Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

High-grade gliomas in adolescents and young adults highlight histomolecular differences from their adult and pediatric counterparts.

BACKGROUND: Considering that pediatric high-grade gliomas (HGGs) are biologically distinct from their adult counterparts, the objective of this study was to define the landscape of HGGs in adolescents and young adults (AYAs). METHODS: We performed a multicentric retrospective study of 112 AYAs from adult and pediatric Ile-de-France neurosurgical units, treated between 1998 and 2013 to analyze their clinicoradiological and histomolecular profiles. The inclusion criteria were age between 15 and 25 years, histopathological HGG diagnosis, available clinical data, and preoperative and follow-up MRI. MRI and tumoral samples were centrally reviewed. Immunohistochemistry and complementary molecular techniques such as targeted/next-generation sequencing, whole exome sequencing, and DNA-methylation analyses were performed to achieve an integrated diagnosis according to the 2016 World Health Organization (WHO) classification. RESULTS: Based on 80 documented AYA patients, HGGs constitute heterogeneous clinicopathological and molecular groups, with a predominant representation of pediatric subtypes (histone H3-mutants, 40%) but also adult subtypes (isocitrate dehydrogenase [IDH] mutants, 28%) characterized by the rarity of oligodendrogliomas, IDH mutants, and 1p/19q codeletion and the relative high frequency of "rare adult IDH mutations" (20%). H3G34-mutants (14%) represent the most specific subgroup in AYAs. In the H3K27-mutant subgroup, non-brainstem diffuse midline gliomas are more frequent (66.7%) than diffuse intrinsic pontine gliomas (23.8%), contrary to what is observed in children. We found that WHO grade has no prognostic value, but molecular subgrouping has major prognostic importance. CONCLUSIONS: HGGs in AYAs could benefit from a specific classification, driven by molecular subtyping rather than age group. Collaborative efforts are needed from pediatric and adult neuro-oncology teams to improve the management of HGGs in AYAs.

In vivo Metabolic Profiles as Determined by 31P and short TE 1H MR-Spectroscopy : No Difference Between Patients with IDH Wildtype and IDH Mutant Gliomas.

PURPOSE: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1H-decoupled 31P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas. METHODS: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1H decoupled 31P MRSI with 3D CSI (3D 31P CSI), 2D 1H CSI and a 1H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites. RESULTS: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pHi) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1H CSI data for tumor and control tissue showed the anticipated results. CONCLUSION: Using 3D-CSI data acquisition, in vivo 31P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.

IDH mutant and 1p/19q co-deleted oligodendrogliomas: tumor grade stratification using diffusion-, susceptibility-, and perfusion-weighted MRI.

PURPOSE: Currently, isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion are proven diagnostic biomarkers for both grade II and III oligodendrogliomas (ODs). Non-invasive diffusion-weighted imaging (DWI), susceptibility-weighted imaging (SWI), and dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI) are widely used to provide physiological information (cellularity, hemorrhage, calcifications, and angiogenesis) of neoplastic histology and tumor grade. However, it is unclear whether DWI, SWI, and DSC-PWI are able to stratify grades of IDH-mutant and 1p/19q co-deleted ODs. METHODS: We retrospectively reviewed the conventional MRI (cMRI), DWI, SWI, and DSC-PWI obtained on 33 patients with IDH-mutated and 1p/19q co-deleted ODs. Features of cMRI, normalized ADC (nADC), intratumoral susceptibility signals (ITSSs), normalized maxim CBV (nCBV), and normalized maximum CBF (nCBF) were compared between low-grade ODs (LGOs) and high-grade ODs (HGOs). Receiver operating characteristic curve and logistic regression were applied to determine diagnostic performances. RESULTS: HGOs tended to present with prominent edema and enhancement. nADC, ITSSs, nCBV, and nCBF were significantly different between groups (all P < 0.05). The combination of SWI and DSC-PWI for grading resulted in sensitivity and specificity of 100.00 and 93.33%, respectively. CONCLUSIONS: IDH-mutant and 1p/19q co-deleted ODs can be stratified by grades using cMRI and advanced magnetic resonance imaging techniques including DWI, SWI, and DSC-PWI. Combined ITSSs with nCBV appear to be a promising option for grading molecularly defined ODs in clinical practice.

Differential hypermethylation of death-associated protein kinase promoter in central neurocytoma and oligodendroglioma.

BACKGROUND: Central neurocytoma and oligodendroglioma are rare tumors of the central nervous system. However, diagnosis between these two types of tumors is challenging due to their many cytological and histological similarities. Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine protein kinase involved in many apoptosis pathways, and repressed expression of DAPK by promoter hypermethylation has been found in a variety of human cancers. The purpose of this study was to assess DAPK protein expression and promoter hypermethylation in central neurocytoma and oligodendroglioma. METHOD: Central neurocytoma and oligodendroglioma samples were obtained from age- and sex-matched patients. DAPK protein expression was performed using immunohistochemical assays in formalin-fixed, paraffin-embedded sections. DAPK promoter hypermethylation was carried out using bisulfite-modified genomic DNA in methylation-specific PCR followed by separation in agarose gels. FINDINGS: A statistically significant difference (P = 0.021) in DAPK promoter hypermethylation between central neurocytoma (76.9%) and oligodendroglioma (20%) was observed. High levels of DAPK protein expression were generally found in oligodendroglioma (90%), compared with 38.5% in central neurocytoma (P = 0.054; not statistically significant). There was an inverse correlation between DAPK protein expression and DAPK promoter hypermethylation in the cohort of 23 patients (P = 0.002). CONCLUSIONS: The results show that DAPK promoter hypermethylation and repressed expression of DAPK protein were more common in central neurocytoma than in oligodendroglioma. Thus, DAPK promoter hypermethylation could be useful for differential diagnosis between these two types of tumors, whereas DAPK protein expression might be less predictive. The role of DAPK promoter hypermethylation in the pathogenesis of central neurocytoma warrants further study.

Benefit from procarbazine, lomustine, and vincristine in oligodendroglial tumors is associated with mutation of IDH.

PURPOSE: Patients with 1p/19q codeleted anaplastic oligodendroglial tumors who participated in RTOG (Radiation Therapy Oncology Group) 9402 lived much longer after chemoradiotherapy (CRT) than radiation therapy (RT) alone. However, some patients with noncodeleted tumors also benefited from CRT; survival curves separated after the median had been reached, and significantly more patients lived ≥ 10 years after CRT than RT. Thus, 1p/19q status may not identify all responders to CRT. PATIENTS AND METHODS: Using trial data, we inquired whether an IDH mutation or germ-line polymorphism associated with IDH-mutant gliomas identified the patients in RTOG 9402 who benefited from CRT. RESULTS: IDH status was evaluable in 210 of 291 patients; 156 (74%) had mutations. rs55705857 was evaluable in 245 patients; 76 (31%) carried the G risk allele. Both were associated with longer progression-free survival after CRT, and mutant IDH was associated with longer overall survival (9.4 v 5.7 years; hazard ratio [HR], 0.59; 95% CI, 0.40 to 0.86; P = .006). For those with wild-type tumors, CRT did not prolong median survival (1.3 v 1.8 years; HR, 1.14; 95% CI, 0.63 to 2.04; P = .67) or 10-year survival rate (CRT, 6% v RT, 4%). Patients with codeleted mutated tumors (14.7 v 6.8 years; HR, 0.49; 95% CI, 0.28 to 0.85; P = .01) and noncodeleted mutated tumors (5.5 v 3.3 years; HR, 0.56; 95% CI, 0.32 to 0.99; P < .05) lived longer after CRT than RT. CONCLUSION: IDH mutational status identified patients with oligodendroglial tumors who did (and did not) benefit from alkylating-agent chemotherapy with RT. Although patients with codeleted tumors lived longest, patients with noncodeleted IDH-mutated tumors also lived longer after CRT.

Acetate supplementation induces growth arrest of NG2/PDGFRα-positive oligodendroglioma-derived tumor-initiating cells.

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.

Oligodendrogliomas: new insights from the genetics and perspectives.

PURPOSE OF REVIEW: Since the discovery, in 1994, of recurrent codeletion of chromosome regions 1p36/19q13 in oligodendrogliomas, genetics has accomplished significant advances improving our knowledge in biology of this tumor type and our clinical management of oligodendroglioma patients. Indeed, 1p36/19q13 has been shown successively to predict increased chemosensitivity and better prognosis, to be associated with frontal location in brain and classic oligodendroglioma morphology, to be mutually exclusive with high-level gene amplification, to be actually whole chromosome arms 1p/19q codeletion, to mediate a t(1;19)(q10;p10) and to be associated with IDH mutations. More recently, pivotal studies, using high-throughput approaches, have provided significant novel insights in the molecular oncogenesis of oligodendrogliomas. RECENT FINDINGS: Capicua homolog (Drosophila) (CIC) and Far Upstream element Binding Protein 1 (FUBP1) have been shown to be frequently mutated in 70 and 40% of 1p/19q codeleted oligodendrogliomas, respectively. The biological and clinical significance of these mutations remains unsettled. Additional recent studies have also demonstrated that 1p/19q codeleted oligodendrogliomas exhibit a proneural transcriptomic profile including overexpression of internexin alpha, a neuronal intermediate filament. Finally, 1p/19q codeleted and IDH-mutated tumors have been shown to be hypermethylated, suggesting a strong link between these both molecular alterations detected in the subgroup of oligodendrogliomas with better prognosis. SUMMARY: Next-generation molecular biology technologies have recently identified recurrent CIC and FUBP1 point mutations in 1p/19q codeleted and IDH-mutated oligodendrogliomas. Their clinical and biological values are under investigation.

A low-frequency variant at 8q24.21 is strongly associated with risk of oligodendroglial tumors and astrocytomas with IDH1 or IDH2 mutation.

Variants at 8q24.21 have been shown to be associated with glioma development. By means of tag SNP genotyping and imputation, pooled next-generation sequencing using long-range PCR and subsequent validation SNP genotyping, we identified seven low-frequency SNPs at 8q24.21 that were strongly associated with glioma risk (P=1×10(-25) to 1×10(-14)). The most strongly associated SNP, rs55705857, remained highly significant after individual adjustment for the other top six SNPs and two previously published SNPs. After stratifying by histological and tumor genetic subtype, the most significant associations of rs55705857 were with oligodendroglial tumors and gliomas with mutant IDH1 or IDH2 (odds ratio (OR)=5.1, P=1.1×10(-31) and OR=4.8, P=6.6×10(-22), respectively). Strong associations were observed for astrocytomas with mutated IDH1 or IDH2 (grades 2-4) (OR=5.16-6.66, P=4.7×10(-12) to 2.2×10(-8)) but not for astrocytomas with wild-type IDH1 and IDH2 (smallest P=0.26). The conserved sequence block that includes rs55705857 is consistently modeled as a microRNA.

Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity.

Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an 'aggrecanase' activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices.

Proteomic analysis of oligodendrogliomas expressing a mutant isocitrate dehydrogenase-1.

Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation.

Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome.

Point mutations of the NADP(+)-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing.

Isocitrate dehydrogenase 1 (IDH1) mutations have recently been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as secondary glioblastomas, whereas primary glioblastomas very rarely contain IDH1 mutations. Furthermore, a specific monoclonal antibody, IMab-1, which recognizes IDH1-R132H-the most frequent IDH1 mutation-has been generated. IMab-1 has been reported to react with the IDH1-R132H protein, but not the wild-type IDH1 or the other IDH1 mutant proteins in Western-blot analysis. However, the importance of immunohistochemistry using IMab-1 has not yet been elucidated. In this study, we compared the findings from IMab-1 immunohistochemistry and direct DNA sequencing using 49 glioma samples. IMab-1 detected 12 out of 49 cases; however, only nine cases were found to be IDH1-R132H by direct DNA sequencing because of a small population of IDH1-R132H mutation-possessing tumor cells, indicating that IMab-1 immunohistochemistry is useful for detecting IDH1-R132H. We conducted immunohistochemical detection in 52 cases of grade III astrocytomas. The median time to progression (TTP) was significantly longer in the cases with the IDH1 mutation (86.7 months) compared to the cases without the IDH1 mutation (wild type, 10.4 months) (p < 0.01). In conclusion, the anti-IDH1-R132H-specific monoclonal antibody IMab-1 is very useful for detecting IDH1-R132H in immunohistochemistry, and predicting the time to progression in grade III anaplastic astrocytomas. Therefore, IMab-1 is likely to be useful for the diagnosis of mutation-bearing gliomas and for determining the treatment strategy of grade III gliomas.

High-throughput immunohistochemical profiling of primary brain tumors and non-neoplastic systemic organs with a specific antibody against the mutant isocitrate dehydrogenase 1 R132H protein.

Isocitrate dehydrogenase 1 (IDH1) mutations are common in grade II-III diffuse gliomas and secondary glioblastomas. The aim of this study is to investigate the staining pattern of mIDH1R132H, an antibody specific to mutant IDH1 protein, in primary brain tumors and non-neoplastic systemic organs. Eight of 13 diffuse astrocytomas, 1 of 6 anaplastic astrocytomas, 9 of 11 oligodendrogliomas, 15 of 22 anaplastic oligodendrogliomas, 6 of 7 oligoastrocytomas, and 5 of 8 anaplastic oligoastrocytomas showed both cytoplasmic and nuclear positivity. Two of 25 atypical meningiomas and 2 of 42 pituitary adenomas were positive for mIDH1R132H. The following non-neoplastic systemic organs showed positivity in the cytoplasm alone: the myocardium, peribronchial glands, interlobular ducts of the salivary gland, gastric fundic gland, columnar epithelia of the large bowel, hepatocytes, centroacinar cells, the intercalated ducts of the pancreas, renal proximal and distal tubules, adrenocortex, ovarian granulosa cells, and the choroid plexus epithelia. It was concluded that the immunopositivity detected in non-neoplastic systemic organs was due to cross-reactivity, because immunohistochemistry with an anti-mitochondrial antibody revealed that the mIDH1R132H staining pattern was identical to that of the mitochondria. Therefore, mIDH1R132H positivity should only be considered to be validated when a cell shows both cytoplasmic and nuclear staining.

Coexpression network analysis identifies transcriptional modules related to proastrocytic differentiation and sprouty signaling in glioma.

Gliomas are primary brain tumors with high mortality and heterogeneous biology that is insufficiently understood. In this study, we performed a systematic analysis of the intrinsic organization of complex glioma transcriptome to gain deeper knowledge of the tumor biology. Gene coexpression relationships were explored in 790 glioma samples from 5 published patient cohorts treated at different institutions. We identified 20 coexpression modules that were common to all the data sets and associated with proliferation, angiogenesis, hypoxia, immune response, genomic alterations, cell differentiation phenotypes, and other features inherent to glial tumors. A collection of high-quality signatures for the respective processes was obtained using cross-data set summarization of the modules' gene composition. Individual modules were found to be organized into higher order coexpression groups, the two largest of them associated with glioblastoma and oligodendroglioma, respectively. We identified a novel prognostic gene expression signature (185 genes) linked to a proastrocytic pattern of tumor cell differentiation. This "proastrocytic" signature was associated with long survival and defined a subgroup of the previously established "proneural" class of gliomas. A strong negative correlation between proastrocytic and proneural markers across differentiated tumors underscored the distinction between these subtypes of glioma. Interestingly, one further novel signature in glioma was identified that was associated with EGFR (epidermal growth factor receptor) gene amplification and suggested that EGF signaling in glioma may be a subject to regulation by Sprouty family proteins. In summary, this integrated analysis of the glioma transcriptome provided several novel insights into molecular heterogeneity and pathogenesis of glial tumors.

IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas.

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.

Cyclooxygenase-2 expression in astrocytes and microglia in human oligodendroglioma and astrocytoma.

Cyclooxygenases (cox) are potent mediators of inflammation and two cox-isoenzymes, cox-1, cox-2, are described to date. Cox-2 is cytokine-inducible in inflammatory cells and enhanced cox-2 expression has been attributed a key role in the development of edema and immunomodulation in pathologically altered brain tissues. In normal cerebral cortex cox-2 is present only in neurons, but not in the glial or vascular endothelial cells. The function of microglia in glioma biology is unclear. Microglia have both neurotrophic and neurotoxic functions and have been shown to release a variety of cytokines. Our preliminary results showed that the expression pattern of cox-2 is predominantly neuronal although glial expression was observed with the correlation of high malignancy. In this study we aimed to assess the phenotypes (astrocyte, microglia) of the cox-2-expressing glial cells in various types of human gliomas and to compare their expression patterns. For this purpose we employed dual immunohistochemistry for cox-2 and GFAP (astrocyte) or LCA-MAC (microglia-macrophage) in archival formalin-fixed, paraffin embedded human tissue diagnosed as oligodendroglioma and/or astrocytoma. The results showed that cox-2 immunoreactivity is up-regulated in the neurons according to the tumor grade. Most of the cox-2 immunoreactive glia were GFAP-positive in anaplastic oligodendrogliomas and at lesser extend in glioblastomas. Cox-2 and LCA co-localization was detected in more glial cells in glioblastomas. It may be speculated that the induction of cox-2 in microglia may contribute to the deleterious effects of prostanoids in cerebral edema formation during the progression of oligodendrogliomas. The detection of cox-2 in astrocytes surrounding the necrotic areas might be important to develop new strategies, such as the usage of cox-2 inhibitors combine with chemotherapy and radiotherapy in the treatment of glioma patients.

Carbonic anhydrase IX in oligodendroglial brain tumors.

BACKGROUND: Carbonic anhydrase IX is a hypoxia-induced enzyme that has many biologically important functions, including its role in cell adhesion and invasion. METHODS: This study was set out to investigate the role of CA IX in a series of 86 oligodendroglial brain tumors (71 primary and 15 recurrent; 48 pure oligodendrogliomas and 40 mixed oligoastrocytomas). RESULTS: 80% of the tumors showed CA IX expression by immunohistochemistry. Tumors with moderate or strong CA IX expression had decreased level of cell proliferation compared to weak or no CA IX expression (median 2.9 vs. 5.8, p = 0.015). CA IX correlated with two antioxidative enzymes, manganese superoxide dismutase (MnSOD) and regulatory gammaglutamylcysteine synthetase (GLCL-R): CA IX expression was significantly higher in MnSOD-positive tumors (p = 0.008) and decreased in GLCL-R-positive tumors (p = 0.044). In Cox multivariate analysis CA IX expression, patient age and histological component (pure oligodendroglioma vs. mixed oligoastrocytoma) showed independent prognostic values (p = 0.009, p = 0.003 and p = 0.022, respectively), CA IX positivity predicting poorer outcome. CONCLUSION: CA IX was proved to be an independent prognostic indicator in oligodendroglial brain tumors, and it also correlates reversely with cell proliferation. It may have a role in the biology of oligodendrogliomas, and most interestingly, as it is mainly expressed in tumor tissue, CA IX could serve as a target molecule for anticancer treatments.

A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

BACKGROUND: Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). RESULTS: MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells.

Mutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron-sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron-sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron-sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models.