COMPLETE LOSS OF SUCCINATE DEHYDROGENASE B (SDHB) IN PEDIATRIC GASTROINTESTINAL STROMAL TUMORS (GIST) Katherine A. Janeway, MD November 7, 2009 Connective Tissue Oncology Society
Jan 13, 2016
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