Ryusuke Hatae

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.

A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

The prognostic impact of TERT mutations has been controversial in IDH-wild tumors, particularly in glioblastomas (GBM). The controversy may be attributable to presence of potential confounding factors such as MGMT methylation status or patients’ treatment. This study aimed to evaluate the impact of TERT status on patient outcome in association with various factors in a large series of adult diffuse gliomas. We analyzed a total of 951 adult diffuse gliomas from two cohorts (Cohort 1, n = 758; Cohort 2, n = 193) for IDH1/2, 1p/19q, and TERT promoter status. The combined IDH/TERT classification divided Cohort 1 into four molecular groups with distinct outcomes. The overall survival (OS) was the shortest in IDH wild-type/TERT mutated groups, which mostly consisted of GBMs (P < 0.0001). To investigate the association between TERT mutations and MGMT methylation on survival of patients with GBM, samples from a combined cohort of 453 IDH-wild-type GBM cases treated with radiation and temozolomide were analyzed. A multivariate Cox regression model revealed that the interaction between TERT and MGMT was significant for OS (P = 0.0064). Compared with TERT mutant-MGMT unmethylated GBMs, the hazard ratio (HR) for OS incorporating the interaction was the lowest in the TERT mutant-MGMT methylated GBM (HR, 0.266), followed by the TERT wild-type-MGMT methylated (HR, 0.317) and the TERT wild-type-MGMT unmethylated GBMs (HR, 0.542). Thus, patients with TERT mutant-MGMT unmethylated GBM have the poorest prognosis. Our findings suggest that a combination of IDH, TERT, and MGMT refines the classification of grade II-IV diffuse gliomas.

MR Imaging-Based Analysis of Glioblastoma Multiforme: Estimation of IDH1 Mutation Status

BACKGROUND AND PURPOSE: Glioblastoma multiforme is highly aggressive and the most common type of primary malignant brain tumor in adults. Imaging biomarkers may provide prognostic information for patients with this condition. Patients with glioma with isocitrate dehydrogenase 1 (IDH1) mutations have a better clinical outcome than those without such mutations. Our purpose was to investigate whether the IDH1 mutation status in glioblastoma multiforme can be predicted by using MR imaging. MATERIALS AND METHODS: We retrospectively studied 55 patients with glioblastoma multiforme with wild type IDH1 and 11 patients with mutant IDH1. Absolute tumor blood flow and relative tumor blood flow within the enhancing portion of each tumor were measured by using arterial spin-labeling data. In addition, the maximum necrosis area, the percentage of cross-sectional necrosis area inside the enhancing lesions, and the minimum and mean apparent diffusion coefficients were obtained from contrast-enhanced T1-weighted images and diffusion-weighted imaging data. Each of the 6 parameters was compared between patients with wild type IDH1 and mutant IDH1 by using the Mann-Whitney U test. The performance in discriminating between the 2 entities was evaluated by using receiver operating characteristic analysis. RESULTS: Absolute tumor blood flow, relative tumor blood flow, necrosis area, and percentage of cross-sectional necrosis area inside the enhancing lesion were significantly higher in patients with wild type IDH1 than in those with mutant IDH1 (P < .05 each). In contrast, no significant difference was found in the ADCminimum and ADCmean. The area under the curve for absolute tumor blood flow, relative tumor blood flow, percentage of cross-sectional necrosis area inside the enhancing lesion, and necrosis area were 0.850, 0.873, 0.739, and 0.772, respectively. CONCLUSIONS: Tumor blood flow and necrosis area calculated from MR imaging are useful for predicting the IDH1 mutation status.

A comprehensive analysis identifies BRAF hotspot mutations associated with gliomas with peculiar epithelial morphology.

Brain tumors harbor various BRAF alterations, the vast majority of which are the BRAF kinase‐activating V600E mutation. BRAF mutations are most frequently detected in certain subtypes of low‐grade glioma, such as pilocytic astrocytoma (PA), pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNT). However, it is unclear whether gliomas harboring BRAF mutations can be invariably regarded as these glioma subtypes or their derivatives. To address this question, we analyzed 274 gliomas in our institutional case series. We performed high‐resolution melting analyses and subsequent direct Sanger sequencing on DNA isolated from snap‐frozen tumor tissues. As expected, BRAF mutations were detected in the aforementioned low‐grade gliomas: in 4/27 PAs, 2/3 PXAs, 4/8 GGs, and 1/6 DNTs. In addition to these gliomas, 1/2 astroblastomas (ABs) and 2/122 glioblastomas (GBs) harbored BRAF mutations. Pathological investigation of the two GBs revealed that one was a GB displaying epithelial features that presumably arose from a precedent GG, whereas the other GB, which harbored a rare G596 A mutation, showed marked epithelial features, including astroblastic rosettes. Our results indicate that in addition to being present in established BRAF‐associated gliomas, BRAF mutations might be associated with epithelial features in high‐grade gliomas, including sheet‐like arrangement of polygonal tumor cells with a plump cytoplasm and astroblastic rosettes, and thus could potentially serve as a genetic marker for these features.

Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.

High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.

Deferred radiotherapy and upfront procarbazine–ACNU–vincristine administration for 1p19q codeleted oligodendroglial tumors are associated with favorable outcome without compromising patient performance, regardless of WHO grade

Recently updated phase III trials revealed the favorable effect of add-on procarbazine-lomustine-vincristine chemotherapy (CT) to radiotherapy (RT) in treating anaplastic oligodendrogliomas with 1p19q codeletion (codel). However, the underlying rationality of deferring RT and upfront CT administration for these tumors is yet to be elucidated. Here, we retrospectively analyzed the long-term outcome of our case series with oligodendroglial tumors treated with deferred RT and upfront procarbazine+nimustine+vincristine (PAV) in the introduction administration. We enrolled 36 patients with newly diagnosed oligodendroglial tumors (17, grade II and 19, grade III) treated during 1999–2012 and followed up for a median period of 69.0 months. Their clinical and genetic prognostic factors were analyzed, and progression-free survival, overall survival (OS), and deterioration-free survival (DFS) were evaluated. Regardless of the WHO grade, the 25 patients with 1p19q codel tumors never received RT initially, and of these 25, 23 received PAV treatment upfront. The 75% OS of patients with 1p19q codel tumor was 135.3 months (did not reach the median OS), indicating a favorable outcome. Multivariate analysis revealed that IDH mutation and 1p19q, not WHO grade, are independent prognostic factors; furthermore, IDH and 1p19q status stratified the cohort into 3 groups with significantly different OS. The DFS explained the prolonged survival without declining performance in patients with both grade II and III 1p19q codel tumors. Deferred RT and upfront PAV treatment for 1p19q codel oligodendrogliomas were associated with favorable outcomes without compromising performance status, regardless of WHO grade.

Prevalence and clinicopathological features of H3.3 G34-mutant high-grade gliomas: a retrospective study of 411 consecutive glioma cases in a single institution

A recurrent glycine-to-arginine/valine alteration at codon 34 (G34R/V) within H3F3A, a gene that encodes the replication-independent histone variant H3.3, reportedly occurs exclusively in pediatric glioblastomas. However, the clinicopathological and biological significances of this mutation have not been completely elucidated; especially, no such data exist for tumor samples from Japanese patients. We analyzed 411 consecutive glioma cases representing patients of all ages. Our results demonstrated that 14 patients (3.4%) harbored H3F3A mutations, of which four had G34R mutations and 10 had K27M mutations. G34R-mutant tumors were located in the parietal region in two patients and the basal ganglia in one patient. One patient showed multi-lobular extension similar to the pattern observed in gliomatosis cerebri. Regarding neuroradiological features, intratumoral calcification was evident in two cases and all cases showed no or scarce contrast enhancement on MRI. Histopathologically, the four G34R-mutant cases included three glioblastomas and one astroblastoma. We have also investigated alterations in histone methylation including H3K27me3, H3K9me3, and H3K4me3 in G34R-mutant samples by immunohistochemistry. These results indicate that G34R-mutant tumors are likely to show extensive infiltration and alterations in global histone trimethylation might also play an important role in G34R mutant tumors.

Detection of proneural/mesenchymal marker expression in glioblastoma: temporospatial dynamics and association with chromatin-modifying gene expression

Proneural and mesenchymal are two subtypes of glioblastoma identified by gene expression profiling. In this study, the primary aim was to detect markers to develop a clinically applicable method for distinguishing proneural and mesenchymal glioblastoma. The secondary aims were to investigate the temporospatial dynamics of these markers and to explore the association between these markers and the expression of chromatin-modifying genes. One hundred thirty-three glioma samples (grade II: 14 samples, grade III: 18, grade IV: 101) were analyzed. We quantified the expression of 6 signature genes associated with proneural and mesenchymal glioblastoma by quantitative reverse transcription-polymerase chain reaction. We assigned proneural (PN) and mesenchymal (MES) scores based on the average of the 6 markers and calculated a predominant metagene (P-M) score by subtracting the MES from the PN score. We used these scores to analyze correlations with malignant transformation, tumor recurrence, tumor heterogeneity, chromatin-modifying gene expression, and HDAC7 expression. The MES score positively correlated with tumor grade, whereas the PN score did not. The P-M score was able to distinguish the proneural and mesenchymal subtypes. It was decreased in cases of tumor recurrence and malignant transformation and showed variability within a tumor, suggesting intratumoral heterogeneity. The PN score correlated with the expression of multiple histone-modifying genes, whereas the MES score was associated only with HDAC7 expression. Thus, we demonstrated a simple and straightforward method of quantifying proneural/mesenchymal markers in glioblastoma. Of note, HDAC7 expression might be a novel therapeutic target in glioblastoma treatment.

Add-on bevacizumab can prevent early clinical deterioration and prolong survival in newly diagnosed partially resected glioblastoma patients with a poor performance status

Purpose The AVAglio trial established the beneficial effect of add-on bevacizumab (BEV) for the treatment of newly diagnosed glioblastomas (nd-GBMs) that led to the approval of BEV for the treatment of these patients in Japan. However, the rationality of using BEV as a first-line treatment for nd-GBMs remains controversial. The purpose of this study was to analyze the outcomes of a case series of nd-GBM patients. Patients and methods The outcomes of 69 nd-GBM patients treated after 2006 were retrospectively analyzed. Clinical and genetic analyses were performed, and estimates of progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan–Meier method. Since add-on BEV therapy was only used for partially resected GBMs (pr-GBMs) after its approval in 2013, the patients were subdivided into 3 treatment groups: Type I, partial removal with temozolomide (TMZ)/BEV and concurrent radiotherapy (CCRT); Type II, partial removal with TMZ and CCRT; and Type III, gross total removal with TMZ and CCRT. Results The PFS rate of Type I patients was significantly higher than that of Type II patients (P=0.014), but comparable to that of Type III patients. Differences in OS rates between Type I and Type II patients were less apparent (P=0.075), although the median OS of Type I patients was ~8 months higher than that of Type II patients (17.4 vs 9.8 months, respectively). The clinical deterioration rate during initial treatment was significantly (P=0.024) lower in Type I than in Type II patients (7.7% vs 47.4%, respectively). Differences in OS rates between Type I and Type II patients with a poor performance status (PS) were significant (P=0.017). Conclusion Our findings suggest that add-on BEV can prevent early clinical deterioration of pr-GBM patients and contribute to a prolonged survival, especially for those with a poor PS.

A Rare Case of Chiari Type-1 Malformation Accompanied by Symptomatic Cerebrospinal Fluid Hypovolemia: Comparison of Congenital Chiari Type-1 Malformation and Acquired Chiari Malformation Secondary to Cerebrospinal Fluid Hypovolemia: Case Report

A 23-year-old woman was injured in a rear-end collision. She had general malaise and posterior neck pain, which were more severe when she was in an upright position. Magnetic resonance imaging (MRI) revealed the presence of cerebellar tonsil descensus and syringomyelia in the spinal cord. Radioisotope (RI) cisternography showed signs of an early accumulation of RI in the bladder, and a delayed accumulation of RI in the cerebral fornix. We considered the possibilities of cerebrospinal fluid (CSF) hypovolemia and congenital Chiari type-1 malformation as being responsible for her headache. To obtain a definitive diagnosis, we performed gadolinium (Gd)-enhanced MR cisternography and found evidence of CSF leakage. We performed an epidural blood patch (EBP), and her symptoms resolved. In 2 years since the episode, her symptoms have not recurred, and additional treatment has not been required. In addition, MRI performed 2 years after the EBP did not reveal any changes. There seems no previous report which described successful differentiation of pre-existing congenital Chiari type-1 malformation from the acquired one caused by symptomatic CSF hypovolemia. Because treatment protocols differ between these two conditions, the establishment of a correct diagnosis is important.