francis hornicek, laura macconail, levi garraway, david harmon,, zhenfeng duan
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/1.jpg)
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
![Page 2: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/2.jpg)
Disclosures
none
![Page 4: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/4.jpg)
McDermott and Settleman, JCO, 2009
![Page 5: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/5.jpg)
Krause and Van Etten, NEJM, 2005
Gene Amplification
Point mutation
Translocation
![Page 6: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/6.jpg)
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.
![Page 7: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/7.jpg)
What we did not do
• Whole gene sequencing• Copy number analysis• RNA expression• microRNA sequencing• Translocation analysis• SNP analysis• Germ-line mutation testing
![Page 8: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/8.jpg)
Tarkkanen, Canc Res 1995
![Page 9: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/9.jpg)
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
![Page 10: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/10.jpg)
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
![Page 11: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/11.jpg)
Assay: OM_v2_1121Samples: 50, 76Gene: CDH1Mutation: A617T
![Page 12: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/12.jpg)
Results
We identified mutations in genes previously known to be altered in osteosarcomas:
– p53 (R273H, Y163C, R273C, and Y163C)
– RB1 (E137).
![Page 13: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/13.jpg)
Results
We did not find canonical mutations in:
– EGFR (166 mutations tested)– PDGF– KIT– BRAF– ALK– MET– RAS
![Page 14: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/14.jpg)
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)
![Page 15: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/15.jpg)
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.
![Page 16: Francis Hornicek, Laura MacConail, Levi Garraway, David Harmon,, Zhenfeng Duan](https://reader035.vdocuments.us/reader035/viewer/2022081519/568140ca550346895dac9726/html5/thumbnails/16.jpg)
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