COMPLETE LOSS OF SUCCINATE DEHYDROGENASE B SDHB IN

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

Pediatric GIST o o o o 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)

Syndromic GIST o Carney Triad n n o GIST, pulmonary chondroma, paraganglioma Not inherited Carney-Stratakis syndrome n n n GIST and paraganglioma Autosomal dominant Incomplete penetrance

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

Succinate Dehydrogenase Gottlieb and Tomlinson, Nature Reviews Cancer, 2005

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

Pediatric wildtype GIST and SDH o o o 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

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

Pediatric wildtype GIST and SDH

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

Pediatric wildtype GIST and SDH KIT mutant o Pediatric KIT wildtype Normal Complex III and IV activity in pediatric GIST

SDH mutation analysis o o 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 n SDHB o n n 6 bp intronic deletion in several cases BUT c. DNA sequencing normal SDHC: No mutations SDHD: No mutations

SNP analysis for SDH deletions n Ch 1 p Ch 11 Ch 1 q D EL ET IO N n 13 pediatric wildtype GISTs 4 KIT mutant GISTs Normal control samples N O n D EL ET IO N o 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 N O o

q. RTPCR for expression of SDH subunits o o o Objective: To determine if there might be epigenetic causes for loss of SDHB expression and SDH function q. RTPCR of SDHB, SDHC, SDHD No significant difference in expression of SDHA, SDHB, and SDHD in pediatric wildtype vs. KIT mutant GISTs SDHC p = 0. 06 (with P 3) p = 0. 004 (without P 3)

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

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