C. Lieu, H. Tran, Z. Jiang, M. Mao, M. Overman, C. Eng,

1. The Association of Alternate VEGF Ligands With Resistance to Anti-VEGF Therapy in Metastatic Colorectal Cancer C. Lieu, H. Tran, Z. Jiang, M. Mao, M. Overman, C. Eng, J. Morris, L. Ellis, J. Heymach, and S. Kopetz Departments of Gastrointestinal Medical Oncology, Surgical Oncology, and Head and Neck/Thoracic Medical Oncology The University of Texas MD Anderson Cancer Center

2. VEGF Biology VEGF-A VEGF-C, VEGF-D PlGF Y Bevacizumab Cell membrane VEGF-R1 (Flt-1) Migration Invasion Survival VEGF-R2 (KDR/Flk-1) Proliferation Survival Permeability VEGF-R3 (Flt-4) Lymphangio- genesis Functions

3. VEGF-A X VEGF-A VEGF-C VEGF-A X Baseline Treatment with bevacizumab VEGF-D PlGF Hypothesis: Alternate VEGF ligands are associated with the resistance to anti-VEGF therapy in patients with metastatic colorectal cancer Angiogenesis Restored Adapted from Bergers et al. Nat Rev Cancer. 2008;8:592-603.

4. Study Design Prospective Clinical Trial Retrospective Validation Cohort The Texas Genetic Consortium database (n = 710) Heterogeneous treatment histories Single cytokine measurement • Phase II trial of FOLFIRI + bevacizumab in patients with metastatic colorectal cancer • Forty-three patients enrolled • Intensive cytokine measurements PD PD PD

5. Study Design Prospective Clinical Trial Retrospective Validation Cohort The Texas Genetic Consortium database (n = 710) Heterogeneous treatment histories Single cytokine measurement • Phase II trial of FOLFIRI + bevacizumab in patients with metastatic colorectal cancer • Forty-three patients enrolled • Intensive cytokine measurements PD PD PD

6. Prospective Cohort: PlGF Increased Prior to Progression Kopetz et al. J Clin Oncol28:453-459

7. Prospective Cohort: VEGF-C Increased Prior to Progression * * * p<0.05 by Mann Whitney U test

8. Prospective Cohort: VEGF-D Minimally Increased at Progression * * p = 0.04

9. Study Design Prospective Clinical Trial Retrospective Validation Cohort The Texas Genetic Consortium database (n = 710) Heterogeneous treatment histories Single cytokine measurement • Phase II trial of FOLFIRI + bevacizumab in patients with metastatic colorectal cancer • Forty-three patients enrolled • Intensive cytokine measurements PD PD PD

10. Retrospective Cohort • Separated patients into three groups: • Patients presenting prior to frontline therapy • Patients treated with chemotherapy without bevacizumab • Patients treated with chemotherapy and bevacizumab • To minimize heterogeneity, samples were matched for: • Metastatic disease sites • Chemotherapy cycles • Time from last chemo to plasma collection • 533 patients were included in the analysis

11. PlGF Elevated After Bevacizumab p < 0.0001 p < 0.0001

12. VEGF-C Elevation Unable to be Confirmed p < 0.0001 p = 0.64

13. Minimal VEGF-D Elevation Confirmed p < 0.0001

14. Summary Prospective Retrospective Conclusions PlGF is elevated after FOLFIRI+B A similar elevation was seen after chemotherapy + bev but not after chemotherapy alone PlGF VEGF-C is elevated after FOLFIRI+B No difference was seen in the second cohort between the two “post-therapy” groups Limitations include heterogeneity and high inter-patient variability VEGF-C Modest elevations in VEGF-D were seen after FOLFIRI+B Elevations were seen in the “post-therapy” groups but not impacted by bevacizumab therapy VEGF-D

15. How long do PlGF and VEGF-D stay elevated after bevacizumab? No temporal change in PlGF and VEGF-D in patients treated with chemotherapy only

16. Limitations of Cytokine Analysis • How well do circulating levels of VEGF ligands reflect the tumor microenvironment? • Or host response? • Difficult to place magnitude of changes into context • What degree of elevation would be necessary to evoke a biologic response • Association vs. Causation • Are alternate VEGF ligands driving resistance to bevacizumab • Return to preclinical models • Clinical trial

17. Large molecule VEGF inhibitors PlGF VEGF-A VEGF-C, VEGF-D Y Bevacizumab Y Aflibercept (VEGF Trap) Ramucirumab (IMC-1121B) Y II VGX-100 Y TB403 CT-322 Y IMC-18F1

18. Phase III VEGF-Trap (Aflibercept) after Bevacizumab 2ndline CRC (after treatment with oxaliplatin-based therapy) N=1200 patients Primary endpoint: OS FOLFIRI + Placebo R FOLFIRI + Aflibercept 4mg/kg “VELOUR” Study Primary endpoint OS met

19. Conclusions • VEGF family ligands other than VEGF itself are associated with bevacizumab-containing chemotherapy resistance in mCRC • Plasma levels of PlGF are increased prior to radiographic progression of disease • Changes in VEGF-C were not able to be validated • Limited by technical concerns in the validation cohort • VEGF-D is minimally increased at the time of progression • Unclear biologic significance • Further study of agents targeting multiple VEGF-ligands are ongoing

20. Acknowledgments • GI Medical Oncology • Scott Kopetz • Karen Mao • Camilla Ziang • James Abbruzzese • Thoracic/H&N Medical Oncology • Hai Tran • John Heymach • Stef Fiorentino • GU Medical Oncology • Gary Gallick • Funding • ASCO Cancer Foundation Young Investigators Award • Circadian Technologies • T32 Training Grant

21. Plasma PlGF in mCRC • In a prospective cohort, plasma PlGF levels are elevated prior to progression and at the time of progression on a bevacizumab regimen • In a validation cohort, compared with untreated patients and patients who have received chemotherapy only, patients who have received chemotherapy and bevacizumab had elevated levels of PlGF • These changes appear specific to patients receiving bevacizumab Lieu et al. ASCO 2011 Abstract #3533

22. Lieu et al. ASCO 2011 Abstract #3533