Blood vessel development is a critical requirement for the progression of solid malignancies. Therapeutic agents inhibiting angiogenesis have become a cornerstone of modern oncologic medicine and have been shown to slow disease progression, extend survival, and provide clinical benefit in a number of disease settings. Antiangiogenic agents have been approved in the United States for the treatment of a variety of cancers in the metastatic setting, including colorectal cancer, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, ovarian cancer, pancreatic neuroendocrine tumors, soft-tissue sarcoma, glioblastoma, and non–small cell lung cancer, and in addition, they are used to treat breast cancer in Europe.1–11 Despite their broad use, major challenges in the development and application of antiangiogenic agents still remain. Many patients do not respond to these agents, and those who do respond eventually develop resistance, and their disease progresses. Extensive efforts to identify and prospectively validate a predictive biomarker of benefit for antiangiogenic agents have proven unsuccessful for a multitude of reasons.12 Clinical trial elements including treatment arm crossover, response analyses, and statistical endpoints can hinder biomarker analysis. The need for biomarkers to guide the use of antiangiogenic agents continues to be paramount.
The search for molecular biomarkers for antiangiogenic therapies has been naturally guided by the pathways that these agents target. As solid tumors grow and require more nutrients than can be provided by the existing vasculature, they become hypoxic and experience metabolic stress. The hypoxic tumor environment up-regulates levels of key transcription factors, including hypoxia-inducible factor (HIF), which initiate proangiogenic signaling cascades. The resulting angiogenic program relies on complex and highly regulated interactions between the tumor and many important stromal elements, including endothelial cells, pericytes, fibroblasts, the extracellular matrix, and infiltrating immune cells.13,14 Any of these elements could yield informative biomarkers, but the vascular endothelial growth factor (VEGF) pathway has been the primary focus of study in this field. The VEGF pathway consisting of the VEGF ligands VEGF-A, VEGF-B, VEGF-C, and VEGF-D and their receptors VEGFR-1 (also called FLT-1), VEGFR-2 (FLT-2/KDR), and VEGFR-3 (FLT-4) is a major signaling axis for coordinating angiogenic processes.15 Vascular endothelial growth factor A secreted by tumor cells binds to VEGFR-2 on the surface of endothelial cells to initiate a host of proangiogenic effects. Several isoforms of VEGF-A exist in the circulation, including the short isoform VEGF-A121 and the longer isoforms VEGF-A145, VEGF-A165, VEGF-A189, and VEGF-A206, which vary in their abundance and binding partners.16,17 Longer VEGF-A isoforms contain a heparin-binding domain that mediates interactions with the extracellular matrix and limits diffusion after secretion, whereas VEGF-A121 lacks this domain, is freely diffusible, and is capable of signaling over greater distances. Isoforms also differ in their affinities for the VEGFR-2 coreceptors neuropilin 1 (NRP-1) and NRP-2. Vascular endothelial growth factor receptor 2 phosphorylation and downstream signaling are increased upon formation of a VEGFR-2/VEGF ligand/NRP-1 complex.18 Vascular endothelial growth factor receptor 2 and NRP-1 do not directly interact, so formation of an activated complex is dependent on the presence of a ligand that can bridge the two coreceptors. Vascular endothelial growth factor A165 can mediate formation of the tripartite complex, whereas VEGF-A121 cannot.19 Given the integral role of NRP-1 signaling through VEGFR-2, it may also serve as a valuable biomarker of VEGFR-2 phosphorylation and activation of the VEGF axis. However, in contexts where shorter VEGF-A isoforms are specifically up-regulated, NRP-1 levels may not accurately reflect VEGFR-2 activity, highlighting the need to assay multiple proteins to provide context for the analysis of any potential biomarker.
While VEGF-A is considered to be the primary driver for angiogenic signaling, other factors are known to regulate this highly coordinated process. The related family members VEGF-C and VEGF-D also bind and activate VEGFR-2, providing potential resistance mechanisms in the context of VEGF-A depletion.20 Additional factors and pathways with roles in angiogenesis include placental growth factor (PlGF), basic fibroblast growth factor, platelet-derived growth factors, angiopoietins, inflammatory pathways including interleukin 6 (IL-6) and IL-8, matrix remodeling, and coagulation.21–23 These factors provide both context-dependent specificity and mechanisms of functional redundancy in the complex task of remodeling and generating new vasculature.
Studies using preserved tumor tissue can provide data on both the tumor and the surrounding stroma, but these studies are often limited to samples collected at the time of diagnosis that may not reflect changes in the disease over the course of treatment. Fresh tumor biopsies can be expensive and challenging to obtain and expose the patient to meaningful risks. These difficulties limit the ability to collect serial tissue samples across the continuum of care in the clinic. In contrast, blood-based biomarkers are appealing because collection of samples is safe, inexpensive, and easily performed along the course of treatment. Examples of circulating biomarkers include circulating tumor cells, circulating endothelial cells, nucleic acids (cfDNA, miRNA exosomal RNA, etc), and protein markers. Protein-specific biomarker assays have the key advantage of reflecting the cumulative interplay between the tumor and microenvironment. Furthermore, using multiplex technologies, numerous target proteins can be assayed at one time while maintaining a high degree of sensitivity and specificity.
Broadly, US Food and Drug Administration–approved modalities of inhibiting tumor angiogenesis can be categorized into two groups of agents: proteins that block binding of VEGF-A to VEGFR-2 (eg, the VEGF-A binders bevacizumab and ziv-aflibercept and the VEGFR-2 binder ramucirumab) and the small molecule anti-VEGFR multikinase inhibitors (axitinib, cabozantinib, pazopanib, regorafenib, sorafenib, and sunitinib). This review describes circulating protein biomarkers with potential predictive value from large phase II or randomized phase III clinical trials of antiangiogenic agents.
The first approved antiangiogenic agent was bevacizumab, a monoclonal antibody that sterically blocks VEGF-A binding to VEGFR-2 on the endothelial cell surface.1 Over the past decade, there has been substantial interest around the role of VEGF-A and VEGFR-2 as potential biomarkers for bevacizumab therapy. Disappointingly, results from multiple large analyses have offered conflicting results and generally failed to show any consistent predictive value. For example, Hegde et al24 measured plasma VEGF-A levels across four randomized phase III trials of bevacizumab in colorectal (AVF2107), lung (ECOG E4599 and AVAiL), and renal (AVOREN) cancers using a standardized analysis approach. This analysis used a pan–VEGF-A assay that did not distinguish between the various VEGF-A isoforms. Pan–VEGF-A plasma levels were prognostic across all tumor types but were not predictive for benefit from bevacizumab in any of the studies evaluated. Similarly, two phase III trials of bevacizumab versus cediranib in colorectal cancer (HORIZON II and III) also observed that higher VEGF-A plasma levels were prognostic of shorter survival times.25
Most reported data do not distinguish among the various VEGF-A isoforms at the protein level, and key differences in isoform levels can go unmeasured or have unappreciated effects on the final data. Several reports using an assay that preferentially detects the shorter VEGF-A isoforms have found that these isoforms may better predict for bevacizumab benefit than pan–VEGF-A levels. These include trials in breast (AVADO),26 pancreatic (AViTA),27 and gastric cancers (AVAGAST)28; in all of these trials, high levels of the short isoform of VEGF-A were potentially predictive of benefit from bevacizumab. These results led to the development of a prospective trial to test the predictive value of small isoform VEGF-A levels in patients with bevacizumab-treated breast cancer. MERiDiAN is a randomized phase III trial of paclitaxel with bevacizumab or placebo for HER2-negative metastatic breast cancer in which plasma VEGF-A levels will be prospectively measured, and patients with high VEGF-A will constitute a prespecified analytic subgroup. Validation of the predictive power of short VEGF-A isoforms in MERiDiAN would be a major clinical advance and could impact the use of many antiangiogenic agents.
However, small isoform VEGF-A levels have not been predictive for bevacizumab in all studies examined. Trials in lung (AVAiL), colorectal (AVF2107), and renal (AVOREN) cancers failed to observe a predictive effect of small isoform VEGF-A in these diseases.29 Furthermore, small isoform VEGF-A failed to predict benefit in a randomized phase III trial of bevacizumab in combination with carboplatin and paclitaxel in refractory metastatic ovarian cancer (GOG-0218), indicating that the predictive power of small isoform VEGF-A may not be as generalizable as initially hoped.30 Further complicating the analysis of VEGF-A in clinical samples is the fact that many commercial assays detect both free VEGF-A and VEGF-A bound to bevacizumab to varying degrees. Reports showing rapid increases in VEGF-A levels after starting bevacizumab treatment are likely observing VEGF-A bound and stabilized by bevacizumab.31,32 However, VEGF-A is up-regulated in response to VEGFR-2 inhibition by other agents.33,34
Importantly, alternative VEGF signaling ligands may provide multiple mechanisms of resistance in the setting of VEGF-A inhibition.35 Specifically, VEGF-C, VEGF-D, and PlGF are known to bind and activate signaling through different VEGFR receptors in a context-dependent manner.36 Vascular endothelial growth factor D and PlGF have been found to be elevated in patients at the time of disease progression in patients with metastatic colorectal cancer treated with FOLFIRI and bevacizumab, highlighting these ligands as potential mechanisms of resistance to VEGF-A inhibition.20 Furthermore, CALGB 80303, a randomized phase III study of gemcitabine with or without bevacizumab in pancreatic cancer, found that low levels of VEGF-D were predictive of benefit from bevacizumab.37 This randomized study also found the ligand Ang-2 to be both prognostic and predictive of outcome. These results identified alternative mechanisms of signaling through the VEGF axis as important factors in determining clinical outcomes on antiangiogenic therapies.
Inflammatory signaling, particularly the IL-6 and IL-8 pathways, have also been implicated in therapeutic responses to bevacizumab in several disease settings. Interleukin 6 is an inflammatory cytokine secreted by T cells that signals through the mitogenic JAK-STAT pathway to stimulate host immune responses through macrophage activation and lymphocyte proliferation. Preclinical work has shown that unregulated activation of the JAK-STAT pathway in myeloid cells correlates with increased numbers of myeloid-derived suppressor cells and enhances tumor growth.38 Interleukin 6 signaling is also able to stimulate expression of HIF and VEGF-A through the downstream transcription factor STAT3.39 Intriguingly, STAT3 has been shown to stimulate VEGF expression via both HIF-dependent and HIF-independent mechanisms.40 Two phase III studies have now identified IL-6 as a potential predictor for anti-VEGF therapy. Interleukin 6 was identified as predictive of benefit from bevacizumab in combination with interferon (CALGB 90206) in patients with metastatic renal cancer.41 High IL-6 was a negative prognostic factor, but paradoxically predictive of benefit from bevacizumab, indicating that while inflammatory signaling may have negative consequences for the patient, it may also potentiate the response to bevacizumab. Furthermore, IL-6 was part of a multivariate model, along with the immune-modulatory cytokine SDF-1, which was predictive of benefit from bevacizumab in patients with advanced pancreatic cancer in CALGB 80303.37 Interleukin 8 is a chemokine secreted by macrophages and endothelial cells that has been identified as potentially predictive in phase II studies of bevacizumab in colorectal cancer42 and hepatocellular carcinoma.43 These lines of evidence establish the contextual overlap between inflammation, immunity, and angiogenesis in human malignancy, while highlighting alternative mechanisms as potential therapeutic targets.
Ziv-aflibercept is a recombinant, soluble receptor antibody containing the ligand-binding domains of VEGFR-1 and VEGFR-2 and differs from bevacizumab by sequestering both VEGF-A and PlGF. Thus far, blocking PlGF in addition to VEGF-A has not provided additional benefit over targeting VEGF-A alone in multiple disease settings.44,45 This may be related to the multiple mechanisms of resistance involving both VEGF-dependent and -independent pathways. Most recently, the baseline biomarker analysis from the VELOUR trial of ziv-aflibercept in combination with FOLFIRI in refractory metastatic colorectal cancer showed multiple potentially predictive markers. Elevated levels of VEGF-A, VEGFR-2 and VEGFR-3, and IL-8 were all potentially predictive of benefit from ziv-aflibercept.46 These results suggest that many of the same mechanisms mediating resistance to bevacizumab may also apply to ziv-aflibercept. However, as analyses of samples from additional phase III trials of ziv-aflibercept are completed, differences in biomarker profiles relating to PlGF binding or other angiogenic pathways may be discovered.
Ramucirumab is a monoclonal antibody that binds VEGFR-2 that has recently demonstrated efficacy in multiple cancers, including second-line metastatic colorectal cancer (RAISE),47 gastric cancer (RAINBOW48 and REGARD49), and refractory lung cancer (REVEL).50 The clinical benefit of VEGFR-2 inhibition in these cancers remains modest and generally of similar magnitude as observed with bevacizumab. Biomarker studies of ramucirumab have shown that levels of VEGF-A and PlGF are increased by treatment.34,51 In addition, increasing levels of soluble VEGFR-1 were associated with poor outcome.52 The mechanisms mediating these effects still remain unclear. Circulating VEGFR-1 acts as a ligand sink for VEGF-A and PlGF, conceivably enhancing the activity of ramucirumab or other agents that block the VEGF-A/VEGFR-2 interaction. This effect has also been observed in trials of sunitinib and cediranib, small molecule tyrosine kinase inhibitors (TKIs) that directly inhibit signaling through VEGFR-2.52 In addition, by directly targeting VEGFR-2, ramucirumab prevents its binding of VEGF-C and VEGF-D, thus blocking this potential mechanism of resistance to bevacizumab. Novel trials with integrated analyses of VEGF-C, VEGF-D, and PlGF levels could be used to tailor optimal use of ramucirumab and other antiangiogenic therapies.
Small molecule TKIs have both advantages and disadvantages compared with the highly specific protein-based agents. A principal advantage is that TKIs are capable of inhibiting multiple signaling pathways, typically including all VEGF receptors. This is relevant, given that angiogenesis involves many signaling axes that are required to coordinate the various actions across multiple cell types. In addition to inhibiting the VEGF axis, TKIs can block alternative pathways of angiogenesis and potential mechanisms of resistance. Negative consequences of TKIs include increased off-target toxicities leading to frequent treatment interruptions, potentially resulting in diminished coverage of the target receptors. Given the compensatory up-regulation of VEGF-A in response to VEGFR-2 inhibition by ramucirumab,34 alternating periods with and without VEGFR-2 inhibition could theoretically lead to receptor hyperstimulation.
Circulating protein biomarkers that have been identified for TKIs include elements of the VEGF-axis,53 the angiopoietin receptor Tie-1,54 and several cytokine-driven pathways.54,55 Importantly, a retrospective analysis of patients with renal cancer in 2 trials found that higher levels of IL-6 were predictive of progression-free survival benefit from pazopanib treatment.56 These results are consistent with data from CALGB 90206, where IL-6 was identified as a significant predictive marker for bevacizumab in first-line renal cancer when given with interferon.41 These overlapping findings may indicate that IL-6 signaling plays an important role in the response of renal cancers to antiangiogenic therapies. Other studies of sunitinib in renal cancer have found that VEGFR-3 levels may be associated with longer survival.55 A randomized phase III trial of sorafenib in hepatocellular carcinoma showed Ang-2 and VEGF-A to be prognostic,57 and similar results have been reported in a smaller study.58 Lastly, a recent report of a phase II trial of tivozanib versus bevacizumab in colorectal cancer (BATON-CRC) found that low levels of soluble NRP-1 predicted for substantial progression-free survival benefit from tivozanib compared with bevacizumab. This suggests that circulating NRP-1 levels may be used in the future to help guide the selection of antiangiogenic agents.59 Despite the widespread clinical use of multitarget TKIs in various disease settings, no circulating protein biomarkers have been prospectively validated.
Blood-based protein biomarkers offer the promise of a minimally invasive, real-time snapshot of patients to give the most accurate representation of their current disease status. With the rapid rise of routine molecular profiling of tumor samples in the clinic, we now have the ability to couple circulating protein biomarker data with other genomic profiling information. This strategy will allow information to be leveraged around critical oncologic pathways intersecting with angiogenesis at the individual patient level to guide therapies or select clinical trials. However, the current lack of prospectively validated biomarkers for antiangiogenic therapies creates significant challenges for the use of circulating protein biomarkers to inform treatment decisions in the clinic.
There are still many practical issues to overcome in the identification of predictive biomarkers for antiangiogenic agents. The general lack of standardization surrounding the integration of biomarker studies into clinical protocols, the assays used to detect targets of interest, and the collection and handling of samples all slow the progress toward identifying and validating candidate predictive markers. Furthermore, the various assays often utilize different monoclonal antibodies that may recognize distinct epitopes, so it is possible for different assays that measure the same target to have important and meaningful differences. These differences may include binding kinetics, levels of interference, off-target competition, and varying degrees of specificity for distinct isoforms or splice variants. Cross-study comparisons are also complicated by biological differences related to tumor types and/or lines of therapy, preanalytic issues such as use of EDTA versus citrated plasma, differences in the magnitude of treatment effect, and variable numbers of patients with samples available for analysis. Without standardization of both the assays used and the types of samples used, it will be difficult to determine whether the observed discrepancies across trials are biological or technical in origin.
It is also crucial to assess multiple proteins simultaneously, as single-marker analyses may never be able to accurately predict patient outcome. Multivariable models may be needed to describe the complex biological processes of tumor angiogenesis and the development of resistance to antiangiogenic agents. However, all too often biomarker studies are underpowered and assess only a limited number of markers. As patient samples are limited, the prospect of depleting a sample must be weighed against the value of future discoveries as new technologies and hypotheses evolve.
The need to evaluate multiple proteins and biological processes is highlighted by recent advances in immuno-oncology. While the VEGF axis has been the focus of biomarker development for antiangiogenic agents, resistance mechanisms can emerge from alternative pathways. The IL-6 and IL-8 inflammatory pathways have been implicated in responses to antiangiogenic therapy and highlight the overlap between angiogenesis and immune modulation. Vascular endothelial growth factor A is known to inhibit immune responses by negatively regulating dendritic cell maturation,60 and high levels of VEGF-A are associated with reduced response to the immune-stimulatory anti-CLTA4 antibody ipilimumab in metastatic melanoma.61 There is also a complex relationship between the tumor and its surrounding stroma. Ferrara and colleagues have shown that myeloid cells in the tumor microenvironment can mediate resistance to antiangiogenic therapy in an IL-17–dependent mechanism.62 This mechanism may involve factors such as STAT3 activation and Bv8, granulocyte-colony stimulating factor, and IL-17 in myeloid-derived suppressor cells, which may in turn implicate additional inflammatory cytokines that are known to be STAT dependent.21,63,64 With the rapid expansion of immune-checkpoint therapies today in the clinic, multiple classically described angiogenic biomarkers may reflect important immune and inflammatory alterations in the tumor microenvironment.
Many of the markers examined to date have been elements of the VEGF pathway with well-described roles in angiogenesis. Particularly promising markers in this class are the alternative VEGFR-2 ligands, VEGF-C, VEGF-D, and PlGF. Ang-2 is classically understood to play important roles in angiogenesis and is a promising prospective biomarker in several disease settings. Recent work has highlighted the important overlap between angiogenesis and immune regulation in the tumor microenvironment. Further work is now required to understand the clinical utility of combining strategies targeting both tumor angiogenesis and tumor immune evasion. Building on the past decade of progress investigating circulating angiogenic proteins in patients treated with antiangiogenic therapies, the future holds numerous challenges and opportunities for novel biomarker development. Importantly, early-phase clinical trials need to emphasize routine, scientifically driven, and fit-for-purpose integration of biomarker analyses. Finally, efforts to include more integral circulating biomarkers in randomized phases II and III clinical trials are necessary for the timely validation of prospective biomarkers to ultimately improve patient care.
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