Download Abstract 847

A GENOME-WIDE APPROACH
TO PREDICT OUTCOME IN
OSTEOSARCOMA
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
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
An Emerging Molecular Paradigm
Analysis of global gene expression
Classification of OSA tumors
Prediction of disease outcome.
Microarray Analysis
PATIENTS
High-grade Intramedullary
63 patients
A
No Metastasis at Diagnosis
46 patients
B
Metastasis at Diagnosis
17 patients
A1
No Metastasis 4 years post Dx.
(29 patients)
A2
Metastasis within 4 years Dx.
(17 patients)
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
 19K cDNA microarrays
Image Acquisition : Axon Scanner
Spot Analysis : GenePix Pro5
Data Storage: IobianTM Gene Traffic
 Statistical Analysis
Quality Control
Reproducibility
Aim 1: Outcome of the Patients Presenting with no Metastases
No Metastases 4 years post Dx
vs
Metastases within 4 years Dx
18981 cDNAs
T-statistic
p<0.001
(BrB Array Tools)
n=50 genes
for tumor classification/clustering
50 Most Significant Genes
No Mets
4 yrs post Dx.
Mets within
4 yrs Dx.
STATISTICAL VALIDATION
Leave-One Out (LOO) cross-validation method
Diagonal Linear Discriminant Analysis
(DLDA)
Class Prediction
Prediction Accuracy 74%
Differentially expressed genes that are higher in metastasis group
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 lower 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
Aim 2: Analysis of gene expression profiles of OSA patients
presenting with metastasis
Metastases at Dx
vs
No Metastases at Dx
18981
cDNAs
T-statistics
p<0.001
(BrB Array Tools)
n=2161 genes
for tumor classification/clustering
DLDA
Class Prediction
94% Prediction Accuracy
MOLECULAR VALIDATION by REAL TIME PCR
Mean Expression Levels in
Tumors of Patients with:
Ge ne
N o M e tastasis at
Diagnosis
M e tastasis at
Diagnosis
p-value
D4, Zinc and
Double PHD
Fingers Family 2
(DPF2)
0.27
0.15
0.0149
Secretory Carrier
Membrane Protein
5 (SCAMP5)
0.36
0.22
0.0966
Signal-Induced
ProliferationAssociated Gene
1 (SIPA1)
0.34
0.21
0.0790
Secreted Protein,
Acidic, CysteineRich (SPARC)
5.02
1.64
0.0041
Hydroxyacylglutat
hione Hydrolase
(HAGH)
0.31
0.21
0.1156
STAM2 was selected as the internal control gene after assessing 6 housekeeping
genes by a statistical model described by Szabo et.al.(2004).
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?
Work in Progress
U2OS, SaOS, HOS Cells
Knock down the DPF2 gene by SiRNA
Drug Treatment
Investigate the effect for the
Apotosis
CONCLUSIONS
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.
FUTURE STUDIES
Identify pathways related to genes in the classifier
Protein-Protein Interactions found by Pathway Studio
for 50 Significant Genes in A1vs A2 groups
FUTURE STUDIES
Online Predicted
Human Interactive Database (OPHID)
Protein-Protein Interactions found in OPHID for
Significant Genes in A vs B groups
Acknowledgement
Mount Sinai Hospital
Orthopedic Surgeons
IL Andrulis
JS Wunder
Hospital for Sick Children
D.Malkin
RS Bell
Vancouver General Hospital
C.Beauchamp
T.Yan
M. Ghert
Mona Gill
S Bull
W He
R Parkes
University of Washington
E.Conrad III
R Kandel
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