Masazumi Fujii

Human Gene Therapy for Malignant Gliomas (Glioblastoma Multiforme and Anaplastic Astrocytoma) by In Vivo Transduction with Human Interferon β Gene Using Cationic Liposomes

Transfer of interferon beta gene via cationic liposomes has been found to induce regression of experimental glioma. We performed a pilot clinical trial of safety and effectiveness of this interferon beta gene therapy in five patients with malignant glioma (glioblastoma multiforme or anaplastic astrocytoma). Transgene expression and antitumor activity were detected in four patients. Two patients showed a partial response (>50% tumor reduction) and two others had stable disease 10 weeks after beginning therapy. One patient could not be evaluated because of previous treatment with gamma-knife therapy. This study suggests the feasibility and safety of interferon beta gene therapy, which may become an important treatment option for patients with malignant glioma.

Human neural stem cells target and deliver therapeutic gene to experimental leptomeningeal medulloblastoma

Medulloblastomas are highly malignant neuroectodermal cerebellar tumors of children. One of the reasons for the difficulty for the treatment of medulloblastomas is their inherent tendency to metastasize through the cerebrospinal fluid (CSF) pathway leading to leptomeningeal dissemination. Recently, genetically modified neural stem cells (NSCs) were shown to have the capability of selectively migrating into glioma mass and delivering therapeutic agents with significant therapeutic benefits. In the present study, we applied the NSC strategy to target medulloblastomas, particularly their leptomeningeal dissemination. We used NSCs that were retrovirally transduced with the cytosine deaminase gene (CD-NSCs). In vitro studies demonstrated that CD-NSCs had sufficient migratory activity toward medulloblastoma cells and exerted a remarkable bystander effect on these cells following the application of 5-fluorocytosine (5-FC). It is noteworthy that neutralization of the hepatocyte growth factor blocked their migration In animal studies using our leptomeningeal dissemination model, CD-NSCs implanted directly into CSF space were shown to distribute diffusely within the disseminated tumor cells and could provide remarkable antitumor effect after intraperitoneal administration of 5-FC. Furthermore, CD-NSC treatment followed by 5-FC administration prolonged survival periods significantly in experimental animals. Our data suggest that the CD-NSC strategy can also be applied to target leptomeningeal dissemination of medulloblastomas.

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O 6-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

Intraoperative Tractography and Motor Evoked Potential (MEP) Monitoring in Surgery for Gliomas Around the Corticospinal Tract

BACKGROUND Our goal is to indicate the importance of combining intraoperative tractography with motor-evoked potential (MEP) monitoring for glioma surgery in motor eloquent areas. METHODS Tumor removal was performed in 28 patients with gliomas in and around the corticospinal tract (CST), in an operation theater equipped with an integrated high-field intraoperative magnetic resonance imaging and a neuronavigation system. Diffusion-tensor imaging-based tractography of the CST was implemented preoperatively and intraoperatively. When the surgically manipulated area came close to the corticospinal pathway, MEP responses were elicited by subcortical stimulation. Responsive areas were compared with the locations of fibers traced by preoperative and intraoperative tractography. Imaging and functional outcomes were reviewed. RESULTS Intraoperative tractography demonstrated significant inward or outward shift during surgery. MEP responses were observed around the tract at various intensities, and the distance between MEP responsive sites and intraoperative tractography was significantly correlated with the stimulation intensity (P < 0.01). The distance from preoperative tractography was not correlated. A more than subtotal resection was achieved in 24 patients (85.7%). Transient motor deterioration was seen in 12 patients (42.8%), and a permanent deficit was seen in 1 patient (3.5%). CONCLUSIONS We found that intraoperative tractography demonstrated the location of the CST more accurately than preoperative tractography. The results of the linear regression between distance and stimulation intensity were informative for guiding approaches to tumor remnants without impinging on the CST. The combination of intraoperative tractography and MEP monitoring can enhance the quality of surgery for gliomas in motor eloquent areas.

A combination of IFN-β and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation

PurposeMethylation of the O6-methyguanine-DNA methyltransferase (MGMT) gene promoter in gliomas has been reported to be a useful predictor of the responsiveness to temozolomide (TMZ). In our previous experiments, we observed that IFN-β sensitized TMZ-resistant glioma cells with the unmethylated MGMT promoter and that the mechanism of action was possibly due to attenuation of MGMT expression via induction of TP53. In this study, (1) we explored the synergistic effect of IFN-β and TMZ in the animal model, and (2) clarified the role of IFN-β induced TP53 in the human MGMT promoter.Methods(1) Nude mice with either subcutaneous T98 (TMZ-resistant) or U251SP (TMZ-sensitive) tumor were treated with IFN-β/TMZ for 5 consecutive days. (2) The MGMT promoter activity was assayed by a luciferase reporter system in Saos2 (p53-null) cells transduced with a p53-adenoviral vector, and T98 glioma cells treated with IFN-β.Results(1) A combination of IFN-β/TMZ had significant synergistic antitumor activity on the growth of both T98 and U251SP tumors. (2) MGMT promoter activity was suppressed by either adenovirally transduced p53 or IFN-β.ConclusionsIt would be appealing to consider a prospective clinical trial in which genetic markers are used for personalized drug selection, eliciting other forms of treatment or inhibition of MGMT for those with MGMT expression. In this context, IFN-β inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ.

A phase I clinical trial of interferon-beta gene therapy for high-grade glioma: novel findings from gene expression profiling and autopsy.

High‐grade gliomas are highly lethal neoplasms representing approximately 20% of all intracranial tumors. Cationic liposome‐mediated interferon‐beta (IFN‐β) gene transfer has been found to induce regression of experimental glioma. We have previously performed a pilot clinical trial to evaluate the safety and effectiveness of this IFN‐β gene therapy in five patients with high‐grade glioma. Two patients showed more than 50% reduction while others had stable disease 10 weeks after treatment initiation.

P16 PROMOTER METHYLATION IN THE SERUM AS A BASIS FOR THE MOLECULAR DIAGNOSIS OF GLIOMAS

OBJECTIVEDeoxyribonucleic acid (DNA) methylation of tumor origin can be detected in the serum/plasma of cancer patients. The aim of this study was to detect aberrant p16 promoter methylation as a potential diagnostic marker in the serum of patients with diffuse glioma to differentiate between gliomas and, particularly, to differentiate those in the brainstem from others; this was done by using the modified methylation-specific polymerase chain reaction technique. METHODSThe methylation-specific polymerase chain reaction was used to detect p16 methylation in the DNA extracted from 20 astrocytic tumors and 20 oligodendroglial tumors and the corresponding serum samples. Serum samples from 10 healthy individuals were used as controls. The association of p16 hypermethylation in the serum DNA of glioma patients with clinicopathological characteristics was analyzed. In addition, the serum DNA in 7 patients with a brainstem tumor (4 gliomas, 1 schwannoma, 1 cavernous angioma, and 1 ependymoma) was analyzed. RESULTSWe found p16 methylation in 12 (60%) of the 20 tissues with astrocytoma, but in only 1 of the tissues with oligodendroglioma. Similar methylations were detected in the serum of 9 (75%) of the 12 patients with aberrant methylation in the tumor tissues. No methylated p16 sequences were detected in the peripheral serum of the patients having tumors without these methylation changes or in the 10 healthy controls. Additionally, p16 promoter methylation in the serum was observed in all brainstem astrocytoma cases, but not in other cases. CONCLUSIONThis assay has potential for use as a serum-based molecular diagnosis technique for diffuse glioma.

Intraoperative subcortical mapping of a language-associated deep frontal tract connecting the superior frontal gyrus to Broca's area in the dominant hemisphere of patients with glioma

OBJECT The deep frontal pathway connecting the superior frontal gyrus to Brocas area, recently named the frontal aslant tract (FAT), is assumed to be associated with language functions, especially speech initiation and spontaneity. Injury to the deep frontal lobe is known to cause aphasia that mimics the aphasia caused by damage to the supplementary motor area. Although fiber dissection and tractography have revealed the existence of the tract, little is known about its function. The aim of this study was to determine the function of the FAT via electrical stimulation in patients with glioma who underwent awake surgery. METHODS The authors analyzed the data from subcortical mapping with electrical stimulation in 5 consecutive cases (3 males and 2 females, age range 40-54 years) with gliomas in the left frontal lobe. Diffusion tensor imaging (DTI) and tractography of the FAT were performed in all cases. A navigation system and intraoperative MRI were used in all cases. During the awake phase of the surgery, cortical mapping was performed to find the precentral gyrus and Brocas area, followed by tumor resection. After the cortical layer was removed, subcortical mapping was performed to assess language-associated fibers in the white matter. RESULTS In all 5 cases, positive responses were obtained at the stimulation sites in the subcortical area adjacent to the FAT, which was visualized by the navigation system. Speech arrest was observed in 4 cases, and remarkably slow speech and conversation was observed in 1 case. The location of these sites was also determined on intraoperative MR images and estimated on preoperative MR images with DTI tractography, confirming the spatial relationships among the stimulation sites and white matter tracts. Tumor removal was successfully performed without damage to this tract, and language function did not deteriorate in any of the cases postoperatively. CONCLUSIONS The authors identified the left FAT and confirmed that it was associated with language functions. This tract should be recognized by clinicians to preserve language function during brain tumor surgery, especially for tumors located in the deep frontal lobe on the language-dominant side.

Evaluation of Resting State Networks in Patients with Gliomas: Connectivity Changes in the Unaffected Side and Its Relation to Cognitive Function

In this study, we investigated changes in resting state networks (RSNs) in patients with gliomas located in the left hemisphere and its relation to cognitive function. We hypothesized that long distance connection, especially between hemispheres, would be affected by the presence of the tumor. We further hypothesized that these changes would correlate with, or reflect cognitive changes observed in patients with gliomas. Resting state functional MRI datasets from 12 patients and 12 healthy controls were used in the analysis. The tumor’s effect on three well-known RSNs including the default mode network (DMN), executive control network (ECN), and salience network (SN) identified using independent component analysis were investigated using dual regression analysis. Scores of neuropsychometric testing (WAIS-III and WMS-R) were also compared. Compared to the healthy control group, the patient group showed significant decrease in functional connectivity in the right angular gyrus/inferior parietal lobe of the ventral DMN and in the dorsolateral prefrontal cortex of the left ECN, whereas a significant increase in connectivity in the right ECN was observed in the right parietal lobe. Changes in connectivity in the right ECN correlated with spatial memory, while that on the left ECN correlated with attention. Connectivity changes in the ventral DMN correlated with attention, working memory, full IQ, and verbal IQ measures. Although the tumors were localized in the left side of the brain, changes in connectivity were observed in the contralateral side. Moreover, these changes correlated with some aspects of cognitive function indicating that patients with gliomas may undergo cognitive changes even in the absence of or before the onset of major symptoms. Evaluation of resting state networks could be helpful in advancing our hodological understanding of brain function in glioma cases.

Neurosurgical robotic system for brain tumor removal

PurposeBrain tumor (e.g., glioma) resection surgery, representing the first step for many treatments, is often difficult and time-consuming for neurosurgeons. Thus, intelligent neurosurgical instruments have been developed to improve tumor removal.MethodsThe concept and robotic structure of intelligent neurosurgical instruments were introduced. These instruments consist of a surgical robot, a master device and operating software. The robot incorporates a surgical motion base and tool manipulator, including a volume control suction tool. Open Core Control software was developed for connecting intelligent neurosurgical instruments through a network connection and integrating the instruments into a system.ResultsMechanical evaluation tests on the components and a preliminary system evaluation were performed. A phantom model was fixed on a head frame, and a tumor-removal procedure was successfully performed using prototype intelligent neurosurgical instruments.ConclusionIntelligent neurosurgical instruments are feasible and suitable for on-going evaluation in practical tasks, including in-vivo animal testing.