1. 1 Osteosarcoma: The addition of muramyl tripeptide to chemotherapy improves overall survival: A report from the Children’s Oncology Group Paul A. Meyers, MD, Cindy Schwartz, MD, Mark Krailo, PhD, John H. Healey, MD, Mark Bernstein, MD, Donna Betcher, Ernest Conrad, MD*, William S. Ferguson, MD, Mark C. Gebhardt, MD, Allen Goorin, MD, Michael Harris, MD, Eugenie Kleinerman, MD, Michael Link, MD, Helen Nadel, MD, Michael Nieder, MD, Judith Sato, MD*, Gene P. Siegal, MD, Michael Weiner, MD, Robert Wells, MD, Richard Womer, MD, Holcombe Grier, MD

2. 2 Spacer Muramyl Tripeptide-Phosphatidyl Ethanolamine (MTP-PE) MTP MDPPE 2005.01

3. 3 L-MTP-PEL-MTP-PE 2003.03 MTP-PE

4. 4 Cytosol Macrophage Administration of MTP NOD2 MDP MTP 1 hour5 hours IL- 1 IL- 8 IL- 6 TNFa Adapted from Strober W., Nature Rev Immunol, 6:9-20, 2006

5. 5 MTP Induces Infiltration of Inflammatory Macrophages into Lung Metastases Kleinerman et al., Cancer Immunol. Immunother., 1992 Without MTPWith MTP 2006.01

6. 6 MacEwen EG et al. J Natl Cancer Inst. 1989;81:935-938. 2119.02 MTP for Osteosarcoma in Dogs

7. 7 Phase 2 Results in Relapsed Osteosarcoma with Lung Metastases 2025.04 Kleinerman ES et al, Am.J. Clin. Onc. 18:93, 1995 Disease-Free Survival1.00.8 0.6 0.4 0.2 0 Years FailTotal Historical control2021 Cohort 21116 Cohort 112 010864212

8. 8 Phase 3 Study Design A Cisplatin Doxorubicin HDMTX 2028.03 B Ifosfamide Doxorubicin HDMTX INDUCTION Cisplatin, Ifosfamide, Doxorubicin, HDMTX 203627 Weeks Cisplatin, Doxorubicin, HDMTX MAINTENANCE A B Cisplatin, Doxorubicin, HDMTX, MTP Cisplatin, Ifosfamide, Doxorubicin, HDMTX, MTP A+ B+ DEFINITIVESURGERYDEFINITIVESURGERY DEFINITIVESURGERYDEFINITIVESURGERY

9. 9 678337341Totals 338171167MTP 340166174 No MTP Totals B: CDDP+Dox+MT X +IFS A: CDDP+Dox+MTX (no IFS) IDM ITT 678337341Totals 338171167MTP 340166174 No MTP Totals B: CDDP+Dox+MT X +IFS A: CDDP+Dox+MTX (no IFS) IDM ITT Phase 3 – 2x2 Study Enrollment 2096.02 Chemotherapy Regimens AB Cisplatin Doxorubicin HDMTX Ifosfamide Cisplatin Doxorubicin HDMTX Total Patients No MTP168163331 MTP163168331 Total patients331 662

13. Analysis of interaction Interaction between assigned chemotherapy and MTP was assessed using the proportional hazards regression. A p-value of 0.10 level or less was considered evidence of a significant interaction. Interaction between assigned chemotherapy and MTP was assessed using the proportional hazards regression. A p-value of 0.10 level or less was considered evidence of a significant interaction.

14. Analysis of Interaction Event free survival: Test of the hypothesis of no interaction (p = 0.102) Event free survival: Test of the hypothesis of no interaction (p = 0.102) MTP Hazard ratio [95% CI] MTP Hazard ratio [95% CI] Regimen A0.99 [0.69, 1.4] Regimen A0.99 [0.69, 1.4] Regimen B0.65 [0.45, 0.93] Regimen B0.65 [0.45, 0.93] All patients0.80 [0.62, 1.0] All patients0.80 [0.62, 1.0]

18. Analysis of Interaction Overall survival: Test of the hypothesis of no interaction (p = 0.60) Overall survival: Test of the hypothesis of no interaction (p = 0.60) MTP Hazard ratio [95% CI] MTP Hazard ratio [95% CI] Regimen A0.76 [0.49, 1.2] Regimen A0.76 [0.49, 1.2] Regimen B0.66 [0.43, 1.0] Regimen B0.66 [0.43, 1.0] All patients0.71 [0.52, 0.96] All patients0.71 [0.52, 0.96]

21. 21 Osteosarcoma: Overall Survival SEER and COG 2006 Data (All Patients) MTP No MTP SEER 1993-2002 SEER 1987-1992 SEER 1981-1986 SEER 1975-1980

22. 22 Validity of Survival Endpoint Threats to the validity of survival –asymmetric follow-up –asymmetric application of effective post relapse intervention Post-relapse intervention for osteosarcoma –No chemotherapy effect on post-relapse survival –No survival post relapse without surgical resection Post-relapse intervention INT-0133 –no between-arm differences 2121.01

23. 23 ConclusionsConclusions The addition of ifosfamide in this dose schedule to cisplatin, doxorubicin, and HDMTX did not improve EFS or overall survival The addition of MTP to chemotherapy resulted in a statistically significant improvement in overall survival and a trend toward better EFS. The survival for patients treated with MTP and chemotherapy was superior to SEER results for the last twenty years.

24. 24 59% 23% 18% N=576; 149 alive Median Follow-up All: 1.2 years Survivors: 4.2 years 38% 15% Recurrent Osteosarcoma: Overall Survival Kempf-Bielack B et al. J Clin Oncol. 2005;23:559-568. Years From Relapse Survival

25. 25 Recurrent Osteosarcoma: Complete Surgery? Ke Kempf-Bielack B et al. J Clin Oncol. 2005;23:559-568. Yes (CR#2) n=339 No (macroscopic rest) n=229 P<.0001 0% 38% Years After Relapse Survival

26. 26 Postrelapse Survival in Osteosarcoma of the Extremities: Prognostic Factors for Long-Term Survival 1 st RelapsesN 5-Year Survival Total162 28% Surgery complete114 39% Surgery incomplete48 0% (at 3 years)

27. 27 Multidrug chemo n=333 None or single agent n=216 P=.012 26% 21% Recurrent Osteosarcoma: Chemotherapy? Ke Kempf-Bielack B et al. J Clin Oncol. 2005;23:559-568. Years Since Relapse Survival

28. 28 Data for post relapse therapy by study arm RegimenReg A without MTP Reg A with MTP Reg B without MTP Reg B with MTP Number of patients with relapse 62587149 Number of patients with data available 51486543 Data available for 86% of relapse patients

29. 29 Site of first metastatic disease Relapse limited to the lungs Reg A without MTP Reg A with MTP Reg B without MTP Reg B with MTP YES18 (38%) 23 (50%) 33 (52%) 20 (48%) NO30 (63%) 23 (50%) 30 (48%) 22 (52%) p= 0.45

30. 30 Surgery following relpase Reg A without MTP Reg A with MTP Reg B without MTP Reg B with MTP Relapse surgery 13 (24%) 16 (31%) 21 (31%) 9 (20%) No relapse surgery 41 (76%) 36 (69%) 47 (69%) 35 (80%) p= 0.22