Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

Differences in signaling pathways and expression level of the phosphoinositide phosphatase SHIP1 between two oncogenic mutants of the receptor tyrosine kinase KIT.

Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KIT(K642E), a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KIT(WT) and the murine homologues of human KIT oncogenic mutants, further referred to as KIT(K641E) and KIT(del559), a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KIT(K641E) and KIT(del559), whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KIT(K641E) only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation.

Gain-of-Function Mutations of Receptor Tyrosine Kinases in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in human gastrointestinal tract. We first found that most GISTs expressed KIT, a receptor tyrosine kinase encoded by protooncogene c-kit and that approximately 90% of the sporadic GISTs had somatic gain-of-function mutations of the c-kit gene. Since both GISTs and interstitial cells of Cajal (ICCs) were double-positive for KIT and CD34, GISTs were considered to originate from ICCs or their precursor cells. We also found that germline gain-of-function mutations of the c-kit gene resulted in familial and multiple GISTs with diffuse hyperplasia of ICCs as the preexisting lesion. Moreover, we found that about half of the sporadic GISTs without c-kit gene mutations had gain-of-function mutations of platelet-derived growth factor receptor alpha (PDGFRA) gene that encodes another receptor tyrosine kinase. Imatinib which is known to inhibit constitutively activated BCR-ABL tyrosine kinase in chronic myelogenous leukemia also inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for metastatic or unresectable GISTs as a molecular target drug. Mutational analyses of c-kit and PDGFRA genes are considered to be significant for prediction of effectiveness of imatinib and newly developed/developing other agents on GISTs. Some mouse models of familial and multiple GISTs have been genetically created, and may be useful for further investigation of GIST biology.

Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Gain-of-function mutations in the juxtamembrane domain of the c-kit gene have been found in several GISTs. In this study, we examined the correlation between the presence of c-kit mutation and prognosis in 124 cases of GIST. DNA samples were extracted from paraffin sections. Exon 11 of the c-kit gene encoding the juxtamembrane domain and exon 17 encoding the kinase domain were amplified by PCR and sequenced. Most GISTs (89%) express the KIT protein, and missense mutations of exon 11 were found in 71 of 124 GISTs (57%). No mutations were detectable in exon 17. These 71 mutation-positive GISTs were larger in size and had more frequently invaded adjacent tissues than did the 53 mutation-negative GISTs. Histologically, the mutation-positive GISTs showed higher mitotic figures and more necrosis and hemorrhage. The patients with mutation-positive GISTs showed more frequent recurrences (P = 0.0005) and higher mortality (P = 0.0001) than did those with mutation-negative GISTs. The c-kit mutation was an independent prognostic factor for overall and cause-specific survival of the patients with GISTs. These results suggest that GISTs may be divided into mutation-positive and -negative subtypes. The prognosis was worse in patients with mutation-positive GISTs than in those with mutation-negative GISTs. Thus, mutation of the c-kit gene may be a good prognostic marker of GISTs.

Wn mutation of c-kit receptor affects its post-translational processing and extracellular expression.

The W locus of mice encodes the c-kit receptor tyrosine kinase. Recently, we characterized a novel mutant allele, Wn, and demonstrated that the c-kit protein synthesized in Wn/Wn cultured mast cells (CMC) was reduced in size and not expressed on their surface (Tsujimura et al., 1993). In this study, we further examined biochemical nature of the mutant form of c-kit protein, by using Wn/Wn CMC and 293T cells transfected with Wn-type c-kit cDNA (c-kitWn). The c-kit product synthesized in Wn/Wn CMC was truncated almost all cytoplasmic domain and was less glycosylated. In c-kitWn-transected cells, both glycosylation and extracellular expression of c-kit protein was also impaired, however, no truncation was detected. These results indicate that Wn-mutant form of c-kit product is insufficient in maturation, which is associated with impairments in the transport to the plasma membrane, and retention of the mutant protein in endoplasmic reticulum is suggested. This is the first demonstration of the c-kit mutation affecting posttranslational processing its product.

Inhibition of attachment between cultured mast cells and fibroblasts by phorbol 12-myristate 13-acetate and stem cell factor.

Cultured mast cells (CMC) derived from the bone marrow of mice express the receptor encoded by the W (c-kit) locus (W receptor), and the WCB6F1(+/+)-3T3 fibroblasts express the ligand encoded by the Sl locus (stem cell factor [SCF]). CMC attach to the fibroblasts through the W receptors and cell-bound SCF. We investigated the effect of phorbol 12-myristate 13-acetate (PMA) and recombinant murine SCF (rmSCF) on the attachment. PMA induced both the internalization and shedding of W receptors, whereas rmSCF induced only the internalization. Moreover, both PMA and rmSCF reduced the expression of c-kit mRNA levels in CMC. Addition of either PMA or rmSCF to the coculture of CMC and fibroblasts resulted in the inhibition of attachment. Since the magnitude of the attachment between CMC and fibroblasts may be manipulated by changing the doses of either PMA or rmSCF, the present experimental system may be useful as a model for the attachment between blood cells and stromal cells.

Effects of loss-of-function and gain-of-function mutations of c-kit on the gastrointestinal tract.

Protooncogene c-kit encodes a receptor tyrosine kinase, KIT. Interstitial cells of Cajal (ICCs) that are important for the autonomous movement of the gastrointestinal tract essentially require the normal function of the KIT for their development. Therefore, germline loss-of-function mutations of the c-kit gene cause deficiency of ICCs that results in disturbed gastrointestinal movement. On the other hand, somatic gain-of-function mutations of the c-kit gene induce gastrointestinal stromal tumors (GIST) that are considered to originate from ICCs. Moreover, germline gain-of-function mutations of the c-kit gene are a cause of familial development of multiple GISTs.

Design for a bioreactor with sunlight supply and operations systems for use in the space environment.

An experiment was carried out to determine the characteristics of an operations system that can support fast cultivation of algae at high densities in the weightlessness of space. The experiment was conducted in glass bioreactor tanks, in which light was supplied by radiator rods connected to optical fiber cables. The illumination areas of the tanks were 2600 cm2, 6000 cm2, and 9200 cm2 per liter of solution. The characteristics of O2-CO2 gas exchange, concentration and separation of chlorella in the growth medium, dialysis of ionic salts in the growth medium, etc. were examined. Chloralla ellipsoidea was used in the experiment, yielding the following results: (1) By increasing the ratio of illumination area to volume, growth rates of up to approximately 0.6 g/L h could be obtained in a highly concentrated solution (one that contains 20 g/L or more of algae). (2) The most suitable proportions of carbon dioxide and oxygen gases for growing algae quickly at high concentrations were found to be 10% CO2 and 10% O2 (by volume). (3) There was a high optimum concentration for fast cultivation, and the data obtained resembled the theoretical curve postulated by P. Behrens et al. (4) It was possible to exchange carbon dioxide and oxygen using gas-permeable membrane modules. (5) It was possible to separate the chlorella from the growth medium and recycle the medium.

[An emergency cesarean section using general anesthesia for a patient with schizophrenia].

A 27 year-old female in 39th week gestation with schizophrenia underwent an emergency Cesarean section using general anesthesia. A diagnosis of schizophrenia was made two years previously, since then oral anti-psychotic drugs such as chlorpromazine had been given to her. In June 1989 she suddenly became excited and generalized muscle rigidity was observed without any triggering episodes. Her excitement was so marked that we had to administer intramuscular levomepromazine 75 mg and diazepam 10 mg to her, but they failed to sedate her adequately. Emergency Cesarean section was scheduled to overcome this situation. Spinal or epidural anesthesia was not indicated because of her vigorous excitement, and anesthesia was induced with thiopental 350 mg and succinylcholine 40 mg. Induction-delivery time was 12 minutes. Pentazocine 30 mg in combination with nitrous oxide was given for the maintenance of anesthesia. Plasma levomepromazine levels were 46.9 ng.ml-1 in the mother and 11.3 ng.ml-1 in the umbilical vein, respectively. The baby's Apgar score was 9 and 1 min and 9 at 5 min after the delivery. The baby developed slight generalized tremor until next day, probably due to effect of levomepromazine given before the Cesarean section. The patient was discharged without any cardiorespiratory trouble and her baby has been doing well so far.

[Sevoflurane anesthesia for a patient with cerebral palsy].

We report anesthetic management of a 20-year-old male patient with cerebral palsy who underwent an operation for retinal detachment of the left eye. Induction and maintenance of anesthesia were smoothly done with inhalation of 1-5% sevoflurane with nitrous oxide and oxygen. Emergence of anesthesia was also smooth except for slight and transient excitement. Anesthetic management of a patient with cerebral palsy was also discussed.

[Plasma morphine levels during its continuous epidural infusion].

We evaluated plasma levels of morphine during its continuous epidural infusion for postoperative analgesia in nine adult patients. A bolus injection of 3 mg of morphine was administered epidurally 3 hours prior to the proposed end of the surgery, and thereafter continuous epidural infusion of morphine was continued at a rate of 0.167-0.042 mg.hr-1 with a pump (CADD-PCA, 5200P, Pharmacia) during and after the surgery until the 3rd postoperative day. The dose of morphine was gradually decreased to 0.021-0.042 mg.hr-1 without reducing the quality of postoperative analgesia. Plasma morphine levels were measured by gas chromatography-mass spectrometry method. Plasma concentrations of morphine were 4.6 +/- 0.7 (Mean +/- SE) ng.ml-1 at the end of surgery and they decreased thereafter to 0.7 +/- 0.1 ng.ml-1, 0.3 +/- 0.1 ng.ml-1 and 0.1 +/- 0.1 ng.ml-1 on the 1st, 2nd and 3rd postoperative days, respectively. Plasma levels of morphine decreased gradually correlating with reduction of infused morphine. Adequate postoperative pain relief was obtained throughout the procedure without any severe complications such as respiratory depression. The analgesic effect of epidural morphine did not parallel with that of plasma concentration. Plasma concentrations of morphine administered by continuous epidural infusion with a pump were estimated to be lower than the minimum analgesic plasma concentration (10-40 ng.ml-1), and the toxic levels of morphine during continuous epidural infusion were not detected by our method.

[Hemodynamic and endocrine responses to prostaglandin E1 induced hypotension during enflurane anesthesia in surgical patients--evaluation by transesophageal echocardiography].

This study was undertaken to find a relationship between cardiac function indices and endocrine functions during prostaglandin E1 induced hypotension in surgical patients. Thirteen patients who underwent either orthopedic or gynecologic surgery were the subjects of the study. Systolic blood pressure decreased to 80 torr with prostaglandin E1 infusion (0.5-2 micrograms.kg-1 x min-1) under enflurane-N2O anesthesia. The cardiac function were judged by transesophageal echo-cardiography. Plasma ANP and ADH levels were measured by radioimmunoassay. Significant reductions in the left atrial diameter and A/R and significant increases in cardiac output were observed, but there were no significant changes in fractional shortening and pulmonary vein flow. Plasma ANP concentrations decreased significantly from 45.3 +/- 5.1 pg.ml-1 (mean +/- SE) of pre-hypotension to 32.6 +/- 2.2 pg.ml-1 of control, but plasma ADH levels increased significantly during hypotension (P < 0.05). We could not find any significant correlation between the cardiac function indices and plasma hormone levels. Transesophageal echocardiography is an excellent monitor during induced hypotension. It is difficult to predict the ANP and ADH levels by measuring cardiac function as judged by TEE.

[Prolonged respiratory depression following general anesthesia in a patient with dystrophia myotonica].

A case of general anesthesia for a 52 year old female with previously undiagnosed dystrophia myotonica was reported. The patient was diagnosed as flaccid paralysis of the bilateral lower extremities but myotonic symptoms were not found preoperatively. The patient underwent duodenal resection to have a benign tumor removed. Anesthesia was induced with thiamylal 250 mg and pancuronium bromide 4 mg intravenously to facilitate tracheal intubation. Anesthesia was maintained with enflurane (0.6-1.0%) in nitrous oxide (50%) and oxygen (50%). The course of anesthesia was uneventful except for a transient hypotension of 80 mmHg systolic for about 10 minutes. The patient did not recover smoothly from anesthesia and prolonged apnea was observed. 90 minutes after the end of surgical procedure, spontaneous ventilation appeared. Then the tidal volume increased gradually but it still remained around 100-150 ml even 3 hours after the end of the operation. She was on ventilator and observed carefully. The endotracheal tube was removed four days after the operation. The patient was examined again by a neurologist and a final diagnosis of dystrophia myotonica was made. Prolonged recovery from anesthesia and postoperative respiratory depression observed in this patient was due to preoperatively undiagnosed dystrophia myotonica. A careful preoperative examination should be made to minimize possible complication related to anesthesia in the disease.

[Endocrine and hemodynamic responses to total body hyperthermia in humans].

We investigated effects of total body hyperthermia (TBH) on endocrine and hemodynamic responses. A total of five treatments were performed in five patients with gastric cancer under neuroleptanesthesia with morphine followed by 0.2 to 0.4% enflurane. TBH was extracorporeally induced with veno-venous shunt incorporating with heat exchanger to keep their temperature between 41.5 degrees C and 42.0 degrees C for three hours. The patients were administered angiotensin to maintain tumor blood flow. Lactated Ringer's solution was administered at the rate of 10 to 15 ml.kg-1.hr-1 for five hours. Plasma cortisol levels decreased significantly to about one third of the control value after heating and the levels recovered to the control value after cooling. Plasma norepinephrine level increased significantly to about 7 to 9 times the control value following TBH, but this hormonal response was insufficient to reveal marked direct hemodynamic effects. The magnitude of fall in SVR was more significant in spite of the administration of angiotensin. Cardiac index increased significantly to about 2.0 to 2.6 fold of control value, but mean arterial blood pressure (MAP) decreased significantly to about two thirds to four fifths of the control value. Morphine relieved the hormonal response in ACTH and cortisol strongly, but morphine suppressed hemodynamics by decreasing SVR. Neither norepinephrine released from sympathetic nerve endings nor even 50 to 200 ng.kg-1.min-1 of angiotensin administered failed to restore SVR or MAP during hyperthermia.

[Anesthetic experience of a patient with blue rubber bleb nevus syndrome].

We report an anesthetic experience of a 63-year-old female patient with blue rubber bleb nevus syndrome who underwent an open reduction of the fracture of the right femur. The syndrome is characterized by bluish nevus scattered throughout the skin of the whole body and angiomas of the gastrointestional tract causing serious bleeding. Anesthesia was induced with thiopental followed by intravenous injection of succinylcholine chloride and maintained with neuroleptanesthesia in nitorous oxide and oxygen. The course of anesthesia and emergence from anesthesia were uneventful. Anesthetic management of patients with blue rubber bleb nevus syndrome was also discussed.

A mouse model of a human multiple GIST family with KIT-Asp820Tyr mutation generated by a knock-in strategy.

Several families exhibiting multiple gastrointestinal stromal tumours (GISTs) and germline c-kit gene mutations at exons 8, 11, 13, or 17 have been reported. These patients also exhibit diffuse hyperplasia of the interstitial cells of Cajal (ICCs) as a pre-existing lesion of multiple GISTs. We generated a mouse model of a family with germline c-kit gene mutation at exon 17, and compared the phenotypes between the mice and humans. The mouse counterpart (KIT-Asp818Tyr) of the human KIT-Asp820Tyr mutation was transmitted into germline by a knock-in strategy. Mating of male and female heterozygotes (KIT-Asp818Tyr/+) resulted in the generation of homozygotes (KIT-Asp818Tyr/KIT-Asp818Tyr). Histological examination revealed that all heterozygotes had both a small KIT-positive mesenchymal tumour at the caecum, consistent with GIST, and KIT-positive diffuse spindle-shaped cell proliferation in the distal oesophagus, stomach, proximal duodenum, and colon consistent with ICC hyperplasia. All homozygotes exhibited a larger caecal tumour and more prominent spindle-shaped cell proliferation compared with the heterozygous mice, and they usually died within 10 weeks after birth, likely due to ileus. The small intestine of both genotypes showed no apparent morphological abnormality, and autonomous contraction of the ileal segments appeared normal. Western blotting demonstrated that the caecal tumours expressed phosphorylated KIT, MAPK, Stat1, and Stat5. These mutant mice are considered to be useful for further investigation of the mechanism of GIST development as a result of ICC hyperplasia and for assessment of the in vivo effects of drugs against molecular targets.

Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors.

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.