nalan gokgoz, taiqiang yan, michelle ghert, mona gill, shelley b bull, robert s bell, jay s wunder,...

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