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Angiogenesis and AntiangiogenicTherapy

What 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 an-

swers. 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 angio-

genesis 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

significant extent on our growing understanding of the biologic factors

relating to angiogenesis. Although the excitement surrounding this

approach is certainly justified by the ubiquity and centrality of angiogen-

esis to the metastatic phenotype, the development of these novel agents iscertainly 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

6 Curr Probl Cancer, January/February 2002



The Process of AngiogenesisBlood vessel formation consists of two related but separate processes,

vasculogenesis and angiogenesis. Vasculogenesis, which occurs duringembryogenesis, 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 capillarylumen 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 regulatedprocesses, governed by specific paracrine signals. Although many regu-

lators of vascularization and angiogenesis have been identified (Table 1),

gene knockout studies in mice have identified 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 endothelialcells 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 fluid exchange across capillaries and in

hemodynamic stress resistance. Unsurprisingly, as discussed below, thesephysiologically important receptor tyrosine kinase complexes, in conjunc-

tion 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



membranes. They tend to be highly irregular and tortuous, with arterio-

venous shunts and blind ends being common. Blood flow 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 support-

ing pericyte structure.3

Regulators of AngiogenesisThe 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

TABLE 1. Natural positive and negative regulators of angiogenesis

Positive regulators

VEGF (vascular endothelial growth factor)

Basic and acidic FGF (fibroblast 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-8Leptin

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 metalloproteinasesInterferon-

Angiopoietin-2

Troponin-1

Retinoic Acid

Interleukin-12

Vasostatin

Prolactin

Prostate specific antigen




in angiogenesis and tumor progression.7 Although it is impossible to

discuss all of these regulators in detail, we will focus on several in eachcategory that have proved especially important from a biologic or

therapeutic standpoint.

Positive Regulators of AngiogenesisVascular 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, suggest-

ing that its occurrence precedes tumor invasion and metastasis.8-10

Similarly, VEGF expression is found in preneoplastic Barrett’s esopha-gus.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 thesetrials, 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,

TABLE 2. Negative prognostic value of VEGF

Investigator PatientsNodal

StatusMethod

High vs low VEGF

(P value)

RFS OS

Toi 1994325 103 N/N IHC .039 NR

Toi 1995326 328 N/N IHC .01 NR

Obermair 199789 89 N/N IMA NS NR

Relf 1997237 64 N/N RNase .03 NR

Linderholm 200113 1307 N/N ELISA .034 .0017

Eppenberger 199847 305 N/N ICMA .001 NR

Gasparini 1999328 301 N IMA .05 .05

Gown 2001329 123 N4 IHC NR .006

Gasparini

330

260 N

IMA .001 .001Eppenberger 199847 190 N ICMA .04 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 significant; NR , not reported.




and poor tumor differentiation. In addition, Linderholm’s data suggest

that VEGF production is associated with an increased risk of brain and

visceral metastasis, a finding confirmed 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 can-

cer,

32

and glioma.

33

The literature surrounding this association is as vastas it is confusing, with great variation in technique, reagents, and scoring.

A significant 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-expres-

sion 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 isbelieved 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 infiltrated with the tumor cells.

Related experiments in an orthotopic model demonstrated that VEGF-C

overexpression resulted in significantly 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 Itis 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,




as discussed above. The regulation of the angiopoietins and Tie-2 are stillbeing 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 significance 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

Fig 1. VEGF and VEGF receptors. In addition to the parent VEGF ligand, several other related ligandshave been identified. In turn, these ligands are recognized by several transmembrane receptortyrosine kinases shown above.




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 v3

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 v3

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, v3 integrin is required

for sustained mitogen-activated protein kinase activity during angiogen-

esis.

55

Integrins may function as endothelial cell survival factors bypreventing 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 v3 integrin promote tumor

regression in preclinical models by inducing apoptosis of tumor blood

vessels.56,57

In spite of these interesting observations, v3 integrin has not been

well studied in human cancers, primarily because of the lack of a readily

available reagent for use with formalin-fixed, paraf fin-embedded tissue.

Gasparini et al have demonstrated that increased vascular expression of

v3 integrin is associated with a greater relapse rate in early stage breast

cancer.82,311,317,328

Negative Regulators of AngiogenesisPerhaps unsurprisingly, the first regulators of angiogenesis to be

discovered were positive regulators. More recently, however, several

negative regulators of angiogenesis have been discovered. The impor-

tance of these negative regulators for normal physiology is self-evident;negative feedback loops are necessary to prevent uncontrolled prolifera-

tion of blood vessels. The importance of negative regulators in cancer is

perhaps less obvious for existing macroscopic tumors, which by definition

have already undergone an “angiogenic switch.” The presence of uncon-

trolled 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 generatedby several proteases. Originally described by O’Reilly 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 isgenerated 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 fiber 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 endostatin’s

growth-inhibitory activity in vivo.68 Endostatin rapidly down-regulates

many genes in exponentially growing endothelial cells, including imme-

diate 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

Endostatin’s 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 (endostatin’s precursor) is associated with smallertumor 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 extracellularmatrix of numerous tissues. Two members of the thrombospondin family,

namely TSP-1 and TSP-2, are naturally occurring inhibitors of angiogen-

esis.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

defined. In several tumors, low thrombospondin levels have been asso-

ciated with either increased measures of angiogenesis or progressivedisease stage and impaired prognosis. These include cervical cancer,77

colorectal cancer,78,79 bladder cancer,80 and glioma.81 In contrast, ele-

vated 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 ProgressionThe 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 Folk-

man84) suggest that new blood vessel formation occurs after initial tumor

initiation but before the development of invasive tumors. Studies from the

clinic have confirmed 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 significantly during the transition from atypical hyper-

plasia 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 MetastasisFrom 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 al

90

in the New England Journal of Medicine. Previousinvestigators 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 increasingMVD 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 conflicting

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 thereforepresumably 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 analy-

sis.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 preser-




vation, investigator experience, and intraobserver and interobserver vari-

ability. Interobserver reproducibility has varied significantly 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 reviewby the College of American Pathologists of prognostic factors considered

MVD to fall into category III (“factors not suf ficiently 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.

TABLE 3. Negative prognostic value of tumor microvessel density in breast cancer

Investigator PatientsNodal

status AntibodyCounting

method

High vs low

MVD

(P value)

RFS OS

Weidner 199190 49 N/N FVIII “Hot spot” .003 NR

Weidner 1992304 165 N/N FVIII “Hot spot” .001 .001

Hall 1992305 87 N/N FVIII “Hot spot” NS NS

Obermair 1994306 106 N/N FVIII “Hot spot” .0002 NR

Fox 1995307 211 N/N CD31 Chalkley NR .05

Toi 1995308 328 N/N FVIII “Hot spot” .00001 NR

Ogawa 1995309 155 N/N FVIII “Hot spot” .025 .01

Axelsson 1995

97

220 N

/N

FVIII “Hot spot” NS NSMorphopoulas 1996310 160 N/N FVIII “Hot spot” NS NS

Gasparini 1998311 531 N/N CD31 “Hot spot” .05 .05

Kumar 1999269 106 N/N CD105 “Hot spot” .0362 .0029

CD34 NS NS

Tynninen 1999312 84 N/N FVIII “Hot spot” NS NR

Hansen 200094 836 N/N CD34 Chalkley .05 NR

Vincent-Salomon313 685 N/N FVIII “Hot spot” NS NS

Bosari 1992314 88 N FV “Hot spot” .01 NF

32 N NS NS

Van Hoef 1993315 93 N FVIII “Hot spot” NS NS

Fox 1994316

109 N CD31 Chalkley .01 .028Gasparini 1994317 254 N CD31 “Hot spot” .0004 .047

Bevilacqua 1995318 211 N CD31 “Hot spot” .0001 .044

Inada 1995319 110 N FVIII “Hot spot” .05 .05

Obermair 1995306 230 N FVIII “Hot spot” .05 NR

Costello 1995320 87 N FVIII “Hot spot” NS NS

Karaiossifidi 1996320 52 N FVIII “Hot spot” .05 NR

Heimann 1996322 167 N CD34 “Hot spot” .018 NR

Ozer 1997323 35 N FVIII “Hot spot” .034 NR

Medri 2000324 378 N FVIII “Hot spot” NS NS

RFS , relapse free survival, OS , overall survival, N

, lymph node positive, N

, lymph nodenegative; NS , not significant; NR , not reported.




Angiogenesis and Tumor DormancyPatients may harbor micrometastatic cancers for prolonged periods

before the emergence of overt metastatic disease. The following possibleexplanations 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 repre-

sent 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 celldivision) 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 cellscontinue 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 suf ficient 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 qualified)

by Guba et al,104 who demonstrated the presence of single dormant

microscopic metastatic cells independent of angiogenesis inhibition (al-

though such inhibition was also noted in their model system).

Angiogenesis in Hematologic MalignanciesOne of the more fascinating outcomes of research involving angiogen-

esis 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 my-

eloma, idiopathic myelofibrosis, and myelodysplatic syndromes.19,106-

110 Increased microvessel density has been correlated with impairedclinical outcome in multiple myeloma107,111 and idiopathic myelofi-

brosis.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 acutemyelogenous leukemia19 and non-Hodgkin’s 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 promy-

elomonocytic leukemia cell line (expressing both VEGF and VEGF

R-2) was inoculated into sublethally irradiated NOD-SCID mice, andthen 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 antibod-

ies 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, bothstromal-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 AntiangiogenicsOncologists 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 mech-

anisms of action.118 Tamoxifen inhibits VEGF-stimulated and FGF-

stimulated embryonic angiogenesis in the chick chorioallantoic mem-

brane model. This effect was not reversed by excess estradiol, suggesting

that the antiangiogenic mechanism is not dependent on estradiol concen-

tration or estrogen receptor content.

119,120

Treatment with tamoxifenresulted 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 tech-

nology 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. Throm-

bospondin-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 treat-

ment 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 confirm the

importance of dose and schedule. In all 3 the combination of low,frequent-dose chemotherapy plus an agent that specifically targets the

endothelial cell compartment controlled tumor growth much more effec-

tively 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 andEMT-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 neuroblas-

toma xenografts during 210 days of therapy.

133

Similar findings havebeen 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.

Specifically, Fennelly et al136 reported that 4 of 13 patients with ovarian

cancer, who had received and subsequently relapsed from prior paclitaxeltherapy, 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-fluorouracil (CMF)

regimens: a classic 28-day regimen incorporating daily oral cyclophos-

phamide for 14 days and a modified 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-fluorouracil), 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 re-

sponse.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. Toiet 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. Exam-

ining 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 tamoxifenand to chemotherapy with 5-fluorouracil, adriamycin, and cyclophos-

phamide (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

Thebelief that VEGFs effects, mediated through the classic receptors Flt-1

and KDR, are limited to endothelial cells is no longer tenable.

Targeting AngiogenesisOur burgeoning understanding of angiogenesis has fostered the devel-

opment of agents targeting specific 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 peer-

reviewed literature; no phase III trials (with the exception of matrix

metalloproteinase inhibitors) have been completed. Rather than an ex-

haustive review of all agents currently in clinical testing, we haveconceptually 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 specifically target endothelial antigens, and natural inhibitors,

which stimulate or mimic substances known to naturally inhibit angio-

genesis. 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 cru-

cial 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 Phase Patient population Combination therapy

Æ-941 (Neovastat) II MyelomaIII Renal cell

III IIIa/IIIb nonsmall cell Platinum-based

chemotherapy RT

Angiostatin I All solid tumors

I All solid tumors RT

Avastin (rhuMab VEGF) I Advanced head and neck 5-fluorouracil

hydroxyurea RT

II Relapsed myeloma Thalidomide

II Colorectal 5-fluorouracil

leucovorin

II Renal cell, cervical, ovarian,lymphoma,

II Hormone-refractory prostate Docetaxel, estramustine

II Hematologic malignancies Cytarabine, mitoxantrone

II Breast vinorelbine

II Ib-IIIa resectable nonsmall

cell

Neoadjuvant paclitaxel

carboplatin

II Inflammatory breast Neoadjuvant docetaxel

doxorubicin

II/III Colorectal 5-fluorouracil

leucovorin CPT-ll

II/III Nonsmall cell Paclitaxel carboplatin

II/III Breast Paclitaxel

III Breast Capectabine

III Advanced colorectal Oxaliplatin 5-

fluorouracil

leucovorin

BMS-275291 I/II HIV-related Kaposi’s

sarcoma

II Ia-IIIa breast Chemotherapy or

tamoxifen

II/III Nonsmall cell Paclitaxel carboplatinCarboxyamodotriazole I All solid tumor and

lymphoma

Paclitaxel

II Renal cell, ovarian

COL-3 I/II Advanced solid tumors,

glioma

II HIV-related Kaposi’s

sarcoma

Celecoxib I/II Cervical Cisplatin 5-fluorouracil

RT


leucovorin CPT-11

EMD-121974 I All solid tumors

I/II Glioma

Endostatin I/II All solid tumors

IM-862 II Ovarian Paclitaxel carboplatin





TABLE 4. (Continued)

Agent Phase Patient population Combination therapy

Interleukin-12 I/III HIV-related Kaposi’ssarcoma Liposomal doxorubicin

Marimastat III Small cell lung

2-methoxyestradiol I Breast, all solid tumors

I Breast Docetaxel

II Hormone-refractory

prostate, myeloma

RPI-4610 II Renal cell

Soy isoflavone II Breast

Squalamine II Ovarian Carboplatin

II Glioma RT

SU-5416 I/II All solid tumors, glioma,hematologic malignancies

I All solid tumors Paclitaxel

I/II Soft tissue sarcoma Neoadjuvant RT

I/II Colorectal CPT-11

II Renal cell Interferon alfa-2b

II Hormone-refractory prostate Dexamethasone

III Colorectal 5-fluorouracil

leucovorin CPT-11

Suramin II Myeloma

Thalidomide I Glioma Topotecan

I/II Melanoma Temozolomide

II Myelodysplastic syndromes,

colorectal, ovarian,

uterine sarcoma,

endometrial, chronic

lymphocytic leukemia, low

grade lymphoma,

hepatocellular

II Ovarian Carboplatin

Hormone-refractory prostate Docetaxel

II Myeloma PrednisoneII Chronic lymphocytic

leukemia

Fludarabine

II Nonsmall cell Carboplatin CPT-11

II Low grade lymphoma,

hepatocellular

Interferon-alfa

II Hepatocellular Doxorubicin

chemoembolization

III Nonsmall cell Carboplatin paclitaxel

RT

III Myeloma Dexamethasone

cyclophosphamide

etoposide cisplatin

III Renal cell Interferon alfa-2b

III Prostate Hormone ablation

RT, Radiation therapy.

Source: http://cancernet.nci.nih.gov




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

identified musculoskeletal syndromes including arthralgia/arthritis, ten-

donitis, 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 timeto 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 chemo-

therapy or concomitantly with tamoxifen. Musculoskeletal toxicity re-

sulted in significant dose reductions and limited chronic administration todoses 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 proinflammatory cytokine and cyto-

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

DNAMicrotubules

Adapted from: Kerbel RS. Clinical trials of antiangiogenic drugs: opportunities, problems, and

assessment of initial results. J Clin Oncol 2001;18(Suppl.):45s-51s.




kine receptor shedding from the cell surface. BMS-275291 preserves the

homeostasis of tumor necrosis factor- and other proinflammatory

cytokine and cytokine receptors hypothesized to play a role in the doselimiting 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 mod-

els153,154; a humanized recombinant version of this antibody has entered

clinical trials. RhuMAb-VEGF was well tolerated and produced theexpected 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 significant 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 dominant-

negative 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 withdose-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-specific

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 specific catalytic activity.Angiozyme is a synthetic ribozyme designed to cleave the messenger

RNA for the VEGF Flt-1 receptor. Preclinical studies confirmed inhibi-

tion 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 acces-

sible tumor samples found variable VEGF Flk-1 and Flt-1 expression

with Angiozyme localized to tumor endothelial cells.167 Multiple disease-

specific 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 significantly

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 ef-

fect.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 (AGM-

1470) inhibits endothelial cell proliferation and entered clinical trials

nearly a decade ago.173-175 Phase I studies found reversible dose-

dependent neurologic toxicity; only one transient objective response was

reported, although several patients had stabilization of disease.176,177 Thehalf-life of TNP470 was extremely short with practically no drug

detectable an hour after treatment suggesting alternate treatment sched-

ules might be required for maximal activity.178,179

Disruption of endothelial cell chemotaxis and migration interferes with

angiogenesis. The integrins, particularly v3 integrin, provide critical

attachment between the migrating endothelial cell and the extracellular

matrix180; v3 integrin also localizes MMP-2 to the membrane of

endothelial cells in the leading podosomes of new vessels providingcarefully targeted matrix destruction.181 Moreover, immunohistochemical

studies of clinical specimens from ocular pathologies suggest that both

v3 and v5 integrins are of importance for endothelial cell function in

angiogenic neovascular disease.182 Antibodies that block v3 integrin

inhibit angiogenesis and tumor growth in vitro56 and in vivo.57 A




humanized monoclonal antibody against v3 integrin, vitaxin was well

tolerated and showed some activity in a phase I trial.183 Phase II trials are

ongoing.Specific antibodies used to characterize the vitronectin receptors v3

integrin and v5 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 v3 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 significant an-tiangiogenic 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 microvas-

culature than in the vasculature of surrounding normal tissues. Antibodies

directed against endoglin decreased tumor growth in mice xeno-

grafts.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 identified. Pharmacokinetics were linear, withdose-proportionate increases in both peak concentration and total expo-

sure. Antibodies to rhu-angiostatin were identified in 2 of 15 patients and

did not appear clinically significant. No objective responses were re-

ported, 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, hasantiangiogenic activity similar to angiostatin.194 Endostatin has a highly

basic region with significant af finity 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 microfilaments in




endothelial cells; a peptide that mimics the endostatin-binding epitope of

tropomyosin blocks endostatin antitumor activity. Thus disruption of

microfilament 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 flow with oxygen 15-labeled positron emission tomog-

raphy (PET) scanning and dynamic computed tomography imaging,

increased tumor apoptosis and decrease in peripheral blood endothelialcell 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 antiangio-

genic drug acting on tumor vasculature. In vitro, 2ME2 exhibits antipro-

liferative 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 fibroblast 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 activa-

tion 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 identified 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, aninactive metabolite, was significant, 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 fibroblast 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 fixed

ratio between the two exists, the vasculotoxins might be expected to havea 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 re-

search: 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 IIsetting 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 underangiogenic signaling. This model considers growth of the tumor vascu-

lature 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 lowerthis plateau tumor size, it is hoped to a level compatible with asymptom-

atic host survival. Importantly, the final 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 angio-

statin) 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 guidetherapy. 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 toAntiangiogenic Therapy

Initial Assumptions and Clinical RealitiesAntiangiogenic 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 malig-

nant 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 fibrosarcoma, 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), suggest-

ing 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

ef ficacy (eg, VEGF-targeting therapies in breast, lung, and colorectalcancers and thalidomide in myeloma), there is currently no suggestion in

the clinical literature that antiangiogenic therapies represent the therapeu-

tic “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 justified 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 onefaced 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 specific to antiangiogenesis resistance suggests possibilities for

ameliorating or bypassing resistance.

Means of Thwarting Resistance to Antiangiogenic Therapy

Use Standard Therapies with Antiangiogenic Intent. Chemotherapeu-

tic 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 primary—target. Recent preclinical studies call these

assumptions into question. Numerous chemotherapeutic agents have

antiangiogenic activity, and this activity may occur at levels far lowerthan 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 thechemotherapeutic 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 significantly from those of “antitumor chemother-

apy,” we may be faced with the dilemma of not knowing which approach

to forego.

Combine Antiangiogenic Agents with Standard Chemotherapy Regi-

mens. An iteration on the use of chemotherapeutic agents as antiangio-genic therapy involves the combination of chemotherapeutics with

antiangiogenic agents. This approach has an extensive preclinical basis

for support, with multiple antiangiogenic agents and multiple chemother-

apeutic 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 ef ficacy.

One angiogenic factor that has been extensively examined with regardto 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 al

124

have recently demonstrated that VEGF protectsendothelial 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 chemother-

apeutic agents.142 It is reasonable to expect that, both from the aspect of

antineoplastic and antiangiogenic activities, the combination of chemo-

therapeutic agents with agents targeting VEGF will allow one to increase

therapeutic ef ficacy.

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 simulta-

neously 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 TNP-

1470,221 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. Forinstance, 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 influences 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 specifically, 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 preclinicalmodels 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 Vitoria-

Petit 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-targetingagent 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 unex-

plored, as is the toxicity of combinations of chemotherapy with antian-

giogenic therapy. At a minimum, it would seem reasonable to require of

an antiangiogenic therapy being considered as adjuvant therapy that it canbe 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 con-

ducted for the matrix metalloproteinase inhibitor marimistat in the

adjuvant breast cancer setting.152

Use Antiangiogenic Therapy as Targeted Therapy. Antiangiogenictherapy has been viewed to date essentially as another form of chemo-

therapy: a general therapy given on a population basis, rather than as a

targeted therapy given to patients with specific 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 patient’s 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 specific molecular targets. Ideally these targets should be

biologically relevant (in the sense of being crucial to the tumor’smalignant phenotype), clinically measurable, and definably correlated

with clinical benefit. Examples that fit 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

v3 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 AngiogenesisCorrelative laboratory studies assessing biologically meaningful inter-

mediate endpoints of angiogenesis are a necessity. Unfortunately, the

correlation between intermediate endpoints, angiogenesis and biologicalactivity 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 newlyformed 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 concentra-

tions of VEGF may primarily reflect 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 inflammatory 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 rheuma-

toid synovial fluid was blocked by antibodies to soluble VCAM-1.258




Messenger RNA for VCAM-1 has been identified in juvenile rheumatoid

arthritis synovium; the level of expression was significantly higher than in

synovium from patients with osteoarthritis.

259

Human dermal microvas-cular endothelial cells transplanted into severe combined immunodefi-

cient mice on biodegradable polymer matrixes differentiate into func-

tional 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 cellcancers.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. Al-

though 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 significantly 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

significantly 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 com-plete 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 andMMP-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 beentested 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 surviv-

al.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-6

279

and tumor necrosis factor- remainunder investigation.280

Imaging Tumor VasculatureAssessment of tumor blood flow by color-flow 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 significant correlation between

CDUS images and microvessel density; increased blood flow 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 vascular-

ity 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 ultrasonog-raphy in 40% of patients.283

Use of ultrasonography to measure breast tumor vasculature has been

hampered by the technical imitations of traditional clinical ultrasonogra-

phy (2 to 10 MHz).284 Low-velocity flow within the central region of a

tumor and increased peripheral perfusion is common. Traditional ultra-

sound can not readily distinguish these regional variations because the

low-velocity flow approximates the tissue motion velocity. Modifications

to clinical ultrasonography including laser Doppler flow measure-ments,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 flow 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 flow approximates the tissue motion

velocity. Modifications to clinical ultrasonography including laser Dopp-

ler flow measurements, use of encapsulated microbubbles as contrastagents, 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 radiophar-

maceuticals 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 indicatorof metabolic activity. Sequential PET imaging with FDG predicted

response to primary chemotherapy in 3 small pilot trials.287-289 Popular

tracers to quantify blood flow 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

reflect relative blood flow. 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 The15O-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 using15O-labeled water found increased blood flow associated with the tumor

compared with surrounding normal breast tissue but did not attempt anycorrelation 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 interac-

tions, and angiogenesis-related gene expression.296

Magnetic resonance imaging (MRI) has been used to evaluate tumor-

associated 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 field by

deoxyhemoglobin.299,300 Intrinsic contrast methods rely solely on intra-




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 antibodydirected against VEGF.303

Future Prospects and ConclusionsAngiogenesis clearly represents a central theme in the genesis and

progression of human cancers. Will it represent a central therapeutic

TABLE 6. Contrast Enhanced MR parameters compared to angiogenesis in breast cancer

Investigator

No.

tumors(B:M)

MR

parameter VEGF MVD

Comparisons with MVD

(unless otherwise

noted)

r P

Buada331 20:51 steepest slope – CD34 0.83 .001

Stomper332 23:25 max. – FVIII NR .02

amplitude

Buckley333 0:40 enhancement – FVIII 0.47 .002

at 1 minute

Hulka334 36:21 extraction – FVIII 0.36 .01

flow product

Ikeda

335

29:61 K

trans

– NR 0.89

.01Knopp336 20:7 K21 IHC CD31 K21/VEGF,

0.52

.05

MVD/VEGF() NS

MVD/VEGF(),

0.71

.07

Matsubayashi337 4:31 rim IHC ND VEGF, NR .008

enhancement

B , benign; M , malignant; NS , not significant; NR , not reported; IHC , immunohistochemistry;

ND , not done; K trans, permeability surface area product per unit volume of tissue; K 21

, vascular

permeability.




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. Tobelieve otherwise would be to assume that angiogenesis is both biologi-

cally crucial and therapeutically unimportant, an unlikely paradox.

The major barriers to the use of antiangiogenic therapies—practical and

psychological—are 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 earlyhopes of a therapy “resistant to resistance.” The presence of well-

established 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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