Grischa Toedt

Publications of Grischa Toedt

• 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.

• FSTL5 is a marker of poor prognosis in non-WNT/non-SHH medulloblastoma.

  Authors: Marc Remke, Thomas Hielscher, Andrey Korshunov, Paul A Northcott, Sebastian Bender, Marcel Kool, Frank Westermann, Axel Benner, Huriye Cin, Marina Ryzhova [......] Andrea Wittmann, Anna Schöttler, André O von Bueren, Andreas von Deimling, Stefan Rutkowski, Wolfram Scheurlen, Andreas E Kulozik, Michael D Taylor, Peter Lichter, Stefan M Pfister

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 09/2011; 29(29):3852-61.

  Integrated genomics approaches have revealed at least four distinct biologic variants of medulloblastoma: WNT (wingless), SHH (sonic hedgehog), group C, and group D. Because of the remarkableIntegrated genomics approaches have revealed at least four distinct biologic variants of medulloblastoma: WNT (wingless), SHH (sonic hedgehog), group C, and group D. Because of the remarkable clinical heterogeneity of group D tumors and the dismal prognosis of group C patients, it is vital to identify molecular biomarkers that will allow early and effective treatment stratification in these non-WNT/non-SHH tumors. We combined transcriptome and DNA copy-number analyses for 64 primary medulloblastomas. Bioinformatic tools were used to discover marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the screening cohort. The prognostic power of follistatin-like 5 (FSTL5) immunopositivity was tested for 235 nonoverlapping medulloblastoma samples on two independent tissue microarrays. Comprehensive analyses of transcriptomic and genetic alterations delineate four distinct variants of medulloblastoma. Stable subgroup separation was achieved by using the 300 transcripts that varied the most. Distinct expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time polymerase chain reaction. Immunopositivity of FSTL5 identified a large cohort of patients (84 of 235 patients; 36%) at high risk for relapse and death. Importantly, more than 50% of non-WNT/non-SHH tumors displayed FSTL5 negativity, delineating a large patient cohort with a good prognosis who would otherwise be considered intermediate or high-risk on the basis of current molecular subgrouping. FSTL5 expression denoted a dismal prognosis both within and across medulloblastoma subgroups. The addition of FSTL5 immunohistochemistry to existing molecular stratification schemes constitutes a reliable and cost-effective tool for prognostication in future clinical trials of medulloblastoma.

• Molecular signatures classify astrocytic gliomas by IDH1 mutation status.

  Authors: Grischa Toedt, Sebastian Barbus, Marietta Wolter, Jörg Felsberg, Björn Tews, Frederic Blond, Michael C Sabel, Stefanie Hofmann, Natalia Becker, Christian Hartmann, Hiroko Ohgaki, Andreas von Deimling, Otmar D Wiestler, Meinhard Hahn, Peter Lichter, Guido Reifenberger, Bernhard Radlwimmer

  International journal of cancer. Journal international du cancer. 03/2011; 128(5):1095-103.

  To identify novel glioma-associated pathomechanisms and molecular markers, we performed an array-based comparative genomic hybridization analysis of 131 diffuse astrocytic gliomas, including 87To identify novel glioma-associated pathomechanisms and molecular markers, we performed an array-based comparative genomic hybridization analysis of 131 diffuse astrocytic gliomas, including 87 primary glioblastomas (pGBIV), 13 secondary glioblastomas (sGBIV), 19 anaplastic astrocytomas (AAIII) and 12 diffuse astrocytomas (AII). All tumors were additionally screened for IDH1 and IDH2 mutations. Expression profiling was performed for 74 tumors (42 pGBIV, 11 sGBIV, 13 AAIII, 8 AII). Unsupervised and supervised bioinformatic analyses revealed distinct genomic and expression profiles separating pGBIV from the other entities. Classifier expression signatures were strongly associated with the IDH1 gene mutation status. Within pGBIV, the rare subtype of IDH1 mutant tumors shared expression profiles with IDH1 mutant sGBIV and was associated with longer overall survival compared with IDH1 wild-type tumors. In patients with IDH1 wild-type pGBIV, PDGFRA gain or amplification as well as 19q gain were associated with patient outcome. Array-CGH analysis additionally revealed homozygous deletions of the FGFR2 gene at 10q26.13 in 2 pGBIV, with reduced FGFR2 mRNA levels being frequent in pGBIV and linked to poor outcome. In conclusion, we report that diffuse astrocytic gliomas can be separated into 2 major molecular groups with distinct genomic and mRNA profiles as well as IDH1 gene mutation status. In addition, our results suggest FGFR2 as a novel glioma-associated candidate tumor suppressor gene on the long arm of chromosome 10.

• Elastic SCAD as a novel penalization method for SVM classification tasks in high-dimensional data.

  Authors: Natalia Becker, Grischa Toedt, Peter Lichter, Axel Benner

  BMC bioinformatics. 01/2011; 12:138.

  Classification and variable selection play an important role in knowledge discovery in high-dimensional data. Although Support Vector Machine (SVM) algorithms are among the most powerfulClassification and variable selection play an important role in knowledge discovery in high-dimensional data. Although Support Vector Machine (SVM) algorithms are among the most powerful classification and prediction methods with a wide range of scientific applications, the SVM does not include automatic feature selection and therefore a number of feature selection procedures have been developed. Regularisation approaches extend SVM to a feature selection method in a flexible way using penalty functions like LASSO, SCAD and Elastic Net.We propose a novel penalty function for SVM classification tasks, Elastic SCAD, a combination of SCAD and ridge penalties which overcomes the limitations of each penalty alone.Since SVM models are extremely sensitive to the choice of tuning parameters, we adopted an interval search algorithm, which in comparison to a fixed grid search finds rapidly and more precisely a global optimal solution. Feature selection methods with combined penalties (Elastic Net and Elastic SCAD SVMs) are more robust to a change of the model complexity than methods using single penalties. Our simulation study showed that Elastic SCAD SVM outperformed LASSO (L1) and SCAD SVMs. Moreover, Elastic SCAD SVM provided sparser classifiers in terms of median number of features selected than Elastic Net SVM and often better predicted than Elastic Net in terms of misclassification error.Finally, we applied the penalization methods described above on four publicly available breast cancer data sets. Elastic SCAD SVM was the only method providing robust classifiers in sparse and non-sparse situations. The proposed Elastic SCAD SVM algorithm provides the advantages of the SCAD penalty and at the same time avoids sparsity limitations for non-sparse data. We were first to demonstrate that the integration of the interval search algorithm and penalized SVM classification techniques provides fast solutions on the optimization of tuning parameters.The penalized SVM classification algorithms as well as fixed grid and interval search for finding appropriate tuning parameters were implemented in our freely available R package 'penalizedSVM'.We conclude that the Elastic SCAD SVM is a flexible and robust tool for classification and feature selection tasks for high-dimensional data such as microarray data sets.

• Recurrent copy number gain of transcription factor SOX2 and corresponding high protein expression in oral squamous cell carcinoma.

  Authors: Kolja Freier, Karl Knoepfle, Christa Flechtenmacher, Susanne Pungs, Frauke Devens, Grischa Toedt, Christof Hofele, Stefan Joos, Peter Lichter, Bernhard Radlwimmer

  Genes, chromosomes & cancer. 09/2009;

  Gene copy number aberrations are involved in oral squamous cell carcinoma (OSCC) development. To delineate candidate genes inside critical chromosomal regions, array-CGH was applied to 40 OSCCGene copy number aberrations are involved in oral squamous cell carcinoma (OSCC) development. To delineate candidate genes inside critical chromosomal regions, array-CGH was applied to 40 OSCC specimens using a microarray covering the whole human genome with an average resolution of 1 Mb. Gene copy number gains were predominantly found at 1q23 (9 cases), 3q26 (11), 5p15 (13), 7p11 (7), 8q24 (17), 11q13 (15), 14q32 (8), 19p13 (8), 19q12 (7), 19q13 (8), and 20q13 (9), whereas gene copy number losses were detected at 3p21-3p12 (15), 8p32 (11), 10p12 (8), and 18q21-q23 (10). Subsequent mRNA expression analyses by quantitative real time polymerase chain reaction found high mRNA expression of candidate genes SOX2 in 3q26.33, FSLT3 in 19p13.3, and CCNE1 in 19q12. Tissue microarray (TMA) analyses in a representative OSCC collection found gene copy number gain for SOX2 in 52% (115/223) and for CCNE1 in 31% (72/233) of the tumors. Immunohistochemical analyses on TMA sections of the corresponding proteins detected high expression of SOX2 in 18.1% (49/271) and of CyclinE1 in 23.3% (64/275) of tumors analyzed. These findings indicate that SOX2 and CCNE1 might be activated via gene copy number gain and participate in oral carcinogenesis. The combination of array-CGH with TMA analyses allows rapid pinpointing of novel promising candidate genes, which might be used as therapeutic stratification markers or target molecules for therapeutic interference. (c) 2009 Wiley-Liss, Inc.

• Outcome prediction in pediatric medulloblastoma based on DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci.

  Authors: Stefan Pfister, Marc Remke, Axel Benner, Frank Mendrzyk, Grischa Toedt, Jörg Felsberg, Andrea Wittmann, Frauke Devens, Nicolas U Gerber, Stefan Joos, Andreas Kulozik, Guido Reifenberger, Stefan Rutkowski, Otmar D Wiestler, Bernhard Radlwimmer, Wolfram Scheurlen, Peter Lichter, Andrey Korshunov

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 05/2009; 27(10):1627-36.

  PURPOSE: Medulloblastoma is the most common malignant brain tumor in children. Current treatment decisions are based on clinical variables. Novel tumor-derived biomarkers may improve the riskPURPOSE: Medulloblastoma is the most common malignant brain tumor in children. Current treatment decisions are based on clinical variables. Novel tumor-derived biomarkers may improve the risk stratification of medulloblastoma patients. PATIENTS AND METHODS: A model for the molecular risk stratification was proposed from an array-based comparative genomic hybridization (array-CGH) screen (n = 80). Fluorescence in situ hybridization (FISH) analyses for chromosome arms 6q, 17p, and 17q and the MYC and MYCN loci were performed in an independent validation set (n = 260). Copy number aberrations were correlated with clinical, histologic, and survival data. RESULTS: Gain of 6q and 17q and genomic amplification of MYC or MYCN were each associated with poor outcome in the array-CGH study (n = 80). In contrast, all patients with 6q-deleted tumors survived. Given these findings, the following hierarchical molecular staging system was defined: (1) MYC/MYCN amplification, (2) 6q gain, (3) 17q gain, (4) 6q and 17q balanced, and (5) 6q deletion. The prognostic value of this staging system was investigated by FISH analysis (n = 260). The addition of molecular markers to clinical risk factors resulted in the identification of a large proportion of patients (72 of 260 patients; 30%) at high risk for relapse and death who would be considered standard risk by application of clinical variables alone. CONCLUSION: Genomic aberrations in medulloblastoma are powerful independent markers of disease progression and survival. By adding genomic markers to established clinical and histologic variables, outcome prediction can be substantially improved. Because the analyses can be conducted on routine paraffin-embedded material, it will be especially feasible to use this novel molecular staging system in large multicenter clinical trials.

• penalizedSVM: a R-package for Feature Selection SVM Classification.

  Authors: Natalia Becker, Wiebke Werft, Grischa Toedt, Peter Lichter, Axel Benner

  Bioinformatics (Oxford, England). 05/2009;

  SUMMARY: Support Vector Machine (SVM) classification is a widely used and one of the most powerful classification techniques. However, a major limitation is that SVM can not perform automatic geneSUMMARY: Support Vector Machine (SVM) classification is a widely used and one of the most powerful classification techniques. However, a major limitation is that SVM can not perform automatic gene selection. To overcome this restriction, a number of penalized feature selection methods have been proposed. In the R package 'penalizedSVM' implemented penalization functions L(1) norm and Smoothly Clipped Absolute Deviation (SCAD) provide automatic feature selection for SVM classification tasks. AVAILABILITY: The R package 'penalizedSVM' is available from the Comprehensive R Archive Network (http://cran.r-project.org/) under GPL-2 or later. CONTACT: natalia.becker@dkfz.de SUPPLEMENTARY INFORMATION: The package contains documentation in the form of manual pages.

• High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-{beta} and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response.

  Authors: Marc Remke, Stefan Pfister, Corinne Kox, Grischa Toedt, Natalia Becker, Axel Benner, Wiebke Werft, Stephen Breit, Shuangyou Liu, Felix Engel, Andrea Wittmann, Martin Zimmermann, Martin Stanulla, Martin Schrappe, Wolf-Dieter Ludwig, Claus R Bartram, Bernhard Radlwimmer, Martina U Muckenthaler, Peter Lichter, Andreas E Kulozik

  Blood. 05/2009;

  Precursor T-cell lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high risk patients as early asPrecursor T-cell lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL, and performed array-based comparative genomic hybridization (array-CGH) and gene expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70/73 (96%) patients. Notably, genomic imbalances predicted to downregulate the TGF-beta or upregulate the PI3K-AKT pathways are identified in 25/73 (34%) and 21/73 (29%) of the patients, suggesting that these pathways play a key role in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9/73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene whose expression is also shown to be downregulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation suggesting a likely functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here thus implicate the TGF-beta and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of T-ALL patients with CASP8AP2 deletions and poor early treatment response.

• BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas.

  Authors: Stefan Pfister, Wibke G Janzarik, Marc Remke, Aurélie Ernst, Wiebke Werft, Natalia Becker, Grischa Toedt, Andrea Wittmann, Christian Kratz, Heike Olbrich [......] Bernhard Radlwimmer, Andreas Kulozik, Torsten Pietsch, Christel Herold-Mende, Astrid Gnekow, Guido Reifenberger, Andrey Korshunov, Wolfram Scheurlen, Heymut Omran, Peter Lichter

  The Journal of clinical investigation. 06/2008; 118(5):1739-49.

  The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis ofThe molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

• Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.

  Authors: Christof Schlaeger, Thomas Longerich, Claudia Schiller, Peter Bewerunge, Arianeb Mehrabi, Grischa Toedt, Jörg Kleeff, Volker Ehemann, Roland Eils, Peter Lichter, Peter Schirmacher, Bernhard Radlwimmer

  Hepatology (Baltimore, Md.). 03/2008; 47(2):511-20.

  Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is characterized by aggressive tumor behavior coupled with poor prognosis. Various etiologies have been linked to HCCHepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is characterized by aggressive tumor behavior coupled with poor prognosis. Various etiologies have been linked to HCC development, most prominently chronic hepatitis B and C virus infections as well as chronic alcohol consumption. In approximately 10% of HCCs, the etiology remains cryptic; however, recent epidemiological data suggest that most of these cryptogenic HCCs develop due to nonalcoholic steatohepatitis. To identify etiology-dependent DNA copy number aberrations and genes relevant to hepatocarcinogenesis, we performed array-based comparative genomic hybridization of 63 HCCs of well-defined etiology and 4 HCC cell lines followed by gene expression profiling and functional analyses of candidate genes. For a 10-megabase chromosome region on 8q24, we observed etiology-dependent copy number gains and MYC overexpression in viral and alcohol-related HCCs, resulting in up-regulation of MYC target genes. Cryptogenic HCCs showed neither 8q24 gains, nor MYC overexpression, nor target gene activation, suggesting that tumors of this etiology develop by way of a distinct MYC-independent pathomechanism. Furthermore, we detected several etiology-independent small chromosome aberrations, including amplification of MDM4 on 1q32.1 and frequent gains of EEF1A2 on 20q13.33. Both genes were overexpressed in approximately half the HCCs examined, and gene silencing reduced cell viability as well as proliferation and increased apoptosis rates in HCC cell lines. CONCLUSION: Our findings suggest that MDM4 and EEF1A2 act as etiology-independent oncogenes in a significant percentage of HCCs.

• Supratentorial primitive neuroectodermal tumors of the central nervous system frequently harbor deletions of the CDKN2A locus and other genomic aberrations distinct from medulloblastomas.

  Authors: Stefan Pfister, Marc Remke, Grischa Toedt, Wiebke Werft, Axel Benner, Frank Mendrzyk, Andrea Wittmann, Frauke Devens, Katja von Hoff, Stefan Rutkowski, Andreas Kulozik, Bernhard Radlwimmer, Wolfram Scheurlen, Peter Lichter, Andrey Korshunov

  Genes, chromosomes & cancer. 09/2007; 46(9):839-51.

  Supratentorial primitive neuroectodermal tumors (stPNETs) and medulloblastomas have long been thought to arise from a common cell type in the subventricular germinal matrix. Because of the infrequentSupratentorial primitive neuroectodermal tumors (stPNETs) and medulloblastomas have long been thought to arise from a common cell type in the subventricular germinal matrix. Because of the infrequent occurrence of stPNETs, little is known about their genetic background. Here, we performed a genome-wide screening for DNA copy-number aberrations in 10 supratentorial PNETs using array-based comparative genomic hybridization (array-CGH). Comparing our findings with data from a previous array-CGH study on 47 medulloblastomas, we identified differences in the frequency of copy-number losses at chromosome regions 1p12-22.1 and 9p, and gains at 19p, all of them more frequently occurring in stPNETs. In contrast to previous reports, we detected chromosome 17 aberrations by array-CGH in 2/10 stPNETs. To validate our findings obtained by array-CGH, we analyzed the loci of interest by fluorescence in situ hybridization in an independent set of 11 stPNETs and found deletions of 9p21 in 5/11 tumors of the second set, three of them being homozygous. All 9p21 deletions were associated with loss of CDKN2A protein expression. Altogether, CDKN2A deletions were detected in 7/21 stPNETs including four homozygous deletions, whereas such deletions were only found in 4/112 medulloblastomas, all of these being heterozygous (P < 0.001). Gains of 19p (14% vs. 0% in medulloblastomas, P = 0.02) were found to be significantly more frequent in stPNETs, whereas gains of 17q (14% vs. 45% in medulloblastomas, P = 0.02) were confirmed to be more frequent in medulloblastomas. These data further support the hypothesis of two different tumor entities of embryonal neuroepithelial tumors with characteristic genetic aberrations. (c) 2007 Wiley-Liss, Inc.

• Identification of novel oligodendroglioma-associated candidate tumor suppressor genes in 1p36 and 19q13 using microarray-based expression profiling.

  Authors: Bjoern Tews, Joerg Felsberg, Christian Hartmann, Annegret Kunitz, Meinhard Hahn, Grischa Toedt, Kai Neben, Lars Hummerich, Andreas von Deimling, Guido Reifenberger, Peter Lichter

  International journal of cancer. Journal international du cancer. 09/2006; 119(4):792-800.

  Loss of heterozygosity (LOH) on chromosomal arms 1p and 19q is the most common genetic alteration in oligodendroglial tumors and associated with response to radio- and chemotherapy as well asLoss of heterozygosity (LOH) on chromosomal arms 1p and 19q is the most common genetic alteration in oligodendroglial tumors and associated with response to radio- and chemotherapy as well as favorable prognosis. Using microsatellite analysis, we previously identified the chromosomal regions 1p36.22-p36.31 and 19q13.3, as candidate tumor suppressor gene regions being commonly deleted in these tumors. To identify genes within these regions that are downregulated in oligodendroglial tumors with LOH 1p/19q, we performed cDNA microarray-based RNA expression profiling of 35 gliomas with known allelic status on 1p and 19q, including 7 oligodendrogliomas and 8 diffuse astrocytomas of World Health Organization (WHO) grade II, as well as 14 anaplastic oligodendrogliomas and 6 anaplastic oligoastrocytomas of WHO grade III. The microarrays used for expression profiling carried approximately 7,000 gene-specific cDNAs, with complete coverage of the genes located in 1p36.13-p36.31 and 19q13.2-q13.33. Microarray analysis identified 8 genes from these regions (MGC4399, SRM, ICMT, RPL18, FTL, ZIN, FLJ10781 and DBP), which all showed significantly lower expression in 1p/19q-deleted gliomas when compared to gliomas without 1p/19q losses. Quantitative real-time reverse transcription-PCR analyses were performed for the MGC4399, ICMT and RPL18 genes and confirmed the microarray findings. In addition, we found that the cytosolic phospholipase A2 (PLA2G4C) gene at 19q13.3 demonstrated significantly lower expression in anaplastic oligodendrogliomas (WHO grade III) when compared to well-differentiated oligodendrogliomas (WHO grade II). Taken together, our study provides a set of interesting novel candidate genes that may play important roles in the pathogenesis of oligodendroglial tumors.

• Isochromosome breakpoints on 17p in medulloblastoma are flanked by different classes of DNA sequence repeats.

  Authors: Frank Mendrzyk, Andrey Korshunov, Grischa Toedt, Frank Schwarz, Bernhard Korn, Stefan Joos, Andreas Hochhaus, Claudia Schoch, Peter Lichter, Bernhard Radlwimmer

  Genes, chromosomes & cancer. 05/2006; 45(4):401-10.

  Medulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20%-25% of all intracranial pediatric tumors. The most frequent chromosomal rearrangement in medulloblastomaMedulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20%-25% of all intracranial pediatric tumors. The most frequent chromosomal rearrangement in medulloblastoma is isochromosome 17, or i(17q). Its frequency suggests that it serves an important role in tumor pathogenesis, possibly mediated by the disruption or permanent activation of a gene at the breakpoint. To address this question, we performed a detailed analysis of chromosome 17 DNA copy number from 18 medulloblastomas previously shown to carry an apparent i(17q). We identified two breakpoint regions, one well within band 17p11.2 (n = 16) and a second within the pericentromeric region (n = 2). To map the breakpoints more precisely, we constructed a tiling-path matrix-CGH array covering chromosomal band 17p11.2 to the centromere and utilized it to delineate two small breakpoint intervals mapping at Mb 19.0 and 21.7 in seven of the medulloblastomas and in nine hematological neoplasias with i(17q). The former interval contains two breakpoint clusters that each colocalize with a pair of head-to-head inverted DNA sequence repeats, and the latter maps close to a region of alpha-satellite repeats. No consensus coding sequence localizes in these regions. Together, these data strongly suggest that the effects of i(17q) in medulloblastoma are mediated by gene-dosage effects of genes on 17p or 17q rather than by the disruption or deregulation of a "breakpoint" gene. Furthermore, we identified artifacts introduced in DNA copy number data by cross-hybridization of low-copy repeat sequences and discuss the challenge these can pose in the interpretation of diagnostic microarrays.

• Gene expression signature predicting pathologic complete response with gemcitabine, epirubicin, and docetaxel in primary breast cancer.

  Authors: Olaf Thuerigen, Andreas Schneeweiss, Grischa Toedt, Patrick Warnat, Meinhard Hahn, Heidi Kramer, Benedikt Brors, Christian Rudlowski, Axel Benner, Florian Schuetz, Bjoern Tews, Roland Eils, Hans-Peter Sinn, Christof Sohn, Peter Lichter

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 05/2006; 24(12):1839-45.

  PURPOSE: Primary systemic therapy (PST) with gemcitabine (G), epirubicin (E), and docetaxel (Doc) has resulted in a pathologic complete response (pCR) in 26% of primary breast cancer patients. ThisPURPOSE: Primary systemic therapy (PST) with gemcitabine (G), epirubicin (E), and docetaxel (Doc) has resulted in a pathologic complete response (pCR) in 26% of primary breast cancer patients. This study was aimed at the identification of a gene expression signature in diagnostic core biopsy tissue samples that predicts pCR. PATIENTS AND METHODS: Core biopsy samples from patients with operable primary breast cancer, T2-4N0-2M0, enrolled onto two phase I and II trials evaluating GEDoc (n = 48) and GE sequentially followed by Doc (GEsDoc; n = 52) as PST were snap frozen and subjected to RNA expression profiling. A signature predicting pCR was discovered in the training set (GEsDoc) applying a support vector machine algorithm, and performance of this classifier was validated on the independent test set (GEDoc) by receiver operator characteristics analysis. RESULTS: We identified a signature consisting of 512 genes, which was enriched in genes involved in transforming growth factor beta and RAS-mediated signaling pathways, that predicts pCR with a sensitivity of 78%, a specificity of 90%, and an overall accuracy of 88% (95% CI, 75% to 95%). Apart from our signature, only HER2 overexpression was an independent predictor of pCR in multivariate analysis. CONCLUSION: In conclusion, our gene expression signature allows prediction of pCR to PST containing G, E, and Doc with unprecedented high overall accuracy and robustness.

• Identification of gains on 1q and epidermal growth factor receptor overexpression as independent prognostic markers in intracranial ependymoma.

  Authors: Frank Mendrzyk, Andrey Korshunov, Axel Benner, Grischa Toedt, Stefan Pfister, Bernhard Radlwimmer, Peter Lichter

  Clinical cancer research : an official journal of the American Association for Cancer Research. 04/2006; 12(7 Pt 1):2070-9.

  PURPOSE: Pathogenesis of ependymomas is still poorly understood and molecular markers for risk-adapted patient stratification are not available. Our aim was to screen for novel genomic imbalances andPURPOSE: Pathogenesis of ependymomas is still poorly understood and molecular markers for risk-adapted patient stratification are not available. Our aim was to screen for novel genomic imbalances and prognostic markers in ependymal tumors. EXPERIMENTAL DESIGN: We analyzed 68 sporadic tumors by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. Novel recurrent genomic gains were validated by fluorescence in situ hybridization using a tissue microarray consisting of 170 intracranial ependymomas. Candidate genes were also tested for mRNA expression by quantitative real-time PCR, and protein expression was determined by immunohistochemistry on the tissue microarray. RESULTS: Chromosomal gain of 1q correlated with pediatric patients (P = 0.004), intracranial ependymomas (P = 0.05), and tumors of grade III (P = 0.002). Gain of 1q21.1-32.1 was associated with tumor recurrence in intracranial ependymomas (P < 0.001). Furthermore, gain of 1q25 as determined by fluorescence in situ hybridization represented an independent prognostic marker for either recurrence-free survival (P < 0.001) or overall survival (P = 0.003). Recurrent gains at 5p15.33 covering hTERT were validated by immunohistochemistry, and elevated protein levels correlated with adverse prognosis (P = 0.01). In addition to frequent gains and high-level amplification of epidermal growth factor receptor (EGFR) at 7p11.2, immunohistochemistry revealed protein overexpression to be correlated with poor prognosis (P = 0.002). EGFR protein status subdivides intracranial grade II ependymomas into two different risk groups (P = 0.03) as shown by multivariate analysis. CONCLUSIONS: Thus, the states of 1q25 and EGFR represent independent prognostic markers for intracranial ependymomas to identify patient subgroups with different risk profiles in further clinical investigations. Moreover, EGFR might serve as therapeutic target for more specific chemotherapy applications.

• Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma.

  Authors: Kolja Freier, Carsten Sticht, Christof Hofele, Christa Flechtenmacher, Daniel Stange, Laura Puccio, Grischa Toedt, Bernhard Radlwimmer, Peter Lichter, Stefan Joos

  Genes, chromosomes & cancer. 03/2006; 45(2):118-25.

  Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. ThoughChromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.

• High-resolution genomic profiling reveals association of chromosomal aberrations on 1q and 16p with histologic and genetic subgroups of invasive breast cancer.

  Authors: Daniel E Stange, Bernhard Radlwimmer, Falk Schubert, Frank Traub, Andreas Pich, Grischa Toedt, Frank Mendrzyk, Ulrich Lehmann, Roland Eils, Hans Kreipe, Peter Lichter

  Clinical cancer research : an official journal of the American Association for Cancer Research. 02/2006; 12(2):345-52.

  PURPOSE: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread,PURPOSE: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread, and epidemiologic features. To elucidate the genetic basis of these differences, we analyzed copy number imbalances that differentiate the histologic subtypes.EXPERIMENTAL DESIGN: High-resolution genomic profiling of 40 invasive breast cancers using matrix-comparative genomic hybridization with an average resolution of 0.5 Mb was conducted on bacterial artificial chromosome microarrays. The data were subjected to classification and unsupervised hierarchical cluster analyses. Expression of candidate genes was analyzed in tumor samples.RESULTS: The highest discriminating power was achieved when combining the aberration patterns of chromosome arms 1q and 16p, which were significantly more often gained in ILC. These regions were further narrowed down to subregions 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. Located within the candidate gains on 1q are two genes, FMO2 and PTGS2, known to be overexpressed in ILC relative to invasive ductal carcinoma. Assessment of four candidate genes on 16p11.2 by real-time quantitative PCR revealed significant overexpression of FUS and ITGAX in ILC with 16p copy number gain. Unsupervised hierarchical cluster analysis identified three molecular subgroups that are characterized by different aberration patterns, in particular concerning gain of MYC (8q24) and the identified candidate regions on 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. These genetic subgroups differed with regard to histology, tumor grading, frequency of alterations, and estrogen receptor expression.CONCLUSIONS: Molecular profiling using bacterial artificial chromosome arrays identified DNA copy number imbalances on 1q and 16p as significant classifiers of histologic and molecular subgroups.

• Patient-based cross-platform comparison of oligonucleotide microarray expression profiles.

  Authors: Joerg Schlingemann, Negusse Habtemichael, Carina Ittrich, Grischa Toedt, Heidi Kramer, Markus Hambek, Rainald Knecht, Peter Lichter, Roland Stauber, Meinhard Hahn

  Laboratory investigation; a journal of technical methods and pathology. 09/2005; 85(8):1024-39.

  The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether inter-platform differences may conceal biologicallyThe comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether inter-platform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general.

• Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization.

  Authors: Boris Zielinski, Sandrine Gratias, Grischa Toedt, Frank Mendrzyk, Daniel E Stange, Bernhard Radlwimmer, Dietmar R Lohmann, Peter Lichter

  Genes, chromosomes & cancer. 08/2005; 43(3):294-301.

  The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on aThe genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33-p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma.

• FACT--a framework for the functional interpretation of high-throughput experiments.

  Authors: Felix Kokocinski, Nicolas Delhomme, Gunnar Wrobel, Lars Hummerich, Grischa Toedt, Peter Lichter

  BMC bioinformatics. 02/2005; 6:161.

  BACKGROUND: Interpreting the results of high-throughput experiments, such as those obtained from DNA-microarrays, is an often time-consuming task due to the high number of data-points that need to beBACKGROUND: Interpreting the results of high-throughput experiments, such as those obtained from DNA-microarrays, is an often time-consuming task due to the high number of data-points that need to be analyzed in parallel. It is usually a matter of extensive testing and unknown beforehand, which of the possible approaches for the functional analysis will be the most informative. RESULTS: To address this problem, we have developed the Flexible Annotation and Correlation Tool (FACT). FACT allows for detection of important patterns in large data sets by simplifying the integration of heterogeneous data sources and the subsequent application of different algorithms for statistical evaluation or visualization of the annotated data. The system is constantly extended to include additional annotation data and comparison methods. CONCLUSION: FACT serves as a highly flexible framework for the explorative analysis of large genomic and proteomic result sets. The program can be used online; open source code and supplementary information are available at http://www.factweb.de.