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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
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
Prediction of disease outcome.
An Emerging Molecular ParadigmAn 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
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
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 ScannerSpot Analysis : GenePix Pro5Data Storage: IobianTM Gene Traffic
19K cDNA microarrays
Statistical Analysis
Quality Control
Reproducibility
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
50 Most Significant Genes50 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 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
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
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-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?
U2OS, SaOS, HOS CellsKnock 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)
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
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