Overexpression of insulin-like growth factor 1 receptor and frequent mutational inactivation of SDHA in wild-type SDHB-negative gastrointestinal stromal tumors

Abstract

Approximately 15% of gastrointestinal stromal tumors (GISTs) in adults and 85% in children lack mutations in KIT and PDGFRA and are known as wild-type GISTs. Wild-type GISTs from adults and children express high levels of insulin-like growth factor 1 receptor (IGF1R) and exhibit stable genomes compared to mutant GISTs. Pediatric wild-type GISTs, GISTs from the multitumor Carney-Stratakis syndrome, and the Carney triad share other clinicopathological properties (e.g., early-onset, multifocal GISTs with epitheliod cell morphology), suggesting a common etiology. Carney-Stratakis is an inherited association of GIST and paragangliomas caused by germline mutations in succinate dehydrogenase (SDH) genes. The connection between defective cellular respiration and GIST pathology has been strengthened by the utilization of SDHB immunohistochemistry to identify SDH deficiency in pediatric GISTs, syndromic GISTs, and some adult wild-type GISTs. SDHB and IGF1R expression was examined in 12 wild-type and 12 mutant GIST cases. Wild-type GISTs were screened for coding-region alterations in SDH genes and for chromosomal aberrations using genome-wide single-nucleotide polymorphism and MIP arrays. SDHB-deficiency, identified in 11/12 wild-type GIST cases, was tightly associated with overexpression of IGF1R protein and transcript. Biallelic inactivation of the SDHA gene was a surprisingly frequent event, identified in 5 of 11 SDHB-negative cases, generally due to germline point mutations accompanied by somatic SDHA allelic losses. As a novel finding, inactivation of the SDHC gene from a combination of a heterozygous coding-region mutation and hypermethylation of the wild-type allele was found in one SDHB-negative case.

Figure 1

KIT, IGF1R and SDHB expression in GIST specimens. IHC expression in an adult wild type (top panel), pediatric wild type (middle panel) and mutant (bottom panel) GIST. Primary antibodies used include KIT (Dako), IGF-1R (Cell Signaling) and SDHB (Abcam). Positive KIT staining is evident throughout tumor tissue in all cases. Strong staining for IGF1R is seen in the wild type GISTs, while SDHB staining is evident in the mutant GIST and in the adjacent normal tissue and epithelial cells in the wild type cases, but absent in wild type GISTs.

Figure 2

RNA expression of IGF1R, CDH2, and ELAVL3 in SDHB-negative and SDHB-positive GISTs as determined by qRT-PCR. Expression levels were normalized to beta-actin. Statistical significance as calculated by the Mann-Whitney test was as follows: IGF1R: P < 0.0001; CDH2: P ~ 0.002; ELAVL3: P ~ 0.003.

Figure 3

Partial sequence chromatograms from amplified genomic DNA from patient blood, and from genomic DNA and cDNA from GISTs. Nucleotide numbering is from the coding regions for SDHA (NM_004168.2) and SDHC (NM_003001.3).

Figure 4

Partial chromatogram of cloned PCR products from the 5’ end of the SDHC gene. Tumor genomic DNA from case 6 was bisulfite-treated prior to amplification and cloning. Arrows show the location of 10 (of 12) CpG dinucleotides (seen in the boxed reference sequence) in the predicted CpG island. The upper chromatogram shows a cloned product with methylation of 11/12 CpGs (the third CpG is un-methylated), while the lower panel shows a fully un-methylated cloned product. The ATG site is indicated as +1.