D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function.

Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation.

The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.

Choice of biological source material supersedes oxidative stress in its influence on DJ-1 in vivo interactions with Hsp90.

DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or naïve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions.

Haplotype-specific expression of the human PDGFRA gene correlates with the risk of glioblastomas.

Aberrant expression of the platelet-derived growth factor alpha-receptor (PDGFRA) gene has been associated with various diseases, including neural tube defects and gliomas. We have previously identified 5 distinct haplotypes for the PDGFRA promoter region, designated H1, H2alpha, H2beta, H2gamma and H2delta. Of these haplotypes H1 and H2alpha are the most common, whereby H1 drives low and H2alpha high transcriptional activity in transient transfection assays. Here we have investigated the role of these PDGFRA promoter haplotypes in gliomagenesis at both the genetic and cellular level. In a case-control study on 71 glioblastoma patients, we observed a clear underrepresentation of H1 alleles, with pH1 = 0.141 in patients and pH1 = 0.211 in a combined Western European control group (n = 998, p < 0.05). Furthermore, in 3 out of 4 available H1/H2alpha heterozygous human glioblastoma cell lines, H1-derived mRNA levels were more than 10-fold lower than from H2alpha, resulting at least in part from haplotype-specific epigenetic differences such as DNA methylation and histone acetylation. Together, these results indicate that PDGFRA promoter haplotypes may predispose to gliomas. We propose a model in which PDGFRA is upregulated in a haplotype-specific manner during neural stem cell differentiation, which affects the pool size of cells that can later undergo gliomagenesis.

The topoisomerase II inhibitor, genistein, induces G2/M arrest and apoptosis in human malignant glioma cell lines.

The protein tyrosine kinase inhibitor, genistein, has been reported to inhibit proliferation and to induce cell death in various non-solid and solid cancer cell lines. Herein, we examined the effects of genistein in several human malignant glioma cell lines. We found that genistein inhibited the proliferation of LN-18, LNT-229, LN-308 and T98G cells at EC50 concentrations of 25-80 microM (72 h of exposure). The growth of a non-neoplastic immortalized human astrocyte cell line, SV-FHAS, was inhibited at similar concentrations. There was a reduction in [3H]-methylthymidine incorporation and a moderate lactate dehydrogenase release as a sign of cell death in genistein-treated glioma cells. Electron microscopy showed morphological changes with mitochondrial swelling and apoptosis in glioma cells treated with high concentrations of genistein. Genistein-induced cytotoxicity was associated with an increased DNA/topoisomerase II complex formation. Furthermore, genistein induced cell cycle arrest in G2/M. There was an increase in the p53 and p21 levels in response to genistein. However, there was no difference in genistein sensitivity between p21-deficient colon carcinoma cells and isogenic control cells. Genistein-induced cell death in LN-18 and LNT-229 was unaffected by the ectopic expression of the preferential caspase 1/8 inhibitor, crm-A, or co-exposure to the pan-specific pseudosubstrate caspase inhibitor, zVAD-fmk. The ectopic expression of the anti-apoptotic BCL-2 protein attenuated the cytotoxic effects of genistein. Moreover, the ectopic expression of temperature-sensitive p53V135A, which acts as a dominant-negative p53 mutant at 38.5 degrees C but assumes p53 wild-type properties at 32.5 degrees C, in LN-18 or LNT-229 cells, had no effect on genistein cytotoxicity at either temperature. Genistein did not act in synergy with CD95 ligand-induced apoptosis or various cancer chemotherapy drugs in cytotoxic or clonogenic cell death assays. Thus, genistein-like protein kinase inhibitors are promising agents for the experimental treatment of malignant gliomas.

Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas.

Glioblastomas frequently carry mutations in the PTEN tumor suppressor gene on 10q23.3. The tumor suppressor properties of Pten are closely related to its inhibitory effect on the phosphatidyl-inositol-3'-kinase (Pi3k)-dependent activation of protein kinase B (Akt) signalling. Here, we report on the analysis of 17 genes related to the Pi3k/Akt signalling pathway for genetic alteration and aberrant expression in a series of 103 glioblastomas. Mutation, homozygous deletion or loss of expression of PTEN was detected in 32% of the tumors. In contrast, we did not find any aberrations in the inositol polyphosphate phosphatase like-1 gene (INPPL1), whose gene product may also counteract Pi3k-dependent Akt activation. Analysis of genes encoding proteins that may activate the pathway upstream of Pi3k revealed variable fractions of tumors with EGFR amplification (31%), PDGFRA amplification (8%), and IRS2 amplification (2%). The protein tyrosine kinase 2 (PTK2/FAK1) gene was neither amplified nor overexpressed at the mRNA level. Investigation of three genes encoding catalytic subunits of Pi3k (PIK3CA, PIK3CD, and PIK3C2B) revealed amplification of PIK3C2B (1q32) in 6 tumors (6%). Overexpression of PIK3C2B mRNA was detected in 4 of these cases. PIK3CD (1p36.2) and PIK3CA (3q26.3) were not amplified but PIK3CD mRNA was overexpressed in 6 tumors (6%). Amplification and overexpression of AKT1 was detected in a single case of gliosarcoma. The IRS1, PIK3R1, PIK3R2, AKT2, AKT3, FRAP1, and RPS6KB1 genes were neither amplified nor overexpressed in any of the tumors. Taken together, our data indicate that different genes related to the Pi3k/Akt signalling pathway may be aberrant in glioblastomas.

Pten signaling in gliomas.

In 1997, the PTEN gene (phosphatase and tensin homolog deleted on chromosome 10) was identified as a tumor suppressor gene on the long arm of chromosome 10. Since then, important progress has been made with respect to the understanding of the role of the Pten protein in the normal development of the brain as well as in the molecular pathogenesis of human gliomas. This review summarizes the current state of the art concerning the involvement of aberrant Pten function in the development of different biologic features of malignant gliomas, such as loss of cell-cycle control and uncontrolled cell proliferation, escape from apoptosis, brain invasion, and aberrant neoangiogenesis. Most of the tumor-suppressive properties of Pten are dependent on its lipid phosphatase activity, which inhibits the phosphatidylinositol-3'-kinase (PI3K)/Akt signaling pathway through dephosphorylation of phosphatidylinositol-(3,4,5)-triphosphate. The additional function of Pten as a dual-specificity protein phosphatase may also play a role in glioma pathogenesis. Besides the wealth of data elucidating the functional roles of Pten, recent studies suggest a diagnostic significance of PTEN gene alterations as a molecular marker for poor prognosis in anaplastic astrocytomas and anaplastic oligodendrogliomas. Furthermore, the possibility of selective targeting of PTEN mutant tumor cells by specific pharmacologic inhibitors of members of the Pten/PI3K/Akt pathway opens up new perspectives for a targeted molecular therapy of malignant gliomas.

Analysis of IDH mutation, 1p/19q deletion, and PTEN loss delineates prognosis in clinical low-grade diffuse gliomas.

BACKGROUND: Grades II and III gliomas have unpredictable rates of progression, making management decisions difficult. Currently, several clinical and radiological characteristics are utilized to predict progression and survival but collectively are suboptimal. METHODS: In this study, we analyzed a set of 108 nonenhancing hemispheric grade II-III gliomas. Demographic variables, including patient age, tumor diameter, extent of resection, and performance status, were combined with molecular data (IDH mutation status [mIDH], 1p/19q codeletion, PTEN deletion, and EGFR amplification). A complete dataset for all variables was compiled for 70 of the 108 patients. Both univariable and multivariable analyses were performed to determine whether the molecular data singly or in combination offer advantages over tumor type and grade for prediction of overall survival (OS) and/or progression-free rate (PFR). RESULTS: Patient age, clinical variables (tumor diameter, extent of resection, performance status), and pathology (tumor type and grade) were not predictive of OS or PFR. IDH mutation status alone was predictive of longer OS and PFR for the entire group of tumors; 1p/19q deletion alone was predictive of OS but not PFR. In the multivariable analysis, none of the clinical or demographic factors were predictive of OS or PFR. IDH mutation status, 1p/19q codeletion, and PTEN deletion were predictive of OS (P = .003, P = .005, P = .02, respectively). Both mIDH (P < .001) and the interaction term of 1p/19q and PTEN (P < .001) were found to be predictive of PFR. CONCLUSIONS: We conclude that the combination of mIDH, 1p/19q codeletion, and PTEN deletion may be particularly effective in discriminating good prognosis from poor prognosis hemispheric gliomas. We propose that such a scheme merits testing on larger prospective cohorts. Should our findings be confirmed, routine clinical analysis of hemispheric gliomas for mIDH, 1p/19q codeletion, and PTEN deletion would be justified.

The NF-κB regulator MALT1 determines the encephalitogenic potential of Th17 cells.

Effector functions of inflammatory IL-17-producing Th (Th17) cells have been linked to autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). However, what determines Th17 cell encephalitogenicity is still unresolved. Here, we show that after EAE induction, mice deficient for the NF-κB regulator MALT1 (Malt1-/- mice) exhibit strong lymphocytic infiltration in the CNS, but do not develop any clinical signs of EAE. Loss of Malt1 interfered with expression of the Th17 effector cytokines IL-17 and GM-CSF both in vitro and in vivo. In line with their impaired GM-CSF secretion, Malt1-/- Th cells failed to recruit myeloid cells to the CNS to sustain neuroinflammation, whereas autoreactive WT Th cells successfully induced EAE in Malt1-/- hosts. In contrast, Malt1 deficiency did not affect Th1 cells. Despite their significantly decreased secretion of Th17 effector cytokines, Malt1-/- Th17 cells showed normal expression of lineage-specific transcription factors. Malt1-/- Th cells failed to cleave RelB, a suppressor of canonical NF-κB, and exhibited altered cellular localization of this protein. Our results indicate that MALT1 is a central, cell-intrinsic factor that determines the encephalitogenic potential of inflammatory Th17 cells in vivo.

Differential proteome analysis of human gliomas stratified for loss of heterozygosity on chromosomal arms 1p and 19q.

Combined deletion of chromosomal arms 1p and 19q is an independent prognostic marker in patients with oligodendroglial brain tumors, including oligodendrogliomas and oligoastrocytomas. However, the relevant genes in these chromosome arms and the molecular mechanisms underlying the prognostic significance of 1p/19q deletion are yet unknown. We used two-dimensional difference gel electrophoresis followed by mass spectrometry to perform a proteome-wide profiling of low-grade oligoastrocytomas stratified for the presence or absence of 1p/19q deletions. Thereby, we identified 22 different proteins showing differential expression in tumors with or without combined deletions of 1p and 19q. Four of the differentially expressed proteins, which are vimentin, villin 2 (ezrin), annexin A1, and glial fibrillary acidic protein, were selected for further analysis. Lower relative expression levels of these proteins in 1p/19q-deleted gliomas were confirmed at the protein level by Western blot analysis and immunohistochemistry. Furthermore, sequencing of sodium bisulfite-treated tumor DNA revealed more frequent methylation of 5'-CpG islands associated with the VIM and VIL2 genes in 1p/19q-deleted gliomas when compared with gliomas without these deletions. In summary, we confirm proteome-wide profiling as a powerful means to identify candidate biomarkers in gliomas. In addition, our data support the hypothesis that 1p/19q-deleted gliomas frequently show epigenetic down-regulation of multiple genes due to aberrant methylation of the 5'-CpG islands.

Identification of novel genes associated with astrocytoma progression using suppression subtractive hybridization and real-time reverse transcription-polymerase chain reaction.

To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low-grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using real-time reverse transcription-PCR on a series of 10 pairs of primary low-grade and recurrent high-grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2-qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfite-modified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells.

Mutation analysis of the Ras pathway genes NRAS, HRAS, KRAS and BRAF in glioblastomas.

Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.

Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target.

We previously reported on the amplification and overexpression of the mouse double minute 4 homolog gene (MDM4) from 1q32 in a subset of malignant gliomas (Riemenschneider et al., Cancer Res 1999;59:6091-6). More recently, amplification and overexpression of the neighboring contactin 2 gene (CNTN2) was reported in individual malignant gliomas without MDM4 amplification (Rickman et al., Cancer Res 2001;61:2162-8). To address the question of whether 1q32 carries 2 independent amplification targets or a common target other than MDM4 and CNTN2, we analyzed primary malignant gliomas for amplification and overexpression of 17 different genes from this region. Our results indicate a single region of amplification that comprises the genes MDM4, GAC1, PIK3C2B and PEPP3, with only MDM4 amplification being invariably associated with overexpression. CNTN2 was found to be coamplified with MDM4 in 3 malignant gliomas but overexpressed in only 1 of these tumors. No CNTN2 amplification was detected in any of 102 malignant gliomas without MDM4 amplification. Our data therefore corroborate the notion that MDM4 is the main amplification target on 1q32 in malignant gliomas. However, coamplification and overexpression of adjacent genes may provide an additional growth advantage in some malignant gliomas with MDM4 amplification.

Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator protein gene in glioblastomas.

The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.

Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas.

Ras signaling is important for the intracellular transduction of mitogenic stimuli from activated growth factor receptors. We have investigated 37 sporadic malignant melanomas (15 primary cutaneous melanomas and 22 melanoma metastases) and 6 melanoma cell lines for mutations in the 3 Ras genes NRAS, KRAS and HRAS. All tumors and cell lines were additionally analyzed for mutation and expression of BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties, as well as for expression of RASSF1A, which encodes a Ras-binding protein with tumor suppressor properties. Mutational analyses identified somatic NRAS mutations in 2 primary melanomas, 4 melanoma metastases and 2 cell lines. One melanoma metastasis showed a somatic KRAS mutation whereas HRAS mutations were not detected. Eight primary melanomas, 6 melanoma metastases and 4 melanoma cell lines carried BRAF mutations affecting the known hot-spot codon 599. None of the tumors or cell lines with BRAF mutation demonstrated NRAS or KRAS mutations. Real-time reverse transcription-PCR showed that 8 melanomas (3 primary tumors, 5 melanoma metastases) had reduced RASSF1A transcript levels of < or =50% relative to benign melanocytic nevi and normal skin. Three melanoma cell lines lacked detectable RASSF1A transcripts. The RASSF1A gene promoter was hypermethylated in these 3 cell lines as well as in 6 of 8 melanomas with reduced RASSF1A mRNA levels. Treatment of the cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in demethylation of the RASSF1A promoter and re-expression of RASSF1A transcripts. Most tumors and all cell lines with RASSF1A promoter methylation additionally carried BRAF or NRAS mutations, suggesting a synergistic effect of these aberrations on melanoma growth. Taken together, 57% of the investigated melanomas and 100% of the melanoma cell lines carried mutations in either NRAS, KRAS or BRAF. In addition, 22% of the melanomas and 50% of the cell lines showed reduced RASSF1A transcript levels. Thus, alterations of Ras pathway genes are of paramount importance in the pathogenesis of sporadic melanomas.

Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC.

Aberrant activation of the Wnt signaling pathway has been reported in different human tumor types, including malignant melanomas. We investigated 37 malignant melanomas (15 primary tumors and 22 metastases) for alterations of 4 genes encoding members of this pathway, i.e., CTNNB1 (beta-catenin gene, 3p22.1), APC (adenomatous polyposis coli gene, 5q22.2), BTRC (beta-transducin repeat-containing protein gene, 10q24.3) and ICAT (inhibitor of beta-catenin and Tcf-4, 1p36.2). Mutational analysis of CTNNB1 identified somatic mutations in 1 primary melanoma and 1 melanoma metastasis from 2 different patients (5%). Both mutations affected the N-terminal degradation box of beta-catenin, which is important for the regulation of beta-catenin homeostasis. Another primary melanoma carried a somatic APC missense mutation within the known mutation cluster region in exon 15. Fourteen tumors (40%) showed LOH at microsatellite markers on 1p36. None of the tumors had lost both copies of the ICAT gene, but 1 melanoma metastasis carried a somatic point mutation altering the translation start codon of ICAT. Real-time RT-PCR showed markedly reduced ICAT transcript levels (

Comprehensive analysis of genomic alterations in gliosarcoma and its two tissue components.

Gliosarcoma is a variant of glioblastoma multiforme characterized by two components displaying gliomatous or sarcomatous differentiation. We investigated 38 gliosarcomas for aberrations of tumor-suppressor genes and proto-oncogenes that are commonly altered in glioblastomas. Amplification of CDK4, MDM2, EGFR, and PDGFRA were found in 11% (4/35), 8% (3/38), 8% (3/38), and 3% (1/35) of the tumors, respectively. Nine of 38 gliosarcomas (24%) carried TP53 mutations. PTEN mutations were identified in 45% (9/20) of the investigated tumors. Twenty gliosarcomas were analyzed by comparative genomic hybridization (CGH). Chromosomal imbalances commonly detected were gains on chromosomes 7 (15/20; 75%), X (4/20; 20%), 9q, and 20q (3/20, 15% each); and losses on chromosomes 10 and 9p (7/20, 35% each), and 13q (3/20, 15%). Five different high-level amplifications were mapped to 4q12-q21 (1 case), 6p21 (1 case), 7p12 (2 cases), proximal 12q (4 cases), and 14q32 (1 case) by CGH. Southern blot and/or differential PCR analyses identified amplification of PDGFRA (4q12), CCND3 (6p21), EGFR (7p12), CDK4 (12q14) and/or MDM2 (12q14.3-q15), and AKT1 (14q32.3) in the respective tumors. Separate analysis of the gliomatous and sarcomatous components of eight gliosarcomas by CGH after microdissection and universal DNA amplification revealed that both components shared 57% of the chromosomal imbalances detected. Taken together, our data indicate that the genomic changes in gliosarcomas closely resemble those found in glioblastomas. However, the number of chromosomes involved in imbalances in gliosarcomas was significantly lower than that in glioblastomas, indicating a higher genomic stability in gliosarcomas. In addition, we provide further support for the hypothesis that the gliomatous and sarcomatous components are derived from a single precursor cell clone, which progressed into subclones with distinct morphological features during tumor evolution. According to our data, gain/amplification of genes on proximal 12q may facilitate the development of a sarcomatous phenotype.

Gliomatosis cerebri: molecular pathology and clinical course.

Gliomatosis cerebri is a rare, diffusely growing neuroepithelial tumor characterized by extensive brain infiltration involving more than two cerebral lobes. Among 13 patients with gliomatosis cerebri (median age, 46 years), biopsies showed features of diffuse astrocytoma (n = 4), oligoastrocytoma (n = 1), anaplastic astrocytoma (n = 5), anaplastic oligoastrocytoma (n = 1), or glioblastoma (n = 2). Molecular genetic investigation showed TP53 mutations in three of seven tumors and both PTEN mutation and epidermal growth factor receptor overexpression in one tumor. Amplification of CDK4 or MDM2 or homozygous deletion of CDKN2A was not detected. Three of 10 patients receiving radiotherapy showed a partial response (one patient) or had stable disease (two patients) lasting for more than 1 year. Four of six patients treated with procarbazine, carmustine, vincristine chemotherapy demonstrated partial remission (one patient), minor response (two patients), or stable disease (one patient). Median survival time from diagnosis was 14 months (range, 4-91+ months). Infratentorial involvement was associated with shorter survival. We conclude that (1) the molecular genetic alterations in gliomatosis cerebri resemble those in diffuse astrocytomas; (2) the prognosis of gliomatosis cerebri is variable but for at least 50% of patients as poor as for glioblastoma; and (3) some patients respond to radiotherapy and/or procarbazine, carmustine, vincristine chemotherapy.