COMPLETE LOSS OF SUCCINATE DEHYDROGENASE B (SDHB) IN PEDIATRIC GASTROINTESTINAL STROMAL TUMORS (GIST)

Katherine A. Janeway, MDNovember 7, 2009Connective Tissue Oncology Society

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

Syndromic GIST

Carney Triad GIST, pulmonary chondroma, paraganglioma Not inherited

Carney-Stratakis syndrome GIST and paraganglioma Autosomal dominant Incomplete penetrance

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

Succinate Dehydrogenase

Gottlieb and Tomlinson, Nature Reviews Cancer, 2005

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

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

SDHB

KIT

PKCθ

Actin

Figure 1.

KIT mutant Pediatric wildtype

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

Pediatric wildtype GIST and SDH

P5

H&E (60x) SDHA SDHB

P5

H&E (60x) SDHA SDHB

P5

H&E (60x) SDHA SDHB

P5

H&E (60x) SDHA SDHB

P5

H&E (60x) SDHA SDHB

P5

H&E (60x) SDHA SDHB

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

Pediatric wildtype GIST and SDH

Normal Complex III and IV activity in pediatric GIST

Pediatric wildtype GIST and SDH

KIT mutant Pediatric KIT wildtype

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

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

Infe

rre

dc

op

yn

um

be

r

0.5

1

1.5

2

2.5

17.2155 17.2214 17.2323 17.2472 17.2473 17.2473 17.2508 17.2555 17.2595 17.2612

Infe

rred

cop

yn

um

ber

Pediatric wildtype GISTKIT mutant GIST

1

1.5

2

2.5

3

159.509 159.524 159.532 159.553 159.602 159.603 159.6051

1.5

2

2.5

3

111.461 111.462 111.487 111.506

SNP position

SDHB

NO DELETIO

N

NO DELETIO

N

NO DELETIO

N

NO DELETIO

N

Ch 1p Ch 1q Ch 11

qRTPCR for expression of SDH subunits

0.001

0.01

0.1

1

M7 M8 M9 M10 P2 P3 P4 P5 P6 P7 P14

Case

Rel

ativ

eE

xpre

ssio

n

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

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

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