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

Arndt and Crist 341 (5): 342,  July 29, 1999

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

Edwin Choy

echoy@partners.org