Transcript Translational Sarcoma Research

HIGH-THROUGHPUT MUTATION PROFILING OF
OSTEOSARCOMA PRIMARY TUMORS AND CELL LINES
IDENTIFIES NEW MUTATIONS IN PREVIOUSLY
UNASSOCIATED ONCOGENES AND TUMOR
SUPRESSOR GENES
Edwin Choy MD PhD
Francis Hornicek, Laura MacConail, Levi
Garraway, David Harmon,, Zhenfeng Duan
Disclosures
none
Arndt and Crist 341 (5): 342, July 29, 1999
McDermott and Settleman, JCO, 2009
Gene Amplification
Point mutation
Translocation
Krause and Van Etten, NEJM, 2005
What we did
• We designed primers and genotyping assays
using MALDI-TOF (matrix-assisted laser
desorption/ionization-time of flight mass
spectrometer; Sequonom iPLEX Genotyping)
• Systematically characterize 1057 mutations in
108 genes across 100 osteosarcoma tumor
samples and cell lines.
What we did not do
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Whole gene sequencing
Copy number analysis
RNA expression
microRNA sequencing
Translocation analysis
SNP analysis
Germ-line mutation testing
Tarkkanen, Canc Res 1995
Results
• 108 genes were analyzed using iPLEX Genotyping.
• Initially observed 19 mutations in 11 genes in at least
one osteosarcoma sample
• To validate these results, we retested all 19 mutations
using hME Genotyping
• confirmed 15 mutations in 8 genes
Genes Analyzed
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ABL1
BRAF
CDK4
EGFR (166
mutations)
ERBB2
ERB4
FGFR
HRAS
NRAS
KRAS
JAK2
KIT
PDGFRA
PIK3CA
RET
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PTPN11
IGF1R
NOTCH1
PTEN
RB1
SRC
EPHA1
NF1
CEBPA
CTNNB1
C-MYC
FLT3
GATA1
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TP53
VHL
ALK
LRP1B
EPHA3
TFDP1
PDPK1
STK11
MINK1
AKT
ABL1
ADAMTSL3
AML1/RUNX1
APC
CDKN2A
Assay: OM_v2_1121
Samples: 50, 76
Gene: CDH1
Mutation: A617T
Results
We identified mutations in genes previously
known to be altered in osteosarcomas:
– p53 (R273H, Y163C, R273C, and Y163C)
– RB1 (E137).
Results
We did not find canonical mutations in:
– EGFR (166 mutations tested)
– PDGF
– KIT
– BRAF
– ALK
– MET
– RAS
Results
We also identified 7 mutations in 5 genes
previously unimplicated in the
pathogenesis of osteosarcomas:
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PIK3CA
KRas
CUBN
CDH1/E-cadherin
CTNNB1/B-catenin
(H1047R, E545K, and H701P)
(G12S)
(I3189V)
(A617T)
(N287S)
Future Directions
– Complete gene sequencing of all five novel genes.
– Prospective genotyping for above novel mutations to
better determine frequency
– Explore efficacy of wnt, PI-3 Kinase, and
farnesyltransferase inhibitors in osteosarcoma cell
lines.
– Further characterize osteosarcoma samples for
downstream signaling elements, i.e. phosphorylated
S6, TCF responsive elements, MAPKinase activation,
etc.
Acknowledgments
Broad Institute of MIT and Harvard
- Eric Lander
- David Altshuler
- Todd Golub
MGH Sarcoma Molecular Biology Lab
– Zhenfeng Duan
– Francis Hornicek
Dana Farber Cancer Institute
– Levi Garraway
– Laura MacConail
Jennifer Hunter Yates Foundation
– David Harmon
Edwin Choy
[email protected]