1. AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS REVEALS DISTINCT CLINICAL PHENOTYPES Sosipatros A. Boikos, Suzanne George, Katherine A. Janeway, Keith J. Killian, Su Young Kim, Michael P. LaQuaglia, Lauren Long, Paul S. Meltzer, Markku M. Miettinen, Karel Pacak, Alberto Pappo, Margarita Raygada, Joshua Schiffman, Constantine Stratakis, Jonathan Trent, Margaret Von Mehren, Christopher B. Weldon, Jennifer Wright, Lee J. Helman On behalf of the Consortium for Pediatric & wildtype GIST Research October 31, 2013 Connective Tissue Oncology Society

2. NO DISCLOSURES

3. Overview Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic.

4. Overview Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic. Correlation of the genetic and epigenetic findings with the clinical phenotype

5. Overview Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic. Correlation of the genetic and epigenetic findings with the clinical phenotype Discussion

6. Wild-Type GIST 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

7. Wild-Type GIST 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA Stomach location, epithelioid histology

8. Wild-Type GIST 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA Stomach location, epithelioid histology May be syndromic (Carney Triad, Stratakis- Carney Syndrome)

9. Wild-Type GIST 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA Stomach location, epithelioid histology May be syndromic (Carney Triad, Stratakis- Carney Syndrome) Tyrosine kinase inhibition is less effective compared to KIT or PDGFRA mutant tumors

10. Wild-Type GIST 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA Stomach location, epithelioid histology May be syndromic (Carney Triad, Stratakis- Carney Syndrome) Tyrosine kinase inhibition is less effective compared to KIT or PDGFRA mutant tumors Mutations in Succinate Dehydrogenase (SDH) genes have been found in some but not in all patients

11. 10 WT GIST clinics; 115 patients have been seen till An international clinic twice a year 115 patients have been seen in 10 clinics since 2008. Objectives of the Wildtype Clinic at NIH To bring together healthcare providers who have the most experience treating and studying GIST To obtain clinical history, response to prior treatments, histopathologic results, radiographic assessments and genetic/molecular analyses Continue long-term follow-up for these patients

12. Summary of Mutation Analysis of 78 Patients with Wild- Type GIST Fully Analyzed for SDH, BRAF, and NF1 Mutations 78 patients

13. NF1 (3) BRAF (3) 78 patients 6 patients Have NF1 or BRAF mutations

14. SDHA (22) SDHB (10) SDHC (11) SDHD (1) 44 Patients Have Mutations in SDHA, B, C, D 78 patients

15. None (28) 28 Patients Have No Detectable Mutation To Date 78 patients

16. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N L511P A454T AA 1 28 40 133 176 206280 SDHB 1 2 3 4 5 6 7 8 U U 2Fe-2S 4Fe-4S c.17_42dup R201fs W200C c189F I127S Splice site S92T R46Q R46X 1 2 3 4 5 U U SDHC 1L 2L 3L 4L 5L 3S 4S R133X p54T Splice Site R15X G75D M1V H127R 1 2 3 4 U U SDHD 1S 2S 3S 4S D118fs Mutations Distributed across all exons-90% Germline 78 patients

17. Wildtype GIST can have negative SDHB staining regardless of the status of mutations in SDH genes Janeway and Kim et al. 2011 PNAS 108:314 NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 78 patients

18. 53 tumors had negative SDHB staining 9 had positive staining NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) 78 patients

19. BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Tumors with SDHA, B, C, D mutations have always negative SDHB staining. NF1 (3) 78 patients

20. BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Tumors with SDHA, B, C, D mutations have always negative SDHB staining. NF1 (3) 78 patients

21. BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Tumors with SDHA, B, C, D mutations have always negative SDHB staining. Tumors with negative SDHB staining and no mutations have probably alterations in SDH pathway. NF1 (3) 78 patients

22. BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Tumors with SDHA, B, C, D mutations have always negative SDHB staining. Tumors with no mutations and negative SDHB staining have probably alterations in SDH pathway. Tumors with BRAF or NF1 mutations had SDHB positivity. Tumors with SDHB positivity and no mutations likely have alterations outside the SDH pathway. NF1 (3) 78 patients

23. Infinium 450 methylation data Killian et al. Cancer Discovery 2013 Succinate Dehydrogenase Mutations lead to a hypermethylator phenotype in Gastrointestinal Stromal Tumor 78 patients

24. NF1 (3) BRAF (3) SDHA (22) 1 st Circle Mutated Genes: Infinium 450 methylation data Killian et al. Cancer Discovery 2013 Negative SDHB staining by IHC correlates with Hypermethylation in 66 tumors 78 patients

25. How SDHB loss is causing hypermethylation? 78 patients Yang M, Pollard PJ Cancer Cell 2013 NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Deviator (66) Centrist (12) 3 rd Circle Methylation

26. Group A (n=12) Positive SDHB IHC Normal Methylation pattern Mutations in NF1, BRAF or other unknown genes 78 patients Group A NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Deviator (66) Centrist (12) 3 rd Circle Methylation

27. Group B (n=22) Negative SDHB staining by IHC Hypermethylation (Deviator) Not known mutations 78 patients Group B NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Deviator (66) Centrist (12) 3 rd Circle Methylation

28. Group C (n=44) Negative SDHB staining by IHC Hypermethylation (Deviator) SDHA, B, C, D mutations 78 patients Group C NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Deviator (66) Centrist (12) 3 rd Circle Methylation

29. Comparison of Group B and C 1. Group B=young age Age 60 40 20 0 B C Years 2. All patients in Group B are females while in Group C 65% are females. We have Carney Triad patients (chondroma, paraganglioma) or Carney-Stratakis Syndrome patients (paraganglioma) in both groups B and C. At the moment we have no statistically significant differences in overall Survival, Recurrence free Survival. 78 patients Group B Group C NF1 (3) BRAF (3) SDHA (22) SDHB (10) SDHC (11) SDHD (1) None (28) 1 st Circle Mutated Genes: 2 nd Circle SDHB staining: Negative (53) Positive (9) Deviator (66) Centrist (12) 3 rd Circle Methylation

30. Conclusions Group AGroup BGroup C Mutated genesNF1, BRAF or other genes Unknown genesSDH genes SDHB expression by ICHnormalno SDH FunctionNormalImpaired Methylation PatternCentristDeviator (hypermethylation) Deviator (hypermethylation) GenderBothFemalesFemale predominance AgeYoung adults, adultsPediatric, young adults Young adults, adults LocationGastric, small bowelGastric 3 distinct groups of Wildtype GIST

31. Group AGroup BGroup C Mutated genesNF1, BRAF or other genes Unknown genesSDH genes SDHB expression by ICHnormalno SDH FunctionNormalImpaired Methylation PatternCentristDeviator (hypermethylation) Deviator (hypermethylation) GenderBothFemalesFemale predominance AgeYoung adults, adultsPediatric, young adults Young adults, adults LocationGastric, small bowelGastric Conclusions Group B tumors have loss of SDHB staining without SDH mutations. We assume these 20 tumors have alterations in SDH pathway.

32. Discussion Should we redefine Wildtype GIST as tumors having SDHB deficiency by IHC? Should we use demethylating agents for SDHB deficient GIST?

33. Acknowledgements Our patients! Dr. Lee Helman lab Lauren Long Yeung Choh Arnaldez Fernanda Erin Gombos Christine Heske Amy McCalla-Martin Anna Nkrumah Wan Xiaolin

35. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD FAD Binding Domain

36. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223Fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A454L R188W T256I A454E A454T R171C T273I

37. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A445L R188W T256I A454E A454T R171C T273I FAD domain mutations

38. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A445L R188W T256I A454E A454T R171C T273I FAD domain mutations Protein-truncating mutations

39. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A445L R188W T256I A454E A454T R171C T273I FAD domain mutations Protein-truncating mutations Mutations disrupting the FAD domain

40. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W c.666delT R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A454L R188W T256I A454E A454T R171C T273I Correlation of Metastasis and site of mutation

41. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 152315402487585 664 FAD bindingCAPFADHelicalC R31X H99Y R171C R188W c.666delT R352X Splice site T256I T273I S445L A454E S505fs R512X K598N A454T SDHA SDHB SDHC SDHD K589N L511P H99A A454L R188W T256I A454E A454T R171C T273I Correlation of Metastasis and site of mutation Similar analysis on SDHB, C and D subunits is ongoing

42. AA 61 85 89 114 119 145 150 158 AA 1 28 40 133 176 206280 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 UU SDHA 1 2 3 4 U U SDHB 1 2 3 4 5 U U SDHC 1 2 3 4 5 6 7 8 U U SDHD 152315402487585 664 FAD bindingCAPFADHelicalC 1S 2S 3S 4S AA 33 44 63 91 95 104 110 139 144 160 1L 2L 3L 4L 5L 3S 4S 2Fe-2S 4Fe-4S R31X H99Y c.17_42dup R171C R188W A223fs R352X Splice site T256I T273I S445L A454E S505fs R512X K598N L511P A454T R201fs W200C c189F I127S Splice site S92T R46Q D118fs R46X R133X p54T Splice Site R15X G75D M1V H127R

43. Female Gender Age at diagnosis Group C SDHx mutations Hypermethylator phenotype Group B No mutations Hypermethylator phenotype Wildtype GIST Phenotype Female Gender Stomach Primary Multifocal Location Paragangliomas Chondromas Carney Triad

44. Female Gender Age at diagnosis Group C SDHx mutations Hypermethylator phenotype Group B No mutations Hypermethylator phenotype Almost all patients in group B are younger and female in comparison with group C Female Gender Stomach Primary Multifocal Location Paragangliomas Chondromas Carney Triad Age 60 40 20 0 B C Years

45. Female Gender Age at diagnosis Group C SDHx mutations Hypermethylator phenotype Group B No mutations Hypermethylator phenotype We have Carney Triad patients (paraganglioma) or Carney-Stratakis Syndrome (chondroma, paraganglioma) in both groups B and C Multifocal Location Paragangliomas Chondromas Carney Triad