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IntroductionIn the 1970s Judah Folkman and colleagues postulated that angiogenesis, the formation of new blood vessels,1 might be a clinically useful process to target in an effort to halt tumour growth. The emerging rationale from this hypothesis was that, first, no tumour can grow without its own blood supply and, therefore, antiangiogenic therapy should be effective for all solid tumour types;2 second, resistance to antiangiogenic therapy would not occur because of the genetic stability of the tumour endothelium and, therefore, anti­angiogenic therapy should render tumours dormant;3,4 and finally, treatment would be safe as proliferating tumour endothelial cells should differentially express target proteins thereby minimising systemic toxicity from antiangiogenic agents (Table 1).5

After more than 10 years of clinical trials, more than 2,000 trials6 and regulatory agency approvals of antiangiogenic agents,7 we now approach a critical point in the field of antiangiogenic therapy, and must face the following questions: as antiangiogenic VEGF inhibitors have not delivered the bene fits envisaged from the initial hypo­theses (Table 1), does that mean that angio­genesis is a target of modest value; that we have not yet developed agents with sufficient antiangiogenic or antivascular effects; or that the vasculature and the tumour popu­lation should be targeted simultaneously? At present, perhaps the most pressing ques­tion facing the field today is: can predictive markers be identified to select the patients most likely to benefit?

The complexity of human cancer genet­ics and epigenetics impacts the benefit that can be achieved with antiangiogenic therapy. Only a minority of patients has disease that responds to single­agent VEGF inhibitors,8,9 and currently we have limited insight into how to select the patients who will benefit. Recent retrospective analyses showing that plasma VEGF concentrations might predict progression­free survival

(PFS) and overall survival in patients with breast, pancreatic and gastric cancers treated with the anti­VEGF monoclonal antibody bevacizumab10,11 have generated optimism in the field and prospective evaluations of the biomarker are ongoing. However, measure ment of VEGF levels does not provide a definitive answer to whether an individual will respond to bevacizumab. It is critical to launch a major, cooperative initiative with new approaches, including molecular, cellular and imaging studies, to identify additional tools that can be used to select patients for VEGF­targeted therapies and the most­appropriate antiangiogenic therapy combinations. In the absence of such an approach, the development of com­bination antiangiogenic regimens will be considerably impaired.

Benefit for some but not allRandomised trials in different epithelial malignancies have reported improved PFS and/or overall survival in patients randomly assigned to receive conventional therapy supplemented with a VEGF inhibitor com­pared with conventional therapy alone.12–15 However, the benefits of these agents are strikingly inconsistent. VEGF inhibitors are active as single agents in renal­cell carci­noma (RCC),15 hepatocellular carcinoma (HCC),14 ovarian,13 and neuroendocrine tumours,9 and glioblastoma (Table 2).16 By contrast, VEGF inhibitors are effec­tive only when combined with cytotoxic chemo therapy in colorectal cancer (CRC),12 non­small­cell lung cancer (NSCLC),17 and breast cancer (although this efficacy is currently contentious18), and ineffective in melanoma,19 pancreatic cancer20,21 and prostate cancer,22 in line with originally reported observations. Nevertheless, VEGF inhibitors have been approved in multiple indications (Table 2) and have become one of the most widely used classes of agents in cancer therapy.

The survival benefits associated with this class of drugs are neither dramatic nor durable, while the promise of minimal toxic­ity has not been observed (Table 1), particu­larly in the case of the low­molecular­weight VEGF receptor tyrosine kinase inhibitors (TKIs). Patients treated with VEGF inhibi­tors frequently experience hypertension and

OPINION

Antiangiogenic therapy—evolving view based on clinical trial resultsGordon C. Jayson, Daniel J. Hicklin and Lee M. Ellis

Abstract | Antiangiogenic therapies that target VEGF or its receptors have become a mainstay of cancer therapy in multiple malignancies. However, the clinical efficacy of these agents is less than originally anticipated and, in most settings, requires the addition of cytotoxic chemotherapy suggesting that, as for other targeted therapies, VEGF inhibitors will require selection of patient subpopulations to achieve maximal clinical benefit. Without the identification and use of predictive biomarkers for VEGF-targeted agents, and other agents that target the vasculature, further improvements in current clinical outcomes are unlikely. Exciting new data presented in 2011 at the ESMO conference showed that retrospective evaluation of plasma concentrations of VEGF-A predicted progression-free survival and/or overall survival benefit from bevacizumab in phase III trials in certain tumour types; prospective evaluation of the assay is required. This endeavour should be followed by further biomarker research, requiring inter-laboratory collaboration and high-quality, adequately powered clinical trials.

Jayson, G. C. et al. Nat. Rev. Clin. Oncol. 9, 297–303 (2012); published online 14 February 2012; doi:10.1038/nrclinonc.2012.8

Competing interestsG. C. Jayson declares an association with the following companies: AstraZeneca, Aveo, Genentech, Merck, Roche. D. J. Hicklin declares an association with the following company: Merck. L. M. Ellis declares an association with the following companies: Genentech, Sanofi-Aventis. See the article online for full details of the relationships.

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proteinuria, which are largely manageable toxic effects, and more rarely experience thromboembolic disease, gastrointestinal perforations, fistula formation, haemor­rhage and leukoencephalopathy, which are obviously more severe toxicities.11,21,22

Clinical studies have also demonstrated that different types of VEGF inhibitors —VEGF­chelating antagonists or VEGFR TKIs—are not interchangeable (Figure 1). No VEGF TKI, when added to chemo­therapy, improved outcomes in patients with advanced­stage and/or metastatic CRC,23,24 breast cancer23 or NSCLC.24,25 In HCC, the TKI sorafenib improved PFS and overall survival,14 whereas another TKI, sunitinib, was found to be too toxic when compared with sorafenib.26 Aflibercept, a soluble VEGF receptor construct, did not benefit patients with NSCLC when combined with chemotherapy,27 whereas bevacizumab did improve treatment­free survival in these patients.17 Recently, aflibercept, when added to chemotherapy, was reported to be of benefit in patients with chemorefractory metastatic CRC.28

Considering the diverse outcomes with VEGF pathway­targeting agents in differ­ent tumour types (Table 2), we must now question their potential mechanisms of action. Many theories have been proposed regarding the mechanism of action of VEGF inhibitors;29 however, given the complexity of tumour biology, it would be naive to attri­bute a single mechanism of action to VEGF inhibitors in multiple tumour types. In RCC, where single­agent activity is observed,15 VEGF inhibitors probably induce vessel regression and prevent vessel growth; that is, have a mostly vascular effect.29 However, in patients with metastatic CRC, single­agent VEGF inhibitors are not effective and

the benefits from cytotoxic–VEGF inhibi­tor combinations are relatively modest.12,30 In metastatic CRC, the mechanism of action of bevacizumab (the only FDA­approved VEGF inhibitor for the treatment of meta­static CRC) is likely due to rapid onset vaso­constriction,31 hypoxia, transient vascular normalisation and other mechanisms.29 The absence of single­agent activity sug­gests that inhibition of angiogenesis or vessel regression may well have a minor role in the mechanism of action of bevacizumab in metastatic CRC.

What is the target cell lineage?VEGF inhibitors are not interchangeable; for example, bevacizumab has shown effi­cacy in CRC and NSCLC, whereas VEGFR TKIs have not; yet all are potent inhibitors of VEGFR2 activity in vitro.32 The infer­ence is that tumour­associated endothelial VEGFR2 might not be the sole target of these agents. However, a significant body of data supports the hypothesis that anti­angiogenic agents have antivascular activ­ity. Biopsies obtained from a small number of patients with breast cancer33 and CRC34 treated with bevacizumab have demon­strated reductions in phospho­VEGFR2, microvessel density, interstitial pressure and improved pericyte coverage, in keeping with the concept of vascular normalisation.35,36 Circulating endothelial cells and their pre­cursors34 are also reduced in patients treated with these agents, and multiple imaging studies—largely based on dynamic contrast­enhanced MRI (DCE­MRI)—have demon­strated reductions in permeability­transfer constants, in keeping with an antivascular effect.37 Thus, there is no doubt that these agents, when active, impact tumour vascu­lar morphology and function. However, the

signalling–receptor complex and signal­ling cascades that lead from VEGFR2 to a vascular phenotype are highly complicated and the interaction with other potentially tractable signalling systems, such as delta­like protein 4–Notch,38 may obscure to some extent the relationship between VEGF and antivascular effects.

Accepting the premise that VEGF inhibi­tors have antiangiogenic activity, the question is whether the sole effect of anti angiogenic agents is mediated through the vascula­ture. This mechanism of action would seem unlikely in view of the previously described data. One hypothesis to explain this would be that certain tumour cells also express VEGFRs, as has been reported in immuno­histochemical and functional studies.39 However, a recent detailed cell­based and tissue­based study challenges that hypo­thesis by demonstrating that the expression of VEGFR2 is almost completely restricted to tumour endoethelium and not tumour cells.40 Tumour biology also varies over time and, therefore, tumour VEGFR expression may be induced transiently through hypoxia or through the plasticity of tumour cells,41 which allows tumour cells to function effec­tively as endothelial cells. Conversely, data have highlighted that endothelial and circu­lating endothelial cells can, under the right conditions, form multi­potent stem cells,42 potentially implicating VEGF biology in a range of cancer­related phenotypes. Other publications, describing a hierarchy of vessels in tumours, raise the possibility that some types of vessels are dependent on VEGF whereas others are not.43,44 Although these arguments do not refute the original antiangiogenic hypotheses, they challenge the concept of a unique and distinct tumour vascular compartment.

If the hypothesis is true that clinically effective antiangiogenic therapy requires activity in endothelial cells and other tumour compartments, then it has implications for how we might manage patients receiving combination regimens who develop pro­gressive disease. In the era of antiangiogenic agent–cytotoxic drug combinations, should we change the antiangiogenic agent and the cytotoxic drug combination at progression or change only one of the components? Limited retrospective clinical data support the concept of maintaining antivascular therapeutics beyond disease progression.45 Although we cannot answer this question conclusively, perhaps we can start to address the following questions: what does vascular progression look like clinically, and can

Table 1 | Predicted and observed clinical effects of antiangiogenic agents

Parameter Anticipated result Actual result based on clinical observations

Tumour response

Induce tumour dormancy in all tumours3,4

Tumour and context dependent:Confirmed RECIST responses to single agent antiangiogenic therapy (RCC,15 pNETs9)Minimal impact as a single agent12 (other solid tumours)Benefit only obtained when combined with cytotoxic therapy (CRC,12 NSCLC,17 breast70 cancer)

Toxicity Minimal toxicity—therapy is limited to affecting ‘activated’ tumour vasculature2–4

Hypertension21,31

ProteinuriaArterial-thromboembolic eventsBowel perforationsFistula formationPosterior leukoencephalopathy

Resistance No resistance to therapy3 Tumours become resistant and progress after initial response12 (when it occurs)

Abbreviations: CRC, colorectal cancer; NSCLC, non-small-cell lung cancer; pNETs, pancreatic neuroendocrine tumours; RCC, renal-cell carcinoma.

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we distinguish vascular progression from tumour compartment progression? We can then determine if these concepts are clini­cally useful. Carefully conducted multiple­laboratory, adequately powered studies and/or trials will be critical if we are to under­stand how to use antiangiogenic agents in the clinic. Indeed, some studies are currently addressing the value of continuing VEGF blockade but changing the chemotherapy backbone after disease progression when receiving combination therapy.44,45

We have argued that VEGF inhibitors are unlikely to be working through inhibition of tumour endothelial VEGFR2 as their sole mechanism of action. Of course, all VEGFR TKIs have intended and unintended targets as they are analogues of ATP and will, therefore, impact a range of tyrosine kinase receptors, hence explaining their off­target toxic effects. Yet, as clinicians, we have only partially defined the impact of these agents on the angiogenic cytokine repertoire in our patients. Perhaps equally importantly, an increasing volume of data has highlighted the importance of the myeloid lineage44 and inflammatory cytokines46 in the regu­lation of VEGF inhibitor efficacy. Of these, G­CSF has been implicated,46 raising con­cerns about the use of G­CSF in patients who are being treated with VEGF inhibi­tors. In addition, because VEGF has a broad role in the immune system47—for example, dendritic­cell maturation,48 and myeloid lineages modulate VEGF biology—this area also requires detailed study. To date, labora­tories focused on vasculature analysis have dominated research in the area. Coordinated international, inter­laboratory efforts (that is, vascular, immune, non­vascular stroma and tumour biology groups) could greatly accelerate and improve our understand­ing of the field allowing us to develop and deliver combinations of mechanism­based antiangiogenic therapeutics, such as inhibi­tors of angiopoietin­1 receptor, c­Met, Notch ligand, Notch, FGF and FGFR, to the right patient. It is, therefore, critical that we elucidate who benefits from VEGF inhibi­tors and in this regard the first reports of predictive biomarkers are just emerging.

Who benefits?Oncology is rapidly moving towards per­sonalised therapies as exemplified by the use of trastuzumab for HER2­amplified breast cancer, imatinib for BCR–ABL­positive chronic myeloid leukaemia and vemurafenib for mutant BRAF melanoma. However, no validated biomarkers exist

to select patients who will benefit from VEGF inhibitors. A number of studies have been undertaken to identify the patients who benefit most from VEGF inhibitors. These investigations have been reviewed,49 and include DCE­MRI and other imaging modalities and measurements of circulat­ing protein and cellular biomarkers, tissue proteins, single­nucleotide polymorphisms, expression arrays50 and early pharmaco­dynamic responses to treatment, for example, hypertension.49 Although initial investigations regarding biomarkers may have been unsuccessful, it is important to point out that the majority of these studies were retro spective and lacked defined hypotheses based on translational research; and many of the initial VEGF inhibitor bio­marker studies may have been flawed by the use of archival primary tumour speci­mens.51 Gene mutations and/or expression in primary tumours and metastases may be discordant.52 Thus, biomarker studies on primary tumours may not be reflective of the biological target. In addition, when pro­spective biomarker studies have been per­formed they are often too underpowered to provide clinically meaningful data.

Despite a global effort involving thou­sands of patients, until October 2011 none of these biomarker studies had provided convincing evidence that the bio markers could predict who will benefit from treat­ment with VEGF inhibitors. Data pre­sented at the European Society for Medical

Oncology (ESMO) annual meeting,10 from a study that assessed biomarkers in pancreatic cancer (AVITA trial53) and gastric cancer (AVAGAST trial54), corroborated those data presented at the San Antonio Breast Cancer Symposium in 2010.11 These important data revealed that a unique anti­VEGF ELISA, which quantified soluble, low­molecular­weight isoforms of VEGF­A, was capable of predicting which patients would benefit from treatment with bevacizumab.11 While the assay was not predictive in all tumour types (RCC, CRC, NSCLC), and does not provide a black and white answer (VEGF levels are divided into quartiles with patients in the highest quartile achieving the great­est benefit), the fact that it was predictive, albeit retrospectively, in patients with breast cancer (PFS), pancreatic cancer (overall survi val) and gastric cancers (overall sur­vival) will potentially change the way we use these agents if these data are confirmed at publication of the peer­reviewed manuscript and validated prospectively.

The aforementioned trials that led to a potential predictive biomarker had assessed bevacizumab, and it is unclear if such a test would help identify which patients should be treated with a VEGFR TKI. However, if a predictive test exists for any of the VEGF inhibitors, how will clinicians use these data? Will we see the widespread incor­poration of these drugs into combination regimens, irrespective of the tumour type, to treat patients whose blood contains

Table 2 | Summary of randomised trial data involving VEGF inhibitors

Cancer type How used or studied Increase in PFS over standard care (months)

Increase RR (%)

FDA approved

Renal-cell carcinoma15 Single agent 3–6 8–30 Yes

Pancreatic neuroendocrine tumour9

Single agent 6 9 Yes

Glioblastoma (phase II)16 Single agent 1–2 15–20 Yes

Hepatocellular carcinoma14

Single agent 1.4–3 2 Yes

Colorectal cancer12,30 In combination with chemotherapy 0–4 0–10 Yes

Non-small-cell lung cancer17

In combination with chemotherapy 0–2 3–15 Yes

Breast cancer70 In combination with chemotherapy 1–6 10–22 Withdrawn

Gastric cancer54 In combination with chemotherapy 1.4 9 No

Prostate cancer22 In combination with chemotherapy 2.4 11 No

Pancreatic cancer21 In combination with chemotherapy 0–1 0–1 No

Melanoma19 In combination with chemotherapy 0–1.4 1–9 No

Ovarian cancer71 In combination with, and after, chemotherapy

1.7–4 NA Pending

Abbreviations: NA, not available; PFS, progression-free survival; RR, response rate.

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elevated concentrations of VEGF as mea­sured through this assay? Conversely, in a patient who has a low plasma concentra­tion of VEGF in this assay, will clinicians and their patients accept exclusion of these drugs in settings where regulatory agen­cies have granted approval? In contrast to the dichotomous results of K­Ras, which predict treatment failure with an anti­EGFR monoclonal antibody in patients with mutant KRAS­containing CRC,55 plasma VEGF is present in a range of concentra­tions. Clearly, prospective evaluation of the assay and validation in settings where beva­cizumab is approved and not approved will be important.

Measurement of plasma VEGF concen­trations might provide some predictive information, but may be confounded by the complex relationship between soluble and matrix­bound VEGF and renal func­tion. Potentially, additional tumour­related biomarkers may augment the predictive power of plasma biomarker tests.56 In parti cular, imaging provides the oppor­tunity to carry out serial studies of the tumour in situ. Although RECIST criteria57 were not developed to assess cytostasis or central cavitation, recent modifications—for example, Choi criteria58 and DCE­MRI

studies59—have been widely implemented to examine this issue. Therefore, it will be attractive to investigate whether, for instance, imaging can supplement the pre­dictive power of the experimental ELISA biomarker test. However, these complex studies are confounded by the pressing need to develop analytical and/or statisti­cal techniques for multi­parameter bio­marker studies that frequently include multiple time points. For example, although VEGF inhibitor­induced time­dependent changes in imaging parameters in patients with CRC liver metastases have been reported,31 clinically meaningful informa­tion is hidden within the mean or median data sets. Rather, it is the individual patient data sets that demonstrate significantly heterogeneous response in magnitude and over time that may be of key importance. That this hetero geneity is of prognostic and pharmacodynamic significance has been shown in multiple studies,37 but remained difficult to quantify until recently.60 On the one hand, merging serial imaging and soluble biomarker data sets for individual patients to determine the predictive power of combined biomarkers remains difficult and requires adequately powered studies. On the other hand, it remains critical to consider at each step whether the costs to both society and the patient of a combina­tion of tests may be too great and whether, ultimately, physicians may adopt a prag­matic approach in which a single predictive test is applied alone.

Resistance to VEGF inhibitorsAt some point during the treatment of patients with advanced­stage disease who are receiving conventional therapy supple­mented with a VEGF inhibitor, disease progression and, therefore, resistance to these agents is likely to occur. At present, it is unknown whether the drugs should be continued beyond initial progression. By analogy with conventional cytotoxic therapy and extending our concept of multi­compartment anticancer therapy, it is likely that within the next 5 years we will switch or augment therapy given at the point of progression or during progression to target alternative angiogenic pathways, for example, the angiopoietin, FGF or c­Met pathways. Whether combination antiangio­genic regimens, such as angiopoietin and VEGF inhibitors, are superior to sequential agents remains to be tested and this could be addressed most efficiently through prospec­tive clinical trials that include insightful and

mechanistic biomarker studies. Predictive biomarkers for each class of antiangiogenic agents are essential for providing optimal benefit for our patients.

Several molecules have been highlighted as potentially mediating intrinsic and/or acquired resistance to VEGF inhibitors. These include FGF2,36 BV8,61 c­Met activa­tion,62 and interleukins and inflammatory mediators.63 Many of these molecules have been detected in trials of multiple drug regi­mens including VEGF inhibitors,64,65 so it is unclear whether these putative biomarkers of resistance represent the response of the disease to the entire regimen or whether the cytokines truly represent vascular resistance mediators. From a scientific perspective, it is critical for investigators to characterize the plasma cytokine repertoire present in patients with disease progression while the patients are still receiving single­agent VEGF inhibitors. Thus, close bio­marker­focused monitoring of patients on maintenance regimens is most likely to be productive. This concept should be applied at the earliest stages to new antiangiogenic agents in development.

Update on the hypothesesThe first postulate was that no tumour can grow without its own blood supply and that antiangiogenic therapy should be effective in all tumour types. Clearly, experimental64 and clinical data65 suggest that this hypothesis is not correct. Experimental data in murine brain metastases from melanoma demon­strate that viable cells that are still in cell cycle can be detected after treatment with a VEGF inhibitor,64 while clinical studies have demonstrated proliferating lung cancer cells in the absence of angiogenesis,65 demon­strating the potential clinical importance of vascular co­option, an unappreciated bio­logical phenotype. Despite the effect that an assay to predict clinical response will have on patient care, in fact the data suggest that universal benefit from VEGF inhibitors is not seen in all tumour types.21,22

Frankly, if a tumour is not angiogenic and its nutrient supply is sustained through co­option of existing vessels, it is unlikely to respond to angiogenic inhibition. There may be subtypes of tumours whose primary oxygen and nutrient supplies are derived from existing vessels, and this situation has been described in tumours growing in vessel­rich organs, such as the lung and liver.66,67

The second hypothesis was that resistance to antiangiogenic therapy will not occur

100 –

90 –

80 –

70 –

60 –

50 –

40 –

30 –

20 –

10 –

0 –

Bevacizumab VEGFR TKIs

Phase II

Phase III

Suc

cess

rat

e (%

)

Figure 1 | Success rate for randomised, phase II or phase III clinical trials of the anti-VEGF monoclonal antibody bevacizumab or the VEGFR TKIs sunitinib, sorafenib and pazopanib. Phase II trials were only included in instances where time-to-progression, progression-free survival or overall survival was the primary end point. Data were derived from TrialTrove clinical trials database, Citeline Intelligent Solutions.6 Abbreviation: TKI, tyrosine kinase inhibitor.

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because of the genetic stability of the endo­thelium and that tumours will be rendered dormant. Again, our clinical experience does not support this hypothesis as, even where we have seen strong initial activity (such as in RCC), tumours eventually become refrac­tory to therapy. Furthermore, some reports have highlighted the potential of the tumour endothelium to acquire genetic defects,68,69 although the clinical significance of these findings is unclear. Adding to this complex­ity, cancer stem cells can acquire endothelial cell phenotypes to function as a component of the tumour vasculature. We are, to date, restricting our discussion to VEGF inhibi­tors, whereas other hitherto undiscovered antiangiogenic agents might be more potent. In addition, the evaluation of radiotherapy–VEGF inhibitor combinations is still at an early stage. Thus, the potential for rendering tumours dormant through as yet unidenti­fied angiogenic mechanisms has not been completely extinguished.

The third hypothesis was that treat­ment would be safe because of the unique

antigens expressed by tumour endothe­lium. Certainly for VEGF receptors, their widespread expression underlies the multi­compartment hypothesis presented in this article. In keeping with this concept is the observation that our patients incur a signifi­cant prevalence of toxic effects, and many of these toxic effects are attributable to targets on quiescent endothelium as demonstrated by the common finding of hypertension in patients receiving VEGF inhibitors.

ConclusionsWe are at a critical point in the develop­ment of antiangiogenic agents with the first reports of a predictive assay for the anti­VEGF antibody, bevacizumab and the potential of agents that target additional vascular targets. However, many open questions remain (Box 1). Whether the proposed multi­compartment theory of tumour treatment is clinically useful and whether combi nation antivascular agents are superior to single­agent VEGF targeted agents will become apparent over the next

few years through the implementation of high quality, inter­laboratory studies that further define mechanisms of action, and the use of translational research to identify robust predictive biomarkers for this class of agents.

Department of Medical Oncology, Christie Hospital and University of Manchester, Wilmslow Road, Manchester Academic Health Sciences Centre, Manchester M20 4BX, UK (G. C. Jayson). Biologics Strategy—Oncology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA (D. J. Hicklin). Departments of Surgical Oncology and Cancer Biology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard‑173, Houston, TX 77230, USA (L. M. Ellis).

Correspondence to: G. C. Jayson gordon.jayson@manchester.ac.uk

1. Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285, 1182–1186 (1971).

2. Langer, R., Brem, H., Falterman, K., Klein, M. & Folkman, J. Isolations of a cartilage factor that inhibits tumor neovascularization. Science 193, 70–72 (1976).

3. Boehm, T., Folkman, J., Browder, T. & O’Reilly, M. S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404–407 (1997).

4. O’Reilly, M. S. et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277–285 (1997).

5. Brem, H. & Folkman, J. Analysis of experimental antiangiogenic therapy. J. Pediatr. Surg. 28, 445–451 (1993).

6. Citeline. TrialTrove [online], http:// informa.citeline.com (2012).

7. National Cancer Institute. Angiogenesis inhibitors—factsheet [online], http:// www.cancer.gov/cancertopics/factsheet/Therapy/angiogenesis-inhibitors (2011).

8. Cannistra, S. A. et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J. Clin. Oncol. 25, 5180–5186 (2007).

9. Raymond, E. et al. Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. N. Engl. J. Med. 364, 501–513 (2011).

10. Jayson, G. C. et al. Evaluation of plasma VEGFA as a potential predictive pan-tumour biomarker for bevacizumab [abstract 804]. Eur. J. Cancer 47 (Suppl. 1), S96 (2011).

11. Miles, D. et al. Plasma biomarker analyses in the AVADO phase III randomized study of first-line bevacizumab + docetaxel in patients with HER2-negative metastatic breast cancer [abstract P2-16-04]. Cancer Res. 70 (Suppl.), 235 (2010).

12. Hurwitz, H. et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, 2335–2342 (2004).

13. Kristensen, G. et al. Result of interim analysis of overall survival in the GCIG ICON7 phase III randomized trial of bevacizumab in women with newly diagnosed ovarian cancer [abstract]. J. Clin. Oncol. 29 (Suppl.), LBA5006 (2011).

Box 1 | Concepts for exploration

Patient selection biomarkers ■ Will the putative bevacizumab predictive biomarker VEGF-A be validated in prospective

studies and can it identify patients that respond to VEGFR tyrosine kinase inhibitors? ■ Will these markers be used routinely to select patients for bevacizumab therapy in the

indications that it is approved for? ■ In diseases where VEGF inhibitors have been approved, will oncologists accept that the

agents do not offer benefit for all patients if predictive biomarker tests suggest no benefit? ■ Combinations of biomarkers should be evaluated for their predictive value in suitably

powered high-quality studies.

Clinical questions ■ Does antiangiogenic therapy work in the pseudo-adjuvant setting, that is, where a complete

response has been achieved? ■ What is the optimum duration of maintenance therapy? ■ Should VEGF inhibitors continue to be administered beyond progression? ■ Can we identify effective second-line antivascular agents on the basis of biomarker assays

that elucidate resistance mechanisms? ■ Should resistance to VEGF inhibitors be addressed through combination with inhibitors

of escape pathways (such as, FGFR inhibitors) or through switching to another agent? ■ To what extent are inflammatory mediators responsible for mediating resistance as opposed

to alternative angiogenic cytokines?

Categorization of angiogenesis or vasculogenesis ■ Can we develop imaging strategies that distinguish vascular co-option; vasculogenesis;

angiogenesis; and the imaging characteristics of tissues containing vessels that are not amenable to VEGF inhibitors?

Validity of the vascular compartment hypothesis ■ Is it useful to think in terms of antitumour and/or antivascular effects in the development

of combination regimens?

Immune versus vascular effects ■ To what extent does the immune and inflammatory system regulate sensitivity for VEGF

inhibitors? ■ Multi-laboratory studies should test the hypothesis that immune effects and vascular effects

are relevant to the clinical activity of VEGF inhibitors. Prospective and sequential evaluation of myeloid (and other immune lineages) infiltration in fresh tumour samples would enable this question to be addressed.

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14. Llovet, J. M. et al. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 (2008).

15. Yang, J. C. 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).

16. Friedman, H. S. et al. Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. J. Clin. Oncol. 27, 4733–4740 (2009).

17. Sandler, A. et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N. Engl. J. Med. 355, 2542–2550 (2006).

18. National Cancer Institute. FDA approval for bevacizumab [online], http://www.cancer.gov/cancertopics/druginfo/fda-bevacizumab (2011).

19. Hauschild, A. et al. Results of a phase III, randomized, placebo-controlled study of sorafenib in combination with carboplatin and paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma. J. Clin. Oncol. 27, 2823–2830 (2009).

20. Kindler, H. L. et al. Gemcitabine plus bevacizumab compared with gemcitabine plus placebo in patients with advanced pancreatic cancer: phase III trial of the Cancer and Leukemia Group B (CALGB 80303). J. Clin. Oncol. 28, 3617–3622 (2010).

21. Van Cutsem, E. et al. Phase III trial of bevacizumab in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. J. Clin. Oncol. 27, 2231–2237 (2009).

22. Kelly, W. K. et al. A randomized, double-blind, placebo-controlled phase III trial comparing docetaxel, prednisone, and placebo with docetaxel, prednisone, and bevacizumab in men with metastatic castration-resistant prostate cancer (mCRPC): survival results of CALGB 90401 [abstract]. J. Clin. Oncol. 28 (Suppl.), LBA4511 (2010).

23. Martin, M. et al. Motesanib, or open-label bevacizumab, in combination with paclitaxel, as first-line treatment for HER2-negative locally recurrent or metastatic breast cancer: a phase 2, randomised, double-blind, placebo-controlled study. Lancet Oncol. 12, 369–376 (2011).

24. Spigel, D. R. et al. Randomized, double-blind, placebo-controlled, phase II trial of sorafenib and erlotinib or erlotinib alone in previously treated advanced non-small-cell lung cancer. J. Clin. Oncol. 29, 2582–2589 (2011).

25. Scagliotti, G. et al. Phase III study of carboplatin and paclitaxel alone or with sorafenib in advanced non-small-cell lung cancer. J. Clin. Oncol. 28, 1835–1842 (2010).

26. Cheng, A. et al. Phase III trial of sunitinib (Su) versus sorafenib (So) in advanced hepatocellular carcinoma (HCC) [abstract]. J. Clin. Oncol. 29 (Suppl.), a4000 (2011).

27. Sanofi-Aventis. Sanofi-Aventis and Regeneron report top-line results from phase III study with aflibercept (VEGF Trap) in second-line non-small cell lung cancer [online], http://www.sanofi.co.uk/l/gb/medias/57180F97-E9EB-4FA1-A508-77091E3164C4.pdf (2011).

28. Van Cutsem, E. et al. Intravenous (IF) aflibercept versus placebo in combination with irinotecan/5-FU (FOLFIRI) for second-line treatment of metastatic colorectal cancer (MCC): results of a multinational phase III trial (EFC10262-VELOUR) [abstract]. Ann. Oncol. 22 (Suppl. 5), aO-0024 (2011).

29. Ellis, L. M. & Hicklin, D. J. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat. Rev. Cancer 8, 579–591 (2008).

30. Saltz, L. B. et al. Bevacizumab in combination with oxaliplatin-based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J. Clin. Oncol. 26, 2013–2019 (2008).

31. O’Connor, J. P. et al. Quantifying antivascular effects of monoclonal antibodies to vascular endothelial growth factor: insights from imaging. Clin. Cancer Res. 15, 6674–6682 (2009).

32. Wedge, S. R. et al. AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res. 65, 4389–4400 (2005).

33. Wedam, S. B. et al. Antiangiogenic and antitumor effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J. Clin. Oncol. 24, 769–777 (2006).

34. Willett, C. G. et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat. Med. 10, 145–147 (2004).

35. Winkler, F. et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell 6, 553–563 (2004).

36. Batchelor, T. T. 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).

37. Jackson, A., O’Connor, J. P., Parker, G. J. & Jayson, G. C. Imaging tumor vascular heterogeneity and angiogenesis using dynamic contrast-enhanced magnetic resonance imaging. Clin. Cancer Res. 13, 3449–3459 (2007).

38. Li, J.-L. et al. DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo. Cancer Res. 71, 6073–6083 (2011).

39. Duff, S. E. et al. Vascular endothelial growth factors and receptors in colorectal cancer: implications for anti-angiogenic therapy. Eur. J. Cancer 42, 112–117 (2006).

40. Smith, N. R. et al. Vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3 are localized primarily to the vasculature in human primary solid cancers. Clin. Cancer Res. 16, 3548–3561 (2010).

41. Ricci-Vitiani, L. et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468, 824–828 (2010).

42. Medici, D. et al. Conversion of vascular endothelial cells into multipotent stem-like cells. Nat. Med. 16, 1400–1406 (2010).

43. Shojaei, F. et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells. Nat. Biotech. 25, 911–920 (2007).

44. Grothey, A. et al. Bevacizumab beyond first progression is associated with prolonged overall survival in metastatic colorectal cancer: results from a large observational cohort study (BRiTE). J. Clin. Oncol. 26, 5326–5334 (2008).

45. US National Library of Medicine. ClinicalTrials.gov [online], http://clinicaltrials.gov/ct2/show/NCT00700102 (2011).

46. Shojaei, F. et al. G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor

refractoriness to anti-VEGF therapy in mouse models. Proc. Natl Acad. Sci. USA 106, 6742–6747 (2009).

47. Motz, G. T. & Coukos, G. The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat. Rev. Immunol. 11, 702–711 (2011).

48. Gabrilovich, D. I. et al. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat. Med. 10, 1096–1103 (1996).

49. Murukesh, N., Dive, C. & Jayson, G. C. Biomarkers of angiogenesis and their role in the development of VEGF inhibitors. Br. J. Cancer 102, 8–18 (2010).

50. Gourley, C. et al. Establishing a molecular taxonomy for epithelial ovarian cancer (EOC) from 363 formalin-fixed paraffin embedded (FFPE) specimens [abstract]. J. Clin. Oncol. 29 (Suppl.), a5000 (2011).

51. Jubb, A. M. et al. Impact of vascular endothelial growth factor-A expression, thrombospondin-2 expression, and microvessel density on the treatment effect of bevacizumab in metastatic colorectal cancer. J. Clin. Oncol. 24, 217–227 (2006).

52. Fabi, A. et al. HER2 protein and gene variation between primary and metastatic breast cancer: significance and impact on patient care. Clin. Cancer Res. 17, 2055–2064 (2011).

53. Van Cutsem, E. et al. Rash as a marker for the efficacy of gemcitabine plus erlotinib-based therapy in pancreatic cancer: results from the AViTA study [abstract]. ASCO Gastrointestinal Cancers Symp. a117 (2009).

54. Kang, Y. et al. AVAGAST: A randomized, double-blind, placebo-controlled, phase III study of first-line capecitabine and cisplatin plus bevacizumab or placebo in patients with advanced gastric cancer (AGC) [abstract]. J. Clin. Oncol. 28 (Suppl. 18), LBA4007 (2011).

55. Khambata-Ford, S. et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J. Clin. Oncol. 25, 3230–3237 (2007).

56. Mitchell, C. L. et al. Identification of early predictive imaging biomarkers and their relationship to serological angiogenic markers in patients with ovarian cancer with residual disease following cytotoxic therapy. Ann. Oncol. 21, 1982–1989 (2010).

57. RECIST. RECIST 1.1 [online], http:// www.recist.com/index.html (2010).

58. Choi, H. et al. Correlation of computed tomography and positron emission tomography in patients with metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of new computed tomography response criteria. J. Clin. Oncol. 25, 1753–1759 (2007).

59. O’Connor, J. P., Jackson, A., Parker, G. J. & Jayson, G. C. DCE-MRI biomarkers in the clinical evaluation of antiangiogenic and vascular disrupting agents. Br. J. Cancer 96, 189–195 (2007).

60. O’Connor, J. P. et al. DCE-MRI biomarkers of tumour heterogeneity predict CRC liver metastasis shrinkage following bevacizumab and FOLFOX-6. Br. J. Cancer 105, 139–145 (2011).

61. Shojaei, F., Singh, M., Thompson, J. D. & Ferrara, N. Role of Bv8 in neutrophil-dependent angiogenesis in a transgenic model of cancer progression. Proc. Natl Acad. Sci. USA 105, 2640–2645 (2008).

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62. You, W.-K. et al. VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet cancer. Cancer Res. 71, 4758–4768 (2011).

63. Hanrahan, E. O. et al. Distinct patterns of cytokine and angiogenic factor modulation and markers of benefit for vandetanib and/or chemotherapy in patients with non-small-cell lung cancer. J. Clin. Oncol. 28, 193–201 (2010).

64. Leenders, W. P. et al. Antiangiogenic therapy of cerebral melanoma metastases results in sustained tumor progression via vessel co-option. Clin. Cancer Res. 10, 6222–6230 (2004).

65. Pezzella, F. et al. Non-small-cell lung carcinoma tumor growth without morphological evidence

of neo-angiogenesis. Am. J. Pathol. 151, 1417–1423 (1997).

66. Stessels, F. et al. Breast adenocarcinoma liver metastases, in contrast to colorectal cancer liver metastases, display a non-angiogenic growth pattern that preserves the stroma and lacks hypoxia. Br. J. Cancer 90, 1429–1436 (2004).

67. Sardari Nia, P., Hendriks, J., Friedel, G., Van Schil, P. & Van Marck, E. Distinct angiogenic and non-angiogenic growth patterns of lung metastases from renal cell carcinoma. Histopathology 51, 354–361 (2007).

68. Hida, K. et al. Tumor-associated endothelial cells with cytogenetic abnormalities. Cancer Res. 64, 8249–8255 (2004).

69. Sheldon, H. et al. New mechanism for notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood 116, 2385–2394 (2010).

70. Miller, K. D. E2100: a phase III trial of paclitaxel versus paclitaxel/bevacizumab for metastatic breast cancer. Clin. Breast Cancer 3, 421–422 (2003).

71. Perren, T. J. et al. A phase 3 trial of bevacizumab in ovarian cancer. N. Engl. J. Med. 365, 2484–2496 (2011).

Author contributionsAll the authors contributed to researching data for the article, discussion of content, and to writing and editing the article prior to submission.

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