1. Markers of Response for the Antiangiogenic Agent Bevacizumab Diether Lambrechts, Heinz-Josef Lenz, Sanne de Haas, Peter Carmeliet, and Stefan J. Scherer J Clin Oncol 31:1219-1230 R4 김승민 /Prof 백선경

2. Introduction Bevacizumab is a humanized monoclonal antibody against the vascular endothelial growth factor A (VEGF-A) the formation of blood vessels (angiogenesis) in tumors. Bevacizumab is currently approved in Europe and USA in combination with standard chemotherapyfor the treatment of (mCRC) and (NSCLC). combination with interferon alfa-2a for renal cell carcinoma(RCC) Combination with standard chemotherapy for advanced ovarian cancer in Europe single agent for recurrent glioblastoma in the USA

3. Introduction In early 2008, bevacizumab also received the US FDA accelerated approval for treatment of metastatic breast cancer(mBC). Subsequent completion of the E2100 trial and publication of other trials in first-line mBC, in which bevacizumab failed to improve overall survival (OS), revealed excess toxicity and less benefit As a result, the US FDA revoked the license for bevacizumab in the setting of firstline mBC. it is increasingly being realized that biomarkers for targeted cancer therapiesare necessary

4. Introduction extensive biomarker programs have been built into numerous clinical studies with bevacizumab. However, a marker that predicts bevacizumab treatment outcome has not yet been validated. the challenges in identifying biomarkers for bevacizumab. identify a set of markers that we consider most promising, either predictive in placebo-controlled studies involving large numbers of patients or have been replicated in several other studies. how to further improve this set of markers and discuss how to implement them into clinical practice

5. IDENTIFYING BIOMARKERS FOR BEVACIZUMAB The search for a biomarker predictive of bevacizumab treatment outcome has proven to be challenging for various reasons. First, angiogenesis is a complex and highly adaptive biologic process. Despite the predominant role of VEGF-A, multiple factors can play an essential role during angiogenesis, PlGF, FGFs, PDGFs, angiopoietins (ANGs), cytokines. other factors that promote proteolytic degradation of the matrix or induce maturation of the vasculature by stimulating pericyte coverage (PDGF-BB, ephrin-B2, and NOTCH) also critically contribute to the process.

6. IDENTIFYING BIOMARKERS FOR BEVACIZUMAB Second, preclinical studies revealed that VEGF-A blockade has heterogeneous effects on tumors, inhibition of vessel expansion regression of pre-existing vessels inhibition of bone marrow–derived cell & endothelial progenitor cell  how the tumor vasculature differs between cancers ?  same cancer at different stages of progression ? (eg, adjuvant or metastatic setting)  different treatment regimens ? Finally, clinical end points fail to accurately identify which patients benefit from bevacizumab.

7. PLASMA MARKERS PREDICTIVE OF BEVACIZUMAB high levels of VEGF-A could indicate VEGF-A dependency of the tumor vasculature. plasma VEGF-A levels are well established as indicators of poor prognosis data related to the predictive effect of pretreatment VEGF-A levels have largely been inconsistent. In the E4599 study for NSCLC, response rates in patients with high VEGF-A levels  higher RR in the bevacizumab arm many other studies failed to observe such effect Recent meta-analysis phase III trials in CRC, NSCLC, and RCC pretreatment VEGF-A levels  prognostic marker > predictive marker

8. PLASMA MARKERS PREDICTIVE OF BEVACIZUMAB pretreatment soluble VEGFR1 (sVEGFR1) levels inversely correlated with outcome of either bevacizumab or anti-VEGFR TKIs in at least five different trials. inverse correlation in patients with rectal cancer after bevacizumab and chemoradiotherapy, BC after bevacizumab with chemotherapy, hepatocellular carcinoma after cediranib, mCRC after vandetanib. baseline levels of most other CAFs failed to predict benefit after bevacizumab, although potentially interesting correlations were observed for interleukin-8 (IL-8) and ANG-2

9. PLASMA MARKERS PREDICTIVE OF BEVACIZUMAB novel enzyme linked immunosorbent assay (ELISA)  short VEGF-A isoforms, promising as a predictive marker. Through alternative RNA splicing, several VEGF-A isoforms are generated, of which the short VEGF-A121 isoform is freely diffusible, because it lacks basic amino acid residues that bind the extracellular matrix (ECM). The longer isoforms, consisting of 165, 189, or 206 amino acids, bind to heparin and heparan sulfate proteoglycans in the ECM Several phase III randomized,mBC or pancreatic cancer high baseline levels of VEGF-A, as measured with this novel ELISA, exhibit improved PFS and/or OS after bevacizumab.

10. PLASMA MARKERS PREDICTIVE OF BEVACIZUMAB Treatment-related changes in CAF might also predict adaptive resistance to anti-angiogenic therapies. Several studies have shown acute increases in circulating VEGF-A levels on delivery of bevacizumab. The magnitude of these changes was proposed as a predictive marker, but findings have not been consistent (Table 2). Because most of the circulating VEGF-A is bound by bevacizumab and cannot be differentiated from unbound VEGF-A, it is not clear how an increase in VEGF-A expression could contribute to resistance

11. PLASMA MARKERS PREDICTIVE OF BEVACIZUMAB bevacizumab quite consistently led to persistent increases in PlGF levels, with patients exhibiting a two-fold increase showing improved clinical benefit. several studies  plasma at disease progression under bevacizumab bFGF, PDGF-BB,VEGF-C,51 and VEGF-D51 increased on progression. Overall, these data confirm that upregulation of alternative proangiogenic signaling pathways may act as a mechanism of evasive resistance against bevacizumab A pending question is whether this compensatory upregulation can efficiently and cost-effectively be used in a clinical setting.

12. CIRCULATING ENDOTHELIAL CELLS AND PROGENITOR CELLS Most CECs exhibit a mature phenotype and represent apoptotic cells derived from the endothelial wall. Tumor angiogenesis driven by VEGF-A depends at least partly on the mobilization of CEPs, growing tumors, formation of a functional vascular bed. Increased concentrations of CEPs may reflect active tumo angiogenesis and could serve as predictive markers for antiangiogenic therapies. Preliminary data correlating changes in CEC levels with response to bevacizumab are conflicting Finally, the number of samples analyzed was small, and data remain to be confirmed in larger studies.

13. PREDICTIVE MARKERS IN THE TUMOR AND ITS ENVIRONMENT Several studies assessed expression of VEGF pathway genes in tumor and stromal cells. studies assessed expression of the VEGFRs (including the co-receptor neuropilin-1 [NRP1]), VEGF ligands, or other angiogenic factors (bFGF, IL-8) by immunohistochemistry. Interestingly, in the mCRC (AGITG MAX), low baseline VEGFR1 expression correlated with improved OS after bevacizumab in AVAGAST, low VEGFR1 correlated with improved PFS but not OS.

14. PREDICTIVE MARKERS IN THE TUMOR AND ITS ENVIRONMENT more than 700 gastric tumors from AVAGAST, low baseline NRP1 prolonged OS in patients receiving bevacizumab. In particular, patients with low NRP1 showed improved OS (HR, 0.75) versus patients with high NRP1 (HR, 1.07). In mBC and CRC, low NRP1 had similar effects on disease progression (Table 3). low NRP1 expression represents one of the most consistent and promising markers identified thus far

15. PREDICTIVE MARKERS IN THE TUMOR AND ITS ENVIRONMENT stromal cells play an important role in mediating response to antiangiogenic therapies. In particular, the presence of Gr-1CD11b myeloid cells renders murine tumors refractory to anti–VEGF-A therapy. Tumor-associated fibroblasts also upregulate PDGF-C expression after delivery of a neutralizing VEGF-A antibody in murine lymphoma models In particular, components of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) pathways were upregulated.

16. IMAGING THE RESPONSE TO BEVACIZUMAB VEGF-A blockade is believed to reduce tumor vascular permeability and perfusion dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), monitors changes in vascular structure and function, represents an attractive biomarker to assess bevacizumab treatment response. additional research is needed. In particular, current challenges involve standardization of DCE-MRI to enable its application in a wider context, such as in multicenter clinical studies.

17. GENETIC SUSCEPTIBILITY AS A BIOMARKER The response of the tumor vasculature to bevacizumab could be considered a host-mediated process influenced by genetic variability Several studies have meanwhile assessed whether (SNPs) in candidate genes predict bevacizumab treatment outcome In the mBC E2100 trial, mutant carriers of the rs699947 and rs1570360 SNPs, which correlate with reduced expression of VEGF-A, predicted favorable median OS in the bevacizumab arm but not in the control arm. In the AVADO trial, it was found that rs699947, but not rs1570360, correlated with PFS in the placebo arm, whereas in patients with mCRC, rs699947 and rs1570360 correlated with OS in the bevacizumab

18. GENETIC SUSCEPTIBILITY AS A BIOMARKER Finally, rs2230054 in CXCR2, which could affect splicing of CXCR2, has been correlated with reduced PFS in at least three independent studies. Recently, the first approaches to systematically cover SNPs in all genes of the VEGF pathway were reported. In particular, up to 158 SNPs located in 14 genes were assessed in the AViTA randomized pancreatic cancer trial. Four SNPs in VEGFR1 correlated highly significantly with both PFS and OS in the bevacizumab arm (per allele HR, 2.1).

19. CONCLUDING REMARKS AND FUTURE DIRECTIONS The most promising markers for bevacizumab treatment outcome short VEGF-A isoforms, VEGFRs (VEGFR1 or NRP1), either in plasma or tumors. there is abundant functional evidence that these markers could indeed determine bevacizumab outcome (Note 1 and Fig 1), none of them has consistently been replicated across different studies involving various cancer types additional biomarker discovery to more consistently predict bevacizumab treatment outcome across cancer types ? prospective translation of existing markers into clinical practice ?

21. CONCLUDING REMARKS AND FUTURE DIRECTIONS future studies should continue to focus on the discovery of novel biomarkers in different tissue types (plasma, DNA, tumor, and so on) future studies should not be limited to testing one or two markers in plasma or genotyping only a few genetic variants. Instead, they should include homogeneous sets of candidate markers to allow for comparison between studies. studies should integrate the individual effects of these markers by using advanced statistical analyses and combine them into a general predictive score.

22. CONCLUDING REMARKS AND FUTURE DIRECTIONS The short VEGF-A isoforms are an example of such biomarkers, because they have been correlated with outcome in three large randomized studies The phase III MERiDiAN trial in mBC (opening in 2012) impact of bevacizumab in patients for plasma short VEGF-A isoforms. first prospective validation of a biomarker for bevacizumab and, if positive, MERiDiAN will result in the clinical application of short VEGF-A isoforms as a biomarker for bevacizumab in mBC. In addition, MERiDiAN will indicate whether prospective validation of other markers replicated in several studies is meaningful (eg, VEGFR1 or NRP1 expression in mCRC or gastric cancer).

23. Conclusions Bevacizumab is the first anti-angiogenic therapy proven to slow metastatic disease progression in patients with cancer. Although it has changed clinical practice, some patients do not respond or gradually develop resistance, resulting in rather modest gains in terms of overall survival. robust biomarkers that can guide selection of patients for whom bevacizumab therapy is most beneficial. short vascular endothelial growth factor A (VEGF-A) isoforms, expression of neuropilin-1 VEGF receptor 1 in tumors or plasma, genetic variants in VEGFA or its receptors

24. Conclusions The current challenge is to expand this first set of markers and to validate it and implement it into clinical practice. A first prospective biomarker study known as MERiDiAN, which will treat patients stratified for circulating levels of short VEGF-A isoforms with bevacizumab and paclitaxel, is planned and will hopefully provide us with new directions on how to treat patients more efficiently.