1. Anti-angiogenic therapy: a new approach to the treatment of metastatic breast cancer Kathy Miller Indiana University School of Medicine, USA

2. VEGF is a central mediator of angiogenesis, the formation of new blood vessels Adapted from Carmeliet P. Nat Med 2003;9:653–60 SMC = smooth muscle cell Vasculogenesis Hemangioblast Endothelial progenitors Artery VEGF Mature vasculature SMC progenitor Angiogenesis VEGF Vein

3. Adapted from Poon RT, et al. J Clin Oncol 2001;19:1207–25 Angiogenesis is involved throughout tumor formation, growth and metastasis Stages at which angiogenesis plays a role in tumor progression Premalignant stage Malignant tumor Tumor growth Vascular invasion Dormant micrometastasis Overt metastasis (Avascular tumor) (Angiogenic switch) (Vascularised tumor) (Tumor cell intravasation) (Seeding in distant organs) (Secondary angiogenesis)

4. VEGF: a candidate for anticancer therapy Tumors rely on their existing vasculature for survival VEGF is a powerful survival factor for the immature vessels that comprise tumor microvasculature 1 The abnormal structure and function of tumor vasculature inhibits the action of conventional therapies 2 inhibition of VEGF normalizes existing vasculature 3 Tumors require new vasculature for growth and metastasis 4 VEGF is a potent stimulator of new vessel growth VEGF has a limited role in healthy adults, 5 so anti-VEGF therapy should have minimal physiological effects 1 Alon T, et al. Nat Med 1995;1:1024–8 2 Netti PA, et al. Proc Natl Acad Sci USA 1999;96:3137–42 3 Dvorak HF, et al. Am J Pathol 1995;146:1029–39 4 Folkman J, N Engl J Med 1971;285:1182–6; 5 Ferrara N, et al. Nat Med 2003;9:669–76

5. Effects of VEGF inhibition: regression of microvasculature Inai T, et al. Am J Pathol 2004;165:35–52 Reduction in tumor blood flow after 1 day of anti-VEGF therapy reduction in vessel patency and blood flow in RIP-Tag2 tumor vessels treated for 1 day with anti-VEGF therapy (B) and a vehicle control (A), as shown by lectin screening over 7 days, this was followed by endothelial cell regression

6. Effects of VEGF inhibition: modification of existing vasculature Abnormal vasculature is ‘normalized’ following VEGF inhibition Inai T, et al. Am J Pathol 2004;165:35–52

7. Effects of VEGF inhibition: inhibition of new vascular development Days after implantation 1 69 Before implantation Control Anti-VEGF therapy Osusky KL, et al. Angiogenesis 2004;7:225–33 Neovascularization in a ‘vascular window’ mouse model following implantation of tumor cells and induction of ischaemia

8. RhuMAb VEGF (bevacizumab) Recombinant humanized monoclonal anti-VEGF antibody developed from murine anti-VEGF MAb A4.6.1 1 93% human, 7% murine recognises all major isoforms of human VEGF, Kd = 8 x 10–10M. Affinity similar to that of the murine antibody RhuMAb VEGF binding is restricted to human, non-human primate and rabbit VEGF terminal half-life 17–21 days 1 Presta LG, et al. Cancer Res 1997;57:4593–9 Rhu = recombinant human

9. Bevacizumab mechanism of action: summary Bevacizumab may act against tumors in three ways regression of existing microvasculature normalization of mature vasculature inhibition of production of new vasculature EARLY BENEFITCONTINUED BENEFIT Regression of existing microvasculature Normalization of surviving microvasculature Inhibition of vessel regrowth and neovascularisation

10. Phase I/II dose-escalation trial of bevacizumab in MBC: study design Primary endpoint: overall response rate Bevacizumab administered i.v. every 2 weeks until PD or maximum of 13 treatments Response assessment on days 70 and 154 patients with stable disease or better were eligible for extension study Dosing cohorts Bevacizumab 3mg/kg every 2 weeks (n=18) PD Bevacizumab 10mg/kg every 2 weeks (n=41) PD Bevacizumab 20mg/kg every 2 weeks (n=16) PD Previously treated MBC (n=75) Cobleigh MA, et al. Semin Oncol 2003;30(5 Suppl. 16):117–24 PD = progression of disease i.v. = intravenous

11. Phase I/II trial of bevacizumab monotherapy in MBC: efficacy Bevacizumab (all doses) (n=75) Overall response rate (%)9.3 Complete response (%) Partial response (%) 1.3 8.0 Median duration of response (months)5.5 Median time to progression (months)2.4 Median survival (months)10.2 Cobleigh MA, et al. Semin Oncol 2003;30(5 Suppl. 16):117–24 The efficacy data for this trial compare favorably with those for other agents as monotherapy in similar patient populations

12. Phase I/II trial of bevacizumab monotherapy in MBC: summary Headache with nausea and vomiting occurred in 4 patients at 20mg/kg and was dose limiting Otherwise bevacizumab was well tolerated, with the now typical events associated with this agent hypertension (23% of patients) minor bleeding (epistaxis, 25.3%) venous thromboembolism (4%) proteinuria (24%) A bevacizumab dose of 10mg/kg every 2 weeks produced the best therapeutic ratio in this population of heavily pretreated patients Cobleigh MA, et al. Semin Oncol 2003;30(5 Suppl. 16):117–24

13. Phase I/II clinical trials of bevacizumab in breast cancer: summary Bevacizumab has been combined with several other therapeutic agents in phase I/II trials in locally advanced/recurrent or MBC vinorelbine 1 docetaxel 2 erlotinib 3 trastuzumab 4 letrozole 5 Efficacy and safety data from these trials suggest that these combinations are worthy of further investigation 1 Burstein HJ, et al. Breast Cancer Res Treat 2002;76:Abstract 446 2 Ramaswamy B, et al. Breast Cancer Res Treat 2003;81:Abstract 224 3 Dickler M, et al. J Clin Oncol 2004;22(15 July Suppl.):Abstract 2001 4 Pegram M, et al. Breast Cancer Res Treat 2004;88:Abstract 3039 5 Traina TA, et al. Breast Cancer Res Treat 2005 94: Abstract 2030

14. Phase III trial of bevacizumab plus capecitabine in MBC (AVF2119g) Primary endpoint: progression-free survival Secondary endpoints: overall response rate, duration of response, overall survival Treatment administration bevacizumab 15mg/kg i.v. every 3 weeks capecitabine 2,500mg/m 2 orally daily for 2 weeks of a 3-week cycle Previously treated MBC (n=462) Capecitabine (n=230) Capecitabine + bevacizumab 15mg/kg every 3 weeks (n=232) PD PD* Miller KD, et al. J Clin Oncol 2005;23:792–9*No cross over was permitted

15. Phase III MBC trial of bevacizumab and capecitabine: efficacy summary Miller KD, et al. J Clin Oncol 2005;23:792–9 Capecitabine alone Bevacizumab + capecitabinep-value Overall response rate (%) Inv19.130.20.006 IRF9.119.80.001 Progression-free survival (months) 4.174.860.857 Overall survival (months)14.515.1– Inv = investigator IRF = independent review facility

16. Phase III MBC trial of bevacizumab and capecitabine: grade 3/4 adverse events *No grade 4 Miller KD, et al. J Clin Oncol 2005;23:792–9 Adverse event Capecitabine (n=215) Bevacizumab + capecitabine (n=229) Hypertension (%)*0.517.9 Proteinuria (%)*00.9 Thrombosis (%)3.75.6 Hand-foot syndrome (%)*24.227.5 Bleeding (%)*0.50.4 CHF/cardiomyopathy (%)1.03.0 Nausea (%)*1.92.6

17. Breast cancer VEGF bFGF TGF  -1 VEGF bFGF TGF  -1 PlGF VEGF bFGF TGF  -1 PlGF PD-ECGF Pleiotrophin VEGF bFGF TGF  -1 PlGF PD-ECGF Adapted from Relf M, et al. Cancer Res 1997;57:963–9 tumor growth bFGF = basic fibroblast growth factor TGF  -1 = transforming growth factor beta-1 PlGF = placental growth factor PD-ECGF = platelet-derived endothelial cell growth factor Progression of angiogenic activity in human tumors

18. Phase III MBC trial of bevacizumab and capecitabine: summary The combination of bevacizumab and capecitabine was well tolerated, with no increase in capecitabine-related toxicities Addition of bevacizumab to capecitabine produced a significant increase in response rate, but did not result in longer progression-free survival, the primary trial endpoint As a tumor progresses, the angiogenic pathways involved become more numerous and redundant; the optimal timing for use of bevacizumab is likely to be at an earlier stage

19. RANDOMIZERANDOMIZE Paclitaxel + bevacizumab Paclitaxel 28-day cycle: Paclitaxel 90 mg/m 2 days 1, 8 and 15 Bevacizumab 10 mg/kg days 1 and 15 Phase III trial of first-line bevacizumab in MBC (E2100): study design Stratify: disease-free interval (DFI) ≤24 months vs >24 months <3 vs ≥3 metastatic sites adjuvant chemotherapy yes vs no estrogen receptor (ER)-positive vs -negative vs status unknown Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

20. Phase III trial of first-line bevacizumab in MBC (E2100): key eligibility criteria Locally recurrent or metastatic breast cancer HER2+ only if prior treatment with trastuzumab No prior chemotherapy regimens for MBC Adjuvant taxane allowed if DFI >12 months ECOG PS 0 or 1 No antitumor therapy within 21 days No CNS metastases (head CT or MR required) No significant proteinuria (>500 mg/24 hr) No therapeutic anticoagulation Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

21. Phase III trial of first-line bevacizumab in MBC (E2100): statistical design Primary endpoint: progression-free survival (PFS) 85% power for a 33% improvement —6 vs 8 months One-sided type I error ≈ 2.5% required 650 eligible patients Final analysis after 546 PFS events Interim analyses after 270 and 425 events Asymmetric boundaries to stop early either for demonstrated benefit or for lack of benefit Safety analyses Grade 4 hemorrhage or hypertension (1% acceptable) Grade 3/4 thrombosis or embolism (5% acceptable) Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

22. Phase III trial of first-line bevacizumab in MBC (E2100): current analysis Study activated December 21, 2001 Closed March 24, 2004 680 eligible patients Most common reasons for ineligibility: Baseline evaluation >4 weeks from entry (11) Hormonal therapy within 3 weeks (10) Data cut-off September 27, 2005 484 events Progression – 426 Death without documented progression – 58 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

23. Phase III trial of first-line bevacizumab in MBC (E2100): patient characteristics Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

24. Phase III trial of first-line bevacizumab in MBC (E2100): response Paclitaxel Pac + bev 339 29.9% 13.8% p<0.0001 All patients 40 30 20 10 0 Overall response rate 341 16% 37.7% Measurable disease 236262 p<0.0001 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

25. Months PFS probability Phase III trial of first-line bevacizumab in MBC (E2100): progression-free survival HR = 0.51 (0.43–0.62) Log rank test p<0.0001 Pac + bev 11.4 months Paclitaxel 6.11 months 484 events reported 1.0 0.8 0.6 0.4 0.2 0.0 0612182430 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

26. Phase III trial of first-line bevacizumab in MBC (E2100): progression-free survival GroupRatio95% CI ER+, PR+ ER+, PR- ER–, PR– No adj chemo Non-taxane Taxane Age 27–49 Age 50–64 Age 65–85 DFI 0–24 months DFI >24 months <3 sites ≥3 sites Overall 0.39 0.86 0.47 0.60 0.51 0.38 0.45 0.44 0.79 0.57 0.47 0.48 0.54 0.51 (0.29, 0.53) (0.52, 1.43) (0.35, 0.63) (0.44, 0.82) (0.39, 0.67) (0.25, 0.59) (0.32, 0.63) (0.33, 0.58) (0.53, 1.17) (0.43, 0.75) (0.37, 0.60) (0.37, 0.61) (0.41, 0.71) (0.43, 0.62) 0.00.51.01.5 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

27. Survival probability Phase III trial of first-line bevacizumab in MBC (E2100): overall survival HR = 0.84 (0.64–1.05) Log rank test p=0.12 275 events reported Months 1.0 0.8 0.6 0.4 0.2 0.0 061218243036 Pac + bev 28.4 months Paclitaxel 25.2 months Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

28. Phase III trial of first-line bevacizumab in MBC (E2100): bevacizumab toxicity NCI-CTC v3.0, worst per patient *p<0.0001; **p=0.02; ***p=0.002 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

29. Phase III trial of first-line bevacizumab in MBC (E2100): other grade 3/4 toxicities ↓LVEF = decreased left ventricular ejection fraction *p=0.05 NCI-CTC v3.0, worst per patient Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

30. Phase III trial of first-line bevacizumab in MBC (E2100): FACT-B 310252198 301220167 Mean and 95% CI Baseline17 weeks33 weeks 95 100 105 110 Pac + bev Paclitaxel FACT-B total Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

31. Phase III trial of first-line bevacizumab in MBC (E2100): FACT-G FACT-G total 75 80 85 311254200 303223168 Baseline17 weeks33 weeks Mean and 95% CI Pac + bev Paclitaxel Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

32. Phase III trial of first-line bevacizumab in MBC (E2100): ongoing correlative studies Circulating markers Serum VCAM-1 Urine VEGF Analysis of primary tumor samples VEGF expression VEGF polymorphisms Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

33. Phase III trial of first-line bevacizumab in MBC (E2100): conclusions and future directions Addition of bevacizumab to paclitaxel Significantly prolongs progression-free survival Increases objective response rate Longer follow-up required to assess impact on overall survival Further studies should Explore the role of bevacizumab in the adjuvant setting Develop methods to identify patients who are most likely to benefit from VEGF-targeted therapies Miller KD, et al. Breast Cancer Res Treat 2005;94:S6 (Abstract 3)

34. E2104 Adjuvant Pilot Trial REGISTERREGISTER Doxorubicin 60 mg/m 2 plus Cyclophosphamide 600 mg/m 2 Bevacizumab 10 mg/kg every 14 days x 4 Arm A: ddBAC >BT >B Arm B: ddAC >BT >B Doxorubicin 60 mg/m 2 plus Cyclophosphamide 600 mg/m 2 every 14 days x 4 Paclitaxel 175 mg/m 2 Bevacizumab 10 mg/kg every 14 days x 4 Bevacizumab 10 mg/kg every 14 days x 18 Bevacizumab 10 mg/kg every 14 days x 22 *Hormone therapy and radiation per standard care

35. Planned phase III trial of bevacizumab plus docetaxel in MBC (AVADO): study design Previously untreated MBC (n=705) Docetaxel + placebo PD Docetaxel + bevacizumab 7.5mg/kg every 3 weeks Docetaxel + bevacizumab 15mg/kg every 3 weeks Docetaxel – 100mg/m 2 every three weeks PD Randomised, double-blind, placebo-controlled, multicentre, phase III trial Primary endpoint: progression-free survival Secondary endpoints: overall response rate, duration of response, time to treatment failure, overall survival, safety, quality of life Recruitment to commence late March 2006

36. Conclusions Bevacizumab is the first anti-angiogenic agent with proven clinical benefit in breast cancer Bevacizumab is well tolerated in patients with breast cancer, with side effects that are easily managed Combination of bevacizumab with other agents does not increase the frequency or severity of chemotherapy- related toxicities Bevacizumab significantly improves progression-free survival and response rate in patients with locally advanced or MBC when used first line in combination with paclitaxel

37. Acknowledgements ECOG Operations Christine Sceppa Pam Cogliano Michelle Parent ECOG Statistical Office Robert Gray Molin Wang Mark Powell CTEP Jeff Abrams Helen Chen Genentech Robert Mass Ginny Langmuir Amy Sing IU Anita Rush-Taylor Robin Zon George Sledge Investigators Patients