Anti-tumor effect of antibody-drug conjugate targeting cell adhesion molecule 1 on GIST cells representing small intestinal GIST.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the alimentary tract. The prognosis depends on the primary site, and small intestinal GISTs have a worse prognosis than gastric GISTs. Molecularly targeted drugs to inhibit tyrosine kinase activity of KIT were used for unresectable or recurrent GISTs. However, secondary resistance to the drugs is often acquired, and treatments based on other mechanisms are needed. Previously, we reported that cell adhesion molecule 1 (CADM1) was highly expressed in most of small intestinal GISTs but not in most of gastric GISTs. In the present study, we examined whether the antibody-drug conjugate (ADC) with anti-CADM1 antibody and monomethyl auristatin E (anti-CAD-ADC) shows anti-tumor effect on CADM1-expressing human GIST cells. The ADC adhibited in this study was previously used for CADM1-expressing human mesothelioma cells and showed anti-tumor effect for them in vitro. GIST-T1 cell line of gastric origin which scarcely expresses CADM1 and GIST-T1 cells transfected with CADM1 cDNA (GIST-T1-CAD cells) which highly expresses CADM1 and represents small intestinal GIST were used. In vitro, anti-CAD-ADC showed remarkable cytotoxic activity on GIST-T1-CAD cells, but control ADC did not. Both anti-CAD-ADC and control ADC did not show anti-tumor effect on original GIST-T1 cells. When GIST-T1-CAD cells were subcutaneously injected to the nude mice, intravenous administration of anti-CAD-ADC showed inhibitory effect for tumor enlargement. Tumor of GIST-T1 cells grew even after anti-CAD-ADC injection. When GIST-T1-CAD cells were injected into peritoneal cavity of the SCID mice, intraperitoneal administration of anti-CAD-ADC showed reduction of the peritoneal tumor. On the other hand, peritoneal tumor grew after control ADC administration. Tissue and organ damage due to administration of anti-CAD-ADC was not apparent by macroscopic and histological examinations in mice. These results indicate that anti-CAD-ADC could have apparent anti-tumor effect on CADM1-expressing human GIST cells both in in vitro and in vivo mouse models.

CADM1 promotes adhesion to vascular endothelial cells and transendothelial migration in cultured GIST cells.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. Small intestinal GISTs appear to be associated with poorer prognosis and higher metastasis rate than gastric GISTs of the same size and mitotic index. Recently, we reported that cell adhesion molecule 1 (CADM1) is expressed specifically in most small intestinal GISTs, but not in most gastric GISTs, suggesting that this difference in CADM1 expression between gastric GISTs and small intestinal GISTs might influence the difference in clinical behavior between them. The aim of the present study was to examine whether high CADM1 expression affected proliferation, migration, invasion, adhesion to endothelial cells and transendothelial migration of cultured GIST cells by comparing original GIST-T1 cells with very low CADM1 expression with GIST-T1 cells with high CADM1 expression induced by CADM1 cDNA transfection (GIST-T1-CAD cells). GIST-T1-CAD cells had reduced ability to proliferate, migrate and invade compared with the original GIST-T1 cells, but showed significantly higher ability to adhere to human umbilical vein endothelial cells and migrate through endothelial cell monolayers. Thus, CADM1 may contribute to higher metastasis rates in small intestinal GISTs facilitating tumor cell adhesion to vascular endothelial cell and transendothelial migration of tumor cells. CADM1 might serve as a potential target for inhibition of metastasis in small intestinal GISTs.

Differential Expression of CADM1 in Gastrointestinal Stromal Tumors of Different Sites and with Different Gene Abnormalities.

Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor of the human gastrointestinal tract, differentiating toward the interstitial cell of Cajal (ICC), arises predominantly in the stomach and small intestine. Small intestinal GISTs appear to have worse prognosis than gastric GISTs. In a pilot study of a cDNA expression chip using several GISTs, we found that Cell Adhesion Molecule 1 (CADM1), which could contribute to tumor growth and infiltration, is expressed more strongly in small intestinal GISTs than gastric GISTs. In the present study, we examined CADM1 expression in GISTs of different sites and with different gene abnormalities using a large number of gastric and small intestinal GISTs. First, immunoblotting confirmed significantly higher CADM1 expression in small intestinal GISTs with exon 11 c-kit mutation than gastric GISTs with exon 11 c-kit mutation. Real-time PCR also revealed that small intestinal GISTs with exon 11 c-kit mutation showed significantly higher CADM1 mRNA than gastric GISTs with exon 11 c-kit mutation. Although most small intestinal GISTs showed high CADM1 mRNA expression regardless of gene abnormality types, different CADM1 expression was detected between gastric GISTs with c-kit mutation and those with PDGFRA mutation. Immunohistochemistry showed that many small intestinal GISTs were CADM1-positive but most gastric GISTs CADM1-negative or -indefinite. In the normal gastric and small intestinal walls, immunoreactivity of CADM1 was detected only in nerves, but neither in gastric ICCs nor small intestinal ICCs, indicating that the high CADM1expression in small intestinal GISTs might be acquired during tumorigenesis. Different CADM1 expression between gastric and small intestinal GISTs might be related to different prognoses between them. Further functional experiments are needed to elucidate the role of CADM1 on GIST biology, and there is a possibility that targeting therapy against CADM1 has a preventive effect for tumor spreading in small intestinal GISTs.

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation.

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology.

Reliability of Isometric Knee Extension Muscle Strength Measurements of Healthy Elderly Subjects Made with a Hand-held Dynamometer and a Belt.

[Purpose] The purpose of this study was to examine the reliability of three isometric knee extension strength measurements (IKE) made with a hand-held dynamometer (HHD) and a belt of healthy elderly living in the community as subjects. [Subjects] The subject cohort consisted of 186 healthy elderly people, aged 65 to 79 years, living in local communities. [Methods] IKE of the leg subjects used to kick a ball was measured. IKE of each subject was measured three times using an HHD-belt at intervals of 30 seconds. The reliability of the larger of the first two measurements (LV2) as well as the third measurement (3V) was investigated. [Results] The intraclass correlation coefficients [ICC (1, 1)] for LV2 and 3V were 0.955. Bland-Altman analysis showed a fixed bias, and the limits of agreement ranged from -5.6 to 4.6. [Conclusion] The ICC results show that the test-retest reproducibility of IKE measurements of healthy elderly subjects using an HHD-belt is high. However, Bland-Altman analysis showed a fixed bias, suggesting the need for three measurements.

Gastrointestinal stromal tumors with exon 8 c-kit gene mutation might occur at extragastric sites and have metastasis-prone nature.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the human gut. Most sporadic GISTs have somatic gain-of-function mutations of the c-kit gene. The mutations are frequently found at exon 11, sometimes at exon 9 and rarely at exon 13 or 17. Recently, exon 8 c-kit gene mutations were reported in very minor proportion of sporadic GISTs. We also found 3 GISTs with exon 8 c-kit gene mutations in approximately 1,000 sporadic GISTs examined. In the present report, we showed the clinicopathological data of those GISTs. One case had a deletion of codon 419 of aspartate, and 2 cases had a substitution of 3 amino acids of codon 417 to codon 419 to tyrosine. The former was the same mutation recently reported in 2 GIST cases, but the latter has not been reported in any GISTs. All three cases occurred at extragastric sites and two of three showed distant metastasis. Since the remaining case was regarded as high risk for recurrence, imatinib adjuvant treatment has been done without evidence of metastasis. Our results confirmed the idea that exon 8 mutations are minor but actually existing abnormalities in sporadic GISTs, and suggested that such GISTs have a feature of extragastric development and a metastasis-prone nature. Since the exon 8 mutations appeared to be really sensitive to imatinib as shown in the present case study, accurate genotyping including exon 8 of the c-kit gene is necessary in GISTs to predict response to imatinib in both the unresectable/metastatic and adjuvant settings.

Extracellular domain c-kit mutation with duplication of Ser501Ala502 found in gastrointestinal stromal tumors is more imatinib- and nilotinib-sensitive than that with duplication of Ala502Tyr503.

The great majority of gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the c-kit gene, which encodes KIT receptor tyrosine kinase. Most of the mutations are located at exon 11, but some are at exon 9 or at other exons. Mutation types at exon 11 vary, while most mutations at exon 9 are a particular duplication of Ala502Tyr503 (KIT-Dup-Ala502Tyr503). Recently a duplication of Ser501Ala502 (KIT-Dup-Ser501Ala502) at exon 9 has been reported in two cases of pediatric mastocytosis and one case of adult mast cell leukemia. Although KIT-Dup-Ser501Ala502 had not been reported in GISTs, we found two GIST cases possessing the mutation in 45 GIST cases with exon 9 c-kit gene mutations, among a total of approximately 500 GIST cases examined. In this report, we briefly summarize clinicopathological findings of the two cases, and characterize the biology of the mutation. When autophosphorylation of KIT-Dup-Ser501Ala502 was examined by transient transfection of c-kit cDNA with Dup-Ser501Ala502 into CHO-K1 cells, KIT-Dup-Ser501Ala502 was ligand-independently activating. The inhibitory effect of selective tyrosine kinase inhibitors, imatinib and nilotinib, on KIT-Dup-Ser501Ala502 was examined and compared with that of KIT-Dup-Ala502Tyr503. Imatinib efficiently inhibited constitutive activation of KIT-Dup-Ser501Ala502 at a concentration of 0.1 µM, whereas it inhibited that of KIT-Dup-Ala502Tyr503 at a concentration of 10 µM. Constitutive activation of KIT-Dup-Ser502Ala503 was not inhibited by nilotinib even at a concentration of 10 µM but that of KIT-Dup-Ala501Tyr502 was almost completely inhibited at a concentration of 1 µM. The results suggest that imatinib and nilotinib could be more effective on GISTs with KIT-Dup-Ser501Ala502 than those with KIT-Dup-Ala502Tyr503. In fact, a patient with KIT-Dup-Ser501Ala502 showed long-term stable disease with administration of the usual dose of 400 mg imatinib. Although mutation sites of KIT-Dup-Ser501Ala502 and KIT-Dup-Ala502Tyr503 are closely located, imatinib- and nilotinib-sensitive KIT-Dup-Ser501Ala502 are distinguishable from KIT-Dup-Ala502Tyr503.

The effect of mastication on reaction latency to unanticipated external disturbances in the standing position.

Previous research has shown that mastication reduces shifts in the center of gravity of persons standing still. The present research was conducted to determine whether mastication improves reactive balance in the standing position in response to unanticipated external disturbances. The subjects were 32 healthy male adults (mean age 21.1 years, standard deviation (SD) 0.7 years). Latency data determined with the Motor Control Test of Computerized Dynamic Posturography (CDP) were compared for the three conditions of mastication status, the direction of translation, and the magnitude of translation, using three-way repeated measures ANOVA and lower-order ANOVA with the three conditions separated. Latency was significantly shorter with mastication than with the lower jaw relaxed (P < 0.00001). Mastication alone, however, cannot be considered significant because of the complex interactions involved among the three conditions. Mastication increases not only static balance but also reactive balance in response to unanticipated external disturbances. Gum chewing may therefore reduce falls among elderly persons with impaired balance.

Characterization of novel germline c-kit gene mutation, KIT-Tyr553Cys, observed in a family with multiple gastrointestinal stromal tumors.

We found a novel type germline mutation at exon 11 of the c-kit gene, which results in a substitution of Tyr to Cys at codon 553 of the c-kit gene product (KIT-Tyr553Cys), in a 68-year-old female patient with multiple gastrointestinal stromal tumors (GISTs). In the present study, we carried out mutational analysis in her family members to determine the carriers and characterized the mutation by introducing the corresponding mutation (murine KIT-Tyr552Cys) into expression vector possessing murine c-kit cDNA. Mutational analysis of peripheral blood leukocytes of her family members revealed that a 44-year-old son had the same mutation, but at present he had neither apparent symptoms nor images of multiple GISTs. By transfection with the expression vector possessing the murine mutant c-kit cDNA, interleukin-3-dependent Ba/F3 murine lymphoid cells started growing autonomously without any growth factors, indicating that the mutation was considered to be of gain-of-function. Imatinib, a small molecule of tyrosine kinase inhibitor, effectively inhibited autophosphorylation of KIT-Tyr552Cys. Nilotinib, another small molecule of the KIT inhibitor, also effectively inhibited autophosphorylation of KIT-Tyr552Cys. In fact, proliferation of Ba/F3 cells expressing KIT-Tyr552Cys was effectively inhibited by both imatinib and nilotinib. These findings indicate that the novel type human KIT-Tyr553Cys mutation is the cause of the present familial and multiple GISTs, and that both imatinib and nilotinib might effectively inhibit the growth of GISTs developing in the patients of this family.

Three-dimensional motion of the upper extremity joints during various activities of daily living.

Highly reliable information on the range of motion (ROM) required to perform activities of daily living (ADL) is important to allow rehabilitation professionals to make appropriate clinical judgments of patients with limited ROM of the upper extremity joints. There are, however, no data available that take full account of corrections for gimbal-lock and soft tissue artifacts, which affect estimation errors for joint angles. We used an electromagnetic three-dimensional tracking system (FASTRAK) to measure the three-dimensional ROM of the upper extremity joints of healthy adults (N=20, age range 18-34) during 16 ADL movement tasks. The ROM required for the performance of each movement was shown in terms of the joint angle at the completion of the task, using a new definition of joint angle and regression analysis to compensate for estimation errors. The results of this study may be useful in setting goals for the treatment of upper extremity joint function.

Evaluation of the age-related changes in movement smoothness in the lower extremity joints during lifting.

The purpose of this study was to analyze age-related movement smoothness changes in the lower extremity joints during load lifting. A total of 10 young and 13 elderly subjects participated in the study. Infrared reflective markers were attached to body landmarks in each subject. While the subjects stood on force plates and lifted a box, the marker displacements and ground reaction forces were measured using a 3D motion analysis system. The jerk square mean value (JSM) was defined as the lower extremity joint movement smoothness index during lifting. JSM represented the average of the square of the joint angle third derivative value, according to the jerk third derivative of the position data. Each subject's JSM values were calculated for the hip, knee and ankle joints. Movement smoothness appeared to decrease as JSM increased. Multiple regression analyses were performed for dependent variables (hip, knee and ankle joint JSM values) and independent variables (age, hand grip strength, sex difference and lifting duration). The level of significance was set at p<0.05. For the hip joint JSM, the regression coefficient for age was significantly positive and that for lifting duration was significantly negative. For the knee joint JSM, the regression coefficient for lifting duration was significantly negative. For the ankle joint JSM, the regression coefficients for age and hand grip strength were significantly positive and that for lifting duration was significantly negative. These results suggest that movement smoothness in the hip and ankle joints during lifting decreases with advancing age.

In vivo effect of imatinib on progression of cecal GIST-like tumors in exon 17-type c-kit knock-in mice.

Two families with a germline Asp820Tyr mutation at exon 17 of the c-kit gene and multiple gastrointestinal stromal tumors (GISTs) have been reported. Recently, we generated a knock-in mouse model of the family, and mice with KIT-Asp818Tyr corresponding to human KIT-Asp820Tyr showed a cecal GIST-like tumor. In this report, we examined the in vivo effect of imatinib on tumor progression in knock-in mice. Imatinib of 100 microg/g body weight was administered to heterozygous (KIT-Asp818Tyr/+) mice orally for 7, 14 and 28 days, and cecal tumors were dissected. Both macroscopic size and the measured volume of cecal tumors were not significantly reduced after a 7-, 14- and 28-day administration of imatinib when compared with those before imatinib administration. Cell proliferation was assessed by Ki-67 immunohistochemistry and the labeling index significantly decreased after imatinib administration, but the value of the index after imatinib was only about half compared with that before imatinib. Western blotting and real-time PCR revealed that KIT expression was almost equivalent, but KIT phosphorylation was significantly but not completely inhibited in tumor tissues after 7, 14 and 28 days of imatinib administration when compared with that before imatinib administration. Phosphorylation of Akt and Stat1 was accordingly inhibited after imatinib administration. Thus, imatinib seemed to inhibit in vivo tumor proliferation but not decrease tumor volume on this mouse model, probably because of an insufficient inhibition of phosphorylation of KIT and its downstream signaling molecules. These results suggested that progression of multiple GISTs in patients with germline Asp820Tyr might be partially controlled by imatinib and that model mice provide an opportunity to examine the effect of various other targeted drugs on in vivo tumor progression.

Effect of occlusion status on the time required for initiation of recovery in response to external disturbances in the standing position.

BACKGROUND: To examine whether occlusion status contributes to improvement of postural balance. METHODS: Thirty healthy adolescents (15 males and 15 females; mean age, 20.3; standard deviation (SD) 1.6 years) with no equilibrium or stomatognathic function abnormalities were examined. Occlusion is a term meaning "jaw clenching." Occlusion status was evaluated by measuring masseter activity using the EMG system. Balancing ability was evaluated using the EquiTest system, which measures sway of the center of gravity produced by rapid movement of force plates as an external disturbance (three intensity levels). The time required for initiation of recovery after application of the disturbance was calculated by measuring displacement of the center of foot pressure. Data were compared according to occlusion status. FINDINGS: Little difference in latency was observed following a small disturbances; however, the greater the disturbance the shorter the latency with occlusion, while without occlusion, latency increased with increasing disturbance. A statistically significant interaction (P<0.001) between occlusion and external disturbance was also found. INTERPRETATION: This study suggested that occlusion contributes to maintenance of postural balance and improvement of stability when unexpected sway occurs in the standing position.

CAD/CAM evaluation of the fit of trans-tibial sockets for trans-tibial amputation stumps.

The purpose of this study was to objectively evaluate the fit of sockets for trans-tibial stumps in order to establish a guideline for use in the automated production of prosthetic sockets. Subjects were 24 trans-tibial amputees. Using a CAD/CAM system, 11 parameters regarding the 3D shape of the stumps and the sockets were objectively evaluated. A correlation was found between the activity level and the upper and lower volumes of the socket, and between the cause of amputation and the upper volume of the socket. It was considered desirable to make the lower part of the socket looser for patients with lower activity levels, to make the upper part tighter for patients with higher activity levels, and to make the upper part looser for amputation patients with peripheral circulatory diseases.

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-alpha (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.

Differential expression of connexin 43 in gastrointestinal stromal tumours of gastric and small intestinal origin.

Gastrointestinal stromal tumours (GISTs) are considered to originate from interstitial cells of Cajal (ICCs). ICCs are classified into several subtypes according to their location or roles. Several reports indicate that GISTs of the small intestine appear to have different clinical and pathological characteristics from gastric GISTs. We previously found using a cDNA expression chip that connexin 43, a component of gap junctions, is expressed specifically in small intestinal GISTs but not in gastric GISTs. To confirm the specificity of connexin 43 expression, we analysed 10 small intestinal GISTs and 15 gastric GISTs by northern blotting, western blotting and immunohistochemistry in this study. Northern blotting was performed in five small intestinal GISTs and five gastric GISTs, and revealed connexin 43 mRNA expression in all of the five small intestinal GISTs, but in none of the gastric GISTs. By western blotting, bands corresponding to connexin 43 were easily detected in all of the five small intestinal GISTs studied but were absent in all five gastric GISTs analysed. Immunohistochemistry showed that all of the 10 small intestinal GISTs were positive for connexin 43 but only one of 15 gastric GISTs, which exhibited a mutation in exon 9 of the KIT gene, was connexin 43-positive. We also examined the localization of connexin 43 in the normal stomach and small intestine. Immunoreactivity for connexin 43 was present in both normal gastric and small intestinal circular muscle layers, but it was unclear which cell type was positive. These results suggest that GISTs are divided into at least two groups, namely the gastric subtype and the small intestinal subtype, through phenotype but not location. Furthermore, these data indicate that the gastric and the small intestinal subtypes of GIST may originate from different subtypes of ICC.

Different inhibitory effect of imatinib on phosphorylation of mitogen-activated protein kinase and Akt and on proliferation in cells expressing different types of mutant platelet-derived growth factor receptor-alpha.

Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the c-kit gene. Previously, we found 2 types of gain-of-function mutation of the PDGFRA gene, Val561 to Asp and Asp842 to Val, in about half of GISTs without c-kit gene mutations. Although the inhibitory effect of imatinib on various types of activating mutant KIT has been well examined, that on the activating mutant PDGFRA has not been fully investigated. In the present study, we examined the effect of imatinib on autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and of Akt and in vitro cell proliferation using murine Ba/F3 cells stably transfected with one of the 2 murine-type mutated PDGFRA cDNAs. Imatinib almost completely inhibited autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and Akt as well as in vitro cell proliferation at the concentration of 0.01 microM in cells expressing mutant PDGFRA with Val561 to Asp. However, in cells expressing mutant PDGFRA with Asp842 to Val, imatinib almost completely inhibited autophosphorylation of mutant PDGFRA and phosphorylation of MAPK and Akt at 1.0 microM. The concentration contributing to complete inhibition of in vitro cell proliferation was 10 microM. Ba/F3 cells expressing mutant PDGFRA are a good model to investigate the mechanism of cell proliferation or growth inhibition by imatinib in mutant PDGFRA-driven 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.