1. Angiogenic Cytokines Are Increased Prior to Disease Progression In Metastatic Colorectal Cancer Patients Treated With Bevacizumab Scott Kopetz, Paulo M. Hoff, Cathy Eng, Michael Overman, Katrina Y. Glover, David Z. Chang, Robert A. Wolff, James L. Abbruzzese, Lee M. Ellis, John V. Heymach The University of Texas, M.D. Anderson Cancer Center, Houston, Texas Centro de Oncologia, Hospital Sírio Libanês, Sao Paulo, Brazil

2. Disclosures Ad hoc consulting –Genentech –Roche

3. Ellis, Hicklin, Clin Cancer Res, 2008 Proposed Mechanisms of Bevacizumab Resistance

4. Purpose: To evaluate circulating levels of cytokines: 1) after treatment with bevacizumab 2) just prior to progressive disease on a bevacizumab- containing regimen Trial: Phase II of FOLFIRI + Bevacizumab –43 previously untreated patients –First cycle consisted of single agent bevacizumab –Response rate of 60% –Progression-free survival of 12.8 months –Overall survival of 30.7 mos. Study Purpose and Methods Kopetz, et al. ASCO ’07 Abs 4089

5. Sample Collection Study Aim 1: Subsequent samples were obtained every two weeks n=40 with evaluable samples at the selected time points Study Methods Bevacizumab FOLFIRI Bevacizumab Cycle 1 Day 1 Cycle 2 Day 15 Cycle 3 Day 29 After BevacizumabAfter FOLFIRI + BevBaseline Cycle 4+ Day 43 Bevacizumab FOLFIRI Every 2 weeks

6. Study Methods Multiplex bead assay, ELISA for panel of 38 cytokines, 6 identified for a priori evaluation: E-Selectin placental growth factor [PlGF]matrix metalloprotease 9 [MMP-9] basic fibroblast growth factor [bFGF]Interleukin 8 [IL-8] hepatocyte growth factor [HGF]Eotaxin / CCL11 platelet derived growth factor-BB [PDGF]stromal derived factor 1a/CXCL12 [SDF-1] stem cell factor/KIT ligand [SCF]monocyte chemotactic protein 3 [MCP-3] Interleukin 1-beta [IL-1β]macrophage colony stimulating factor [M-CSF] granulocyte colony stimulating factor [G-CSF] Primary Cytokines Exploratory Cytokines Comparisons by nonparametric Wilcoxon paired test –p<0.05 for a priori primary cytokines, multiple comparison adjusted for exploratory cytokines.

7. Cytokine Modulation after Single-agent Bevacizumab, and after FOLFIRI + Bevacizumab p<0.05; † p<0.05 after multiple comparison correction Error bars represent a nonparametric estimate of SD * * * † † † † † † There were no significant changes in the other cytokines

8. Study Aim 2: Evaluate Modulation of Cytokines Prior to Progression Prior to Progression RECIST Measurements for Representative Patient “Angiogenic Activity”? Biologic resistance to FOLFIRI + Bevacizumab should precede clinical progression

9. PlGF and bFGF are Increased Prior to Progression ULN = upper limits of normal for healthy controls

10. Individual Patients May Have Greater Increases in PlGF and bFGF Prior to Progression PlGFbFGF

11. HGF but not PDGF is Increased Prior to Progression ULN = upper limits of normal for healthy controls

12. Exploratory Analysis: Prior to Progression, Increased Cytokines Related to Myeloid Cell Activation/Recruitment * p<0.05 after multiple comparison correction; ** p<0.05, # p<0.10

13. Limitations of Analysis Clinical progression after a combination regimen may not reflect resistance to the individual components –Grothey et al. JCO 2008 Cytokine changes are associated with resistance, but not necessarily causative –At clinical progression, general pro-inflammatory state may obscure relevant changes Plasma levels may not reflect tumor microenvironment and paracrine/autocrine effects

14. Conclusions PlGF, bFGF, and HGF were increased prior to progression on a bevacizumab-containing regimen –PlGF increased after single-agent bevacizumab, an peaked prior to progression There is considerable variation in the magnitude of these changes between patients Myeloid lineage activators and chemotactic cytokines are increased prior to progression

15. Implication of Findings Markers for early detection of resistance Provides therapeutic targets to reverse resistance to anti-angiogenic therapy Individualize continued anti-angiogenic therapy after progression PlGF signaling may be inhibited by VEGF tyrosine kinase inhibitors bFGF receptor tyrosine kinase inhibitors are in clinical development Absence of alternate angiogenic signaling at progression may represent opportunity for continued benefit from bevacizumab

16. Acknowledgements John Heymach Lab Hai Tran Kathy McKee Shaoyu Yan Department of Biostatistics Jeff Morris Sijin Wen Support from: NIH U54 CA-090810 (Abbruzzese, PI; Ellis, Co-PI) Department of GI Medical Oncology Rosni Adinin Andrea Burden Renitta Boudreaux Susie Bullock Yvonne Lassere Gail Bland Clinical trial support from Genentech