1. 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 The Association of Alternate VEGF Ligands With Resistance to Anti-VEGF Therapy in Metastatic Colorectal Cancer

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

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

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

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

6. Prospective Cohort: PlGF Increased Prior to Progression Kopetz et al. J Clin Oncol 28:453-459

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

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

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

10.  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 Retrospective Cohort

11. PlGF Elevated After Bevacizumab p < 0.0001

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

13. p < 0.0001 Minimal VEGF-D Elevation Confirmed

14. Summary PlGF VEGF-C VEGF-D ProspectiveRetrospectiveConclusions 1)PlGF is elevated after FOLFIRI+B 2)A similar elevation was seen after chemotherapy + bev but not after chemotherapy alone 1)VEGF-C is elevated after FOLFIRI+B 2)No difference was seen in the second cohort between the two “post-therapy” groups 3)Limitations include heterogeneity and high inter-patient variability 1)Modest elevations in VEGF-D were seen after FOLFIRI+B 2)Elevations were seen in the “post- therapy” groups but not impacted by bevacizumab therapy

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

18. Phase III VEGF-Trap (Aflibercept) after Bevacizumab 2 nd line CRC (after treatment with oxaliplatin-based therapy) N=1200 patients Primary endpoint: OS R FOLFIRI + Placebo 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