Guido Reifenberger

Publications of Guido Reifenberger

• Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial.

  Authors: Wolfgang Wick, Michael Platten, Christoph Meisner, Jörg Felsberg, Ghazaleh Tabatabai, Matthias Simon, Guido Nikkhah, Kirsten Papsdorf, Joachim P Steinbach, Michael Sabel, Stephanie E Combs, Jan Vesper, Christian Braun, Jürgen Meixensberger, Ralf Ketter, Regine Mayer-Steinacker, Guido Reifenberger, Michael Weller

  The lancet oncology. 05/2012;

  BACKGROUND: Radiotherapy is the standard care in elderly patients with malignant astrocytoma and the role of primary chemotherapy is poorly defined. We did a randomised trial to compare the efficacyBACKGROUND: Radiotherapy is the standard care in elderly patients with malignant astrocytoma and the role of primary chemotherapy is poorly defined. We did a randomised trial to compare the efficacy and safety of dose-dense temozolomide alone versus radiotherapy alone in elderly patients with anaplastic astrocytoma or glioblastoma. METHODS: Between May 15, 2005, and Nov 2, 2009, we enrolled patients with confirmed anaplastic astrocytoma or glioblastoma, age older than 65 years, and a Karnofsky performance score of 60 or higher. Patients were randomly assigned 100 mg/m(2) temozolomide, given on days 1-7 of 1 week on, 1 week off cycles, or radiotherapy of 60·0 Gy, administered over 6-7 weeks in 30 fractions of 1·8-2·0 Gy. The primary endpoint was overall survival. We assessed non-inferiority with a 25% margin, analysed for all patients who received at least one dose of assigned treatment. This trial is registered with ClinicalTrials.gov, number NCT01502241. FINDINGS: Of 584 patients screened, we enrolled 412. 373 patients (195 randomly allocated to the temozolomide group and 178 to the radiotherapy group) received at least one dose of treatment and were included in efficacy analyses. Median overall survival was 8·6 months (95% CI 7·3-10·2) in the temozolomide group versus 9·6 months (8·2-10·8) in the radiotherapy group (hazard ratio [HR] 1·09, 95% CI 0·84-1·42, p(non-inferiority)=0·033). Median event-free survival (EFS) did not differ significantly between the temozolomide and radiotherapy groups (3·3 months [95% CI 3·2-4·1] vs 4·7 [4·2-5·2]; HR 1·15, 95% CI 0·92-1·43, p(non-inferiority)=0·043). Tumour MGMT promoter methylation was seen in 73 (35%) of 209 patients tested. MGMT promoter methylation was associated with longer overall survival than was unmethylated status (11·9 months [95% CI 9·0 to not reached] vs 8·2 months [7·0-10·0]; HR 0·62, 95% CI 0·42-0·91, p=0·014). EFS was longer in patients with MGMT promoter methylation who received temozolomide than in those who underwent radiotherapy (8·4 months [95e% CI 5·5-11·7] vs 4·6 [4·2-5·0]), whereas the opposite was true for patients with no methylation of the MGMT promoter (3·3 months [3·0-3·5] vs 4·6 months [3·7-6·3]). The most frequent grade 3-4 intervention-related adverse events were neutropenia (16 patients in the temozolomide group vs two in the radiotherapy group), lymphocytopenia (46 vs one), thrombocytopenia (14 vs four), raised liver-enzyme concentrations (30 vs 16), infections (35 vs 23), and thromboembolic events (24 vs eight). INTERPRETATION: Temozolomide alone is non-inferior to radiotherapy alone in the treatment of elderly patients with malignant astrocytoma. MGMT promoter methylation seems to be a useful biomarker for outcomes by treatment and could aid decision-making. FUNDING: Merck Sharp & Dohme.

• Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells.

  Authors: Caroline Happold, Patrick Roth, Wolfgang Wick, Natalie Schmidt, Ana-Maria Florea, Manuela Silginer, Guido Reifenberger, Michael Weller

  Journal of neurochemistry. 05/2012;

  Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients with methylation of the O(6)Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients with methylation of the O(6) -methyl-guanine DNA methyltransferase (MGMT) promoter. However, all patients eventually suffer from tumor progression because their tumors become resistant to TMZ. Here, we modelled acquired TMZ resistance in glioma cells in vitro to identify underlying molecular mechanisms. To this end, the glioma cell lines LNT-229, LN-308 and LN-18 were exposed repetitively to increasing concentrations of TMZ to induce a stable resistant phenotype (R) defined by clonogenic survival assays. The molecular mechanisms mediating acquired resistance were assessed by immunoblot, PCR and flow cytometry. Rescue experiments were performed with siRNA-mediated candidate gene silencing. We found in LN-18 cells constitutively expressing MGMT a strong up-regulation of MGMT levels in TMZ-resistant cells. TMZ resistance in the MGMT-negative cell lines LNT-229 and LN-308 was not associated with de novo expression of MGMT. Instead, we found a down-regulation of several DNA mismatch-repair proteins in resistant LNT-229 cells. A TMZ-resistant phenotype was also achieved by silencing selected DNA mismatch repair proteins in parental LNT-229 cells. No obvious mechanism of resistance was identified in the third cell line, LN-308, except for reduced methylation of LINE-1 repetitive elements. In conclusion, we demonstrate that different molecular mechanisms may contribute to the development of acquired TMZ resistance in glioma cells, indicating the need to develop distinct strategies to overcome resistance. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

• Corrigendum: Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

  Authors: Jeremy Schwartzentruber, Andrey Korshunov, Xiao-Yang Liu, David T W Jones, Elke Pfaff, Karine Jacob, Dominik Sturm, Adam M Fontebasso, Dong-Anh Khuong Quang, Martje Tönjes [......] Olaf Witt, Cindy Zhang, Pedro Castelo-Branco, Peter Lichter, Damien Faury, Uri Tabori, Christoph Plass, Jacek Majewski, Stefan M Pfister, Nada Jabado

  Nature. 03/2012;

• Nos2 inactivation promotes the development of medulloblastoma in ptch1 mice by deregulation of gap43-dependent granule cell precursor migration.

  Authors: Daniel Haag, Petra Zipper, Viola Westrich, Daniela Karra, Karin Pfleger, Grischa Toedt, Frederik Blond, Nicolas Delhomme, Meinhard Hahn, Julia Reifenberger, Guido Reifenberger, Peter Lichter

  PLoS genetics. 03/2012; 8(3):e1002572.

  Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrantMedulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.

• Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

  Authors: Jeremy Schwartzentruber, Andrey Korshunov, Xiao-Yang Liu, David T W Jones, Elke Pfaff, Karine Jacob, Dominik Sturm, Adam M Fontebasso, Dong-Anh Khuong Quang, Martje Tönjes [......] Olaf Witt, Cindy Zhang, Pedro Castelo-Branco, Peter Lichter, Damien Faury, Uri Tabori, Christoph Plass, Jacek Majewski, Stefan M Pfister, Nada Jabado

  Nature. 02/2012; 482(7384):226-31.

  Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. ToGlioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

• Prospective cohort study of radiotherapy with concomitant and adjuvant temozolomide chemotherapy for glioblastoma patients with no or minimal residual enhancing tumor load after surgery.

  Authors: Walter Stummer, Thomas Meinel, Christian Ewelt, Peter Martus, Olga Jakobs, Jörg Felsberg, Guido Reifenberger

  Journal of neuro-oncology. 02/2012;

  Survival of glioblastoma patients has been linked to the completeness of surgical resection. Available data, however, were generated with adjuvant radiotherapy. Data confirming that extensiveSurvival of glioblastoma patients has been linked to the completeness of surgical resection. Available data, however, were generated with adjuvant radiotherapy. Data confirming that extensive cytoreduction remains beneficial to patients treated with the current standard, concomitant temozolomide radiochemotherapy, are limited. We therefore analyzed the efficacy of radiochemotherapy for patients with little or no residual tumor after surgery. In this prospective, non-interventional multicenter cohort study, entry criteria were histological diagnosis of glioblastoma, small enhancing or no residual tumor on post-operative MRI, and intended temozolomide radiochemotherapy. The primary study objective was progression-free survival; secondary study objectives were survival and toxicity. Furthermore, the prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation was investigated in a subgroup of patients. One-hundred and eighty patients were enrolled. Fourteen were excluded by patient request or failure to initiate radiochemotherapy. Twenty-three patients had non-evaluable post-operative imaging. Thus, 143 patients qualified for analysis, with 107 patients having residual tumor diameters ≤1.5 cm. Median follow-up was 24.0 months. Median survival or patients without residual enhancing tumor exceeded the follow-up period. Median survival was 16.9 months for 32 patients with residual tumor diameters >0 to ≤1.5 cm (95% CI: 13.3-20.5, p = 0.039), and 13.9 months (10.3-17.5, overall p < 0.001) for 36 patients with residual tumor diameters >1.5 cm. Patient age at diagnosis and extent of resection were independently associated with survival. Patients with MGMT promoter methylated tumors and complete resection made the best prognosis. Completeness of resection acts synergistically with concomitant and adjuvant radiochemotherapy, especially in patients with MGMT promoter methylation.

• Inflammatory demyelinating brain lesions heralding primary CNS lymphoma.

  Authors: Leila Husseini, Andreas Saleh, Guido Reifenberger, Hans-Peter Hartung, Bernd C Kieseier

  The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques. 01/2012; 39(1):6-10.

  Brain biopsy plays a crucial role in the exploration of suspect white matter lesions in the differential diagnosis of primary central nervous system lymphoma (PCNSL) and inflammatory demyelination.Brain biopsy plays a crucial role in the exploration of suspect white matter lesions in the differential diagnosis of primary central nervous system lymphoma (PCNSL) and inflammatory demyelination. We present the case of a previously healthy, immunocompetent woman, aged fifty-nine, who developed a histologically confirmed demyelinating white matter lesion months prior to the manifestation of a PCNSL. Similar cases of "sentinel lesions" preceding a PCNSL have been reported. In a literature review, we compared the diagnostic features that may be useful to differentiate a PCNSL from inflammatory demyelinating disease in older age. We conclude that the occurrence of large, contrast-enhancing cerebral lesions in older patients with a relapsing-remitting disease course and steroid-resistant vision disorders should lead to the consideration of a PCNSL.

• The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis.

  Authors: Iris Augustin, Violaine Goidts, Angelika Bongers, Grainne Kerr, Gordon Vollert, Bernhard Radlwimmer, Christian Hartmann, Christel Herold-Mende, Guido Reifenberger, Andreas von Deimling, Michael Boutros

  EMBO molecular medicine. 12/2011; 4(1):38-51.

  Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described,Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.

• Predictive impact of MGMT promoter methylation in glioblastoma of the elderly.

  Authors: Guido Reifenberger, Bettina Hentschel, Jörg Felsberg, Gabriele Schackert, Matthias Simon, Oliver Schnell, Manfred Westphal, Wolfgang Wick, Torsten Pietsch, Markus Loeffler, Michael Weller

  International journal of cancer. Journal international du cancer. 12/2011;

  O(6) -methylguanine-DNA-methyltransferase (MGMT) promoter methylation identifies a subpopulation of glioblastoma patients with more favorable prognosis and predicts a benefit from alkylating agentO(6) -methylguanine-DNA-methyltransferase (MGMT) promoter methylation identifies a subpopulation of glioblastoma patients with more favorable prognosis and predicts a benefit from alkylating agent chemotherapy (CT). Little is known about its prevalence and clinical significance in older glioblastoma patients. We studied 233 glioblastoma patients aged 70 years or more (144 males, 89 females, median age: 74 years, range: 70.0-86.6 years), who were prospectively enrolled in the German Glioma Network, for MGMT promoter methylation by methylation-specific PCR (MSP) in all patients and DNA pyrosequencing in 166 patients. MGMT data were correlated with patient outcome. Median progression-free survival (PFS) was 4.8 months (95% CI: 4.3-5.3) and median overall survival (OS) was 7.7 months (95% CI: 6.3-9.0). MGMT promoter methylation was detected by MSP in 134 patients (57.5%). For the whole cohort, PFS was 5.2 versus 4.7 months (p = 0.207) and OS was 8.4 versus 6.4 months (p = 0.031) in patients with versus without MGMT promoter methylation. Patients with MGMT methylated tumors had longer PFS when treated with radiotherapy (RT) plus CT or CT alone compared to patients treated with RT alone. Patients with MGMT unmethylated tumors appeared to derive no survival benefit from CT, regardless of whether given at diagnosis together with RT or as a salvage treatment. Patients treated with RT plus CT or CT alone demonstrated longer OS when pyrosequencing revealed >25% MGMT methylated alleles. Taken together, MGMT promoter methylation may be a useful biomarker to stratify elderly glioblastoma patients for treatment with versus without alkylating agent CT.

• 5-aminolevulinic acid (5-ALA)-induced fluorescence in intracerebral metastases: a retrospective study.

  Authors: Marcel A Kamp, Philipp Grosser, Jörg Felsberg, Philipp J Slotty, Hans-Jakob Steiger, Guido Reifenberger, Michael Sabel

  Acta neurochirurgica. 11/2011; 154(2):223-8; discussion 228.

  Microsurgical, circumferential stripping of intracerebral metastases often proves to be insufficient to prevent local tumor recurrence. We were interested in the potential impact of 5-aminolevulinicMicrosurgical, circumferential stripping of intracerebral metastases often proves to be insufficient to prevent local tumor recurrence. We were interested in the potential impact of 5-aminolevulinic acid (5-ALA)-induced-fluorescence (5-AIF) as a diagnostic tool for the resection of intracerebral metastases. A retrospective analysis was performed for 52 patients who underwent 5-AIF-guided resection for intracerebral mass lesions that histologically corresponded to metastases from tumors outside the central nervous system. The presence of ALA fluorescence in the tumor was determined in each patient. In 42 patients, fluorescence of the resection cavity after tumor removal was additionally recorded. Data were correlated with neuropathological findings in tissue specimens. A total of 32 of the 52 metastases (62%) exhibited 5-AIF in tumor parts. All 5-AIF-positive metastases exhibited an inhomogeneous fluorescence pattern. 5-AIF was neither associated with the histological type nor with the site of origin of the metastases. Residual fluorescence of the resection cavity was detected after macroscopically complete white light resection in 24 patients with 5-AIF positive metastases. Residual tumor tissue was histologically confirmed in 6 of 18 patients with available tissue specimens from such 5-AIF positive areas (33%). The majority of metastases (62%) were 5-AIF positive, suggesting a potential impact of 5-AIF for improved visualization of metastatic tumor tissue within the brain. However, residual 5-AIF after macroscopically complete resection of a metastasis needs to be interpreted with caution because of the limited specificity for detection of residual tumor tissue.

• Unraveling the glioma epigenome: from molecular mechanisms to novel biomarkers and therapeutic targets.

  Authors: Bastian Malzkorn, Marietta Wolter, Markus J Riemenschneider, Guido Reifenberger

  Brain pathology (Zurich, Switzerland). 09/2011; 21(6):619-32.

  Epigenetic regulation of gene expression by DNA methylation and histone modification is frequently altered in human cancers including gliomas, the most common primary brain tumors. In diffuseEpigenetic regulation of gene expression by DNA methylation and histone modification is frequently altered in human cancers including gliomas, the most common primary brain tumors. In diffuse astrocytic and oligodendroglial gliomas, epigenetic changes often present as aberrant hypermethylation of 5'-cytosine-guanine (CpG)-rich regulatory sequences in a large variety of genes, a phenomenon referred to as glioma CpG island methylator phenotype (G-CIMP). G-CIMP is particularly common but not restricted to gliomas with isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) mutation. Recent studies provided a mechanistic link between these genetic mutations and the associated widespread epigenetic modifications. Specifically, 2-hydroxyglutarate, the oncometabolite produced by mutant IDH1 and IDH2 proteins, has been shown to function as a competitive inhibitor of various α-ketoglutarate (α-KG)-dependent dioxygenases, including histone demethylases and members of the ten-eleven-translocation (TET) family of 5-methylcytosine (5mC) hydroxylases. In this review article, we briefly address (i) the basic principles of epigenetic control of gene expression; (ii) the most important methods to analyze focal and global epigenetic alterations in cells and tissues; and (iii) the involvement of epigenetic alterations in the molecular pathogenesis of gliomas. Moreover, we discuss the promising roles of epigenetic alterations as molecular diagnostic markers and novel therapeutic targets, and highlight future perspectives toward unraveling the "glioma epigenome."

• Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes.

  Authors: S Delic, N Lottmann, K Jetschke, G Reifenberger, M J Riemenschneider

  Neuropathology and applied neurobiology. 07/2011; 38(2):201-12.

  S. Delic, N. Lottmann, K. Jetschke, G. Reifenberger and M. J. Riemenschneider (2012) Neuropathology and Applied Neurobiology38, 201-212 Identification and functional validation of CDH11, PCSK6 andS. Delic, N. Lottmann, K. Jetschke, G. Reifenberger and M. J. Riemenschneider (2012) Neuropathology and Applied Neurobiology38, 201-212 Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes Aims: The molecular mechanisms underlying the infiltrative growth of glioblastomas, the most common primary tumours of the central nervous system in adults, are still poorly understood. We aimed to identify and functionally validate novel glioma invasion-associated candidate genes. Methods: Microarray-based expression analysis was applied to identify differentially expressed genes in microdissected infiltrating glioma cells in vivo. Promising candidate genes were selected by the invasion-associated gene ontology terms cell adhesion, endocytosis, extracellular matrix and cell migration and validated in vitro by invasion assays and in situ by immunohistochemistry. Results: We identified 180 up-regulated and 61 down-regulated genes (fold change: ≥2; P < 0.01) in the infiltration zone relative to more central cell-rich tumour areas of malignant astrocytic gliomas (n = 11). Twenty-seven of these genes matched to invasion-related gene ontology terms. From these, we confirmed the genes encoding cadherin-11 (CDH11), proprotein convertase subtilisin/kexin type 6 (PCSK6) and SH3-domain GRB2-like 3 (SH3GL3) as novel glioma invasion-associated candidate genes, with knockdown of PCSK6 and SH3GL3 inhibiting glioma cell invasion, while inhibition of CDH11 promoted glioma cell invasion in vitro. Immunohistochemistry on glioblastoma tissue sections revealed expression of CDH11 and PCSK6 protein in glioma cells of more central, cell-rich tumour areas, with only weak or absent CDH11 immunoreactivity but consistent PCSK6 staining in infiltrating glioma cells. Conclusion: Using molecular profiling of microdissected primary tumour tissue specimens followed by functional in vitro analysis, we identified and validated CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes that likely contribute to the invasive phenotype of malignant gliomas.

• DNA hypermethylation and histone modifications downregulate the candidate tumor suppressor gene RRP22 on 22q12 in human gliomas.

  Authors: Natalie Schmidt, Sonja Windmann, Guido Reifenberger, Markus J Riemenschneider

  Brain pathology (Zurich, Switzerland). 06/2011; 22(1):17-25.

  RRP22 (Ras-related protein on chromosome 22) has been suggested as a candidate tumor suppressor in human cancers. Investigating a panel of 70 human gliomas, we found a frequent decrease in the RRP22RRP22 (Ras-related protein on chromosome 22) has been suggested as a candidate tumor suppressor in human cancers. Investigating a panel of 70 human gliomas, we found a frequent decrease in the RRP22 mRNA expression levels (67%), preferentially in high-grade gliomas [World Health Organization (WHO) grades III and IV] as compared with low-grade gliomas (WHO grade II). Moreover, reduced RRP22 mRNA expression was associated with shorter overall survival in 180 glioblastoma patients included in the National Institutes of Health Repository for Molecular Brain Neoplasia Data (NIH REMBRANDT) database. Decreased RRP22 expression levels were in part explained by 5'-CpG island hypermethylation and increased by the treatment with the demethylating agent 5-aza-2'-deoxycytidine in glioblastoma cell lines. In addition, the in vitro treatment with the histone deacetylase inhibitor trichostatin A alone resulted in RRP22 reexpression as well as a significant increase in the levels of RRP22 promoter DNA bound to pan-acetylated histone H3 and H4. Moreover, in primary human glioblastomas, we observed an increase of H3K9me3-bound and a decrease of pan-Ac-H3-bound RRP22 in comparison with non-neoplastic brain tissue, consistent with a heterochromatinization of the RRP22 promoter. Taken together, our findings demonstrate that both 5'-CpG island hypermethylation and histone modifications contribute to the frequent and prognostically unfavorable transcriptional downregulation of RRP22 in malignant gliomas.

• SOCS3 promoter methylation is mutually exclusive to EGFR amplification in gliomas and promotes glioma cell invasion through STAT3 and FAK activation.

  Authors: Carina Lindemann, Oliver Hackmann, Sabit Delic, Natalie Schmidt, Guido Reifenberger, Markus J Riemenschneider

  Acta neuropathologica. 05/2011; 122(2):241-51.

  The suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of theThe suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of the JAK/STAT signaling pathway. We investigated 60 human gliomas of different histological types for SOCS3 alterations and found frequent SOCS3 promoter hypermethylation and transcriptional downregulation. However, SOCS3 promoter hypermethylation was virtually absent in primary glioblastomas, which are characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. Assessment of the relationship between SOCS3 and EGFR aberrations revealed that SOCS3 promoter hypermethylation was inversely related to both the EGFR gene dosage as well as the EGFR protein expression, thus suggesting SOCS3 inactivation as a mechanism substituting for EGFR activation in a subset of gliomas. In support of this hypothesis, stable shRNA-mediated SOCS3 knock-down in U251 glioblastoma cells resulted in an activation of EGFR-related signaling pathways, i.e. an increase in the activation levels of STAT3, FAK and to a lesser extent MAPK, while the AKT phosphorylation levels remained unaffected. Functionally, SOCS3-depletion caused strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. In summary, our findings suggest that SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation.

• NOA-05 phase 2 trial of procarbazine and lomustine therapy in gliomatosis cerebri.

  Authors: Martin Glas, Oliver Bähr, Jörg Felsberg, Katja Rasch, Dorothee Wiewrodt, Martin Schabet, Matthias Simon, Horst Urbach, Joachim P Steinbach, Johannes Rieger, Rolf Fimmers, Michael Bamberg, Thomas Nägele, Guido Reifenberger, Michael Weller, Ulrich Herrlinger

  Annals of neurology. 05/2011; 70(3):445-53.

  The NOA-05 multicenter trial was performed to analyze the efficacy of primary chemotherapy with procarbazine and lomustine (PC) in patients with gliomatosis cerebri (GC) and to define clinical,The NOA-05 multicenter trial was performed to analyze the efficacy of primary chemotherapy with procarbazine and lomustine (PC) in patients with gliomatosis cerebri (GC) and to define clinical, imaging, and molecular factors influencing outcome. Thirty-five patients with previously untreated GC were treated with up to six 56-day courses of 110mg/m(2) lomustine on day 1 and 60mg/m(2) procarbazine on days 8 to 21. The primary endpoint was the rate of patients without therapy failure (defined as progressive disease, death from any cause, or termination of PC therapy before the end of course 4) at 8 months after the beginning of PC chemotherapy. The failure-free survival rate at 8 months was 50.3%. Median progression-free survival was 14 months. At progression, 12 patients received salvage radiotherapy. Median overall survival was 30 months. Multivariate analysis revealed isocitrate dehydrogenase 1 (IDH1) gene mutation (hazard ratio [HR], 0.11; 95% confidence interval [CI], 0.02-0.58) and initial presentation without a bilateral symmetrical infiltration pattern on magnetic resonance imaging (HR 0.07, 95%CI 0.01-0.54) as independent prognostic factors associated with prolonged survival. IDH1 mutation was significantly associated with MGMT promoter methylation and an oligodendroglial tumor component. PC chemotherapy is effective in GC. With the NOA-05 trial being the first prospective multicenter trial in GC, PC chemotherapy can be regarded as a promising option for the primary therapy of these tumors.

• Molecular markers in low-grade gliomas: predictive or prognostic?

  Authors: Christian Hartmann, Bettina Hentschel, Marcos Tatagiba, Johannes Schramm, Oliver Schnell, Clemens Seidel, Robert Stein, Guido Reifenberger, Torsten Pietsch, Andreas von Deimling, Markus Loeffler, Michael Weller

  Clinical cancer research : an official journal of the American Association for Cancer Research. 05/2011; 17(13):4588-99.

  To investigate whether TP53 mutation, 1p/19q codeletions, O(6)-methylguanylmethyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutation predict natural course ofTo investigate whether TP53 mutation, 1p/19q codeletions, O(6)-methylguanylmethyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutation predict natural course of disease or response to radiotherapy or chemotherapy or both in low-grade glioma patients. Cohort A consisted of 89 patients with diffuse astrocytoma World Health Organization (WHO) grade II (n = 40), oligoastrocytoma (n = 23), or oligodendroglioma (n = 26) who did not receive radiotherapy or chemotherapy after first operation and were monitored until progression [progressive disease (PD); n = 59] and beyond or until the end of follow-up (n = 30). Cohort B consisted of 50 patients with WHO grade II gliomas who received radiotherapy or chemotherapy at diagnosis. Tumors were analyzed for TP53 mutations, 1p/19q codeletions, MGMT promoter methylation, and IDH1 mutations. Median progression-free survival (PFS) in cohort A was 4.1 years (95% CI: 3.1-5.1). No molecular marker was prognostic for PFS after surgery alone, using multivariate adjustment for histology, age, and extent of resection. IDH1 mutations were associated with prolonged survival from the diagnosis of PD in oligoastrocytomas (OA II)/oligodendrogliomas (O II) and with overall survival (OS) in all tumors. 1p/19q codeletion and IDH1 mutation were prognostic for PFS and OS in cohort B. None of the parameters are sensitive prognostic biomarkers in WHO grade II glioma patients who do not receive radiotherapy or chemotherapy after surgery. Limitations of this study include the selection of patients with favorable outcome, the nonrandomized allocation of treatment, and the insufficient sample size to distinguish between effects of radiotherapy versus chemotherapy. Regardless of histology, IDH1 mutation status is the strongest prognostic marker for OS.

• Aberrant expression of retinoic acid signaling molecules influences patient survival in astrocytic gliomas.

  Authors: Benito Campos, Franz-Simon Centner, Justo Lorenzo Bermejo, Ramadan Ali, Katharina Dorsch, Feng Wan, Jörg Felsberg, Rezvan Ahmadi, Niels Grabe, Guido Reifenberger, Andreas Unterberg, Jürgen Burhenne, Christel Herold-Mende

  The American journal of pathology. 05/2011; 178(5):1953-64.

  Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss ofUndifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis.

• Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma.

  Authors: Genevieve Schindler, David Capper, Jochen Meyer, Wibke Janzarik, Heymut Omran, Christel Herold-Mende, Kirsten Schmieder, Pieter Wesseling, Christian Mawrin, Martin Hasselblatt, David N Louis, Andrey Korshunov, Stefan Pfister, Christian Hartmann, Werner Paulus, Guido Reifenberger, Andreas von Deimling

  Acta neuropathologica. 03/2011; 121(3):397-405.

  Missense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene. Initially described inMissense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene. Initially described in melanoma, colon and papillary thyroid carcinoma, these alterations have also been observed in primary nervous system tumors albeit at a low frequency. We analyzed exon 15 of BRAF spanning the V600 locus by direct sequencing in 1,320 adult and pediatric tumors of the nervous system including various types of glial, embryonal, neuronal and glioneuronal, meningeal, adenohypophyseal/sellar, and peripheral nervous system tumors. A total of 96 BRAF mutations were detected; 93 of the V600E type and 3 cases with a three base pair insertion between codons 599 and 600. The highest frequencies of BRAF (V600E) mutations were found in WHO grade II pleomorphic xanthoastrocytomas (42/64; 66%) and pleomorphic xanthoastrocytomas with anaplasia (15/23; 65%), as well as WHO grade I gangliogliomas (14/77; 18%), WHO grade III anaplastic gangliogliomas (3/6) and pilocytic astrocytomas (9/97; 9%). In pilocytic astrocytomas BRAF (V600E) mutation was strongly associated with extra-cerebellar location (p = 0.009) and was most frequent in diencephalic tumors (4/12; 33%). Glioblastomas and other gliomas were characterized by a low frequency or absence of mutations. No mutations were detected in non-glial tumors, including embryonal tumors, meningiomas, nerve sheath tumors and pituitary adenomas. The high mutation frequencies in pleomorphic xanthoastrocytomas, gangliogliomas and extra-cerebellar pilocytic astrocytomas implicate BRAF (V600E) mutation as a valuable diagnostic marker for these rare tumor entities. Future clinical trials should address whether BRAF (V600E) mutant brain tumor patients will benefit from BRAF (V600E)-directed targeted therapies.

• Association of stem cell-related markers and survival in astrocytic gliomas.

  Authors: Feng Wan, Christel Herold-Mende, Benito Campos, Franz-Simon Centner, Christine Dictus, Natalia Becker, Frauke Devens, Carolin Mogler, Jörg Felsberg, Niels Grabe, Guido Reifenberger, Peter Lichter, Andreas Unterberg, Justo Lorenzo Bermejo, Rezvan Ahmadi

  Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals. 03/2011; 16(2):136-43.

  To study the clinical relevance of undifferentiated tumour cells in astrocytic gliomas we employed a large tumour tissue microarray (n=283) with corresponding clinical data and analyzed theTo study the clinical relevance of undifferentiated tumour cells in astrocytic gliomas we employed a large tumour tissue microarray (n=283) with corresponding clinical data and analyzed the expression of Nestin and Sox-2, which mark undifferentiated stem- and progenitor cells in the normal brain. Both markers were expressed abundantly and staining of nestin significantly increased with WHO grade. Further, nestin and Sox-2 immunoreactivity was significantly associated with tumour cell proliferation and nestin expression was independently associated with poor patient survival. Our findings suggest that immature glioma cells are involved in tumour growth and tumour progression and significantly impact on patient prognosis.

• Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas.

  Authors: Jörg Felsberg, Niklas Thon, Sabina Eigenbrod, Bettina Hentschel, Michael C Sabel, Manfred Westphal, Gabriele Schackert, Friedrich Wilhelm Kreth, Torsten Pietsch, Markus Löffler, Michael Weller, Guido Reifenberger, Jörg C Tonn

  International journal of cancer. Journal international du cancer. 03/2011; 129(3):659-70.

  Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). WeEpigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care.