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Using exome-wide next generation sequencing to identify genetic changes in wild type GIST

Using exome-wide next generation sequencing to identify genetic changes in wild type GIST. Martin G. Belinsky Ph.D. Kinase mutations in adult and pediatric GIST. What gene(s) are mutated? Effect of mutation? Targeted therapeutically?. Imatinib mesylate. Tarn and Godwin 2005.

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Using exome-wide next generation sequencing to identify genetic changes in wild type GIST

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  1. Using exome-wide next generation sequencing to identify genetic changes in wild type GIST Martin G. Belinsky Ph.D.

  2. Kinase mutations in adult and pediatric GIST What gene(s) are mutated? Effect of mutation? Targeted therapeutically? Imatinib mesylate Tarn and Godwin 2005

  3. Mutant | Wild type Mutant | Wild type Exome-sequencing: focus on wild type GIST Genome-stable High IGF1R

  4. Approach: NexGen Exome Sequencing • Exome = the ~ 1% of the genome formed by exons, the coding portion of genes • Exome-capture with Agilent Human All Exon SureSelect library • High-throughput sequencing on Illumina Genome Analyzer IIX • Data transformation and analysis through Fox Chase Institute for Personalized Medicine, Biostatistics and Bioinformatics Facility

  5. Paternal germline Maternal germline Patient germline GIST Pilot study: germline plus tumor DNA

  6. 69,239 72,003 69,444 71,050 Filter data-> Substitution: G->A Sequence variants vs. reference genome Number of variants Reference sequence (~30 million bases)

  7. Total exome variants 71,050 Causes protein change 15,002 Not in variant databases 4,191 Not in FCCC controls 448 Data filtering: tumor example

  8. Paternal germline Maternal germline Patient germline What variants are in tumor only? GIST Biological query 1: somatic mutations

  9. Not found in patient 54 Not found in parents 24 Discard duplicates 18 Visual inspection 3 All confirmed Filtering GIST sequence variants:biological query Total exome variants 71,050 Causes protein change 15,002 Not in variant databases 4,191 Not in FCCC controls 448

  10. Tumor Patient Tumor Patient Sanger sequencing (conventional) Mutations are confirmed by conventional sequencing Tviewer

  11. 3 confirmed somatic mutations MAP-kinase pathway, cell proliferation Cell cycle regulation Mitotic spindle assembly checkpoint

  12. Query 2: inherited gene variations: cancer susceptibility?

  13. Paternal germline Maternal germline Patient germline GIST What variants are found only in patient germline? Query 3: de novo mutations

  14. Results of de novo analysis

  15. Summary and Directions • Confirmation...somatic mutations in GIST tumors, and potential disease-susceptibility variants and mutations in patient germline DNA • Validation… in larger set of wild type GIST • Modeling..effect of mutation on protein structure and function • Testing…effects on cell signaling and cell growth • Translation… to the clinic

  16. Acknowledgements Institute for Personalized Medicine • Jeff Boyd • Biao Luo • Beth Wirchansky • Caitlyn Forsythe • Lucinda Hughes Andy Godwin Lisa Vanderveer Meg von Mehren Lori Rink Alexis Cordero Biosample Repository • Joellen Weaver • Bioinformatics • Suraj Peri • Alla Lapidus • Michael Slifker • Olga Tchuvatkina • Cytogenetics • Joe Testa • Jianming Pei • Funding • GCRF • FCCC • SFA • NIH • RTOG

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