PDF file 114K, described the legends of all the supplementary figures

PDF file 1160K, AM is a growth factor survival for LECs

PDF file 392K, Vascular network stabilization and induction of lymphangiogenesis and neoangiogenesis modulated by the AM/AMR signaling pathway

PDF file 42K, AM is involved in many functions of PC3 cells in vitro

PDF file 507K, Expression of CLR, RAMP2, and RAMP3 in Du145 cells

PDF file 45K, {alpha}AM inhibits AM-mediated functions on LECs in vitro

PDF file 97K, AM induced activation of MAPK pathway and Normal Vascularization is not disrupted by {alpha}AM treatment

PDF file 169K, Characterization of polyclonal anti-AM antibody ({alpha}AM)

The microRNA-200 (miR-200) family is frequently down-regulated in tumors, including pancreatic adenocarcinomas (PDACs). In this study we have examined the mechanisms involved in the loss of miR-200s in tumoral pancreatic cells. Whereas miR-200 gene promoters appear methylated in mature miR-200 deficient cell lines, miR-200 precursors are detected in nuclear but not cytoplasmic compartment of these cells, indicating that promoter hypermethylation is not sufficient to explain the deficit of mature miR-200s. The ribonuclease Monocyte Chemotactic Protein-induced Protein-1 (MCPiP1) may counteract Dicer1 in miRNA maturation process. MCPiP1/Dicer1 mRNA and protein ratios appear higher in miR-200 deficient compared to miR-200 proficient cells, suggesting that MCPiP1 may compete with Dicer1 in mature miR-200 deficient cells. Inhibition of MCPiP1 allows the detection of miR-200 precursors in cytoplasm of miR-200 deficient cells, confirming its involvement in the loss of miR-200s. Also, reversion of MCPiP1/Dicer1 ratio by over-expression of Dicer1 in miR-200 deficient cells leads to the recovery of mature miR-200s. Finally, whereas human malignant pancreatic tissues (PDACs) express lower miR-200 levels than non malignant tissues (non-MPDs), MCPiP1/Dicer1 ratio appears higher in PDACs, when compared to non-MPDs, supporting the hypothesis that MCPiP1/Dicer1 ratio is determinant in regulating miR-200 maturation process in a subset of tumoral pancreatic cells.

Glioblastoma remains the most frequent and aggressive primary brain tumor in adults and is characterized by extensive angiogenesis.1 Recently, bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGFA), was associated with valuable, but heterogeneous, activity in patients with recurrent high-grade gliomas.2,3 In addition to the standard of care for newly diagnosed glioblastoma, the use of bevacizumab as a first-line treatment has been associated with prolonged progression-free survival (PFS) and maintenance of functional independence, but no difference has been observed in overall survival (OS).4,5 Given this heterogeneous activity and the uncertain impact on OS, a predictive biomarker of bevacizumab activity remains an unmet medical need. In this context, we recently reported that before bevacizumab administration, a baseline of high and low plasma levels of MMP2 and MMP9, respectively, were associated with a high response rate and prolonged PFS and OS in recurrent high-grade gliomas treated with bevacizumab.6 Moreover, we did not observe any correlation between these 2 biomarkers and the survival of patients when the plasma levels were measured before administration of cytotoxic agents without antiangiogenic therapy, suggesting a predictive—rather than a prognostic—value of the biomarkers.6 Considering the potential role of biomarkers in treatment monitoring, it is of interest to analyze the successive change in the plasma levels of these markers during bevacizumab treatment. We retrospectively included all patients with recurrent high-grade gliomas who had been referred to both of our institutions (APHM, Marseille, and APHP, Paris). They were treated with a combination of bevacizumab (10 mg/kg on days 1 and 15) and irinotecan until progression, and their plasma samples at each cycle and at progression were available as part of the ongoing studies (APHM Tumor Bank, authorization number 2013–1786). Plasma samples were collected before bevacizumab administration and then every 14 days, on day 1 and day 15 (before bevacizumab administration) of each cycle until progression. All patients provided written informed consent in accordance with our institutional and national guidelines and the Declaration of Helsinki. The plasma levels of MMP2 and MMP9 were assessed using ELISA kits (R&D Systems). Changes were analyzed using the paired-samples t test, and correlations were analyzed using the Spearman test. Forty-one patients were included. Patient and treatment characteristics are reported in Table 1. The plasma level of MMP2 significantly increased during bevacizumab treatment from baseline (B) to the last point prior to progression (PTP) (P = .001) and decreased at the time of progression (P) (P = .033); B, PTP, and P mean plasma levels of MMP2 were 210.4 ng/mL, 245.9 ng/mL, and 220.4 ng/mL, respectively (Fig. 1). In contrast, the plasma level of MMP9 decreased during bevacizumab treatment (P = .034) and tended to increase at P (not significant); B, PTP, and P mean plasma levels of MMP9 were 194.6 ng/mL, 113.0 ng/mL, and 151.0 ng/mL, respectively. Finally, the plasma levels of MMP2 and MMP9 were inversely correlated at B and at P (P < .001 and P = .011, respectively). A trend was observed at the last cycle before progression (P = .065). Table 1. Patient characteristics at the time of bevacizumab + irinotecan administration Fig. 1. Variations of MMP2 (A) and MMP9 (B) mean plasma levels during bevacizumab treatment from baseline until progression. In this retrospective bicentric study, we showed that the plasma level of MMP2, and to a lesser extent that of MMP9, varied during treatment and at the time of progression. We previously reported that these markers could potentially allow initial patient selection for treatment.6 In the present study, the change in the levels of these biomarkers provided additional information on tumor response events during bevacizumab treatment. In particular, considering the challenge of assessing progression during the bevacizumab treatment, the decrease in the plasma levels of MMP2 observed at P suggests that this biomarker can help in monitoring bevacizumab treatment. This predictive value, observed at B in our previous work and at P in the present study demonstrating the changes observed during bevacizumab treatment, reinforces the potential value of these markers and the need of their assessment in a prospective trial. Variations in the plasma levels of MMP2 were previously reported for the multitargeted antiangiogenic tyrosine kinase inhibitor cediranib.7 In a phase 2 trial conducted in patients with recurrent glioblastoma, the use of cediranib induced a transient decrease in the plasma level of MMP2 between 8 and 24 hours after the first administration. While the baseline level of MMP2 had no impact on patient outcome, the increase in the plasma level of MMP2 8 hours after the administration of cediranib was associated with a reduced PFS and OS. The plasma level of MMP9 at B and changes during treatment were not correlated to patient outcome. It is not known if these differences in variations and impact are related to the differences in the activity or the mechanism of action between cediranib and bevacizumab. Finally, MMP2 and MMP9 appeared to be inversely correlated at both B and P. This inverse correlation suggests a biological interaction of these 2 proteases in the tumor vascularization process and is in line with their inverse predictive values that were also observed at B.6 MMP2 was reported to be particularly implicated in angiogenesis through the promotion of VEGFA expression and its coexpression with VEGFR2.8,9 Indeed, in the absence of MMP2 expression, tumor vessel density as well as VEGFA and VEGFR2 expressions decreased dramatically, suggesting an important role of MMP2 in the angiogenic process. In contrast, MMP9 is required for tumor vasculogenesis but not for angiogenesis.10 Indeed, MMP9 appeared to be particularly implicated in the recruitment of circulating endothelial and myeloid precursors, an alternative vascularization process that was in part independent of the VEGF pathway.10 This study on MMP2 and MMP9 changes during bevacizumab treatment provides additional support for considering the potential value of these biomarkers in predicting bevacizumab activity in patients with high-grade glioma. Adequate prospective studies in this regard are warranted.

BACKGROUND: We have previously showed that a high MMP2, and to a lesser extend a low MMP9 plasma level were associated to a high response rate, a prolonged PFS and OS in recurrent GB treated with bevacizumab, but not with cytotoxic agents, (Tabouret and col. Neuro-Oncol 2013). In order to further explore the optimal timing of bevacizumab administration, we analyzed potential differences of MMP2/MMP9 plasma levels and tumor RNA in patients with newly diagnosed and recurrent GB. METHODS: Plasma was collected before radiotherapy in newly diagnosed GB patients (pts) (Pop ND) and in a distinct population of pts at the time of recurrence (Pop RD). MMP2 and MMP9 plasma levels were assessed using ELISA. In a third population with paired initial and recurrent GB tumor (Pop NDT and RDT respectively), MMP2 and MMP9 RNA were analyzed using quantitative RT PCR. Correlations were analyzed using the Mann Withney U test, the Spearman correlation and the T-test. RESULTS: MMP plasma levels were tested in 44 ND and 76 RD pts. Mean levels of ND and RD MMP2 levels were 196.4 pg/ml (± 9.6) and 218.2 pg/ml (± 9.6). Mean levels of ND and RD MMP9 levels were 365.4 pg/ml (± 50.7) and 302.0 pg/ml (± 34.9). No significant difference was observed between ND and RD plasma levels of MMP9 and MMP2. In ND pts, no correlation was found between MMP2 and MMP9 plasma levels, neither between these plasma markers and KPS or age. In NR pts, MMP2 and MMP9 plasma levels were inversely correlated (p < 0.001). Paired MMP9 and MMP2 RNA were available in NDT and RDT for 29 patients. No difference was observed between NDT and RDT expressions of MMP9 and MMP2. At initial diagnosis, MMP9 and MMP2 were 95.7-fold and 15.9-fold over-expressed compared with normal brain. At recurrence, MMP9 and MMP2 were 80.6-fold and 25.9-fold over-expressed. MMP2 and MMP9 RNA were correlated in NDT (p = 0.001) but not in RDT. CONCLUSIONS: In this retrospective study, the prebiomarkers of BEV activity, MMP2 and MMP9, did not show significant change between GB initial presentation and recurrence both for plasma levels and tumor RNA. These results may suggest that BEV could be similarly active for pts with newly diagnosed and recurrent GB. Adequate prospective studies are warranted.

2074 Background: We have previously showed that a high MMP2, and to a lesser extend a low MMP9 plasma levels were associated to a high response rate, a prolonged PFS and OS in recurrent GB treated with bevacizumab (BEV), but not with cytotoxic agents, (Tabouret and col. Neuro-Oncol 2013). In order to further explore the optimal timing of bevacizumab administration, we analyzed potential differences of MMP2/MMP9 plasma levels and tumor RNA in patients with newly diagnosed and recurrent GB. Methods: Plasma was collected before radiotherapy in newly diagnosed GB patients (pts) (Pop ND) and in a distinct population of pts at the time of recurrence (Pop RD). MMP2 and MMP9 plasma levels were assessed using ELISA. In a third population with paired initial and recurrent GB tumors (Pop NDT and RDT respectively), MMP2 and MMP9 RNA were analyzed using quantitative RT PCR. Correlations were analyzed using the Mann Whitney U test, the Spearman corelation and the T-test. Results: MMP plasma levels were tested in 44 ND and 76 RD pts. Mean levels of ND and RD MMP2 levels were 196.4 pg/ml (± 9.6) and 218.2 pg/ml (± 9.6). Mean levels of ND and RD MMP9 levels were 365.4 pg/ml (± 50.7) and 302.0 pg/ml (± 34.9). No significant difference was observed between ND and RD plasma levels of MMP9 and MMP2. In ND pts, no correlation was found between MMP2 and MMP9 plasma levels, neither between these plasma markers and KPS or age. In NR pts, MMP2 and MMP9 plasma levels were inversely correlated (p<0.001). Paired MMP9 and MMP2 RNA were available in NDT and RDT for 29 pts. No difference was observed between NDT and RDT expressions of MMP9 and MMP2. At initial diagnosis, MMP9 and MMP2 were 95.7-fold and 15.9-fold over-expressed compared with normal brain. At recurrence, MMP9 and MMP2 were 80.6-fold and 25.9-fold over-expressed. MMP2 and MMP9 RNA were correlated in NDT (p=0.001) but not in RDT. Conclusions: In this retrospective study, the prebiomarkers of BEV activity, MMP2 and MMP9, did not show significant change between GB initial presentation and recurrence both for plasma levels and tumor RNA. These results may suggest that BEV could be similarly active for pts with newly diagnosed and recurrent GB. Adequate prospective studies are warranted.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is among the deadliest of human cancers. Dysregulation of microRNAs (miRNAs) expression is an important step in tumor progression as miRNAs can act as tumor suppressors or oncogenes and may affect cell sensitivity to chemotherapy. Whereas the oncogenic miR21 has been shown to be overexpressed in gliomas, the expression and function of the tumor-supressor miR200a in GBMs remains unknown. In this study, we show that miR21 is upregulated in grade IV (GBMs) vs. grade II-III (LGs) gliomas, confirming that miR21 expression level is correlated with tumor grade, and that it may be considered as a marker of tumor progression. Conversely, miR200a is demonstrated for the first time to be downregulated in GBMs compared with LGs, and overexpression of miR200a in GBM cells is shown to promote TMZ-sensitivity. Interestingly, miR200a but not miR21 expression level is significantly higher in TMZ-responsive vs. -unresponsive tumoral glial cells in primary culture. Furthermore, miR200a appears negatively correlated with the expression of the DNA repair enzyme O (6)-methylguanine methyltransferase (MGMT), and the inhibition of MGMT activity results in an increase of miR200a expression in GBM cells. Taken together, these data strongly suggest that miR200a is likely to act as a crucial antitumoral factor regarding glioma progression. Interplay between miR200a and MGMT should be considered as potential mechanism involved in therapeutic response.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is among the deadliest of human cancers. Dysregulation of microRNAs (miRNAs) expression is an important step in tumor progression as miRNAs can act as tumor suppressors or oncogenes and may affect cell sensitivity to chemotherapy. Whereas the oncogenic miR21 has been shown to be overexpressed in gliomas, the expression and function of the tumor-supressor miR200a in GBMs remains unknown. In this study, we show that miR21 is upregulated in grade IV (GBMs) vs. grade II–III (LGs) gliomas, confirming that miR21 expression level is correlated with tumor grade, and that it may be considered as a marker of tumor progression. Conversely, miR200a is demonstrated for the first time to be downregulated in GBMs compared with LGs, and overexpression of miR200a in GBM cells is shown to promote TMZ-sensitivity. Interestingly, miR200a but not miR21 expression level is significantly higher in TMZ-responsive vs. -unresponsive tumoral glial cells in primary culture. Furthermore, miR200a appears negatively correlated with the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT), and the inhibition of MGMT activity results in an increase of miR200a expression in GBM cells. Taken together, these data strongly suggest that miR200a is likely to act as a crucial antitumoral factor regarding glioma progression. Interplay between miR200a and MGMT should be considered as potential mechanism involved in therapeutic response.

Antiangiogenic agents that target the vascular endothelial growth factor (VEGF) pathway have been successfully developed and approved in the vast majority of cancers. As a consequence, an increasing use of antiangiogenic agents has been observed, leading to cost issues and reassessment of their benefit. However, activity on tumor response, and survival benefit of these agents, varies mostly among tumor types and with agents tested. Bevacizumab, a monoclonal antibody against VEGF, was the first antiangiogenic agent that has demonstrated a benefit on progression-free survival (PFS) with or without impact on survival, in patients with advanced and metastatic cancer.1 Glioblastoma multiforme (GBM) is a devastating disease, characterized by a highly angiogenic and invasive phenotype, suggesting a potential role for antiangiogenic strategies. Preclinical data as well as high levels of intratumoral VEGF expression have supported the evaluation of agents that target the VEGF pathway.2 Among them, bevacizumab has been recently approved by the FDA for patients with recurrent glioblastoma, based on a high response rate (RR) with prolonged PFS compared with historical controls3 and is under investigation in the first-line setting.4 Biomarkers able to predict response to antiangiogenic agents and particularly to bevacizumab are an unmet medical need for patients with cancer. The ideal biomarker should be easy to measure on multiple points upon treatment and standardized in their analysis.5 Baseline levels and/or variation of numerous intratumoral or circulating candidate prebiomarkers have been explored. However, to date their predictive significance has been generally weak and rarely confirmed among studies or compared with a cytotoxic treated population. In situ prebiomarkers such as VEGF, VEGF receptor (VEGFR) 2, and carbonic anhydrase 9, as well as plasma prebiomarkers such as VEGF, VEGFR1, intercellular adhesion molecule 1, and interleukin (IL)-6 and -8, have been reported to predict bevacizumab benefit, but this predictive value is generally weak and restricted to one end point (response, PFS, or overall survival [OS]).6 With other antiangiogenic agents such as cediranib in GBM and vandetanib in non–small-cell lung carcinoma, various prebiomarkers, including VEGF, VEGFR2, placenta growth factor (PlGF), basic fibroblast growth factor (bFGF), and matrix metalloproteinase (MMP) 2, present transition variations that have been related to either progression or survival.7,8 Given the remarkable but inconsistent activity of bevacizumab in GBM, we explored the value of selected plasma prebiomarkers to predict response and survival in patients treated with bevacizumab for recurrent high-grade glioma (HGG).

BACKGROUND A predictive marker of bevacizumab activity is an unmet medical need. We evaluated the predictive value of selected circulating prebiomarkers involved in neoangiogenesis and invasion on patient outcome in recurrent high-grade glioma treated with bevacizumab. METHODS Analyzed in plasma were a set of 11 prebiomakers of interest (vascular endothelial growth factor receptor [VEGF]; VEGF receptor 2; basic fibroblast growth factor; stromal cell derived factor 1; placenta growth factor; urokinase-type plasminogen activator; plasminogen activator inhibitor 1; matrix metalloproteinases 2, 7, and 9; and adrenomedulline), using ELISA, at baseline and 2 weeks after bevacizumab initiation in a prospective cohort of 26 patients (Cohort 1). Correlations were validated in a separate retrospective cohort (Cohort 2; n = 50) and tested in cohort patients treated with cytotoxic agents without bevacizumab (Cohort 3; n = 34). Dosages were correlated to objective response, progression-free survival (PFS), and overall survival (OS). RESULTS In Cohort 1, high MMP2 baseline level was associated with a probability of objective response of 83.3% versus 15.4% for low MMP2 level (P = .001). In multivariate analysis, baseline level of MMP2 correlated with PFS (hazard ratio, 3.92; 95% confidence interval [CI]:1.46-10.52; P = .007) and OS (hazard ratio, 4.62; 95% CI: 1.58-13.53; P = .005), as decrease of VEGF (P = .038 for PFS and P = .013 for OS) and MMP9 (P = .016 for PFS and P = .025 for OS). In Cohort 2, MMP2, but not MMP9, confirmed its predictive significance. In Cohort 3, no association was found between MMP2, MMP9, and outcome. CONCLUSION In patients with recurrent high-grade glioma treated with bevacizumab, but not with cytotoxic agent, high MMP2 plasma levels are associated with prolonged tumor control and survival. MMP2 should be tested in randomized clinical trials that evaluate bevacizumab efficacy, and its biological role reassessed.

Abstract Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

2024 Background: Predictive marker of Bev activity is an unmet medical need. We evaluated predictive value of selected circulating prebiomarkers involved in neoangiogenesis and invasion on patient outcome in recurrent HGG treated with Bev. Methods: A set of eleven prebiomakers of interest (VEGF, VEGF-R2, bFGF, SDF1, PlGF, uPA, PAI1, MMP2, MMP7, MMP9, and adrenomedulline) were analyzed in plasma, using ELISA, at baseline from Bev initiation in a prospective cohort of 26 patients (Cohort1). Correlations were validated in a separate retrospective Bev treated cohort (Cohort2; n = 50) and then tested in a cohort of patients treated with cytotoxic agents without Bev (Cohort3; n = 34). Dosages were correlated to OR, PFS, and OS. MMP2 and MMP9 were then analyzed at multiple time points up to progression. Results: In cohort1, high MMP2 baseline level was associated with an OR rate of 83.3% for high levels versus 15.4% for low MMP2 levels (p = 0.001). In multivariate analysis, MMP2 baseline level was correlated with PFS (hazard-ratio (HR), 3.92; 95% confidence-interval (CI):1.46-10.52; p = 0.007) and OS (HR, 4.62; 95%CI 1.58-13.53; p = 0.005), as MMP9 (p = 0.016 for PFS and p = 0.025 for OS). Similar results were found in cohort2 for MMP2, (MMP2: p<0.001 for OR; p = 0.009 for PFS; p = 0.009 for OS) but not for MMP9. In cohort3, no association was found between MMP2, MMP9 and outcome. Significant changes in MMP2 and MMP9 plasma levels were observed during treatment. MMP2 increased after Bev initiation (p = 0.002), and decreased at progression (p = 0.002) while MMP9 initially decreased (p = 0.007) then increased at progression (p = 0.031). Conclusions: In patients with recurrent HGG treated with bevacizumab, but not with cytotoxic agents, high MMP2 plasma levels are associated with prolonged tumor control and survival while changes over time may reflect tumor control. MMP2 should be tested in randomized clinical trials that evaluate bevacizumab efficacy, and its biological role should be reassessed.

ABSTRACT Background Tumor EGFR and KRas mutations are both predictive and prognostic biomarkers in patients with advanced NSCLC. We analyzed the correlation between these biomarkers and treatment outcomes in a phase III trial of 3rd/4th line sorafenib in patients with NSCLC. Methods The global, randomized, placebo-controlled MISSION trial enrolled 703 patients with advanced relapsed/refractory NSCLC of predominantly non-squamous histology. The primary study endpoint was overall survival (OS). EGFR and KRas mutations were analyzed in archival tumor samples and in circulating tumor DNA isolated from plasma. Results Tumor and/or plasma mutation data were available from 347 patients (49%). EGFR and KRas mutations were detected in 89 (26%) and 68 (20%) patients, respectively, and were well balanced between treatment arms. Analysis of the interaction between EGFR mutation status and treatment effect on survival suggested that patients with EGFR mutations (mEGFR) benefitted from sorafenib, while those with wild-type EGFR (wtEGFR) did not (p = 0.023). Median OS was two-fold longer in mEGFR patients receiving sorafenib versus placebo (423 vs 197 days, HR 0.48, p = 0.002). There was no significant difference in OS between patients with wtEGFR receiving sorafenib or placebo (253 vs 256 days, HR 0.92, p = 0.559). An interaction was also seen between EGFR mutation status and the sorafenib effect on PFS (p = 0.015). Patients with mEGFR treated with sorafenib had better outcomes compared to placebo based on Cox regression analysis (HR 0.27, p  Conclusion Post-hoc analyses of efficacy outcomes in MISSION suggest that advanced NSCLC patients with EGFR mutations may derive a survival benefit from receiving 3rd/4th line sorafenib. These results must be interpreted with caution due to the small, non-representative nature of the genetic biomarker subpopulation analyzed in this trial. Further prospective investigation may be warranted. Disclosure T.S.K. Mok: Honoraria: AstraZeneca, Roche, Eli Lilly, Merck Serono, Eisai, BMS, BeiGene, AVEO, Pfizer, Taiho, Boehringer Ingelheim, and GSK Biologicals Speaker: Astrazeneca, Roche, Eli Lilly, Boehringer Ingelheim, and Merck Serono Research funding: Astrazeneca. L. Paz-Ares: Dr. Paz-Ares has received honoraria from Bayer HealthCare Pharmaceuticals, Lilly, Roche and Pfizer. Y. Wu: Dr. Wu has received lecture fees from Roche, AstraZeneca, Eli Lilly, and Pfizer. V. Hirsh: Member of the steering committee for the MISSION trial. C. Lathia: Dr. Lathia is an employee of Bayer HealthCare. T.J. Ong: Dr. Ong is an employee of, and owns shares in, Bayer HealthCare. C. Pena: Dr. Pena is an employee of Bayer HealthCare. All other authors have declared no conflicts of interest.