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 Tarn and Godwin 2005 What gene(s) are mutated? Effect of mutation? Targeted therapeutically? Imatinib mesylate

3. Exome-sequencing: focus on wild type GIST Mutant | Wild type 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. Pilot study: germline plus tumor DNA Maternal germline Paternal germline Patient germline GIST

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

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

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

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

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

11. 3 confirmed somatic mutations 1.MAP-kinase pathway, cell proliferation 2.Cell cycle regulation 3.Mitotic spindle assembly checkpoint

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

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

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