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OBJECTIVE An autologous formalin-fixed tumor vaccine (AFTV) derived from resected glioblastoma (GBM) tissue can be used against unidentified tumor antigens. Thus, the authors conducted a multicenter double-blind phase IIb trial to investigate the efficacy of an AFTV. METHODS Eligible patients were adults with supratentorial GBMs, 16–75 years of age, with Karnofsky Performance Scale (KPS) scores ≥ 60%, and no long-term steroid administration. An AFTV comprising fixed paraffin-embedded tumor tissue with immune adjuvants or an identical placebo without fixed tumor tissue was injected intradermally over three courses before and after chemoradiotherapy. The primary and secondary end points were overall survival (OS), progression-free survival (PFS), and 3-year survival rate. RESULTS Sixty-three patients were enrolled. The average patient age was 61 years. The median KPS score was 80%, and the median resection rate was 95%. The full analysis set of 57 patients indicated no significant difference in OS (p = 0.64) for the AFTV group (median OS 25.6 months, 3-year OS rate 38%) compared with the placebo group (31.5 months and 41%, respectively) and no difference in PFS (median PFS 13.3 months in both groups, p = 0.98). For patients with imaging-based total tumor removal, the 3-year PFS rate was 81% in the AFTV group versus 46% in the placebo group (p = 0.067), whereas the 3-year OS rate was 80% versus 54% (p = 0.16), respectively. Similar results were obtained in the p53-negative subgroups. Severe adverse effects were not observed. CONCLUSIONS The AFTV may have potential effects in certain patient subgroups. A phase III study for patients with total tumor removal remains warranted to confirm these findings. Clinical trial registration no.: UMIN000010602 (UMIN Clinical Trials Registry)

Supplementary Figure 5 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Recent findings have shown that malignant tumors contain cancer-initiating cells (CIC), which self-renew and are tumorigenic. However, CICs have not been characterized properly due to lack of specific markers. We recently established a mouse glioma cell line, NSCL61, by overexpressing an oncogenic HRasL61 in p53-deficient neural stem cells. Using limiting dilution assays, we show that only 2 of 24 NSCL61 clones retained their tumorigenicity in vivo, although the others also expressed oncogenic HRasL61 and could proliferate in culture. A comparison of the gene expression profiles of tumorigenic and nontumorigenic clones showed that the tumorigenic clones had lost Sox11 expression. We show that overexpression of sox11 prevented tumorigenesis of NSCL61s by inducing their neuronal differentiation accompanied with decreased levels of plagl1. We also show that overexpression of plagl1 abolished neuronal commitment of nontumorigenic cells and induced them to become tumorigenic. Moreover, we show that human glioma-initiating cells lost sox11 expression, and overexpression of sox11 prevented their tumorigenesis in vivo. Together with the clinical evidence showing that downregulation of sox11 mRNA correlates with a significant decrease in survival, these findings suggest that Sox11 prevents gliomagenesis by blocking the expression of oncogenic plagl1. [Cancer Res 2009;69(20):7953–9]

Supplementary Figure 3 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figure 4 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figure 8 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figure 2 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figure 6 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figures 1-5, Table 1 from NKX2.2 Suppresses Self-Renewal of Glioma-Initiating Cells

Supplementary Figure 1 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Methods, Figure Legends 1-8 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Supplementary Figure 7 from Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16Ink4a/p19Arf led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches. Cancer Res; 71(3); 1135–45. ©2010 AACR.

Supplementary Material and Methods

Supplementary Table S1-S4

Isocitrate dehydrogenase-mutant low-grade gliomas (IDHmut-LGG) grow slowly but frequently undergo malignant transformation, which eventually leads to premature death. Chemotherapy and radiotherapy treatments prolong survival, but can also induce genetic (or epigenetic) alterations involved in transformation. Here, we developed a mathematical model of tumor progression based on serial tumor volume data and treatment history of 276 IDHmut-LGGs classified by chromosome 1p/19q codeletion (IDHmut/1p19qcodel and IDHmut/1p19qnoncodel) and performed genome-wide mutational analyses, including targeted sequencing and longitudinal whole-exome sequencing data. These analyses showed that tumor mutational burden correlated positively with malignant transformation rate, and chemotherapy and radiotherapy significantly suppressed tumor growth but increased malignant transformation rate per cell by 1.8 to 2.8 times compared with before treatment. This model revealed that prompt adjuvant chemoradiotherapy prolonged malignant transformation-free survival in small IDHmut-LGGs (≤ 50 cm3). Furthermore, optimal treatment differed according to genetic alterations for large IDHmut-LGGs (> 50 cm3); adjuvant therapies delayed malignant transformation in IDHmut/1p19qnoncodel but often accelerated it in IDHmut/1p19qcodel. Notably, PI3K mutation was not associated with malignant transformation but increased net postoperative proliferation rate and decreased malignant transformation-free survival, prompting the need for adjuvant therapy in IDHmut/1p19qcodel. Overall, this model uncovered therapeutic strategies that could prevent malignant transformation and, consequently, improve overall survival in patients with IDHmut-LGGs.Significance:A mathematical model successfully estimates malignant transformation-free survival and reveals a link between genetic alterations and progression, identifying precision medicine approaches for optimal treatment of IDH-mutant low-grade gliomas.

Supplementary Figures S1-S8

Purpose Pineal parenchymal tumors of intermediate differentiation (PPTIDs), which were recognized in the 2007 World Health Organization (WHO) classification, are rare, accounting for less than 1% of all central nervous system tumors. This rarity and novelty complicate the diagnosis and treatments of PPTID. We therefore aimed to evaluate the clinicopathological significance of this tumor. Methods At 11 institutions participating in the Kyushu Neuro-Oncology Study Group, data for patients diagnosed with PPTID were collected. Central pathology review and KBTBD4 mutation analysis were applied to attain the diagnostically accurate cohort. Results PPTID was officially diagnosed in 28 patients: 11 (39%) with WHO grade 2 and 17 (61%) with WHO grade 3 tumors. Median age was 49 years, and the male:female ratio was 1:2.1. Symptom was headache in 50% (14/28). Surgery was attempted in all 28 patients, and gross total resection (GTR) was achieved in 46% (13/28). Adjuvant radiotherapy and chemotherapy were administered to, respectively, 82% (23/28) and 46% (13/28). The 5-year progression-free and overall survival rates were 64.9% and 70.4% respectively. Female sex (p = 0.02) and GTR (p < 0.01) were found to be independent prognostic factors. Initial and second recurrences were most often leptomeningeal (67% and 100% respectively). 80% (20/25) of patients harbored a KBTBD4 mutation. Conclusions Female sex and GTR were independent prognostic factors in our patients with PPTID. Leptomeningeal recurrence was observed to be particularly characteristic of this tumor. The rate of KBTBD4 mutation observed in our cohort was acceptable and this could prove the accuracy of our PPTID cohort.

We evaluated the efficacy and safety of bevacizumab beyond progression (BBP) in Japanese patients with newly diagnosed glioblastoma and explored predictors of response to bevacizumab. This phase II study evaluated a protocol-defined primary therapy by radiotherapy with concurrent and adjuvant temozolomide plus bevacizumab, followed by bevacizumab monotherapy, and secondary therapy (BBP: bevacizumab upon progression). Ninety patients received the protocol-defined primary therapy (BBP group, n = 25). Median overall survival (mOS) and median progression-free survival (mPFS) were 25.0 and 14.9 months, respectively. In the BBP group, in which O6-methylguanine-DNA methyltransferase (MGMT)-unmethylated tumors predominated, mOS and mPFS were 5.8 and 1.9 months from BBP initiation and 16.8 and 11.4 months from the initial diagnosis, respectively. The primary endpoint, the 2-year survival rate of the BBP group, was 27.0% and was unmet. No unexpected adverse events occurred. Expression profiling using RNA sequencing identified that Cluster 2, which was enriched with the genes involved in macrophage or microglia activation, was associated with longer OS and PFS independent of the MGMT methylation status. Cluster 2 was identified as a significantly favorable independent predictor for PFS, along with younger age and methylated MGMT. The novel expression classifier may predict the prognosis of glioblastoma patients treated with bevacizumab.

Resumen: Este artículo presenta la construcción y validación de la dimensión “entorno de enseñanza” como concepto clave en este trabajo, que hace referencia a la formación del estudiantado y las interrelaciones que se establecen entre este y el profesorado. Para su análisis semántico, el instrumento fue examinado por siete expertos. Para el análisis de consistencia se aplicó una prueba preliminar a 92 estudiantes de la Universitat de València (España) y a 37 de la Universidad Federal de Paraíba (Brasil). En el análisis semántico, el instrumento fue adecuado en términos de coherencia y validez para obtener informaciones sobre el entorno de enseñanza. En el análisis de consistencia, se constató un grado de contradicción G2 cercano a 0 (0.07). El grado de certeza G1 (0.75) indica que hay concordancia entre las proposiciones, como elemento importante para estudios sobre la permanencia en la educación superior.

Introduction Chromosomal instability, the cell condition in which chromosome mis-segregation occur at a high frequency during cell division, has been considered to be involved in the molecular mechanisms that give rise to the complex genetic background of glioma. However, most of this phenomenon has been based on researches using cell line, and there have been few studies of chromosomal instability in clinical specimens of gliomas. Methods Primary cell culture was obtained from 11 glioma specimens (eight Glioblastoma (GBM), one Anaplastic PXA (aPXA), one Astrocytoma, and one Ependymoma), which was removed at our hospital, and chromosomes of up to five cells per case were analyzed by the SKY method. Chromosome instability was quantified by two parameters, one was AS (Aneuploidy score), which means the number of gain or loss of whole of the chromosome, and the other was SS: Structural abnormality score, which means the number of chromosome structural abnormality per cell. In addition, the phenotypes, which were characteristic of chromosomal instability were observed individually. Results Each quantitative value was as follows: GBM; AS:2.30±0.51 /SS:1.64±0.38, aPXA; AS :1.40±1.33 / SS:8.20±0.99, Astrocytoma and Ependymoma; AS:0.00 /SS:0.00, suggesting that chromosomal instability was associated with GBM and aPXA. Chromosome 7 amplification was most frequent in GBM (57%), and Mosaic loss of chromosome Y was also observed in 60% of males. Some characteristic karyotypes which suggest the phenomenon of Chromothripsis or Double minute were also observed. The karyotype concordance rate in the cases with p53 mutation was 60%, and that with p53 wild type was 100%, indicating that the p53 mutation increased the genotype heterogeneity in the same specimen. Discussion In clinical specimens of gliomas, aneuploidy and structural abnormalities were identified in GBM and aPXA, suggesting that chromosomal instability contributes to their cellular phenotype and malignancy.

BackgroundPediatric neoplastic diseases account for about 10% in fever of unknown origin (FUO), and most neoplastic diseases cases are leukemia, lymphoma, and neuroblastoma. Brain tumors are rarely reported as the cause of FUO, although craniopharyngioma, metastatic brain tumor, and Castleman's disease have been reported. We report a case of intracranial mesenchymal tumor (IMTs) with FET-CREB fusion gene, whose had inflammatory phenotype without neurological signs.Case presentationA 10-year-old girl was admitted with 2 months history of intermittent fever and headache, whereas her past history as well as her family history lacked special events. Sepsis work-up showed no pathological organism and empirical antibiotics therapy was not effective. Bone marrow examination showed a negative result. CSF examination showed elevated protein as well as cell counts and head MRI showed a hyper vascular mass lesion with contrast enhancement in the left cerebellar hemisphere. The patient underwent tumor excision, which made the intermittent fever disappear. Pathological examinations resemble those of classic angiomatoid fibrous histiocytoma (AFH) but the morphologic features are distinct from the AFH myxoid variant, then we performed break-apart fluorescence in situ hybridization (FISH) and confirmed the tumor harbored rare EWSR1-CREM fusion gene (the Ewing sarcoma breakpoint region 1 gene (EWSR1), cAMP response element binding (CREB) family gene). Consequently, we diagnose as IMT with EWSR1-CREM fusion. Elevated serum concentration of IL-6 was normalized after tumor resection, which suggested the fever could be caused by tumor-derived IL-6.ConclusionsThis is the first case of IMT with EWSR1-CREM fusion that showed paraneoplastic symptoms associated with the IL-6/STAT3 signaling pathway. Although brain tumors are rarely diagnosed as a responsible disease for FUO, they should be considered as a cause of unknown fever even in the absence of abnormal neurological findings. (285 words)