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Heinrich & Corless Laboratories GIST Research Updates: May 2012 GIST Research Updates: May 2012 • Corless – Will discuss new technologies for DNA sequencing – The goal is to further subclassify GISTs and better understand the genes that contribute to malignancy • Heinrich – Will discuss studies aimed at understanding how to kill persistent GIST cells that imatinib and sunitinib cannot always control ‘Next Generation’ DNA Sequencing • Traditional DNA sequencing is limited to just one part of one gene at a time • Second- and third-generation DNA sequencers are now available at OHSU • We have been using these new technologies to study GISTs Second-Generation DNA Sequencing Sequencing GIST ‘Exomes’ • Human genome consists of 3.2 billion letters (A, C, T, G) • Genes comprise only ~2% of our DNA (about 64 million letters) • We ‘pre-select’ the genes, so that only the 64 million letters of interest get sequenced – this is the ‘exome’ • Cost: $2,000 per sample GIST Exome Sequencing Specimen Summary Genotype Sequencing completed Sequencing in progress Patients Specimens Patients Specimens KIT exon 9 5 8 1 1 KIT exon 11 9 13 PDGFRA 3 3 Wildtype 1 1 BRAF 1 1 18 25 5 Pts Specimens 4 6 3 3 1 1 8 10 1 3 unknown TOTAL Available 11 Additional matched normal specimens: 19 completed; 5 in progress; 8 available Sequencing results: Mutant genes by group Apoptosis, autophagy , 6 Cell adhesion, 5 Cell cycle, 1 DNA damage response, 4 Epigenetic regulation, 8 •25 specimens from 18 patients •285 non-synonymous mutations •276 unique genes GTPase regulation (Ras family) , 6 Golgi, intracellular transport , 7 Kinase, adaptor, RTK regulation , 15 Ribosome biogenesis, RNA Polymerase, RNA splicing, 13 Transcription factor, 15 unknown, analysis in progress, 173 Tumor suppressor, TP53-related , 6 Ubiquitin , 9 Misc potentially interesting , 8 GIST Analysis Using Third-Generation DNA Sequencing • The Knight Labs have partnered with Ion Torrent/Life Tech on the development of genotyping panels using thirdgeneration DNA sequencing technology • Sequencing is performed on a semiconductor chip Sequencing Directly On A Chip Traditional Sequence 3rd Generation Sequence ACTGGTCCTGCTGGTTAG ACTGGTCCTGCTGGTTAG ACTGGTCCTGCTGGTTAG ACTGGTCCTGCTGGTTAG ACTGGTCCTGCTGGTTAG ACTGGTCCAGCTGGTTAG ACTGGTCCAGCTGGTTAG ACTGGTCCAGCTGGTTAG ACTGGTCCAGCTGGTTAG ACTGGTCCAGCTGGTTAG Knight Diagnostic Labs GIST Panel AKT1 AKT2 AKT3 ATM BRAF CDKN2A HRAS KIT KRAS MAP2K1 NF1 NRAS PDGFRA PIK3CA PTEN PTPN11 SDHA SDHAF1 SDHAF2 SDHB SDHC SDHD TP53 Common GIST mutations Important in WT GISTs Predicting GIST prognosis Rare GIST mutations of research interest