1. Mutated p53 correlates with extreme sensitivity to Yondelis  in low passaged cell lines explanted from chemonaive sarcoma patients 11th Annual CTOS Meeting November 19–21, 2005 V. Moneo, B.G. Serrelde, M. Taron, J. Fominaya, J.F. Martinez-Leal, C. Blanco-Aparicio, L. Romero, M. Sánchez-Beato,J.C Cigudosa, J.C Tercero, M.A. Piris, J. Jimeno, A. Carnero CNIO Dpt. Of Molecular Biology, Madrid, Spain PharmaMar R&D, Colmenar Viejo, Madrid, Spain

2. Proposed mechanisms of action of ET-743 Interference DNA repair pathways Interaction with transcription factors and DNA binding proteins Binding to the minor groove of DNA ET-743 (Yondelis) Ecteinascidia turbinata

3. Objective Remissions and Tumor Control in patients with Progressive STS Treated with Yondelis PFS-6 CR+PR

4. Yondelis Long Lasting Objective Response in a patient with Anthracycline Resistant High Grade Liposarcoma Courtesy of Dr G Demetri

5. Generation and molecular characterization of the low passage sarcoma cell lines - In vitro Cytotoxicity assays - MTT assay 96h - Clonogenic assay - Q-RT-PCR: mRNA expression - Western: protein expression - full gene p-53 sequencing

6. Sarcoma primary cell line panel ET-743 vs Doxorubicin IC 50

7. Molecular characterization sarcoma cell lines Apaf-1 p73 PTEN p85  -actin E-cad APC  -cat MSH-2 MLH-1  -actin p 21 cip1 p 27 kip1 p16 INK4a p14 ARF p15 INK4b RS0306 CA1010 SW 872 SR2103/ASR2103/B SR2205 SR2410SR2910 A673 LS0904 SR0312SR0406 RS0306 CA1010 SW 872SR2103/1ASR2103/1B SR2205 SR2410SR2910 A673 LS0904 SR0312 SR0406 RSSSSSSSSRRRRSSSSSSSSRRR mRNA expression

8. Cyc D1 cdk4 Tubulina p27 MDM2 MUT p53 RS0306 CA1010 SW 872 SR2103/A SR2103/B SR2205 SR2410 SR2910 A673 LS0904 SR0312 SR0406 RSSSSSSSSRRR S A. Carnero, AARC-04 Protein expression Molecular characterization sarcoma cell lines

9. Sarcoma primary cell line panel p53 mutational status Mut. p53

10. -20123456 30 40 50 60 70 80 90 100 110 120 P53+/+ ET-743 (log pM) P53-/- 4.92 16.63 Expt 1 3.22 11.30 Expt 2 5.05P53 null 15.89P53 wt Expt 3HCT116 IC50 (nM) Impact of p53 knock-out on cytotoxicity of Yondelis

11. Conclusions This primary sarcoma panel has established a set of cell lines with differential extreme sensitivity to Yondelis (IC50 < 1nM).Such concentration range is reacheable in plasma well below the recommended dose. Yondelis lacks complete cross-resistance with doxorubicin. The extreme sensitivity to Yondelis is correlated with mp53 genotype(p<0.01). This data might be of clinical relevance since it can provide a tool to characterize the response/resistance to Yondelis  in patients. Correlative studies in tumor samples from sarcoma patients treated with Yondelis are ongoing.