Molecular characterization of pediatric gastrointestinal stromal tumors

Abstract

Purpose: Pediatric gastrointestinal stromal tumors (GIST) are rare and occur preferentially in females as multifocal gastric tumors, typically lacking mutations in KIT and PDGFRA. As KIT oncoprotein is consistently overexpressed in pediatric GIST, we sought to investigate the activation of KIT downstream targets and alterations of KIT/PDGFRA gene copy number, mine novel therapeutic targets by gene expression, and test tyrosine kinase receptor activation by proteomic profiling.

Experimental design: Seventeen pediatric GISTs were investigated for KIT/PDGFRA genotype and biochemical activation of KIT downstream targets. The transcriptional profile of 13 nodules from 8 pediatric patients was compared with 8 adult wild-type (WT) GISTs, including 3 young adults. The drug sensitivity of second-generation kinase inhibitors was tested in murine Ba/F3 cells expressing human WT KIT, as well as in short-term culture of explants of WT GIST cells.

Results: A KIT/PDGFRA WT genotype was identified in all 12 female patients, whereas two of five males had either a KIT exon 11 or PDGFRA exon 18 mutation. KIT downstream targets were consistently activated. Pediatric GISTs showed a distinct transcriptional signature, with overexpression of BAALC, PLAG1, IGF1R, FGF4, and NELL1. In vitro studies showed that nilotinib, sunitinib, dasatinib, and sorafenib are more effective than imatinib against WT KIT.

Conclusions: Rare cases of pediatric GIST may occur in male patients and harbor activating KIT/PDGFRA mutations. Pediatric GISTs show distinct transcriptional signature, suggesting a different biology than WT GIST in adults. In vitro drug screening showed that second-generation kinase inhibitors may provide greater clinical benefit in pediatric GIST.

Fig. 1

Histologic appearance of pediatric GIST. Epithelioid gastric GIST in a 14-year-old female patient showing a uniform plasmacytoid appearance (A, H&E, ×200); immunoreactive with CD117 antibody (B, ×200); and who developed bilateral pulmonary chondromas, diagnostic of Carney's triad (C, patient 6, ×40). D, KIT exon 11 – mutated small-bowel GIST showing a pure spindle cell morphology in a 17-year-old male (H&E, ×100, patient 17). E and F, PDGFRA D842V – mutated gastric GIST showing an epithelioid phenotype, with focal nuclear pleomorphism and binucleated forms (H&E, ×200) and distinct areas of calcification and ossification (H&E, ×100, patient 16).

Fig. 2

Unsupervised hierarchical clustering of 13 pediatric GISTs and 5 adult WT GISTs showing distinct clustering of the two groups (Genespring GX 7.3).

Fig. 3

Western blotting of 5 pediatric tumors and 2 adult KIT exon 11-mutant GIST. A and B, tumors show activation of KIT and its downstream signaling targets. PDGFRA and PDGFRB are not activated in any of the pediatric cases tested.

Fig. 4

The phospho-RTK array highlights phosphorylated kinases in tumor lysates from a pediatric (A), young adult (B), and an adult WT GIST (C). Western blotting of the same three GIST tumors (D) showing similar phosphorylation patterns of KIT and EGFR (an EGFR-mutated lung cancer sample used as positive control). The columns on the right show the mRNA raw expression values of the phosphorylated tyrosine kinases, obtained from the DNA microarray studies on the same three tumors.

Fig. 5

Apoptosis assay (A) and biochemical assays (B) on WT KIT – transfected Ba/F3 cells showed nilotinib- and dasatinib-induced apoptosis of ~40% of cells at 100 nmol/L, whereas the remaining drugs, except imatinib, showed significant apoptosis at only 1,000 nmol/L. Biochemical assays (B) showed nilotinib- and dasatinib-induced loss of KIT activation, whereas KIT activation persisted with imatinib. In contrast, all inhibitors were effective against KIT^V559D – transfected Ba/F3 cells, with dasatinib being the most potent. This finding is also reflected by apoptosis (C) and biochemical (D) assays.

Fig. 6

Western blot assay on short-term culture of WT GIST cells shows loss of KIT activation with dasatinib treatment, which is retained in the imatinib-treated cells.