Predicting benefit from antiangiogenic

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1 Predicting benefit from antiangiogenic agents in malignancy Adrian M. Jubb*, Adam J. Oates, Scott Holden and Hartmut Koeppen Abstract A high probability of benefit is desirable to justify the choice of anti-angiogenic therapy from an ever-expanding list of expensive new anticancer agents. However, biomarkers of response to cytotoxic agents are not optimal for predicting benefit from anti-angiogenic drugs. This discussion will focus on both preclinical and clinical research to identify biomarkers for anti-angiogenic therapies that can inform dosing, early clinical benefit, initial drug choice, emerging resistance and second-line treatments. Intussusception The taking up or receiving of one part within another. In reference to angiogenesis, intussusception is a specific term for the division of vessels by transluminal invagination and pillar formation. *Academic Unit of Pathology, Leeds Institute for Molecular Medicine, University of Leeds, UK. Departments of BioOncology and Pathology, Genentech Inc., California, USA. Department of General Surgery, St James s Hospital, Leeds, UK. Correspondence to A.J. adrianjubb@gmail.com doi: /nrc1946 Published online 13 July 2006 Angiogenesis, the sprouting or intussusception of parent blood vessels to form new vessels, is essential for the growth of large tumours 1. Several signalling pathways are involved, although current understanding places greater importance on the vascular-endothelialgrowth-factor (VEGF) pathway 2. VEGF is a ligand produced by tumour cells and associated stroma that functions predominantly on the VEGF receptor 2 (VEGFR2, also known as kinase insert domain receptor (KDR)) to stimulate endothelial migration and proliferation 2. VEGF signalling can be inhibited by antibodies and small molecules that inactivate VEGF or its receptor(s) 2. For example, bevacizumab (Avastin; Genentech and Roche) is a humanized monoclonal antibody that binds the VEGF ligand and prevents receptor binding and signal transduction 2. Bevacizumab has recently been approved as an adjunct to first-line cytotoxic treatment of metastatic colorectal cancer, and is currently being evaluated in other malignancies 3. In addition, many other antiangiogenic agents (TABLE 1) that target the VEGF pathway and other pathways have entered the clinic (for example, PTK787 (vatalanib; Novartis and Schering), ZD6474 (zactima), ZD6126 (AstraZeneca), BAY (sorafenib; Bayer and Onyx), SU5416 (semaxanib), SU11248 (sunitinib) and AG (Pfizer)). Parallel to the progress of these clinical trial programmes, researchers have begun to search for objective measures that indicate pharmacological responses to anti-angiogenic drugs. Such biomarkers should have the potential to better inform dosage, early clinical benefit and the development of resistance. However, anti-angiogenic therapy, either alone or in combination with cytotoxic drugs, has a new mechanism of action, and biomarkers that have previously been shown to be useful for anti-tumour drugs alone are not necessarily relevant. Therefore, new biomarkers that are pertinent to angiogenesis must be identified and validated. The aim of this Review is to critically evaluate the potential of biomarkers to predict benefit from antiangiogenic therapies in clinical trials. In addition, the utility of preclinical models to identify clinically relevant biomarkers is discussed. Angiogenesis 2, the development of anti-angiogenic agents 4 and predictive biomarkers of cytotoxic chemotherapeutics 5 have been comprehensively reviewed elsewhere and will not be discussed further. The need for biomarkers A successful clinical trial programme has shown that the addition of bevacizumab to first-line chemotherapy in unselected populations is significantly beneficial. Why, therefore, is there a need for new objective measures to indicate pharmacological responses to anti-angiogenic drugs? Optimal biological dose. With cytotoxic drugs, administration of the maximum tolerated dose (that is, the upper limit of the therapeutic range) is usually associated with maximum clinical benefit. However, there is evidence to suggest that the maximum tolerated dose of anti-angiogenic agents is not necessarily the most effective 6. For example, in a phase II trial of bevacizumab in colorectal cancer, dosing at 5 mg per kg seemed to be more efficacious than 10 mg per kg (REF. 7), but the toxicity of bevacizumab is dose-dependent 7. It has been suggested that higher doses of anti-angiogenic drugs might lead to excessive vascular pruning, which could reduce the penetration of cytotoxic drugs into solid tumours, thereby potentially reducing efficacy 6. Therefore, for certain indications the optimal biological dose is 626 AUGUST 2006 VOLUME 6

2 At a glance The addition of anti-angiogenic agents to cytotoxic chemotherapy prolongs patient survival in specific types of cancer. Objective response is a poor estimate of probable benefit from the addition of antivascular-endothelial-growth-factor (VEGF ) therapy in colorectal cancer. The cost and choice of cancer therapy are increasing dramatically. Therefore, a high likelihood of benefit is needed to justify the use of a targeted anti-angiogenic agent. Anti-angiogenic therapies induce a number of biological and physiological changes in preclinical models of cancer. Similar observations have been reported in human disease, and might function as early indicators of survival benefit. Resistance to anti-vegf therapy has been noted in human and animal models. Understanding the mechanisms of resistance will be essential when choosing secondline therapies. The optimal biological dose of anti-angiogenic therapy is likely to be lower than the maximum tolerated dose used for cytotoxic chemotherapy. Biomarkers could be needed to guide adequate dose selection and maximize potential benefit. A better understanding of the mechanism of anti-angiogenic therapy is essential to guide the development of biomarkers, drug choice and dosage, and improve survival. probably lower than the maximum tolerated dose, and could spare patients from unnecessary toxicity. Furthermore, tumour uptake of certain anti-angiogenic drugs varies between patients and tumour types 8. Careful titration of drug dosage, to achieve equivalent tumour exposure, is likely to result in increased efficacy. Early surrogate measures of benefit. Clinical trials for cytotoxic drugs are often powered to show an improvement in the objective response rate (the disappearance of all known disease or a decrease in total tumour size of 50% for at least 4 weeks, without the appearance of new lesions). However, anti-angiogenic drugs are cytostatic in action, and tumour shrinkage is likely to be an insensitive estimate of efficacy 9. A survival benefit from the addition of bevacizumab to cytotoxic chemotherapy is apparent in metastatic colorectal cancer irrespective of whether patients achieve an objective response 9 (FIG. 1). Strategies that discontinue antiangiogenic drugs in patients without an objective tumour response can compromise clinical benefit. To appropriately characterize the treatment effect of antiangiogenic agents, new measures of objective vascular response are needed. Resistance. Anti-angiogenic therapy targets endothelial cells that are presumed to be genetically stable, and that were initially thought unlikely to develop resistance. However, it is apparent that the median survival benefit associated with the addition of bevacizumab to first-line chemotherapy is subsequently lost with the acquisition of resistance and disease progression in metastatic colorectal cancer 3. In a large randomized controlled trial, the progression-free survival curves for Cytostatic The slowing or cessation of cellular replication in response to an intervention, such as a drug. Table 1 Mechanisms of action of the drugs discussed in this article Drug AG BAY Bevacizumab Cetuximab DC101 Erlotinib SU5416 (semaxanib) BAY (sorafenib) SU6668 SU11248 (sunitinib) Trastuzumab PTK787 (vatalanib) ZD6474 (zactima) ZD6126 ZD1839 Mechanism of action Small-molecule tyrosine kinase inhibitor against all vascular-endothelial-growth-factor receptors (VEGFRs), platelet-derived growth factor receptor-β (PDGFRβ) and KIT Small-molecule tyrosine kinase inhibitor against VEGFR2 and PDGFR Monoclonal antibody against VEGF. US food and drug administration (FDA) approval was granted on 26 February 2004 for the first-line treatment of patients with metastatic colorectal cancer (in combination with intravenous 5-fluorouracil-based chemotherapy) Monoclonal antibody against epidermal growth factor receptor (EGFR). FDA approval granted 12 February 2004 Monoclonal antibody against VEGFR2 A small-molecule tyrosine kinase inhibitor against EGFR. FDA approval granted 18 November 2004 Small-molecule tyrosine kinase inhibitor against VEGFR1, VEGFR2, KIT and FMS-like tyrosine kinase 3 (FLT3). Withdrawn from clinical trials because of a lack of efficacy Small-molecule tyrosine kinase inhibitor against Raf kinases, VEGFR2, PDGFRβ, FLT3 and KIT. FDA approval granted 20 December 2005 for the treatment of patients with advanced renal-cell carcinoma Small-molecule tyrosine kinase inhibitor against VEGFR2 and PDGFRβ Small-molecule tyrosine kinase inhibitor against VEGFR2, PDGFRβ, FLT3 and KIT. FDA approval granted 26 January 2006 for the treatment of gastrointestinal stromal tumour after disease progression or resistance to imatinib mesylate Monoclonal antibody against human epidermal growth factor receptor 2 (HER2). FDA approval granted 25 September 1998 Small-molecule tyrosine kinase inhibitor against VEGFR1, VEGFR2, VEGFR3, PDGFRβ and KIT Small-molecule tyrosine kinase inhibitor against VEGFR2, EGFR and RET A tubulin-binding vascular-disrupting agent A small-molecule tyrosine kinase inhibitor against EGFR NATURE REVIEWS CANCER VOLUME 6 AUGUST

3 n Hazard ratio (95% CI) All patients ( ) Responders ( ) Non-responders ( ) Progression-free survival (%) Forest plot Figure 1 Progression-free survival benefit from the addition of bevacizumab to first-line chemotherapy (irinotecan, 5-fluorouracil and leucovorin) in metastatic colorectal cancer. Data are shown as Forest plots, showing the hazard ratio for progression-f ree survival associated with the addition of bevacizumab to IFL (irinotecan, 5-fluorouracil and leucovorin), compared with IFL and a placebo. When taking all patients into account, the addition of bevacizumab was associated with a decreased hazard ratio (that is, a longer progression-free survival). Patients were then divided into two subsets: those who achieved an objective response (responders; disappearance of all known disease or a decrease in total tumour size of 50% for at least 4 weeks, without the appearance of new lesions) and those who did not achieve an objective response (non-responders) 9. Both subsets had similar baseline characteristics. A similar incremental survival benefit associated with the addition of bevacizumab can be seen in both responders and non-responders (interaction P value = 0.73) 9. These analyses indicate that objective response is not an appropriate measure of the efficacy of bevacizumab in metastatic colorectal cancer. CI, confidence interval; n, number of patients. RIP1-Tag2 A transgenic mouse strain that expresses the simian virus 40 large T antigen (Tag) under the rat insulin II promoter (RIP) in pancreatic islet cells. Carcinomas develop in the pancreatic islets and progress through characteristic stages. bevacizumab and placebo-treated groups eventually meet 3 (FIG. 2). Furthermore, although bevacizumab is an effective first-line agent, its addition to second-line and third-line chemotherapy for metastatic breast cancer failed to confer a significant survival benefit in unselected patients 10. The mechanism of tumour resistance is not yet clear, but several hypotheses have been proposed. First, increasing redundancy of angiogenic factors has been shown with cancer progression, and could circumvent drugs that target specific pathways 11. Second, treatmentresponsive increases in VEGF and other pro-angiogenic Months Initial response Disease progression IFL + bevacizumab IFL + Placebo Figure 2 Kaplan Meier progression-free survival curves showing escape from anti-angiogenic therapy in metastatic colorectal cancer. The median survival benefit of 4.4 months does not persist, with an equivalent fraction of each treatment group (placebo plus IFL (irinotecan, 5-fluorouracil and leucovorin) versus bevacizumab plus IFL) showing progression-free survival at 20 months. Reproduced and adapted with permission from REF Massachusetts Medical Society. All rights reserved. factors could contribute to tumour escape from antiangiogenic treatments 12 (FIG. 3). Third, maturation of the tumour vasculature might render vessels relatively resistant to the effects of therapies that target the VEGF signalling pathway 13 (FIG. 4). Biomarkers that can predict initial or emergent pharmacological resistance, by any or all of these mechanisms, could better inform the choice of anti-angiogenic therapy and decisions about second-line interventions. Model for biomarker-led therapy. It is probable that several different biomarkers will be required to address the unique problems posed by anti-angiogenic drugs. First, biomarkers are needed to help determine the best choice of drug(s) from an ever-expanding list of expensive, targeted agents 14. Such biomarkers will probably derive from molecular and histological analyses of tumour biopsies and resections. Biomarkers are also needed to predict dosing and ongoing benefit, which necessitates frequent, repetitive measurements. So, non-invasive biomarkers will be essential to verify an ongoing benefit from the drug regimen of choice. Should resistance develop, repeat analysis of tumour biopsies after treatment will inform of changes in the angiogenic expression profile of the tumour and give detailed information on vascular histology. These data might then be used to guide the choice of second-line therapy, with appropriate monitoring. In this manner, the intelligent administration of anti-angiogenic drugs could improve outcomes (FIG. 5). Biomarkers to guide drug selection Choice of first-line therapy. The efficacy of first-line anti-angiogenic therapy is likely to depend on several factors, including the tumour stage, the nature of the tumour vascular bed and the origin and genotype of the neoplastic cells. Regarding tumour stage, preclinical and clinical data suggest that the expression of pro- and anti-angiogenic factors changes with tumour progression. Although VEGF seems to be important in the early stages of tumorigenesis 2, progression is often accompanied by altered expression of other angiogenic factors 11. For example, advanced human breast cancers can express at least six different pro-angiogenic factors, including VEGF, acidic and basic fibroblast growth factors (afgf and bfgf), transforming growth factor β1 (TGFβ1), platelet-derived growth factor (PDGF), placental growth factor (PLGF) and pleiotrophin 11. Tumour cells express distinct levels of angiogenic factors 15,16, and the vasculature expresses distinct markers 17 at different stages of tumorigenesis in the RIP1 Tag2 mouse model of pancreatic islet cancer. For example, WNT2 seems to be expressed by the vasculature of neoplastic islets, but not by the vasculature of precursor hyperplastic islets that have activated the angiogenic switch 17. Indeed, discrete anti-angiogenic agents are differentially effective against RIP1 Tag2 neoplasia depending on the tumour stage 18. The mechanism of action of certain drugs provides clues as to which angiogenic mechanisms are important at different stages of tumorigenesis. For example, the release of VEGF, following the remodelling of the extracellular matrix by matrix 628 AUGUST 2006 VOLUME 6

4 VEGF VEGF VEGF VEGF Blood vessels Anti-bFGF Tumour cell Anti-VEGF Anti-VEGF Anti-VEGF PDGFs bfgf PDGFs bfgf Anti-PDGF + anti-vegf Chemo-naive tumour Initial response to first-line anti-angiogenic therapy Induction of different pro-angiogenic factors Vascular remodelling, leading to tumour escape and resistance to anti-vegf therapy Response to second-line anti-angiogenic therapy Figure 3 A putative mechanism of acquired resistance to anti-vegf therapy. Early tumours show expression of vascular endothelial growth factor (VEGF) and associated vasculature. With anti-vegf therapy (such as an antibody to VEGF (bevacizumab), an antibody to VEGF receptor 2 (DC101) or small-molecule inhibitors of VEGF receptor 2 (SU5416)), the vasculature of the tumour decreases and tumour growth is limited. An early increase in VEGF levels might be observed. With continued anti-angiogenic therapy, increased production of other pro-angiogenic molecules (such as basic-fibroblast growth factor (bfgf) and platelet-derived growth factor (PDGF)) has been reported. Treatment with anti-bfgf agents (for example, bfgf TRAP) or anti-pdgf and VEGF agents (such as small-molecule inhibitors of both VEGFR2 and PDGFRβ (SU6668)) result in a renewed anti-angiogenic effect 20,32,39. Reproduced and adapted with permission from REF 68. Pericytes The fibroblastic and smooth muscle-like cells that are found in close contact with endothelium in small blood vessels. They function as regulators of blood vessel formation and function, contributing in particular to vascular integrity. metalloproteinase 9 (MMP9) (REF. 19), is reported to be a component of the RIP1 Tag2 angiogenic switch 15,16,19. Early islet growth might be impaired by the inhibition of MMP9 or VEGF 16,18,19. Nevertheless, the inhibition of VEGF is not effective against established β-cell islet tumours 16,20, which suggests that the vasculature matures with increased pericyte coverage and a wider spectrum of survival signals, thereby reducing dependence on VEGF 20. Perhaps the same is true for human cancer? If so, the question arises: might biomarkers indicate which angiogenic agent will be most effective at an individual tumour stage? The success of targeted therapies, such as trastuzumab (Genentech), is often dependent on the expression of the drug s target by the tumour 21. Given that bevacizumab is a monoclonal antibody with a well-defined target, VEGF 2, it is logical that VEGF expression might predict benefit. However, in retrospective subset analyses, VEGF expression by the primary tumours of metastatic, treatment-refractory breast cancers 22 or metastatic colorectal cancers did not predict benefit from the addition of bevacizumab 23. The reasons for this are not entirely clear. Perhaps VEGF expression by primary tumours is not representative of metastatic disease, but detailed research indicates that they are equivalent 24. Most colorectal cancers express some VEGF 23, and it could be that high versus low levels of expression are not clinically important. Similarly, the expression of both pro-angiogenic (for example, VEGF) and anti-angiogenic (such as thrombospondin II (THBS2)) factors by the tumour stroma did not predict benefit 23. It is apparent that the effects of combination anti-angiogenic and cytotoxic chemotherapy are more complex than originally thought, and there is still much to learn about the mechanism of action of bevacizumab. Indeed, it might be necessary to look at several components of a pro-angiogenic pathway to identify tumours that are more likely to benefit from anti-angiogenic therapy (for example, tumours most likely to benefit would be expected to show ligand overexpression, receptor phosphorylation and the expression of downstream effectors). Wedam and colleagues explored the possibility that activation and/or phosphorylation of VEGFR2, the receptor that mediates the proliferative and migratory effects of VEGF on endothelial cells 2, might predict benefit from bevacizumab 25. However, in their study of inflammatory breast cancer, the authors reported that the tumour cells themselves expressed phosphorylated VEGFR2 (REF. 25). This observation prompted the hypothesis that treatment with bevacizumab has a direct anti-tumour effect in inflammatory breast cancer, with an increase in tumour cell apoptosis and a decrease in the expression of phosphorylated VEGFR2. However, the results of this study are preliminary, and these observations might not apply to other tumour types where bevacizumab has a strictly anti-angiogenic mechanism of action. NATURE REVIEWS CANCER VOLUME 6 AUGUST

5 PECAM αsma β PDGF PDGFR Control Anti-VEGF Anti-VEGF/topo Figure 4 Vascular remodelling and increased expression of PDGF pathway components in xenografts that recur following anti-vegf therapy. Relatively small calibre vessels can be seen in control tumours by immunostaining for the endothelial marker, platelet-endothelial cell adhesion molecule (PECAM, also known as CD31). Following anti-vegf (vascular endothelial growth factor) therapy with bevacizumab or bevacizumab with topotecan (topo), vascular remodelling can be observed with an increase in vessels with large-calibre lumens and many layers of vascular mural cells (immunostaining for α-smooth muscle actin (αsma)). In association with vascular remodelling, an increase in the expression of PDGFβ and PDGFR can be observed in the vasculature (arrows) by in situ hybridization. Reproduced with permission from REF 13. Fenestrated endothelium Endothelial fenestrations are anatomical apertures (with or without a structural diaphragm) that span the width of the cells that line the intimal surface of the cardiovascular and lymphatic systems. The tumour vasculature is the target of anti-angiogenic drugs, which has led to the hypothesis that quantitative or qualitative measures of the vasculature might serve as biomarkers of efficacy. Histological microvessel density has been proposed to predict benefit from angiogenic drugs as it represents the summation of pro- and antiangiogenic influences on the tumour, and has been observed to decrease following anti-angiogenic treatment of rectal cancer 26,27. Nonetheless, a retrospective review of overall microvessel density in the primary cancers of treated colorectal cancer metastases did not predict the survival benefit associated with the addition of bevacizumab to chemotherapy 23. Furthermore, in inflammatory and locally-advanced breast cancer no change in histological microvessel density was reported following combination treatment with bevacizumab and cytotoxic chemotherapy 25. However, these results must be considered preliminary, as there is no standardized method for the measurement of microvessel density and there is considerable potential for selection and observer bias. In addition to quantitative measures of vascularity, the vascular phenotype has been proposed to predict benefit from anti-angiogenic drugs. The vascular beds that support individual tissues are distinct in their structure and function 1, and have different rates of endothelial proliferation 28. However, the tumour vasculature is diverse, and contains a mixture of immature and mature vessels 1. In preclinical studies, therapies that target the VEGF pathway have differential efficacy depending on the organ that supports a tumour s growth. For example, fenestrated endothelium (a phenotype induced by VEGF signalling) 29,30, immature vessels (endothelium without associated pericytes) 26 and endothelium with a high proliferation rate (indicated by angiopoietin-2 expression) 26,27,31 show the greatest reduction after anti- VEGF therapy. Anti-angiogenic therapies that target other pathways might be more efficacious against other types of vessel. For example, mature vasculature with associated pericytes seem to respond better to therapies directed against PDGF signalling 17,20,32. Tumour cell genotype has been proposed to influence susceptibility to anti-angiogenic drugs in tightly controlled preclinical experiments. Yu and colleagues used p53-null and p53-wild-type HCT116 colorectal cancer cell lines to show that p53 status can influence benefit from therapies that target the VEGF pathway 33. Although xenografts of both cell lines responded to an antibody against VEGFR2, the growth delay was significantly greater in p53-wild-type cells 33. Similarly, Ras status has been reported to influence VEGF expression and, theoretically, could influence benefit from anti-vegf therapies 34,35. However, evidence from the clinic so far indicates that p53, KRAS and BRAF status are not relevant to the clinical efficacy of bevacizumab 36. Perhaps this is due to the relative complexity of human cancer compared with preclinical models, which might be resolved by more thorough analyses such as expression profiling. However, to our knowledge there are no publically available data on the use of expression profiles in predicting benefit from anti-angiogenic drugs in solid malignancies. Similarly, there are no clinical or preclinical data in the literature that describe changes in the gene-expression profile after short-term or long-term anti-angiogenic therapy. Further work in this area is essential to test this hypothesis. Much of the clinical work to date has failed to identify biomarkers that could assist in the choice of antiangiogenic therapies 23,36,37. Nonetheless, there is good evidence from preclinical studies that this approach is valid 17,20,32,33. However, it is important to be cautious in the extrapolation of preclinical data to human disease (TABLE 2). Preclinical experiments are often designed to look at the influence of well-defined biological phenomena on drug efficacy. By contrast, human disease is more complex, and might require multiple markers to accurately predict efficacy. Publication bias is also likely to favour preclinical models that show a desired effect, which could be misleading in the clinic. For example, endothelial cell proliferation is notably higher in mice than humans 28,38. Therefore, an anti-angiogenic therapy might have a reduced benefit in human cancer compared with preclinical models. Future work will need to validate preclinical findings in the clinic and improve models to better represent the clinical disease. 630 AUGUST 2006 VOLUME 6

6 First-line Tumour biopsy or resection Tissue biomarker Baseline dynamic biomarker Choice of drug Selection of likely optimal biological dose Dynamic biomarker Dynamic biomarker Cycle 1 Change in biomarker indicating efficacy Cycle 2 Change in biomarker indicating efficacy Cycle 3 No change No change Second-line Tissue biopsy or resection Choice of drug Tissue biomarker Figure 5 A model indicating how biomarkers might guide the use of antiangiogenic therapy in cancer treatment. Tissue biomarkers that measure treatment-induced changes in tumour vasculature and angiogenic gene expression might be useful in the initial selection of anti-angiogenic drugs to target individual tumours. However, they are invasive and are not suited to ongoing estimates of clinical benefit. Dynamic biomarkers of objective vascular response, such as plasma proteins or vascular imaging, are better suited to repetitive measurements and might identify emerging resistance. Nonetheless, such biomarkers are unlikely to identify the changes that underlie resistance to therapy, and cannot be used to guide second-line treatment. Where resistance is observed, tissue-biomarkers can help to select the most appropriate second-line therapy. Choice of second-line therapies. Tumour escape from combination first-line cytotoxic chemotherapy with bevacizumab is evident in metastatic colorectal cancer 3. Biomarkers of tumour escape are likely to involve different angiogenic pathways, and might be useful in choosing second-line anti-angiogenic therapy. Further preclinical work in RIP1 Tag2 (REF. 20) and xenograft 32 tumours has shown that the initial effect (vascular regression and impaired tumour growth) of the inhibition of VEGF signalling (with a VEGFR2 inhibitor, SU5416) is followed by vascular remodelling and resistant tumour growth. In both experiments, tumour relapse was associated with the increased expression of PDGFRβ and other factors 20,32. Paracrine PDGF signalling is thought to occur between pericytes and adjacent endothelial cells, providing survival signals to endothelium in mature vessels and circumventing the requirement for VEGF 13. Treatment of relapsed tumours could not be achieved by the inhibition of VEGFR2 alone; the combined inhibition of both PDGFRβ and VEGFR2 was needed to inhibit the growth of advanced tumours 20,32. In another study that used the RIP1 Tag2 model, the functional blockade of mouse VEGFR2 with an antibody (DC101; ImClone Systems) was able to transiently inhibit tumour growth 39. Subsequent tumour escape was associated with an increase in bfgf expression and was unresponsive to anti-vegfr2 therapy 39. However, the management of these resistant neoplasms with anti-bfgf therapy (bfgf TRAP) was associated with tumour shrinkage or growth delays 39. These results support the investigation of intelligent, multistage inhibition of angiogenesis in mouse models of cancer, which could translate into similar models in human cancer. Initial response to tumour-targeted therapies, such as cetuximab (a monoclonal antibody against epidermal growth factor receptor (EGFR); ImClone Systems and Merck), is followed by relapse in certain preclinical tumours 40. The emergence of resistance is accompanied by increased angiogenesis, which is caused in part by increased VEGF expression 40. Therefore, biomarkers of angiogenesis could also guide the use of anti-angiogenic drugs following first-line treatment with tumourtargeted therapies. In addition, biomarkers can inform intelligent combinations of anti-angiogenic drugs with other targeted agents. This hypothesis is supported by the observation that the combined inhibition of VEGFR2 (with vatalanib) and EGFR (with the small-molecule inhibitor ZD1839) has a co-operative anti-tumour effect in preclinical models 41. In summary, the use of tissue biomarkers to select efficacious anti-angiogenic drugs is still in its infancy. Preclinical data must be validated in human cancer to objectively assess the relevance of putative biomarkers. Nevertheless, there are several promising avenues of research, and it is likely that there are many biomarkers of benefit from anti-angiogenic drugs. Guiding dosage and assessing ongoing benefit Research into the discovery and validation of noninvasive, dynamic biomarkers is at a more advanced stage than that for biomarkers to guide drug selection. Three distinct classes of biomarkers are evident: the measurement of circulating proteins concerned with angiogenesis, estimations of circulating endothelial cells and/or progenitors, and vascular imaging. Circulating proteins. At present, most anti-angiogenic drugs in the clinic target the VEGF pathway. Therefore, it is reasonable to assume that biomarkers relating to the levels and activity of VEGF signalling might offer relevant pharmacodynamic information. VEGF protein can be observed in the serum and plasma of patients with various solid malignancies 42,43. Indeed, high levels of circulating VEGF are associated with the presence and NATURE REVIEWS CANCER VOLUME 6 AUGUST

7 Table 2 Comparison of preclinical models and human cancer Preclinical models High index of endothelial proliferation Putatively, a greater proportion of tumour-associated endothelium is derived from the bone marrow Aggressive, fast-growing tumours; associated vasculature is often immature Relatively simple model systems Published effects of anti-angiogenic drugs often demonstrate tumour shrinkage or growth delays Objective response or stable disease appear to be good measures of clinical benefit; publication bias (?) Metronomic scheduling Chronic administration of chemotherapy at relatively low, non-toxic doses, on a frequent schedule of administration, with no prolonged drug-free breaks. Human disease Lower index of endothelial proliferation A small proportion of tumour-associated endothelium is derived from the bone marrow Typically slower growing tumours; vasculature has had a longer time to mature Complex; more varied genetic background of the tumour host; increased genetic variability among tumours Anti-angiogenic drugs are typically cytostatic Objective response is a poor measure of clinical benefit stage of neoplastic disease 42,43. Perhaps circulating VEGF levels could guide dosing and provide an early indication of benefit. In a retrospective subset analysis, pretreatment plasma levels of VEGF protein did not correlate with benefit from the addition of bevacizumab in metastatic colorectal cancer 37. Similarly, pretreatment plasma VEGF levels were not predictive of benefit from bevacizumab and gemcitabine in advanced pancreatic cancer 44. However, plasma VEGF levels have been reported to increase following treatment with bevacizumab 27, sunitinib (a small-molecule inhibitor of VEGFR2, PDGFRβ, FLT3 and KIT) 45,46, BAY (a small-molecule inhibitor of VEGFR2 and PDGFRβ) 47 or vatalanib 12. In preclinical models 48 and human disease 12, the treatment-associated increase in plasma VEGF occurs in a dose-dependent fashion. Moreover, results from xenograft studies have indicated that plasma VEGF levels, following the inhibition of VEGFR2, are associated with benefit both in terms of tumour size and survival 48. In patients with advanced colorectal cancer, the increase in circulating VEGF following treatment with vatalanib was positively associated with the probability of achieving nonprogressive disease 12. Indeed, the equivalent dose of vatalanib that was associated with the greatest change in plasma VEGF levels in mice carrying xenografts has been reported to be the most efficacious dose in human metastatic colorectal cancer 49. A better understanding of the mechanism responsible for this increase could permit accurate titration of the optimal biological dose and enable changes in the concentration of circulating VEGF to be used as an early biomarker of efficacy. In contrast to antibody or small-molecule drugs, the treatment of patients with an antisense oligonucleotide directed against VEGF mrna results in a decrease in circulating VEGF levels 50. The decrease in circulating VEGF levels showed a nonsignificant negative correlation with an increasing dose of antisense oligonucleotide 50. Determining the threshold of reduction in VEGF levels that correlates with the best outcome and lowest toxicity might indicate the optimal biological dose. Other circulating angiogenic factors have also been seen to change with anti-angiogenic therapy. For example, plasma levels of bfgf increase following treatment with vatalanib, but are not associated with objective response 12. Plasma PLGF (a VEGFR1 ligand) levels are reported to increase after treatment with sunitinib or bevacizumab 27,46. Data from the initial preclinical studies indicate that PLGF signalling through VEGFR1 could help to overcome the effects of anti-vegf therapy 51,52, but this has not yet been verified. By contrast, circulating levels of soluble VEGFR2 are thought to decrease following treatment with sunitinib 45,46 or BAY (REF. 47). The significance of these observations in terms of survival benefit or dosing is not clear. Several other circulating markers (for example, soluble TIE2) do not seem to change with vatalanib treatment in humans 12. Given the complexity of the proteins in circulation, a large-scale analysis is warranted for biomarker discovery. Serum proteomics is a sensitive measure of many different proteins within complex samples. Salmon and colleagues investigated whether serum proteomics could discriminate between radiologically defined responders and progressors in patients with non-small-cell lung cancer receiving combination treatment with erlotinib and bevacizumab 53. A prediction model was developed from this series, and is currently being validated in a new multicentre randomized phase II trial 53. Hopefully, subsequent circulating biomarker identification will yield new tools to predict benefit from treatment combinations of bevacizumab and erlotinib. Circulating endothelial cells. Angiogenesis relies on the formation of new blood vessels by endothelial cells, either by the division of endothelial cells in situ (angiogenic sprouting) or by the recruitment of new endothelial cells from the bone marrow 2. The relative contributions of these sources to tumour-associated angiogenesis vary considerably between animal models and humans, between tumour types and, probably, according to the method of measurement used In humans, it seems that bone-marrow-derived endothelial cells contribute relatively little to the tumour vasculature 55,57, although both distal endothelial recruitment 26,58 and angiogenic sprouting 26,29 are susceptible to anti-angiogenic therapy. Angiogenic sprouting is difficult to assess without biopsy or resection of the tumour material. However, levels of circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) can be readily measured without invasive procedures. In preclinical models, levels of CECs and CEPs are sensitive to pro-angiogenic (for example, VEGF, bfgf and TIE2) and anti-angiogenic (for example, thrombospondin I and anti-vegfr2) influences 58. In the clinic, Willett and colleagues have reported a pervasive decrease in viable CECs 3 days after the treatment of a small series of locally advanced rectal cancers with a single infusion of bevacizumab 26. Twelve days after treatment the number of viable endothelial cells returned to baseline levels 26. Although not specifically anti-angiogenic, metronomic scheduling of 632 AUGUST 2006 VOLUME 6

8 cytotoxic chemotherapy has been reported to function through an anti-angiogenic mechanism 59. Recently, achieving an objective response or stable disease with metronomic chemotherapy for breast cancer has been associated with a post-treatment increase in non-viable CECs 59. So, it would seem that a decrease in viable CECs, or an increase in non-viable CECs (resulting in an overall increase in total CECs compared with the baseline), might function as early biomarkers of efficacy for anti-angiogenic therapy. One proposed mechanism for the observed changes is that antiangiogenic treatments damage and/or kill endothelial cells, either in circulation or in tumour-associated blood vessels, with subsequent release into the circulation 59. In support of this theory, Rugo and colleagues have reported a statistically significant association between a decrease or no change in viable CECs (at 3 weeks after treatment versus the baseline) and prolonged progression-free survival in metastatic breast cancer treated with bevacizumab and erlotinib (9-week follow up) 60. Norden-Zfoni and colleagues performed a similar analysis on metastatic, imatinib-resistant gastrointestinal stromal tumours treated with sunitinib. Patients with an objective response showed a greater rate of increase in mature CECs (it is not clear whether these are viable, non-viable or total CECs) after 6 20 days of therapy than patients with progressive disease 61. Nevertheless, the interaction between between CECs, host angiogenic factors, cytotoxic chemotherapy and anti-angiogenic therapy is a complex one 58. Current understanding of the mechanisms that regulate CECs and their role in angiogenesis is at a very early stage. Moreover, there is some dispute as to the markers that best define the CEC population 62,63. Although early data are promising, further work is needed to better characterize CECs and CEPs and their value as biomarkers of angiogenesis and anti-angiogenesis. Vascular imaging. As anti-angiogenic therapies function on the tumour vasculature, the investigation of associated changes in tumour haemodynamics with imaging techniques could yield reliable biomarkers that predict benefit. Imaging modalities are relatively non-invasive and can assess a larger volume of the tumour than histology, which reduces the potential for observer bias 64. Many imaging techniques are also able to measure objective responses to cytotoxic drugs. Although tumour size is not always relevant to the efficacy of anti-angiogenic treatments, combinations of anti-angiogenic and cytotoxic drugs make it advantageous to simultaneously assess both tumour vascularity (a marker of anti-angiogenic efficacy) and size (a marker of cytotoxic efficacy). There are many imaging modalities available for investigating tumour vascularity, which have been comprehensively reviewed elsewhere 64. However, very few techniques have been evaluated in conjunction with anti-angiogenic therapies in the clinic. There is good evidence to support a potential role for three techniques: dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 8,25,26,65,66, dynamic computed tomography (CT) 26 and positron emission tomography (PET) 8,26. DCE-MRI is a magnetic resonance imaging acquisition strategy that involves several scans over a set volume during the injection of a contrast agent (such as gadolinium-dtpa) to measure constants that are surrogates for blood flow, capillary permeability, capillary surface area and/or extravascular space 64. The effects of many anti-angiogenic agents have been evaluated by DCE-MRI in phase I and II clinical trials, including bevacizumab 25, vatalanib 66, ZD6126 (a tubulin-binding vascular disruptive agent) 65 and AG (a smallmolecule inhibitor of the VEGFRs, PDGFRβ and KIT) 67. Although a standardized method for DCE-MRI has not been decided on and the measured constants are not well defined, the outcomes of these studies are similar. All agents showed an early (within hours or days) dose response effect on DCE-MRI measures of vascularity, which indicates that this method has the potential to function as a marker of early benefit and could help to choose the most appropriate dose of drug 25, Data from Morgan and colleagues 66 indicated that radiographic responses to vatalanib were associated with post-treatment reductions in the bidirectional transfer constant, Ki (a measure of vascularity and permeability), of colorectal cancer liver metastases. Only reductions in Ki of >40% were associated with reductions in tumour size. However, this phase I study did not indicate whether patients with stable or progressive disease, but with smaller reductions in Ki, benefited from anti-angiogenic therapy in terms of prolonged survival 66. Dynamic CT assessment of tumour perfusion is at a similar stage of development to DCE-MRI, and has been shown by Willett and colleagues to indicate an anti-vascular response to bevacizumab within 12 days 26. However, the analysis of tumour metabolism using 18-fluorodeoxyglucose (FDG)-PET did not show an appreciable change at day 12 (REF. 26). Only at the second evaluation (6 7 weeks after completion of the regimen) were decreases in FDG uptake appreciable 26. Although preliminary, these data suggest FDG-PET changes are not immediate enough to function as useful biomarkers of benefit in the context of anti-angiogenic treatments. PET scanning might also be used to examine the pharmacokinetics of appropriately labelled antiangiogenic drugs. Jayson and colleagues have reported marked heterogeneity in the distribution and clearance of an antibody against VEGF (HuMV833) both between and within patients and between and within individual tumours 8. Therefore, determining the optimal biological dose of immunotherapy for each patient might necessitate measuring tumour uptake of the labelled drug, or calculating tumour vascular parameters 8. Whether PET is the best method to inform optimal biological dosing has yet to be determined. In summary, vascular imaging shows promise as a measure of objective vascular responses. However, further evidence is required to determine whether objective vascular responses are informative in terms of patient survival. NATURE REVIEWS CANCER VOLUME 6 AUGUST

9 Summary and concluding remarks It is not yet clear which of the many known and putative effects of anti-angiogenic therapies are most relevant to patient survival 6. The intelligent selection of biomarkers that will best predict a survival benefit requires a better understanding of the mechanism of action of individual anti-angiogenic drugs, and of combination therapy with anti-angiogenic and cytotoxic drugs. Indeed, the most appropriate biomarker will probably vary between different drugs, different tumours and the use of anti-angiogenic monotherapy versus combination therapy. However, there are insufficient follow-up periods and patient numbers in many biomarker studies to determine which mechanism of action is most relevant to overall survival. Nevertheless, there is a growing list of candidate biomarkers, which has paralleled the preclinical and clinical evaluation of anti-angiogenic drugs. Future trial protocols should either screen for potential biomarkers in any of the above categories, or should attempt to validate putative biomarkers from preclinical models. Although anti-angiogenic drugs are efficacious in unselected populations, increasing market competition between targeted therapies is likely to drive the growth of individualized chemotherapy, with a central role for biomarkers. Indeed, biomarkers could become essential to justify the cost of targeted therapies, by increasing the likelihood of benefit to a level that is acceptable to patients and clinicians. 1. Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, (2000). 2. Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, (2004). 3. Hurwitz, H. et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, (2004). 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