Secondary Glioblastomas Research Articles

Quantitative Proteomics Reveals Fundamental Regulatory Differences in Oncogenic HRAS and Isocitrate Dehydrogenase (IDH1) Driven Astrocytoma

Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.

Rapid Progression of Gliomatosis Cerebri to Secondary Glioblastoma, Factors That Affect the Progression Rate: A Case Report

Rapid Progression of Gliomatosis Cerebri to Secondary Glioblastoma, Factors That Affect the Progression Rate: A Case Report

The role of IDH1/2 mutations in the pathogenesis of secondary glioblastomas

ABSTRACT Diffuse astrocytoma and glioblastoma (GBM) constitute a group of diffusely infiltrating astrocytic neoplasms, which more commonly arise in cerebral hemispheres of adults. The prevalence of malignant transformation of diffuse astrocytoma to anaplastic astrocytoma and GBM varies from 40% to 75% of cases in different series. Distinct genetic abnormalities are related to neoplastic progression, and the cells prone to glial neoplasm development include progenitor cells, stem cells, or differentiated cells. Primary GBM arises typically de novo, with no previous history of a lower-grade precursor lesion. Secondary GBM evolves from low-grade astrocytoma, is predominant in younger patients, and frequently exhibits isocitrate dehydrogenase (IDH) 1 and 2 mutations. IDH1/2 mutated gliomas have been associated with a better prognosis when compared to IDH-wildtype gliomas. IDH mutations are rare in primary GBM and gliomas arising in children. Evidence suggests that IDH mutations lead to a hypermethylation phenotype and represent early events in tumoral transformation of the central nervous system (CNS) due to the production of the oncometabolite 2-hydroxyglutarate. IDH1 mutations precede tumor protein p53 (TP53) mutations in around 63% of cases of diffuse astrocytomas, and result in loss in 10q and 19q chromosomes. Primary GBMs are also associated with alterations in cell proliferation [epidermal growth fator receptor (EGFR) amplification/mutation, platelet derived growth factor receptor alpha (PDGFRA) amplifications, neurofibromin 1 (NF1) mutations], abnormal apoptotic index [cyclin dependent kinase inhibitor 2A (CDKN2A) homozygous deletion and TP53 mutation], and aberrant progression in G1/S phase of the cell cycle.

Transgenic mouse models of Idh -mutated neural stem cells: an appropriate model for low grade glioma?

The large majority of diffuse gliomas, classified by World Health Organization (WHO) guidelines as grade II and III, and the grade IV gliomas that have progressed from these lower grade cancers (the so-called secondary glioblastomas) carry heterozygous hotspot mutations in IDH1 or IDH2 , the genes encoding the metabolic enzymes isocitrate dehydrogenase 1 (IDH1, cytosolic/peroxisomal) or IDH2 (the mitochondrial variant) (1,2). Besides glioma, the occurrence of these mutations is limited to a small number of cancer types, including acute myeloid leukemia (AML), enchondromas, chondrosarcoma and hepatic cholangiocarcinoma (3).

A stiff path to aggressiveness

Whether a glioblastoma-associated IDH1 mutant can prevent the hypoxic induction of HIF1α depends on the stiffness of the extracellular matrix.

MTSS1 is epigenetically regulated in glioma cells and inhibits glioma cell motility

Epigenetic silencing by DNA methylation in brain tumors has been reported for many genes, however, their function on pathogenesis needs to be evaluated. We investigated the MTSS1 gene, identified as hypermethylated by differential methylation hybridization (DMH). Fifty-nine glioma tissue samples and seven glioma cell lines were examined for hypermethylation of the MTSS1 promotor, MTSS1 expression levels and gene dosage. GBM cell lines were treated with demethylating agents and interrogated for functional consequences of MTSS1 expression after transient transfection. Hypermethylation was significantly associated with IDH1/2 mutation. Comparative SNP analysis indicates higher incidence of loss of heterozygosity of MTSS1 in anaplastic astrocytomas and secondary glioblastomas as well as hypermethylation of the remaining allele. Reversal of promoter hypermethylation results in an increased MTSS1 expression. Cell motility was significantly inhibited by MTSS1 overexpression without influencing cell growth or apoptosis. Immunofluorescence analysis of MTSS1 in human astrocytes indicates co-localization with actin filaments. MTSS1 is down-regulated by DNA methylation in glioblastoma cell lines and is part of the G-CIMP phenotype in primary glioma tissues. Our data on normal astrocytes suggest a function of MTSS1 at focal contact structures with an impact on migratory capacity but no influence on apoptosis or cellular proliferation.

Somatic retrotransposition is infrequent in glioblastomas.

BackgroundGliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals.ResultsWe found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions.ConclusionsThese findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0077-5) contains supplementary material, which is available to authorized users.

EXTH-47. RAPID CONVERSION OF MUTANT IDH1 FROM DRIVER TO PASSENGER IN A MODEL OF HUMAN GLIOMAGENESIS

Missense mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. While mutant IDH1 expression is thought to drive the gliomagenesis process, the extent to which it remains a viable therapeutic target remains unknown. To address this question we exposed immortalized (p53/pRb-deficient), untransformed human astrocytes to the mutant IDH1 inhibitor AGI-5198 prior to, concomitant with, or at intervals after, introduction of transforming mutant IDH1, then measured effects on 2-HG levels, histone methylation (H3K4me3, H3K9me2, H3K9me3 or H3K27me3) and growth in soft-agar. Addition of AGI-5198 prior to, or concomitant with, introduction of mutant IDH1 blocked all mutant IDH1-driven changes including cellular transformation. Addition at time intervals as short as 4 days following introduction of mutant IDH1 also suppressed 2-HG levels, but had minimal effects on histone methylation, and lost the ability to suppress clonogenicity in a time-dependent manner. Furthermore, in two different models of mutant IDH1-driven gliomagenesis, AGI-5198 exposures that abolished production of 2-HG also failed to decrease histone methylation, adherent cell growth, or anchorage-independent growth in soft-agar over a prolonged period. These studies show although mutant IDH1 expression drives gliomagenesis, mutant IDH1 itself rapidly converts from driver to passenger.

MPTH-22. SERUM 2-HYDROXYGLUTARATE (2HG) LEVELS ARE CORRELATED WITH TISSUE 2HG LEVELS IN ISOCITRATE DEHYDROGENASE 1 AND 2 GENES (IDH1/2)-MUTATED GLIOMAS BUT NOT IN IDH1/2-WILDTYPE GLIOMAS

Mutations in isocitrate dehydrogenase 1 and 2 genes (IDH1/2) frequently occur in lower-grade gliomas and secondary glioblastomas. Mutant IDH1/2 proteins gain a new ability to produce the oncometabolite 2-hydroxyglutarate (2HG), however, the clinical impact of 2HG is not clear. In this study, we analyzed 2HG levels in both serum and tumor tissue and evaluated the role of 2HG in diffuse gliomas. We measured 2HG concentrations in 123 serums and in 78 tumor tissues by using liquid chromatography-tandem mass spectrometry method. IDH1/2 mutations status were determined by direct sequencing and/or immunohistochemistry. O-6-methylguanine DNA methyltransferase (MGMT) promoter methylations were analyzed using pyrosequencing and defined the mean level of methylation at the 16 CpG sites.Serum levels of 2HG in 42 patients with IDH-Mut tumors did not differ from those in 81 patients with IDH-WT tumors (median: 19.9 ng/mL for IDH-Mut vs 22.8 ng/mL for IDH-WT, p=0.86), however, tissue levels of 2HG in 28 patients with IDH-Mut tumors were significantly higher than those in 50 patients with IDH-WT tumors (median: 4.9 ug/mg for IDH-Mut vs 0.09 ug/mg for IDH-WT, p<0.0001). In 28 patients with IDH-Mut tumors, serum levels of 2HG were significantly correlated with tissue levels of 2HG (R2 = 0.39, p=0.0004), but in 50 patients with IDH-WT tumors, serum and tissue levels of 2HG did not show the relationship (R2 = 0.067, p=0.070). Tissue levels of 2HG showed a significant correlation with MGMT promoter methylations (R2= 0.15, p=0.0024).Our results suggest that tumor-derived 2HG is released in serum in the patients with IDH1/2 Mut tumors, however, their amounts are too small to detect the difference of serum 2HG levels in between patients with IDH-Mut and IDH-WT tumors. Accumulation of 2HG in tumor tissue may have an impact on MGMT promoter methylations.

A new functional IDH2 genetic variant is associated with the risk of lung cancer.

Recently, mutations in isocitrate dehydrogenase 1/2 (IDH1/2) were discovered in 70% of low-grade glioma and secondary glioblastoma multiforme. The discovery of an oncogenic function and the identification of onco-metabolites of IDH1/2 support new roles for metabolism in cancer. For example, some evidence indicates that IDH2 might also exhibit oncogenic functions by promoting cellular metabolism and cancer cell growth. We examined the association between IDH2 rs11540478 and lung cancer risk in 262 lung cancer patient cases and 602 healthy control subjects and also investigated the biological function of rs11540478 in vivo. We found that a higher risk was observed in lung cancer patient carriers of rs11540478 TT and CT compared with CC carriers (OR = 1.44; 95%CI = 1.04-2.00; P = 0.03). The frequency of IDH2 rs11540478 TT and CT carriers was decreased in healthy individuals between the ages of 50-77 compared to those aged 30-49 (OR = 0.67; 95%CI = 0.47-0.96; P = 0.03). Functional analysis showed the effect of rs11540478 on IDH2 expression and lung cancer cell viability, with higher IDH2 expression and cell viability among T allele compared with C allele. IDH2 mRNA was higher in peripheral blood lymphocytes from lung cancer patients compared to healthy subjects. Herein, for the first time we identified IDH2 rs11540478 as a new susceptibility locus for lung cancer. The effect of rs11540478 on mRNA expression of IDH2 and lung cancer cell viability might provide new insight for the genetic basis of lung cancer. © 2016 Wiley Periodicals, Inc.

Rapid Conversion of Mutant IDH1 from Driver to Passenger in a Model of Human Gliomagenesis.

Agents that target mutant IDH may be effective for a narrow time and may require further optimization or additional therapeutics in glioma. Mol Cancer Res; 14(10); 976-83. ©2016 AACR.

Non-invasive detection of 2-hydroxyglutarate in IDH-mutated gliomas using two-dimensional localized correlation spectroscopy (2D L-COSY) at 7 Tesla

BackgroundMutations in the isocitrate dehydrogenase enzyme are present in a majority of lower-grade gliomas and secondary glioblastomas. This mis-sense mutation results in the neomorphic reduction of isocitrate dehydrogenase resulting in an accumulation of the “oncometabolite” 2-hydroxyglutarate (2HG). Detection of 2HG can thus serve as a surrogate biomarker for these mutations, with significant translational implications including improved prognostication. Two dimensional localized correlated spectroscopy (2D L-COSY) at 7T is a highly-sensitive non-invasive technique for assessing brain metabolism. This study aims to assess tumor metabolism using 2D L-COSY at 7T for the detection of 2HG in IDH-mutant gliomas.MethodsNine treatment-naïve patients with suspected intracranial neoplasms were scanned at 7T MRI/MRS scanner using the 2D L-COSY technique. 2D-spectral processing and analyses were performed using a MATLAB-based reconstruction algorithm. Cross and diagonal peak volumes were quantified in the 2D L-COSY spectra and normalized with respect to the creatine peak at 3.0 ppm and quantified data were compared with previously-published data from six normal subjects. Detection of 2HG was validated using findings from immunohistochemical (IHC) staining in patients who subsequently underwent surgical resection.Results2HG was detected in both of the IDH-mutated gliomas (grade III Anaplastic Astrocytoma and grade II Diffuse Astrocytoma) and was absent in IDH wild-type gliomas and in a patient with breast cancer metastases. 2D L-COSY was also able to resolve complex and overlapping resonances including phosphocholine (PC) from glycerophosphocholine (GPC), lactate (Lac) from lipids and glutamate (Glu) from glutamine (Gln).ConclusionsThis study demonstrates the ability of 2D L-COSY to unambiguously detect 2HG in addition to other neuro metabolites. These findings may aid in establishing 2HG as a biomarker of malignant progression as well as for disease monitoring in IDH-mutated gliomas.

P08.68 The Ran signaling pathway is upregulated dependent from glioma grade and radiochemotherapy

AbstractIntroduction:Numerous studies on different non-brain cancers indicate Ran (Ras-related nuclear protein), a key protein in the nucleoplasmic transport during interphase and the regulation of mitotic spindle formation, to be involved in tumorigenesis, cancer progression and tumor cell survival. It further is predictive for poor outcome.Overexpression of Ran has been reported in glioblastoma [GBM] tissue. To further evaluate the role of the Ran signaling pathway in astrocytoma, we measured the expression of its key components Ran, RCC1, RANBP1 and RANBP2 in different types of brain tumor tissue.Materials and Methods:Using qRT-PCR and Western blot analysis the gene and protein expression was analyzed in 80 tissue samples. The tissue subtypes were astrocytomas WHO II°, anaplastic astrocytomas, primary and secondary glioblastomas and tissue samples after recurrence subsequent to radiochemotherapy. Peritumoral brain tissue was used as a control. For PCR analysis at least 10 glioma probes and for Western blot analysis at least 6 glioma probes were used per group.Results:Ran protein overexpression compared to non-neoplastic brain was detected in anaplastic astrocytomas (90%), primary glioblastoma (140%) and secondary glioblastoma (170%). With increasing WHO grade there was an increase of Ran mRNA expression and chemo-radiation therapy led to further enhanced expression in primary and secondary glioblastoma (from 1.36 to 2.25 and from 2.08 to 3.08 arbitrary unites [AU], respectively). There was also an increase in gene expression of RCC1, RANBP1 and RANBP2 analogue to that of Ran: showing a steady increase with higher WHO grading. Correspondingly to Ran, gene expression of RCC1 increased in primary and secondary glioblastoma recurrent after radiochemotherapy from 3.19 to 3.87 AU and from 4.14 to 10.11 AU respectively as well as that of RANBP2 from 2.36 to 5.14 AU and from 2.77 to 4.74 AU respectively.Discussion:Our data clearly show a grade dependent upregulation of key components of the Ran pathway in astrocytomas and a further increase after radiochemotherapy. Therefore, this pathway may represent a future target for glioblastoma therapy.

OS2.6 Identification of IDH1R132H-specific T cell receptors from a humanized mouse model and from glioma patients treated with an IDH1R132H-specific peptide vaccine

AbstractCentral to clinically relevant immunotherapy of gliomas are the discovery of tumor-specific antigens and the development of therapeutic strategies that effectively exploit them. One such example is the mutation in the gene encoding for isocitrate dehydrogenase 1 (IDH1). The point mutation IDH1R132H is the most frequent one in grade II and grade III gliomas and secondary glioblastomas. We have previously shown that a peptide vaccine encoding the IDH1R132H epitope evokes an MHC II restricted CD4+ T Helper-1 cell (TH1) immune response and is therapeutic in an MHC-humanized A2.DR1 mouse tumor model.To determine the clonality in the T-cell receptor (TCR) repertoire of the IDH1R132H specific T-cells, a T-cell line was developed from splenocytes of vaccinated A2.DR1 mice by re-stimulations with autologous, naive irradiated and p132–142 (IDH1R132H)-loaded splenocytes. The cells were then sorted for IDH1R132H specificity by tetramer staining. Deep sequencing analyses of the variable V-J or V-D-J regions of the TRA and TRB genes, which code for the hyper-variable regions of the TCR-alpha and TCR-beta chains, respectively, revealed multiple dominant TRA and TRB sequences. Full length TCRs were constructed by pairing the two most dominant TRA and TRB sequences and cloned into expression vectors for functional validation.In addition to identifying the murine HLA-DR1-restricted TCR, we have analysed IDH1R132H-specific TCRs from a patient with a progressive IDH1R132H-mutated astrocytoma, who has been treated with an IDH1(R132H) vaccine on a compassionate use basis. Single T-cells from this patient, who had a high baseline mutation-specific T-cell response, specifically responding to IDH1(R132H), were expanded in vitro and subjected to single-cell-sorting and subsequent TCR sequencing. 11 different TCR sequences have been identified.The long-term goal is to develop an adoptive T-cell therapy using autologous IDH1R132H-specific T cells.

Concordance analysis and diagnostic test accuracy review of IDH1 immunohistochemistry in glioblastoma.

The study investigated isocitrate dehydrogenase (IDH) 1 immunohistochemistry (IHC) positive rate and concordance rate between IDH1 IHC and molecular test in glioblastoma. The current study included 1360 glioblastoma cases from sixteen eligible studies. Meta-analysis, including subgroup analysis by antibody clones and cut-off values, for IDH1 IHC positive rate was conducted. In addition, we performed a concordance analysis and diagnostic test accuracy review between IDH1 IHC and molecular tests. The estimated rates of IDH1 IHC were 0.106 [95% confidence interval (CI) 0.085-0.132]. The IDH1 IHC positive rate of primary and secondary glioblastomas was 0.049 (95% CI 0.023-0.99) and 0.729 (95% CI 0.477-0.889), respectively. The overall concordance rate between IDH1 IHC and molecular test was 0.947 (95% CI 0.878-0.978). In IDH1 IHC-positive and negative subgroups, the concordance rate was 0.842 (95% CI 0.591-0.952) and 0.982 (95% CI 0.941-0.995), respectively. The pooled sensitivity and specificity for IDH1 IHC were 1.00 (95% CI 0.82-1.00) and 0.99 (95% CI 0.96-1.00), respectively. IDH1 IHC is an accurate test for IDH1 mutation in glioblastoma patients. Further cumulative studies for evaluation criteria of IDH1 IHC will determine how to best apply this approach in daily practice.

PROX1 is a novel pathway-specific prognostic biomarker for high-grade astrocytomas; results from independent glioblastoma cohorts stratified by age and IDH mutation status.

PROX1 is a transcription factor with an essential role in embryonic development and determination of cell fate. In addition, PROX1 has been ascribed suppressive as well as oncogenic roles in several human cancers, including brain tumors. In this study we explored the correlation between PROX1 expression and patient survival in high-grade astrocytomas. For this purpose, we analyzed protein expression in tissue microarrays of tumor samples stratified by patient age and IDH mutation status. We initially screened 86 unselected high-grade astrocytomas, followed by 174 IDH1-R132H1 immunonegative glioblastomas derived from patients aged 60 years and older enrolled in the Nordic phase III trial of elderly patients with newly diagnosed glioblastoma. Representing the younger population of glioblastomas, we studied 80 IDH-wildtype glioblastomas from patients aged 18-60 years. There was no correlation between PROX1 protein and survival for patients with primary glioblastomas included in these cohorts. In contrast, high expression of PROX1 protein predicted shorter survival in the group of patients with IDH-mutant anaplastic astrocytomas and secondary glioblastomas. The prognostic impact of PROX1 in IDH-mutant 1p19q non-codeleted high-grade astrocytomas, as well as the negative findings in primary glioblastomas, was corroborated by gene expression data extracted from the Cancer Genome Atlas. We conclude that PROX1 is a new prognostic biomarker for 1p19q non-codeleted high-grade astrocytomas that have progressed from pre-existing low-grade tumors and harbor IDH mutations.

IDH1 and IDH2 mutations as novel therapeutic targets: current perspectives.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations define distinct subsets of cancers, including low-grade gliomas and secondary glioblastomas, chondrosarcomas, intrahepatic cholangiocarcinomas, and hematologic malignancies. Somatic point mutations in IDH1/2 confer a gain-of-function in cancer cells, resulting in the accumulation and secretion in vast excess of an oncometabolite, the D-2-hydroxyglutarate (D-2HG). Overproduction of D-2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. Indeed, high levels of D-2HG inhibit α-ketoglutarate-dependent dioxygenases, including histone and DNA demethylases, leading to histone and DNA hypermethylation and finally a block in cell differentiation. Furthermore, D-2HG is a biomarker suitable for the detection of IDH1/2 mutations at diagnosis and predictive of the clinical response. Finally, mutant-IDH1/2 enzymes inhibitors have entered clinical trials for patients with IDH1/2 mutations and represent a novel drug class for targeted therapy.

Multicentric Glioma Develops via a Mutant IDH1-Independent Pathway: Immunohistochemical Study of Multicentric Glioma

Multicentric gliomas are very rare. Due to differences in their tumor types they remain enigmatic. We focused on the pathogenesis of multicentric gliomas and compared their immunoprofile with that of solitary gliomas. This retrospective study included 6 males and 8 females with multicentric glioma (8 glioblastomas, 2 anaplastic astrocytomas, 4 diffuse astrocytomas). Their age ranged from 27 to 75 years and all were treated between 2004 and June 2015. The expression of mutant isocitrate dehydrogenase 1 (IDH1), α-thalassemia X-linked intellectual disability (ATRX), p53, phosphatase and tensin homolog (PTEN), and epidermal growth factor receptor (EGFR) was examined immunohistochemically; for 1p19q analysis we used fluorescence in situ hybridization (FISH). In all patients, immunohistochemical staining was negative for mutant IDH1 and cytoplasmic PTEN; only 1 patient (7.1%) manifested nuclear PTEN positivity. FISH for 1p19q codeletion was negative in all 9 examined samples; 5 of 14 specimens (35.7%) were p53-positive, 9 (64.3%) were EGFR-positive, and 4 (28.6%) were ATRX-negative. The MIB-1 labeling index was 0.9-15.6% for grades II and III, and ranged between 17.3 and 52.4% for glioblastoma. Our results suggest that the pathogenesis of multicentric gliomas is different from the mutant IDH1-R132H pathogenesis of lower-grade glioma and secondary glioblastomas. More studies are needed to confirm the molecular mechanisms underlying the pathogenesis of multicentric glioma.

Abstract 1259: MutT homolog 1 (MTH1) is upregulated in glioblastoma multiforme: its inhibition by siRNA or crizotinib results in impaired cell migration and tumor growth in vivo

Abstract Introduction: MutT homolog1 (MTH1) is required for the survival of cancer cells. These are characterized by irregular redox formation resulting in the production of reactive oxygen species which damage the deoxynucleoside triphosphate pool and subsequently the DNA. MTH1 inhibits the incorporation of oxidized bases into the DNA thus avoiding apoptosis of cancer cells. Our aim was to analyze the pathophysiological role of MTH1 in glioma. Materials and Methods: MTH1 expression was analyzed using quantitative PCR and Western Blot analysis in human glioma tissue (n = 80). We compared peritumoral tissue with grade II, III, and IV gliomas. Moreover, we compared primary to secondary GBM and initial tumors with recurrent tumors after temozolomide treatment. U87 cell migration was tested after siRNA knock-down of MTH1 and treatment with the MTH1 inhibitor crizotinib by scratch assays. After orthotopic implantation of MTH1-siRNA treated U87 into rats, tumor size was measured after 8 days. Results: In RT-PCR and Western blot we found a significantly higher expression of MTH1 in primary and secondary glioblastoma than in lower grade brain tumor. Immunofluorescence staining of histological sections of glioblastoma showed also a higher level of MTH1 than in lower grade tumors. By nucleofection, we transfected siRNA MTH1 into the U-87 glioblastoma cells and a slower migration was investigated applying migration assay. Furthermore, treatment with the drug crizotinib inhibited MTH1 and decreased U-87 cell migration. The xenograft study showed a slower tumor growth when U87 cells were transfected with MTH1 siRNA compared to normal U87. Conclusion: These results show that MTH1 plays an essential role in migration and proliferation of glioma cells resulting in clinically observed malignization of glioma and disease progression in recurrent glioblastoma. Moreover, MTH1 can be targeted by Crizotinib indicating a role for potential future therapies. Citation Format: Marco Timmer, Sara Gerdi Kannampuzha, Gabriele Röhn, Roland Goldbrunner. MutT homolog 1 (MTH1) is upregulated in glioblastoma multiforme: its inhibition by siRNA or crizotinib results in impaired cell migration and tumor growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1259.

Abstract 1002: IDH1 promotes tumor growth and resistance to targeted therapies in the absence of mutation

Abstract Metabolic abnormalities of cancers provide opportunities for novel tumor-specific therapies. Isocitrate dehydrogenases (IDHs) are enzymes of the tricarboxylic acid (TCA) cycle that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and the reduction of NADP+ to NADPH. Oncogenic mutations in two IDH-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico analysis of The Cancer Genome Atlas (TCGA) data combined with wet-bench analysis of tumor extracts indicate that non-mutated IDH1 mRNA and protein is commonly overexpressed in primary GBM. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs survival of animal subjects bearing patient-derived xenografts (PDXs). On molecular levels, diminished IDH1 activity results in reduced α-KG and NADPH production, which is paralleled by deficient metabolic flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common mechanism of metabolic adaptation of GBM to support macromolecular synthesis, aggressive growth, and therapy resistance. Citation Format: Alexander H. Stegh. IDH1 promotes tumor growth and resistance to targeted therapies in the absence of mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1002.