1. Bevacizumab: Antiangiogenic therapy for breast cancer: where do we stand? Fortunato Ciardiello Division of Medical Oncology, Department of Clinical and Experimental Medicine, Second University of Naples, Italy

2. VEGF SurvivalMigration Proliferation ANGIOGENESIS Endothelial cell Binding and activation of VEGF receptor Release VEGF H2O2H2O2 PDGF IGF-1 TGF  IL-6 bFGF Hypoxia  COX-2  NO  Oncogenes  VEGF as a key mediator of angiogenesis Upstream activators of VEGF synthesis Downstream signaling pathways Antibodies Tyrosine kinase inhibitors

3. Rationale for anti-VEGF therapy in breast cancer VEGF expression is increased in many tumour types including breast cancer 1 Positive correlation between VEGF levels and poor clinical outcome, including patient survival 2 VEGF levels correlate with response to chemo/radiotherapy 3 Anti-VEGF treatment inhibits growth of human breast tumour xenografts in animals 4 1 Brown LF, et al. Hum Pathol 1995;26:86–91 2 Linderholm B, et al. J Clin Oncol 2000;18:1423–31 3 Gasparini G, et al. Cancer J Sci Am 1999;5:101–11 4 Borgstrom P, et al. Anticancer Res 1999;19:4203–14 VEGF = vascular endothelial growth factor

4. Phase II trials of Bevacizumab plus chemotherapy in MBC

5. Phase II trial of Bevacizumab plus vinorelbine in refractory breast cancer (AVF2324s) Two-stage design 19 patients recruited.  6 responses required for a further 18 patients to be recruited Primary endpoint: response rate Secondary endpoints include time to progression and safety Treatment administration Bevacizumab 10mg/kg i.v. every 2 weeks vinorelbine 25mg/m 2 i.v. weekly. Dose adjusted following ANC assessment Bevacizumab (10mg/kg every 2 weeks) + vinorelbine PD ANC = absolute neutrophil count Refractory breast cancer Burstein HJ, et al. Breast Cancer Res Treat 2002;79: S115 (Abstract 446)

6. Phase II trial of Bevacizumab plus vinorelbine in refractory breast cancer (AVF2324s): efficacy Data from 54 evaluable patients Burstein HJ, et al. Breast Cancer Res Treat 2002;79: S115 (Abstract 446)

7. Phase II trial of Bevacizumab plus vinorelbine in refractory breast cancer (AVF2324s): safety Data from 55 evaluable patients Burstein HJ, et al. Breast Cancer Res Treat 2002;79: S115 (Abstract 446)

8. Phase II trial of Bevacizumab plus vinorelbine in refractory breast cancer(AVF2324s): conclusions Bevacizumab plus vinorelbine has clinical activity 31% of patients had an objective response several patients had responses of >1 year, which is encouraging This combination was well tolerated side effects relating to Bevacizumab included hypertension and epistaxis Studies in less heavily pretreated patients may be warranted Burstein HJ, et al. Breast Cancer Res Treat 2002;79: S115 (Abstract 446)

9. Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): study design Primary endpoints: response rate, overall survival and toxicity Secondary endpoints: correlative studies baseline plasma VEGF soluble activated endothelial cell markers and adhesion molecules microvessel density by CD31 immunohistochemistry tumour and endothelial cell apoptosis by TUNEL assay Bevacizumab (10mg/kg every 2 weeks) + docetaxel X 6 PD Metastatic breast cancer (n=27) Bevacizumab alone Docetaxel 35mg/m 2 weekly for 3 weeks of a 4-week cycle Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

10. Metastatic disease measurable by RECIST 0–1 prior chemotherapy regimens for metastatic disease ECOG PS 0–2 At least 6 months since prior taxane therapy No brain metastases No major surgical procedure or significant traumatic injury within 28 days Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): eligibility criteria Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

11. Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): efficacy summary Responsen (%) Complete response0 (0) Partial response14 (52) Stable disease9 (33) Overall response14 (52) Withdrawn due to toxicity prior to 2 cycles2 (7) Median progression-free survival (months) [95% CI] 7.5 [6.2–8.3] Median duration of response (months) [95% CI] 6.0 [4.6–6.5] Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

12. Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): Avastin-related toxicity Bevacizumab plus docetaxel (n=27) Grade 2Grade 3Grade 4 Hypertension, n (%) 4 (14.8) 1 (3.7)0 Proteinuria, n (%)11 (40.7)00 Epistaxis, n (%) 1 (3.7)00 Thromboembolic events, n (%)00 2 (7.4) Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

13. Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): other toxicities Bevacizumab plus docetaxel (n=27) Grade 2Grade 3Grade 4 Dyspnoea, n (%)18 (66.7)1 (3.7)0 Eye tearing, n (%)15 (55.6)00 Fatigue, n (%)19 (70.3) 4 (14.8)0 Leukopenia, n (%) 3 (11.1) 6 (22.2)1 (3.7) Neutropenia, n (%) 2 (7.4) 4 (14.8)1 (3.7) Infection, n (%) 4 (14.8)01 (3.7) Neuropathy, n (%) 3 (11.1)2 (7.4)0 Stomatitis, n (%) 9 (33.3)2 (7.4)0 Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

14. Phase II trial of Bevacizumab plus weekly docetaxel in MBC (AVF2326s): summary Efficacy data from this first reported clinical trial of Avastin and docetaxel are encouraging the response rate of 52% shows that this is an active combination Toxicity was acceptable: the only grade 4 adverse event attributable to Avastin was venous thromboembolism in two patients most toxicity was consistent with the safety profile of weekly docetaxel This regimen is worthy of further investigation in a randomised phase III trial Ramaswamy B, et al. Clin Cancer Res 2006;12:3124–9

15. A phase II trial is investigating Avastin 15mg/kg plus docetaxel 75mg/m 2 every 3 weeks in treatment-naïve MBC Primary endpoint: time to progression 43 of 75 patients have been enrolled 21 have had at least one assessment Response rate is 40% The most common grade 3/4 adverse events to date are neutropenia, febrile neutropenia and hypertension LVEF decline seen in one patient This regimen is well tolerated and appears active in this patient population Ongoing phase II trial of Bevacizumab plus docetaxel in MBC (AVF3110s) Chan D, et al. J Clin Oncol 2006;24(20 June suppl.):605s (Abstract 13047)

16. Phase III trials of Bevacizumab plus chemotherapy in MBC

17. Phase III trial of Bevacizumab plus Xeloda ® in MBC (AVF2119g) Primary endpoint: progression-free survival Secondary endpoints: overall response rate, duration of response and overall survival Treatment administration Bevacizumab 15mg/kg i.v. every 3 weeks Xeloda 2,500mg/m 2 orally daily for 2 weeks of a 3-week cycle Previously treated MBC (n=462) Xeloda (n=230) Xeloda + Avastin 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

18. Phase III trial of Bevacizumab and chemotherapy in relapsed/refractory MBC (AVF2119g) Inclusion criteria prior anthracycline and taxane treatment —one or two prior chemotherapy regimens for MBC or —relapse within 12 months of completing anthracycline- and taxane- containing adjuvant therapy ECOG PS 0 or 1 Exclusion criteria antitumour therapy within 21 days anticoagulation therapy CNS metastases (head CT or MRI required) Miller KD, et al. J Clin Oncol 2005;23:792–9

19. Phase III MBC trial of Bevacizumab and chemotherapy (AVF2119g): patient characteristics

20. Phase III MBC trial of Bevacizumab and chemotherapy (AVF2119g): progression-free survival *Determined by independent review facility where available 012345678910111213141516 1.0 0.8 0.6 0.4 0.2 0 Progression-free survival (months)* Proportion progression-free 4.17 4.86 Xeloda alone (n=230) (median progression-free survival = 4.17 months) Xeloda + Bevacizumab (n=232) (median progression-free survival = 4.86 months) HR=0.98; p=0.857 Miller KD, et al. J Clin Oncol 2005;23:792–9

21. Phase III MBC trial of Bevacizumab and chemotherapy (AVF2119g): duration of survival Proportion surviving Duration of survival (months) 1.0 0.8 0.6 0.4 0.2 0 Xeloda alone (n=230) (median survival = 14.5 months) Xeloda + Bevacizumab (n=232) (median survival = 15.1 months) 012345678910111213141516 17 18 19 Miller KD, et al. J Clin Oncol 2005;23:792–9

22. Phase III MBC trial of Bevacizumab and chemotherapy (AVF2119g): efficacy summary Miller KD, et al. J Clin Oncol 2005;23:792–9 Inv = determined by investigators IRF = determined by independent review facility

23. Phase III MBC trial of Bevacizumab and chemotherapy (AVF2119g): grade 3/4 adverse events *No grade 4 Miller KD, et al. J Clin Oncol 2005;23:792–9

24. Phase III trial of Bevacizumab in first-line MBC (E2100) This trial focuses on a less heavily pretreated population than AVF2119g Primary endpoint: progression-free survival (PFS) Other endpoints: overall response rate, overall survival, quality of life, correlative studies Previously untreated MBC (n=722) Paclitaxel (n=354) Paclitaxel + Bevacizumab 10mg/kg every 2 weeks (n=368) PD* PD *No cross over will be permitted Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3)

25. Phase III trial of Bevacizumab in first-line MBC (E2100): eligibility criteria Locally recurrent or MBC HER2+ only if prior treatment with Herceptin (trastuzumab) or contraindication No prior chemotherapy regimens for MBC adjuvant taxane allowed if disease-free interval >12 months ECOG PS 0 or 1 No antitumour therapy within 21 days No CNS metastases (head CT or MRI required) No significant proteinuria (>500mg/24 hours) No therapeutic anticoagulation HER = human epidermal growth factor receptor CT = computed tomography MRI = magnetic resonance imaging Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3)

26. Phase III trial of Bevacizumab in first-line MBC (E2100): patient characteristics 56 (29-84) 42 43 65 18 59 5 55 (27-85) 42 43 64 18 64 4 Median age, range (years) Disease-free interval <24 months (%) >3 sites (%) Adjuvant chemotherapy (%) Taxane (%) ER+ (%) HER2+ (%) Paclitaxel + Bevacizumab (n=341) Paclitaxel (n=339) Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3) ER = oestrogen receptor

27. 0.0 0.2 0.4 0.6 0.8 1.0 Months Progression-free survival proportion 0612182430 Phase III trial of Bevacizumab in first-line MBC (E2100): progression-free survival HR = 0.51 (0.43-0.62) Log rank test p<0.0001 Bevacizumab + paclitaxel: 11.4 months Paclitaxel: 6.11 months 484 events reported (89% of required events) Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3) HR = hazard ratio 6.1111.4

28. Phase III trial of Bevacizumab in first-line 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)

29. Phase III trial of Bevacizumab in first-line MBC (E2100): overall response rate Overall response rate (%) 339341262236 p<0.0001 Paclitaxel Bevacizumab + paclitaxel 13.8 29.9 16.0 37.7 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3) 0 10 20 30 40 All patientsMeasurable disease

30. 0.0 0.2 0.4 0.6 0.8 1.0 Months Overall survival proportion 0 61218243036 Phase III trial of Bevacizumab in first-line MBC (E2100): overall survival Bevacizumab + paclitaxel: 28.4 months Paclitaxel: 25.2 months HR = 0.84 (0.64, 1.05) Log rank test: p=0.12 275 events reported (57% of required events) Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3)

31. Phase III trial of Bevacizumab in first-line MBC (E2100): NCI-CTC grade 3 and 4 toxicities NCI-CTC v3.0, worst per patientNCI-CTC = National Cancer Institute common toxicity criteria Paclitaxel (n=332) Paclitaxel + Bevacizumab (n=350) Grade 3Grade 4Grade 3Grade 4 Hypertension* (%)2015<1 Thromboembolic events (%) 2220 Bleeding † (%)002<1 Proteinuria § (%)0011 *p<0.0001; † p=0.02; § p=0.002 Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3)

32. Phase III trial of first-line Bevacizumab in MBC (E2100): summary Addition of Bevacizumab to paclitaxel significantly increased progression-free survival, the primary endpoint of the trial Addition of Bevacizumab to paclitaxel significantly increased overall response rate in all patients and in patients with measurable disease Overall survival data are preliminary, after only 57% of required events The combination of Bevacizumab and paclitaxel was well tolerated, with no unexpected side effects reported Miller KD, et al. Breast Cancer Res Treat 2005;94:S6(Abstract 3)

33. Ongoing and future trials of Bevacizumab in MBC

34. Planned phase III trial of Bevacizumab plus docetaxel in MBC (AVADO): study design 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 commenced March 2006 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

35. AVADO: key eligibility criteria Chemonaïve locally recurrent or metastatic breast cancer Aged ≥18 years, female ECOG performance status 0–1 HER2-negative; documented oestrogen/progesterone receptor status Prior adjuvant chemotherapy permitted if relapse ≥6 months since last dose (≥12 months if taxane based) No uncontrolled hypertension

36. Ongoing trials of Bevacizumab in breast cancer: RIBBON 1 (AVF3694g) Primary endpoint: progression-free survival Chemotherapy regimen is determined by investigator prior to randomisation Trial already open in USA and will be opening in other countries during 2006 *Continuation or cross over to Avastin after confirmation of PD is allowed at the discretion of the investigator Taxane-based or anthracycline-based or Xeloda + Avastin 15mg/kg every 3 weeks Taxane-based or anthracycline-based or Xeloda + placebo Previously untreated MBC (n=950) PD* Randomise 2:1 2 1

37. Planned trial of docetaxel and Herceptin with or without Bevacizumab (AVEREL) Primary endpoint: progression-free survival Secondary endpoints: response rate, duration of response, overall survival, safety Start date: Q3 2006 Previously untreated HER2+ MBC (n=320) Docetaxel + Herceptin + Avastin 15mg/kg every 3 weeks PD PD* * No cross-over permitted

38. Bevacizumab in MBC: summary Bevacizumab monotherapy has activity in patients with MBC Bevacizumab plus paclitaxel significantly prolongs progression-free survival, and significantly improves response rate Ongoing trials are evaluating Bevacizumab with other chemotherapy agents and in other settings, including the adjuvant and neoadjuvant settings