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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.

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francis hornicek laura macconail levi garraway david harmon zhenfeng duan

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

slide5

Gene Amplification

Point mutation

Translocation

what we did
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
What we did not do
  • Whole gene sequencing
  • Copy number analysis
  • RNA expression
  • microRNA sequencing
  • Translocation analysis
  • SNP analysis
  • Germ-line mutation testing
results
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
Genes Analyzed
  • 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
  • ABL1
  • BRAF
  • CDK4
  • EGFR (166 mutations)
  • ERBB2
  • ERB4
  • FGFR
  • HRAS
  • NRAS
  • KRAS
  • JAK2
  • KIT
  • PDGFRA
  • PIK3CA
  • RET
slide11

Assay: OM_v2_1121

Samples: 50, 76

Gene: CDH1

Mutation: A617T

results12
Results

We identified mutations in genes previously known to be altered in osteosarcomas:

  • p53 (R273H, Y163C, R273C, and Y163C)
  • RB1 (E137).
results13
Results

We did not find canonical mutations in:

  • EGFR (166 mutations tested)
  • PDGF
  • KIT
  • BRAF
  • ALK
  • MET
  • RAS
results14
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
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
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
Edwin Choy

echoy@partners.org