Koji Isozaki

Gain-of-function mutations of platelet-derived growth factor receptor α gene in gastrointestinal stromal tumors

BACKGROUND & AIMS Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of c-kit receptor tyrosine kinase (KIT) gene, but some GISTs do not. We investigated the cause of GISTs without KIT mutations. Because GISTs apparently expressed platelet-derived growth factor receptor (PDGFR) alpha, we examined whether GISTs without KIT mutations had a mutation of PDGFR alpha. METHODS Whole coding region of PDGFR alpha complementary DNA (cDNA) was sequenced in GISTs with or without KIT mutations. Mutant PDGFR alpha cDNA was transfected into 293T human embryonic kidney cells, and autophosphorylation of PDGFR alpha was examined. Proliferation of Ba/F3 murine lymphoid cells stably transfected with mutant PDGFR alpha cDNA was estimated by tritium thymidine incorporation. Wild-type KIT cDNA was cotransfected with mutant PDGFR alpha cDNA, and immunoprecipitation by anti-KIT antibody was performed. Inhibitory effect of Imatinib mesylate on activated PDGFR alpha was examined. RESULTS We found 2 types of constitutively activated mutations of PDGFR alpha, Val-561 to Asp or Asp-842 to Val, in 5 of 8 GISTs without KIT mutations but not in 10 GISTs with KIT mutations. Stable transfection of each mutation induced autonomous proliferation of Ba/F3 cells. Constitutively activated mutant PDGFR alpha bound and activated the cotransfected wild-type KIT. The constitutive activation of PDGFR alpha with Val-561 to Asp was inhibited effectively by Imatinib mesylate but that of PDGFR alpha with Asp-842 to Val was inhibited only weakly, even at the concentration of 10 micromol/L. CONCLUSIONS The gain-of-function mutations of PDGFR alpha appear to play an important role in development of GISTs without KIT mutations.

Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

nature genetics volume 19 august 1998 323 Gastrointestinal stromal tumour (GIST) is the most common mesenchymal tumour of the human gastrointestinal tract. Most GISTs are solitary, and gain-offunction mutations of the KIT protooncogene have been found in these tumours1. We report here a family with multiple GISTs: affected members all have a KIT mutation occurring between the transmembrane and tyrosine kinase domains, which is also the region where mutations have been found in solitary GISTs (ref. 1). The KIT mutation in this family was detected not only in tumours but also in leukocytes, indicating that GISTs constitute a familial cancer syndrome2. Development of multiple GISTs was found in a 60-year-old Japanese woman (Fig. 1a, case 5). Her nephew (case 10) also suffered from multiple benign GISTs. Analysis of the family pedigree revealed many family members suffering from symptoms attributable to development of multiple GISTs (Fig. 1a), including case 9 (a niece of case 5) who underwent surgery for benign and malignant GISTs. The benign GISTs obtained from cases 5, 9 and 10, and the malignant GIST from case 9, all expressed the KIT protein (Fig. 1b–e). DNA was extracted from paraffin-embedded specimens of the tumours3, and the mutation was investigated using single-strand conformation polymorphism analysis4 (SSCP). SSCP of tumours from cases 5 and 10 showed wild-type and mutant bands at exon 11 (Fig. 1f). Direct sequencing of the mutant bands of exon 11 showed deletion of one of two consecutive valine residues (codon 559 and 560, GTTGTT) which are located between the transmembrane and tyrosine kinase domains. Unfortunately, DNA samples suitable for SSCP and direct sequencing were not obtained from tumours of case 9. Next, we obtained DNA from peripheral leukocytes of cases 5 and 10 and their family members. The valine deletion was detected in leukocyte DNA from cases 5, 10 and 15, but not in DNA from other family members. Case 15 is 22 years old and has so far had no abdominal symptoms. We investigated the function of the mutant KIT protein by introducing an analogous mutation into mouse Kit cDNA and transfecting it into the interleukin-3 (IL-3)-dependent Ba/F3 mouse lymphoid cell line1,5–7. Evidence was found for the constitutive phosphorylation and kinase Familial gastrointestinal stromal tumours with germline mutation of the KIT gene

Gain-of-function mutation at the extracellular domain of KIT in gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the human gastrointestinal tract. Previous studies of GISTs found gain‐of‐function mutations of the c‐kit gene, which encodes a receptor tyrosine kinase (KIT). All the mutations were confined to exon 11, which encodes the juxtamembrane domain. By further examination of the whole coding region of c‐kit complementary DNA in 35 GISTs, two were found to show the identical mutation at exon 9, which encodes the extracellular domain. The aims of the present study were to examine the frequency of the extracellular domain mutation and to determine whether the mutation is a gain‐of‐function type or not. Genomic DNA was extracted from paraffin‐embedded tissues of 133 GISTs and exon 9 of the c‐kit gene was amplified by polymerase chain reaction. Screening of the mutation was carried out by single‐strand conformation polymorphism analysis and direct sequencing was done. Mutant c‐kit cDNA was transfected into 293T human embryonic kidney cells and the magnitude of autophosphorylation of the mutant KIT was examined with or without the ligand of KIT, stem cell factor (SCF). In total, seven GIST cases (approximately 5%) were found with the identical mutation at exon 9. The mutant KIT exhibited constitutive autophosphorylation without SCF stimulation. The prognosis of the patients with the extracellular domain mutation was comparable to that of the patients with the juxtamembrane domain mutation. Copyright

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors

BACKGROUND & AIMS The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. METHODS Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. RESULTS The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. CONCLUSIONS Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws Ws mutant rats

BACKGROUND & AIMS Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats. METHODS The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach. RESULTS The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats. CONCLUSIONS The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.

Absence of c-kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patients

Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c‐kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c‐kit gene mutations and that sporadic GISTs from non‐NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c‐kit gene mutations and ten sporadic GISTs from ten non‐NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c‐kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax‐embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c‐kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non‐NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c‐kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c‐kit gene mutations and none of the ten GISTs derived from non‐NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non‐NF1 patients. Copyright

Pathology of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract. It was found that most GIST expressed KIT, a receptor tyrosine kinase encoded by protooncogene c‐kit. In normal gastrointestinal wall, KIT is expressed by interstitial cells of Cajal (ICC), which are a pacemaker for autonomous gastrointestinal movement. Because both GIST and ICC are double‐positive for KIT and CD34, and because familial and multiple GIST appear to develop from diffuse hyperplasia of ICC, GIST are considered to originate from ICC or their precursor cells. It was also found that approximately 90% of the sporadic GIST have somatic gain‐of‐function mutations of the c‐kit gene, and that the patients with familial and multiple GIST have germline gain‐of‐function mutations of the c‐kit gene. These facts strongly suggest that the c‐kit gene mutations are a cause of GIST. Approximately half of the sporadic GIST without c‐kit gene mutations were demonstrated to have gain‐of‐function mutations in platelet‐derived growth factor receptor‐α (PDGFRA) gene that encodes another receptor tyrosine kinase. Because KIT is immunohistochemically negative in a minority of GIST, especially in PDGFRA gene mutation‐harboring GIST, mutational analyses of c‐kit and PDGFRA genes may be required to diagnose such GIST definitely. Imatinib mesylate was developed as a selective tyrosine kinase inhibitor. It inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for KIT‐positive metastatic or unresectable GIST as a molecular target drug. Confirmation of KIT expression by immunohistochemistry is necessary for application of the drug. The effect of imatinib mesylate is different in various types of c‐kit and PDGFRA gene mutations, and the secondary resistance against imatinib mesylate is often acquired by the second mutation of the identical genes. Mutational analyses of c‐kit and PDGFRA genes are also significant for prediction of effectiveness of drugs including newly developed agents.

Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa.

BACKGROUND & AIMS Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been recently identified as a member of the EGF family. EGF receptors to which HB-EGF can bind have been detected in some types of gastric epithelial cells. The aim of this study was to investigate whether HB-EGF is produced in gastric epithelial cells to maintain normal gastric mucosa. METHODS Gene expression and production of HB-EGF protein were investigated using Northern hybridization and immunohistochemistry, and the types of cells producing this protein were determined in human gastric mucosa. RESULTS HB-EGF messenger RNA was detected in the body and antrum. Immunohistochemical staining showed that HB-EGF was localized mainly in parietal cells of fundic glands and in gastrin cells of pyloric glands. Also, the immunoreactivity of EGF receptors was observed in parietal cells and gastrin cells and faintly in surface epithelial cells and mucous neck cells of the proliferative zone. CONCLUSIONS The results suggest that HB-EGF is synthesized mainly in parietal cells and gastrin cells and may act in an autocrine and/or paracrine manner in the regulation of proliferation and differentiation of the gastric mucosal cells through their surface EGF receptors.

Deficiency of KIT-positive cells in the colon of patients with diabetes mellitus.

Diabetes mellitus is a well‐known cause of gastrointestinal dysmotility. The pathogenesis of diabetic gastroenteropathy is mainly considered to be a neuropathy, but the cause of dysmotility remains unknown. Interstitial cells of Cajal (ICC), which express c‐kit receptor tyrosine kinase (KIT), are considered to be pacemaker cells for the gastrointestinal movement. Therefore, we investigated a possible involvement of ICC in the pathogenesis of diabetic gastroenteropathy in humans.

c-kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors.

Background and Aim:  Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the human gut. They frequently have gain‐of‐function mutations of the c‐kit gene, which encodes a receptor, tyrosine kinase. The mutations were found at exon 11 in most cases, and either at exon 9 or at exon 13 in rare cases. Recently, we found a family with multiple GIST and a gain‐of‐function mutation at exon 17. The family was the first reported GIST case with c‐kit gene mutation at exon 17 including sporadic GIST. Although we previously reported that the c‐kit gene mutation at exon 17 was not detected in 124 sporadic GIST by single‐strand conformation polymorphism (SSCP) analysis, the mutation at exon 17 observed in the familial GIST was detectable by the use of direct sequencing but not by our SSCP method. In the present study, we examined the mutations at exon 17 and exon 13 by using direct sequencing.