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Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

Alterations of the EGFR gene occur frequently in human gliomas where the most common is an in-frame deletion of exons 2-7 from the extracellular domain, resulting in a truncated mutant receptor (deltaEGFR or de 2-7 EGFR). We previously demonstrated that introduction of deltaEGFR into human U87MG glioblastoma cells (U87MG.deltaEGFR) conferred remarkably enhanced tumorigenicity in vivo. Here, we show by cell-mixing experiments that the enhanced tumorigenicity conferred by deltaEGFR is attributable to a growth advantage intrinsic to cells expressing the mutant receptor. We analyzed the labeling index of the proliferation markers Ki-67 and bromodeoxyuridine and found that tumors derived from U87MG.deltaEGFR cells had significantly higher labeling indexes than those of tumors derived from U87MG cells that were either naive, expressed kinase-deficient mutants of deltaEGFR, or overexpressed exogenous wild-type EGFR. We also utilized terminal deoxynucleotidyl transferase-mediated nick end-labeling assays and showed that the apoptotic index of U87MG.deltaEGFR tumors was more than 4-fold lower than that of parental U87MG tumors. This decrease in cell death was inversely correlated with the expression level of Bcl-X(L), a negative regulator of apoptosis, which was more than 3-fold higher in U87MG.deltaEGFR-derived tumors than in those derived from parental cells. Similar observations were obtained in vitro in serum-free conditions. These results suggest that deltaEGFR exerts its pronounced enhancement of glioblastoma tumorigenicity by stimulating proliferation and inhibiting apoptosis and that the effects are directly attributable to its constitutively active signal.

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.

Background: Two rare cases of triple primary malignant neoplasms (PMN), including malignant brain tumors, which were glioblastoma multiformes, are described., Methods: The clinical characteristics and underlying genetic alterations in triple or more PMN, including malignant brain tumors are discussed with intensive review of the literature., Results: The first patient, a 77-year-old male, suffered metachronously from tubular adenocarcinoma of the stomach, transitional cell carcinoma of the bladder, and glioblastoma in the brain. This glioblastoma had loss of heterozygosity in exons 7-8 in p53 gene. The second patient, a 68-year-old male, developed papillary adenocarcinoma of the lung, adenocarcinoma of the rectum, and glioblastoma in the brain during a period of 7 years. In 42 such cases described in the literature, age distribution demonstrated two characteristic peaks, one in the third decade and the other over 50 years of age. The younger group consisted mainly of Turcot's syndrome, and of a case of Li-Fraumeni familial cancer syndrome. On the other hand, neither of these hereditary cancer syndromes were contained in the elder group. Regarding the site of PMN, colorectal cancers were associated most frequently with malignant brain tumors, followed by stomach cancers, and thyroid cancers. Malignant brain tumors, mostly glioblastoma multiforme, tend to occur as the last tumor of triple or more PMN., Conclusions: These results suggest that genetic background might play an important role in tumorigenesis of PMN in the younger group, whereas epigenetic factors would be more important in the older group. Characteristic organ association and factors influencing carcinogenesis, such as aging, environmental carcinogens, and underlying genetic alterations in these tumors are further discussed.

Background: Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy. The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance. Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs., Methods: We present a 45-year-old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy. In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor., Results: Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance. On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low. Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable. Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression. For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis. Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16., Conclusions: The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy.

We present a 24-year-old woman with multifocal and heterochronous recurrence of malignant astrocytomas that were consecutively treated by linac-based sterotactic radiotherapy. The patient had previously received multimodalities of treatment for malignant astrocytomas in the right occipital lobe consisting of surgical resection on four occasions, conventional external-beam irradiation, immunological therapy, and systemic chemotherapy. Thereafter she developed relatively small and well-demarcated recurrent lesions in eight different sites, most of which were located in eloquent and deep-seated regions. She underwent ten fractionated linac-based stereotactic radiotherapy courses with a median total dose of 42 Gy, seven fractions of 6 Gy each in 15 days, experiencing neither any adverse effects nor neurologic deterioration. Three out of 10 treated lesions responded well with subsequent reduction in size, and 80% of them did not increase at least for 6 months, although 66.7% of the lesions eventually exhibited enlargement of the enhanced areas, some of which were presumed as radiation necrosis. These clinical results suggest that the fractionated linac-based stereotactic radiotherapy had significant effects on tumor growth inhibition and attainment of acceptable patient performance status.

The authors report a 57-year-old man with an ossified and calcified epidural hematoma found incidentally 40 years after incurring a severe head injury. Since the introduction of computed tomography (CT) scan, few such cases have been described in the literature. A characteristic intracranial "double-outlined" contour on plain skull x-ray films and CT scans represented bone formation and calcification of the hematoma capsule adjacent to the dura.

Retroflexion of the hypoplastic fused cerebrum is a rare condition in holoprosencephaly. We describe a new case of this entity in a 7-year-old boy with alobar holoprosencephaly whi developed retroflexion of the holosphere 7 years after a ventriculo-peritoneal shunt was placed. The radiological findings revealed retroflexion of the cerebrum anchored to the cranial vault between the precerebral space and the dorsal sac, which were open to each other. In patients with holoprosencephaly, the possibility of retroflexion of the holosphere should be considered after placement of a ventriculo-peritoneal shunt for associated hydrocephalus.

A 22-year-old woman harboring recurrent malignant astrocytoma presented with intracranial hypertension by tumor hemorrhage during repeated administration of lymphokine-activated killer (LAK) cells via Ommaya's reservoir. She first suffered from the tumor located at the right occipital lobe at the age of 13. The tumor regressed completely by subtotal removal of the tumor, followed by external irradiation. Nine years later, however, the occipital tumor recurred and was subtotally resected. Pathological diagnosis was astrocytoma grade 3. Postoperatively, LAK cells induced from her peripheral blood lymphocytes incubated with interleukin-2 and anti-CD3 antibody were injected into the tumor cavity via Ommaya's reservoir for eight times. At the end of the LAK therapy, the tumor regrew with massive hemorrhage in the tumor cavity causing intracranial hypertension. At the reoperation, thick granulation tissue covered the surface of the recurrent tumor and dense deposits of clot were noted around the tip of the Ommaya's tube. Histologically the superficial layer of the tumor was infiltrated with macrophages and lymphocytes, mostly CD3-positive T cells, accompanied with capillary hyperplasia. Viable astrocytoma cells were abundant beneath the granulation layer. It should be considered that in local LAK therapy granulation tissue formation with hypervascularization at the surface of the tumor cavity may lead to tumor bleeding as well as resistance to the treatment.

Small-field radiotherapy based on a 6-MeV linac and a conventional head mold is investigated as an alternative to radiosurgery with stereotactic frames. The system requires no additional device and allows fractionated treatment. The dose distributions obtained are comparable to those reported with a Gamma Unit. Overall positioning errors are within 2 mm. Using this approach, seven patients with brain tumors who could not have been treated otherwise, underwent fractionated radiotherapy with total accumulated doses ranging from 70 to 108 Gy. The treatment was tolerated well with no acute toxicity or adverse effect encountered during the follow-up period of 8-14 months. All of the patients remained free from disease progression in the treated volumes. Although the follow-up is brief, the preliminary results suggest that this is a simple and inexpensive but effective system for the treatment of small intracranial malignancies.

Application of flow cytometric DNA analysis was tried to determine the sensitivity of ACNU, one of nitrosourea derivatives which had been used very commonly for malignant brain tumors, to tumor cells. To evaluate the degree of chemo-sensitivity of ACNU, factor B was introduced, indicating the accumulated cells in SG2M phases after ACNU treatment as the percentage of cells that was previously in the SG2M phases and it revealed that ACNU sensitive cell clones gave much larger values of Factor B than ACNU resistant ones at the concentration of 10 micrograms/ml ACNU treatments. Twenty clinical materials obtained by operation were examined by measuring the degree of values of this Factor B, and the findings indicated that this method would be applicable as a clinical test for chemo-sensitivity of malignant brain tumors, after some improvement of the method.

CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy camptothecin, is a well-known DNA topoisomerase I inhibitor. SN-38 is a metabolite of this compound. Both emit fluorescence when activated by a laser beam. With a confocal laser scanning microscope (CLSM), we determined the intracellular distribution of CPT-11 and SN-38 and the chronological changes in drug-treated PC-7, a cell line of human non-small cell lung cancer, and its CPT-11 resistant variant, PC-7/CPT cells. There were many more granules in the cytoplasm in PC-7/CPT than in the parent cell line (PC-7). The granule formation of the resistant cell could indicate a different drug metabolism in the cytoplasm from that of the parent cell. This technique would provide a new way of investigating the mechanism of resistance of cancer cells to anticancer drugs.

Northern blot analysis with O6-methylguanine-DNA methyltransferase (MGMT) cDNA as a probe was used to analyze the MGMT activity regulating drug resistance of human cells to chloroethylnitrosoureas (CENUs). By this method, the expression levels of MGMT mRNA in six human glioma cell lines and 12 human brain tumor tissues from surgical specimens were determined. These MGMT mRNA levels were compared with the SD10 values of the tumor cells, estimated by cell survival assay, which indicated their resistance to the anticancer drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Human brain tumors that were highly resistant to ACNU, such as glioblastoma Gbl1 and metastatic brain tumor Col1 with SD10 values (microM) of above 100, expressed markedly increased amounts of 0.95 kb MGMT mRNA. In contrast, tumor cells such as U-87MG, U-251MG, U-343MG, U-373MG and SF-126 with SD10 values of under 14 indicating low resistance to ACNU scarcely synthesized any MGMT mRNA. These results indicated that the level of expression of MGMT mRNA in human brain tumors determined by Northern blot analysis truly reflects their cellular resistance to ACNU. Thus the Northern method with MGMT cDNA probe reported here is a practical and reliable method for estimation of cellular resistance to CENUs such as ACNU and for screening the chemotherapeutic response to CENUs of human brain tumors.

A case of primary malignant lymphoma of the skull base is reported. A 62-year-old man was admitted with headache and diplopia. Computed tomography and magnetic resonance imaging showed an abnormal mass in the right parasellar region. The final diagnosis after surgery was primary malignant lymphoma of the skull base. Primary malignant lymphomas sometimes occur in the central nervous system but those in the skull base are exceptional. The present paper presents just such a rare case of malignant lymphoma of the skull base. Diagnosis and combined therapy consisting of surgery, radiotherapy and chemotherapy for the disease are discussed, and the literature on recent clinical problems of central nervous system lymphomas is reviewed.

This experiment was designed to compare sensitive and insensitive children under psychological stress during performance on a motor skill. A new index for sensitivity based on the psychological and physiological data (heart rate) was introduced. The subjects were 50 Japanese children. Although experimental groups received competitive instruction and were given the mirror-drawing task, a control group did not receive such instruction. The sensitive group made fewer errors than the insensitive group and the control group. These results suggest that the combined index is a more useful measure of psychological stress than a single index.

A 6-month-old boy having intraspinal lipoma with lumbosacral subcutaneous lipoma and occult spinal dysraphism is described. CT scan and magnetic resonance imaging (MRI) are demonstrated. On admission, the patient showed no neurological deficits. Lumbosacral hemangioma and subcutaneous lipoma were noticed on physical examination. A plain spinal roentogenogram revealed occult spinal dysraphism extending from L 4 level down to the sacral region. A plain CT scan disclosed a round-shaped low density area surrounded by a relatively high density area corresponding to the neural tissue in the spinal canal. MRI (1.5-T superconducting system) with sagittal views clearly showed an oval-shaped high signal intensity (SI) area-an intraspinal lipoma-with the neural tissue running longitudinally within and on its surface. The lipoma appeared to be attached to the spinal cord at L 1/2 level. On operation, we found an extramedullary lipoma which had a certain connection to the sacral epidural adipose tissue with an opening of the dural sac, as far as the subcutaneous lipoma. Tethered cord and thickened filum terminale were not identified. Generally, lipoma is demonstrated as a low density area of approximately -90 H.U. on a CT scan. In MRI, we can obtain a high contrast between the lipoma, the spinal cord, and the cerebrospinal fluid, since they are shown as a high, an intermediate, and a low SI area respectively, on T1-weighted spin echo images. Furthermore, sagittal section of MRI is regarded as a potent diagnostic modality to get the precise anatomy of the spinal cord lesion. These short spin echo images can be taken in a relatively brief time. It is a great advantage for patients with spinal lipoma, who are usually infants. MRI is considered to be a quite useful modality to diagnose spinal lipoma, and is able to lead to an early diagnosis non-invasively and accurately, facilitating appropriate surgical treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to investigate the effects of psychological stress upon attention as subjects performed on the MFF test. Thirty-four undergraduate students served as subjects. They participated in two experimental conditions: "stress condition" and "non-stress condition". Main sources of stress were competitive instruction and anxiety caused by unfamiliar experimental setting. Heart rate and state anxiety were measured as the indices of those stressors. Their performance on the MFF test was measured in terms of reaction time and the number of correct responses in each condition. The main results were as follows. Reaction time in stress condition was found significantly shorter than that in non-stress condition. However, there was no significant difference in the number of correct responses between these two conditions. Besides, there was no linear relationship found between either heart rate or state anxiety scores and MFF test performance. These results suggest that attention measured by MFF test differs in its reaction time, depending upon whether stressful instruction exists or not, but it holds constancy in its correct responses throughout the experiment.