Quantification of O6-methylguanine-DNA methyltransferase mRNA in human brain tumors.

O6-Methylguanine-DNA methyltransferase (MGMT) is strongly involved in drug resistance mechanism of tumor cells to chloroethylnitrosoureas (CENUs), because it removes and repairs CENU-induced O6-alkylguanine-DNA by accepting the alkyl group at a cysteine moiety. MGMT activity and MGMT mRNA expression are good indicators for detection of sensitive cells or resistant cells to CENUs. In the present study, we applied a non-radioactive reverse transcription-polymerase chain reaction (RT-PCR) method on quantitative measurement of MGMT mRNA expression. Estimated levels of MGMT mRNA expression determined by this RT-PCR method were consistent with the actual doses of MGMT mRNA. This relationship was noted at a wide range from 10 fg to 10 pg. The relative expression levels of MGMT mRNA estimated from kinetic analysis correlated well with MGMT activity determined using 3H-methyl-nitrosourea-treated DNA substrate in brain tumor cells (P<0.001 with a correlation coefficient of 0.997). The RT-PCR method facilitated quantitative measurements in even a small amount of biopsy specimens obtained by stereotactic brain surgery.

Time course of dorsal root axon regeneration into transplants of fetal spinal cord: I. A light microscopic study.

Cut dorsal root axons regenerate into intraspinal transplants of fetal spinal cord and establish synaptic connections there. The aims of the present study were to describe the progression of dorsal root growth within the transplants and the maturation of transplant morphology and to determine whether the regenerated dorsal root axons persist within the transplants or eventually withdraw. Embryonic (E) day 14 spinal cord was grafted into the lumbar enlargement of adult Sprague-Dawley rats, and the L4 or L5 dorsal root was cut and juxtaposed to the transplants. The morphology of the transplants was examined from 1 day to over 1 year after surgery, and the regenerated dorsal roots were labeled with immunohistochemical methods to study the subset that contains calcitonin gene-related peptide (CGRP). Embryonic spinal cord transplants survived and grew within the host spinal cord in over 90% of the animals. Transplant volume increased and the morphology of the transplants matured over the first 12 weeks and then did not change for 48-60 weeks. During the first week the transplants were composed of dissociated neurons, glia, and hematogenous cells with considerable extracellular space between them. Subsequently, the grafted neurons became densely aggregated, and non-neuronal elements such as inflammatory cells and myelin debris disappeared. CGRP-immunoreactive dorsal roots began to regenerate into the transplants within 24 hours, formed dense bundles by 4 days, and were still present at 60 weeks, the longest survival period examined. Myelination of axons within transplants began at 2 weeks. Quantitative analysis showed that the area of the transplants occupied by CGRP-labeled axons and the distribution area of the labeled axons within the transplants increased until 12 weeks and persisted unchanged for over 48 weeks. These results indicate that regenerated dorsal root axons are permanently maintained within transplants of embryonic spinal cord and suggest that the transplants can contribute to the permanent restoration of damaged intraspinal neural circuits.

Long-term positron emission tomography evaluation of slowly progressive gliomas.

Non-invasive positron emission tomography (PET) was performed to identify changes in blood flow and metabolism, specific to early stages of tumour occurrence or recurrence. 2 patients with slowly progressive gliomas from early to late stages of tumour development were analysed by serial PET measurements of circulation and metabolism using 15O-gas and 18F-fluorodeoxyglucose. PET revealed a persistent depression of oxygen metabolism, as indicated by the regional oxygen extraction fraction or metabolic rate of oxygen, in the regions where tumours were later found. Abnormal blood flow and metabolism may precede the morphological changes detected by computed tomography (CT) in patients with gliomas.

Innovative approach in the diagnosis of gliomatosis cerebri using carbon-11-L-methionine positron emission tomography.

A case of gliomatosis cerebri was studied with positron emission tomography (PET). Carbon-11-L-methionine (11C-Met) accumulated in the diffusely infiltrative tumorous area more widely and accurately than the lesion detected by conventional x-ray computerized tomography (CT) or magnetic resonance (MR) imaging. Autopsy findings three months after the time of the PET study showed good anatomical correspondence between the extent of densely aggregated tumor cells and the region with high uptake of 11C-Met. PET may offer an innovative approach in the delineation of gliomatosis cerebri, which has not been clearly recognized by CT or MR.

Measurement of O-6-methylguanine-DNA methyltransferase activity using oligonucleotides and restriction enzyme in human brain tumors.

We determined the levels of MGMT activity in 46 human brain tumors, using oligonucleotides containing O-6-methylguanine at a PvuII recognition site, and correlated MGMT activity with chemotherapeutic effectiveness in 14 patients with high-grade gliomas who received ACNU chemotherapy. The results characterize MGMT activities among different types and within individual types of human brain tumors. A certain fraction of gliomas exhibited a low value of MGMT activity. Tumors showing a low MGMT activity responded well to ACNU and resulted in prolongation in the time to tumor progression. Thus, the low MGMT activity of gliomas may provide a theoretical basis for application of CENU chemotherapy.

Malignant gliomas treated with split course radiotherapy plus chemotherapy.

Twenty-five adult patients with supratentorial malignant gliomas (malignant astrocytoma and glioblastoma multiforme) verified histologically from 1979 to 1986 postoperatively received combined modalities of split course radiotherapy (sRT), nimustine hydrochloride (ACNU), and tegafur (FT). The initial course of radiation consisted of 30 Gy whole brain in 15 fractions five days a week. After a one-week interruption, local radiation with a limited field including a tumor and a margin was boosted to a total dose of about 50-60 Gy. The results were compared with historical control comprising malignant gliomas which received continuous course of radiotherapy (cRT) with or without other chemotherapy before 1979 and after 1986. The median survival time (MST) of malignant gliomas was 18 months for sRT plus chemotherapy, 12 months for cRT alone, and 13 months for cRT plus chemotherapy. No differences between the Kaplan-Meier survival curves were significant by the generalized Wilcoxon test. Tumor histology, Karnofsky performance status, and patient age related well with survival.

Sequential PET studies in neuro-Behçet's syndrome.

A case of neuro-Behcet's syndrome is presented with sequential positron emission tomography (PET) studies. Regional cerebral blood flow (rCBF) and oxygen consumption (rCMRO2) were decreased in the brain lesion; however, on follow-up studies 3 months after steroid therapy rCBF and rCMRO2 had increased in the lesion, which demonstrated the reversibility of this disease. Such monitored improvement may accurately reflect the early stage of the disease and its response to steroid therapy.

Central neurocytoma with clinically malignant behavior.

We describe two cases of central neurocytoma that did not show histopathologic features of anaplasia but did show tumor dissemination after surgery and radiation therapy. CT and MR imaging before surgery depicted extraventricular extension of the tumors. The importance of radiologic findings is stressed.

Superselective angio-CT of brain tumors.

PURPOSE: To determine the efficacy of superselective angio-CT in the diagnosis of astrocytoma. METHODS: Nineteen patients with astrocytoma had superselective angio-CT before chemotherapeutic agents were administered via superselective intraarterial infusion. CT was performed after contrast material was delivered through a microcatheter, which had been advanced into the feeding arteries of the tumor. Superselective angio-CT scans were compared with digital subtraction angiograms, conventional contrast-enhanced CT scans, and contrast-enhanced T1-weighted MR images. RESULTS: Superselective angio-CT scans depicted contrast enhancement of the tumor in all of the patients and the medullary veins of the tumors in 32% of the patients. Digital subtraction angiograms showed tumor stains in 68% of the patients and the medullary veins in only 5%. Conventional CT scans and MR images showed contrast enhancement of the tumor in 89% of the patients. Superselective angio-CT scans confirmed the proper position of the catheter tip for the infusion of a chemotherapeutic agent. CONCLUSIONS: Superselective angio-CT can be used to depict contrast enhancement of tumors and the vascular structures that are characteristic of astrocytomas.

Intramedullary spinal cord germinoma: case report and review of the literature.

We discuss the case of a patient with a recurring intramedullary spinal cord germinoma of the lower thoracic spinal cord, which was successfully excised. A primary intramedullary spinal cord germinoma is very rare, and only four other cases have been reported in the literature. All five cases are reviewed regarding the appearance of the germinomas, their neuroradiological features, and their histopathological findings. We also discuss treatment choices for germinomas of the spinal cord.

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.

Cross-resistance pattern in brain tumour cells resistant to antitumour chloroethylnitrosoureas.

We tested acquired resistance and cross-resistance of brain tumour cells after repeated treatments of antitumour agents including chloroethylnitrosoureas (CENUs) in clinical use for brain tumour chemotherapy. Within ten repeated 2-day incubation periods with either 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) or methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyr anoside (MCNU), brain tumour cells (9L) developed high degrees of resistance to these drugs, as evidenced by about 3- and 6-fold increases, respectively, at the 10% survival dose (SD10). The resistance has unchanged with time after termination of challenging treatments. Repeated challenges with bleomycin (BLM), cis-diaminedichloroplatinum (II) (CDDP), and methotrexate (MTX) did not develop a significant resistance. ACNU-resistant (9L/ACNU) cells showed complete cross-resistance to MCNU, and MCNU-resistant (9L/MCNU) cells to ACNU. Drug sensitivity to other DNA-damaging agents (BLM, NCS, CDDP, etoposide) than CENUs fluctuated to a lesser extent among 9L parent, 9L/ACNU, and 9L/MCNU cells. These data suggest clinical disadvantage of prolonged adjuvant CENU chemotherapy and advantage of combined CENU chemotherapy with other agents than CENUs in brain tumours.

Microspectrofluorometric evaluation of single- and double-stranded DNA in short-term cultured human glioma cells.

Seven cerebral gliomas in short-term culture were studied by microspectrofluorometry and acridine orange staining to assess their nuclear content of single- and double-stranded DNA. Benign gliomas showed a diploid DNA pattern, whereas malignant gliomas revealed a higher frequency of DNA aneuploidy and hyperploidy. The content of single-stranded DNA remained relatively low in benign gliomas; however, that in malignant gliomas varied widely. After ACNU treatment, the double-stranded DNA histograms showed S-phase-specific accumulation in all cases but 1 with increasing concentrations of ACNU. The single-stranded DNA content decreased considerably in 2 cases, which responded well to chemotherapy and showed clinical amelioration.

Positron emission tomographic evaluation of histological malignancy in gliomas using oxygen-15 and fluorine-18-fluorodeoxyglucose.

HEADTOME III, a high resolution PET, has been employed using 15O and 18F labelled pharmaceuticals to evaluate histological malignancy of gliomas preoperatively. PET study was applied on eighteen preoperative gliomas including two recurrent cases. Haemocirculatory and metabolic indices of regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), oxygen extraction fraction (rOEF), cerebral metabolic rates for oxygen (rCMRO2) and glucose (rCMRGI) were measured in the viable portion of the tumour, and the contralateral grey and white matter. In the tumour region, rCBF and rCBV were variable and unrelated to grades of tumour malignancy. rCMRO2 and rOEF values reduced significantly (p less than 0.01) relative to the contralateral brain tissue. The average rCMRGI values was 3.00 +/- 1.06 mg 100 ml-1 min-1 (mean +/- SD) for 7 low grade gliomas (grade II), and 5.91 +/- 3.61 mg 100 ml-1 min-1 for 11 high grade gliomas (grade III and IV). These results would support that anaerobic glycolysis increased in the metabolism of gliomas with malignancy. In comparison with normal volunteers, rCBF, rCMRO2, and rCMRGI values in the contralateral grey matter of gliomas were markedly reduced (p less than 0.01, p less than 0.05, p less than 0.01, respectively) possibly due in part to raised intracranial pressure and depressed cerebral functional activity, so that rOEF was increased to a level of approximately 0.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Indications for differential diagnosis of nontumor central nervous system diseases from tumors. A positron emission tomography study.

To accurately differentiate nontumor central nervous system (CNS) diseases from brain tumors, we retrospectively evaluated the cerebral circulation and metabolism in patients with nontumor CNS diseases using positron emission tomography (PET). Regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), oxygen extraction fraction (rOEF), the metabolic rates of oxygen (rCMRO2), and of glucose (rCMRGI), and the uptake of 11C-methyl-L-methionine (11C-Met) were visually evaluated in lesions and compared with values for the contralateral white matter regions. PET findings were correlated with those of x-ray computed tomography (CT) and magnetic resonance imaging (MRI), and were analyzed for nontumor CNS diseases and cerebral gliomas. rCBF and rCBV were changeable from disease to disease or from stage to stage of disease progression. rOEF and rCMRO2 remained low in 5 and 6, respectively, of 9 nontumor CNS diseases examined, whereas these parameters were increased in CNS infections such as brain abscess. Overall, noteworthy was the locally increased rOEF and rCMRO2 in the patients with a brain abscess in contrast to the values for patients with gliomas. rCMRGI reflected biological characteristics of each disease, and correlated with cell density, whether reactive glial cells or inflammatory cells. 11C-Met was accumulated at a certain stage of nontumor CNS diseases, which implied uptake of the tracer as a result of disruption of the blood-brain barrier as well as metabolic incorporation.

Case report: saccular aneurysm of the intraorbital ophthalmic artery.

A saccular aneurysm of the intraorbital portion of the ophthalmic artery is extremely rare. We report this entity in a 54-year-old man who also had a dural arteriovenous malformation (AVM) at the base of the anterior cranial fossa. The aneurysmal formation at this unusual site may be explained by a haemodynamic stress that derived from the presence of the dural AVM.

Case report: radiation-induced vasculopathy implicated by depressed blood flow and metabolism in a pineal glioma.

A case of radiation-induced vasculopathy of a pineal glioma was presented with haemodynamic and metabolic changes before and after radiotherapy. After radiation of 60 Gy with conventional fractionation (1.8-2.0 Gy daily, 5 days per week), regional blood flow, oxygen extraction fraction, metabolic rate of oxygen, kinetic metabolic rate of glucose and the rate constants (K2, K3) were markedly depressed (20% or greater) compared with the pre-irradiated study. 7 months after radiotherapy, the patient developed transient transient episodes of both right and left upper limb convulsion, terminating in generalized convulsion. When she developed status epilepticus, computed tomography showed extensive low density areas in the territory supplied by the right middle cerebral and the right posterior cerebral arteries. Cerebral angiography revealed diffuse stenosis at both carotid bifurcations and at the origins of the right posterior communicating and posterior cerebral arteries. Haemodynamic and metabolic depression therefore implicated radiation-induced vasculopathy in the present case.

Tumor extent of slowly progressive oligodendroglioma determined by 18F-fluorophenylalanine positron emission tomography.

We report the use of positron emission tomography (PET) and 18F-fluorophenylalanine (18F-Phe) to determine the extent of a slowly progressive oligodendroglioma, which gradually grew over 10 years after the onset of convulsions. The tumor exhibited intense accumulation of 18F-Phe and was easily distinguished from the surrounding brain tissue. The tumor lesion indicated by 18F-phe PET was more extensive than the lesion detected by computerized tomography (CT) and was comparable to the hypointense lesion detected by T2-weighted magnetic resonance imaging (MRI). The tumor, a histologically verified oligodendroglioma, was extensively resected, and its extent corresponded to the high uptake region of 18F-Phe. The accuracy of the 18F-Phe PET technique for determining the extent of a tumor is valuable and will aid in the selection of an appropriate therapy modality for the management of oligodendrogliomas.

Synthesized image processing in clinical neurosurgery.

In an effort to achieve efficient image management in clinical neurosurgery utilizing a local image filing system (EFPACS, Fuji Electric Co., Ltd), adequate image-processing and display techniques were developed. One is a method whereby the anatomical location of the thalamic and basal ganglion lesions is determined by automatically superimposing these lesions defined by magnetic resonance imaging (MRI) directly onto the Schaltenbrand-Wahren's human brain atlas. Horizontal, coronal and sagittal MR images are initially obtained based on the intercommissural line and a perpendicular erected on the midcommissural point as the basic reference coordinates. Precise superimposition is accomplished by the use of these reference axes. This imaging technique may offer the potential for help in anatomical identification of small intracranial lesions. Another technique described in the current communication is automated synthesis of two cerebral angiograms. By simply indicating two reference points, nasion and inion, with a cursor on the display screen, two films are automatically superimposed and displayed as a single synthesized image, featuring two different vascular phases simultaneously by a positive(vein)-negative(artery) mode. This technique was applied to patients with complete occlusion of the middle cerebral artery with or without sufficient blood flow through collateral circulation and was found useful in evaluating basic hemodynamics on a single angiographic image.

Establishment of the two glioma cell lines: YH and AM.

We have established two human glioma cell lines, which were designated as YH cells and AM cells. The two cell lines have maintained morphological appearance as seen in the primary culture and immunohistochemically expressed glial fibrillary acidic protein (GFAP) and S-100 protein. Population doubling time for YH cells and AM cells indicated 30 hours and 25 hours, respectively, in an exponential phase of culture.