CHEMOTHERAPY, BIOLOGICAL THERAPY & CANCER VACCINES Stefano Iacobelli, M.D. Medical Oncology

1. CHEMOTHERAPY, BIOLOGICAL THERAPY & CANCER VACCINES Stefano Iacobelli, M.D. Medical Oncology University G. D’Annunzio Medical School Chieti, Italy

2. COLORECTAL CARCINOMA • GLOBAL SCENARIO • 2nd leading cause of death from cancer • In Western countries! • ~1,000,000 new cases and ~ 500,000 deaths/year • At diagnosis: • 70-80% localized, resectable disease • 50% stage II • 35% stage III • ~40% relapse and die after potentially curative • surgery (~400,000/yr)

3. Early disease (adjuvant setting) • Advanced disease • Standard (Conventional) Therapy • Chronomodulated CT • Cancer vaccines

4. ADJUVANT THERAPY DIAGNOSIS Adjuvant therapy “Chemotherapy (or other type of therapy) administered after potentially curative surgery” Aim: to kill micrometastases; to increase DFS & OS SURGERY TIME Resectable (< 8 cm3) (< 1010 cells) Visibible to imag. procedures (>0.5 cm3) (> 108 cells) Not visible (< 0.5 cm3) (< 108 cells)

5. Rationale Cytotoxic chemotherapy acts mainly on proliferating cells; 2. Tumor cell growth kinetics of “gompertzian” type, i.e. the growth fraction diminishes as tumor dimensions increase; 3. The growth fraction of the micrometastatic cells is higher than that of larger tumor masses; 4. Micrometastatic tumor cells are highly sensitive to cytotoxic drugs

6. Adjuvant therapy in the ‘90s

7. 5FU + LEVAMISOLE Moertel CG et al: Levamisole and Fluorouracil for adjuvant therapy of resected colon cancer. N Engl J Med 322: 352-358, 1990 1296 pts, stage II-III Results at 3 years: Reduction of recurrences by 41% Reduction of mortality by 33% NCI Clinical update, October 1990:“… the therapeutic option of postsurgical observation (control arm) is no longer justifiable” NIH Consensus Statement, JAMA 1990, “…postoperative chemotherapy with 5FU and Levamisole for 12 months improves survival in stage III patients and is reccommended”

8. 5FU+Leucovorin vs 5FU+Levamisole • NCCTG-NCIC (O’Connel et al. JCO 16: 295-300, 1998) • 915 pts.5FU+Levamisole (6 or 12 mo.) • 5FU+Leucovorin (6 or 12 mo.) • Int 0089 (Haller DG et al. ASCO 1998) • 3759 pts5FU+Levamisole (12 mo.) • 5FU+high dose Leucovorin (6 mo.) • 5FU+low dose Leucovorin (6 mo.) • 5FU+low dose Leucovorin+Levamisole (6 mo.) • NSABP CO-4 ( Wolmark N. et al. ASCO 1996) • 2151 pts.5FU+Levamisole (12 mo.) • 5FU+high dose Leucovorin (6 mo.) • 5FU+high dose Leucovorin+Levamisole (6 mo.) • QUASAR (QUASAR Collaborative Group. Lancet 355: 1588-1596, 2000) • (Kerr DJ et al. Ann Oncol 11: 947-955, 2000) • 4927 pts. 5FU+high dose Leucovorin • 5FU+low dose Leucovorin + levamisole + placebo + levamisole + placebo

9. RESULTS • adjuvant chemotherapy in completely resected stage III colon • cancer improves long-term survival; • 5FU+Leucovorin for 6 mo. is equivalent or superior to 5FU + • Levamisole for 12 mo; • the addition of Levamisole to 5FU/Leucovorin confers no survival • benefit; • low-dose Leucovorin is equally efficacious as high-dose when • combined with 5FU; ………

10. “STANDARD” REGIMENS IN THE ’90s (Many doses and schedules – same results) MAYO CLINIC: Leucovorin 20 mg/m2 iv bolus dd 1, 5 5FU 425 mg/m2 iv bolus dd 1, 5 every 4wks NCCTG- MAYO CLINIC: Leucovorin 100 mg/m2 iv in 15’ dd 1, 5 5FU 400 mg/m2 iv in 15’ dd 1, 5 every 4wks ROSWELL PARK: Leucovorin 250 mg/m2 iv in 2 h weekly x 6 5FU 600 mg/m2 iv bolus weekly x 6 MACHOVER: Leucovorin 100 mg/m2 iv bolus dd 1, 5 5FU 370 mg/m2 iv in 15’ dd 1, 5 every 4wks DE GRAMONT - LVFU2 Leucovorin 100 mg/m2 2h infusion dd 1, 2 every 2 wks, for 24 or 36 wks 5FU 400 mg/m2 IV bolus 5FU 600 mg/m2 22h infusion

11. Should STAGE II Patients be treated?

12. Stage III vs Stage II • 318 stage II pts , 5FU+Levamisole vs surgery alone. • 7 yrs DFS: 79% vs 71% (p= 0.10) • 7 yrs OS: 72% vs 72% Moertel et al., 1990

13. IMPACT B2 Pooled analysis of 5 phase III trials (1016 stage II pts) Treatment arms: surgery vs 5FU + Leucovorin 5 yrs OVS: 80% vs 82% (p= 0.13) 8 yrs OVS: 63% vs 79% (p= 0.057) 5 yrs EFS: 73% vs 76% (p= 0.13) 8 yrs EFS: 63% vs 73% (p= 0.061) “Data does not support the routine use of 5FU+Leuc in all patients with B2 colon cancer” J Clin Oncol 17: 1356-1363, 1999

14. NSABP trials (J Clin Oncol 17: 1349-1355, 1999) Comparison: INFERIOR treatment Vs SUPERIOR treatment arm of each trial “Pts with Dukes’ B colon cancer benefit from adjuvant chemotherapy and should be presented with this option”

15. Next page…….. OXALIPLATIN

16. FOLFOX in the adjuvant treatment of colorectal cancer (MOSAIC trial, André et al. NEJM 350: 2343, 2004)

17. Kaplan-Meier Estimates of DFS in the Group Given FL and the Group Given FL plus Oxaliplatin, According to the Intention to Treat Andre, T. et al. N Engl J Med 2004;350:2343-2351

18. Kaplan-Meier Estimates of DFS in the Group Given FL and the Group Given FL plus Oxaliplatin, According to the Stage of Disease and the Intention to Treat Andre, T. et al. N Engl J Med 2004;350:2343-2351

19. Incidence of Neurosensory Symptoms during Treatment and Follow-up in the Group Given Fluorouracil, Leucovorin, and Oxaliplatin Andre, T. et al. N Engl J Med 2004;350:2343-2351

20. MOSAIC trial • CONCLUSIONS • FOLFOX4 is safe in adjuvant colon cancer • - Median relative dose-intensity of oxaliplatin 81% • - All cause mortality under treatment 0.5% in both arms • - G3 sensory neuropathy observed in 12.4% of patients, • decreasing to 1.1% at one year follow-up. • FOLFOX4 is the first combination to demonstrate superiority over • LV/5FU in adjuvant colon cancer • -3-year Disease Free Survival: 78.2% vs 72.9%, (p=0.002) • - Reduction in the risk of relapse by 23% • Stage II patients: Statistically significant benefit

21. What next • Oral fluoropyrimidine prodrugs (capecitabine, UFT) ? • Irinotecan ? • COX-2 inhibitors ? • Monoclonal antibodies (i.e. Bevacizumab, Cetuximab) ?

22. ADVANCED DISEASE

23. 5 active agents in metastaticCRC : • 5-FU, Irinotecan, Oxaliplatin • Bevacizumab, Cetuximab • 120 possible combinations and permutations, increasing to 720 with Capecitabine*

24. Oxaliplatin Regimens-1st line therapy OS (months) RR TTP/PFS (mo) FOLFOX4 16.2 50.7% 9 LVFU2 14.7 22.3% 6.2 OHP + 5 FU Chrono 19.1 53% 8.7 5 FU Chrono 19.4 16% 6.1 FUFOX 21.4 49.1% 7.8 Mayo - Regimen 16.1 22.6% 5.3 De Gramont et al. J Clin Oncol18:2938-2947, 2000; Giacchettiet al. J Clin Oncol 18: 136-147, 2000;

25. Irinotecan Regimens-1st line therapy OS (months) RR TTP/PFS (mo) Bolus 5 FU 12.6 28% 4.3 Bolus 5 FU/Irinotecan 14.8 50% 7 Inf 5 FU 14.1 31% 4.4 Inf 5 FU/Irinotecan 17.4 49% 6.7 AIO 16.9 32% 6.4 AIO/Irinotecan 20.1 54% 8.5 Saltz LB, et al. N Engl J Med. 2000;343:905-914; Douillard JY, et al. Lancet. 2000;355:1041-1047 Koehne ASCO 2003

26. Irinotecan-Oxaliplatin Sequences Arm A Arm B FOLFIRI FOLFOX FOLFOX FOLFIRI N 109 89 111 69 RR 56% 15% 54% 4% PFS 8.5 4.2 8.0 2.5 PFS overall 14.2 10.9 OS 21.5 20.6 Tournigand et al, J Clin Oncol 15:229-37, 2004

27. Novel Drugs(Targeted therapy) • Anti-EGFR • Anti-VEGF

28. Anti-EGFR Approaches MAbs Ligand Ligand K K K K TKIs Signal transduction Signal transduction Modified after Tabernero, 2003

29. EGFR TKI Ligand • Chemical class: quinazoline • Orally bioavailable • Selective inhibitors of EGFR TK • Competitive inhibitor of ATP • Inhibit ligand-induced cell growth R R û û K K TKI TKI F Erlotinib Gefitinib Cl HN O HN O N O O N N O O N N O Moyer JD, et al. Cancer Res 1997;57:4838–48

30. BEVACIZUMAB Recombinant humanized monoclonal antibody • 93% human; 7% murine • High affinity. Kd = 0.8 nM • No single agent DLT • Safely administered in combination with CT (FU-FA; FOLFOX, IFL; Carbo/Tax; Doxo) • Toxicity: hypertension, proteinuria, hemorrage and thrombosis, GI perforation VEGF VEGF VEGF VEGF VEGF

31. Bevacizumab prolongs survival in first-line colorectal cancer (CRC) Results of a randomised phase III trial (AVF2107g) of bevacizumab in combination with bolus irinotecan, 5-fluorouracil, leucovorin

32. IFL + bevacizumab IFL + placebo AVF2107g: survival Median survival (months)IFL + bevacizumab: 20.3 (95% CI: 18.46–24.18) IFL + placebo: 15.6 (95% CI: 14.29–16.99) vs Hazard ratio (HR) = 0.66 (95% CI: 0.54–0.81)p=0.00003 1.0 0.8 0.6 0.4 0.2 0 Probability of survival 15.6 20.3 0 10 20 30 40 Survival (months) CI = confidence interval Hurwitz H, et al. N Engl J Med 2004;350:2335–42

33. IFL + bevacizumab IFL + placebo AVF2107g: progression-free survival 1.0 0.8 0.6 0.4 0.2 0 Median progression-free survival (months)IFL + placebo: 6.24 (95% CI: 5.59–7.66)IFL + bevacizumab: 10.55 (95% CI: 9.03–11.04)HR = 0.54 (95% CI: 0.45–0.66) p<0.00001 Probability of being progression-free 6.2 10.6 0 10 20 30 Progression-free survival (months) Hurwitz H, et al. N Engl J Med 2004;350:2335–42

34. Cetuximab Monotherapy and Cetuximab plus Irinotecan in Irinotecan-Refractory Metastatic Colorectal Cancer David Cunningham, M.D., Yves Humblet, M.D., Ph.D., Salvatore Siena, M.D., David Khayat, M.D., Ph.D., Harry Bleiberg, M.D., Ph.D., Armando Santoro, M.D., Danny Bets, M.Sc., Matthias Mueser, M.D., Andreas Harstrick, M.D., Chris Verslype, M.D., Ph.D., Ian Chau, M.B., B.S. and Eric Van Cutsem, M.D., Ph.D. N Engl J Med 351;4:337-345. 2004 HR = 0.54, P<0.001 Median TTP: 4.1 vs 1.5 mos

35. 1.0 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 16 18 20 months ECOG trial E3200, phase III, 2nd lineSurvival Median: 10.7 vs 12.5 months HR=0.74 FOLFOX4 + bevacizumab 10mg/kg FOLFOX4 Probability p=0.0024 Giantonio B, et al. ASCO GI Symposium 2005

36. 5-FU/LV 12–14 months IFL or FOLFIRI ~15–16 months 5-FU/LV + bevacizumab 18.3 months FOLFOX4 or CAPEOX 19–20 months IFL + bevacizumab 20.3 months FOLFOX6  FOLFIRI 20.6 months IFL + BV  OX 25.1 months Median overall survival in first-line metastatic CRC Best supportive care ~4–6 months 0 6 12 18 24 Median overall survival (months)

37. MetCRC Key Messages • Use of all 3 active CT agents (Irinotecan, 5-FU, oxaliplatin) increases survival • Sequence may be important to optimize survival • Addition of new targeted agents (BEVACIZUMAB, CETUXIMAB) to Irinotecan or oxaliplatin regimens further enhances efficacy

38. Where Are We Going? • How do we better select patients? • Combination of targeted agents? • Chronotherapy?

40. Implications des rythmes biologiques en cancérologie • Neuronal rhythms 0.01-10 s • Hearth rhythm 1s • Oscillations of calcium 1 s - 30 min • Biochemical Oscillations 1-20 min • Mitotic cycle 10 min - 24 h • Circadian Rhythm 20 - 28 h • Hormonal rhythms 10 min - 3 h • Ovarian cycle 28 d Different Biological Rythms D ’après Goldbeter in : Biochemical oscillations and cellular rhythms. The molecular bases of periodic and chaotic behaviour (1997)

41. Some Rhythms in Blood

42. 145 133 121 109 97 85 0 21 23 01 03 05 07 09 11 13 15 17 19 DNA Synthesis in Human Rectal Mucosa Rhythm Characteristics from least-squares fit:24h cosine: P-value: 0.006 Amplitude: 13% (3%, 24%) Acrophase: 07:04h Circadian incorporation of tritiated thymidine into rectal mucosal cell DNA (dpm/µg DNA) of 16 healthy men (24 series total) from Buchi, et al., Gastroenterology 1999: 101: 410-415. Best-fitting 24 hour cosine Rectal Mucosal DNA (Percent of 24h Mean) 2h Mean ± SE N of values: 26 19 24 23 19 24 37 19 24 24 19 24 Time of Biopsy

43. Système circadien SNC, hormones, peptides, mediateurs Epiphyse Coordination centrale NPV NPY Mélatonine SCN Glutamate ? TGF EGF Arb. units ? Métabolisme 11 23 7 23 7 Time (h) Prolifération Cycle activité-repos Oscillateurs périphériques (genes de l’horloge) Lévi, Lancet Oncol 2001

44. Circadian rhythms are endogenous and genetically fixed * “Clock genes” hClock, hPer1, hBmal…… * * Mutations of the clock genes are associated with marked alterations of circadian rhythms and higher incidence of cancer in animals

45. Interacting Molecular Loops in the Mammalian Circadian Clock 25% of human genome, including cell cycle, apoptosis, and survival Shearman et al., Science 2004, 288, 1013-1018

46. Night shift work, light at night, and risk of breast cancer.Davis S, Mirick DK, Stevens RG. J Natl Cancer Inst,2000 Researchers search for link between circadian rhythms, breast cancer. Pillittere D, Miller M. Cancer, 2001 Rotating night shifts and risk of breast cancer in women participating in the nurses' health study.Schernhammer et al J Natl cancer Inst, 2001 • Night-shift work and risk of colorectal cancer in the nurses' • health study.Schernhammer et al. • J Natl Cancer Inst. 2003

47. THE ACTIGRAPH Piezo-electric accelerometer; Number of wrist accel./minute; Allows continuous recording over a long time period.

48. HEALTHY SUBJECT (ACTIGRAPHY PARAMETERS) Activity counts (arbitrary units) Activity counts (arbitrary units) Parameters: r24 :0,73 I<O :100 O>I :99,0 Mean:165,8 Acro.:17:13 Activity counts (arbitrary units) Activity counts AVERAGED WAVEFORM 00:00 00:00 00:00

49. ACTIGRAPHY IN CANCER PATIENTS 23:00 07:00 23:00 07:00 r24 I<O 0,77 100 -0,06 97,5 0,29 50,8 23:00 07:00