KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors

Abstract

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms arising in the gastrointestinal tract. GISTs express the KIT receptor tyrosine kinase, and many cases have activating mutations in the KIT juxtamembrane region. We now report an analysis of KIT cDNA and genomic sequences in eight GISTs that lack juxtamembrane region mutations. Six cases contained heterozygous exon 9 mutations in which six nucleotides, encoding Ala-Tyr, were duplicated. The other two cases contained homozygous exon 13 missense mutations, resulting in substitution of Glu for Lys(642), that were associated with constitutive KIT tyrosine phosphorylation. Sequence analysis of DNAs from nonneoplastic companion tissues revealed that both the exon 9 and exon 13 mutations were somatic. These are the first descriptions, in any tumor, of mutations in KIT exons encoding the C-terminal end of the extracellular domain and the first part of the split kinase domain. These findings indicate that KIT may be activated by mutations in at least three domains-extracellular, juxtamembrane, and kinase-in GISTs.

Figure 1.

Genomic evaluations of KIT by sequencing (A and B) and fluorescence in situ hybridization (C). A: Sequence analysis of total genomic DNA demonstrates a heterozygous exon 9 6-bp insertion/duplication in a GIST (top). Sequence analysis of cloned PCR products, from the same GIST as shown at top, demonstrates the wild-type (middle) and mutant (bottom) alleles. B: Sequence analysis of total genomic DNA demonstrates a homozygous exon 13 A>G missense mutation. The GIST sequence is at bottom, and the wild-type sequence, obtained from adjacent nonneoplastic tissue, is at top. C: Fluorescence in situ hybridization demonstrates nondeleted KIT, in the same GIST as in B, consistent with loss of the chromosome 4 homolog containing the wild-type KIT allele and duplication of the chromosome 4 homolog containing the mutant KIT allele. FITC signals (green) identify the KIT loci at chromosome band 4q12, and rhodamine signals (red) identify the chromosome 4 pericentromeric region.

Figure 2.

Demonstration of high-level, ligand-independent, KIT tyrosine phosphorylation in a GIST cell line, established from case 7, expressing the KIT K642E oncoprotein. Immunoprecipitations and Western blotting were with KIT and phosphotyrosine antibodies, respectively. K642E mutant KIT is tyrosine phosphorylated in GIST cells cultured in 15% fetal bovine serum (second lane), serum-free media with stem cell factor 100 ng/ml (third lane), and serum-free media without stem cell factor (fourth lane). Low-level KIT activation, in a malignant peripheral nerve sheath tumor cell line (ST88-14), is not detected on this 10-second film exposure (first lane).