Review

Medscape Continuing Medical Education online
Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians the opportunity to earn CME credit. Medscape, LLC is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide CME for physicians. Medscape, LLC designates this educational activity for a maximum of 1.0 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To receive credit, please complete the post-test.

Learning objectives

Upon completion of this activity, participants should be able to:

1. Identify 4 antiangiogenesis drugs approved since 2004 for human use in patients with solid cancers.
2. List markers of clinical progression of cancer.
3. Describe tumors targeted by the 4 approved anti-angiogenesis drugs in clinical trials.
4. Discuss challenges in the development of validated biomarkers of antiangiogenic activity, as well as their potential role in cancer treatment.
5. Identify functional imaging techniques for assessing antiangiogenesis drugs.

Competing interests

The authors, the Journal Editor L Hutchinson and the CME questions author D Lie declared no competing interests.

To complete the questions online and earn continuing education credits, you must be a registered user on Medscape.com. If you are not registered on Medscape.com please click on the New Users: Free Registration link on the top left-hand side of the website to register. Registration is free. For questions regarding the content of this activity, contact the accredited provider for this CME activity: CME@medscape.net. For technical assistance, contact CME@webmd.net.

Bevacizumab

Bevacizumab is a humanized anti-VEGF monoclonal antibody that prevents VEGF from binding to its receptors, thereby inhibiting angiogenesis.^{5, 12} Evidence of antitumor activity was first observed in a randomized, double-blind, phase II trial that compared placebo with bevacizumab (3 mg/kg or 10 mg/kg) in pretreated patients with renal cell carcinoma (RCC). Bevacizumab prolonged TTP in the high-dose group but not in the low-dose group. A recent, randomized, double-blind, phase III study compared bevacizumab and interferon (IFN)-2a with placebo and IFN-2a as first-line therapy, and showed that the bevacizumab combination improved PFS compared with IFN-2a alone.¹³ In patients with advanced colorectal cancer (CRC), bevacizumab in combination with chemotherapy extended TTP and OS leading to approval for clinical use.¹⁴ Pretreatment circulating levels of VEGF did not predict response to bevacizumab in CRC.¹⁵ Subsequently, bevacizumab was shown to improve PFS in metastatic breast cancer, OS in patients with CRC who had received previous treatment and, in combination with chemotherapy, OS in patients with non-small-cell lung cancer.¹⁶

Sorafenib

The small-molecule-kinase inhibitor sorafenib targets wild-type and mutated B-Raf, VEGFR2, VEGFR3, PDGFR-, c-KIT, FLT-3 and p38.⁶ It induces growth arrest and apoptosis of endothelial cells and some tumor cell types. Activity was first reported in a phase II randomized discontinuation trial in patients with RCC, whereby PFS was prolonged compared with placebo (24 weeks versus 6 weeks). Patients were treated for 12 weeks (run-in treatment) and at the end of that period those with stable disease were subsequently randomized to sorafenib or placebo.¹⁷ In a phase III, randomized, double-blind, first-line placebo-controlled trial, sorafenib prolonged PFS of patient with metastatic RCC and was approved by the FDA and European Medicines Evaluation Agency (EMEA) for the treatment of advanced and metastatic RCC.¹⁸ Retrospective analysis showed that high basal VEGF levels (>131 pg/ml) correlated with a poor prognosis and a trend towards greater PFS benefit in sorafenib versus placebo-treated patients.¹⁹

Sunitinib

Sunitinib is a tyrosine-kinase inhibitor (TKI) that targets VEGFR1, VEGFR2 and VEGFR3, PDGFR-/, FLT-3, c-KIT and c-RET.⁷ Sunitinib showed evidence of activity in a phase I trial in many tumors, including thyroid and neuroendocrine cancers, soft tissue sarcoma, gastrointestinal stromal tumor and RCC.²⁰ In a phase II trial in patients with cytokine-refractory RCC, the overall response rate was 34% and median PFS was 8.3 months.²¹ A phase III trial in patients with metastatic RCC showed that sunitinib improved PFS and overall response rate compared with IFN as first-line therapy, and sunitinib was subsequently approved for the treatment of advanced and metastatic RCC.²² Retrospective analysis from a phase II trial in bevacizumab-refractory RCC, suggests that circulating levels of VEGF and sVEGFR3 might be predictive of response to sunitinib: responding patients had lower basal levels of sVEGFR3 (P <0.0318) and a trend towards a greater decrease upon treatment levels (P <0.10) than nonresponding patients.²³

Temsirolimus

Temsirolimus inhibits mTOR, an Akt-target kinase downstream of VEGFR2, which controls cell proliferation, cellular metabolism and survival.²⁴ Activity was demonstrated in a phase I trial in patients with RCC, and was confirmed in phase II studies, improving outcomes when administered either as single agent or in combination with IFN.²⁵ A three-arm, randomized, phase III trial compared temsirolimus, IFN and a combination of the two drugs as first-line treatment in aggressive RCC. Patients who received temsirolimus alone had longer OS and PFS than did patients in either of the other two treatment arms.²⁶

Other studies of antiangiogenic agents

Some of the kinases targeted by sunitinib and sorafenib such as PDGFR, p38, FLT-3, c-KIT, c-RET, are active in cancer cells and promote tumor-cell proliferation, survival and motility. mTOR, the temsirolimus target, is often activated in cancer and has a central role in regulating metabolic events, in particular protein synthesis, which is essential for tumor growth.²⁴ Thus, in addition to antiangiogenic effects, these drugs may have direct effects on cancer cells that contribute to their global antitumor activities.

The use of anti-VEGF agents in CRC has provided varied results. While bevacizumab, in combination with irinotecan, fluorouracil and leucovorin induced a significant increase in OS in patients with advanced, previously untreated CRC,¹⁴ the pan-VEGFR TKI vatalanib (PTK787), combined with the FOLFOX4 regimen, did not produce a survival benefit. In two phase I trials, vatalanib decreased vascular permeability and vascularity, as measured by dynamic contrast-enhanced (DCE) MRI in patients receiving 750 mg/d.²⁷ Although this effect occurred only in patients with liver metastases, it was interpreted as validation of vatalanib activity and was used to support the case for further clinical development. The phase III trial, however, failed to demonstrate efficacy and survival advantage. The reason for this negative result is unclear. A possible explanation might be that the dose and schedule determined in the early trials on the basis of DCE MRI (pharmacodynamic biomarker) and applied to the phase III study were not optimum.

What can we learn from these studies? First, all four approved drugs demonstrated activity in RCC and target the VEGF/VEGFR pathway, albeit to different extents. RCC is the prototype cancer with VEGF-driven angiogenesis owing to frequent mutations in the von Hippel Lindau (VHL) tumor suppressor gene, a negative regulator of hypoxia-inducible-factor-1 (HIF-1) activity and VEGF expression.²⁸ These findings suggest that the match between the targeted pathway and the cancer chosen may be relevant or possibly critical to the success of these drugs. Second, biomarkers are dispensable when clinical results obtained in phase I/II trials are already convincing. By contrast, when the tested agent doesn't achieve tumor control during an early stage of clinical development, a pharmacodynamic biomarker reporting biological activity would constitute an essential requirement to pursue clinical development. The use of a putative pharmacodynamic biomarker that is irrelevant to the main biological activity of the drug or representative of activity in only a specific patient subpopulation, however, might compromise further development.

Circulating angiogenic factors and related molecules

Pretreatment blood levels of VEGF have been tested in many studies. In general, elevated levels are indicative of a poor prognosis, and therefore prognostic, but do not predict response to antiangiogenic drugs, including bevacizumab.^{15, 33, 34} Rising levels of circulating factors (e.g. VEGF and placenta growth factor) were observed in response to antiangiogenic drugs or chemotherapy, possibly reflecting treatment-induced tumor hypoxia.^{7, 35, 36, 37} The practical utility of using drug-induced increases in circulating factors as surrogate biomarkers remains to be demonstrated, and their use might be confounded by increases associated with tumor resistance or escape.

Microvessel density and endothelial-signaling events

Microvessel density (MVD) at regions of intense angiogenesis (i.e. 'hot spots') has prognostic but not predictive value in many cancers.³⁸ MVD does not provide information on the functionality (perfusion) of the tumor vessels. MVD of primary tumors in patients with metastatic CRC did not predict response to bevacizumab.³⁴ The phosphorylation statuses of ERK and AKT in tumor endothelial cells have been explored as biomarkers of antiangiogenic therapy. Phosphorylation of both kinases was observed in angiogenic vessels and was attenuated by treatment with SU6668.³⁹ Since tissue-based biomarkers provide direct information on events at tumor site, they are highly valuable; however, they are impractical as pharmacodynamic biomarkers for routine clinical use and might be applied only to selected studies.

Circulating cells

Increased numbers of circulating endothelial cells (CEC) and bone-marrow-derived circulating endothelial cell progenitors (CECP) are observed in the blood of patients with cancer.^{40, 41} In mice, CECP—mobilized by tumor-derived factors that include VEGF—have proangiogenic activities.¹ Their increased frequency correlates with angiogenesis, and the levels of these cells return to normal following antiangiogenic treatments.⁴² In patients with cancer, elevated CEC numbers returned to normal after tumor removal or following complete remission after chemotherapy.^{41, 43} Bevacizumab reduced the frequency of viable CEC and CECP in patients with rectal cancer,³⁶ while ZD6474, a VEGFR2/EGFR TKI, caused an increase in mature CECs but not CECP, possibly reflecting therapy-induced endothelial cell detachment from tumor vessels.⁴⁴ In patients with metastatic gastrointestinal stromal tumors, those demonstrating a clinical benefit had greater increases in CECs. Sunitinib was reported to cause a greater increase in CEC in patients with gastrointestinal stromal tumor, and was associated with clinical benefits compared with patients with progressive disease.⁴⁵ Further studies are needed to establish the potential clinical value of CEC and CECP as biomarkers of angiogenesis.⁴⁰ The low frequency of these cells in samples, the sophisticated methodology required for their detection, and the difficulty in standardizing protocols may limit the widespread application of this approach in clinical practice. Peripheral blood lymphocytes have been used to demonstrate drug effects on selected signaling pathways. Monitoring ERK phosphorylation in lymphocytes obtained from sorafenib-treated patients and from lymphocytes stimulated in vitro with a phorbol ester provided useful information on the dose necessary to achieve ERK inhibition in vivo.⁴⁶

Functional genomics and proteomics

Approaches based on genomics and proteomics have provided insights into the mechanistic processes of angiogenesis, and facilitated the identification of novel candidate therapeutic targets and allowed the monitoring of drug activities.⁴⁷ Gene-expression profiling was used to identify genes expressed in tumor endothelial cells,^{48, 49} determine transcripts induced by VEGF,⁵⁰ define the angiogenic stage of endothelial cells,⁵¹ and characterize CEC in patients.^{52, 53} Specific gene-expression signatures have been reported in endothelial cells and blood lymphocytes in response to antiangiogenic drugs, such as the VEGFR2 inhibitor SU5416, endostatin, and brivanib alaninate, a VEGFR2/FGFR-1 inhibitor.^{54, 55, 56} This approach should be further explored for its potential to monitor tumor angiogenesis and antiangiogenic drug activity in the clinic. Proteomics-based approaches, although still in their infancy, have been used to identify angiogenesis-related proteins that are expressed in tumor vessels,⁵⁷ tumor interstitial fluid,⁵⁸ and cultured endothelial cells,⁵⁹ and to profile the secretome of endothelial cells.⁶⁰ Proteomic techniques have been applied to profile the substrate selectivity of antiangiogenic drugs, such as SU6668,⁶¹ and to study therapy-induced effects on endothelial cells.⁶² Proteomic analysis of serum proteins is considered a promising approach to identify angiogenesis biomarkers.^{63, 64} While promising, these approaches still face many challenges before entering routine clinical consideration, including the need to obtain high-quality material and the requirement of costly and sophisticated technologies.

Dynamic contrast-enhanced MRI

This technique uses gadolinium-chelate-based agents that cause a decrease of T1 (time of realignment of hydrogen nuclei in the magnetic field) at low concentration and a decrease of T2 (time of loss of transverse magnetism) at high concentration.⁷⁰ T2 imaging is used early during the arterial input to calculate MTT, rBV and rBF, while T1 imaging is used at later time points to estimate K^trans (Figure 1A). DCE-MRI has been used in multiple studies to monitor the effects of antiangiogenic drugs.⁷¹ The area under the curve and K^trans have been used to monitor treatment-induced changes in vascularity and permeability.^{65, 72} While K^trans has been used to measure biological activity (i.e. a pharmacodynamic marker) its value in predicting clinical response (i.e. a surrogate marker) remains to be demonstrated. The main limitations of DCE-MRI include quantification and reproducibility. Quantification by use of pharmacokinetic modeling necessitates calibration to determine the relationship between concentration of the contrast agent and signal intensity;⁷³ however, there is no simple relationship between contrast concentration and signal-intensity, especially at high contrast concentration. The nonlinear signal-intensity correlation is probably responsible for much of the variability in measurements observed with this technique. In addition, data acquisition is highly dependent on the machine characteristics, making comparisons across different platforms difficult.

Dynamic contrast-enhanced CT

This imaging technique uses iodine-based contrast agents to generate a linear relationship between contrast concentration and measured signal. It allows calculation of MTT, rBV, rBF and K^trans (Figure 1B). DCE-CT has not yet been systematically included in clinical studies, in part because of the lack of common protocols and methodologies for data acquisition, standardization and analysis.^{74, 75} There are some reports, however, that suggest the feasibility of this approach.⁷⁶ DCE-CT was used to assess tumor vascularity in rectal cancer and measure perfusion changes after chemoradiotherapy. The results revealed that tumors with high pretherapy rBF values tended to have a poor response to therapy.⁷⁷ The routine use of DCE-CT might be limited by the high X-ray exposure required.

Positron-emission tomography

H₂¹⁵O PET is considered the reference technique for quantification of rBF and vascular permeability.^{70, 78} Injected radioactive water can freely diffuse in the tissue, and a time-concentration curve reflecting radioactive oxygen accumulation can be determined by PET scanning. The requirement for a nearby cyclotron for probe preparation, owing to the short half-life of radioactive oxygen, is a major obstacle to the widespread clinical use of this technique. PET scanning by use of ¹¹C-methionine uptake coupled with DCE-MRI revealed a correlation between vascularity and methionine uptake in brain gliomas.⁷⁹ Combining imaging techniques could allow studies to concomitantly monitor the direct effects of antiangiogenic drugs on tumor vasculature and their secondary effects on tumor metabolism. In addition, PET can be applied to measure tumor hypoxia.⁸⁰

Contrast-enhanced ultrasound

Contrast-enhanced ultrasound is an attractive technique to measure perfusion. The contrast agent consists of phospholipid-based microbubbles that encapsulate an inert gas, which when exposed to an ultrasound pulse, generate nonlinear resonances that allow enhanced representation of the vasculature.⁸¹ Semiquantitative perfusion measurements can be derived from the time-concentration curve, while quantitative measurements can be obtained by local destruction of the microbubbles with a high-pressure acoustic pulse and measurement of the following signal recovery by low pressure acoustic pulses⁸² (Figure 2). The time-intensity curve in each region of interest depends on perfusion and is fitted to a kinetic model to determine volume flow rate.⁸² The accuracy of contrast-enhanced ultrasound for microvascular perfusion measurement and perfusion changes following therapy has been documented both in experimental models^{83, 84} and in patients with cancer.^{85, 86, 87} Contrast-enhanced ultrasound has several advantages over DCE-MRI and DCE-CT: measured values strictly refer to the intravascular compartment and are not confounded by extravascular diffusion; repeat measurements can be taken with equipment that is easily accessible and has limited costs; and patients are not exposed to ionizing radiation.

Figure 2 Measurement of perfusion index in a hepatocellular carcinoma by contrast-enhanced ultrasound-based imaging.

Microbubbles are injected intravenously and after reaching a steady-state they are destroyed by a high-pressure acoustic pulse (white arrow). A low acoustic pressure is applied to obtain a time-concentration curve in a region of interest determined by the user (red circle, region of interest in tumor tissue; yellow circle, region of interest in healthy liver tissue; the blue region indicates the areas of the image processed to compensate for respiration-induced liver movements to obtain a still image). Perfusion parameters may be automatically calculated by software or by postprocessing on a stand-alone work station, as in this example (software supplied by Bracco Research SA, Geneva).⁹¹ A perfusion index representing relative blood flow (rBF) can be computed from the raw data. Upper left image—raw image. Upper right image—, parametric-map showing he perfusion index within the yellow region, which is indicated by the solid white arrow.

Full figure and legend (35K)Figures & Tables indexDownload Power Point slide (240K)

In conclusion, imaging techniques allow measurement of tumor vascular parameters and have been employed in many clinical studies, but their impact on decisions during drug development has remained modest. This outcome might change if imaging analyses can be associated with analyses of molecular and biological biomarkers and compared with PFS and OS to test their value as surrogate markers. Future developments will include molecular-imaging-based approaches that allow expression levels or activity of specific molecular targets, such as vascular integrins, VEGFRs or matrix metalloproteinases, to be monitored.⁸⁸ Since the number of targets on angiogenic endothelial cells is small, only PET and single-photon-emission CT techniques have sufficient sensitivity for targeted imaging.⁶⁵

Monitoring angiogenesis

While many parameters reflecting specific aspects of angiogenesis can be measured in patients, none has been unambiguously validated. It might be necessary to combine different approaches in order to obtain robust quantification of angiogenesis. Thus, future studies should be designed to test multiple surrogate biomarkers in parallel.

Monitoring drug activity

Biomarkers of efficacy are less relevant in phase III studies aimed at validating treatments, because improved TTP or OS are the critical end points at this stage of development. Biomarkers will be most useful as surrogate end points in phase I and phase II studies to facilitate the identification of active versus inactive drugs, and make the transition to phase III clinical studies more rapid (Figure 3). If a drug does not provide evidence of antitumor activity on the basis of standard criteria at an early stage of development, there are three possibilities: first, the drug has no effect on the target; second, the drug modulates the target, but does not elicit the desired biological activity; third, the drug exerts a biological activity, but this does not translate into a therapeutic effect. Nowadays, biological activity is considered the minimal requirement to proceed with drug development when clinical responses are not measurable. Therefore, monitoring drug effects through a pharmacodynamic biomarker, is the only way to obtain valuable information for 'go/no–go' decisions in phase I/II trials. This approach might possibly ensure that biologically active drugs reach large, phase III trials, while preventing the progression of biologically inactive drugs.

Figure 3 Rationale for the use of biomarkers in the development of antiangiogenic therapies.

Abbreviations: EC, endothelial cell; CEC, circulating endothelial cells; CECP, circulating endothelial cell progenitors; CT, computed tomography; DCE-MRI, dynamic contrast-enhanced magnetic resonance imaging; FACS, fluorescence activated cell sorting; IHC, immunohistochemistry; OS, overall survival; RT-PCR, reverse transcription-polymerase chain reaction; PET, positron emission tomography; TTP, time to progression; US, ultrasound.

Full figure and legend (40K)Figures & Tables indexDownload Power Point slide (244K)

Predicting response

Not every patient responds equally well to antiangiogenic drugs, which is not surprising considering the multiplicity of angiogenic mechanisms and the intrinsic heterogeneity of tumor biology. In clinical practice, a biomarker able to predict response to therapy would be extremely valuable. Such a biomarker could act as a surrogate marker of clinical outcome and would be useful for identifying those patients responding to therapy.

Definition of optimum biological dose

The activity and toxicity profiles associated with antiangiogenic drugs are different from those associated with chemotherapy. Antiangiogenic drugs are expected to cause cytostasis, rather that cytotoxicity and tumor regression. The optimum biological dose for these drugs might be lower than the maximal-tolerated doses. Moreover, standard criteria used to define doses for cytotoxic drugs do not apply to this class of drugs. Pharmacodynamic biomarkers could provide important information for the definition of a minimally active dose or a range of doses, which, in conjunction with clinical data, could define the dose escalation process and determine the optimum biological dose.

Design of combination therapies

Antiangiogenic drugs enhance the activity of standard chemotherapy, while combinations of drugs targeting complementary angiogenic pathways are expected to enhance responses. Early biological data may provide information that is useful to the selection of drugs and combination strategy. In the case of available antiangiogenic drugs, surrogate biomarkers could help to improve their use and to design novel combinatorial regimens.

Resistance to antiangiogenesis therapies

Patients treated with antiangiogenic drugs who show an initial antitumor response eventually show disease progression, suggesting that tumors develop resistance to this class of drugs.¹⁴ Resistance under anti-VEGF therapy was demonstrated experimentally.⁸⁹ It will be important to detect resistance during treatment so patients can be given the most appropriate second-line treatment. The mechanisms responsible for resistance to antiangiogenic drugs are likely to be complex and are yet to be defined.¹

Translational research

Our understanding of the mechanisms of angiogenesis and their modification by antiangiogenic treatments in human cancer are still rudimentary. Analysis is mostly focused in understanding the role of individual molecules or pathways, while we lack an integrated view and understanding of the functional association between apparently distinct events and their modification during angiogenesis and antiangiogenesis therapy. For example, a growing amount of evidence indicates that tumors react to therapy by upregulating angiogenic factors and mobilizing bone-marrow-derived CECP.^{7, 35, 36, 37, 90} In spite of the obvious clinical relevance of these observations, we still have little knowledge of how tumors adapt to, and possibly escape from, angiogenesis inhibition. To address these and many other outstanding questions, it will be important to associate clinical studies not only with pharmacodynamic measurements, but also with relevant preclinical experimental models.

In short, biomarkers of angiogenesis should still be considered essential elements of clinical drug development. In phase I/II trials they are needed as pharmacodynamic biomarkers to validate the supposed biological effect of the drug, and define the optimum biological dose and schedule. At late stages of development (phase III) or in clinical practice, biomarkers are needed to identify responding patients early in the course of treatment (predictive biomarkers), to detect patients developing resistance and to guide the development of novel combination therapies.

The natural intra-patient and inter-patient variability of candidate biomarkers currently under investigation should be taken into account during future studies. Data collection and analysis should help to identify and quantify these variabilities and other putative confounding factors. In the case of simple biomarkers (e.g. serum measurements, blood pressure), the natural variability can be easily determined by taking multiple measurements. In the case of more complex analyses, such as DCE-MRI, multiple assessments could be very burdensome and financially prohibitive.