International Scholarly Research NetworkISRN Cell BiologyVolume 2012, Article ID 587259, 11 pagesdoi:10.5402/2012/587259

Review Article

Molecular Biomarkers of Response toAntiangiogenic Therapy for Cancer

Dan G. Duda

Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital,Harvard Medical School, Boston, MA 02114, USA

Correspondence should be addressed to Dan G. Duda, duda@steele.mgh.harvard.edu

Received 8 October 2012; Accepted 30 October 2012

Academic Editors: D. Arnoult and I. K. Koutna

Copyright © 2012 Dan G. Duda. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Antiangiogenic therapy for cancer has gone from an intriguing hypothesis in the 1970s to an accepted treatment approach formany cancer types. It has also become a standard of care for certain eye diseases. Yet, despite the use of molecularly targeted drugswith well defined targets, to date there are no biomarkers to guide the use of antiangiogenic therapy in patients. The mechanismsof action of these drugs are also being debated. This paper discusses some of the emerging biomarker candidates for this type ofcancer therapy, which have provided mechanistic insight and might be useful in the future for optimizing cancer treatment.

1. Antiangiogenic Therapy

Approval of an anti-vascular endothelial growth factor(VEGF) blocking antibody (bevacizumab or Avastin, Genen-tech, South San Francisco, CA, USA) in combination withchemotherapy for metastatic colorectal cancer in 2004 repre-sented a paradigm shift in cancer therapy. For the first time,an agent targeting the tumor stroma (i.e., the vasculature), asopposed to directly targeting the malignant cells proved to bea viable anticancer treatment option.

Over the last decade, the United States Food and DrugAdministration has approved eight anti-angiogenic agentsfor cancer treatment, and three anti-angiogenic agents forwet age-related macula degeneration therapy (Table 1). Alarge number of other anti-angiogenic agents are in latephases of clinical development (phase III clinical trials). Allthe approved anti-angiogenic drugs target VEGF signaling.Some are blocking the ligand, VEGF, for example, beva-cizumab, aflibercept (Zaltrap/Eylea, Sanofi-Aventis, Paris,France, and Regeneron Pharmaceuticals, Tarrytown, NY,USA), ranibizumab (Lucentis, Genentech, South San Fran-cisco, CA, USA), and pegaptanib (Macugen, OSI Pharma-ceuticals, Long Island, NY, USA). Others are inhibiting theactivity of the VEGF tyrosine kinase receptors (VEGFR1,VEGFR2), for example, sorafenib and regorafenib (Nexavarand Stivarga, Bayer Healthcare Pharmaceuticals, Leverkusen,

Germany, and Onyx Pharmaceuticals, South San Francisco,CA, USA), sunitinib (Sutent) and axitinib (Inlyta) (PfizerInc., New York, NY, USA), pazopanib (Votrient, GlaxoSmith-Kline, Brentford, Middlesex, UK), and vandetanib (Zactima,Astra Zeneca Pharmaceuticals, Alderley Park, Cheshire, UK).Anti-VEGF therapy has become a standard of care formetastatic colo-rectal cancer (in first, second, and thirdline of treatment), advanced non-small cell lung cancer,renal cell carcinoma, hepatocellular carcinomas, glioblas-toma, gastrointestinal stromal tumor (GIST), pancreaticneuroendocrine tumor, and medullary thyroid cancer [1–13] (Table 1). Given these developments, anti-angiogenictherapy represents one of the most exciting areas in cancerresearch and clinical oncology [14–23]. But beyond theproof-of-the-principle efficacy for anti-angiogenic therapy inthese advanced cancers, this success brought critical chal-lenges. First, this therapy has not shown the same benefit inother advanced cancers (e.g., breast cancer, ovarian cancer,prostate cancer, and pancreatic ductal adenocarcinoma)[24]. Second, in the patients who respond to anti-angiogenictherapy, the benefit is transient (i.e., the tumors acquire resis-tance to anti-angiogenic treatment). Other patients do notrespond at all (i.e., the tumors are inherently resistant to anti-angiogenic treatment). Third, similar to most other anti-cancer drugs, anti-angiogenic agents may induce significant

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Table 1: Anti-angiogenic drugs approved by the United States Food and Drug Administration (2004–2012).

Anti-VEGF drug Approved indication

Bevacizumab

Metastatic colorectal cancer (with chemotherapy)

Metastatic nonsquamous non-small cell lung cancer (with chemotherapy)

Metastatic breast cancer (with chemotherapy)

Recurrent glioblastoma (monotherapy)

Metastatic renal cell carcinoma (with IFNα)

SunitinibMetastatic renal cell carcinoma (monotherapy)

Gastrointestinal stromal tumors (monotherapy)

Pancreatic neuroendocrine tumors (monotherapy)

SorafenibMetastatic renal cell carcinoma

Unresectable hepatocellular carcinoma

PazopanibMetastatic renal cell carcinoma

Advanced soft tissue sarcoma

Vandetanib Advanced medullary thyroid cancer

Axitinib Advanced renal cell carcinoma

Regoranfenib Metastatic colorectal cancer

AfliberceptMetastatic colorectal cancer (with chemotherapy)

Wet age-related macula degeneration

Pegaptanib Wet age-related macula degeneration

Ranibizumab Wet age-related macula degeneration

side effects [25]. And finally, treatment with current anti-angiogenic agents is extremely costly [26, 27]. How do wetackle these problems? One potential solution for optimizingtreatment with current anti-angiogenic agents is to identifyuseful biomarkers (see Box 1 for definitions) in order to (1)select (or exclude) patients for treatment with a specific anti-angiogenic drug; and (2) detect early the escape from anti-VEGF therapy.

The former would allow a “more personalized” treatment(for patients more likely to benefit) and/or a sparing of afraction of patients (those unlikely to respond) from the sideeffects and the high cost of these treatments. The latter wouldallow us to devise better combinatorial treatment strategiesto extend the benefit of anti-angiogenic agents. Biomarkershave been used for “molecularly targeted” agents, but sofar only for those that directly attack the cancer cells, forexample, human epidermal growth factor receptor 2 (HER2)expression or amplification for anti-HER2 therapy in breastcancer, RAS mutation for anti-epidermal growth factorreceptor (EGFR) therapy, and BRAFV600 mutation for BRAFinhibitors in melanoma [57–59]. But despite agreement thatbiomarker discovery and validation are a major priorityin the tumor angiogenesis field and the widespread use ofanti-angiogenic drugs in the clinic, there are currently novalidated biomarkers for use in the clinic for treatmentof patient with any type of cancer or for the treatmentfor wet age-related macula degeneration. Among the manyreasons for this situation the most notable are (1) tumorheterogeneity, (2) the incomplete understanding of themechanism(s) of action that lead to benefit of anti-VEGFtherapy in some but not all advanced cancers, and (3) the

differential targeting of VEGF pathway and off-target effectsby the current anti-angiogenic drugs and by the agents inclinical development. I will summarize here the currentprogress on our mechanistic understanding of anti-VEGFtherapy and on the current status molecular and cellularbiomarker discovery and will provide a perspective on thefuture directions in this field.

2. The Angiogenic Balance

Over four decades of research on angiogenesis have unrav-eled many of the underpinnings of tumor angiogenesis ingeneral and VEGF pathway in particular [60–64]. VEGF isa key proangiogenic molecule in developmental neovascu-larization as well as in physiological and pathological angio-genesis [62, 65–67]. VEGF exerts its effect by binding thetwo tyrosine kinase (TK) receptors VEGFR-1 (FLT1) andVEGFR-2 (KDR) as well as the non-TK receptors neuropilin1 (NRP-1) and NRP-2 [60]. Of these, VEGF interaction withVEGFR2 is thought to convey most of the critical pro-angiogenic signals [18]. However, VEGF interaction withVEGFR-1 and NRP-1 in cancer cells or in nonendothelialstromal cells (e.g., in myeloid cells such as macrophages)may be critical for the growth of tumors that depend onthis pathway for survival and, through indirect mechanisms,to angiogenesis in tumors [60]. During development and inphysiological conditions, the effects of VEGF are finely tunedand counterbalanced by anti-angiogenic molecules such asthe soluble form of VEGFR-1 (sVEGFR-1/sFLT-1) or throm-bospondins (TSP-1 and -2), which ensures stabilization andmaturation of the vasculature [68]. In tumors, oncogene or

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According to current US Food and Drug Administration draft guidance, a biological marker, or biomarker, isdefined as a characteristic that is objectively measured and evaluated as an indicator of normal biologicprocesses, pathogenic processes, or biological responses to a therapeutic intervention [28]. Such characteristicsmay include genetic differences, either inherited by the individual in the germline or residing in the tumor,or both; changes in RNA, protein, or metabolite levels as a consequence of the disease or of the therapeutic process;changes in physiologic or systemic parameters, such as blood pressure; or anatomical parameters, such astumor growth, stasis, or shrinkage [28].

When considering biomarker research, it is important to be aware of the different types of biomarkers thatcan be identified and the limitations posed by certain types of studies. Biomarkers that can be used before treatmentinclude prognostic markers, which predict patient outcome regardless of treatment, and predictive markers,which provide information about the effect of a specific therapeutic intervention, usually compared to another.The majority of clinical studies of anti-angiogenic agents to date have identified mainly potential prognosticrather than predictive biomarkers because the studies were either too small to show a statistical differencebetween treatment arms with respect to the biomarker, or because they were early stage trials that includedonly one treatment arm [29]. This situation is changing as many larger trials of anti-angiogenic cancer therapiesare now incorporating preplanned biomarker analyses. Pharmacodynamic biomarkers are used duringtreatment to monitor its course, and/or to detect resistance or drug toxicity. Ideally, both predictive andpharmacodynamic biomarkers should reflect modulation of an identified biological target of the therapy inquestion.While this requirement is more straightforward for agents that target oncogenic pathways in cancercells, it may be difficult to attain for biomarkers of anti-angiogenic agents, since their exact mechanisms ofaction are not yet well defined [30].

In addition to their functional characteristics, biomarkers should be robust, reliable, reproducible, feasible foruse in a clinical setting, and carefully validated as to specificity and sensitivity. The US Food and Drug Administrationis currently taking an active role in setting standards for biomarker development and pharmacogenomicbiomarkers have been incorporated into drug labels for multiple oncologic therapies, both targeted and untargeted.Such guidance will prove increasingly important as cancer treatment becomes more personalized and as new therapiesare developed that are designed to hit ever more specific targets in tumorigenic, angiogenic, and genetic pathways.

Reproduced from Duda, [31].

Box 1: Defining biomarkers.

hypoxia-driven VEGF overexpression leads to dysregulatedangiogenesis and an abnormal vasculature [60]. Here, thebalance is tipped toward pro-angiogenesis. In genetic modelsin mice, overexpression of VEGFR-1 and sVEGFR-1 led to a“normalization” of the tumor vasculature [69]. Conversely,overexpression of sVEGFR1 may lead to hypertension (e.g.,preeclampsia) or defects in developmental angiogenesis [70–72]. Beyond VEGF, other VEGF family members can bindthe VEGFRs and participate in angiogenesis: PlGF, VEGF-B,VEGF-C, and VEGF-D. The role of VEGFR-1 and its moreselective ligand placental growth factor (PlGF) is currentlyunclear, but may be particularly important in certain malig-nancies [60]. Similarly, VEGF-C and VEGF-D might playa role during new blood vessel formation [73]. First, theycan bind to VEGFR2, and second, their cognate receptorVEGFR3 is expressed on “tip” cells (specialized endothelialcells responsible for vessel sprouting) [60, 73]. In addi-tion, other angiogenesis modulators (positive or negative)may affect the angiogenic balance, for example, the pro-angiogenic molecules basic fibroblast growth factor (bFGF orFGF-2), angiopoietin 1 (Ang-1), Ang-2, and endoglin or theendogenous angiogenesis inhibitors TSP-1 and TSP-2 [60].These angiogenic molecules are produced by the cancer cellsand by the stromal cells alike. The latter include activatedtumor-activated fibroblasts and bone marrow derived cellsrecruited by the tumor—most notably tumor infiltrating

macrophages, neutrophils, and myeloid derived suppressorcells [35, 74, 75].

Could we exploit all this knowledge for biomarker dis-covery? The answer is likely yes, provided that biomarkerstudies will be biology driven and prospectively validated[28–31, 76]. The systemic and imaging biomarkers may alsoplay a crucial role in discovery of biomarkers for anti-an-giogenic therapy and are discussed in detail elsewhere [30,77, 78]. Here, I will discuss in the next three sections thecandidate biomarkers belonging to VEGF family and to otherangiogenic pathways, and the cellular biomarkers.

3. Molecular and Cellular BiomarkerCandidates for Antiangiogenic Therapy

Tissue-based biomarkers are ideal because they reflect thechanges occurring in a tumor during treatment, but obtain-ing biopsies is difficult due to the invasive nature of theprocedure. Circulating molecular and cellular biomarkersfound in blood are a minimally invasive alternative that canbe used repeatedly over the course of treatment with an anti-angiogenic agent. Whereas changes in blood circulation mayreflect the systemic effects of anti-VEGF therapy, the impactof these changes on tumor response or escape remainsunclear and will need to be established in mechanistic studiesin preclinical models [31].

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3.1. VEGF Family Members as Circulating Biomarkers. VEGFexpression is usually elevated both in the tumors as well asin the circulation of cancer patients and is often an indicatorof poor prognosis. All the anti-angiogenic drugs that havereceived or are pending approval from the US Food and DrugAdministration target VEGF signaling—either by blockingthe ligand (bevacizumab, aflibercept) or by inhibiting thetyrosine kinase receptors (sorafenib, sunitinib, vandetanib,pazopanib, axitinib, and regorafenib). Thus, the naturalchoice for a biomarker has been VEGF itself. However, todate the results have been highly inconsistent [30]. HighVEGF levels are almost invariably associated with pooroutcomes in correlative studies [56], which is indicative of itsprognostic biomarker value. In some cancers (e.g., breastcancers or HCC) the levels of circulating VEGF in plasmacorrelated with outcome of anti-VEGF therapy [30, 56, 79].However, in other cancers neither the intra-tumoralnor the circulating VEGF associated with outcome ofbevacizumab treatment [80, 81]. For example, a recent meta-analysis across four randomized phase III trials ofbevacizumab with chemotherapy or immunotherapy inmetastatic colorectal cancer, advanced non-small cell lungcancer, and advanced renal cell carcinoma showed thathigher baseline levels of circulating VEGF were associatedwith shortened progression free survival and overall survivalregardless of bevacizumab treatment [82]. This indicates thatcirculating VEGF levels may be prognostic but not predictivebiomarkers for bevacizumab containing regimens. Moreover,the authors did not find a good correlation between bloodcirculating VEGF concentration and intratumor expressionof VEGF [82]. On the other hand, more recent studieshave measured shorter isoforms of VEGF (e.g., VEGF121),which do not bind to the extracellular matrix components(i.e., heparin), and have found intriguing correlations withoutcome [83]. However, other studies failed to detect asignificant correlation for short isoforms of VEGF [84].Thus, the clinical significance of circulating or tissue VEGFlevels remains to be clarified, as most of the efforts to useVEGF itself as a predictive biomarker have thus far beendisappointing. Current ongoing efforts to measure distinctVEGF isoforms or VEGF fragments may yield additionalinsight and resurrect interest in research on VEGF aspredictive biomarker.

3.1.1. Other VEGF Family Members. In addition to VEGF-A(or VEGF), the VEGF family includes VEGF-B, VEGF-C,VEGF-D, and PlGF. These VEGF family members may playa role in tumor angiogenesis [60]. Currently available antian-giogenic drugs affect these factors in a differential manner(i.e., they are not affected by bevacizumab but may beblocked by aflibercept or receptor tyrosine kinase inhibitors)[60]. Of interest, some of these factors have been shown to beupregulated in response to anti-VEGF therapy both inpatients and in preclinical models [30]. The most consistentchange has been the increase in circulating levels of plasmaPlGF, which has been reported essentially for all anti-VEGF drugs and experimental agents, irrespective of theirmechanism of VEGF inhibition [30, 85]. This has led to

the hypotheses that (1) PlGF change may have pharmaco-dynamic biomarker value and (2) that PlGF increase maymediate resistance to anti-VEGF agents that do not block thismolecule (e.g., bevacizumab). Both of these hypotheses needto be further validated prospectively. Of interest, the increasein PlGF may be due to systemic effects, as tumor-derivedPlGF may actually be decreased after bevacizumab treatment[86]. Similarly, VEGF-C and VEGF-D have been proposed asescape biomarkers for bevacizumab in metastatic colorectalcancer patients in other exploratory studies [87].

3.1.2. Soluble VEGF Receptors. As discussed above, there are3 VEGF tyrosine kinase receptors in the plasma membrane,known as VEGFR-1 (FLT-1), VEGFR-2 (KDR), and VEGFR-3 (FLT-4). In addition to the plasma membrane receptors,soluble receptors are present in blood circulation—as a resultof alternative splicing or possibly due to plasma membranereceptor shedding [30].

Of these soluble receptors, sVEGFR-1 has clear biologicalactivity. This has led our group to conduct extensive stud-ies of circulating sVEGFR-1—an endogenous blocker ofVEGF and PlGF and a factor linked with “vascular nor-malization”—as biomarker or response to anti-VEGF agents[88]. Our hypothesis has been that circulating plasmasVEGFR-1 is a “negative” biomarker that could be used topredict response to anti-VEGF therapies in cancer. Specifi-cally, we propose that cancer patients with preexisting highlevels of circulating sVEGFR-1 (i.e., in whom VEGF pathwayis endogenously suppressed) are resistant to bevacizumaband other anti-VEGF treatments. Indeed, we have shown inexploratory studies that patients with higher plasma levelsof sVEGFR-1 have a poor outcome after treatment withbevacizumab, sunitinib, vandetanib, and cediranib [88–94].Collectively, these results suggest that anti-VEGF therapymay not have a beneficial effect in patients with highsVEGFR-1 levels. In further support of this, we also foundthat patients with higher sVEGFR-1 levels in circulationexperienced fewer side effects from anti-VEGF treatments[88, 92, 93]. Finally, polymorphisms in the FLT1 gene that areassociated with higher VEGFR1 expression have also beenassociated with poor outcome of bevacizumab containingregimens in phase III studies (see below) [95]. If confirmed inlarger studies, plasma sVEGFR1 may potentially allow strat-ification of cancer patients to regimens that include anti-VEGF therapy.

Soluble VEGFR2, which is an abundant protein inhuman plasma, has also been extensively studied. Multiplestudies have shown that anti-VEGFR tyrosine kinase inhi-bitors but not bevacizumab induce a significant decreasein plasma sVEGFR-2 levels (summarized in [30]). Thesame result has been reported for circulating sVEGFR-3(i.e., a decrease in plasma sVEGFR3 after treatment withtyrosine kinase inhibitors that block VEGFR-3). The pres-ence of this signature has been associated with improvedoutcomes in some studies, but its value as a predictive orpharmacodynamic biomarkers is currently unknown [29–31].

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Chronic inflammation is a potential precursor and promoter of carcinogenesis in many cancers [32–35].In many cancers, nuclear factor kappa B (NF-κB) is involved in tumor initiation and progressionmediated via STAT3 activation [36–38]. Inflammatory cytokines induced by NF-κB pathway activationmight affect angiogenesis directly via endothelial cells, or indirectly by cancer cells or recruitment and/oractivation of inflammatory cells [39–46]. Interleukin (IL)-1α has a critical roleby recruitment of inflammatory cells [47, 48]. Tumor necrosis factor (TNF)-α can also promote tumorprogression by different pathways: direct effect on tumor cells, induction of CXCR4, and stimulation ofepithelial-mesenchymal transition [49]. TNF-α promotes cell survival and angiogenesis or induceendothelial cell apoptosis, vascular disruption, and increased permeability. IL-6 is also inducedby activation of NF-κB and other transcription factors (C/EPBb and AP-1) and modulates inflammationvia IL-6R and gp130. Vascular smooth muscle cells, T lymphocytes, and macrophages secrete IL-6to stimulate immune responses and promote inflammation. IL-6 may also have anti-inflammatory effectsby inhibition of TNF-α and IL-1, and activation of IL-1Ra and IL-10. The proliferative and survival effects ofIL-6 are mediated by STAT3 [34]. Moreover, IL-8 may have a role in cancer cell invasion [50, 51].IL-8 can promote tumorigenesis and angiogenesis through CXCR1 and CXCR2, and the Duffy antigenreceptor for cytokines, which has no defined intracellular signaling capabilities [52]. Overexpressionof VEGF induces the expression of the CXCR4 ligand—stromal cell derived factor 1 alpha (SDF1α) orCXCL12— and SDF1α and CXCR4 may drive cell migration and angiogenesis by VEGF-independentmechanisms [53, 54]. Stem Cell Factor (also known as SCF or KIT-ligand) is a cytokine that binds tothe c-KIT receptor (CD117), primarily expressed by early hematopoietic precursors. While c-KITexpression is rarely detectable in the cancer cells, both SCF and c-KIT could be expressed duringcarcinogenesis, for example, in cholangiocarcinomas [55].

Box 2: Inflammatory molecules and their potential role in liver cancer angiogenesis. Abbreviations: AP-1, activator protein 1; C/EPB, CAAT/enhancer binding protein; CXCR, C- X-C-chemokine receptor; STAT, signal transducers and activators of transcription. Reproduced from[56].

3.2. Other Soluble Plasma Biomarker Candidates

3.2.1. Soluble Basement Membrane Components. Collagen IVis one of the main constituents of vascular basement mem-branes. In glioblastomas, there is an excessive deposition ofbasement membranes, which more than doubles the thick-ness tumor blood vessels compared to normal brain bloodvessels [96, 97]. Vascular normalization after anti-VEGFtherapy results in normalization of the vascular basementmembrane—that is, a reduction in thickness—as seen inmice and in patients [96–98]. Thus, we tested the hypothesisthat proteolytic degradation of these membranes couldrelease soluble collagen IV in blood circulation and that thisbiomarker could be used as a measure of therapeutic efficacy.Indeed, we found that recurrent glioblastoma patients whohad an increased in plasma collagen IV levels after anti-VEGFtherapy had an increase in progression-free survival [99].If validated, either alone or in combination with imagingbiomarkers of vascular normalization, the change in solublecollagen IV may potentially allow an early assessment of drugactivity and stratification of glioblastoma patients to anti-VEGF therapies [99].

3.2.2. Inflammatory Factors. In addition to VEGF familymembers, many biomarker studies have focused on inflam-matory cytokines and chemokines because they may exertpro-angiogenic effects either directly or indirectly (viamodulation of bone marrow derived cell recruitment incirculation and infiltration in tumors) (Box 2).

A comprehensive study was conducted in patients withadvanced non-small cell lung cancer who were treated with

vandetanib plus chemotherapy, vandetanib alone, or chemo-therapy alone. Interestingly, the patterns of changes insoluble biomarkers in each of the three study arms weredistinct [100]. Specifically, an increased risk of disease pro-gression was associated with increases in a different markerin each arm, increased plasma VEGF levels for vandetanibmonotherapy versus increase in plasma Interleukin (IL)-8concentration for combination therapy. IL-8 may act as aVEGF-independent pro-angiogenic pathway [51] and hasbeen associated with poor prognosis in hepatocellular car-cinoma patients treated with sunitinib [94]. Other notablecandidates for biomarkers of tumor evasion from anti-VEGFtherapy are the stromal-cell-derived factor 1 alpha (SDF1α,also referred to as CXCL12) and IL-6. We have found associ-ations between increased plasma SDF1α after treatment andpoor outcome in studies of anti-VEGF agents in recurrentglioblastoma (cediranib), sarcoma (sorafenib), and breastcancer (bevacizumab) patients [89, 92, 101–103]. Moreover,increased plasma SDF1α and plasma IL-6 have been associ-ated with poor outcomes in locally advanced rectal cancerand advanced hepatocellular carcinoma patients treated withbevacizumab, chemoradiation, and sunitinib, respectively[94, 104]. These potential resistance biomarkers may drivethe design of trials anti-VEGF agents.

3.2.3. Other Circulating Factors or Soluble Receptors. Finally,recent studies have reported significant changes or associa-tions with outcome for other circulating factors and/or theirsoluble receptors. Some of the findings have been moreconsistent, for example, the transient decrease in plasma

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Ang-2 after anti-VEGF therapy [89, 90, 92]. Others appearedto be more agent/disease specific, for example, changes andcorrelations between circulating bFGF, platelet derivedgrowth factor (PDGF)-BB, soluble (s)Tie2, soluble intercel-lular adhesion molecule 1 (sICAM-1), and matrix metallo-proteinase (MMP)-2, MMP-9, and MMP-10 [87, 89, 90, 92,94, 100, 105]. All of these biomarkers will require additionalstudy and prospective validation.

3.3. Tissue Based Biomarkers. Whenever available—for example,when serial biopsies can be performed or when tissuesare obtained at surgery or autopsy—tumor specimens havebeen invaluable for conducting correlative studies andgaining mechanistic insights into the effects of anti-VEGFtherapies. These studies have been quite limited because ofthe invasive and costly nature of these procedures and the dif-ficulty in standardizing immunohistochemical procedures.

As mentioned previously, intratumoral levels of VEGFhave not been so far shown to predict survival outcome ofanti-VEGF therapy [81, 82], although correlations withresponse rates have been reported [106, 107]. Given thedisappointing data reported so far, and considering the lim-itations of tissue VEGF evaluation, this biomarker does notappear promising.

However, these intriguing results raised critical ques-tions. If neither circulating nor tissue VEGF correlate withoutcome of anti-VEGF agents, then what is the mechanism ofaction that leads to a benefit after treatment with these drugs?While multiple groups are actively exploring various mecha-nisms involving the vasculature, stroma, immune system, orcancer cells themselves, several emerging data are standingout. Tumor microvascular density has been often evaluatedboth as a predictive biomarker and as a pharmacodynamicmarker of anti-angiogenic therapy with anti-VEGF agents.Indeed, two studies found a decrease in vascular density afterbevacizumab treatment in rectal and breast cancer [92, 104,108]. But other studies did not find a significant change[109]. This effect was associated with increased apoptoticrate in cancer cells but interestingly, did not change theproliferation rate of cancer cells [108, 109]. One explanationfor this paradoxical finding is that the remaining vasculatureafter anti-VEGF therapy is more “normal” structurally andfunctionally [110–113]. The association between microvas-cular density and survival remains unclear, with most studiesreporting a lack of correlation [81].

In a study of serial biopsies from rectal cancers, our grouphas reported that while bevacizumab did no change VEGFor VEGFR expression in the cancer cells, this anti-VEGFtreatment decreased PlGF and increased SDF1α and itsreceptor (CXCR4) expression in the rectal cancer cells [86].Of interest, increased plasma SDF1α levels during treatmentin these patients correlated with distant disease progressionpointing toward SDF1α/CXCR4 axis as a potential escapemechanism from anti-VEGF therapy [86, 102].

While enticing, these hypotheses on the mechanism ofaction of anti-VEGF agents remain to be further confirmedin patients, as our understanding of the dynamics of VEGFRregulation and the interactions between receptor subtypes in

tumor tissue is not well enough advanced to allow the use ofthese levels as biomarkers of therapeutic efficacy.

Finally, genetic studies of tumor samples have also gener-ated mixed results. While establishing the mutational statusin various cancers has made a crucial impact on the devel-opment and use of anti-cancer agents, for example, KRASmutation for cetuximab treatment in metastatic colorectalcancer and BRAF mutation for vemurafenib treatment inmelanoma, it has failed so far to impact the developmentor the use of anti-VEGF drugs. For example, P53, KRAS, orBRAF mutations in metastatic colorectal cancer did notassociate with bevacizumab-chemotherapy treatment out-come in metastatic colorectal cancer [114]. Many studieshave focused on single nucleotide polymorphisms (SNPs)in VEGF family genes as well as other genes [115–118].Some reports found significant correlations between certainVEGF and VEGFR2 genes with survival or risk of developinghypertension after bevacizumab treatment in metastaticbreast and colorectal cancer [117, 119]. However, thesefindings have not been yet reproduced by other studies. Morerecently, SNPs in VEGFR1 were shown to associate withsurvival after treatment with bevacizumab based regimens in2 phase III studies in advanced pancreatic adenocarcinomaand metastatic renal cell carcinoma [95]. These SNPs wereassociated with higher VEGFR1 expression [95]. TheseVEGFR1 SNPs correlated with a poor outcome, which is inline with the finding that high circulating sVEGFR1 isassociated with poor outcome after anti-VEGF therapy (seeabove) [88–94]. Also, a consistent finding appears to be theassociation between SNPs in CXCR2 and IL8 genes and out-come after anti-VEGF therapies [115, 116, 118, 120]. Onceagain, this suggests an important role that inflammatorycytokines and their receptors may play in the outcome ofanti-VEGF therapy. These data strongly suggest that SNPevaluation could be used in the future to predict outcomeof anti-VEGF therapy. Moreover, the evaluations of genepolymorphisms have the great advantage of being morefeasible as they are minimally invasive and less expensive anddo not necessarily require tumor tissue. However, only moreextensive investigation and validation of the current leadcandidates could potentially provide a biomarker for anti-VEGF therapy.

4. Challenges, Conclusions, andFuture Perspective

One major challenge for the interpretation of molecularbiomarker studies in general is that a vast amount of datawas generated in single arm studies, that is, in which allpatients received the same therapy. This makes the distinc-tion between prognostic and predictive biomarkers impos-sible. Another challenge is that while bevacizumab andaflibercept are specific inhibitors of VEGF pathways, all theanti-angiogenic tyrosine kinase inhibitors are promiscuous,inhibiting multiple, and non-angiogenic tyrosine kinases aswell as angiogenic ones [121, 122]. Therefore, it can be diffi-cult to know whether a given biochemical or physiologicaleffect is the result of anti-angiogenic activity or due toeffects on other oncogenic targets (e.g., c-KIT inhibition by

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sunitinib in gastrointestinal stromal tumors or EGFR andRET inhibition by vandetanib in advanced medullary thy-roid cancer). Even for bevacizumab/aflibercept studies, theinterpretation is confounded by the fact that most studiesincluded concurrent chemotherapeutic drugs, making itdifficult to tease out the effects of each type of therapy.

In summary, identifying and validating predictive bio-markers of response and gaining the ability to stratifycancer patients to currently approved anti-angiogenic drugsremain major priorities in oncology. A number of potentialbiomarkers have emerged from correlative clinical studiesthat warrant further study in large randomized trials. Somesuch trials are now underway and their results will becritical for advancement of this field, not only for biomarkerdiscovery but also for further elucidation of the specificmechanisms of action of these important new therapies.

Conflict of Interests

The author does not have any direct financial relation withthe commercial identities mentioned in this paper.

Acknowledgments

The research of the author is supported by US NationalInstitutes of Health Grants P01-CA080124, R01-CA159258,Federal Share National Cancer Institute Proton Beam Pro-gram Income, and the American Cancer Society Grant120733-RSG-11-073-01-TBG. This paper uses some updatedinformation from [31, 56].

References

[1] A. L. Cheng, Y. K. Kang, Z. Chen et al., “Efficacy and safety ofsorafenib in patients in the Asia-Pacific region with advancedhepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial,” The Lancet Oncology, vol. 10,no. 1, pp. 25–34, 2011.

[2] T. F. Cloughesy, M. D. Prados, P. Wen et al., “non-comparative clinical trial of the effect of bevacizumab aloneor in combination with irinotecan (CPT-11) on 6-monthprogression free survival in recurrent, treatment-refractoryglioblastoma,” Journal of Clinical Oncology, vol. 26, abstract2010, 2008.

[3] G. D. Demetri, A. T. van Oosterom, C. R. Garrett et al.,“Efficacy and safety of sunitinib in patients with advancedgastrointestinal stromal tumour after failure of imatinib: arandomised controlled trial,” The Lancet, vol. 368, no. 9544,pp. 1329–1338, 2006.

[4] B. Escudier, T. Eisen, W. M. Stadler et al., “Sorafenib inadvanced clear-cell renal-cell carcinoma,” The New EnglandJournal of Medicine, vol. 356, no. 2, pp. 125–134, 2007.

[5] B. J. Giantonio, P. J. Catalano, N. J. Meropol et al., “Beva-cizumab in combination with oxaliplatin, fluorouracil, andleucovorin (FOLFOX4) for previously treated metastaticcolorectal cancer: results from the Eastern CooperativeOncology Group Study E3200,” Journal of Clinical Oncology,vol. 25, no. 12, pp. 1539–1544, 2007.

[6] H. Hurwitz, L. Fehrenbacher, W. Novotny et al., “Beva-cizumab plus irinotecan, fluorouracil, and leucovorin for

metastatic colorectal cancer,” The New England Journal ofMedicine, vol. 350, no. 23, pp. 2335–2342, 2004.

[7] J. M. Llovet, S. Ricci, V. Mazzaferro et al., “Sorafenib inadvanced hepatocellular carcinoma,” The New England Jour-nal of Medicine, vol. 359, no. 4, pp. 378–390, 2008.

[8] R. J. Motzer, T. E. Hutson, P. Tomczak et al., “Sunitinib versusinterferon alfa in metastatic renal-cell carcinoma,” The NewEngland Journal of Medicine, vol. 356, no. 2, pp. 115–124,2007.

[9] B. I. Rini, S. Halabi, J. E. Rosenberg et al., “Phase III trialof bevacizumab plus interferon alfa versus interferon alfamonotherapy in patients with metastatic renal cell carci-noma: final results of CALGB 90206,” Journal of ClinicalOncology, vol. 28, no. 13, pp. 2137–2143, 2010.

[10] A. Sandler, R. Gray, M. C. Perry et al., “Paclitaxel-carboplatinalone or with bevacizumab for non-small-cell lung cancer,”The New England Journal of Medicine, vol. 355, no. 24, pp.2542–2550, 2006.

[11] C. N. Sternberg, I. D. Davis, J. Mardiak et al., “Pazopanib inlocally advanced or metastatic renal cell carcinoma: resultsof a randomized phase III trial,” Journal of Clinical Oncology,vol. 28, no. 6, pp. 1061–1068, 2010.

[12] E. Van Cutsem, J. Tabernero, R. Lakomy et al., “Addition ofaflibercept to fluorouracil, leucovorin, and irinotecanimproves survival in a phase III randomized trial in patientswith metastatic colorectal cancer previously treated with anoxaliplatin-based regimen,” Journal of Clinical Oncology, vol.30, no. 28, pp. 3499–3506, 2012.

[13] S. A. Wells Jr., B. G. Robinson, R. F. Gagel et al., “Vandetanibin patients with locally advanced or metastatic medullarythyroid cancer: a randomized, double-blind phase III trial,”Journal of Clinical Oncology, vol. 30, pp. 134–141, 2012.

[14] D. G. Duda, T. T. Batchelor, C. G. Willett, and R. K. Jain,“VEGF-targeted cancer therapy strategies: current progress,hurdles and future prospects,” Trends in Molecular Medicine,vol. 13, no. 6, pp. 223–230, 2007.

[15] D. G. Duda, R. K. Jain, and C. G. Willett, “Antiangiogenics:the potential role of integrating this novel treatment modalitywith chemoradiation for solid cancers,” Journal of ClinicalOncology, vol. 25, no. 26, pp. 4033–4042, 2007.

[16] L. M. Ellis, “Antiangiogenic therapy at a crossroads: clinicaltrial results and future directions,” Journal of Clinical Oncol-ogy, vol. 21, no. 23, supplement, pp. 281s–283s, 2003.

[17] F. A. L. M. Eskens, “Angiogenesis inhibitors in clinical devel-opment; where are we now and where are we going?” BritishJournal of Cancer, vol. 90, no. 1, pp. 1–7, 2004.

[18] N. Ferrara, K. J. Hillan, H. P. Gerber, and W. Novotny,“Discovery and development of bevacizumab, an anti-VEGFantibody for treating cancer,” Nature Reviews Drug Discovery,vol. 3, no. 5, pp. 391–400, 2004.

[19] A. Grothey and E. Galanis, “Targeting angiogenesis: progresswith anti-VEGF treatment with large molecules,” NatureReviews Clinical Oncology, vol. 6, no. 9, pp. 507–518, 2009.

[20] V. L. Heath and R. Bicknell, “Anticancer strategies involvingthe vasculature,” Nature Reviews Clinical Oncology, vol. 6, no.7, pp. 395–404, 2009.

[21] R. K. Jain, “Antiangiogenic therapy for cancer: current andemerging concepts,” Oncology, vol. 19, no. 4, supplement, pp.7–16, 2005.

[22] R. K. Jain, D. G. Duda, J. W. Clark, and J. S. Loeffler, “Lessonsfrom phase III clinical trials on anti-VEGF therapy forcancer,” Nature Clinical Practice Oncology, vol. 3, no. 1, pp.24–40, 2006.

8 ISRN Cell Biology

[23] L. S. Rosen, “VEGF-targeted therapy: therapeutic potentialand recent advances,” Oncologist, vol. 10, no. 6, pp. 382–391,2005.

[24] E. Van Cutsem, D. Lambrechts, H. Prenen, R. K. Jain, and P.Carmeliet, “Lessons from the adjuvant bevacizumab trial oncolon cancer: what next?” Journal of Clinical Oncology, vol.29, no. 1, pp. 1–4, 2011.

[25] H. M. W. Verheul and H. M. Pinedo, “Possible molecularmechanisms involved in the toxicity of angiogenesis inhibi-tion,” Nature Reviews Cancer, vol. 7, no. 6, pp. 475–485, 2007.

[26] T. Fojo and C. Grady, “How much is life worth: cetuximab,non-small cell lung cancer, and the $440 billion question,”Journal of the National Cancer Institute, vol. 101, no. 15, pp.1044–1048, 2009.

[27] A. T. Fojo and D. R. Parkinson, “Biologically targeted cancertherapy and marginal benefits: are we making too much oftoo little or are we achieving too little by giving too much?”Clinical Cancer Research, vol. 16, no. 24, pp. 5972–5980, 2010.

[28] Biomarkers Definitions Working Group, “Biomarkers andsurrogate endpoints: preferred definitions and conceptualframework,” Clinical pharmacology and therapeutics, vol. 69,no. 3, pp. 89–95, 2001.

[29] N. Murukesh, C. Dive, and G. C. Jayson, “Biomarkers ofangiogenesis and their role in the development of VEGFinhibitors,” British Journal of Cancer, vol. 102, no. 1, pp. 8–18, 2010.

[30] R. K. Jain, D. G. Duda, C. G. Willett et al., “Biomarkers ofresponse and resistance to antiangiogenic therapy,” NatureReviews Clinical Oncology, vol. 6, no. 6, pp. 327–338, 2009.

[31] D. G. Duda, “Targeting tumor angiogenesis: biomarkers ofangiogenesis and antiangiogenic therapy in cancer,” Angio-genesis Foundation e-publication, 2011, http://www.angio.org/cme/biom.php.

[32] L. M. Coussens and Z. Werb, “Inflammation and cancer,”Nature, vol. 420, no. 6917, pp. 860–867, 2002.

[33] A. Mantovani, P. Allavena, A. Sica, and F. Balkwill, “Cancer-related inflammation,” Nature, vol. 454, no. 7203, pp. 436–444, 2008.

[34] W. E. Naugler and M. Karin, “The wolf in sheep’s clothing:the role of interleukin-6 in immunity, inflammation andcancer,” Trends in Molecular Medicine, vol. 14, no. 3, pp. 109–119, 2008.

[35] J. W. Pollard, “Tumour-educated macrophages promotetumour progression and metastasis,” Nature Reviews Cancer,vol. 4, no. 1, pp. 71–78, 2004.

[36] G. He, G. Y. Yu, V. Temkin et al., “Hepatocyte IKKβ/NF-κB inhibits tumor promotion and progression by preventingoxidative stress-driven STAT3 activation,” Cancer Cell, vol.17, no. 3, pp. 286–297, 2010.

[37] E. J. Park, J. H. Lee, G. Y. Yu et al., “Dietary and geneticobesity promote liver inflammation and tumorigenesis byenhancing IL-6 and TNF expression,” Cell, vol. 140, no. 2,pp. 197–208, 2010.

[38] E. Pikarsky, R. M. Porat, I. Stein et al., “NF-κB functions asa tumour promoter in inflammation-associated cancer,”Nature, vol. 431, no. 7007, pp. 461–466, 2004.

[39] M. Belakavadi and B. P. Salimath, “Mechanism of inhibitionof ascites tumor growth in mice by curcumin is mediated byNF-kB and caspase activated DNase,” Molecular and CellularBiochemistry, vol. 273, no. 1-2, pp. 57–67, 2005.

[40] T. P. Hamsa and G. Kuttan, “GAntiangiogenic activity ofberberine is mediated through the downregulation ofhypoxia-inducible factor-1, VEGF, and proinflammatory

mediators,” Drug and Chemical Toxicology, vol. 35, pp. 57–70, 2012.

[41] J. Rhode, S. Fogoros, S. Zick et al., “Ginger inhibits cellgrowth and modulates angiogenic factors in ovarian cancercells,” BMC Complementary and Alternative Medicine, vol. 7,article 44, 2007.

[42] A. Shibata, T. Nagaya, T. Imai, H. Funahashi, A. Nakao, andH. Seo, “Inhibition of NF-κB activity decreases the VEGFmRNA expression in MDA-MB-231 breast cancer cells,”Breast Cancer Research and Treatment, vol. 73, no. 3, pp. 237–243, 2002.

[43] L. Veschini, D. Belloni, C. Foglieni et al., “Hypoxia-indu-cible transcription factor-1 alpha determines sensitivity ofendothelial cells to the proteosome inhibitor bortezomib,”Blood, vol. 109, no. 6, pp. 2565–2570, 2007.

[44] M. Wu, C. Huang, X. Li et al., “LRRC4 inhibits glioblastomacell proliferation, migration, and angiogenesis by downregu-lating pleiotropic cytokine expression and responses,” Journalof Cellular Physiology, vol. 214, no. 1, pp. 65–74, 2008.

[45] W. Zhang, X. D. Zhu, H. C. Sun et al., “Depletion of tumor-associated macrophages enhances the effect of sorafenibin metastatic liver cancer models by antimetastatic andantiangiogenic effects,” Clinical Cancer Research, vol. 16, no.13, pp. 3420–3430, 2010.

[46] J. D. Zhao, J. Liu, Z. G. Ren et al., “Maintenance of Sorafenibfollowing combined therapy of three-dimensional conformalradiation therapy/intensity-modulated radiation therapy andtranscatheter arterial chemoembolization in patients withlocally advanced hepatocellular carcinoma: a phase I/IIstudy,” Radiation Oncology, vol. 5, no. 1, article 12, 2010.

[47] Y. Carmi, E. Voronov, S. Dotan et al., “The role of macro-phage-derived IL-1 in induction and maintenance of angio-genesis,” Journal of Immunology, vol. 183, no. 7, pp. 4705–4714, 2009.

[48] T. Sakurai, G. He, A. Matsuzawa et al., “Hepatocyte necrosisinduced by oxidative stress and IL-1α release mediatecarcinogen-induced compensatory proliferation and livertumorigenesis,” Cancer Cell, vol. 14, no. 2, pp. 156–165, 2008.

[49] G. Germano, P. Allavena, and A. Mantovani, “Cytokines asa key component of cancer-related inflammation,” Cytokine,vol. 43, no. 3, pp. 374–379, 2008.

[50] F. Kubo, S. Ueno, K. Hiwatashi et al., “Interleukin 8 inhuman hepatocellular carcinoma correlates with cancer cellinvasion of vessels but not with tumor angiogenesis,” Annalsof Surgical Oncology, vol. 12, no. 10, pp. 800–807, 2005.

[51] Y. Mizukami, W. S. Jo, E. M. Duerr et al., “Induction ofinterleukin-8 preserves the angiogenic response in HIF-1α-deficient colon cancer cells,” Nature Medicine, vol. 11, no. 9,pp. 992–997, 2005.

[52] D. J. Brat, A. C. Bellail, and E. G. Van Meir, “The role ofinterleukin-8 and its receptors in gliomagenesis and tumoralangiogenesis,” Neuro-Oncology, vol. 7, no. 2, pp. 122–133,2005.

[53] M. Grunewald, I. Avraham, Y. Dor et al., “VEGF-inducedadult neovascularization: recruitment, retention, and role ofaccessory cells,” Cell, vol. 124, no. 1, pp. 175–189, 2006.

[54] W. Li, E. Gomez, and Z. Zhang, “Immunohistochemicalexpression of stromal cell-derived factor-1 (SDF-1) andCXCR4 ligand receptor system in hepatocellular carcinoma,”Journal of Experimental and Clinical Cancer Research, vol. 26,no. 4, pp. 527–533, 2007.

[55] T. Mansuroglu, P. Ramadori, J. Dudás et al., “Expression ofstem cell factor and its receptor c-Kit during the development

ISRN Cell Biology 9

of intrahepatic cholangiocarcinoma,” Laboratory Investiga-tion, vol. 89, no. 5, pp. 562–574, 2009.

[56] A. X. Zhu, D. G. Duda, D. V. Sahani, and R. K. Jain, “HCCand angiogenesis: possible targets and future directions,”Nature Reviews Clinical Oncology, vol. 8, no. 5, pp. 292–301,2011.

[57] P. B. Chapman, A. Hauschild, C. Robert et al., “Improvedsurvival with vemurafenib in melanoma with BRAF V600Emutation,” The New England Journal of Medicine, vol. 364,no. 26, pp. 2507–2516, 2011.

[58] C. S. Karapetis, S. Khambata-Ford, D. J. Jonker et al., “K-ras mutations and benefit from cetuximab in advancedcolorectal cancer,” The New England Journal of Medicine, vol.359, no. 17, pp. 1757–1765, 2008.

[59] D. J. Slamon, B. Leyland-Jones, S. Shak et al., “Use ofchemotherapy plus a monoclonal antibody against her2 formetastatic breast cancer that overexpresses HER2,” The NewEngland Journal of Medicine, vol. 344, no. 11, pp. 783–792,2001.

[60] P. Carmeliet and R. K. Jain, “Molecular mechanisms andclinical applications of angiogenesis,” Nature, vol. 473, no.7347, pp. 298–307, 2011.

[61] H. F. Dvorak, “Vascular permeability factor/vascularendothelial growth factor: a critical cytokine in tumorangiogenesis and a potential target for diagnosis andtherapy,” Journal of Clinical Oncology, vol. 20, no. 21, pp.4368–4380, 2002.

[62] N. Ferrara, H. P. Gerber, and J. LeCouter, “The biology ofVEGF and its receptors,” Nature Medicine, vol. 9, no. 6, pp.669–676, 2003.

[63] J. Folkman, “Tumor angiogenesis: therapeutic implications,”The New England Journal of Medicine, vol. 285, no. 21, pp.1182–1186, 1971.

[64] J. Folkman, “Angiogenesis: an organizing principle for drugdiscovery?” Nature Reviews Drug Discovery, vol. 6, no. 4, pp.273–286, 2007.

[65] P. Carmeliet, “Angiogenesis in life, disease and medicine,”Nature, vol. 438, no. 7070, pp. 932–936, 2005.

[66] P. Carmeliet, V. Ferreira, G. Breier et al., “Abnormal bloodvessel development and lethality in embryos lacking a singleVEGF allele,” Nature, vol. 380, no. 6573, pp. 435–439, 1996.

[67] N. Ferrara, K. Carver-Moore, H. Chen et al., “Heterozygousembryonic lethality induced by targeted inactivation of theVEGF gene,” Nature, vol. 380, no. 6573, pp. 439–442, 1996.

[68] R. K. Jain, “Molecular regulation of vessel maturation,”Nature Medicine, vol. 9, no. 6, pp. 685–693, 2003.

[69] M. Mazzone, D. Dettori, R. Leite de Oliveira et al., “Het-erozygous deficiency of PHD2 restores tumor oxygenationand inhibits metastasis via endothelial normalization,” Cell,vol. 136, no. 5, pp. 839–851, 2009.

[70] J. C. Chappell, S. M. Taylor, N. Ferrara, and V. L. Bautch,“Local guidance of emerging vessel sprouts requires solubleFlt-1,” Developmental Cell, vol. 17, no. 3, pp. 377–386, 2009.

[71] R. J. Levine, S. E. Maynard, C. Qian et al., “Circulatingangiogenic factors and the risk of preeclampsia,” The NewEngland Journal of Medicine, vol. 350, no. 7, pp. 672–683,2004.

[72] S. E. Maynard, J. Y. Min, J. Merchan et al., “Excess placentalsoluble fms-like tyrosine kinase 1 (sFlt1) may contributeto endothelial dysfunction hypertension, and proteinuria inpreeclampsia,” Journal of Clinical Investigation, vol. 111, no.5, pp. 649–658, 2003.

[73] T. Tammela, G. Zarkada, E. Wallgard et al., “BlockingVEGFR-3 suppresses angiogenic sprouting and vascularnetwork formation,” Nature, vol. 454, no. 7204, pp. 656–660,2008.

[74] D. Hanahan and L. M. Coussens, “Accessories to the crime:functions of cells recruited to the tumor microenvironment,”Cancer Cell, vol. 21, pp. 309–322, 2012.

[75] C. Murdoch, M. Muthana, S. B. Coffelt, and C. E. Lewis,“The role of myeloid cells in the promotion of tumourangiogenesis,” Nature Reviews Cancer, vol. 8, no. 8, pp. 618–631, 2008.

[76] J. W. Park, R. S. Kerbel, G. J. Kelloff et al., “Rationale forbiomarkers and surrogate end points in mechanism-drivenoncology drug development,” Clinical Cancer Research, vol.10, no. 11, pp. 3885–3896, 2004.

[77] J. M. Collins, “Imaging and other biomarkers in earlyclinical studies: one step at a time or re-engineering drugdevelopment?” Journal of Clinical Oncology, vol. 23, no. 24,pp. 5417–5419, 2005.

[78] S. M. Galbraith, “Antivascular cancer treatments: Imagingbiomarkers in pharmaceutical drug development,” BritishJournal of Radiology, vol. 76, no. 1, pp. S83–S86, 2003.

[79] D. W. Miles, S. L. de Haas, L. Dirix et al., “Plasmabiomarker analyses in the AVADO phase III randomizedstudy of first-line bevacizumab + docetaxel in patients withhuman epidermal growth factor receptor (HER) 2-negativemetastatic breast cancer,” Cancer Research, vol. 70, abstractP2-16-04, 2010.

[80] A. Dowlati, R. Gray, D. H. Johnson, J. H. Schiller, J. Brahmer,and A. B. Sandler, “Prospective correlative assessment ofbiomarkers in E4599 randomized phase II/III trial of carbo-platin and paclitaxel ± bevacizumab in advanced non-smallcell lung cancer (NSCLC),” Journal of Clinical Oncology, vol.24, p. 7027, 2006.

[81] A. M. Jubb, H. I. Hurwitz, W. Bai et al., “Impact of vascularendothelial growth factor-A expression, thrombospondin-2expression, and microvessel density on the treatment effectof bevacizumab in metastatic colorectal cancer,” Journal ofClinical Oncology, vol. 24, no. 2, pp. 217–227, 2006.

[82] C. Bernaards, P. Hegde, D. Chen et al., “Circulating vas-cular endothelial growth factor (VEGF) as a biomarker forbevacizumab-based therapy in metastatic colorectal, non-small cell lung, and renal cell cancers: analysis of phase IIIstudies,” Journal of Clinical Oncology, vol. 28, supplement,abstract 10519, no. 15, 2010.

[83] E. Van Cutsem, S. de Haas, Y. K. Kang et al., “Bevacizumab incombination with chemotherapy as first-line therapy inadvanced gastric cancer: a biomarker evaluation from theAVAGAST randomized phase III trial,” Journal of ClinicalOncology, vol. 30, pp. 2119–2127, 2012.

[84] G. C. Jayson, S. de Haas, P. Delmar et al., “Evaluationof plasma VEGFA as a potential predictive pan-tumourbiomarker for bevacizumab,” European Journal of Cancer, vol.47, article S96, 2011.

[85] N. S. Horowitz, R. T. Penson, D. G. Duda et al., “Safety,efficacy and biomarker exploration in a phase II studyof bevacizumab, oxaliplatin and gemcitabine in recurrentMüllerian carcinoma,” Clinical Ovarian Cancer, vol. 4, no. 1,pp. 26–33, 2011.

[86] L. Xu, D. G. Duda, E. di Tomaso et al., “Direct evidence thatbevacizumab, an anti-VEGF antibody, up-regulates SDF1α,CXCR4, CXCL6, and neuropilin 1 in tumors from patientswith rectal cancer,” Cancer Research, vol. 69, no. 20, pp. 7905–7910, 2009.

10 ISRN Cell Biology

[87] C. H. Lieu, H. T. Tran, Z. Jiang et al., “The association ofalternate VEGF ligands with resistance to anti-VEGF therapyin metastatic colorectal cancer,” Journal of Clinical Oncology,vol. 29, supplement, abstract 3533, 2011.

[88] D. G. Duda, C. G. Willett, M. Ancukiewicz et al., “Plasmasoluble VEGFR-1 is a potential dual biomarker of responseand toxicity for bevacizumab with chemoradiation in locallyadvanced rectal cancer,” Oncologist, vol. 15, no. 6, pp. 577–583, 2010.

[89] T. T. Batchelor, D. G. Duda, E. di Tomaso et al., “Phase IIstudy of cediranib, an oral pan-vascular endothelial growthfactor receptor tyrosine kinase inhibitor, in patients withrecurrent glioblastoma,” Journal of Clinical Oncology, vol. 28,no. 17, pp. 2817–2823, 2010.

[90] E. R. Gerstner, K. E. Emblem, A. S. Chi et al., “Effectsof cediranib, a VEGF signaling inhibitor, in combination withchemoradiation on tumor blood flow and survival in newlydiagnosed glioblastoma,” Journal of Clinical Oncology, vol. 30,supplement, abstract 2009, 2012.

[91] J. A. Meyerhardt, M. Ancukiewicz, T. A. Abrams et al., “PhaseI study of cetuximab, irinotecan, and vandetanib (ZD6474)as therapy for patients with previously treated metastasticcolorectal cancer,” PLoS ONE, vol. 7, Article ID e38231, 2012.

[92] S. M. Tolaney, D. G. Duda, Y. Boucher et al., “A phase IIstudy of preoperative (preop) bevacizumab (bev) followedby dose-dense (dd) doxorubicin (A)/cyclophosphamide(C)/paclitaxel (T) in combination with bev in HER2-negativeoperable breast cancer (BC),” Journal of Clinical Oncology,vol. 30, supplement, abstract 1026, 2012.

[93] A. X. Zhu, M. Ancukiewicz, J. G. Supko et al., “Clinical, phar-macodynamic (PD), and pharmacokinetic (PK) evaluationof cediranib in advanced hepatocellular carcinoma (HCC):a phase II study (CTEP, 7147),” Journal of Clinical Oncology,vol. 30, supplement, abstract 4112, 2012.

[94] A. X. Zhu, D. V. Sahani, D. G. Duda et al., “Efficacy, safety,and potential biomarkers of sunitinib monotherapy inadvanced hepatocellular carcinoma: a phase II study,” Journalof Clinical Oncology, vol. 27, no. 18, pp. 3027–3035, 2009.

[95] D. Lambrechts, B. Claes, P. Delmar et al., “VEGF pathwaygenetic variants as biomarkers of treatment outcome withbevacizumab: an analysis of data from the AViTA andAVOREN randomised trials,” The Lancet Oncology, vol. 13,no. 7, pp. 724–733, 2012.

[96] W. S. Kamoun, C. D. Ley, C. T. Farrar et al., “Edema controlby cediranib, a vascular endothelial growth factor receptor-targeted kinase inhibitor, prolongs survival despite persistentbrain tumor growth in mice,” Journal of Clinical Oncology,vol. 27, no. 15, pp. 2542–2552, 2009.

[97] F. Winkler, S. V. Kozin, R. T. Tong et al., “Kinetics of vascularnormalization by VEGFR2 blockade governs brain tumorresponse to radiation: role of oxygenation, angiopoietin-1,and matrix metalloproteinases,” Cancer Cell, vol. 6, no. 6, pp.553–563, 2004.

[98] E. di Tomaso, M. Snuderl, W. S. Kamoun et al., “Glioblas-toma recurrence after cediranib therapy in patients: lackof “rebound” revascularization as mode of escape,” CancerResearch, vol. 71, no. 1, pp. 19–28, 2011.

[99] A. G. Sorensen, T. T. Batchelor, W. T. Zhang et al., “A “vascu-lar normalization index” as potential mechanistic biomarkerto predict survival after a single dose of cediranib in recurrentglioblastoma patients,” Cancer Research, vol. 69, no. 13, pp.5296–5300, 2009.

[100] E. O. Hanrahan, H. Y. Lin, E. S. Kim et al., “Distinct patternsof cytokine and angiogenic factor modulation and markers of

benefit for vandetanib and/or chemotherapy in patients withnon-small-cell lung cancer,” Journal of Clinical Oncology, vol.28, no. 2, pp. 193–201, 2010.

[101] T. T. Batchelor, A. G. Sorensen, E. di Tomaso et al.,“AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor,normalizes tumor vasculature and alleviates edema inglioblastoma patients,” Cancer Cell, vol. 11, no. 1, pp. 83–95,2007.

[102] D. G. Duda, S. V. Kozin, N. D. Kirkpatrick, L. Xu, D. Fuku-mura, and R. K. Jain, “CXCL12 (SDF1α)-CXCR4/CXCR7pathway inhibition: an emerging sensitizer for anticancertherapies?” Clinical Cancer Research, vol. 17, no. 8, pp. 2074–2080, 2011.

[103] C. P. Raut, Y. Boucher, D. G. Duda et al., “Effects of sorafenibon intra-tumoral interstitial fluid pressure and circulatingbiomarkers in patients with refractory sarcomas (NCI pro-tocol 6948),” PLoS ONE, vol. 7, Article ID e26331, 2012.

[104] C. G. Willett, D. G. Duda, E. Di Tomaso et al., “Efficacy,safety, and biomarkers of neoadjuvant bevacizumab, radia-tion therapy, and fluorouracil in rectal cancer: a multidisci-plinary phase II study,” Journal of Clinical Oncology, vol. 27,no. 18, pp. 3020–3026, 2009.

[105] S. Kopetz, P. M. Hoff, J. S. Morris et al., “Phase II trialof infusional fluorouracil, irinotecan, and bevacizumab formetastatic colorectal cancer: efficacy and circulating angio-genic biomarkers associated with therapeutic resistance,”Journal of Clinical Oncology, vol. 28, no. 3, pp. 453–459, 2010.

[106] D. Foernzler, P. Delmar, M. Kockx, J. Cassidy, L. Saltz,and S. Scherer, “Tumor tissue based biomarker analysis inNO16966: a randomized phase III study of first-line beva-cizumab in combination with oxaliplatin-based chemother-apy in patients with mCR,” Gastrointestinal Cancers Sympo-sium Proceedings Abstract 374, 2010.

[107] S. X. Yang, S. M. Steinberg, D. Nguyen, T. D. Wu, Z. Mod-rusan, and S. M. Swain, “Gene expression profile and angio-genic marker correlates with response to neoadjuvant beva-cizumab followed by bevacizumab plus chemotherapy inbreast cancer,” Clinical Cancer Research, vol. 14, no. 18, pp.5893–5899, 2008.

[108] C. G. Willett, Y. Boucher, D. G. Duda et al., “Surrogate mark-ers for antiangiogenic therapy and dose-limiting toxicities forbevacizumab with radiation and chemotherapy: continuedexperience of a phase I trial in rectal cancer patients,” Journalof Clinical Oncology, vol. 23, no. 31, pp. 8136–8139, 2005.

[109] S. B. Wedam, J. A. Low, S. X. Yang et al., “Antiangiogenic andantitumor effects of bevacizumab in patients with inflamma-tory and locally advanced breast cancer,” Journal of ClinicalOncology, vol. 24, no. 5, pp. 769–777, 2006.

[110] S. Goel, D. G. Duda, L. Xu et al., “Normalization of thevasculature for treatment of cancer and other diseases,”Physiological Reviews, vol. 91, no. 3, pp. 1071–1121, 2011.

[111] R. K. Jain, “Normalizing tumor vasculature with anti-angio-genic therapy: a new paradigm for combination therapy,”Nature Medicine, vol. 7, no. 9, pp. 987–989, 2001.

[112] R. K. Jain, “Normalization of tumor vasculature: an emergingconcept in antiangiogenic therapy,” Science, vol. 307, no.5706, pp. 58–62, 2005.

[113] R. K. Jain, “Taming vessels to treat cancer,” Scientific Ameri-can, vol. 298, no. 1, pp. 56–63, 2008.

[114] W. L. Ince, A. M. Jubb, S. N. Holden et al., “Association ofk-ras, b-raf, and p53 status with the treatment effect ofbevacizumab,” Journal of the National Cancer Institute, vol.97, no. 13, pp. 981–989, 2005.

ISRN Cell Biology 11

[115] A. Gerger, A. El-Khoueiry, W. Zhang et al., “Pharmacoge-netic angiogenesis profiling for first-line Bevacizumab plusoxaliplatin-based chemotherapy in patients with metastaticcolorectal cancer,” Clinical Cancer Research, vol. 17, pp. 5783–5792, 2011.

[116] L. lo Giudice, M. Di Salvatore, Astone et al., “Polymorphismsin VEGF, eNOS, COX-2, and IL-8 as predictive markers ofresponse to bevacizumab,” Journal of Clinical Oncology, vol.28, supplement, abstract e13502, 2010.

[117] F. Loupakis, A. Ruzzo, L. Salvatore et al., “Retrospectiveexploratory analysis of VEGF polymorphisms in the predic-tion of benefit from first-line FOLFIRI plus bevacizumab inmetastatic colorectal cancer,” BMC Cancer, vol. 11, article247, 2011.

[118] A. M. Schultheis, G. Lurje, K. E. Rhodes et al., “Polymor-phisms and clinical outcome in recurrent ovarian cancertreated with cyclophosphamide and bevacizumab,” ClinicalCancer Research, vol. 14, no. 22, pp. 7554–7563, 2008.

[119] B. P. Schneider, M. Wang, M. Radovich et al., “Association ofvascular endothelial growth factor and vascular endothelialgrowth factor receptor-2 genetic polymorphisms with out-come in a trial of paclitaxel compared with paclitaxel plusbevacizumab in advanced breast cancer: ECOG 2100,” Jour-nal of Clinical Oncology, vol. 26, no. 28, pp. 4672–4678, 2008.

[120] W. W. Zhang, J. E. Cortes, H. Yao et al., “Predictors of pri-mary imatinib resistance in chronic myelogenous leukemiaare distinct from those in secondary imatinib resistance,”Journal of Clinical Oncology, vol. 27, no. 22, pp. 3642–3649,2009.

[121] D. G. Duda, M. Ancukiewicz, and R. K. Jain, “Biomarkersof antiangiogenic therapy: how do we move from candidatebiomarkers to valid biomarkers?” Journal of Clinical Oncol-ogy, vol. 28, no. 2, pp. 183–185, 2010.

[122] A. X. Zhu, D. G. Duda, D. V. Sahani, and R. K. Jain, “Devel-opment of sunitinib in hepatocellular carcinoma: rationale,early clinical experience, and correlative studies,” CancerJournal, vol. 15, no. 4, pp. 263–268, 2009.

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