high-throughput mutation profiling of osteosarcoma primary tumors and cell lines identifies new...
TRANSCRIPT
HIGH-THROUGHPUT MUTATION PROFILING OF OSTEOSARCOMA PRIMARY TUMORS AND CELL LINES
IDENTIFIES NEW MUTATIONS IN PREVIOUSLY UNASSOCIATED ONCOGENES AND TUMOR
SUPRESSOR GENES
Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan
Edwin Choy MD PhD
Disclosures
none
McDermott and Settleman, JCO, 2009
Krause and Van Etten, NEJM, 2005
Gene Amplification
Point mutation
Translocation
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
• 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• ABL1• BRAF• CDK4• EGFR (166
mutations)• ERBB2• ERB4• FGFR• HRAS• NRAS• KRAS• JAK2• KIT• PDGFRA• PIK3CA• RET
• PTPN11• IGF1R• NOTCH1• PTEN• RB1• SRC• EPHA1• NF1• CEBPA
• CTNNB1
• C-MYC
• FLT3
• GATA1
• TP53• VHL• ALK• LRP1B• EPHA3• TFDP1• PDPK1• STK11• MINK1• AKT
• ABL1
• ADAMTSL3
• AML1/RUNX1
• APC
• CDKN2A
Assay: OM_v2_1121Samples: 50, 76Gene: CDH1Mutation: 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:
– PIK3CA (H1047R, E545K, and H701P)– KRas (G12S)– CUBN (I3189V) – CDH1/E-cadherin (A617T) – CTNNB1/B-catenin (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.
AcknowledgmentsBroad 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
