VEGF is a highly conserved, homodimeric, secreted, heparin-binding glycoprotein whose dominant isoform has a molecular weight of 45,000.234,235 VEGF produces a number of biologic effects, including endothelial cell mitogenesis and migration, induction of proteinases
leading to remodeling of the extracellular matrix, increased vascular permeability and vasodilation, immune modulation via inhibition of antigen-presenting dendritic cells, and maintenance of survival for newly formed blood vessels by inhibiting endothelial cell apoptosis. VEGF expression is regulated by hypoxia via molecular pathways similar to those regulating erythropoietin gene expression.236 The biologic effects of VEGF are mediated through binding and stimulation of two receptors on the surface of endothelial cells: Flt-1 (fms-like tyrosine kinase) and KDR (kinase domain region). Although multiple angiogenic factors are commonly expressed by invasive human breast cancers, the 121-amino acid isoform of VEGF predominates.237 VEGF has been measured in sera and is typically detected at higher levels in patients with breast cancer than in healthy volunteers.238,239 Higher serum concentrations have also been found in those patients with stage III compared with stage I or II breast cancer.240 Serum concentrations of VEGF may primarily reect platelet count rather than tumor burden, limiting interpretation of these results.241-243 VEGF can also be easily measured in urine with urinary levels unaffected by platelet count.244 Elevated urinary VEGF concentrations predicted recurrence of bladder cancer in one reported study by Crew et al (J Urol 1999;161: 799-804). Additionally, urine VEGF levels increased in patients with progressive disease during treatment with thalidomide.245 As urine VEGF excretion may be affected by renal function, normalization on the basis of urine creatinine has been suggested.246,247 Although previous attempts to correlate single pretreatment VEGF levels with response to therapy248 and the ability of serial measurements to predict response249 in patients with metastatic breast cancer have been disappointing, neither of these preliminary studies directly assessed angiogenesis. Tumorigenesis and progression to metastatic disease are accompanied by changes in the expression of cell adhesion molecules.250 Though the major physiologic role of VCAM-1 appears to be in the adhesion of leukocytes to endothelium during acute inammatory states, a role in the adhesion of malignant cells during the process of metastasis has been demonstrated.251,252 VCAM-1 is transiently expressed on endothelial cells in response to stimulation by various cytokines, including VEGF,253 interleukin-3, interleukin-4,254 and tumor necrosis factor- .255-257 VCAM-1 plays an integral role in angiogenesis. Human recombinant soluble VCAM-1 induces chemotaxis of human endothelial cells in vitro and angiogenesis in vivo in the rat corneal micropocket assay. Both the in vitro chemotactic activity and the in vivo angiogenic activity of rheumatoid synovial uid was blocked by antibodies to soluble VCAM-1.258
Messenger RNA for VCAM-1 has been identied in juvenile rheumatoid arthritis synovium; the level of expression was signicantly higher than in synovium from patients with osteoarthritis.259 Human dermal microvascular endothelial cells transplanted into severe combined immunodecient mice on biodegradable polymer matrixes differentiate into functional microvessels that anastomose with the mouse vasculature. The newly formed microvessels express multiple markers of angiogenesis including CD31, CD34, intercellular adhesion molecule 1, and VCAM-1.260 Soluble VCAM-1 has been detected in the sera of patients with rheumatoid arthritis,261 and pancreatic, colon, prostate, and kidney cell cancers.262-264 Increased concentrations of serum soluble VCAM-1 were detected in patients with breast cancer compared with patients with benign breast disease, further supporting its role.265,266 In a recently reported pilot study, serum VCAM-1 levels were more tightly correlated with breast cancer microvessel density than serum VEGF levels. Although initial levels in patients with known metastatic disease were not predictive of response, levels quickly rose in patients whose disease progressed but decreased or remained stable in responding patients.249 Similar results were found in a phase II study evaluating razoxane, an antiangiogenic topoisomerase II inhibitor, in patients with metastatic renal cell carcinoma. Serum VCAM-1 levels and urinary VEGF levels rose signicantly after one cycle in patients with progressive disease but not in those with stable disease.244 Endoglin (CD105), a receptor for transforming growth factor 1 and 3 in vascular endothelial cells, is signicantly up-regulated in areas of active angiogenesis.186,267 Endoglin endothelial mesenchymal signaling and is required for normal vascular development.268 Antiendoglin antibodies have produced complete remissions in xenograft models.187,188 Endoglin expression in breast tumors is associated with a poor prognosis269; plasma levels of soluble endoglin correlate with metastasis in many solid tumor including breast cancer.270,271 Changes in endoglin levels with antiangiogenic therapy has not been reported. The MMP family of enzymes degrade molecules of the basement membrane and extracellular matrix and are critical for the process of cellular movement and invasion.272 The gelatinases (MMP-2 and MMP-9) have received particular attention as a result of their ability to degrade type IV collagen and the correlation of expression with tumor angiogenesis.145 Overall expression of the gelatinases has been associated with grade and stage of breast cancer273,274; increased serum levels are found in patients with metastatic disease. Given the central role of these
peptides in the angiogenic process, decreases in serum levels should theoretically correlate with changes in tumor MVD and may predict response to antiangiogenic therapy. This hypothesis has not yet been tested prospectively. FGF is also commonly produced by breast cancers and can be measured in tumor cytosols,50 serum, and urine. Similar to VEGF, FGF levels are increased in patients with breast cancer238,275 and may predict survival.276,277 The correlation of serial FGF measurements with changes in tumor microvasculature has not been reported but was not predictive of response to other systemic treatments in one pilot study.278 Other soluble factors including interleukin-6279 and tumor necrosis factor- remain under investigation.280
Imaging Tumor Vasculature

Assessment of tumor blood ow by color-ow Doppler ultrasonography (CDUS) did not correlate with tumor microvessel density in one small study,281 but nonetheless was an independent predictor of survival.282 Other investigators, however, found a signicant correlation between CDUS images and microvessel density; increased blood ow as detected by CDUS was predictive of lymph node metastasis in this pilot study.281 The potential of this technique to monitor the effect of therapy was shown by investigators at the Royal Marsden Hospital. Thirty-four patients with large or centrally located breast tumors were followed with clinical examination, B-mode ultrasonography, and CDUS. Changes in vascularity as measured by CDUS images were concordant with changes in tumor size at more than 75% of the time points assessed. Perhaps more striking, changes were apparent by CDUS at least 4 weeks before any change in tumor size was detected by physical examination or B-mode ultrasonography in 40% of patients.283 Use of ultrasonography to measure breast tumor vasculature has been hampered by the technical imitations of traditional clinical ultrasonography (2 to 10 MHz).284 Low-velocity ow within the central region of a tumor and increased peripheral perfusion is common. Traditional ultrasound can not readily distinguish these regional variations because the low-velocity ow approximates the tissue motion velocity. Modications to clinical ultrasonography including laser Doppler ow measurements,285 use of encapsulated microbubbles as contrast agents,286 and high-frequency Doppler limitations of traditional clinical ultrasonography (2 to 10 MHz) are anticipated.284 Low-velocity ow within the central region of a tumor and increased peripheral perfusion is common. Traditional ultrasonography can not readily distinguish these regional
variations because the low-velocity ow approximates the tissue motion velocity. Modications to clinical ultrasonography including laser Doppler ow measurements, use of encapsulated microbubbles as contrast agents, and high-frequency Doppler scanning (20 to 100 MHz) to improve quantitative measurement of the tumor microvasculature are intriguing but as yet unproven in the clinic. Positron Emission Tomography (PET) uses small doses of radiopharmaceuticals to depict and quantify biochemistry rather than measure static anatomic structures. The most commonly used agent in clinical PET imaging is 2-(18F)Fluoro-2-Deoxyglucose (FDG). As a glucose analog, FDG measures the uptake and phosphorylation of glucose as an indicator of metabolic activity. Sequential PET imaging with FDG predicted response to primary chemotherapy in 3 small pilot trials.287-289 Popular tracers to quantify blood ow include technetium 99m technetium sestamibi, thallium 201, and 15O-labeled water. The 99mTc sestamibi and 201Tl are taken up so avidly by most tissues that levels predominantly reect relative blood ow. Although 99mTc sestamibi and 201Tl have gained widespread acceptance for assessment of myocardial perfusion, markedly increased uptake in tumors has also been demonstrated.290,291 Yoon et al292 evaluated 99mTc sestamibi uptake and washout in 31 patients with untreated primary breast cancer. Both early and late tumor-to-normal breast ratios correlated well with tumor MVD.292 The 15 O-labeled water has a short half-life (2 minutes), mandating cyclotron production near the PET facility. However, 15O-labeled water can quantify perfusion (milliliters/gram/minute) in all body tissues including breast cancer. A small pilot study in patients with breast cancer using 15 O-labeled water found increased blood ow associated with the tumor compared with surrounding normal breast tissue but did not attempt any correlation with histologic study or microvessel density.293 Absolute tumor-associated blood volume can be calculated by use of carbon 11-labeled carbon monoxide, either as an inhaled gas or mixed with whole blood ex vivo.294 The 15O-labeled oxygen has been used to measure changes in tumor oxygen metabolism during therapy.295 Several novel PET imaging strategies are currently in development to quantify tumor hypoxia, proliferation index, apoptosis, receptor/ligand interactions, and angiogenesis-related gene expression.296 Magnetic resonance imaging (MRI) has been used to evaluate tumorassociated vasculature with either intrinsic or contrast-enhanced methods. Intrinsic methods use pulse sequences that detect the motion of water in the vascular bed297,298 or the distortion of the magnetic eld by deoxyhemoglobin.299,300 Intrinsic contrast methods rely solely on intra38 Curr Probl Cancer, January/February 2002
TABLE 6. Contrast Enhanced MR parameters compared to angiogenesis in breast cancer No. tumors (B:M) 20:51 23:25 0:40 36:21 29:61 20:7 MR parameter steepest slope max. amplitude enhancement at 1 minute extraction ow product Ktrans K21 Comparisons with MVD (unless otherwise noted) r Buada Stomper332 Buckley333 Hulka334 Ikeda335 Knopp336
331
Investigator
VEGF
MVD
P .001 .02 .002 .01 .01 .05 NS .07 .008
IHC
CD34 FVIII FVIII FVIII NR CD31
0.83 NR 0.47 0.36 0.89 K21/VEGF, 0.52 MVD/VEGF( ) MVD/VEGF( ), 0.71 VEGF, NR
Matsubayashi337
4:31
rim enhancement
IHC
ND
B, benign; M, malignant; NS, not signicant; NR, not reported; IHC, immunohistochemistry; ND, not done; Ktrans, permeability surface area product per unit volume of tissue; K21, vascular permeability.
vascular red blood cells and are not affected by changes in vascular permeability but have a lower contrast-to-noise ratio limiting resolution. Dynamic contrast enhanced MRI with small molecular contrast media that equilibrate quickly between blood and the extracellular space is dependent on the tumor MVD, vascular permeability (K21) and the transfer constant (Ktrans permeability-surface area product per unit volume of tissue). Such quantitative MRI parameters have been correlated with tumor MVD and VEGF expression in several studies (Table 6). Macromolecular contrast agents quantitatively assay microvascular hyperpermeability and therefore produce an increased signal-to-noise ratio. In a pilot study of 32 patients, tumors with the most intense early signal enhancement and rapid contrast washout (high signal enhancement ratio) had the highest MVD.301 A similar contrast-enhanced method correlated well with changes in tumor necrosis and increasing vascular permeability after treatment with tamoxifen302 or a monoclonal antibody directed against VEGF.303
Future Prospects and Conclusions

Angiogenesis clearly represents a central theme in the genesis and progression of human cancers. Will it represent a central therapeutic
approach in years to come? Although it is too early to claim victory for the antiangiogenics, it is not too soon to predict the successful integration of some form of antiangiogenic therapy into routine clinical practice. To believe otherwise would be to assume that angiogenesis is both biologically crucial and therapeutically unimportant, an unlikely paradox. The major barriers to the use of antiangiogenic therapiespractical and psychologicalare real. For many human cancers we lack a good sense of which angiogenic factors are crucial (crucial in the sense that the tumor cannot survive or grow without the factor). For many tumors the redundancy of proangiogenic factors may prove a barrier to individual treatments. The existence of resistance will result in the death of early hopes of a therapy resistant to resistance. The presence of wellestablished vascular networks in large tumors may render our normal fashion of testing novel agents (beginning with the most refractory tumors and progressing to the smallest, least treated tumors) a prescription for therapeutic failure. Antiangiogenic therapies may have hidden toxicities that may be related to mechanism or drug class, toxicities that may only become apparent with prolonged exposure. These barriers, although real, should not blind us to the promise of this therapy. We are still in the beginning of a revolution, not at its end.304-337
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Angiogenesis and Antiangiogenic Therapy

Curr Probl Cancer 2002;26:1-60. 0147-0272/2002/$35.00 0 56/1/122105

Curr Probl Cancer, January/February 2002

The Process of Angiogenesis

Positive Regulators of Angiogenesis

Negative Regulators of Angiogenesis

Angiogenesis in Carcinogenesis and Tumor Progression

Angiogenesis and Metastasis

OS NR .001 NS NR .05 NR .01 NS NS .05 .0029 NS NR NR NS NF NS NS .028 .047 .044 .05 NR NS NR NR NR NS

Angiogenesis and Tumor Dormancy

Angiogenesis in Hematologic Malignancies

Therapeutic Implications of Angiogenesis

Angiogenesis as Predictive Factor

Platinum-based chemotherapy RT 5-uorouracil hydroxyurea Thalidomide 5-uorouracil leucovorin

Angiostatin Avastin (rhuMab VEGF)

I/II II II/III I II I/II II

Chemotherapy or tamoxifen Paclitaxel carboplatin Paclitaxel

Cisplatin 5-uorouracil RT 5-uorouracil leucovorin CPT-11

EMD-121974 Endostatin IM-862

Paclitaxel carboplatin 5-uorouracil

Curr Probl Cancer, January/February 2002

RPI-4610 Soy isoavone Squalamine SU-5416

Renal cell Prostate

RT, Radiation therapy. Source: http://cancernet.nci.nih.gov

Curr Probl Cancer, January/February 2002

Conceptual Approaches to Antiangiogenic Therapy

Potential Strategies for Thwarting Resistance to Antiangiogenic Therapy

Means of Thwarting Resistance to Antiangiogenic Therapy

Soluble Measures of Angiogenesis

Imaging Tumor Vasculature

P .001 .02 .002 .01 .01 .05 NS .07 .008

CD34 FVIII FVIII FVIII NR CD31

Future Prospects and Conclusions

147. 148. 149.

Curr Probl Cancer, January/February 2002

194. 195. 196. 197. 198.

Curr Probl Cancer, January/February 2002

213. 214. 215.

217. 218. 219. 220.

Curr Probl Cancer, January/February 2002

275. 276. 277. 278.

Curr Probl Cancer, January/February 2002

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