1. Pediatric GIST Genetic progression mechanisms KIT/PDGFRA transforming roles Katherine Janeway, MD

2. CharacteristicAdult GISTPediatric GIST AgePeak 60 Gender distributionEqual Morphology70% spindle cell KIT IHCPositive (except PDGFRA mutant) KIT/PDGFRA genotype >85% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual Morphology70% spindle cell KIT IHCPositive (except PDGFRA mutant) KIT/PDGFRA genotype >85% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell KIT IHCPositive (except PDGFRA mutant) KIT/PDGFRA genotype >85% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) KIT/PDGFRA genotype >85% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) Positive KIT/PDGFRA genotype >85% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) Positive KIT/PDGFRA genotype >85% mutant15% mutant Genetic alterations14q, 22q, 1p loss ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) Positive KIT/PDGFRA genotype >85% mutant15% mutant Genetic alterations14q, 22q, 1p loss? ModelsCell lines, xenografts, transgenics TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) Positive KIT/PDGFRA genotype >85% mutant15% mutant Genetic alterations14q, 22q, 1p loss? ModelsCell lines, xenografts, transgenics None TherapyImatinib, sunitinib, others CharacteristicAdult GISTPediatric GIST AgePeak 60Median 12 Gender distributionEqual75% female Morphology70% spindle cell70% epithelioid KIT IHCPositive (except PDGFRA mutant) Positive KIT/PDGFRA genotype >85% mutant15% mutant Genetic alterations14q, 22q, 1p loss? ModelsCell lines, xenografts, transgenics None TherapyImatinib, sunitinib, others?

3. Objectives To determine whether the incidence of KIT / PDGFRA mutations in a large group of pediatric patients is similar to that reported previously in smaller patient groups To correlate KIT / PDGFRA genotype with the activation status of KIT, PDGFRA and downstream signaling intermediates To define the chromosomal aberrations in pediatric GIST in relation to those seen in adult GIST

4. Methods Patients 27 patients age less than 25 with confirmed GIST  Subset of 15 patients had cryopreserved specimens KIT and PDGFRA mutation analysis KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were PCR amplified and screened for mutations by high performance liquid chromatography The entire KIT coding sequence was PCR amplified from cDNA and sequenced using the ABI 3730 xl sequencer

5. SNP assay DNAs were isolated from cryopreserved tumor and analyzed using an Affymetrix 10K SNP array at the DFCI Microarray Core Facility Western blotting Whole cell lysates from cryopreserved tumors were separated by gel electrophoresis using 4 to 12% Bis- Tris gels and blotted to nitrocellulose membranes. Immunostains were detected by enhanced chemiluminesence. Methods

6. KIT and PDGFRA genotyping Mutation analysis Three of 27 patients (11%) with KIT or PDGFRA mutation  KIT exon 11 homozygous deletion VV559-560 (17 yo)  KIT exon 9 heterozygous AY502-503 tandem duplication (22 yo)  PDGFRA exon 18 D842V point mutation (14 yo) Four patients with GISTs lacking mutations on genomic DNA sequencing also lacked KIT mutations upon sequencing of the entire coding region from cDNA

10. Conclusions Most pediatric GISTs are KIT/PDGFRA - wildtype  Our 27 cases plus 31 previously published genotyped cases: 15% of pediatric GISTs harbor KIT or PDGFRA mutations PediatricAdult KIT exon 11 5% 66% KIT exon 9 9% 10% PDGFRA 3% 7%

11. Mechanisms of genetic progression are significantly different in pediatric KIT-wildtype GISTs versus pediatric and adult KIT-mutant GISTs  Biologically different tumors despite KIT expression and activation Conclusions

12. Pediatric KIT-wildtype GISTs display KIT expression and activation at levels comparable with KIT-mutant pediatric and adult GISTs  Mechanism is unclear Therapeutic inhibitors of KIT activation, particularly wildtype-KIT activation and inhibitors of signaling molecules downstream of KIT have the potential to be active in pediatric KIT-wildtype GIST Conclusions

13. Thanks! Brigham and Women’s Hospital  Jonathan Fletcher  Bernadette Liegl Oregon Health Sciences University  Mike Heinrich  Chris Corless Children’s Hospital, Boston  Antonio Perez-Atayde  Harry Kozakewich Big Papi