1. HIGH-THROUGHPUT MUTATION PROFILING OF OSTEOSARCOMA PRIMARY TUMORS AND CELL LINES IDENTIFIES NEW MUTATIONS IN PREVIOUSLY UNASSOCIATED ONCOGENES AND TUMOR SUPRESSOR GENES Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan Edwin Choy MD PhD

2. Disclosures none

3. Arndt and Crist 341 (5): 342, July 29, 1999

4. McDermott and Settleman, JCO, 2009

5. Krause and Van Etten, NEJM, 2005 Gene Amplification Point mutation Translocation

6. What we did We designed primers and genotyping assays using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometer; Sequonom iPLEX Genotyping) Systematically characterize 1057 mutations in 108 genes across 100 osteosarcoma tumor samples and cell lines.

7. What we did not do Whole gene sequencing Copy number analysis RNA expression microRNA sequencing Translocation analysis SNP analysis Germ-line mutation testing

8. Tarkkanen, Canc Res 1995

9. Results 108 genes were analyzed using iPLEX Genotyping. Initially observed 19 mutations in 11 genes in at least one osteosarcoma sample To validate these results, we retested all 19 mutations using hME Genotyping confirmed 15 mutations in 8 genes

10. Genes Analyzed ABL1 BRAF CDK4 EGFR (166 mutations) ERBB2 ERB4 FGFR HRAS NRAS KRAS JAK2 KIT PDGFRA PIK3CA RET PTPN11 IGF1R NOTCH1 PTEN RB1 SRC EPHA1 NF1 CEBPA CTNNB1 C-MYC FLT3 GATA1 TP53 VHL ALK LRP1B EPHA3 TFDP1 PDPK1 STK11 MINK1 AKT ABL1 ADAMTSL3 AML1/RUNX1 APC CDKN2A

11. Assay: OM_v2_1121 Samples: 50, 76 Gene: CDH1 Mutation: A617T

12. Results We identified mutations in genes previously known to be altered in osteosarcomas: –p53 (R273H, Y163C, R273C, and Y163C) –RB1 (E137).

13. Results We did not find canonical mutations in: –EGFR (166 mutations tested) –PDGF –KIT –BRAF –ALK –MET –RAS

14. Results We also identified 7 mutations in 5 genes previously unimplicated in the pathogenesis of osteosarcomas: –PIK3CA (H1047R, E545K, and H701P) –KRas (G12S) –CUBN (I3189V) –CDH1/E-cadherin (A617T) –CTNNB1/B-catenin (N287S)

15. Future Directions –Complete gene sequencing of all five novel genes. –Prospective genotyping for above novel mutations to better determine frequency –Explore efficacy of wnt, PI-3 Kinase, and farnesyltransferase inhibitors in osteosarcoma cell lines. –Further characterize osteosarcoma samples for downstream signaling elements, i.e. phosphorylated S6, TCF responsive elements, MAPKinase activation, etc.

16. Acknowledgments Broad Institute of MIT and Harvard -Eric Lander -David Altshuler -Todd Golub MGH Sarcoma Molecular Biology Lab –Zhenfeng Duan –Francis Hornicek Dana Farber Cancer Institute –Levi Garraway –Laura MacConail Jennifer Hunter Yates Foundation –David Harmon

17. Edwin Choy echoy@partners.org