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THE LANCET Oncology Vol 2 December 2001726

Angiogenesis, the formation of new blood vessels fromexisting vasculature, is essential to the late stages ofcarcinogenesis, allowing tumours to grow beyond 1–2 mm in diameter, invade surrounding tissue, andmetastasise. However, more than two decades ago,angiogenesis that preceded neoplastic transformationwas seen. Indeed, it can be detected in inflammatoryand infectious diseases that increase the risk ofdeveloping cancer. Recent advances in fluorescenceendoscopy and histological assessment suggest that,for certain cancers, the degree of new blood-vesselformation may differ between the early and late stagesof carcinogenic progression. The association betweenangiogenesis and cancer occurrence, and ease ofdetection of this process in accessible tissues early incarcinogenesis, mean that angiogenesis fulfils thecriteria for a biomarker of the effectiveness ofchemopreventive intervention. There is also someevidence that biochemical assays of angiogenic growthfactors may offer similar potential as surrogatebiomarkers. Many natural and syntheticchemopreventive agents in development or in clinicaluse inhibit new vessel formation in vivo. Validation ofangiogenesis as a biomarker for the effectiveness ofchemoprevention should further the advancement ofsome chemopreventive agents. Lancet Oncol 2001; 2: 726–32

Angiogenesis, the formation of new blood vessels fromexisting vasculature (Figure 1), has been associated withneoplasia since the experiments of Greene, published in1941.1 Angiogenesis is critical to the transition ofpremalignant lesions in a hyperproliferative state to themalignant phenotype, which leads to tumour growth andmetastasis.2 The intensity of angiogenesis, as assessed bycounting of microvessels in neoplastic tissue (Figure 2), actsas a prognostic factor for many solid tumours such asbreast, prostate, and ovarian cancers,3–5 and very earlycancers of the endometrium and lung.6,7 Similarly,expression of angiogenic growth factors is associated withprognosis for lung and other cancers.8–10 Angiogenesisenables tumours to grow larger than 1–2 mm in diameter,invade surrounding tissue, and travel to distant sites in thebody (metastasise).2 In this review, we argue thatangiogenesis is not only relevant to preneoplastic stages ofcarcinogenesis, but also fulfils the criteria for a surrogatebiomarker of chemopreventive intervention in its ownright. In fact, angiogenesis is already targeted bychemopreventive agents at various stages of drugdevelopment or in clinical practice.

HistologyIncreased vascularity precedes neoplasia. In 1982, Ziche andGullino11 showed that several cell lines, including humanBALB/c fibroblasts, C57BL-MG epithelium, and Syriangolden hamster embryo cells, acquired the ability to inducenew capillary formation before neoplastic transformationoccurred. In these experiments, the cells were cultured invitro and then injected into rabbit cornea; capillaryformation was measured by slit-lamp stereomicroscopy andconfirmed by histology. Similar observations were made inhamster models of chemically induced carcinogenesis.12,13

Collectively, these data supported the idea that theacquisition of angiogenic capability by a previously non-angiogenic cell population may be associated with anincreased risk of neoplastic transformation. Around thesame time, Brem and colleagues reported that preneoplasticlesions of the human breast, when implanted onto the rabbitiris, stimulated the formation of capillaries that were ‘leaky’to fluorescein dye.14 Chodak and colleagues reported similarobservations using atypical cells from biopsies ofpremalignant human bladder lesions implanted onto rabbitirises.15 Using slit-lamp stereomicroscopy to investigate

Review Angiogenesis and chemoprevention

RAS, WPS, and KJO’B are at the Department of Oncology,University of Leicester Royal Infirmary, Leicester, UK. ALH isdirector of the Imperial Cancer Research Fund Medical OncologyUnit, Churchill Hospital, Oxford, UK. AGD is Director, Division ofOncology, Gastroenterology, Endocrinology and Metabolism, StGeorge’s Hospital Medical School, London, UK.

Correspondence: Dr Ricky Sharma, Oncology Department,University of Leicester, Leicester Royal Infirmary, Leicester, LE1 5WW, UK. Tel: + 44 (0) 116 252 5541. Fax: + 44 (0) 116 2525616. Email: ras20@le.ac.uk

Angiogenesis as a biomarker andtarget in cancer chemoprevention

Ricky A Sharma, Adrian L Harris, Angus G Dalgleish, William P Steward, and Kenneth J O’Byrne

Figure 1. Growing tubules immunostained with CD 31 representingimmature vessel formation.

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THE LANCET Oncology Vol 2 December 2001 727

normal skin, Polverini and colleagueshad already shown that new capillaryformation occurs during delayedhypersensitivity responses inguineapigs.16 Thus, before relevantgrowth factors and specific geneticabnormalities had been identified, itwas recognised that the ‘switch’ to anangiogenic phenotype involvedoverexpression of one or morepositive regulators of angiogenesis,mobilisation of proteins from theextracellular matrix, recruitment ofhost cells such as macrophages, or acombination of these processes.2

More recently, histological markersof angiogenesis have been linked todysplasia in human beings (Figure 2).Carcinoma in situ (CIS) denotes severedysplasia of epithelial tissue beforeinvasion of the basement membrane.17

In the cervix, significant increases inmicrovessel density (MVD) are seen asnormal cells progress through cervicalintraepithelial neoplasia stages I, II,and III to invasive squamous-cellcarcinoma.18 Similarly, increasedangiogenesis has been confirmedhistologically in lung metaplasia,dysplasia, and CIS,19,20 in breast ductalCIS,21 and in Barrett’s oesophagus incomparison with normal tissue.22

Recent advances in fluorescenceendoscopy have improved thesensitivity and reproducibility ofangiogenesis detection in premalignant tissue.23 Thistechnique has been used to identify abnormal areas ofbronchial epithelium in smokers who are at high risk ofdeveloping lung cancer. Immunohistochemical analysis ofthese areas shows a unique lesion, consisting of capillaryblood vessels close to, and projecting into, metaplastic ordysplastic squamous bronchial epithelium, known asangiogenic squamous dysplasia (ASD). MVD wassubstantially increased, compared with normal mucosa, inhyperplastic and metaplastic epithelium, and in ASD.Interestingly, ASD was not detected in normal, non-smokingcontrols. The presence of this lesion in high-risk smokerssuggests that unusual patterns of neovascularisation may beuseful biomarkers for the identification of early stages ofbronchial carcinogenesis.23

Unlike cervix and lung tissues, colonic epithelium doesnot undergo squamous metaplasia before the developmentof intraepithelial neoplasia, but instead becomeshyperplastic directly.17 In hyperproliferative human colonicepithelium, angiogenesis has been detected at the same timeas the earliest signs of focal dysplasia, and MVD increasesgradually through the transition from low-grade to high-grade dysplasia and cancer.24 In rats with chemically induced,multistage skin carcinogenesis, the angiogenic ‘switch’ is avery early event, occurring during focal hyperplasia.25

Other features that predispose individuals to thedevelopment of malignant disease have also been associatedwith angiogenesis. Examples include many viral infections,including human herpes virus 8 (HHV8), hepatitis C, andhuman immunodeficiency virus (HIV), and several chronicinflammatory conditions associated with the developmentof tumours, including cigarette smoking, asbestos exposure,and liver cirrhosis (Figure 2).26–33 These observations suggesta role for angiogenesis in carcinogenesis, before the onset ofmetaplasia and hyperplasia. They also imply that theprocesses resulting in chronic immune activation provide anenvironment that facilitates the development of malignantdisease.34 Interestingly, MVD in normal liver tissue canpredict tumour recurrence in patients undergoingpotentially curative resection for hepatocellular carcinoma.27

Angiogenic growth factors An alternative approach to studying angiogenesis involvesgrowth factors necessary for new blood-vessel formation. Inestablished malignant disease, angiogenic growth factors maybe released directly, by tumour cells themselves, or indirectly,via recruitment of other cells such as macrophages andendothelial cells.2 The initial release of these substances, orevidence of complex formation between angiogenic growthfactors and high-affinity receptors, can be referred to as an

ReviewAngiogenesis and chemoprevention

Figure 2. Immunohistochemical demonstration of angiogenesis in human tissues in a potentiallypreneoplastic phase or at different stages of carcinogenesis. Panels A, B, and C are sections fromcirrhotic liver, a colorectal adenoma, and a superficial bladder cancer, respectively. They are stainedwith an antibody to CD34 (QB-END/10) to show blood vessels. In Panel D, an epithelioidmesothelioma is shown stained with an antibody to CD31, which labels endothelial cells and variouscellular components of whole blood.

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THE LANCET Oncology Vol 2 December 2001728

angiogenic ‘switch’.2 This stage can beinduced by several processes, includinghypoxia, or may result from alterationsin the genes (mutations or changes inexpression) involved in the promotionand progression stages of car-cinogenesis, eg RAS and P53.35,36 Thecapacity of malignant cells to induceangiogenesis is associated with theproduction of these growth factors,37

and peripheral-blood concentrationsmay correlate with MVD in someprimary tumours.38

The expression of angiogenicgrowth factors may also be increasedin preneoplastic tissue (Figure 3),although interpretation of experi-mental results is complicated by theconstitutive expression of growthfactors in normal epithelia34 andinducible expression in acute disease.39

In rats with chemically inducedcancer, concentrations of vascularendothelial growth factor (VEGF) areincreased in simple hyperplasia of thebladder40 and pituitary gland;41 VEGFproduction may be a rate-limiting stepin the development of premalignantskin lesions.42 Similar evidence fromtransgenic mice with skin cancersuggests a role for matrix metal-loproteinase 9 (MMP9).43 In hamsterbuccal pouch keratinocytes, studies ofthe angiogenic growth factor,transforming growth factor �1 (TGF�1), imply that angio-inhibitoryactivity is lost at the earliest stages ofcarcinogenesis.44 High expression ofVEGF, especially under hypoxicconditions has been seen in studies ofliver cirrhosis and hepatitis B virusinfection, which both predispose individuals tohepatocarcinogenesis.27,32 Similarly, HIV and HHV 8, bothimplicated in the development of Kaposi’s sarcoma, causethe production of factors that promote angiogenesis.30,31

There is also convincing evidence in CIS of humancervix, oesophagus, and breast tissues that overexpression ofVEGF or platelet-derived endothelial-cell growth factor(PDEGF) occurs.18,22,45,46 In human bronchus, significantincreases in VEGF expression have been detected inspecimens representative of the transition from normalepithelium through moderate dysplasia to CIS.20 Highconcentrations of growth factors have been found indysplastic adenomas, which are precursors of colorectalcancer.24,34 In one study, VEGF expression was absent in mildto moderately dysplastic adenomas of the colorectum,present in the majority of CIS, and present in all colorectalcarcinomas invading the submucosa.47

Similarly, increasing levels of expression of MMPs andtissue inhibitors of MMP (TIMPs) have been described in

many lesions as they progress from dysplasia through CISand to invasive carcinoma, including those of the breast,lung, and prostate.34,47,48 Increased expression of MMP 2 andmembrane-type MMPs, which both facilitate tumourinvasion and angiogenesis, has been shown in colorectalpreneoplasia.37 This association is particularly strong foradenomas that overexpress the cyclo-oxygenase COX 2enzyme.34

COX 2 is the inducible form of the enzyme that catalysesprostaglandin and thromboxane formation fromarachidonic acid. COX 2, and its isozyme COX 1, areinvolved in the induction of angiogenesis in humanendothelial cells cultured with colon cancer cells.37

Overexpression of COX 2 has been implicated in thetumorigenesis of cancers of the colon, rectum, stomach,lung, breast, and head and neck.34 COX activity seems toconfer a survival advantage to transformed cells through theinhibition of apoptosis, enhanced cell proliferation, reducedcell-to-cell adhesion, increased adherence of cells to the

Review Angiogenesis and chemoprevention

PRO

GR

ESS

ION

PR

OM

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ON

Normalepithelium

Predisposingconditions

Metaplasia

Hyperplasia

Mild-moderatedysplasia

Severedysplasia

Invasivecarcinoma

Metastasis

Smoking

Viralinfection

Cirrhosis

MICROVESSEL DENSITY (MVD)

VEGF

MMPs

INC

REA

SED

EXP

RES

SIO

N

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Intraepithelialneoplasia

Intra-mucosalcapillaryloops

Carcinomain situ

MVD

MVD

Figure 3. Multistep model of carcinogenesis (modified from 60). Whereas metaplasia andhyperplasia may be reactive and frequently regress, dysplasia is characterised by more advancedmolecular changes and persists for many years. Black arrows indicate detection at the earliest stageof the multistep model, and for some measures of angiogenesis (broken lines) increasing expressionhas been observed within or across subsequent stages (see text). VEGF, vascular endothelial growthfactor; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of matrix metalloproteinases.

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THE LANCET Oncology Vol 2 December 2001 729

extracellular matrix, and the inductionof angiogenesis (see Figure 3).34,49

Moreover, prostaglandin E2, theformation of which is catalysed byCOX 2, is itself an angiogenic growthfactor in vivo50 and also inducessynthesis of angiogenic growth factorssuch as VEGF in cultured non-malignant cell lines such as synovialcells and osteoblasts.51,52 As well asbeing constitutively expressed in solidtumours, COX 2 expression is seen inmany premalignant lesions, includingcolorectal adenomas and atypicaladenomatous hyperplasia of the lung.52

COX 2 also modulates the activity ofgrowth regulatory peptides, such asTGF �.49 The growth promoting andpro-angiogenic effects of TGF � areseen when epithelial cells have beenexposed to other cytokines that induce COX 2 expression,such as epidermal growth factor.49 Recent findings on thelocalisation of COX 2 in adenomas that develop in mice subject to intestinal carcinogenesis53 provide aninteresting link with Polverini and colleagues’ early findings on the importance of activated macrophages in inducing angiogenesis.54

The genetic changes that characterise invasive cancer canbe found in preneoplastic lesions. The main function of thewild-type P53 protein in normal mammalian cells is themaintenance of DNA integrity and genomic stability by wayof cell-cycle arrest, which allows cells to repair damagedDNA. Its other functions include the implementation ofapoptosis and inhibition of angiogenesis. Concentrations ofmutated P53 are associated with MVD in human bladdercancer and breast CIS,55,56 and in some cancers they havebeen linked with VEGF expression.57 The presence of wild-type P53 reduces MMP 2 concentrations in humanmelanoma cells, with associated reductions in invasivepotential.58 Loss of wild-type P53 has been associated withpreneoplasia of the bronchial tree20 and with humanpapillomavirus infection that predisposes to neoplasia of theuterine cervix; in these cases, angiogenesis increases with thedegree of histological abnormality.18 VEGF expression hasalso been linked with DNA microsatellite instability59

and with regulatory proteins linked to carcinogenesis, such as Ki-67.9

Relevance to chemopreventionCollectively, these data indicate that histologicalmeasurements of angiogenesis, perhaps supported bycorresponding biochemical features, could representbiomarkers of dysplasia and invasive malignant disease. Incertain tissues, such as the cervix and lung, there isaccumulating evidence not only that expression of thesemarkers differs between normal tissue and early cancers, butalso that angiogenesis may increase during metaplasia,hyperplasia, and mild to moderate dysplasia.18,19,20,24,34 Thisincrease in new blood vessels may not apply to allpremalignant disorders,23 and the presence of angiogenesis

in early carcinogenesis may just be a circumstantial event, orepiphenomenon, rather than an essential causative factor perse. Such epiphenomena may represent useful biomarkers ofthe carcinogenic process.60 Neovascularisation can also bedetected in disorders that increase risk of developing cancer,such as inflammation of the lung, liver cirrhosis, andchronic viral infections. These features fulfil the first fourcriteria of potential efficacy (see Table 1) for a biomarker ofcancer chemoprevention, defined as the inhibition,retardation, or reversal of carcinogenic processes bychemical means.60 Such ‘surrogate’ biomarkers are necessaryin chemoprevention trials because of the long time beforemalignant disease develops, and the need for monitoringstudy participants at a mechanistic level. Use of biomarkersenables clinical advancement of chemopreventive agentsbefore definitive data on cancer incidence or mortality areavailable.17 Validation of angiogenesis as a chemopreventivebiomarker will require clarification of its role in the earlieststages of carcinogenesis, assessment of its reproducibility in chemical and genetic models, and pilot studies in high-risk volunteers.61

Screening tests are not developed if no managementstrategies exist for the disease detected. The biomarkermeasured in chemoprevention must have the potential formodification by therapeutic interventional agents. In thisregard, angiogenesis is a particularly attractive biomarker.Potentially non-toxic antiangiogenic drugs being developedby cancer chemotherapy pharmacologists are being appliedat early stages of carcinogenesis with promising results.62

ReviewAngiogenesis and chemoprevention

Table 1. Criteria used to judge the potential efficacy of

chemopreventive biomarkers (modified from ref 75)

● Variability of expression between phases of the carcinogenetic process, ienormal, premalignant, and malignant (preferably increasing withcarcinogenetic progression)

● Ability to be detected early in the carcinogenetic pathway

● Association with risk of developing cancer or recurrence of tumour

● Presence in tissues that are easily accessible for many biopsies

● Potential for modification by a chemopreventive agent

Growth factors eg EGF

Inflamation eg bacterial lipopolysaccharide

Tumour promoters eg phorbol 12-myristate 13-acetate

Angiogenic growth factorseg VEGF

NORMALADHESION

ANGIOGENESIS

APOPTOSIS

PROLIFERATION

COX 2INDUCTION

PGE2

Figure 4. Simplified representation of the potential role of cyclo-oxygenase induction in activatedmacrophages in inducing angiogenesis in preneoplastic lesions. EGF, endothelial growth factor;PGE2, prostaglandin E2; VEGF, vascular endothelial growth factor.

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THE LANCET Oncology Vol 2 December 2001730

Furthermore, many chemopreventive agents at variousstages of development appear to inhibit angiogenesis.Epidemiological evidence suggests that aspirin63 andcaptopril64 use is associated with a lower risk of malignantdisease. Aspirin is significantly antiangiogenic in vivo,37

although its chemopreventive effectiveness may depend oncellular factors such as P53 status.65 Captopril also hasantiangiogenic activity, via inhibition of zinc-dependentendothelial-cell gelatinase MMP activity.66 Tamoxifen andcelecoxib, which were recently licensed for certain well-defined populations of patients by the US Food and DrugAdministration for the chemoprevention of breast andcolorectal cancers, respectively,61 also have antiangiogenicproperties in xenograft models.37,67 Finally, the dietaryconstituents and potential cancer chemopreventives,selenium, N-acetyl cysteine, vitamin D3, curcumin,genistein and related flavonoids, and the tea constituentepigallocatechin-3-gallate have all been shown to inhibitangiogenesis in vivo without causing toxic effects.68–73 Thepleiotropic activity of each of these phytochemicals at morethan one level within cellular pathways61 may avoidpotentially proangiogenic consequences of selectiveinhibition of one angiogenic growth factor, as shownrecently in transgenic mice with abnormalities of MMP 7and MMP 9 production.74 Current clinical trials of naturaland synthetic agents as chemopreventives60 are not atpresent measuring biomarkers of angiogenesis.

ConclusionAngiogenesis seems to be present in many disorders thatpredispose individuals to cancer and during the stages ofcarcinogenesis that precede CIS. Improvements inendoscopic techniques, histological assessment, and assaysof angiogenic growth factors will allow further clarificationof these findings. Early indications suggest that angiogenesismay fulfil the criteria for a suitable biomarker of the efficacyof chemopreventive intervention. Most notably, itsassociation with risk of cancer occurrence or recurrence, itsdetectability in accessible tissues early in carcinogenesis, andits differential expression during various stages ofcarcinogenesis, support this idea. Furthermore, theangiogenic process may be modified by potentialchemopreventive agents such as aspirin, celecoxib,tamoxifen, and epigallocatechin-3-gallate. We suggestmonitoring angiogenesis as a biomarker in metaplastic andhyperplastic lesions in preneoplastic or premalignantconditions such as Barrett’s oesophagus, colorectaladenomas, and ASD. Validation of this biomarker may

facilitate the development of new synthetic drugs andpromising phytochemicals in cancer chemoprevention.

AcknowledgmentsWe acknowledge the support of the Imperial Cancer Research Fund,the Institute of Cancer Studies, Leicester, UK, and the UniversityHospitals of Leicester Research Funding Group. RAS would like tothank Clare Winfield for renumbering the references and Sue Spriggsfor checking their accuracy. KOB thanks Jonathan Goddard, John Edwards, and Diana Cullen for help with Figure 1.

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Review Angiogenesis and chemoprevention

Search strategy and selection criteriaPublished data for this review were identified by searchesof Web of Science and MEDLINE among papers publishedin peer-reviewed journals (English language only) from1966 to January 2001. Keywords used: angiogenesis,neovascularisation, chemoprevention, cancer, carcino-genesis, neoplasia, malignancy, premalignancy, andpreneoplasia. We have attempted to select original articlesrather than reviews or commentaries, where possible.

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49 Saha D, Datta PK, Sheng H, et al. Synergistic induction ofcyclooxygenase-2 by transforming growth factor-beta1 andepidermal growth factor inhibits apoptosis in epithelial cells.Neoplasia 1999; 1: 508–17.

50 Benefield J, Petruzzelli GJ, Fowler S, Taitz A, Kalkanis J, Young MR.Regulation of the steps of angiogenesis by human head and necksquamous cell carcinomas. Invasion Metastasis 1996; 16: 291–301.

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52 Hosomi Y, Yokose T, Hirose Y, et al. Increased cyclooxygenase 2(COX-2) expression occurs frequently in precursor lesions ofhuman adenocarcinoma of the lung. Lung Cancer 2000; 30:73–81.

53 Hull MA, Booth JK, Tisbury A, et al. Cyclooxygenase 2 isupregulated and localized to macrophages in the intestine of Minmice. Br J Cancer 1999; 79: 1399–1405.

54 Polverini PJ, Cotran RS, Gimbone MA Jr, Unanue ER. Activatedmacrophages induce vascular proliferation. Nature 1977; 269:804–06.

55 Bochner BH, Esrig D, Groshen S, et al. Relationship of tumorangiogenesis and nuclear p53 accumulation in invasive bladdercancer. Clin Cancer Res 1997; 3: 1615–22.

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