1. COMPLETE LOSS OF SUCCINATE DEHYDROGENASE B (SDHB) IN PEDIATRIC GASTROINTESTINAL STROMAL TUMORS (GIST) Katherine A. Janeway, MD November 7, 2009 Connective Tissue Oncology Society

2.  Definition: GIST diagnosed at age 18 years or less  85% “wildtype”, lacking mutations in KIT or PDGFRA  KIT is expressed and activated  Underlying oncogenic event is not known  Less sensitive to tyrosine kinase inhibition than KIT or PDGFRA mutant tumors  Better understanding of biology is needed  A proportion of pediatric patients with GIST have additional tumors (syndromic GIST) Pediatric GIST

3. Syndromic GIST  Carney Triad GIST, pulmonary chondroma, paraganglioma Not inherited  Carney-Stratakis syndrome GIST and paraganglioma Autosomal dominant Incomplete penetrance

4.  Familial paraganglioma 10% of paragangliomas Caused by germline mutations in SDHB, SDHC, SDHD  Germline SDHB mutations can be associated with pheochromocytoma and renal cell carcinoma  Classic tumor suppressor Mutations missense in conserved AAs or nonsense Tumors have loss of the normal allele  Sporadic paraganglioma 10% of patients with germline mutations in SDH Amar L JCO 23(34): 8812, 2005 Succinate Dehydrogenase and paraganglioma

5. Succinate Dehydrogenase Gottlieb and Tomlinson, Nature Reviews Cancer, 2005

6. SDH Mutations in Carney-Stratakis Syndrome  11 patients from 9 families with Carney-Stratakis Diad Age 9 to 37 at GIST diagnosis Germline mutation or deletion of SDHB, SDHC or SDHD McWhinney, et al NEJM, 2007

7. Pediatric wildtype GIST and SDH  Objective: To determine whether pediatric wildtype GISTs express SDHB  Western blot with whole cell lysates from 3 KIT mutant GISTs and 8 pediatric wildtype GISTs  Stain with antibodies for SDHB, Kit, PKCθ and actin

8. Pediatric wildtype GIST and SDH  Objectives: Determine whether loss of SDHB restricted to pediatric wildtype GIST  Evaluated additional GIST subtypes for SDHB loss  Larger number of KIT mutant GISTs evaluated for SDHB loss  Immunohistochemistry of SDHA and SDHB 6 pediatric wildtype cases 5 NF-1 associated cases 15 KIT mutant cases  Exon 11: 9  Exon 13: 2  Exon 9: 2 SDH mutant pheo VHL mutant pheo

9. Pediatric wildtype GIST and SDH

10.  Objective: To determine whether SDH / complex II functions normally in pediatric wildtype GIST Pediatric wildtype GIST and SDH

11.  Normal Complex III and IV activity in pediatric GIST Pediatric wildtype GIST and SDH KIT mutantPediatric KIT wildtype

12. SDH mutation analysis  Objective: To determine if loss of SDHB expression and SDH function is due to mutations in SDHB, SDHC or SDHD  Genomic DNA sequencing of 14 pediatric wildtype GISTs and 2 pediatric KIT mutant GISTs SDHB  6bp intronic deletion in several cases BUT cDNA sequencing normal SDHC: No mutations SDHD: No mutations

13. SNP analysis for SDH deletions  Objective: To determine if loss of SDHB expression and SDH function is due to deletions in SDHB, SDHC or SDHD  Affymetrix 250 K SNP (Sty) array 13 pediatric wildtype GISTs 4 KIT mutant GISTs Normal control samples NO DELETION Ch 1p Ch 1q Ch 11

14. qRTPCR for expression of SDH subunits  Objective: To determine if there might be epigenetic causes for loss of SDHB expression and SDH function  qRTPCR of SDHB, SDHC, SDHD  No significant difference in expression of SDHA, SDHB, and SDHD in pediatric wildtype vs. KIT mutant GISTs p = 0.06 (with P3) p = 0.004 (without P3) SDHC

15. Conclusions  Pediatric wildtype GISTs lack SDHB expression and complex II activity In contrast to KIT-mutant GISTs  Loss of SDHB expression and complex II activity is not due to mutations or deletions of SDHB, SDHC or SDHD  Loss of SDHB expression and complex II activity may be due to epigenetic events (methylation) causing decreased SDHC expression Work is ongoing

16. Acknowledgements  Brigham and Women’s Hospital Jonathan Fletcher Vania Nose  Hopital Robert Debre, Paris Pierre Rustin  University of Texas Health Science Center Patricia Dahia  Broad Institute Jordi Barretina  Dana Farber Cancer Institute Angela Lai