Deficient homologous recombination DNA repair appears to cluster sarcoma patients sensitive to trabectedin (ET-743, Yondelis ® ) Maki RG, Taron M, van Oosterom AT, Schöffski P, Tercero JC, Fernandez Sousa-Faro JM, Jimeno JM, Rosell R Memorial Sloan Kettering Cancer Center, New York, NY; Hospital Germans Trias i Pujol, Badalona, Barcelona, SPAIN UZ Gasthuisberg, Leuven, BELGIUM PharmaMar Research and Development, Madrid, SPAIN

2 E. turbinata Introduction ET-743 (trabectidin, Yondelis ® ) is a marine-derived compound in phase II-III development for solid tumors Binds to the minor groove of DNA Interacts with transcription factors and DNA binding proteins Disorganizes of the microtubule network Perturbs the cell cycle Interferes with DNA repair pathways Van Kesteren et al. Anticancer Drugs. 2002; 13:381

3 ET-743 induces long-lasting responses and tumor control in a clinically relevant proportion of patients. 26%29%OS > 2 years (percent) MEDIAN OS (months) 22%20%PFS > 6 months 15 (24%)41 (22%)TUMOR CONTROL 5 (8%)13 (7%)SD 4 (6%)14 (8%)MR 6 (10%)14 (8%)PR Dox / Ifos Resistant (n=63) Full Cohort (n=183) PARAMETER JA López et al. Proc. ASCO 2003; Abstr A. Le Cesne et al, JCO 2005; 23:576 ET-743 sarcoma program: combined phase II results

4 Supervivencia Global–ET-743 STS Median Survival 10.3 months 29% of pts alive > 2 yrs ET-743 phase II sarcoma program: Overall survival

5 Identify and validate molecular markers that correlate with sensitivity or resistance to ET-743 –Select the group of STS patients who will most benefit from ET-743 Marker + Marker - Objective

6 Human STS cell lines explanted from chemotherapy-naïve patients 12 low passage sarcoma cell lines sensitive or resistant to ET-743 underwent gene expression profile (CNIO Oncochip; > 6700 cancer related genes) STS cell lines 10nM ET-743 0h 6h12h24h72h MOLECULAR PROFILE OF ET-743 I. Identification of genes in vitro associated with ET-743 sensitivity or resistance

7 51EWING SARCOMASR OSTEOSARCOMASR LIPOSARCOMASR2103/B 450.7LIPOSARCOMASR2103/A >3000.5LIPOSARCOMASW872 >3009LEIOMYOSARCOMALS not done > Doxorubicin (nM) >100WILMS TUMORRS0306 1EWING SARCOMAA673 > ET-743 (nM) MPNST FIBROUS TUMOR LEIOMYOSARCOMA TUMOR HISTOLOGY SR0406 SR2410 SR2205 CA1010 CELL LINE Chemotherapy sensitivity profile of low passage sarcoma cell lines

8 Genes differentially expressed in ET-743 sensitive and resistant cell lines Cell cycle control –TP53 Inhibition of transcription and the DNA damage response –JUNB, ATF3, CS-1, SAT, ID2, GADD45B DNA repair –BRCA1, XPD, RAD17, p31, p53DINP1

9 Takebayashi Y. et al. Nature Med 2001; 7:961 II. DNA repair genes appear central to the function of ET-743: gene deletion experiments Intact nucleotide excision repair increases cytotoxicity Deficient double stranded break repair increases cytotoxicity

10 III. Examining genes involved in DNA repair and how they are implicated in patient responses to ET-743 Source material: tumor samples from 45 heavily pre- treated STS patients, subsequently treated with ET-743 Analyze genes important in DNA repair by mRNA expression and / or analysis of single nucleotide polymorphisms (SNPs) (a) NER pathway: ERCC1 and XPD mRNA expression and SNP analysis (b) BRCA1 mRNA expression

11 # PtsPRMR SD (*) PD patients, 8 not evaluable; Median Duration of PR or MR: 13.3 months (*) 4 / 14 SD achieved PFS > 6 mo 10 / 45 PD achieved PFS > 6 mo Best ET-743 response in patients providing tumor samples

12 Overall survivalProgression-free survival Median: 11.8 monthsMedian: 1.6 months PFS > 6 months: 26 % 73% of this group were compassionate use patients: Histology: Leiomyosarcoma31% Osteogenic sarcoma22% Synovial sarcoma16% Prior chemo: Median # RegimensMedian # Agents 2 (0-6)2 (0-10) Comparison of this cohort to the larger cohort of analyzed patients

13 Paraffin embedded tumor tissue RNA extractionDNA extraction Quantitation of RNA expression level Identification of polymorphic DNA alleles Reverse transcription Q-RT-PCR Allele discrimination RT-PCR Gln NTC Gln/G ln Lys/G ln Lys/Ly s Lys Gln XPD 751 Gln Lys XPD 751 Gln Lys XPD 751 Lys Experimental design

14 Polymorphism Patients PR Lys751Lys 14 3 Asp312Asp 15 3 Gln751Gln 5 0 Asn312Asn 7 0 There is a trend in the association between Lys751Lys and Asp312Asp genotypes in XPD gene and better clinical response to ET-743 in sarcoma patients (but p=NS by Fisher’s exact test). XPD polymorphisms associated with responses to ET-743

15 high low high low Cut-off expression level = 5.86Cut-off expression level = 1.95 Patients with high levels of expression of both ERCC1 and XPD show trend toward better tumor control rates ( PFS > 6 months ) p=NS by Fisher’s exact test PFS by ERCC1PFS by XPD High or low ERCC1 and XPD expression and their association with overall survival

16 Parameter ERCC1 mRNA Expression Levels < median > median PR + MR / Total (%) 2 / 19 (11%) 4 / 19 (21%) PFS > 6 months 3 / 19 (16%) 6 / 19 (32%) Patients with high levels of ERCC1 expression showed a trend toward better RR (21% vs. 11%) and a higher rate of patients achieving PFS > 6 months (32% vs 16%) (p = NS by Fisher exact test) ERCC1 expression and sensitivity to ET-743

17 Parameter BRCA1 expression levels < median > median PR + MR (%) PFS > 6 months (%) 4 / 17 (24%) 6 / 17 (35%) 1 / 17 (6%)* 1 / 17 (6%) ++ Patients with low BRCA1 mRNA expression levels demonstrated a trend towards higher PR+MR and tumor control rates *p = p = 0.08 BRCA1 expression and ET-743 sensitivity

18 Median survival: BRCA1 < median: 16.8 months BRCA1 > median: 6.0 months Total population: 11.8 months Phase II pivotal: 10.3 months PFS > 6 month rate: BRCA1 < median: 41 % BRCA1 > median: 6 % Total population : 26 % Phase II pivotal: 20 % p = 0.02p = 0.06 Low SurvivalPFS High Kaplan-Meier survival curves by BRCA1 expression

19 Conclusions Preclinical analysis of cell lines sensitive or resistant to ET-743 identifies a set of genes that underscores the importance of DNA repair (intact NER) in the mechanism of action of ET-743. Low expression of BRCA1 is associated with better objective response, higher rate of progression free survival at 6 months, and a statistically significant improved median survival of sarcoma patients treated with ET-743. High expression levels of XPD or ERCC1 may be associated with improved clinical outcome on ET-743 Lys751Lys and Asp312Asp genotypes in the XPD gene also appear to be associated with a higher rate of response to ET-743. Analysis of a larger group of tumors from patients sensitive and resistant to ET-743 will be necessary to confirm the relationship between DNA repair genes and ET-743 sensitivity.

20 Positano, Italy Thank you for the opportunity to present.