Nalan Gokgoz, Taiqiang Yan, Michelle Ghert, Mona Gill, Shelley B Bull, Robert S Bell, Jay S Wunder, Irene L Andrulis Mount Sinai Hospital and Samuel Lunenfeld Research Institute Toronto, Ontario, Canada A GENOME-WIDE APPROACH TO PREDICT OUTCOME IN OSTEOSARCOMA

 Treatment involves (neo)adjuvant chemotherapy and wide surgical resection  Patients without Metastases at Diagnosis:  5 year disease-free survival 50-78%  Patients with Metastases at Diagnosis:  5 year disease-free survival 10-20%.  Few accurate clinical predictors of outcome  Molecular markers ( e.g. p53, RB, cdk4,SAS): not prognostic OSTEOSARCOMA

 Prediction of disease outcome. An Emerging Molecular Paradigm Microarray Analysis Analysis of global gene expression  Classification of OSA tumors

High-grade Intramedullary 63 patients No Metastasis at Diagnosis 46 patients No Metastasis 4 years post Dx. (29 patients) Metastasis within 4 years Dx. (17 patients) Metastasis at Diagnosis 17 patients PATIENTS AB A1 A2

TUMOR SAMPLES 63 fresh frozen, primary,high-grade intramedullary osteosarcoma samples Tumor specimens from open biopsies obtained prior to chemotherapy. Tumor specimen chosen based on frozen section histological analysis. Minimum follow-up 4years or metastasis

Clinical Charactersitics of Patients Presenting with Non-metastatic OSA

Microarray Analysis Image Acquisition : Axon Scanner Spot Analysis : GenePix Pro5 Data Storage: Iobian TM Gene Traffic  19K cDNA microarrays  Statistical Analysis Quality Control Reproducibility

No Metastases 4 years post Dx vs Metastases within 4 years Dx cDNAs T-statistic p<0.001 (BrB Array Tools) n=50 genes for tumor classification/clustering Aim 1: Outcome of the Patients Presenting with no Metastases

50 Most Significant Genes No Mets 4 yrs post Dx. Mets within 4 yrs Dx.

Diagonal Linear Discriminant Analysis (DLDA) Class Prediction Leave-One Out (LOO) cross-validation method STATISTICAL VALIDATION Prediction Accuracy 74%

 RB1-inducible coiled-coil 1 (RB1CC1)  HBV preS1-transactivated protein 4 (PS1TP4)  Hypothetical protein FLJ11184 (FLJ11184)  Yippee-like 3 (Drosophila) (YPEL3)  AP1 gamma subunit binding protein 1 (AP1GBP1)  Protein phosphatase 2, regulatory subunit B', beta isoform (PPP2R5B)  Tubulin folding cofactor A (TBCA)  EP400 N-terminal like (EP400NL)  GTP-binding protein 10 (putative) (GTPBP10)  Melanoma cell adhesion molecule (MCAM)  Potassium channel tetramerisation domain containing 20 (KCTD20)  Pentatricopeptide repeat domain 3 (PTCD3)  Adenosine deaminase-like (ADAL)  Leucine rich repeat containing 3B (LRRC3B)  Flotillin 2 (FLOT2)  12 ESTs Differentially expressed genes that are higher in metastasis group

 Adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2)  Hypothetical protein MGC39715 (MGC39715)  DIP2 disco-interacting protein 2 homolog B (Drosophila) (DIP2B)  PHD finger protein 19 (PHF19)  Solute carrier family 6 (neurotransmitter transporter, creatine), member 8 (SLC6A8)  Ras-associated protein Rap1 (RBJ)  Muscleblind-like (Drosophila) (MBNL1)  Fc fragment of IgG, low affinity IIIa, receptor (CD16a) (FCGR3A)  Glial cells missing homolog 2 (Drosophila) (GCM2)  Chromosome 9 open reading frame 123C9orf123 Chromosome 2 open reading frame 29 (C2orf29)  Phospholipase D2 (PLD2)  Ribosomal protein L27a (RPL27)  Hypothetical protein LOC (LOC339400)  Chromosome 12 open reading frame 49 (C12orf49)  Platelet-activating factor acetylhydrolase 2, 40kDa (PAFAH2)  Solute carrier family 5 (sodium-dependent vitamin transporter), member 6(SLC5A6)  7 ESTs Differentially expressed genes that are lower in metastasis group

Metastases at Dx vs No Metastases at Dx cDNAs n=2161 genes for tumor classification/clustering T-statistics p<0.001 (BrB Array Tools) DLDA Class Prediction 94% Prediction Accuracy Aim 2: Analysis of gene expression profiles of OSA patients presenting with metastasis

STAM2 was selected as the internal control gene after assessing 6 housekeeping genes by a statistical model described by Szabo et.al.(2004). MOLECULAR VALIDATION by REAL TIME PCR

DPF2 (Requiem)  member of the d4 domain family with a Kruppel type zinc-finger  Functions as a transcription factor for the apoptotic response  Induction of apoptosis by extracellular signals  Examples: Deprivation of survival factors in myeloid cells Drug treatment in OS cells?

U2OS, SaOS, HOS Cells  Knock down the DPF2 gene by SiRNA Work in Progress  Drug Treatment  Investigate the effect for the Apotosis

 The use of this genome-wide approach identified a number of genes that may play a role in osteosarcoma.  Genes and pathways not previously implicated in osteosarcoma have been elucidated by this study. CONCLUSIONS

FUTURE STUDIES Protein-Protein Interactions found by Pathway Studio for 50 Significant Genes in A1vs A2 groups Identify pathways related to genes in the classifier

Protein-Protein Interactions found in OPHID for Significant Genes in A vs B groups FUTURE STUDIES Online Predicted Human Interactive Database (OPHID)

Acknowledgement Mount Sinai Hospital Orthopedic Surgeons IL Andrulis JS Wunder RS Bell T.Yan M. Ghert Mona Gill Hospital for Sick Children D.Malkin Vancouver General Hospital C.Beauchamp S Bull W He R Parkes R Kandel University of Washington E.Conrad III Royal Orthopedic Hospital R.Grimer Memorial Sloan-Kettering J.Healey Mayo Clinic M.Rock/ L.Wold

Acknowledgements Ontario Cancer Research Network (OCRN) National Cancer Institute of Canada (NCIC) Canadian Institute of Health Research (CIHR) Interdisciplinary Health Research Team (IHRT) in Musculoskeletal Neoplasia Rubinoff-Gross Chair in Orthopaedic Oncology at Mount Sinai Hospital, University of Toronto