Séverine Tabone-Eglinger

KIT mutations induce intracellular retention and activation of an immature form of the KIT protein in gastrointestinal stromal tumors.

Purpose: Gastrointestinal stromal tumors (GIST) are frequently associated with gain-of-function mutations of KIT, which can be inhibited by imatinib both in vitro and in vivo. The survival of patients with GIST, following imatinib therapy, has been correlated with the nature of mutations but not with KIT expression. Experimental Design: Subcellular localization, activation, and trafficking of the mature and the immature forms of KIT were investigated in GIST samples and in NIH3T3 cells infected with two different GIST-type exon 11–mutated human KIT cDNA. Results: Paranuclear dot expression of KIT was more frequent in GISTs with homozygous KIT mutations than in those with heterozygous (P = 0.01) or no mutations (P < 0.01). Activation of the immature 125 kDa form of KIT was detected in most GISTs with KIT mutations but not in GISTs without KIT mutations. In NIH3T3 cells, mutant KIT was mainly retained within endoplasmic reticulum and Golgi compartments in an immature constitutively phosphorylated form, whereas the wild-type KIT was expressed at the plasma membrane, in a mature nonphosphorylated form. Imatinib-induced inhibition of the phosphorylation of immature and mature mutant KIT proteins resulted in the restoration of KIT expression at the cell surface. Conclusions: These results show that GIST-type KIT mutations induce an activation-dependent alteration of normal maturation and trafficking, resulting in the intracellular retention of the activated kinase within the cell. These observations likely account for the absence of correlation between response to imatinib and KIT expression using immunohistochemistry and may deserve to be investigated in other tyrosine kinase–activated tumors.

Frequent EGFR Positivity and Overexpression in High-Grade Areas of Human MPNSTs.

Malignant peripheral nerve sheath tumours (MPNSTs) are highly malignant and resistant. Transformation might implicate up regulation of epidermal growth factor receptor (EGFR). Fifty-two MPNST samples were studied for EGFR, Ki-67, p53, and survivin expression by immunohistochemistry and for EGFR amplification by in situ hybridization. Results were correlated with clinical data. EGFR RNA was also quantified by RT-PCR in 20 other MPNSTs and 14 dermal neurofibromas. Half of the patients had a neurofibromatosis type 1 (NF1). EGFR expression, detected in 86% of MPNSTs, was more frequent in NF1 specimens and closely associated with high-grade and p53-positive areas. MPNSTs expressed more EGFR transcripts than neurofibromas. No amplification of EGFR locus was observed. NF1 status was the only prognostic factor in multivariate analysis, with median survivals of 18 and 43 months for patients with or without NF1. Finally, EGFR might become a new target for MPNSTs treatment, especially in NF1-associated MPNSTs.

GIST with homozygous KIT exon 11 mutations

Sir, We found the article of Lasota et al about gastrointestinal stromal tumors (GIST) with homozygous KIT exon 11 mutations of great interest. Indeed, most of the GISTs have heterozygous mutations, and only few studies have focused on homozygosity of KIT mutations in these tumors. The authors analyzed a series of 32 patients, and obtained data suggesting that most of these homozygous mutations result from a loss of KIT wild-type allele and duplication of the mutant allele. Could these complex mutations result from a mitotic recombination? We would like to have more precision concerning the definition of homozygous deletion of KIT. Indeed, the authors said that mutations were detected by PCR amplification and direct sequencing of PCR products of tumor DNA. However, tumor DNA is always contaminated by variable

Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

Objective Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations. Materials and Methods Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples. Results Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway. Conclusion Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways.

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild‐type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation‐dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra‐cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra‐cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface.

Histologie et pathologie moléculaire des tumeurs stromales gastrointestinales (GIST)

Resume Les tumeurs stromales gastrointestinales (GIST) sont les sarcomes les plus frequents du tube digestif. Ces tumeurs peuvent se developper a partir de tous les segments du tractus digestif, depuis l’œsophage jusqu’a l’anus, ou exceptionnellement a partir du mesentere et du peritoine. Les circonstances de decouverte les plus frequentes sont des douleurs abdominales, des saignements digestifs, des troubles du transit ou la presence d’une masse abdominale palpable. Toutefois ces tumeurs restent longtemps peu symptomatiques. De ce fait, 10 a 20 % sont de decouverte fortuite et 15 a 50 % sont decouvertes a un stade metastatique. Macroscopiquement, les GIST sont generalement des tumeurs bien limitees, a developpement extra-parietal, de consistance ferme, de couleur chair de poisson avec de frequents remaniements hemorragiques. Les GIST se caracterisent histologiquement par une proliferation importante de cellules le plus souvent fusiformes ou epithelioides. Le diagnostic doit etre confirme par l’immunohistochimie, avec une positivite de KIT dans 95 % des cas, ou par la biologie moleculaire, avec des mutations de KIT ou PDGFRA dans 85 % des cas. Toute GIST est a priori maligne, et le risque de recidive ou de metastase est actuellement evalue en fonction de la taille tumorale et l’activite mitotique. Des metastases se developpent chez 30 a 50 % des patients atteints de GIST. Celles-ci sont totalement resistantes aux chimiotherapies conventionnelles, mais l’utilisation des therapies ciblees avec des anti-oncogenes tels le Glivc ® (Imatinib Mesylate), constitue l’un des progres therapeutiques majeurs de ces dernieres annees en cancerologie.