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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
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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
OSTEOSARCOMAOSTEOSARCOMA
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Prediction of disease outcome.
An Emerging Molecular ParadigmAn Emerging Molecular Paradigm
Microarray Analysis
Analysis of global gene expression
Classification of OSA tumors
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High-grade Intramedullary
63 patients
No Metastasis at Diagnosis
46 patients
Metastasis at Diagnosis
17 patients
No Metastasis 4 years post Dx.
(29 patients)
Metastasis within 4 years Dx.
(17 patients)
PATIENTS
A B
A1
A2
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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
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Clinical Charactersitics of Patients Presenting with
Non-metastatic OSA
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Microarray Analysis
Image Acquisition : Axon ScannerSpot Analysis : GenePix Pro5Data Storage: IobianTM Gene Traffic
19K cDNA microarrays
Statistical Analysis
Quality Control
Reproducibility
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No Metastases 4 years post DxNo Metastases 4 years post Dxvs vs
Metastases within 4 years Dx Metastases within 4 years Dx
18981 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
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50 Most Significant Genes50 Most Significant Genes
No Mets
4 yrs post Dx.
Mets within
4 yrs Dx.
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Diagonal Linear Discriminant Analysis
(DLDA)
Class Prediction
Leave-One Out (LOO) cross-validation method
STATISTICAL VALIDATION
Prediction Accuracy 74%
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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
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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 LOC339400 (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
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Metastases at Dx Metastases at Dx vs vs
No Metastases at DxNo Metastases at Dx
18981
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
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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
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DPF2 (Requiem)
member of the d4 domain family with a Kruppel type zinc-fingerFunctions as a transcription factor for the apoptotic response
Induction of apoptosis by extracellular signalsExamples: Deprivation of survival factors in myeloid cells
Drug treatment in OS cells?
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U2OS, SaOS, HOS CellsKnock down the DPF2 gene by SiRNA
Work in Progress
Drug Treatment
Investigate the effect for the Apotosis
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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
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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
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Protein-Protein Interactions found in OPHID for Significant Genes in A vs B groups
FUTURE STUDIES Online Predicted
Human Interactive Database (OPHID)
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AcknowledgementAcknowledgementMount Sinai HospitalOrthopedic Surgeons
IL Andrulis
JS Wunder
RS Bell
T.YanM. GhertMona 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
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AcknowledgementsAcknowledgements
• 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