1. 1 Highlights in the management of colorectal cancer “Therapeutic antibodies: Bevacizumab “ Roma, 1-2 February 2007 Vincenzo Adamo Oncologia Medica e Terapie Integrate A.O.Universitaria, Policlinico “G.Martino” Messina

2. 2 Angiogenesis Ferrara N. Kidney Int 1999;56:794–814 Inhibitors Thrombospondin (TSP) Angiostatin Endostatin Vasostatin Heparin Prolactin Growth hormone Canstatin Tumstatin Interferon-a (IFN-a) Activators VEGF Acid-FGF Basic-FGF TGF-a, b EGF TNF-a Angiogenin IL-8 Angiopoietin-1, 2 is the growth of new blood vessels from existing vessels and plays an a role in tumor development

3. 3 The Angiogenic Switch is necessary... for Tumor Growth and Metastasis Somatic mutation Small avascular tumor Tumor secretion of angiogenic factors stimulates angiogenesis TAF Rapid tumor growth and metastasis Carmeliet and Jain. Nature. 2000; 407:249; Bergers and Benjamin. Nat Rev Cancer. 2003; 3:401. Tumor is dormant Neovascularization: Allows rapid tumor growth by providing oxygen, nutrients, and waste removal Facilitates metastasis Angiogenic switch

4. 4 Angiogenesis: role of VEGF Key mediator of angiogenesis Stimulates growth of endothelial cells Also known as VEGF-A Related molecules are VEGF-B, C & D, placental growth factor (PIGF) Homodimeric glycoprotein Molecular weight: 45,000Da Binds VEGF receptor-2 and heparin Four molecular species VEGF 121 VEGF 165* VEGF 189 VEGF 206 Ferrara N, et al. Endocr Rev 1997;18:4–25 *Predominant molecular species

5. 5 Angiogenesis: VEGF family, receptors, signal transduction and its effects Shibuya M. Cell Struct Funct 2001;26:25–35 VEGF binding to VEGF receptor-2 activates a signalling cascade resulting in cellular effects Cation channel  Permeability – P P– – P P– – P P– VEGF receptor-1 VEGF receptor-2 VEGF-C VEGF-D VEGF receptor-3 DAG PLC Protein kinase C Raf-1 MAPK Proliferation, migration  Permeability SAPK/ JNK Apoptosis Survival Calcium release Ca 2+ Proliferation Migration IP 3 PLC P13K Protein kinase B VEGF-A VEGF-B PlGF VEGF-A

6. 6 Anti-VEGF antibody Bevacizumab  Recombinant humanised monoclonal anti-VEGF antibody 93% human, 7% murine  Recognises all major isoforms of human VEGF  RhuMAb VEGF binding is restricted to human  Bevacizumab binds VEGF, preventing interaction with its receptors and activation of downstream signalling pathways  This ultimately leads to vascular regression, leaving the tumour dormant Bevacizumb – P P– VEGF X Growth Proliferation Migration Survival X

7. 7 Agents targeting the VEGF pathway VEGF VEGF receptor-2 Cation channel  Permeability Antibodies inhibiting VEGF (e.g. bevacizumab) Antibodies inhibiting VEGF receptors Soluble VEGF receptors (VEGF-TRAP) Small-molecules inhibiting VEGF receptors (TKIs) (e.g. PTK-787) Ribozymes (Angiozyme) – P P– – P P– – P P– Migration, permeability, DNA synthesis, survival Lymphangiogenesis Angiogenesis ???

8. 8 Angiogenesis: therapeutic rational to inhibition of VEGF A.Prevent tumor angiogenesis: ► Inhibition of tumor and metastasis growth B.Reduce tumoral vascularization and normalize vascular permeability and tumoral interstitial pressure ► easier penetration of the tumor by chemotherapeutic agents C.Induce vascular regression ►possible tumoral dormancy Jain RK. Nat Med 2001;7:987–9 Willett CG, et al. Nat Med 2004;10:145–7

9. 9 Bevacizumab in clinical development: overview Combined with IFL in previously untreated mCRC 5mg/kg every 2 weeks (n=813) Hurwitz H, et al. 2004 Dose-escalation trial in solid malignancies. Safety and pharmacokinetics (n=25) Gordon MS, et al. 2001 Phase I Phase II Phase III 5-FU = 5-fluorouracil; LV = leucovorin; mCRC = metastatic colorectal cancer; FOLFOX = 5-FU/LV + oxaliplatin; XELOX = Xeloda + oxaliplatin; IFN = interferon; CP = carboplatin/paclitaxel; CG = cisplatin/gemcitabine; RCC = renal cell cancer; MBC = metastatic breast cancer; IFL = irinotecan/5-fluorouracil/leucovorin; NSCLC = non-small cell lung cancer; PC = pancreatic cancer Combined with Xeloda  in previously treated MBC 15mg/kg every 3 weeks (n=462) Miller KD, et al. 2005 Combined with chemotherapy in metastatic cancers 3mg/kg every week (n=12) Margolin K, et al. 2001 Combined with either FOLFOX4 or XELOX in previously untreated mCRC (n=1,920) AVOREN: combined with IFN-  2a in metastatic RCC 10mg/kg every 2 weeks (n=638) AVANT: combined with FOLFOX4 or XELOX in stage II/III colon cancer 5mg/kg every 2 weeks or 7.5mg/kg every 3 weeks (n=3,450) AVAIL: combined with CG in previously untreated stage IIIb, IV or recurrent NSCLC 7.5 or 15mg/kg every 3 weeks (n=830) Combined with CP in previously untreated NSCLC (n=99) Johnson DH, et al. 2004 Monotherapy in previously treated RCC 3 or 10mg/kg every 2 weeks (n=116) Yang JC, et al. 2003 Combined with 5-FU/LV in previously untreated mCRC 5 or 10mg/kg every 2 weeks (n=104) Kabbinavar F, et al. 2003 Monotherapy in previously treated MBC 3, 10 or 20mg/kg every 2 weeks (n=75) Cobleigh MA, et al. 2003 Combined with 5-FU/LV in previously untreated mCRC 5mg/kg every 2 weeks (n=209) Kabbinavar F, et al. 2005 Combined with gemcitabine in unresectable PC 10mg/kg every 2 weeks Kindler HL, et al. 2004 Combined with Tarceva TM + gemcitabine in previously untreated PC 5mg/kg every 2 weeks (n=600)

10. 10 Bevacizumab with 5-FU/LV

11. 11 Phase II trial of Avastin in mCRC (AVF0780g): study design Primary endpoints time to progression response rate i.v. 5-FU 500mg/m 2 and LV 500mg/m 2 were given weekly for the first 6 weeks of an 8-week cycle (Roswell Park regimen) Bevacizumab was given every 2 weeks Tumour assessments were at the end of each 8-week cycle Kabbinavar F, et al. J Clin Oncol 2003;21:60–5 5-FU/LV + Avastin (10mg/kg) (n=33) 5-FU/LV + Avastin (5mg/kg) (n=35) 5-FU/LV (n=36) High-dose Avastin (10mg/kg every 2 weeks) Previously untreated metastatic CRC PD

12. 12 Phase II trial of Avastin in metastatic CRC (AVF0780g): time to progression Blinded review by independent evaluators Median (months) Control: 5.2 Avastin 5mg/kg: 9.0 (HR=0.46; p=0.005) Avastin 10mg/kg: 7.2(HR=0.66; p=0.217) 1.0 0.8 0.6 0.4 0.2 0 02468101214 Avastin 5mg/kg (n=35) Avastin 10mg/kg (n=33) Control (n=36) Time to progression (months) Proportion progression-free 5.27.29.0 Roche data on file

13. 13 Phase II trial of Avastin in metastatic CRC (AVF0780g): adverse events All events Not adjusted for treatment duration Kabbinavar F, et al. J Clin Oncol 2003;21:60–5

14. 14 Bevacizumab plus IFL (AVF2107g) in metastatic CRC

15. 15 Phase III trial of IFL ± Avastin in mCRC (AVF2107g): study design IFL bolus 5-FU 500mg/m 2 LV 20mg/m 2 + irinotecan 125mg/m 2 given 4/6 weeks Hurwitz H, et al. N Engl J Med 2004;350:2335–42 5-FU/LV bolus 5-FU 500mg/m 2 LV 500mg/m 2 given 6/8 weeks Avastin 5mg/kg every 2 weeks May receive Avastin past disease progression No Avastin past disease progression May receive Avastin past disease progression Previously untreated metastatic CRC Bolus IFL + placebo (n=411) Bolus IFL + Avastin (n=402) 5-FU/LV + Avastin (n=110) Arm closed to enrolment

16. 16 Phase III trial of IFL ± Avastin in mCRC (AVF2107g): survival Median survival (months) IFL + placebo: 15.6 (95% CI: 14.3–17.0) vs IFL + Avastin: 20.3 (95% CI: 18.5–24.2) HR=0.66 (95% CI: 0.54–0.81) p<0.001 CI = confidence interval HR = hazard ratio Probability of survival 1.0 0.8 0.6 0.4 0.2 0 010203040 Survival (months) IFL + Avastin IFL + placebo 15.6 20.3 Hurwitz H, et al. N Engl J Med 2004;350:2335–42

17. 17 Phase III trial of IFL ± Avastin in mCRC (AVF2107g): PFS Median progression-free survival (months) IFL + placebo: 6.2 (95% CI: 5.6–7.7) IFL + Avastin: 10.6 (95% CI: 9.0–11.0) HR=0.54 (95% CI: 0.45–0.66) p<0.001 Probability of being progression free 1.0 0.8 0.6 0.4 0.2 0 0102030 Progression-free survival (months) 6.2 10.6 IFL + Avastin IFL + placebo Hurwitz H, et al. N Engl J Med 2004;350:2335–42 AVF2107g

18. 18 Phase III trial of IFL ± Avastin in mCRC (AVF2107g): Avastin-related toxicity NB: not adjusted for different time on therapy *p<0.05 Hurwitz H, et al. N Engl J Med 2004;350:2335–42

19. 19 First-line Phase II trial of Avastin ± 5- FU/LV in mCRC (AVF2192g) Multicentre, double-blind, randomised, controlled trial in 209 patients not eligible for first-line irinotecan Previously untreated metastatic CRC (n=209) 5-FU/LV (n=105) 5-FU/LV + Bevacizumab 5mg/kg every 2 weeks (n=104) PD Primary endpoint: duration of survival Secondary endpoints include overall response rate Kabbinavar FF, et al. J Clin Oncol 2005;23:3697–705

20. 20 Phase II trial of Avastin ± 5-FU/LV in mCRC (AVF2192g): survival 1.0 0.8 0.6 0.4 0.2 0 010203040 Probability of survival 5-FU/LV + Avastin 5-FU/LV + placebo Duration of survival (months) HR=0.79, p=0.160 vs control Median survival: 12.9 vs 16.6 months Kabbinavar FF, et al. J Clin Oncol 2005;23:3697–705 12.916.6 AVF2192g

21. 21 Phase II trial of Avastin ± 5-FU/LV in mCRC (AVF2192g): progression-free survival HR=0.50, p=0.0002 vs control Median progression-free survival: 5.5 vs 9.2 months 1.0 0.8 0.6 0.4 0.2 0 Probability of being progression free 0102030 Progression-free survival (months) 5-FU/LV + Avastin 5-FU/LV + placebo 5.59.2 Kabbinavar FF, et al. J Clin Oncol 2005;23:3697–705

22. 22 Phase II trial of Avastin ± 5-FU/LV in mCRC (AVF2192g): Avastin-related toxicity NB: not adjusted for different time on therapy Kabbinavar FF, et al. J Clin Oncol 2005;23:3697–705

23. 23 FDA Approval BEVACIZUMAB used in combination with intravenous 5-FU– based chemotherapy, is indicated for first-line treatment of patients with metastatic carcinoma of the colon or rectum.

24. 24 Avastin plus FOLFIRI

25. 25 Phase II trial of Avastin plus FOLFIRI (AVIRI): study design Primary endpoint: progression-free survival Secondary endpoints: overall survival, overall response rate, duration of response and safety Recruitment complete Patients with previously untreated metastatic CRC (n=209) Avastin 5mg/kg every 2 weeks + FOLFIRI Sobrero A, et al. J Clin Oncol 2006;24:157s (Abstract 3544)

26. 26 Phase II trial of Avastin plus FOLFIRI (AVIRI): efficacy Sobrero A, et al. J Clin Oncol 2006; 24: 157s (Abstract 3544) The incidence of Bevacizumab related side effects appears to be generally similar to that in other Beva-trials Estimated 6 month progression free survival rate is 82%

27. 27 Phase II trial of Avastin plus FOLFIRI (US): preliminary results Phase II trial of Avastin (5mg/kg every 2 weeks) plus FOLFIRI in 23 patients with metastatic CRC 1 1 Kopetz S, et al. J Clin Oncol 2006:24:165s (Abstract 3579) Generally well tolerated: less diarrhoea and hospitalisations than that observed with IFL plus Avastin; similar level of hypertension and proteinuria *Observed at a median of 4.1 (2.2–8.0) months

28. 28 Avastin plus Capecitabine alone or combo CAPIRI/XELIRI

29. 29 Preclinical evidence for the use of Avastin with Xeloda-based therapy Shen BQ, et al. Proc AACR 2004;45 (Abstract 2203) *Sub-maximum effective doses Combining Avastin and Xeloda resulted in a greater duration of tumour inhibition than with either agent alone Mean tumour volume (mm 3 ) 2,000 1,750 1,500 1,250 1,000 750 500 250 0 Control Avastin* Xeloda* Avastin* + Xeloda* 071421283542495663 Day

30. 30 Ongoing/planned trials of Xeloda plus Avastin in first-line mCRC MAX (ML18513); randomised phase II/III trial (n=333) patients with previously untreated metastatic CRC will be randomised to receive one of three regimens until disease progression —Xeloda —Avastin 7.5mg/kg every 3 weeks plus Xeloda —Avastin 7.5mg/kg every 3 weeks plus Xeloda plus mitomycin C Several other trials of Avastin plus Xeloda are planned, including ML18524, open label study comparing the effect of three chemotherapy regimens (n=300) ML18799, phase II trial (n=80) ML19823, phase II trial in elderly patients (n=60)

31. 31 Phase I/II trial of Avastin plus Xeloda and irinotecan (XELIRI) Responsen=13 (%) Complete response1 (8) Partial response10 (77) Stable disease2 (15) Therapy was well tolerated epistaxis was reported in one patient, proteinuria <1g/day in two patients and granulocytopenia grade 3 in one patient Kocakova I, et al. J Clin Oncol 2006;24:616s (Abstract 13504) Patients (n=14) were treated with Avastin 7.5mg/kg every 3 weeks plus XELIRI; 13 patients are evaluable for response

32. 32 Avastin with oxaliplatin-based regimens

33. 33 Phase III trial of second-line Avastin plus FOLFOX4 (E3200): study design Primary endpoint: duration of survival Secondary endpoint: overall response rate Exclusion criteria: CNS metastases ; active cardiovascular disease Previously treated metastatic CRC (n=822) Oxaliplatin/5-FU/LV (n=290) Avastin monotherapy 10mg/kg every 2 weeks (n=243) Oxaliplatin/5-FU/LV + Avastin 10mg/kg every 2 weeks (n=289) PD Arm closed to enrolment Giantonio BJ, et al. J Clin Oncol 2005;23:1s (Abstract 2)

34. 34 Phase III trial of second-line Avastin (E3200): progression-free survival Probability of being progression free 1.0 0.8 0.6 0.4 0.2 0 Progression-free survival (months) 02468101214161820 MedianTotal 2737.2 2734.8 2292.7 A: FOLFOX4 + Avastin B: FOLFOX4 C: Avastin HR=0.64 A vs B: p<0.0001 B vs C: p<0.0001 Giantonio BJ, et al. J Clin Oncol 2005;23:1s (Abstract 2) 2.77.24.8 A: FOLFOX4 + Avastin C: Avastin B: FOLFOX4

35. 35 Phase III trial of second-line Avastin (E3200): overall survival MedianTotal A: FOLFOX4 + Avastin28912.9 B: FOLFOX429010.8 C: Avastin24310.2 HR = hazard ratio Probability of survival 1.0 0.8 0.6 0.4 0.2 0 Time (months) HR=0.76 A vs B: p=0.0018 B vs C: p=0.95 10.212.9 10.8 0369121518212427303336 A: FOLFOX4 + Avastin C: Avastin B: FOLFOX4 Giantonio BJ, et al. J Clin Oncol 2005;23:1s (Abstract 2)

36. 36 Phase III trial of second-line Avastin (E3200): grade 3/4 toxicity FOLFOX4 + Avastin (n=287) FOLFOX4 (n=284) Avastin (n=234) Grade 3Grade 4Grade 3Grade 4Grade 3Grade 4 Hypertension (%) 512<170 Bleeding (%) 3<1 020 Neuropathy (%)16<19 Vomiting (%) 913<150 Bowel perforation (%)1 01.3 Venous thrombosis 913401 Cardiac ischaemia 120110 Giantonio BJ, et al. J Clin Oncol 2005;23:1s (Abstract 2)

37. 37 Phase III study ongoing: XELOX ± bevacizumab vs FOLFOX4 ± bevacizumab (NO16966C) Factorial study 2 x 2 Previously untreated metastatic CRC (n=1920) Bevacizumab 5 mg/kg every 2 weeks (n=330) Placebo (n=330) Bevacizumab 7,5 mg/kg every 3 weeks (n=330) Placebo (n=330) FOLFOX4 (n=300) XELOX (n=300) PRO Primary endpoints Time to progression with XELOX (± bevacizumab) equivalent to FOLFOX4 (± bevacizumab) Time to progression with bevacizumab + XELOX/FOLFOX superior to XELOX/FOLFOX+placebo Cassidy J.; ESMO 2006; Saltz LB ASCO GI 2007

38. 38 Ongoing phase III trial of XELOX ± Avastin vs FOLFOX4 ± Avastin (NO16966C): preliminary data The study met both primary endpoints: XELOX= FOLFOX4 as similar activity in terms of PFS Bevacizumab + XELOX or FOLFOX4 significantly improved PFS compared with chemotherapy alone No new safety signals related to Beva have been reported Cassidy J. ESMO 2006 ; Saltz LB ASCOGI 2007 1.0 0.8 0.6 0.4 0.2 0.0 HR = 0.70 [ 97.5% CI 0.58–0.83 ] p < 0.0001 XELOX / FOLFOX + bevacizumab 289 events XELOX / FOLFOX + placebo 347 events PFS estimate 05 10 152025 8.5 11.5

39. 39 TREE-2: phase II trial of first-line with various oxaliplatin-based regimens First-line metastatic CRC (n=223) mFOLFOX6 + Avastin 5mg/kg every 2 weeks (n=75) XELOX + Avastin 7.5mg/kg every 3 weeks (n=74) bFOL + Avastin 5mg/kg every 2 weeks (n=74) PD Primary endpoint: grade 3/4 toxicity during the first 12 weeks of therapy Secondary endpoints include overall response rate, time to progression and overall survival Hochster HS, et al. J Clin Oncol 2006;24:148s (Abstract 3510)

40. 40 TREE-1 versus TREE-2: TTP * 0510 Months 152025 1.0 0.8 0.6 0.4 0.2 0 Probability 0510 Months 152025 Probability TREE-1TREE-2 mFOLFOX bFOL XELOX mFOLFOX + Avastin bFOL + Avastin XELOX + Avastin mFOLFOX (n=49) bFOL (n=50) XELOX (n=48) Median TTP (months)8.76.95.9 95% CI 6.5–9.84.2–8.0 5.1–7.4 Avastin + mFOLFOX (n=71) bFOL (n=70) XELOX (n=72) Median TTP (months)9.98.310.3 95% CI 7.9–11.76.6–9.98.6–12.5 1.0 0.8 0.6 0.4 0.2 0 Hochster HS, et al. J Clin Oncol 2006;24:148s (Abstract 3510)

41. 41 TREE-1 versus TREE-2: overall survival mFOLFOX (n=49) bFOL (n=50) XELOX (n=48) Median OS (months) 19.217.917.2 95% CI14.2–24.211.5–24.612.5–22.3 1.0 0.8 0.6 0.4 0.2 0.1 0.3 0.5 0.7 0.9 0510 Survival time (months) 152025303540 0 Probability 1.0 0.8 0.6 0.4 0.2 0.1 0.3 0.5 0.7 0.9 0510 Survival time (months) 152025303540 0 Probability TREE-1TREE-2 Avastin + mFOLFOX (n=71) bFOL (n=70) XELOX (n=72) Median OS (months) 26.020.727.0 95% CI18.0–NE18.8–25.321.8–NE mFOLFOX bFOL XELOX mFOLFOX + Avastin bFOL + Avastin XELOX + Avastin Hochster HS, et al. J Clin Oncol 2006;24:148s (Abstract 3510)

42. 42 TREE-1 versus TREE-2: conclusions TREE-2 shows that combining Avastin with oxaliplatin- containing regimens is well tolerated and does not significantly alter the regimens’ toxicity profiles 1 The addition of Avastin to oxaliplatin-containing regimens improves response rates, TTP and OS compared to TREE-1 XELOX tolerability improved with the lower Xeloda dose (850mg/m 2 bid) used in TREE-2 Bolus 5-FU-based regimens (bFOL) appear to be less active than infusional 5-FU (FOLFOX) or Xeloda-containing regimens Hochster HS, et al. J Clin Oncol 2006;24:148s (Abstract 3510)

43. 43 First-line randomised phase II Avastin + XELIRI or XELOX in mCRC: AIO study preliminary data Previously untreated metastatic CRC XELOX + Bevacizumab 7.5mg/Kg, arm A XELIRI + Bevacizumab 7.5mg/Kg, arm B Conclusions: In pts with untreated mCRC, both regimens, XELOX+B and XELIRI+B are well-tolerated without difference for severe AE in both treatment arms (except neuropathy) and can be safely administered on an outpatient basis. So far, both regimens show similarly high tumor control and response rates. M. Geissler. Et al. ASCO GI 2007 abs335 ML18405 trial TOXICITYARM AARM B diarrhea16.911, 8 hand-foot-syndrome7,92,4 peripheral neuropathy12,40,0 venous thrombosis3,42,4 grade 3 hypertension2,31,2 GI perforation1,11,2

44. 44 Avastin in first-line treatment of metastatic CRC Metastatic CRC IFL/FOLFIRI/ XELIRI 5-FU or Xeloda FOLFOX or XELOX Irinotecan + cetuximab FOLFOX BSC/FOLFOX/ FOLFIRI FOLFIRI5-FU/LV First-line Second-line Avastin Avastin has shown consistent efficacy improvements in metastatic CRC, regardless of the regimen with which it is combined

45. 45 Safety profile of Bevacizumab

46. 46 Bevacizumab-related events in CRC trials to date: overview  hypertension (most common event)  proteinuria  arterial thrombosis  effects on wound healing  bleeding  GI perforation Kabbinavar F, et al. J Clin Oncol 2003;21:60–5 Hurwitz H, et al. N Engl J Med 2004;350:2335–42 Giantonio BJ, et al. ASCO GI Symposium; 27–29 2005; Hollywood, Fl. Abstract 169a./www.asco.org. 2005. Kabbinavar FF, et al. J Clin Oncol 2005..

47. 47 Bevacizumab related toxicity Kabbinavar JCO/03 Kabbinavar JCO/05 Hurwiz NEJM Giantonio JCO/05 Tree 2 Avastin Dosage 5 & 10 MG/KG 5 MG/KG 10 MG/KG5 MG/KG Schedule ROSWELL PARK ROSWELL PARK IFLFOLFOX 4FOLFOX 6 Ipertension11%16% (G3)11.0%(G3 %) 5% (G3) 1% (G4) Proteinuria23%1% (G3)3.1% (G3) 0.8% (G4) 1% (G4) Trombosis26%18%19.4% Bleeding G.I. perforation 6%3% (G3) 2% (G4) 1.5%3% (G3) < 1% (G4) 2.8% Neuropaty ??16% (G3)

48. 48 BRiTE: US observation study & First-BEAT: International observational study (ex-US)

49. 49 BRiTE: study overview BRiTE = Bevacizumab Regimens Investigation of Treatment Effects and Safety Hedrick E, et al. J Clin Oncol 2006; 24(Suppl.):155s (Abstract 3536) Avastin plus chemotherapy PD Previously untreated metastatic, locally advanced and unresectable CRC (n=1,968) Chemotherapy regimen and Avastin dose/schedule at investigator discretion Patients are followed for up to 3 years and clinical data updated every 3 months Objectives safety: incidence of adverse events possibly related to Avastin efficacy: time to progression, response rate and overall survival

50. 50 MO18024 study 381 centres in 41 participating countries Choice of chemotherapy at physician’s discretion Patients are followed every 3 months Primary endpoint: safety; secondary endpoint: efficacy First patient enrolled June 2004, enrolment complete February 2006 First-BEAT: study overview BEAT = Bevacizumab Expanded Access Trial PD Berry S, et al. J Clin Oncol 2006;24(Suppl.): 154s (Abstract 3534) Standard 5-FU containing therapy + Avastin (5mg/kg every 2 weeks or 7.5mg/kg every 3 weeks) Patients with previously untreated metastatic CRC (1,927)

51. 51 mCRC Treated with Bevacizumab chemotherapy combo : Survival Results from the BRiTE registry M. Kozloff, ASCO GI 2007; abs 66

52. 52 Bevacizumab related toxicity Kabbinavar JCO/03 Kabbinavar JCO/05 Hurwiz NEJM Giantonio JCO/05 Tree 2 Avastin dosage 5 & 10 MG/KG 5 MG/KG 10 MG/KG5 MG/KG Schedule ROSWELL PARK ROSWELL PARK IFLFOLFOX 4FOLFOX 6 Ipertension 11%16% (G3)11.0%(G3)5% (G3) 1% (G4) Proteinuria 23%1% (G3)3.1% (G3) 0.8% (G4) 1% (G4) Trombosis 26%18%19.4% Bleeding G.I. perforation 6%3% (G3) 2% (G4) 1.5%3% (G3) < 1% (G4) 2.8% Neuropaty ?? 16% (G3) BEAT 1927 pts BRITE 1968 pts 0.4%16.4% 0.7%1.5% 1.3% 1.4% 2.2%

53. 53 21.5 Van Custem/Hoff JCO 2000 Doulliard Lancet 2000 14.8 17.4 25.1 19.5 20.3 Saltz NEJM 2000 Douillard Lancet 2000 Saltz NEJM 2000 Goldberg JCO 2004 Hurwitz NEJM 2004 Douillard Lancet 2000 Tournigand JCO 2004 Months 1234567891011141512131617181920 222123242526 median overall survival (months) 12.6 14.1 Supportive Care 5-FU bolus 5-FU infusion Irinotecan/5-FU bolus Irinotecan/5-FU infusion Oxaliplatin + 5-FU infusion Irinotecan/5-FU inf. followed by oxaliplatin/inf. 5-FU Irinotecan/5-FU bolus/bevacizumab followed by oxaliplatin Irinotecan/5-FU bolus/bevacizumab Therapeutical progress in metastatic Colorectal cancer FolFox/XeloX- bevacizumab Kozloff ASCO GI 2007 27,1

54. 54 Avastin alone or in combination ongoing trials

55. 55 DREAM study “ the intermittent chemotherapy ” mFOLFOX7 x6 XELOX4 x6 mFOLFOX7 x6 XELOX4 x6 Avastin Previously untreated pts with mCRC (n=640) AvastinAvastin + Tarceva ® Avastin Avastin + Tarceva Primary endpoint: progression-free survival Secondary endpoints: overall survival and duration of disease control mFOLFOX7 or XELOX4: Avastin 5mg/kg every 2 wks ± Tarceva 100mg/day During chemotherapy pause: Avastin 7.5mg/kg every 3 wks ± Tarceva 150mg/day

56. 56 Ongoing phase IV optimisation trial (CONcePT) in first-line mCRC Primary endpoint: time to treatment failure Secondary endpoints include toxicity and quality of life (QoL) mFOLFOX7 + Avastin CONTINUOUS oxaliplatin ‘treat-to-failure’ ± intravenous Ca/Mg mFOLFOX7 + Avastin INTERMITTENT oxaliplatin Patients with metastatic CRC (n=532) 2x2 randomised, multicentre study

57. 57 CALGB/SWOG 80405: Study design Patients with untreated mCRC (N = 2289)* Patient/physician choice: mFOLFOX6 or FOLFIRI Cetuximab 400 mg/m 2 IV Day 1, then 250 mg/m 2 once weekly Cetuximab 400 mg/m 2 IV Day 1, then 250 mg/m 2 once weekly Bevacizumab 5 mg/kg IV Every 2 wks Bevacizumab 5 mg/kg IV Every 2 wks *144 patients enrolled as of June 2006. Primary endpoint: OS Secondary endpoints: PFS, RR

58. 58 Avastin in the neoadjuvant setting

59. 59 Gruenberger B, et al. J Clin Oncol 2006;24(Suppl):157s (Abstract 3546) 5 cycles of XELOX + Avastin every 2 weeks Avastin 5mg/kg day 1 + Xeloda 3,500mg/m 2 /day days 1–7 + oxaliplatin 85mg/m 2 day 1 Sixth cycle of XELOX alone Patients undergo liver resection w/wo colon resection Avastin + XELOX restarted 5 wks after surgery(6 cycles) AAAAA SURGERY 5 weeks = no treatment = XELOX Neoadjuvant Avastin in patients with CRC: single centre, non-randomised trial – patient eligibility

60. 60 34 patients enrolled to date 19 patients have undergone surgery following Avastin + XELOX —remaining patients have not completed therapy 14/19 patients underwent liver resection alone and four had synchronous primary tumour resection —one patient not resected due to extrahepatic disease 16 patients responded —3 complete responses, 13 partial response and 3 stable disease Neoadjuvant Avastin in patients with CRC: single centre, non-randomised trial – status Gruenberger B, et al. J Clin Oncol 2006;24(suppl):157s (Abstract 3546)

61. 61 Complication following hepatic surgery in pts receiving neoadjuvant beva for CRC liver mts Two subgroups of patients were identified, those who received neoadjuvant BV and CTX (group 1) and those who received neoadjuvant CTX only (group 2). There were 82 patients in group 1 and 45 in group Complication Bevacizumab UseInterval from Beva…. to Surgery Y (N = 82) N (N = 45) p-value<60 days (N = 41) >60 days (N = 36) p-value Any41 (50%)19 (42%)0.4023 (56%)16 (44%)0.31 Hepatobiliary4 (5%)5 (11%)0.203 (7%)1 (3%)0.39 Wound23 (28%)11 (24%)0.6613 (32%)10 (28%)0.71 These data suggest that the use of BV with neoadjuvant CTX for pts with CRC liver metastases is not associated with an increase in surgical complications. Kesmodel SB, et. Al. ASCO GI 2007 abs 234

62. 62 Avastin in the adjuvant setting

63. 63 Trials of Avastin in the adjuvant setting NSABP = The National Surgical Adjuvant Breast and Bowel Project

64. 64 AVANT: study design Randomised, open-label study Primary endpoint: disease-free survival Secondary endpoints: overall survival and safety Surgery for high risk stage II + stage III colon cancer (n=3,450) FOLFOX4 FOLFOX4 + Avastin (5mg/kg every 2 weeks) XELOX + Avastin (7.5mg/kg every 3 weeks) Avastin alone (7.5mg/kg every 3 weeks) Avastin alone (7.5mg/kg every 3 weeks) Observation Duration of treatment phases 24 weeks

65. 65 24 weeks NSABP C-08: study design Primary endpoint: disease-free survival Secondary endpoints include survival and tolerability Trial design 90% power for 25% reduction in risk of progression after 5 years 82% power for 25% reduction in risk of death after 7 years mFOLFOX6 alone mFOLFOX6 + Avastin 5mg/kg every 2 weeks Dukes’ C colon cancer (n=2,714) Avastin 5mg/kg every 2 weeks Observation

66. 66 Conclusive considerations 1.Bevacizumab improves OS, PFS and RR, when used in combination with standard chemotherapy regimens in pts with mCRC 2.Bevacizumab-containing regimens are well tolerated and does not significantly alter the regimens’ toxicity profiles 3.Bevacizumab therapy is the essential basis of first-line treatment regimens 4.Beva-monotherapy after irinotecan or oxaliplatin chemotherapy is warrant of further evaluation 5.Interesting the beva-capecitabine combination in mCRC 6.No mature date for adjuvant or neoadjuvant therapeutical modality with bevacizumab

67. 67 The End Stop Here

68. 68 BRiTE: first-line chemotherapy regimens used on study 60 50 40 30 20 10 0 Patients (%) Hedrick E, et al. J Clin Oncol 2006;24(June 20 Suppl.):155s (Abstract 3536) FOLFIRI FOLFOX Bolus 5-FU/LV IFL XELOX Infusional 5-FU/LV FLOXOther FOLFIRI = 5-FU/LV + irinotecan; XELIRI = Xeloda + irinotecan; IROX = irinotecan + oxaliplatin; FLOX = 5-FU/LV + oxaliplatin

69. 69 First-BEAT: most commonly used chemotherapy regimens 30 25 20 15 10 5 0 MonotherapyOxaliplatinIrinotecanOther/missing (15%)(48%)(33%)(3%) 5-FU bolus 8% 5-FU infusion 59% Xeloda 30% Other 3% Percentage (%) Berry S, et al. J Clin Oncol 2006;24(Suppl.):154s (Abstract 3534)

70. 70 CONcePT: intermittent oxaliplatin Stage 1 Avastin 5mg/kg CI 5-FU/LV Oxaliplatin 85mg/m 2 Stage 2 Avastin 5mg/kg CI 5-FU/LV Stage 3 Avastin 5mg/kg CI 5-FU/LV Oxaliplatin 85mg/m 2 CI = continuous infusion *Cumulative dose x8 cycles, months 5–8 x8 cycles, months 1–4 Oxaliplatin 680mg/m 2 * x8 cycles, months 9–12 Oxaliplatin 1,360mg/m 2 * ??????

71. 71 Commonly expressed oncogenes influence VEGF expression adapted from Kerbel R, et al. Nat Rev Cancer 2002;2:727–39

72. 72 Correlation of VEGF expression with metastatic disease Takahashi Y, et al. Cancer Res 1995;55:3964–8 Patients with metastases (%) 5 34 93 Intensity of VEGF staining 100 80 60 40 20 0 0–1+2+3+ 1/19 10/29 13/14 n=62

73. 73 Proteinuria  Proteinuria is a common event occurring in 23.3% of all Bevacizumab-treated patients  majority has been grade 1 proteinuria  Grade 3 proteinuria and nephrotic syndrome are rare  events are possibly linked to hypertension  Improves after Bevacizumab treatment is stopped  Recommendations —proteinuria should be monitored by dipstick urinalysis prior to starting and during Bevacizumab therapy —therapy should be stopped in patients with grade 4 proteinuria (nephrotic syndrome) Bevacizumab Summary of Product Characteristics ??????

74. 74 Arterial thromboembolic events * : results of a pooled analysis Independent risk factors for arterial thromboembolic events included history of prior arterial thromboembolic events such as stroke or heart attack age of 65 years or older Arterial events included in analysis CVA (stroke), transient ischaemic attack, subarachnoid haemorrhage myocardial infarction, angina (unstable angina), arterial thrombosis and other arterial thromboembolic events Chemotherapy alone Bevacizumab + chemotherapy Arterial thromboembolic event (%) 2.0 (15/741) 4.5 (45/1,004) Mortality (%)0.4 (3/741)0.8 (8/1,004) CVA = cerebral vascular accident * Pooled analysis of five randomised trials Roche data on file ??????

75. 75 Bleeding  Patients treated with Bevacizumab may have an increased risk of developing tumour-associated haemorrhage  Epistaxis is the most common bleeding event associated with Bevacizumab in patients with metastatic CRC  easily managed using standard first-aid techniques  Bevacizumab is contraindicated in patients with untreated CNS metastases  Recommendations  Bevacizumab should be permanently discontinued in patients who experience grade 3 or 4 bleeding during therapy Bevacizumab Summary of Product CharacteristicsCNS = central nervous system ??????

76. 76 Wound healing Bevacizumab therapy may adversely affect the wound healing process Major surgical procedures in CRC patients treated with Bevacizumab may uncommonly lead to wound healing complications 1,2 No evidence that Bevacizumab increases the risk of complications in patients who have undergone surgery >28 days prior to Bevacizumab therapy 2 Bevacizumab therapy should be discontinued 3 in patients who experience wound healing complications during therapy until the wound is fully healed for at least 28 days following major surgery or until the surgical wound is fully healed prior to elective surgery 1 Hurwitz H, et al. J Clin Oncol 2004;22(July 15 Suppl.): Abstract 3702 2 Scappaticci F, et al. J Clin Oncol 2004;22(July 15 Suppl.): Abstract 3530 3 Bevacizumab Summary of Product Characteristics ??????

77. 77 GI perforation: incidence and mortality rate 1 Kabbinavar FF, et al. J Clin Oncol 2005. In press. 2 Giantonio BJ, et al. J Clin Oncol 2004;22(July 15 Suppl.): Abstract 3017 3 Giantonio BJ, et al. Presented at: 2005 Gastrointestinal Cancers Symposium; 27–29 January 2005; Hollywood, Fl. Abstract 169a. Available at: http://www.asco.org. Accessed 15 February 2005 4 Hurwitz H et al. N Engl J Med 2004;350:2335–42 (Roche data on file) ??????

78. 78 SI PUO’ RIDURRE IL TEMPO D’INFUSIONE NELLE PRIME SOMMINISTRAZIONI? Simplification of bevacizumab (bev) administration: Do we need 90, 60, or even 30 minute infusion times? 5 mg/kg, 10 mg/kg and 15 mg/kg “ … Administration of the initial dose of bev over 30 minutes appears to be safe and well-tolerated. This has been the standard initial infusion time at MSKCC for 5 mg/kg, 10 mg/kg and 15 mg/kg doses of bev. Based on our favorable experience with 30 minute infusions of 15 mg/kg (0.5 mg/kg/minute), as of Nov '05 we have changed our institutional guidelines such that all non-protocol patients receiving bev are initiated at the infusion rate of 0.5 mg/kg/minute. … “ Saltz et al. Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings Part I. Vol 24, No. 18S

79. 79 Ongoing and planned trials of Avastin in CRC Data from ongoing and planned trials are expected to support and investigate optimisation of Avastin use with chemotherapy optimisation of chemotherapy use with Avastin incorporation of targeted agents with Avastin activity of Avastin across multiple lines of therapy —first-line use is currently recommended to maximise benefit Studies are ongoing to examine continued efficacy after progression

80. 80 Potential mechanisms of action of bevacizumab on tumor vasculature. 1.Owing to high levels of proangiogenic molecules produced locally, such as VEGF, tumors make the transition from in situ carcinoma to frank carcinoma (1). At this stage, tumors become hypervascular, but the vessels are leaky and the blood flow is spatially and temporally heterogeneous. This leads to increased interstitial fluid pressure (IFP) and focal hypoxia, creating barriers to delivery and efficacy of therapeutics. The proposed mechanism of action of the VEGF-specific antibody bevacizumab is twofold: 2.inhibition of new-vessel formation and killing of immature tumor vessels (2); 3.and transient normalization of the remaining vasculature by decrease in macromolecular permeability (and thus the IFP) and hypoxia, and improvement of blood perfusion (3). 4.Another effect of bevacizumab may be the direct killing of cancer cells in subsets of tumors in which the cells express VEGF receptors. 5.Regardless of the mechanisms involved, monotherapy with bevacizumab is not curative because it cannot kill all cancer cells, and in the longer term leads to a vasculature that is inefficient for drug delivery (4), and to tumor relapse using alternative pathways for neovascularization. ??no