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Markers of Response for the AntiangiogenicAgent BevacizumabDiether Lambrechts, Heinz-Josef Lenz, Sanne de Haas, Peter Carmeliet, and Stefan J. Scherer

Diether Lambrechts and Peter Carmeliet,

Vesalius Research Center, Flanders Insti-

tute for Biotechnology (VIB), and Univer-

sity of Leuven, Leuven, Belgium; Heinz-

Josef Lenz, Keck School of Medicine,

University of Southern California, Los

Angeles; Stefan J. Scherer, Genentech,

South San Francisco, CA; and Sanne de

Haas, F. Hoffmann-La Roche, Basel,

Switzerland.

Published online ahead of print at

www.jco.org on February 11, 2013.

Supported by the Fund for Scientific

Research Flanders (D.L.), the Agency for

Innovation by Science and Technology,

the Stichting tegen Kanker, Grant No.

KULPFV/10/016SymBioSys from the

University of Leuven, and by the Seventh

Framework Programme of the European

Community for Research (AngioPredict).

Authors disclosures of potential con-

flicts of interest and author contribu-

tions are found at the end of this

article.

Corresponding author: Diether

Lambrechts, PhD, Vesalius Research

Center, VIB, Herestraat 49, bus 912,Leuven, Belgium B-3000; e-mail:

[email protected].

2013 by American Society of Clinical

Oncology

0732-183X/13/3199-1/$20.00

DOI: 10.1200/JCO.2012.46.2762

A B S T R A C T

Bevacizumab is the first antiangiogenic therapy proven to slow metastatic disease progression inpatients with cancer. Although it has changed clinical practice, some patients do not respond orgradually develop resistance, resulting in rather modest gains in terms of overall survival. A majorchallenge is to develop robust biomarkers that can guide selection of patients for whombevacizumab therapy is most beneficial. Here, we discuss recent progress in finding suchmarkers, including the first results from randomized phase III clinical trials evaluating the efficacyof bevacizumab in combination with comprehensive biomarker analyses. In particular, thesestudies suggest that circulating levels of short vascular endothelial growth factor A (VEGF-A)

isoforms, expression of neuropilin-1 and VEGF receptor 1 in tumors or plasma, and geneticvariants in VEGFA or its receptors are strong biomarker candidates. The current challenge is toexpand this first set of markers and to validate it and implement it into clinical practice. A firstprospective biomarker study known as MERiDiAN, which will treat patients stratified forcirculating levels of short VEGF-A isoforms with bevacizumab and paclitaxel, is planned and willhopefully provide us with new directions on how to treat patients more efficiently.

J Clin Oncol 31. 2013 by American Society of Clinical Oncology

INTRODUCTION

Bevacizumab is a humanized monoclonal antibody

against the vascular endothelial growth factor A

(VEGF-A), a key factor inducing the formation of

blood vessels (angiogenesis) in tumors.1 Bevaci-

zumab is currently approved in Europe and the

United States in combination with standard chem-

otherapy for the treatment of metastatic colorectal

cancer (mCRC)2 and nonsmall-cell lung cancer

(NSCLC).3 The drug is also approved in combi-

nation with interferon alfa-2a for renal cell car-

cinoma (RCC),4-6 with standard chemotherapy

for advanced ovarian cancer in Europe,7,8 and

as a single agent for recurrent glioblastoma in

the United States.9

In early 2008, bevacizumab also received theUS Foodand Drug Administrationsaccelerated ap-

proval for treatment of metastatic breast cancer

(mBC). Its efficacy and safety were shown in a mul-

ticenter trial (E2100) that randomly assigned

women with mBC to conventional chemotherapy

alone or in combination with bevacizumab.10 The

trial showed that bevacizumab significantly im-

proved progression-free survival (PFS). Adverse ef-

fects were moderate and manageable. Subsequent

completion of the E2100 trial and publication of

other trials in first-line mBC, in which bevacizumab

failed to improve overall survival (OS), revealed ex-

cess toxicity and less benefit in terms of PFS than

expected on the basis of E2100.11,12 As a result, the

US Food and Drug Administration revoked the li-

cense for bevacizumab in the setting of first-line mBC.13

This failure needs to be seen in the context of a

moregeneraldebate, inwhich it is increasingly being

realized that biomarkers for targeted cancer thera-

pies are necessary, because only a subset of patients

respond, and the overall clinical benefit is limited.14

Considering the highcost of these therapies, predic-

tive markers are not only a clinical necessity but are

also an economic requirement. With this in mind,

extensive biomarker programs have been built into

numerous clinical studies with bevacizumab. How-

ever, a marker that predicts bevacizumab treatment

outcome has not yet been validated. What are the

reasons for these problems? And what can be done

to move forward? Here, we try to provide an answer

to these questions. First, we describe the challenges

in identifying biomarkers for bevacizumab. Then,

we identify a set of markers that we consider most

promising, either because they were predictive in

placebo-controlled studies involving large numbers

of patients or have been replicated in several other

studies. Finally, we also speculate on how to further

improve this set of markers and discuss how to im-

plement them into clinical practice.

JOURNAL OF CLINICAL ONCOLOGY R E V I E W A R T I C L E

2013 by American Society of Clinical Oncology 1

http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2012.46.2762The latest version is atPublished Ahead of Print on February 11, 2013 as 10.1200/JCO.2012.46.2762

Copyright 2013 by American Society of Clinical Oncology2013 from 128.118.88.48Information downloaded from jco.ascopubs.org and provided by at Penn State Hershey Medical Center on February 18,

Copyright 2013 American Society of Clinical Oncology. All rights reserved.
http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2012.46.2762http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2012.46.2762http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2012.46.2762http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2012.46.2762


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CHALLENGES IN IDENTIFYING BIOMARKERSFOR BEVACIZUMAB

The search for a biomarker predictive of bevacizumab treatment out-

come has proven to be challenging for various reasons. First, angio-

genesis is a complex and highly adaptive biologic process. Despite the

predominant role of VEGF-A, multiple other factors can play an

essential role during angiogenesis, including the placental growth fac-

tor (PlGF), fibroblast growth factors (FGFs) and platelet-derived

growth factors (PDGFs), angiopoietins (ANGs), and various cyto-

kines.1 Inaddition, other factorsthatpromote proteolyticdegradation

of the matrix or induce maturation of the vasculature by stimulating

pericyte coverage (PDGF-BB, ephrin-B2, and NOTCH) also critically

contribute to the process. As a result, theactivity of bevacizumab may

be compensated by at least a dozen alternative angiogenic signals.

Second, preclinical studies revealed that VEGF-A blockade has

heterogeneous effects on tumors, ranging from inhibition of vessel

expansion and regression of pre-existing vessels to inhibition of bone

marrowderived cell and/or endothelial progenitor cell recruitment

to the vascular wall.15,16 However, in humans, studies are often hin-

dered by the inability to perform serial tumor biopsies, thereby pre-venting histologic analysis of vascular changes. A seminal study in

rectal carcinoma revealed that a single infusion of bevacizumab rap-

idly decreased tumor perfusion, vascular volume, microvascular den-

sity, and interstitial fluid pressure (consistent with a reduction in

vascular permeability) and increased the fraction of vessels with peri-

cytes.17 The net result was a more functional and normal vasculature,

with the potential for improved delivery and efficacy of chemothera-

peutic agents.18,19 In patients with malignant glioma, bevacizumab

and the pan-VEGFreceptor(VEGFR) tyrosine kinase inhibitor (TKI)

cediranib also exhibited features of vessel normalization, lead-

ing to reduced peritumoral edema and therapeutic effects of

both drugs.20,21 Normalization of the tumor vasculature may,

however, be transient and context dependent and has yet to beconfirmed inmany of thecommon humantumors.22 Insightson how

the tumor vasculature differs between cancers, the same cancer at

different stages of progression (eg, adjuvant vmetastatic setting), or

after different treatment regimensand, importantly, how these differ-

ences could influence vessel normalization after bevacizumab, are

thus still limited.23,24

Finally, clinical end points fail to accurately identify which pa-

tients benefit frombevacizumab. First, objective responserates do not

predict the magnitude of PFS or OS benefit from bevacizumab ther-

apyin some studiesof mCRC,25,26whereasother studiesobservedthat

bevacizumab induces clear improvements in PFS without increasing

objective response rates.27 Second, because patients may not partici-

pate if the experimental drug is not offered as either first-line or

second-line therapy, some studies include a crossover option on dis-

ease progression. This crossover rate may be high (for instance, up to

37% in the Avastin and Docetaxel in Metastatic Breast Cancer

[AVADO] trial) thereby also compromising OS as an end point for

biomarker studies with bevacizumab.11,12 Finally, distinguishing pa-

tient subgroups with differential effectsof bevacizumab is challenging

on the basis of PFS alone, since bevacizumab is effective only in

combination with chemotherapy, which by itself may also differen-

tially affect survival.

Although several of the issues we raised apply to targeted thera-

pies in general, differences in the tumor vasculature and limited in-

sights into how it reacts to VEGF inhibition are particularly relevant

for antiangiogenic therapies.28 Because of these numerous challenges,

a single biomarker may not suffice to reliably predict bevacizumab

treatment outcome across cancers. The following is a discussion of

various biomarkers that have been identified as predictors of bevaci-

zumab treatment outcome.

PLASMA MARKERS PREDICTIVE OF BEVACIZUMABTREATMENT OUTCOME

Many studies have measured circulating angiogenic factors (CAFs) to

predict outcome of bevacizumab treatment (Table 1). Most studies

included VEGF-A as an obvious candidate, because high levels of

VEGF-A could indicate VEGF-A dependency of the tumor vascula-

ture. Although increased tumor or plasma VEGF-A levels are well

established as indicators of poor prognosis, data related to the predic-

tive effect of pretreatment VEGF-A levels have largely been inconsis-

tent.42 In the E4599 study for NSCLC, response rates in patients with

high VEGF-A levels were significantly higher in the bevacizumab arm

than in the placebo arm.30 Single-arm studies involving cancer of thebreast, ovary, and endometrium observed similar correlations, but

manyother studiesfailed to observe such effect (Data Supplement). A

recent meta-analysis of1,816patientsparticipating inphase IIItrialsin

CRC, NSCLC, and RCC confirmed that pretreatment VEGF-A levels

serve as a prognostic rather than predictive marker (Table 1).31 In

contrast, pretreatment soluble VEGFR1 (sVEGFR1) levels inversely

correlated with outcome of either bevacizumab or anti-VEGFR

TKIs in at least five different trials. In particular, there was an

inverse correlation in patients with rectal cancer after bevaci-

zumab and chemoradiotherapy,32 BC after bevacizumab with

chemotherapy,33 hepatocellular carcinoma after cediranib,43

and mCRC after vandetanib.44

Conversely, baseline levels ofmost other CAFs failed to predict benefit after bevacizumab (Data

Supplement), although potentially interesting correlations were

observed for interleukin-8 (IL-8) and ANG-2 (Table 1).

Recent data suggest that a novel enzyme linked immunosorbent

assay (ELISA) with a preference to detect short VEGF-Aisoforms may

be more promising as a predictive marker. Through alternative RNA

splicing, several VEGF-A isoforms are generated, of which the short

VEGF-A121 isoform is freely diffusible, because it lacks basic amino

acid residues that bind the extracellular matrix (ECM). The longer

isoforms, consisting of 165, 189, or 206 amino acids, bind to heparin

and heparan sulfate proteoglycans in the ECM. Because of

differential affinities to the ECM, VEGF-A isoforms lay down a

spatial VEGF-A gradient, with VEGF-A121 diffusing over longdistances, VEGF-A165 reaching distant and nearby target cells,

and ECM-bound VEGF-A189 providing guidance cues over

short ranges.45 High circulating levels of short VEGF-A iso-

forms provide a more specific readout of the tumor-secreted

VEGF-A. Several phase III randomized trials revealed that patients

with mBC (AVADO trial)as well as patients with pancreatic cancer

(Avastin and Tarceva in Advanced Pancreatic Cancer [AViTA]

trial) who express high baseline levels of VEGF-A, as measured

with this novel ELISA, exhibit improved PFS and/or OS after

bevacizumab.13,37,38 Likewise, in the randomized Avastin in Ad-

vanced Gastric Cancer (AVAGAST) trial, for which plasma was

Lambrechts et al

2 2013 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

2013 from 128.118.88.48Information downloaded from jco.ascopubs.org and provided by at Penn State Hershey Medical Center on February 18,



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Table 1. Circulating Angiogenic Factors As Predictive Biomarkers for Bevacizumab Treatment Outcome

Protein Cancer Type ReferenceStudy

AcronymSample

Size Phase Study DetailsCorrelation With

Outcome

VEGF-A Colorectal Goede et al29 34 All patients receivedbevacizumab combined witheither FOLFIRI, FOLFOX,XELIRI, or XELOX

No (OR/PFS/OS)

Lung Leighl et al34 AVAiL 358 III Three arms, receiving either 0,

7.5, or 15 mg/kgbevacizumab, eachcombined with cisplatin-gemcitabine

No (PFS), no (OS)

Lung Mok et al35 BO21015 287 II All patients receivedbevacizumab carboplatin-paclitaxel or cisplatin-gemcitabine

Yes (PFS), trend(OR)

Lung Dowlati et al30 ECOG E4599 160 II/III Two arms, receiving eitherbevacizumab or placebo, eachcombined with carboplatin-paclitaxel

Yes (OR), no (OS)

Colorectal/lung/renal cell

Bernaards et al31 1,816 Different phase IIIclinical studies

See Jayson et al38 for detailson individual studies

No (PFS/OS)

Short VEGF-Aisoforms

Colorectal Jayson et al38 AVF2107g 398 III Two arms, receiving eitherbevacizumab or placebo,each combined withirinotecan fluorouracil

leucovorin

No (PFS/OS)

Lung Jayson et al38 AVAiL 859 III Three arms, receiving 0, 7.5,or 15 mg/kg bevacizumab,each combined withcisplatin-gemcitabine

Trend (PFS), no(OS)

Breast Miles et al37 AVADO 396 III Three arms, receiving 0, 7.5,or 15 mg/kg bevacizumab,each combined withdocetaxel

Yes (PFS/OS)

Pancreatic Van Cutsem et al36 AViTA 225 III Two arms, receiving eitherbevacizumab or placebo,each combined withgemcitabine-erlotinib

Trend (PFS), yes(OS)

Gastric Van Cutsem et al39 AVAGAST 712 III Two arms, receiving eitherbevacizumab or placebo,each combined withcapecitabine-cisplatin

Trend (PFS), yes(OS)

Renal cell Jayson et al38

AVOREN 404 III Two arms, receiving eitherbevacizumab or placebo, eachcombined with IFN-2a

No (PFS/OS)

sVEGFR1 Rectal Willett et al32 32 I/II Single arm, receiving fourcycles of therapy consistingof bevacizumab for eachcycle; fluorouracil in cycles 2to 4; external-beamirradiation and surgery aftertherapy

Yes (tumor stage)

Breast Tolaney et al33 104 Preoperative trial with a run-inof single-agent bevacizumabfollowed by ddACTchemotherapy

Yes (pathologicresponse)

IL-8 Hepatocellular Boige et al40 43 II All patients receivedbevacizumab

Yes (PFS/OS)

Colorectal Kopetz et al42 43 II All patients receivedbevacizumab FOLFIRI

Yes (PFS)

ANG2 Hepatocellular Kaseb et al41 40 II All patients receivedbevacizumab erlotinib

No (PFS), yes(OS)

Colorectal Goede et al29 34 All patients receivedbevacizumab combined witheither FOLFIRI, FOLFOX,XELIRI, or XELOX

Yes (OR/PFS/OS)

NOTE. All peer-reviewed publications available from PubMed and abstracts presented at international meetings were screened, but only a limited number of studiesare included in this table. Preference was given to studies assessing a large number of patients, studies including a placebo-controlled arm, and markers for whichconsistent findings were reported in several studies.

Abbreviations: ANG2,angiopoietin 2; AVADO, Avastin andDocetaxel in Metastatic BreastCancer; AVAGAST, Avastin in Advanced Gastric Cancer; AVAiL,Avastin in Lung Cancer;AViTA, Avastin and Tarceva in Advanced Pancreatic Cancer; AVOREN, Avastin and Roferon in Renal Cell Carcinoma; ddACT, dose-dense doxorubicin cyclophosphamidepaclitaxel;ECOG, Eastern CooperativeOncologyGroup; FOLFIRI, folinic acidfluorouracil irinotecan;FOLFOX,folinic acidfluorouraciloxaliplatin; IFN-2a, interferonalfa-2a;IL-8, interleukin-8; OR, objective response; OS, overall survival; PFS, progression-free survival; sVEGFR1, soluble vascular endothelial growth factor receptor 1; VEGF-A, vascularendothelial growth factor A; XELIRI, capecitabine irinotecan; XELOX, capecitabine oxaliplatin.

Biomarkers for Antiangiogenic Therapies

www.jco.org 2013 by American Society of Clinical Oncology 3




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available from 712 patients or 92% of the study population, pa-

tients with high baseline plasma VEGF-A levels exhibited im-

proved OS (hazard ratio [HR], 0.72) relative to patients with low

VEGF-A levels (HR, 1.01).39 In the randomized studies for CRC,

NSCLC, and RCC, no such correlation was observed (Table 1),

possibly because citrated plasma rather than EDTA plasma was

collected in these trials.38

Treatment-related changes in CAF might also predict

adaptive resistance to antiangiogenic therapies. Several studies

have shown acute increases in circulating VEGF-A levels on

delivery of bevacizumab.32,46,47 The magnitude of these changes

was proposed as a predictive marker, but findings have not been

consistent (Table 2). Because most of the circulating VEGF-A is

bound by bevacizumab and cannot be differentiated from

Table 2. Changes in Expression of Circulating Angiogenic Factors During Bevacizumab Treatment

Protein Cancer Type ReferenceStudy

AcronymSample

Size Phase Study DetailsChange During

Bevacizumab Treatment

VEGF-A Colorectal Willett et al32 NCI#5642 32 II All patients receivedbevacizumab fluorouracil

Increased at different timepoints after start ofbevacizumab treatment

Breast Baar et al46 49 II Two arms,docetaxel bevacizumab

Increased at weeks 17 to30 after start ofbevacizumab treatment;no increase inchemotherapy-only arm

Hepatocellular Boige et al40 43 II All patients received

bevacizumab as asingle agent

Decreased at day 3 after

start of bevacizumabtreatment

Melanoma Fuerstenberger et al49 SAKK 50/07 60 II All patients receivedbevacizumab temozolomide

Decreased at 2 weeksafter start ofbevacizumab treatment

Ovarian Smerdel et al50 38 All patients receivedbevacizumab

Decreased from cycle 2 to4 of bevacizumabtreatment

PlGF Colorectal Kopetz et al42 43 II All patients receivedbevacizumab FOLFIRI

Increased gradually untilprogression (weeks 2 to4 PD)

Loupakis et al52 25 II All patients receivedbevacizumab FOLFOXIRI

Increased at weeks 8 to24 after start ofbevacizumab treatment,then normalized at PD

Willett et al32 NCI #5642 32 II All patients receivedbevacizumab

fluorouracil

Increased at different timepoints after start of

bevacizumab treatmentVEGF-C Colorectal Lieu et al51 42 II All patients received

bevacizumab FOLFIRI

Increased prior to and atprogression

VEGF-D Colorectal Lieu et al51 42 II All patients receivedbevacizumab FOLFIRI

Increased at progression

bFGF Lung Dowlati et al30 ECOG E4599 160 II/III Two arms, receivingeitherbevacizumab orplacebo, eachcombined withcarboplatin-paclitaxel

Increased after cycle 2(similar increase inplacebo arm)

Colorectal Kopetz et al43 43 II All patients receivedbevacizumab FOLFIRI

Unchanged at weeks 2 to4 after start ofbevacizumab treatment,

but increased prior toand at progression

PDGF-BB Colorectal Kopetz et al42 43 II All patients receivedbevacizumab FOLFIRI

Unchanged, but increasedprior to and atprogression

SDF1 Colorectal Kopetz et al42 43 II All patients receivedbevacizumab FOLFIRI

Unchanged, but increasedprior to progression


Abbreviations: bFGF, basic fibroblast growth factor; ECOG, Eastern Cooperative Oncology Group; FOLFIRI, folinic acid fluorouracil irinotecan; FOLFOX, folinicacid fluorouracil oxaliplatin irinotecan; FOLFOXIRI, irinotecan oxaliplatin fluorouracil folinic acid; NCI, National Cancer Institute; PD, progressive disease;PDGF-BB, platelet-derived growth factor BB; PlGF, placental growth factor; SAKK, Schweizerische Arbeitsgemeinschaft fur Klinische Krebsforschung; SDF1, stromalcell-derived factor 1; VEGF-A, vascular endothelial growth factor A.

Lambrechts et al





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unbound VEGF-A, it is not clear how an increase in VEGF-A

expression could contribute to resistance. Conversely, bevaci-

zumab quite consistently led to persistent increases in PlGF

levels,32,48 with patients exhibiting a two-fold increase showing

improved clinical benefit.53 Interestingly, in a cediranib study,

levels of PlGF and basic FGF (bFGF) were also associated with

radiographic response or survival.53 A landmark study in

NSCLC measured 31 CAFs and investigated the relationship

between CAF changes and tumor shrinkage during treatment

with the anti-VEGFR TKI pazopanib.54 Pazopanib induced sig-

nificant changes in eight CAFs, of which changes in plasma

sVEGFR2 and IL-4 correlated significantly with tumor shrink-

age. Finally, several studies collecting plasma at disease progres-

sion under bevacizumab observed that levels of circulating

bFGF,48 PDGF-BB,48 VEGF-C,51 and VEGF-D51 increased on

progression. Overall, these data confirm that upregulation of

alternative proangiogenic signaling pathways may act as a

mechanism of evasive resistance against bevacizumab (Table 2;

Data Supplement) and may identify patients that develop adap-

tive resistance. A pending question is whether this compensa-

tory upregulation can efficiently and cost-effectively be used ina clinical setting.

CIRCULATING ENDOTHELIAL CELLS AND PROGENITOR CELLS

Most circulating endothelial cells (CECs) exhibit a mature phenotype

and represent apoptotic cells derived from the endothelial wall. A

subpopulation of CECsconsistsof circulatingendothelial progenitors

(CEPs)that are derived from bone marrow and exhibit a proliferative

potential.55 Tumor angiogenesis driven by VEGF-A depends at least

partly on the mobilization of CEPs, which integrate into growing

tumors and contribute to the formation of a functional vascular bed.

Increased concentrations of CEPs may reflect active tumor angiogen-esis and could serve as predictive markers for antiangiogenic thera-

pies.58 Preliminary data correlating changes in CEC levels with

responseto bevacizumabare conflicting (Data Supplement), probably

because of the different cancers analyzed and the various methodolo-

giesused. In particular,there is continuing debate about theideal CEC

marker that should be used for flow cytometry.55 In addition, chem-

otherapy backbones might differentially affect the tumor vasculature

and influence the fluctuations in CEC and CEP levels.55 Finally, the

number of samples analyzed was small, and data remain to be con-

firmed in larger studies.

PREDICTIVE MARKERS IN THE TUMOR ANDITS ENVIRONMENT

Several studies assessed expression of VEGF pathway genes in tumor

and stromal cells. VEGF-A expression on tumor cells was studied by

using immunohistochemistry, real-time polymerase chain reaction,

and in situ hybridization, but few positive correlations were observed

(Data Supplement). Because microvascular density correlates with

VEGF-A expression and serves as a surrogate marker of tumor angio-

genesis, it was also investigated. In the NO16966 randomized study, a

higher density of CD31 vessels was associated with greater benefit

frombevacizumab.These findings wereconfirmed in one study56 but

not in other smaller single-arm studies (Data Supplement). Other

studies assessed expression of the VEGFRs (including the co-receptor

neuropilin-1 [NRP1]), VEGF ligands, or other angiogenic factors

(bFGF,IL-8)by immunohistochemistry (DataSupplement). Interest-

ingly, in the mCRC Australian Gastro-Intestinal Trials Group testing

Mitomycin, Avastin, Xeloda (AGITG MAX), low baseline VEGFR1

expression correlated withimproved OS after bevacizumab (Table 3),

whereas in AVAGAST, low VEGFR1 correlated with improved PFS

(HR,0.67v0.89) butnot OS.39 Furthermore,in morethan 700 gastrictumors from AVAGAST, low baseline NRP1 expression correlated

with reduced OS in patients receiving placebo but with pro-

longed OS in patients receiving bevacizumab.60 In particular,

patients with low NRP1 showed improved OS (HR, 0.75) versus

patients with high NRP1 (HR, 1.07). In mBC and CRC, low

NRP1 had similar effects on disease progression (Table 3).

Notably, the latter studies involved large tumor sets, indicating

that low NRP1 expression represents one of the most consistent

and promising markers identified thus far.58,59

Emerging evidence also suggests that stromal cells play an

important role in mediating response to antiangiogenic therapies.

In particular, the presence of Gr-1

CD11b

myeloid cells rendersmurine tumors refractoryto antiVEGF-A therapy.61 Myeloid cells

provide a rich reserve of angiogenic molecules and possess potent

immunosuppressive activity, both of which favor tumor progres-

sion.62,63 Tumor-associated fibroblasts also upregulate PDGF-C

expression after delivery of a neutralizing VEGF-A antibody in

murine lymphoma models, thereby ensuring the continued forma-

tion of tumor vessels.64 In xenograft lung adenocarcinoma models,

gene expression changes associated with acquired resistance to

bevacizumab occur predominantly in stromal cells.65 In particular,

components of the epidermal growth factor receptor (EGFR) and

fibroblast growth factor receptor (FGFR) pathways were upregu-

lated. Few human studies have assessed expression changes in

tumor-associated cells during bevacizumab therapy. A seminalstudy in rectal cancer revealed that bevacizumab upregulates stro-

mal cellderived factor 1 (SDF-1), its receptor CXCR4, and

CXCL6 in tumor cells and also upregulates ANG-1 but downregu-

lates NRP1 in tumor-associated macrophages.66 Notably, similar

observations were reported for hepatocellular carcinoma treated

with sunitinib, an anti-VEGFR TKI.67 Overall, these studies high-

light the critical role of stromal cells in regulating resistance to

bevacizumab. It is less clear, however, how to evaluate the presence

of stromal cells or themarkers that they express in a routine clinical

setting during treatment.

IMAGING THE RESPONSE TO BEVACIZUMAB

Because VEGF-A blockade is believed to reduce tumor vascular per-

meability and perfusion, dynamic contrast-enhanced magnetic reso-

nance imaging (DCE-MRI), which monitors changes in vascular

structure and function, represents an attractive biomarker to assess

bevacizumab treatment response. The majority of DCE-MRI studies

performed so far involved phase I dose-escalation studies in patients

who had already received extensive treatment with chemotherapy or

in investigator-led trials that test a single or narrow dose range of

bevacizumab.68 Nevertheless, statistically significant changes in mi-

crovascular physiology, involving mainly reductions in the volume






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transfer constant Ktrans, were observed after bevacizumab mono-therapy in at least nine studies. Similar results have been reported in

studies involving sunitinib and pazopanib.69 Although these data are

clearly encouraging, additional research is needed. In particular, cur-

rent challenges involve standardization of DCE-MRI to enable its

application in a wider context, such as in multicenter clinical studies.

Whetherchangesintumor blood flowmeasuredby DCE-MRIpredict

outcome of bevacizumab in combination with chemotherapy still

needs to be assessed.

GENETIC SUSCEPTIBILITY AS A BIOMARKER FORBEVACIZUMAB OUTCOME

Unlike tumor cells, in whichgenes are mutated, deleted,or amplified,

tumor endothelial cells are genetically stable. The response of the

tumor vasculature to bevacizumab could therefore be considered a

host-mediated process influenced by genetic variability in the host

DNA. Several studies have meanwhile assessed whether single nucle-

otide polymorphisms (SNPs) in candidate genes predictbevacizumab

treatment outcome (Data Supplement).

In the mBC E2100trial, mutant carriersof thers699947 and

rs1570360 SNPs, which correlate with reduced expression of

VEGF-A,69 predicted favorable median OS in the bevacizumab

arm but not in the control arm.70 Surprisingly, neither SNP

predicted superior PFS for either arm. In the AVADO trial, it

was found that rs699947, but not rs1570360, correlated with

PFS in the placebo arm,71 whereas in patients with mCRC,

rs699947 and rs1570360 correlated with OS in the bevacizumab

arm.72 Findings for both SNPs are thus inconsistent. Severalother SNPs in VEGFA, including rs833061 or rs3025039, have

been proposed as predictive markers, but except for rs499946,

which is located near rs699947 and which was confirmed in a

meta-analysis of five studies,73 most SNPs have not been con-

firmed (Table 4). Various SNPs in other angiogenic factors have

been assessed, but only SNPs in VEGFR2, IL8, and CXCR2 were

replicated in at least one other study. In particular, the

rs2305948 SNP in VEGFR2, which encodes a Val273Ile substi-

tution that reduces binding of VEGF-A to VEGFR2 in mutant

carriers,75 has been correlated with reduced bevacizumab treat-

ment outcome.76 The mutant A allele of rs4073 in the IL8

Table 3. In Situ Biomarkers in Tumor or Stroma Predictive of Bevacizumab Treatment Outcome

MarkerCancer

Type ReferenceStudy

AcronymSample

Size Phase Study DetailsQuantification

Method

Association ofBiomarker With

Clinical Outcome

VEGFR1 Colorectal Foernzler et al59 NO16966 247 III 2 2 factorialdesign: XELOX vFOLFOX, andbevacizumab vplacebo

IHC on tumor No (PFS/OS)

Weickhardt et al57 AGITG MAX 268 III Three arms, receivingeitherbevacizumab,mitomycin, orplacebo, eachcombined withcapecitabine

IHC on tumor No (PFS), yes (OS);low VEGFR1increases benefitfrombevacizumab

Gastric Van Cutsemet al39

AVAGAST 763 III Two arms, receivingeither bevacizumabor placebo, eachcombined withcapecitabine-cisplatin

IHC on tumor Yes (PFS), no (OS)

NRP1 Colo rectal Foernzler et al59 NO16966 247 III 2 2 factorialdesign: XELOX vFOLFOX, andbevacizumab v

placebo

IHC on tumor Low NRP1increases benefitfrombevacizumab

Gastric Van Cutsemet al39

AVAGAST 763 III Two arms, receivingeither bevacizumabor placebo, eachcombined withcapecitabine-cisplatin

IHC on tumor Low NRP1 isnegativeprognostic andpositivepredictive for OS

Breast Jubb et al58 AVF2119g 223 III Two arms, receivingeither bevacizumabor placebo, eachcombined withcapecitabine

IHC on tumor Trend towardimproved PFS inlow NRP1-expressingpatients


Abbreviations: AGITG MAX, Australasian Gastro-Intestinal Trials Group Mitomycin, Avastin, Xeloda trial; AVAGAST, Avastin in Advanced Gastric Cancer; FOLFOX,folinic acid fluorouracil oxaliplatin; IHC, immunohistochemistry; N/R, not reported; NRP1, neuropilin-1; OS, overall survival; PFS, progression-free survival;

VEGFR1, vascular endothelial growth factor receptor 1; XELOX, capecitabine oxaliplatin.

Lambrechts et al





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Table 4. Genetic Markers Evaluated As Predictive Biomarkers for Bevacizumab Treatment Outcome

GeneCancer

Type Reference Study Acronym Sample Size Phase Study Details Genetic VariantAssociation WithClinical Outcome

VEGFA Colorectal Loupakiset al74

218 (111 withbevacizumab)

All patients receivedFOLFIRIbevacizumab

2578A/C (rs699947), 460C/T(rs833061), 634G/C(rs2010963), 936C/T(rs3025039)

460C increases PFS/OS in bevacizumabpatients

Hansen

et al83

Nordic ACT 218 III Two arms receiving either

FOLFOX/XELOX orFOLFIRI/XELIRIbevacizumab

Five SNPs (not further

specified)

No (OR)

Koutraset al72

209 III All patients receivedbevacizumab FOLFIRI or XELIRI

634G/C (rs2010936), 936C/T(rs3025039), 1154G/A(rs1570360), 2578A/C(rs699947)

2578A and 1154Aincrease OS butnot PFS

Gergeret al76

119 All patients receivedFOLFOX/XELOX bevacizumab

634G/C (rs2010936), 936C/T(rs3025039), 1154G/A(rs1570360), -460C/T(rs833061), 2578A/C(rs699947)

No (response/PFS/OS)

Lung Zhanget al81

ECOG 4599 133 (66 withbevacizumab)

II/III Two arms, receivingeither bevacizumab orplacebo, each combinedwith carboplatin-paclitaxel

634G/C -634G/C correlateswith PFS inbevacizumabpatients

Breast Schneideret al70


III Two arms, receivingpaclitaxelbevacizumab


2578A and 1154Aincrease OS butnot PFS inbevacizumabpatients

Mileset al71

AVADO 336 (231 withbevacizumab)

III Three arms, receivingeither 0, 7.5, or 15 mg/kg bevacizumab, eachcombined withdocetaxel


2578C increasesPFS in placeboarm; 1154Aincreases PFS inbevacizumab arm(trend)

Various Lambrechtset al73

NO16966, AVAiL,AViTA,AVOREN,AVADO

1,348 (669 withbevacizumab)

Various regimens (seeEscudier et al, 5, Mileset al,12 and Saltzet al 27 for details)

158 SNPs in VEGF pathway rs699946-A allelecorrelates withimproved PFS inbevacizumabpatients

VEGFR1 Pancreatic Lambrechtset al82

AViTA 154 (77 withbevacizumab)

III Two arms, receivinggemcitabine-erlotinibbevacizumab

158 SNPs in VEGF pathway rs9582036-A alleleincreases PFS andOS in bevacizumab

arm but not inplacebo arm

Colorectal Hansenet al83

Nordic ACT 218 III Two arms receiving eitherFOLFOX/XELOX orFOLFIRI/XELIRIbevacizumab

rs9582036 ( intronic) rs9582036-A alleleincreases RR tobevacizumab

Breast Mileset al71

AVADO 336 (231 withbevacizumab)

III Three arms, receivingeither 0, 7.5, or 15 mg/kg bevacizumabcombined withdocetaxel

rs9554316, rs9582036 (bothintronic)

No (PFS/OS)

VEGFR2 Colorectal Gergeret al76


889G/A Yes (RR), G alleleincreases RR

Breast Schneideret al70


III Two arms, receivingpaclitaxelbevacizumab

889G/A, 1416A/T No (PFS/OS)

Various Lambrechtset al73

NO16966, AVAiL,AViTA,AVOREN,AVADO

1,348 (669 withbevacizumab)

Various regimens (seeEscudier et al, 5, Mileset al,12 and Saltz et al27 for details)

158 SNPs in VEGF pathway rs11133360-T allelecorrelates withimproved PFS inbevacizumabpatients

IL8 Colorectal Giudiceet al78

35 All patients receivedchemotherapy (notspecified) bevacizumab

251T/A TT carriers haveimproved OR

(continued on following page)






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promoter, which correlates with increased IL-8 production af-

ter stimulation with lipopolysaccharide,77 is associated with

poor response to bevacizumab and pazopanib.78-80 Finally,

rs2230054 in CXCR2, which could affect splicing of CXCR2,76

has been correlated with reduced PFS in at least three indepen-

dent studies.76,79,81

In nearly all studies, limited numbers of SNPs were selected

on the basis of candidate gene approaches. As a result, selected

SNPs differ between studies, leading to heterogeneous data sets

and few possibilities for assessing data consistency. Recently,

the first approaches to systematically cover SNPs in all genes of

the VEGF pathway were reported.82 In particular, up to 158

SNPs located in 14 genes were assessed in the AViTA random-

ized pancreatic cancer trial. Four SNPs in VEGFR1 correlated

highly significantly with both PFS and OS in the bevacizumab

arm (per allele HR, 2.1). No effect was observed in the placebo

arm. Fine mapping of this locus identified rs7993418, a synon-

ymous SNP affecting tyrosine 1213 in the tyrosine kinase do-

main of VEGFR1, as the functional variant underlying the

association.82 Mutant carriers of the rs7993418 allele increased

VEGFR1 expression by almost 20% and exhibited worse out-

come after bevacizumab, thereby confirming previous observa-tions for VEGFR1 expression in plasma or tumors. Intriguingly,

this association was also replicated in patients with RCC,

NSCLC, and CRC but not in those with BC.71,72

Overall, although interesting SNPs have been identified, several

questions remain. First, interesting SNPs still need to be replicated in

as many trials as possible. Second, with the exception of the VEGFR1

locus, predictive effects of most individual SNPs have been rather

modest,raising thequestion of whetherSNPsare sufficientlyinforma-

tive to assist with patient selection. One possible way to increase their

predictive effect would be to consolidate effects of individual SNPs

into a combined predictive score.

CONCLUDING REMARKS AND FUTURE DIRECTIONS

The most promising markers for bevacizumab treatment outcome

consist of circulating short VEGF-A isoforms and modified expres-

sion of VEGFRs (VEGFR1 or NRP1), either in plasma or tumors.

Although there is abundant functional evidence that these markers

could indeed determine bevacizumab outcome (Note 1 and Fig 1),

none of them has consistently been replicated across different

studies involving various cancer types. Perhaps this is not surpris-ing, since biomarkers for bevacizumab may differ between cancer

types. The question is whether additional biomarker discovery to

more consistently predict bevacizumab treatment outcome across

cancer types is still needed or whether prospective translation of

existing markers into clinical practice should be considered

a priority.

Ideally, all biomarkers that most consistently replicate in a

particular cancer type should be considered for prospective valida-

tion. Currently, this strategy is not feasible because there are too

few large-scale biomarker studies in the same cancer that assess the

same panel of markers. Therefore, future studies should continue

to focus on the discovery of novel biomarkers in different tissue

types (plasma, DNA, tumor, and so on) and should not be limited

to testing one or two markers in plasma or genotyping only a few

genetic variants. Instead, they should include homogeneous sets

of candidate markers to allow for comparison between studies.

Furthermore, since it is becoming obvious that single biomark-

ers may not be sufficient to predict the complex phenotype of

response to bevacizumab, studies should integrate the individ-

ual effects of these markers by using advanced statistical analy-

ses and combine them into a general predictive score. Possibly,

such integrated analyses will generate more robust predictions

that are valid across cancers. Markers in plasma, DNA, or tumor

tissue might even have to be combined to obtain accurate

Table 4. Genetic Markers Evaluated As Predictive Biomarkers for Bevacizumab Treatment Outcome

GeneCancer

Type Reference Study Acronym Sample Size Phase Study Details Genetic VariantAssociation WithClinical Outcome

Ovarian Schultheiset al79

PH-II-45 53 II All patients receivedcyclophosphamide bevacizumab

251T/A TT carriers haveincreased RR

CXCR2 Lung Zhanget al81


II/III Two arms, receivingeither bevacizumab or

placebo, each combinedwith carboplatin-paclitaxel

785C/T Yes (PFS inbevacizumab

patients)

Ovarian Schultheiset al79

PH-II-45 53 II All patients receivedcyclophosphamide bevacizumab

785C/T Yes (PFS), C alleleincreases PFS

Colorectal Gergeret al76


785C/T Yes (RR), C alleleincreases RR


Abbreviations: ACT, doxorubicin cyclophosphamide paclitaxel; AVADO, Avastin and Docetaxel in Metastatic Breast Cancer; AVAiL, Avastin in Lung Cancer;AViTA, Avastin and Tarceva in Advanced Pancreatic Cancer; AVOREN, Avastin and Roferon in Renal Cell Carcinoma; CXCR2, chemokine receptor 2; ECOG,Eastern Cooperative Oncology Group; FOLFIRI, folinic acid fluorouracil irinotecan; FOLFOX, folinic acid fluorouracil oxaliplatin; IL8, interleukin-8; OR,objective response; OS, overall survival; PFS, progression-free survival; RR, response rate; SNP, single nucleotide polymorphism; VEGF, vascular endothelialgrowth factor; VEGFA, vascular endothelial growth factor A; VEGFR1, VEGF receptor 1; VEGFR2, VEGF receptor 2; XELIRI, capecitabine irinotecan; XELOX,capecitabine oxaliplatin.

Lambrechts et al





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predictive models at the risk of compromising their clini-

cal applicability.

Obviously, it will remain challenging to discover which set of

individual markers has the highest sensitivity and specificity to

predict outcome of treatment with bevacizumab. A potential solu-

tion might be to explore biomarkers in relation to vascular re-

sponses determined by DCE-MRI as an alternative end point of

treatment response. Another possibility is to focus on cancers in

which chemotherapy does not significantly contribute to the out-

come of bevacizumab (eg, RCC, which is highly dependent on

VEGF for disease progression) or in which bevacizumab is given as

monotherapy. One could also focus on identifying markers for

anti-VEGFR TKIs that are delivered as monotherapies and for

which objective responses can be directly attributed to the TKI.

Most TKIs are not limited to inhibiting VEGFRs, and predictive

markers maytherefore only partlyoverlap with those predictive for

bevacizumab outcome. Finally, one could consider screening

for markers in studies that assess bevacizumab for the trea tment

of age-related macular degeneration.84 In the latter, VEGF-A

inhibition counteracts the excessive growth of leaky blood ves-

sels leading to prevention and reversal of vision loss. Because

neoangiogenesis in age-related macular degeneration differs

from tumor angiogenesis, only a limited number of markers

might overlap. International collaborative efforts will soon re-

lease the mutation and methylation profiles of thousands of

tumors allowing the identification of novel molecular subtypes.

VEGFR2/NRP1

complex

Short VEGF-isoform

(VEGF121)

VEGF-isoform 165

(VEGF165)

Long VEGF-isoform

(VEGF189)

Circulating angiogenic factors

(IL-8, PDGF-C, VEGF-C, VEGF-D,

bFGF, etc.)

Transmembrane VEGFR1

Soluble VEGFR1

(sVEGFR1)

Macrophage,

myeloid cell

Tumor cell

Endothelial cell

Pericyte

Leaky tumorvasculature

Recruitment ofbone marrowderived

myeloid cells

Resistance to Bevacizumab

Response to Bevacizumab

Short VEGFisoformdriven chaotic tumorvasculature

Diffusion of shortVEGF-isoforms

High NRP1expression

A

B

Long VEGFisoform

driven tumorvasculature

Compensation byproangiogenic

factors

Pericytecoverage

Well-perfusedtumor vasculature

High VEGFR1 andsVEGFR1 expression

Limited diffusion oflong VEGF isoforms

Macrophageand myeloid

cell infiltration

Low NRP1expression

Low VEGFR1 andsVEGFR1 expression

Fig 1. (A) Characteristics of a tumor respon-

sive to bevacizumab based on current bio-

marker data. High expression of the short

vascular endothelial growth factor A (VEGF-A)

isoform VEGF121 leads to a chaotic vessel

structure (highly irregular diameter and very

leaky), naked tumor vessels (no pericyte cov-

erage), and a low perfusion index. Expression

of VEGFR1 on tumors, vessels, and macro-

phages is low. Soluble VEGFR1 (sVEGFR1)

expression in plasma is low. Neuropilin-1

(NRP1) expression is low on tumor cells.

There are few stromal cells, such as macro-

phages and fibroblasts, that secrete VEGF-A

present in the tumor. (B) Characteristics of atumor resistant to bevacizumab based on cur-

rent biomarker data. High expression of long

VEGF-A isoforms (VEGF165 and VEGF189)

leads to a less chaotic vessel structure (nor-

mal diameter and not leaky) and to tumor

vessels that are covered with some pericytes

andhave a high perfusionindex. Expression of

VEGFR1 on tumors, vessels, and macro-

phages is high. sVEGFR1 expression in

plasma is high. Expression of NRP1 on tumor

cells is also high. There aremany stromal cells

in the tumor, including macrophages and fi-

broblasts. These will secrete various other

angiogenic molecules, including interleukin-8

(IL-8), basic fibroblast growth factor (bFGF),

platelet-derived growth factor BB (PDGF-BB),

PDGF-C, VEGF-C, and VEGF-D.






10/13

Because these novel subtypes canindirectly modulate tumor angiogen-

esis, it will be necessary to assess them as markers of treatment

outcome. Clinical trials in BC are among the first to take molecular

subtyping into accountBEATRICE is restricted to triple-

negative BCwhereas the AVADO and AVEREL trials are limited

to human epidermal growth factor receptor (HER)

negative and HER-positive patients, respectively. In other cancers,

some initial subtyping has retrospectively been performed, for

instance, by genotyping KRAS and BRAFmutations in CRC, but ithas failed to reveal any differential effects.85 Many other subtypes,

such as microsatellite instability or hypermethylator phenotypes,

have largely been neglected and will need to be assessed in

the future.

Conversely, there might already be sufficient evidence to pro-

spectively validate existing biomarkers. In particular, markers that

have been replicated in several different cancers are eligible. The

short VEGF-A isoforms are an example of such biomarkers, be-

cause they have been correlated with outcome in three large ran-

domized studies, but they failed to replicate in a few other studies

(possibly because plasma was not appropriately collected). The

phase III MERiDiAN trial in mBC (opening in 2012) will evalu-ate the impact of bevacizumab in patients stratified for plasma

short VEGF-A isoforms. This trial represents the first prospec-

tive validation of a biomarker for bevacizumab and, if positive,

MERiDiAN will result in the clinical application of short

VEGF-A isoforms as a biomarker for bevacizumab in mBC. In

addition, MERiDiAN will indicate whether prospective valida-

tion of other markers replicated in several studies is meaningful

(eg, VEGFR1 or NRP1 expression in mCRC or gastric cancer).

Meanwhile, in a commitment to discover and validate other bio-

marker candidates, extensive biomarker programs should con-

tinue in several indications. As long as these markers have not

prospectively been validated, the available clinical trial data pro-

vide the most compelling evidence for prescribing bevacizumab.

AUTHORS DISCLOSURES OF POTENTIAL CONFLICTSOF INTEREST

Altho ugh all autho rs completed the disclosure declaratio n, the follow ingauthor(s) and/or an authors immediate family member(s) indicated a

financial or other interest that is r elevant to t he subje ct matter underconsideration in this article. Certain relationships marked with a Uare those for which no compensation was received; those relationships

marked with a C were compensated. For a detailed description of thedisclosure categories, or for more information about ASCOs conflict ofinterest policy, please refer to the Author Disclosure Declaration and theDisclosures of Potential Conflicts of Interest section in Information

for Contributo rs.Employment or Leadership Position: Sanne de Haas, F.Hoffmann-La Roche (C); Stefan J. Scherer, F. Hoffmann-La Roche(C) Consultant or Advisory Role: Heinz-Josef Lenz, Bayer (C),Genentech/Roche (C), Merck (C), sanofi-aventis (C); PeterCarmeliet, F. Hoffmann-La Roche (C) Stock Ownership: NoneHonoraria: Diether Lambrechts, F. Hoffman-La Roche; Heinz-JosefLenz, Bayer, Genentech/Roche, Merck, sanofi-aventis; PeterCarmeliet, Roche Research Funding: Diether Lambrechts, F.Hoffman-La Roche; Heinz-Josef Lenz, Bayer, Genentech/Roche, F.

Hoffmann-La Roche; Peter Carmeliet, F. Hoffmann-La Roche ExpertTestimony: None Other Remuneration: None Other: DietherLambrechts, patent EP 10744513.2 (publication No. EP 2462242)(U), patent US 13/388,840 (publication No. US 2012-0195858) (U);Sanne de Haas, patent EP 10744513.2 (publication No. EP 2462242)(U), patent US 13/388,840 (publication No. US 2012-0195858) (U);Peter Carmeliet, patent EP 10744513.2 (publication No. EP 2462242)(U), patent US 13/388,840 (publication No. US 2012-0195858) (U);Stefan J. Scherer, patent EP 10744513.2 (publication No. EP 2462242)(U), patent US 13/388,840 (publication No. US 2012-0195858) (U)

AUTHOR CONTRIBUTIONS

Conception and design: Diether Lambrechts, Sanne de Haas, Peter

Carmeliet, Stefan J. Scherer

PRECLINICAL EVIDENCE SUPPORTING PROMISING BIOMARKERS FOR BEVACIZUMAB

High Plasma Levels of Short VEGF-A Isoforms

Because total body production of VEGF-A eclipses VEGF-A production from tumors,86 plasma VEGF-A is unlikely to provide a

sensitive index of tumor-secreted VEGF-A. A modified ELISA that favors detection of short VEGF-A isoforms might, because

these isoforms diffuse over long distances, be more sensitive in detecting vascular dependence of the tumor.

Reduced VEGFR1 Expression in Plasma or Tumors

VEGFR1 triggers angiogenesis either directly by transmitting intracellular signals or indirectly by inducing trans-phosphorylation

of VEGFR2.

87

VEGFR1 expression is upregulated in tumors in which it contributes to tumor survival. A neutralizing PlGF antibody suppresses angiogenesis and recruitment of inflammatory cells in tumor models.88

HighsVEGFR1 levelssequestertumor-derived VEGF-A, thereby limiting thebenefits of VEGF-Aneutralization through bevacizumab.89

Aflibercept, a fusion protein composed of VEGFR1 and VEGFR2 ligand-binding components fused to the fragment crystallizable

portion of human immunoglobulin G1 (IgG1), inhibits all VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) and prolongs PFS in

second-line mCRC.90 Importantly, approximately 30% of patients received prior bevacizumab, thereby illustrating the clinical

relevance of VEGFR1 signaling in the context of VEGF inhibition.

Low NRP1 Expression in Tumors

Antibodies blocking the binding between VEGF-A and NRP1 slow tumor growth in mice.91

A combination of anti-NRP1 and antiVEGF-A antibodies enhances tumor growth and vascular density reduction in mice.92

VEGF-A stimulates cancer stemness and renewal through NRP1.93

Lambrechts et al





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Collection and assembly of data: Diether Lambrechts, Heinz-Josef Lenz,Sanne de Haas, Stefan J. SchererData analysis and interpretation: All authors

Manuscript writing: All authors

Final approval of manuscript: All authors

REFERENCES

1. Carmeliet P, Jain RK: Molecular mechanisms

and clinical applications of angiogenesis. Nature

473:298-307, 2011

2. Hurwitz H, Fehrenbacher L, Novotny W, et al:

Bevacizumab plus irinotecan, fluorouracil, and leuco-

vorin for metastatic colorectal cancer. N Engl J Med

350:2335-2342, 2004

3. S an dl er A , Gr ay R , P er ry MC , e t a l:

Paclitaxel-carboplatin alone or with bevacizumab

for non-small-cell lung cancer. N Engl J Med 355:

2542-2550, 2006

4. Escudier B, Bellmunt J, Negrier S, et al: Phase

III trial of bevacizumab plus interferon alfa-2a in

patients with metastatic renal cell carcinoma

(AVOREN): Final analysis of overall survival. J Clin

Oncol 28:2144-2150, 2010

5. Escudier B, Pluzanska A, Koralewski P, et al:

Bevacizumab plus interferon alfa-2a for treatment ofmetastatic renal cell carcinoma: A randomised, double-

blind phase III trial. Lancet 370:2103-2111, 2007

6. Yang JC, Haworth L, Sherry RM, et al: A

randomized trial of bevacizumab, an anti-vascular

endothelial growth factor antibody, for metastatic

renal cancer. N Engl J Med 349:427-434, 2003

7. Burger RA, Brady MF, Bookman MA, et al: Incor-

poration of bevacizumab in the primary treatment of

ovarian cancer. N Engl J Med 365:2473-2483, 2011

8. Perren TJ, Swart AM, Pfisterer J, et al: A

phase 3 trial of bevacizumab in ovarian cancer.

N Engl J Med 365:2484-2496, 2011

9. Kreisl TN, Kim L, Moore K, et al: Phase II trial

of single-agent bevacizumab followed by bevaci-

zumab plus irinotecan at tumor progression in recur-

rent glioblastoma. J Clin Oncol 27:740-745, 200910. Miller K, Wang M, Gralow J, et al: Paclitaxel plus

bevacizumab versus paclitaxel alone for metastatic

breast cancer. N Engl J Med 357:2666-2676, 2007

11. Robert NJ, Dieras V, Glaspy J, et al: RIBBON-1:

Randomized, double-blind, placebo-controlled, phase

III trial of chemotherapy with or without bevacizumab

for first-line treatment of human epidermal growth

factor receptor 2-negative, locally recurrent or meta-

static breast cancer. J Clin Oncol 29:1252-1260, 2011

12. Miles DW, Chan A, Dirix LY, et al: Phase III study

of bevacizumab plus docetaxel compared with placebo

plus docetaxel for the first-line treatment of human

epidermal growth factor receptor 2-negative metastatic

breast cancer. J Clin Oncol 28:3239-3247, 2010

13. Rugo HS: Inhibiting angiogenesis in breast

cancer: The beginning of the end or the end of thebeginning? J Clin Oncol 30:898-901, 2012

14. Potti A, Schilsky RL, Nevins JR: Refocusing

the war on cancer: The critical role of personalized

treatment. Sci Transl Med 2:28cm13, 2010

15. Potente M, Gerhardt H, Carmeliet P: Basic

and therapeutic aspects of angiogenesis. Cell 146:

873-887, 2011

16. Ellis LM, Hicklin DJ: VEGF-targeted therapy:

Mechanisms of anti-tumour activity. Nat Rev Cancer

8:579-591, 2008

17. Willett CG, Boucher Y, di Tomaso E, et al:

Direct evidence that the VEGF-specific antibody

bevacizumab has antivascular effects in human rec-

tal cancer. Nat Med 10:145-147, 2004

18. Muggia F, Safra T, Borgato L, et al: Pharmacoki-

netics (PK) of pegylated liposomal doxorubicin (PLD)

given alone and with bevacizumab (B) in patients with

recurrent epithelial ovarian cancer (rEOC). J Clin Oncol

28:406s, 2010 (suppl; abstr 5064)

19. Jain RK: Normalizing tumor vasculature withanti-angiogenic therapy: A new paradigm for combi-

nation therapy. Nat Med 7:987-989, 2001

20. Batchelor TT, Sorensen AG, di Tomaso E, et al:

AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor,

normalizes tumor vasculature and alleviates edema in

glioblastoma patients. Cancer Cell 11:83-95, 2007

21. Fischer I, Cunliffe CH, Bollo RJ, et al: High-

grade glioma before and after treatment with radia-

tion and Avastin: Initial observations. Neuro Oncol

10:700-708, 2008

22. Goel S, Duda DG, Xu L, et al: Normalization of

the vasculature for treatment of cancer and other

diseases. Physiol Rev 91:1071-1121, 2011

23. Van Cutsem E, Lambrechts D, Prenen H, et al:

Lessons from the adjuvant bevacizumab trial on

colon cancer: What next? J Clin Oncol 29:1-4, 201124. Carmeliet P, Jain RK: Principles and mechanisms

of vessel normalization for cancer and other angiogenic

diseases. Nat Rev Drug Discov 10:417-427, 2011

25. Grothey A, Hedrick EE, Mass RD, et al:

Response-independent survival benefit in meta-

static colorectal cancer: A comparative analysis of

N9741 and AVF2107. J Clin Oncol 26:183-189, 2008

26. Chun YS, Vauthey JN, Boonsirikamchai P, et

al: Association of computed tomography morpho-

logic criteria with pathologic response and survival in

patients treated with bevacizumab for colorectal

liver metastases. JAMA 302:2338-2344, 2009

27. Saltz LB, Clarke S, Daz-Rubio E, et al: Bevaci-

zumab in combination with oxaliplatin-based chem-

otherapy as first-line therapy in metastatic colorectal

cancer: A randomized phase III study. J Clin Oncol26:2013-2019, 2008

28. Bottsford-Miller JN, Coleman RL, Sood AK:

Resistance and escape from antiangiogenesis ther-

apy: Clinical implications and future strategies. J Clin

Oncol 30:4026-4034, 2012

29. Goede V, Coutelle O, Neuneier J, et al: Iden-

tification of serum angiopoietin-2 as a biomarker for

clinical outcome of colorectal cancer patients

treated with bevacizumab-containing therapy. Br J

Cancer 103:1407-1414, 2010

30. Dowlati A, Gray R, Sandler AB, et al: Cell adhesion

molecules, vascular endothelial growth factor, and basic

fibroblast growth factor in patients with non-small cell

lung cancer treated with chemotherapy with or without

bevacizumab: An Eastern Cooperative Oncology Group

Study. Clin Cancer Res 14:1407-1412, 2008

31. Hegde PS, Jubb AM, Chen D, et al: Predictive

impact of circulating vascular endothelial growth factor in

4 phase IIItrials evaluating bevacizumab.Clin CancerRes

[epub ahead of print on November 20, 2012]

32. Willett CG, Duda DG, di Tomaso E, et al:

Efficacy, safety, and biomarkers of neoadjuvant be-

vacizumab, radiation therapy, and fluorouracil in rec-

tal cancer: A multidisciplinary phase II study. J Clin

Oncol 27:3020-3026, 2009

33. Tolaney SM, Duda DG, Boucher Y, et al: A

phase II study of preoperative (preop) bevacizumab

(bev) followed by dose-dense (dd) doxorubicin (A)/

cyclophosphamide (C)/paclitaxel (T) in combination

with bev in HER2-negative operable breast cancer

(BC). J Clin Oncol 30:55s, 2012 (suppl; abstr 1026)

34. Leighl N, Reck M, de Haas S, et al: Analysis of

biomarkers (BMs) in the AVAiL phase III randomised

study of first-line Bevacizumab (Bv) with cisplatin-

gemcitabine (CG) in patients (pts) with non-smallcell lung

cancer (NSCLC). Eur J Cancer 7:558, 2009 (abstr 9172)

35. Mok T, Gorbunova V, Juhasz E, et al: Biomarkeranalysis in BO21015, a phase II randomised study

of first-line bevacizumab (BEV) combined with

carboplatin-gemcitabine (CG) or carboplatin-paclitaxel

(CP) in patients (pts) with advanced or recurrent non-

squamous non-small cell lung cancer (NSCLC). Eur J

Cancer 47:S592, 2011 (suppl 1; abstr 9003)

36. Van Cutsem E, Jayson G, Dive C, et al:

Analysis of blood plasma factors in the AVITA phase

III randomized study of bevacizumab (bev) with

gemcitabine-erlotinib (GE) in patients (pts) with met-

astatic pancreatic cancer (mPC). Eur J Cancer 47:

S95-S96, 2011 (suppl 1; abstr 803)

37. Miles DW, de Haas SL, Dirix L, et al: Plasma

biomarker analyses in the AVADO phase III random-

ized study of first-line bevacizumab docetaxel in

patients with human epidermal growth factor recep-tor (HER) 2-negative metastatic breast cancer. Can-

cer Res 70, 2010 (suppl 2; abstr P2-16-04)

38. Jayson GC, de Haas S, Delmar P, et al: Evaluation

of plasma VEGF-A as a potential predictive pan-tumour

biomarker for bevacizumab. Proceedings of the 2011

European Multidisciplinary Cancer Congress, Stockholm,

Sweden, September 23-27, 2011 (abstr 804)

39. Van Cutsem E, de Haas S, Kang YK, et al:

Bevacizumab in combination with chemotherapy as

first-line therapy in advanced gastric cancer: A bio-

marker evaluation from the AVAGAST randomized

phase III trial. J Clin Oncol 30:2119-2127, 2012

40. Boige V, Malka D, Bourredjem A, et al: Efficacy,

safety, and biomarkers of single-agent bevacizumab ther-

apy in patients with advanced hepatocellular carcinoma.

Oncologist 17:1063-1072, 201241. Kaseb AO, Garrett-Mayer E, Morris JS, et al:

Efficacy of bevacizumab plus erlotinib for advanced hep-

atocellular carcinoma and predictors of outcome: Final

results of a phase II trial. Oncology 82:67-74, 2012

42. KopetzS, HoffPM,Morris JS, etal: Phase IItrial of

infusional fluorouracil, irinotecan, and bevacizumab for

metastatic colorectal cancer: Efficacy and circulating an-

giogenic biomarkers associated with therapeutic resis-

tance. J Clin Oncol 28:453-459, 2010

43. Zhu AX, Ancukiewicz M, Supko JG, et al: Clinical,

pharmacodynamic (PD), and pharmacokinetic (PK) evalu-

ation of cediranib in advanced hepatocellular carcinoma

(HCC): A phase II study (CTEP 7147). J Clin Oncol

30:266s, 2012 (suppl; abstr 4112)

44. Meyerhardt JA,AncukiewiczM, AbramsTA, et al:

Phase I study of cetuximab, irinotecan, and vandetanib

(ZD6474) as therapy for patients with previously treated

metastastic colorectalcancer. PLoS One 7:e38231, 2012

45. Ruiz de AlmodovarC, Lambrechts D, Mazzone M,

et al: Role and therapeutic potential of VEGF in the

nervous system. Physiol Rev 89:607-648, 2009

46. Baar J, Silverman P, Lyons J, et al: A

vasculature-targeting regimen of preoperative do-

cetaxel with or without bevacizumab for locally

advanced breast cancer: Impact on angiogenic bio-

markers. Clin Cancer Res 15:3583-3590, 2009

47. Horn L, Dahlberg SE, Sandler AB, et al: Phase

II study of cisplatin plus etoposide and bevacizumab

for previously untreated, extensive-stage small-cell

lung cancer: Eastern Cooperative Oncology Group

Study E3501. J Clin Oncol 27:6006-6011, 2009






12/13

48. Buysschaert I, Schmidt T, Roncal C, et al: Genet-

ics, epigenetics and pharmaco-(epi)genomics in angio-

genesis. J Cell Mol Med 12:2533-2551, 2008

49. Fuerstenberger G, Boneberg E, Simcock M,

et al: Predictive and prognostic potential of angio-

genic serum factors and circulating endothelial cells

in metastatic melanoma patients receiving temozo-

lamide plus bevacizumab (SAKK 50/07). J Clin Oncol

28:632s, 2010 (suppl; abstr 8585)

50. Smerdel MP, Steffensen KD, Waldstrm M,

et al: The predictive value of serum VEGF in multi-

resistant ovarian cancer patients treated with bev-

acizumab. Gynecol Oncol 118:167-171, 2010

51. Lieu CH, Tran HT, Jiang Z, et al: The associa-

tion of alternate VEGF ligands with resistance to

anti-VEGF therapy in metastatic colorectal cancer.

J Clin Oncol 29:229s, 2011 (suppl; abstr 3533)

52. Loupakis F, Cremolini C, Fioravanti A, et al: Phar-

macodynamic and pharmacogenetic angiogenesis-

related markers of first-line FOLFOXIRI plus

bevacizumab schedule in metastatic colorectal cancer.

Br J Cancer 104:1262-1269, 2011

53. Batchelor TT, Duda DG, di Tomaso E, et al:

Phase II study of cediranib, an oral pan-vascular

endothelial growth factor receptor tyrosine kinase

inhibitor, in patients with recurrent glioblastoma.

J Clin Oncol 28:2817-2823, 201054. Nikolinakos PG, Altorki N, Yankelevitz D, et al:

Plasma cytokine and angiogenic factor profiling identifies

markers associated with tumor shrinkage in early-stage

non-small cell lung cancer patients treated with pazo-

panib. Cancer Res 70:2171-2179, 2010

55. Bertolini F, Shaked Y, Mancuso P, et al: The

multifaceted circulating endothelial cell in cancer:

Towards marker and target identification. Nat Rev

Cancer 6:835-845, 2006

56. Pohl A, El-Khoueiry A, Yang D, et al: Pharma-

cogenetic profiling of CD133 is associated with

response rate (RR) and progression-free survival

(PFS) in patients with metastatic colorectal cancer

(mCRC), treated with bevacizumab-based chemo-

therapy. Pharmacogenomics J 10.1038/tpj.2011.61

[epub ahead of print on January 10, 2012]57. Weickhardt AJ, Williams D, Lee C, et al: Vascular

endothelial growth factors (VEGF) and VEGF receptor

expression as predictive biomarkers for benefit with

bevacizumab in metastatic colorectal cancer (mCRC):

Analysis of the phase III MAX study. J Clin Oncol 29:

228s, 2011 (suppl; abstr 3531)

58. Jubb AM, Miller KD, Rugo HS, et al: Impact of

exploratory biomarkers on the treatment effect of

bevacizumab in metastatic breast cancer. Clin Can-

cer Res 17:372-381, 2011

59. Foernzler D, Delmar P, Kockx M, et al: Tumor

tissue based biomarker analysis in NO16966: A

randomized phase III study of first-line bevacizumab

in combination with oxaliplatin-based chemotherapy

in patients with mCRC. 2010 Gastrointestinal Can-

cers Symposium, January 22-24, 2010 (abstr 374)60. ShahM, Kang Y, Ohtsu A,et al: Tumor and blood

plasma biomarker analyses in the AVAGAST phase III

randomized study of first-line bevacizumab capecit-

abine/cisplatin in patients with advanced gastric cancer.

Ann Oncol 21:S176, 2010 (suppl 8)

61. Shojaei F, Wu X, Malik AK, et al: Tumor refracto-

riness to anti-VEGF treatment is mediated by CD11b

Gr1myeloid cells. Nat Biotechnol 25:911-920, 2007

62. Carmeliet P: Angiogenesis in life, disease and

medicine. Nature 438:932-936, 2005

63. Loges S, Schmidt T, Carmeliet P: Mecha-

nisms of resistance to anti-angiogenic therapy and

development of third-generation anti-angiogenic

drug candidates. Genes Cancer 1:12-25, 2010

64. Crawford Y, Kasman I, Yu L, et al: PDGF-C

mediates the angiogenic and tumorigenic properties

of fibroblasts associated with tumors refractory to

anti-VEGF treatment. Cancer Cell 15:21-34, 2009

65. Cascone T, Herynk MH, Xu L, et al: Upregu-

lated stromal EGFR and vascular remodeling in

mouse xenograft models of angiogenesis inhibitor-

resistant human lung adenocarcinoma. J Clin Invest

121:1313-1328, 2011

66. Xu L, Duda DG, di Tomaso E, et al: Direct

evidence that bevacizumab, an anti-VEGF antibody,

up-regulates SDF1alpha, CXCR4, CXCL6, and neu-

ropilin 1 in tumors from patients with rectal cancer.

Cancer Res 69:7905-7910, 2009

67. Zhu AX, Sahani DV, Duda DG, et al: Efficacy,

safety, and potential biomarkers of sunitinib mono-

therapy in advanced hepatocellular carcinoma: A

phase II study. J Clin Oncol 27:3027-3035, 2009

68. OConnor JP, Jackson A, Parker GJ, et al: Dy-

namic contrast-enhanced MRI in clinical trials of antivas-

cular therapies. Nat Rev Clin Oncol 9:167-177, 2012

69. Lambrechts D, Storkebaum E, Morimoto M, et al:

VEGF is a modifier of amyotrophic lateral sclerosis inmice and humans and protects motoneurons against

ischemic death. Nat Genet 34:383-394, 2003

70. Schneider BP, Wang M, Radovich M, et al:

Association of vascular endothelial growth factor

and vascular endothelial growth factor receptor-2

genetic polymorphisms with outcome in a trial of

paclitaxel compared with paclitaxel plus bevaci-

zumab in advanced breast cancer: ECOG 2100.

J Clin Oncol 26:4672-4678, 2008

71. Miles DW, DeHaas SL, RomieuG, et al: Polymor-

phism analysis in the AVADO randomised phase III trial

of first-line bevacizumab (BEV) combined with docetaxel

in HER2-negative metastatic breast cancer (mBC). Eur J

Cancer 47:S176, 2011 (suppl 1; abstr 1423)

72. Koutras AK, Antonacopoulou AG, Eleftheraki AG,

et al: Vascular endothelial growth factor polymorphismsand clinical outcome in colorectal cancer patients treated

with irinotecan-based chemotherapy and bevacizumab.

Pharmacogenomics J 12:468-475, 2012

73. Lambrechts D, Delmar P, Miles DW, et al: Single

nucleotide polymorphism analysis and outcome in

advanced-stage cancer patients treated with bevaci-

zumab.Eur J Cancer 47:S173, 2011 (suppl 1; abstr 1414)

74. Loupakis F, Ruzzo A, Salvatore L, et al: Retro-

spective exploratory analysis of VEGF polymor-

phisms in the prediction of benefit from first-line

FOLFIRI plus bevacizumab in metastatic colorectal

cancer. BMC Cancer 11:247, 2011

75. Wang Y, Zheng Y, Zhang W, et al: Polymor-

phisms of KDR gene are associated with coronary

heart disease. J Am Coll Cardiol 50:760-767, 2007

76. Gerger A, El-Khoueiry A, Zhang W, et al:Pharmacogenetic angiogenesis profiling for first-line

bevacizumab plus oxaliplatin-based chemotherapy

in patients with metastatic colorectal cancer. Clin

Cancer Res 17:5783-5792, 2011

77. Hull J, Thomson A, Kwiatkowski D: Associa-

tion of respiratory syncytial virus bronchiolitis with

the interleukin 8 gene region in UK families. Thorax

55:1023-1027, 2000

78. lo Giudice L, Di Salvatore M, Astone A, et al:

Polymorphisms in VEGF, eNOS, COX-2, and IL-8 as

predictive markers of response to bevacizumab.

J Clin Oncol 28:97e, 2010 (suppl; abstr e13502)

79. Schultheis AM, Lurje G, Rhodes KE, et al:

Polymorphisms and clinical outcome in recurrent

ovarian cancer treated with cyclophosphamide and

bevacizumab. Clin Cancer Res 14:7554-7563, 2008

80. Xu CF, Bing NX, Ball HA, et al: Pazopanib efficacy

in renal cell carcinoma: Evidence for predictive genetic

markers in angiogenesis-related and exposure-related

genes. J Clin Oncol 29:2557-2564, 2011

81. Zhang W, Dahlberg SE, Yang D, et al: Genetic

variants in angiogenesis pathway associated with clinical

outcome in NSCLC patients (pts) treated with bevaci-

zumab in combination with carboplatin and paclitaxel:

Subset pharmacogenetic analysis of ECOG 4599. J Clin

Oncol 27:414s, 2009 (suppl; abstr 8032)

82. Lambrechts D, Claes B, Delmar P, et al: VEGF

pathway genetic variants as biomarkers of treat-

ment outcome with bevacizumab: An analysis of

data from the AViTA and AVOREN randomised

trials. Lancet Oncol 13:724-733, 2012

83. Hansen TF, Christensen RD, Andersen RF, et

al: The predictive value of single nucleotide polymor-

phisms in the VEGF system to the efficacy of

first-line treatment with bevacizumab plus chemo-

therapy in patients with metastatic colorectal can-

cer: Results from the Nordic ACT trial. Int JColorectal Dis 27:715-720, 2012

84. Rosenfeld PJ, Brown DM, Heier JS, et al:

Ranibizumab for neovascular age-related macular

degeneration. N Engl J Med 355:1419-1431, 2006

85. Price TJ, Hardingham JE, Lee CK, et al: Im-

pact of KRAS and BRAF gene mutation status on

outcomes from the phase III AGITG MAX trial of

capecitabine alone or in combination with bevaci-

zumab and mitomycin in advanced colorectal can-

cer. J Clin Oncol 29:2675-2682, 2011

86. Rudge JS, Holash J, Hylton D, et al: VEGF trap

complex formation measures production rates of

VEGF, providing a biomarker for predicting effica-

cious angiogenic blockade. Proc Natl Acad Sci U S A

104:18363-18370, 2007

87. Autiero M, Waltenberger J, Communi D, et al:Role of PlGF in the intra- and intermolecular cross

talk between the VEGF receptors Flt1 and Flk1. Nat

Med 9:936-943, 2003

88. Fischer C, Mazzone M, Jonckx B, et al: FLT1 and

its ligands VEGFB and PlGF: Drug targets for anti-

angiogenic therapy? Nat Rev Cancer 8:942-956, 2008

89. Mazzone M, Dettori D, Leite de Oliveira R, et

al: Heterozygous deficiency of PHD2 restores tumor

oxygenation and inhibits metastasis via endothelial

normalization. Cell 136:839-851, 2009

90. Van Cutsem E, Tabernero J, Lakomy R, et al:

Addition of aflibercept to fluorouracil, leucovorin,

and irinotecan improves survival in a phase III ran-

domized trial in patients with metastatic colorectal

cancer previously treated with an oxaliplatin-based

regimen. J Clin Oncol 30:3499-3506, 201291. Bagri A, Tessier-Lavigne M, Watts RJ: Neuropilins

in tumor biology. Clin Cancer Res 15:1860-1864, 2009

92. Pan Q, Chanthery Y, Liang WC, et al: Blocking

neuropilin-1 function has an additive effect with anti-

VEGF to inhibit tumor growth. Cancer Cell 11:53-67,

2007

93. Beck B, Driessens G, Goossens S, et al: A

vascular niche and a VEGF-Nrp1 loop regulate the

initiation and stemness of skin tumours. Nature

478:399-403, 2011

Lambrechts et al





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Acknowledgment

We thank Bart Claes for his helpful and critical comments.



2013 f 128 118 88 48Information downloaded from jco.ascopubs.org and provided by at Penn State Hershey Medical Center on February 18,

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