Amplification of COPS3 in High-grade Osteosarcoma: Relationship to TP 53 Mutation and Patient Outcome

1. Amplification of COPS3 in High-grade Osteosarcoma: Relationship to TP53 Mutation and Patient Outcome Yan T, Wunder JS, Gokgoz N, Eskandarian S, Bell RS, Andrulis IL Samuel Lunenfeld Research Institute Mount Sinai Hospital, Toronto, Canada

2. Introduction: COPS3 • COPS3 maps to 17p11.2, a region frequently amplified in high-grade osteosarcoma, raising the possibility that COPS3 is an oncogene • COPS3 encodes the third subunit of a highly conserved eight-subunit complex, the COP9 signalosome (CSN) • Evidence suggests that CSN modulates a wide variety of cellular processes, including cell cycle progression, cell proliferation and differentiation

3. Previous Studies: COPS3 and Osteosarcoma • Henriksen et al. reported 6/19 (32%) primary osteosarcomas with increased COPS3 gene copy number (Oncogene, 2003) • van Dartel et al. reported 4/9 (45%) primary high-grade osteosarcomas had COPS3 amplification (Cancer Genetics and Cytogenetics, 2004)

4. COPS3 and TP53 COPS3 COPS3 and P53 • CSN targets p53 for 26S-proteasome degradation, and may result in a phenotype similar to mutational inactivation of p53 • COPS3 amplification and TP53 mutation may be mutually exclusive events in osteosarcoma

5. Hypotheses • COPS3 serves as an oncogene that is important in osteosarcoma development and progression • COPS3 alterations may be related to patient outcome • COPS3 amplification and TP53 mutation may be mutually exclusive events

6. Goals • To examine the incidences of COPS3 amplification in a large sample of high-grade osteosarcoma • To correlate COPS3 amplification with patient outcome

7. Goals • We previously showed that TP53 mutation is important in osteosarcoma tumorigenesis but is not prognostic • To evaluate the relationship between COPS3 alterations, TP53 mutation and patient outcome

8. Materials and Methods • 172 frozen high-grade osteosarcoma specimens with corresponding clinical outcome data • 153 of 172 specimens had TP53 mutation data, including 123 wildtype and 30 mutation

9. Materials and Methods • Quantitative PCR and Real-time PCR (TaqMan) for COPS3 gene copy number detection • COPS3 amplification levels normalized to a control gene • Data analysis: Chi-square 2x2 test

10. 1355 1654 847 Placenta 835 571 AS (97) 2.7 1.7 3.8 1.4 1 COPS3 Amplification by Quantitative PCR Cops3(161) Correlation between PCR and Real-time PCR: 0.96

11. Results COPS3 amplification status • COPS3 gene amplification was identified in 50/172 (29%) osteosarcomas • Relative Gene copy number increase: • 2~3-fold - 20 3~5-fold - 14 5~10-fold - 9 > 10-fold - 7

12. Outcome Metastases COPS3 Amplification 31/50 (62%) No-amplification 53/122 (43%) COPS3 Amplification is Associated with Metastasis Chi-square test, p=0.03

13. COPS3 Amplification No-amplification TP53 8 22 Mutation Wildtype 32 91 Are COPS3 Amplification and TP53 Mutation Mutually Exclusive Events in Osteosarcoma? • There was no difference in the frequency of COPS3 amplification between TP53 mutated (27%) and TP53 wildtype (26%) groups (p=1.0)

14. Conclusions • COPS3 is amplified in osteosarcoma • COPS3 amplification correlates with poor patient outcome • COPS3 amplification and TP53 mutation can occur together in the same tumor • Preliminary data suggests patients whose tumors have both COPS3 amplificationand TP53 alterations may have a worse prognosis • - 6/8 patients with both alterations developed metastases

15. Discussion1: Why do COPS3 Amplification and TP53 Mutations Occur in the Same Tumor? • COPS3 and TP53 alterations may not be redundant, such that a double-hit may produce a worse phenotype • COP9-signalosome (CSN) has many cancer-related roles besides targeting p53 for degradation • CSN regulates proteasomal degradation of cell cycle regulators P21, P27 and anti-apoptotic protein NF-κB • CSN stabilizes c-Jun and contributes to activation of AP-1 which is involved in tumorigenesis

16. Discussion2: Why might concomitant occurrence of COPS3 amplification and TP53 mutation correlate with poor patient outcome? In human cells, TP53 mutation does not always lead to centrosome amplification so other mechanisms are likely involved.

17. Discussion2: Why might concomitant occurrence of COPS3 amplification and TP53 mutation correlate with poor patient outcome? • COP9-signalosome may affect centrosome amplification and chromosome instability • It is tempting to hypothesize that COPS3 amplification together with p53 mutation may induce centrosome amplification and chromosomal instability more efficiently

18. Future Directions • Survival Analysis based on COPS3 amplification • Fluorescence in Situ Hybridization (FISH) to detect centrosome amplification, and determine the relationship between centrosome amplification, COPS3 amplification and TP53 mutation

19. Acknowledgement Mount Sinai Hospital I.Andrulis/J.Wunder/R.Bell/R.Kandel N.Gokgoz/K.Eppert/S.Eskandarian S.Bull/A.Davis/M.Blackstein Hospital for Sick Children D.Malkin Vancouver General Hospital C.Beauchamp University of Washington E.Conrad III Mayo Clinic M.Rock/ L.Wold Royal Orthopaedic Hospital R.Grimer Memorial Sloan-Kettering J.Healey