Masahiro Mizoguchi

Mutational landscape and clonal architecture in grade II and III gliomas

Grade II and III gliomas are generally slowly progressing brain cancers, many of which eventually transform into more aggressive tumors. Despite recent findings of frequent mutations in IDH1 and other genes, knowledge about their pathogenesis is still incomplete. Here, combining two large sets of high-throughput sequencing data, we delineate the entire picture of genetic alterations and affected pathways in these glioma types, with sensitive detection of driver genes. Grade II and III gliomas comprise three distinct subtypes characterized by discrete sets of mutations and distinct clinical behaviors. Mutations showed significant positive and negative correlations and a chronological hierarchy, as inferred from different allelic burdens among coexisting mutations, suggesting that there is functional interplay between the mutations that drive clonal selection. Extensive serial and multi-regional sampling analyses further supported this finding and also identified a high degree of temporal and spatial heterogeneity generated during tumor expansion and relapse, which is likely shaped by the complex but ordered processes of multiple clonal selection and evolutionary events.

MiRNA-196 Is Upregulated in Glioblastoma But Not in Anaplastic Astrocytoma and Has Prognostic Significance

Purpose: MicroRNAs (miRNA) are short noncoding RNAs that can play critical roles in diverse biological processes. They are implicated in tumorigenesis and function both as tumor suppressors and as oncogenes. The clinical significance of miRNA expression profiles in malignant gliomas remains unclear. Experimental Design: In this study, we examined the expression levels of 365 mature human miRNAs in 12 malignant gliomas, including 8 glioblastomas and 4 anaplastic astrocytomas, using TaqMan real-time quantitative PCR arrays. A validation study was done to corroborate a subset of the results, including expression levels of miR-196a, -196b, -21, and -15b, by analyzing 92 malignant gliomas by conventional real-time PCR. We modeled the relationship between the expression levels of these miRNAs and the survival rate of 39 glioblastoma patients by Kaplan-Meier method and multivariate analysis. Results: Expression profiles in glioblastomas and anaplastic astrocytomas suggested that 16 miRNAs were candidate markers associated with the malignant progression of gliomas. Among them, miR-196a showed the most significant difference (P = 0.0038), with miR-196b also having a high significance (P = 0.0371). Both miRNAs showed increased expression levels in glioblastomas relative to both anaplastic astrocytomas and normal brains in the validation study. Furthermore, patients with high miR-196 expression levels showed significantly poorer survival by the Kaplan-Meier method (P = 0.0073). Multivariate analysis showed that miR-196 expression levels were an independent predictor of overall survival in all 39 glioblastoma patients (P = 0.021; hazard ratio, 2.81). Conclusions: Our results suggest that miR-196 may play a role in the malignant progression of gliomas and may be a prognostic predictor in glioblastomas. Clin Cancer Res; 16(16); 4289–97. ©2010 AACR.

Perfusion Imaging of Brain Tumors Using Arterial Spin-Labeling : Correlation with Histopathologic Vascular Density

BACKGROUND AND PURPOSE: We investigated the relationship between tumor blood-flow measurement based on perfusion imaging by arterial spin-labeling (ASL-PI) and histopathologic findings in brain tumors. MATERIALS AND METHODS: We used ASL-PI to examine 35 patients with brain tumors, including 11 gliomas, 9 meningiomas, 9 schwannomas, 1 diffuse large B-cell lymphoma, 4 hemangioblastomas, and 1 metastatic brain tumor. As an index of tumor perfusion, the relative signal intensity (SI) of each tumor (%Signal intensity) was determined as a percentage of the maximal SI within the tumor per averaged SI within normal cerebral gray matter on ASL-PI. Relative vascular attenuation (%Vessel) was determined as the total microvessel area per the entire tissue area on CD-34–immunostained histopathologic specimens. MIB1 indices of gliomas were also calculated. The differences in %Signal intensity among different histopathologic types and between high- and low-grade gliomas were compared. In addition, the correlations between %Signal intensity and %Vessel or MIB1 index were evaluated in gliomas. RESULTS: Statistically significant differences in %Signal intensity were observed between hemangioblastomas versus gliomas (P < .005), meningiomas (P < .05), and schwannomas (P < .005). Among gliomas, %Signal intensity was significantly higher for high-grade than for low-grade tumors (P < .05). Correlation analyses revealed significant positive correlations between %Signal intensity and %Vessel in 35 patients, including all 6 histopathologic types (rs = 0.782, P < .00005) and in gliomas (rs = 0.773, P < .05). In addition, in gliomas, %Signal intensity and MIB1 index were significantly positively correlated (rs = 0.700, P < .05). CONCLUSION: ASL-PI may predict histopathologic vascular densities of brain tumors and may be useful in distinguishing between high- and low-grade gliomas and in differentiating hemangioblastomas from other brain tumors.

Amide proton transfer imaging of adult diffuse gliomas: correlation with histopathological grades.

BACKGROUND Amide proton transfer (APT) imaging is a novel molecular MRI technique to detect endogenous mobile proteins and peptides through chemical exchange saturation transfer. We prospectively assessed the usefulness of APT imaging in predicting the histological grade of adult diffuse gliomas. METHODS Thirty-six consecutive patients with histopathologically proven diffuse glioma (48.1 ± 14.7 y old, 16 males and 20 females) were included in the study. APT MRI was conducted on a 3T clinical scanner and was obtained with 2 s saturation at 25 saturation frequency offsets ω = -6 to +6 ppm (step 0.5 ppm). δB0 maps were acquired separately for a point-by-point δB0 correction. APT signal intensity (SI) was defined as magnetization transfer asymmetry at 3.5 ppm: magnetization transfer ratio (MTR)asym = (S[-3.5 ppm] - S[+3.5 ppm])/S0. Regions of interest were carefully placed by 2 neuroradiologists in solid parts within brain tumors. The APT SI was compared with World Health Organization grade, Ki-67 labeling index (LI), and cell density. RESULTS The mean APT SI values were 2.1 ± 0.4% in grade II gliomas (n = 8), 3.2 ± 0.9% in grade III gliomas (n = 10), and 4.1 ± 1.0% in grade IV gliomas (n = 18). Significant differences in APT intensity were observed between grades II and III (P < .05) and grades III and IV (P < .05), as well as between grades II and IV (P < .001). There were positive correlations between APT SI and Ki-67 LI (P = .01, R = 0.43) and between APT SI and cell density (P < .05, R = 0.38). The gliomas with microscopic necrosis showed higher APT SI than those without necrosis (P < .001). CONCLUSIONS APT imaging can predict the histopathological grades of adult diffuse gliomas.

Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell‐signaling pathways. Nox‐family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real‐time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell‐growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells.

Expression of neurofibromatosis 2 protein in human brain tumors: An immunohistochemical study

Abstract The neurofibromatosis 2 (NF2) gene-encoded protein, named merlin, may function as a molecular linkage connecting cytoskeleton and plasma membrane. Merlin is thought to play a crucial role as a tumor suppressor not only in hereditary NF2-related tumors, but also in sporadic tumors such as schwannomas, meningiomas and gliomas. Using a merlin-expression vector system, we raised specific antiserum against merlin. We observed the intracellular distribution of merlin in cultured glioma cells, and further investigated merlin expression in 116 human brain tumors. Immunofluorescence microscopy revealed that merlin was localized beneath the cell membrane and concentrated at cell-to-cell adhesion sites, where actin filaments are densely associated with plasma membrane. By immunohistochemistry, none of the schwannomas from either NF2 patients or sporadic cases showed any immunoreactivity, while normal Schwann cells of cranial nerves were immunopositive. In meningiomas, merlin expression was frequently seen in the meningothelial subtype (8/10, 80%), but no expression could be detected in either the fibrous or the transitional variant. Most normal astrocytes were negative; however, reactive astrocytes often expressed merlin. Glioblastomas and anaplastic astrocytomas were found to be strongly positive, and focal positive staining was observed in fibrillary and pilocytic astrocytomas. Thus, the loss of merlin appears to be integral to schwannoma formation and the differential pathogenesis of meningioma subtypes. However, merlin alterations do not appear to play a critical role in either the tumorigenesis or malignant transformation of neoplastic astrocytes.

Associations between microRNA expression and mesenchymal marker gene expression in glioblastoma

The subclassification of glioblastoma (GBM) into clinically relevant subtypes using microRNA (miRNA)- and messenger RNA (mRNA)-based integrated analysis has been attempted. Because miRNAs regulate multiple gene-signaling pathways, understanding miRNA-mRNA interactions is a prerequisite for understanding glioma biology. However, such associations have not been thoroughly examined using high-throughput integrated analysis. To identify significant miRNA-mRNA correlations, we selected and quantified signature miRNAs and mRNAs in 82 gliomas (grade II: 14, III: 16, IV: 52) using real-time reverse-transcriptase polymerase chain reaction. Quantitative expression data were integrated into a single analysis platform that evaluated the expression relationship between miRNAs and mRNAs. The 21 miRNAs include miR-15b, -21, -34a, -105, -124a, -128a, -135b, -184, -196a-b, -200a-c, -203, -302a-d, -363, -367, and -504. In addition, we examined 23 genes, including proneural markers (DLL3, BCAN, and OLIG2), mesenchymal markers (YKL-40, CD44, and Vimentin), cancer stem cell-related markers, and receptor tyrosine kinase genes. Primary GBM was characterized exclusively by upregulation of mesenchymal markers, whereas secondary GBM was characterized by significant downregulation of mesenchymal markers, miR-21, and -34a, and by upregulation of proneural markers and miR-504. Statistical analysis showed that expression of miR-128a, -504, -124a, and -184 each negatively correlated with the expression of mesenchymal markers in GBM. Our functional analysis of miR-128a and -504 as inhibitors demonstrated that suppression of miR-128a and -504 increased the expression of mesenchymal markers in glioblastoma cell lines. Mesenchymal signaling in GBM may be negatively regulated by miR-128a and -504.

Results of a long-term follow-up after neuroendoscopic biopsy procedure and third ventriculostomy in patients with intracranial germinomas

OBJECT The authors report the results of long-term follow-ups in 12 patients with intracranial germinomas who underwent neuroendoscopic procedures before chemotherapy and radiotherapy, and discuss the usefulness and safety of these procedures. METHODS Between January 1996 and December 2005 at Kyushu University Hospital, 12 patients with intracranial germinomas underwent neuroendoscopic biopsy procedures involving a flexible fiberscope. Eight patients simultaneously underwent endoscopic third ventriculostomy (ETV) for existing obstructive hydrocephalus. All patients received chemotherapy and radiotherapy postoperatively, according to the regimen promulgated by the Japanese Pediatric Brain Tumor Study Group. The patients were followed for an average of 78.6 months (range 15-134 months), and a retrospective study was conducted. RESULTS Germinomas were histologically verified in all patients. No postoperative deaths or permanent morbidity was related to the neuroendoscopic procedures. No other cerebrospinal fluid diversion, such as that achieved with a ventriculoperitoneal shunt, was needed for the management of hydrocephalus. A complete response to postoperative chemotherapy and radiotherapy was achieved in all cases. Only one patient had a recurrent lesion in the spinal cord 6 years after the initial treatment; however, this patient had undergone only the neuroendoscopic biopsy procedure without ETV. CONCLUSIONS Neuroendoscopic procedures can permit a precise histological diagnosis of intracranial germinomas and are safe and effective in the management of hydrocephalus associated with these tumors. The risk of tumor dissemination due to the neuroendoscopic procedures appears to be minimal when the appropriate chemotherapy and radiotherapy are provided postoperatively.

Clinical implications of microRNAs in human glioblastoma

Glioblastoma (GBM) is one of the most common and dismal brain tumors in adults. Further elucidation of the molecular pathogenesis of GBM is mandatory to improve the overall survival of patients. A novel small non-coding RNA molecule, microRNA (miRNA), appears to represent one of the most attractive target molecules contributing to the pathogenesis of various types of tumors. Recent global analyses have revealed that several miRNAs are clinically implicated in GBM, with some reports indicating the association of miRNA dysregulation with acquired temozolomide (TMZ) resistance. More recent studies have revealed that miRNAs could play a role in cancer stem cell (CSC) properties, contributing to treatment resistance. In addition, greater impact might be expected from miRNA-targeted therapies based on tumor-derived exosomes that contain numerous functional miRNAs, which could be transferred between tumor cells and surrounding structures. Tumor-derived miRNAs are now considered to be a novel molecular mechanism promoting the progression of GBM. Establishment of miRNA-targeted therapies based on miRNA dysregulation of CSCs could provide effective therapeutic strategies for TMZ-resistant GBM. Recent progress has revealed that miRNAs are not only putative biological markers for diagnosis, but also one of the most promising targets for GBM treatment. Here in, we summarize the translational aspects of miRNAs in the diagnosis and treatment of GBM.

Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ) is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma (GBM). Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been described as the main modulator to determine the sensitivity of GBM to TMZ, a subset of GBM does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR), and the base excision repair (BER) pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break repair and double-strand break repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.