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Supplemental Table/Figure Legends

Gene expression signatures for immune and stromal cell populations.

Summary of functional signatures.

Summary of cases, clinical variables and expression of immune signatures.

Immunologic treatment strategies are under investigation for high-grade gliomas. Determining relevant immunologic pathways is required for invigorating a tumor-specific immune response. We therefore investigated the immunologic phenotypes within different subgroups of high-grade gliomas, with a focus on rare genetic subgroups of pediatric and adolescent patients to identify potentially targetable mechanisms. We gathered published gene-expression data from 1,135 high-grade glioma patients and applied a machine-learning technique to determine their transcriptional (mesenchymal, classic, neural, and proneural) and mutational [K27, G34, IDH, and wild type (WT)] subtypes. Gene signatures of infiltrating immune cells and functional immune pathways were evaluated in correlation to histologic diagnosis, age, and transcriptional and mutational subgroups. Our analysis identified four distinct microenvironmental signatures of immune cell infiltration (immune 1–4), which can be stratified into vascular, monocytic/stromal, monocytic/T-cell–, and antigen-presenting cell (APC)/natural killer (NK) cell/T-cell–dominated immune clusters. Immune cell expression profiles correlated with transcriptional and mutational subgroups but were independent of age and histologic diagnosis. By including functional pathways and correlating the expression of immunostimulatory and -inhibitory receptor–ligand interactions, we were able to define the immunologic microenvironment and identify possible immunologic subtypes associated with poor prognosis. In addition, comparison of overall survival with the immunologic landscape and with checkpoint molecules revealed correlations within the transcriptional and mutational subgroups, highlighting the potential application of PD-1/PD-L1 checkpoint inhibition in K27-mutated tumors. Our study shows that transcriptional and mutational subgroups are characterized by distinct immunologic tumor microenvironments, demonstrating the immunologic heterogeneity within high-grade gliomas and suggesting an immune-specific stratification for upcoming immunotherapy trials.

Supplementary Figures 1-4

Suppl. Table S1 - A summary of the human cohort used for DNA methylation-profiling_DRG3003

Suppl. Table S3 - Differentially expressed genes of ST-EPN-RELA and cluster 2 - 4

Suppl. Table S2 - Copy number alterations for all 613 tumors

Supplementary Figures and methods

Suppl. Table S6 - Primer list

Suppl. Table S4 - Histopathological evaluation of 25 cases

Suppl. Table S5 - GO analysis for C11orf95-target models specific DEGs

Suppl. Table S7 - Antibody list

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA–RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation–based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion–positive tumors.Significance:ZFTA–RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion–positive tumors, such as GLI2.This article is highlighted in the In This Issue feature, p. 2113

Purpose: The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell-cycle progression. It is involved in the development of multiple organs, and we have previously reported on its important role for the mitotic entry of cerebellar granule neuron precursors. Constitutive expression of FoxM1 is required for the growth of multiple cancer types. This study aimed to determine its role in medulloblastoma, the most frequent malignant brain tumor in childhood that can derive from cerebellar granule neuron precursors.Experimental Design: We evaluated the expression of FoxM1 together with its prognostic value in two independent series of human medulloblastoma samples using immunohistochemistry (n = 43) and gene expression arrays (n = 193). The functional impact of FoxM1 expression was characterized by knockdown experiments in four human medulloblastoma cell lines, and the thiazole antibiotic siomycin A was tested to downregulate FoxM1 and inhibit tumor cell growth.Results: FoxM1 was highly expressed in all subtypes of medulloblastoma. Importantly, expression levels of FoxM1 significantly correlated with unfavorable clinical outcome in univariate analysis (P = 0.0005), and FoxM1 was identified as an independent prognostic marker by multivariate analysis (P = 0.037). Knockdown of FoxM1 in medulloblastoma cell lines resulted in a significant decrease of cell viability which was caused by a failure in mitotic spindle formation and caspase-dependent mitotic catastrophe. Siomycin A significantly inhibited the expression of FoxM1 and the growth of medulloblastoma cells.Conclusions: FoxM1 may be used as an additional prognostic marker and may represent a potential novel target to treat patients suffering from medulloblastoma. Clin Cancer Res; 17(21); 6791–801. ©2011 AACR.

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Introduction: Genome-wide methylation analyses recently revealed novel epigenetic pineoblastoma (PBL) subtypes, but so far few data are available on recurrent cytogenetic alterations due to the rarity of these neoplasms. Therefore, our aim was to shed further light onto the molecular and genetic characteristics underlying the pathogenesis of pineoblastoma subtypes. Experimental procedures: Cytogenetic alterations of tumor samples of 60 patients with a diagnosis of PBL confirmed by reference neuropathology and methylation array profiling (450K or EPIC BeadChips (Illumina)) were analyzed by high-resolution genome-wide molecular inversion probe analysis. 52 cases could be screened for mutations by next-generation DNA panel sequencing. Survival data of 41 patients were available. All patients had surgery and older patients (≥4y) received RT followed by maintenance chemotherapy (n=37); infants ( Results: Of the recently published epigenetic consensus PBL subtypes (Liu et al., 2021), our cohort consisted of 37 PBL-miRNA1 (1A, 30; 1B, 7), 17 PBL-miRNA2, 2 PBL-MYC/FOXR2, and 4 PBL-RB1 samples. PBL-miRNA subtype tumors had characteristic alterations in microRNA-processing genes; DICER1 mutations (n=14; 4 in Grp1A/1 in Grp1B/9 in Grp2) were most abundant, followed by homozygous deletions of the DROSHA locus (n=11; 8/1/2) and DROSHA mutations (n=9; 3/3/3). Most frequent cytogenetic aberrations in these subtypes were chromosome (chr.) 7 gains (n=25) and chr. 14 losses (n=20, with all but one of miRNA2 cases). DICER1 mutations were significantly associated with chr. 14 losses (p Conclusion: The epigenetically defined PBL subtypes were characterized by distinct cytogenetic and mutational events. Although we could not demonstrate a prognostic impact of these events, this might be due to the small sample size. A possible prognostic role for a superior (e.g. tetraploidy) or inferior outcome (e.g. OTX2 gains) need studies in larger cohorts and ideally in prospective clinical trials. Citation Format: Tobias Goschzik, Mathias Yuan, Martin Mynarek, Elke Pfaff, Evelyn Dörner, David T. Jones, Stefan M. Pfister, Stefan Rutkowski, Torsten Pietsch. Recurrent genetic alterations in epigenetic pineoblastoma subtypes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3561.

s: AACR Special Conference on the Advances in Pediatric Cancer Research; September 17-20, 2019; Montreal, QC, Canada In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, Group 4), each associated with distinct genomic features and clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically and clinically relevant subtypes. However, owing to differences in patient cohorts and analytical methods, estimates of subtype number and definition have been inconsistent, especially within Group 3 and Group 4. Herein, we aimed to reconcile the definition of Group 3/Group 4 MB subtypes through the analysis of a series of 1,501 medulloblastomas with DNA methylation profiling data, including 852 with matched transcriptome data. Using multiple complementary bioinformatic approaches, we compared the concordance of subtype calls between published cohorts and analytical methods, including assessments of class definition confidence and reproducibility. While lowest-complexity solutions continued to support the original consensus subgroups of Group 3 and Group 4, our analysis most strongly supported a definition comprising eight robust Group 3/Group 4 subtypes (Types I-VIII). Subtype II was consistently identified across all component studies, while all others were supported by multiple class-definition methods. Regardless of analytical technique, increasing cohort size did not further increase the number of identified Group 3/Group 4 subtypes. Summarizing the molecular and clinicopathologic features of these eight subtypes indicated enrichment of specific driver gene alterations and cytogenetic events among subtypes and identified highly disparate survival outcomes, further supporting their biologic and clinical relevance. Collectively, this study provides continued support for consensus groups 3 and 4, while enabling robust derivation of, and categorical accounting for, the extensive intertumoral heterogeneity within Groups 3 and 4, revealed by recent high-resolution subclassification approaches. Further, these findings provide a basis for application of emerging methods (e.g., proteomics/single-cell approaches) that may additionally inform medulloblastoma subclassification. Outputs from this study will help shape definition of the next generation of medulloblastoma clinical protocols and facilitate the application of enhanced molecularly guided risk stratification to improve outcomes and quality of life for patients and their families. Citation Format: Tanvi Sharma, Edward C. Schwalbe, Daniel W. Williamson, Martin Sill, Volker Hovestadt, Martin Mynarek, Stefan Rutkowski, Giles W. Robinson, Amar Gajjar, Florence Cavalli, Vijay Ramaswamy, Michael D. Taylor, Janet C. Lindsey, Rebecca M. Hill, Natalie Jager, Andrey Korshunov, Debbie Hicks, Simon Bailey, Marcel Kool, Lukas Chavez, Paul A. Northcott, Stefan M. Pfister, Steven C. Clifford. Second-generation molecular subgrouping of medulloblastoma: An international meta-analysis of Group 3 and Group 4 subtypes [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B73.

Purpose: The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell-cycle progression. It is involved in the development of multiple organs, and we have previously reported on its important role for the mitotic entry of cerebellar granule neuron precursors. Constitutive expression of FoxM1 is required for the growth of multiple cancer types. This study aimed to determine its role in medulloblastoma, the most frequent malignant brain tumor in childhood that can derive from cerebellar granule neuron precursors. Experimental Design: We evaluated the expression of FoxM1 together with its prognostic value in two independent series of human medulloblastoma samples using immunohistochemistry (n = 43) and gene expression arrays (n = 193). The functional impact of FoxM1 expression was characterized by knockdown experiments in four human medulloblastoma cell lines, and the thiazole antibiotic siomycin A was tested to downregulate FoxM1 and inhibit tumor cell growth. Results: FoxM1 was highly expressed in all subtypes of medulloblastoma. Importantly, expression levels of FoxM1 significantly correlated with unfavorable clinical outcome in univariate analysis (P = 0.0005), and FoxM1 was identified as an independent prognostic marker by multivariate analysis (P = 0.037). Knockdown of FoxM1 in medulloblastoma cell lines resulted in a significant decrease of cell viability which was caused by a failure in mitotic spindle formation and caspase-dependent mitotic catastrophe. Siomycin A significantly inhibited the expression of FoxM1 and the growth of medulloblastoma cells. Conclusions: FoxM1 may be used as an additional prognostic marker and may represent a potential novel target to treat patients suffering from medulloblastoma. Clin Cancer Res; 17(21); 6791–801. ©2011 AACR.

Introduction: Standard-risk medulloblastoma (SR-MB; 50-60% of patients) is currently defined by the absence of high-risk (e.g. metastatic disease, large-cell/anaplastic histology, MYC amplification) disease features. 75-85% survival rates are achieved, however the identification and validation of novel prognostic biomarkers will be essential to improve risk-adapted therapies, aimed at increased survival and reduced treatment-related late-effects. Experimental procedures: We undertook comprehensive analysis of the pan-European HIT-SIOP-PNET4 prospective clinical trial (2001-2006; age 4-21 years at diagnosis), encompassing central clinical and radiological review, and annotation of molecular pathological features to the WHO (2016) classification. Methods were developed and/or adapted to assess methylation-dependent molecular subgroup (MassArray), copy number aberrations (molecular inversion probe array) and mutational status in scant archival material previously refractory to analysis (n=136). Independent prognostic markers/predictive models for non-WNT/non-SHH patients (n=91) were identified by multivariate analyses, and validated in a representative independent cohort (n=70). Results: WNT (n=28; 21%) and Group 4 (n=76; 56%) tumors were enriched in SR-MB compared to published disease-wide estimates. Favorable-risk WNT disease was confirmed in patients Conclusion: A favorable-risk WCA phenotype identifies a large proportion (42%) of non-WNT/non-SHH SR-MB patients for whom therapy de-escalation should be considered in future biomarker-driven risk-adapted clinical trials; remaining patients (58%; 68% 5-year PFS) might benefit from more intensive and/or novel therapies. Citation Format: Tobias Goschzik, Ed C. Schwalbe, Debbie Hicks, Dominique Figarella-Branger, Francois Doz, Stefan Rutkowski, Goran Gustafsson, Birgitta Lannering, Torsten Pietsch, Steve C. Clifford. A whole chromosome aberration phenotype in non-WNT/non-SHH tumors predicts outcome within standard-risk medulloblastomas from the HIT-SIOP-PNET4 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-324.

Purpose: To investigate the therapeutic role of adjuvant vaccination with autologous mature dendritic cells (DC) loaded with tumor lysates derived from autologous, resected glioblastoma multiforme (GBM) at time of relapse. Experimental Design: Fifty-six patients with relapsed GBM (WHO grade IV) were treated with at least three vaccinations. Children and adults were treated similarly in three consecutive cohorts, with progressively shorter vaccination intervals per cohort. Feasibility and toxicity were assessed as well as effect of age, extent of resection, Karnofsky Performance Score, and treatment cohort on the progression-free (PFS) and overall survival (OS) using univariable and multivariable analysis. Results: Since the prevaccine reoperation, the median PFS and OS of the total group was 3 and 9.6 months, respectively, with a 2-year OS of 14.8%. Total resection was a predictor for better PFS both in univariable analysis and after correction for the other covariates. For OS, younger age and total resection were predictors of a better outcome in univariable analysis but not in multivariable analysis. A trend to improved PFS was observed in favor of the faster DC vaccination schedule with tumor lysate boosting. Vaccine-related edema in one patient with gross residual disease before vaccination was the only serious adverse event. Conclusion: Adjuvant DC-based immunotherapy for patients with relapsed GBM is safe and can induce long-term survival. A trend to PFS improvement was shown in the faster vaccination schedule. The importance of age and a minimal residual disease status at the start of the vaccination is underscored.

Atypical Teratoid Rhabdoid Tumors (ATRTs) are the most common malignant embryonal brain tumors arising in younger children that are distinctly lethal cancers for which effective therapies are lacking. Although ATRTs exhibit substantial clinical heterogeneity, exome studies reveal a relatively bland coding genome with only recurrent alterations of SMARCB1. Despite apparent genomic/genetic homogeneity, we recently reported that ATRTs comprise at least two transcriptional subclasses that correlate with different clinical features and treatment outcomes. However, the biological mechanisms and basis for molecular and therapeutic heterogeneity in ATRTs remained unclear. In this study, we integrated whole genome, exome, RNAseq as well as genome wide methylation and nucleosomal profiling analyses to comprehensively define the genomic and epigenomic landscape of ATRT sub-groups and identify sub-group specific therapeutic targets. Integration of multiplatform genomic analyses revealed novel recurrent genetic alterations in upto 20% of ATRTs. We observed predominantly structural coding events that targeted genes with functions in neural development and epigenetic regulation including BCR, MKL1 and EP300, thus suggesting greater complexity to the ATRT genome than previously appreciated. Global methylation (162) and gene expression analyses (90) of primary tumors indicated further segregation of ATRTs into three epigenetic sub-groups (group 1, 2A and 2B) that correlated with distinct lineage enriched gene expression profiles, global and SMARCB1 specific genotypes and different anatomic tumor locations and age at diagnosis. Group 1 ATRT exhibited enrichment of neurogenic/NOTCH signaling loci (ASCL1, FABP7, MYCN, C1ORF61, HES5/6, DLL1) and were predominantly supra-tentorial tumors arising in children at a median age of 24 months. In contrast Group 2A tumors arose predominantly in infra-tentorial locations in the youngest patients, while group 2B tumors were characteristically spinal in location. BMP signaling and mesenchymal differentiation genes (BMP4, BAMBI, PDGFRB) were commonly enriched in group 2A and B tumors; Group 2B tumors were additionally characterized by enrichment of MYCC, HOXB & C gene clusters. Remarkably, ATAC-seq analyses revealed distinct chromatin landscape associated with each ATRT sub-group, that correlated strikingly with sub-group specific therapeutic response in ATRT cell lines to a panel of signaling (NOTCH, BMP, Dasatinib) and epigenetic (EZH2, G9a, BRD4) inhibitors. Significantly, we discovered that differential methylation of a novel, PDGFRβ associated enhancer element confers robust sensitivity to tyrosine kinase inhibitors Dasatinib and Nilotinib in group 2 ATRTs, and suggest these as novel agents for this highly lethal ATRT sub-type. Citation Format: Jonathon Torchia, Shengrui Feng, King Ching Ho, Louis Letourneau, Daniel Picard, Tiffany S. Chan, Alexandre Vasiljevic, Dong Anh Khuong Quang, Brian Golbourn, Dalia Barsyte-Lovejoy, Constanze Zeller, Patrick Sin-Chan, Natalia R. Agamez, Mei Lu, Lucie Lafay-Cousin, Joseph D. Norman, Maryam Fouladi, Lindsey M. Hoffman, Stefan Rutkowski, Torsten Pietsch, Alexander R. Judkins, Eric Bouffet, James T. Rutka, Cynthia E. Hawkins, Cheryl H. Arrowsmith, Daniel De Carvalho, Nada Jabado, Annie Huang. Integrated (epi)genomic analyses identify subgroup-specific therapeutic targets in CNS rhabdoid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-179.

Primitive neuroectodermal tumors of central nervous system (CNS-PNET) which represents 3-7% of all pediatric brain tumors, are a rare group of neoplasms occurring in the CNS and include supratentorial PNETs (s-PNET), medulloepitheliomas and ependymoblastomas. Whereas ependymoblastomas harbour chromosome 19q13.41 amplification, express the neural stem cell marker LIN28 and show aggressive clinical behaviour, the biological mechanisms and molecular alterations contributing to the pathogenesis of s-PNETs remain so far poorly understood. Expression analyses and limited CGH studies indicated that s-PNETs may represent genetically a heterogeneous group with frequent but multiple copy number aberrations. In order to find possible specific molecular and cytogenetic alterations suitable for molecular risk stratification and predictive for the overall survival, we performed a multiplex ligation-dependent probe amplification (MLPA) and molecular inversion probe (MIP) analysis on DNA samples of 25 s-PNET cases of patients (median age 5.35 years; range, 2.41 - 17.28 years). The majority of patients were enrolled in the clinical GPOH HIT2000 trial. MLPA and MIP analysis revealed large losses of genomic material of chromosomes 3, 4, 5, 13, while frequent gains were observed at chromosomes 1, 17, 19, 20 and 22. Recurrent regions of gains were found at chromosome 7q11.2, 7q22.1, 12q13 and losses at 16q23.1, 6p21.1, and 11q11. High-copy number gains (amplifications) were found in particular at chromosome 2p24.3 (N-Myc) (in 6 cases) and 4q21 (in 2 cases). None of the chromosomal alterations identified was found significantly associated with the age of the patients at diagnosis, presence of tumor metastases or presence of postoperative residual tumor. In contrast, patients with tumors harbouring 2p gain or N-Myc amplification showed shorter overall survival (OS) (p=0.0029 and p=0.0012, respectively).These parameters were independent from the presence of metastases, which was indeed the most important clinical factor for unfavourable OS (p=0.0112) in our series. In the era of the personalized neuro-oncology, the identification of molecular marker associated to patients outcome may represent the further significant step towards the patients's risk stratification and the improvement of therapeutic strategies also for patients suffering from s-PNETs. Citation Format: Marco Gessi, Andre' von Bueren, Andras Treszl, Anja zur Muehlen, Stefan Rutkowski, Torsten Pietsch. Chromosome 2p gain and N-Myc amplification predict poor outcome of patients with LIN28 negative supratentorial primitive neuroectodermal tumors of the central nervous system (CNS-PNET). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2013-4702

In contrast to histopathological grading which varied in respect to its prognostic value between different clinical trials, extent of resection was found to be consistently associated to the clinical outcome. In retrospective series, gain of genetic material of chromosome arm 1q was identified to predict worse outcome. This marker was mainly assessed by FISH analysis which typically showed failure rates of 15-20 % in archival material. To validate this marker in a homogenously treated patient cohort, we analysed chromosome 1qin 209 consecutive cases enrolled into the multicenter trial HIT2000 (postoperative chemotherapy and irradiation, in a sequence depending on age and extent of resection) in which formalin-fixed, paraffin embedded material was available for DNA extraction. By using multiplex ligation-dependent probe amplification (MLPA) for 5 markers located on chromosome 1q and control markers, we were able to analyse 206/209 samples (> 98 %) and found a gain of chromosome 1q in 35 cases (17 %). 135/206 ependymomas were located in the infratentorial region. 35 cases were diagnosed as WHO grade II ependymoma, 171 as anaplastic ependymoma (WHO grade III). Interestingly, only 2 of the 35 WHO grade II ependymomas had 1q gain (5.7%). At a median follow-up of 3.9 years for survivors, patients with tumors showing 1q gain had a significantly lower 4-year overall survival (OS) (±SE) of 58% +/- 9% compared to patients lacking this marker (89% +/- 3%, p=0.002). The distribution of this marker was similar when comparing patients with supra- and infratentorial tumors. Multivariable analysis demonstrated that residual tumor and infratentorial localization were independent risk factors for event-free survival, and gain of chromosome 1q for OS, respectively. In conclusion, we validated chromosomal 1q gain to be a useful independent genetic marker for risk stratification of pediatric ependymoma patients which can be evaluated by MLPA representing a robust, reliable and cost-efficient method. Citation Format: Torsten Pietsch, Evelyn Doerner, Anja zur Muehlen, Natalia Velez-Char, Monika Warmuth-Metz, Rolf-Dieter Kortmann, Katja von Hoff, Carsten Friedrich, Stefan Rutkowski, Andre O. von Bueren. Chromosome 1q gain as genetic marker for risk stratification of pediatric ependymoma patients - validation as an adverse prognostic marker in the German multicenter HIT2000 trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-82. doi:10.1158/1538-7445.AM2013-LB-82

Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four major subgroups, which are now called WNT, SHH, Group 3 and Group 4. A better understanding of each of these molecular subtypes is urgently warranted to improve treatment strategies and the overall survival of patients and ultimately also the quality of life for those that survive medulloblastoma. We used the data of seven independent studies on medulloblastoma for further characterization of these molecular subtypes. All cases (n = 550) were analyzed by expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 408) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy number aberrations, demographics, and survival. Interestingly, the data also showed how different medulloblastomas are between infants, children and adults. In infants, for instance, almost all medulloblastomas are classified as SHH or Group 3, whereas in adults most medulloblastomas are of the SHH subtype and almost never of Group 3. Recent next generation sequencing data (whole genome and exome) generated in our laboratory for a large series of pediatric and adult medulloblastomas show that the spectrum of genetic mutations is also very different, not only between the molecular subtypes, but also between the different age categories. All these data clearly show that medulloblastoma is not one disease. Results from these molecular analyses will form the basis for prospective multi-center studies and will have an impact on how the different variants of medulloblastoma will be treated in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2492. doi:1538-7445.AM2012-2492

Medulloblastoma is a malignant brain tumor of childhood that comprises at least four molecularly distinct subgroups. We have previously described that cerebellar granule neuron precursors may give rise to the subgroup with a molecular fingerprint of Sonic hedgehog (Shh) signaling. Other recent data indicate that precursor cells within the dorsal brain stem may serve as cellular origins for Wnt-associated medulloblastomas. In order to see whether Shh-associated medulloblastomas are also able to develop in the dorsal brainstem, we analyzed two lines of transgenic mice with constitutive Shh signaling in hGFAP- and Math1-positive brainstem precursor populations, respectively. Our results show that in both of these lines, medulloblastomas arise from granule neuron precursors of the cochlear nuclei, a derivative of the auditory lower rhombic lip. This region is distinct from derivatives of precerebellar lower rhombic lip where medulloblastoma arise in mice with constitutive-active Wnt signaling. With respect to their histology and the expression of appropriate markers, Shh tumors perfectly resemble the human counterparts. Moreover, we find that in a series of 63 human desmoplastic medulloblastomas, 19 (30%) have a very close contact to the cochlear nuclei on MRI imaging. In conclusion, we suggest that precursors of the rhombic lip which may either develop into cerebellar or into cochlear granule neurons, may give rise to Shh-associated medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1446. doi:1538-7445.AM2012-1446

Brain tumors are the most common cause of cancer-related death in childhood. Ependymomas, are the third most common pediatric brain tumor. The disease remains incurable for about 45% of patients even after gross total resection and radiotherapy. Despite showing a very homogeneous histological picture, ependymomas display distinct molecular behavior, which supports the existence of several independent entities of the disease. We examined two non-overlapping cohorts of 102 and 75 ependymomas by mRNA expression profiling, on two different array platforms (Affymetrix, Agilent). When performing multiple statistical clustering methods (unsupervised consensus NMF and consensus HCL), we could consistently identify three major clusters, including two subgroups of posterior fossa (PF) ependymoma, a variant common in children and associated with heterogeneous clinical outcome. Subgroup-specific chromosome aberrations of PF tumors were detected by aCGH, and biological signaling pathways distinguishing PF subgroups were identified by gene set enrichment analysis and visualized in Cytoscape. We validated the most significantly classifying markers of each subgroup by immunohistochemistry on a tissue microarray containing an independent set of 265 PF ependymomas. Our findings delineate two subgroups of PF ependymoma (groups A and B) which are demographically, transcriptionally, genetically, and clinically distinct. Group A patients are younger, have laterally located tumors with a balanced genome, more frequently develop secondary metastases and are much more likely to have an extremely poor outcome as compared with group B patients. Based on a multi-variate Cox proportional-hazards model, our identified markers have the strongest independent prognostic value among demographic and molecular variables with Hazard ratios of 8.45 (PFS) and 10.55 (OS). Prognostic significance and predictive impact is being validated in the GPOH HIT2000 Ependymoma study. The identification of two distinct subgroups of PF ependymoma, and markers applicable for their clinical distinction, will allow for better prognostication of individual cases, independent of age, level of resection and WHO grade, and also for stratification in future ependymoma clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1432. doi:1538-7445.AM2012-1432

Current integrated genomic approaches indicate distinct biological variants in medulloblastoma. Comprehensive molecular classification strategies utilize cytogenetic or immunohistochemical biomarkers to refine risk stratification. Novel complementary markers may ameliorate outcome prediction particularly in intermediate or high-risk medulloblastomas. We combined transcriptome and DNA copy-number analysis for 64 primary tumors. Bioinformatic tools were applied to investigate marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the entire screening cohort. Immunohistochemical markers were used to determine molecular subtypes in adult and pediatric medulloblastoma samples (n=235). Immunopositivity of FSTL5 was correlated with molecular and prognostic subgroups for 235 non-overlapping medulloblastoma samples on two independent tissue microarrays (TMA). Unsupervised cluster analyses of transcriptome profiles revealed four distinct molecular variants: WNT, SHH, Group C, and Group D. Stable subgroup separation was achieved using only 300 most varying transcripts. Specific distribution of clinical and molecular characteristics was noted for each cluster. Notably, Group C tumors were exclusively present in pediatric medulloblastomas as determined by immunohistochemistry. Delimited expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time PCR. FSTL5 transcripts were most up-regulated in Group C and Group D tumors with unfavorable prognosis, whereas WNT medulloblastomas showed marked down-regulation. Immunopositivity of FSTL5 identified a large proportion of patients (84 of 235 patients; 36%) at high risk for relapse and death in particular in patients with WNT/SHH-independent tumors. Multivariate analysis revealed that FSTL5 immunopositivity constitutes an independent prognostic marker in pediatric and adult patient cohorts (p Comprehensive analyses of transcriptome and genetic alterations unravel four distinct disease variants. By addition of FSTL5 immunohistochemistry, existing molecular stratification schemes can effectively be complemented and sub-classification of WNT/SHH-independent tumors substantially optimized. This approach may ultimately define clear risk groups to individualize treatment intensities in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3447. doi:10.1158/1538-7445.AM2011-3447

Background: This study was designed to confirm the previously observed favorable survival rates and prognostic factors in young children with non-metastatic medulloblastoma (MB) treated by postoperative chemotherapy alone. Methods: Patients diagnosed between January 2001 and December 2005 and younger than 4 years received three cycles of postoperative systemic multiagent chemotherapy and intraventricular methotrexate. In case of complete remission, treatment was terminated after two additional cycles of chemotherapy. Otherwise secondary surgery, radiotherapy and consolidation chemotherapy were recommended. Results: The 5-year event-free survival (EFS) and overall survival (OS) (±SE) rates of 45 patients were 57 ± 8% and 80 ± 6% (median follow-up: 4.5 years, median age: 2.5 years). Nineteen patients with desmoplastic / nodular MB variants had better EFS and OS (5-year rates: 90 ± 7% and 100 ± 0%) compared to 23 patients with classic MB (30 ± 11% and 68 ± 10%; p< 0.001 for EFS, p=0.008 for OS). Five-year EFS and OS rates of 3 children with anaplastic MB were 33 ± 27%. Desmoplastic / nodular histology was an independent prognostic factor for EFS. Twenty-nine of 30 (97%) patients without postoperative residual tumor remained in continuous complete remission. Conclusions: Our results confirm histology of MB variants as a strong prognostic factor in this age-group. Sustained tumor control and favorable survival rates can be achieved by this chemotherapy regimen in young children with desmoplastic / nodular MB variants. For children with non-desmoplastic / non-nodular MB variants, where predominantly local relapses lead to less favorable survival rates, local radiotherapy has been introduced after chemotherapy since 2006. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3463. doi:10.1158/1538-7445.AM2011-3463

The European research project Kids Cancer Kinome (KCK) is focusing on the investigation of aggressive childhood cancers. The competence of nine research centers is combined to explore the human protein kinase family in different highly malignant pediatric tumors (medulloblastoma, Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, ALL) to validate novel drug targets and develop novel therapeutic options. Medulloblastomas belong to the most frequent malignant brain tumors in children and represent about 20% of all pediatric CNS cancers. Analyzing mRNA expression profiles (Affymetrix microarrays) we discovered a series of kinases considerably overexpressed in primary medulloblastoma tumors samples and medulloblastoma cell lines compared to non-neoplastic tissues. We found a significant mRNA overexpression of PLk1 and Aurora kinases A and B in medulloblastoma tumor samples and cell lines. For further investigation we selected these kinases which are all involved in cell cycle control. Defects in the process of cell division are believed to be associated with tumorigenesis. Using the small molecules inhibitors ZM447439 and VX680 for Aurora kinases and BI2536 and GW843682 for PLK1 inhibition we evaluated the effects on medulloblastoma cell proliferation and apoptosis. Incubation of medulloblastoma cells with the inhibitors for 72h resulted in a concentration dependent decrease of the proliferative activity as measured by MTS proliferation assays and an increased apoptosis determined by Caspase-Glo 3/7 assay. In a second approach, we transduced cell lines with lentiviral particles carrying shRNA targeting PLK1 or Aurora kinases A and B. The specific downregulation of the kinases mediated by the lentiviral shRNA was confirmed by Western blotting and had a negative effect on the viability of the medulloblastoma cells. We observed that the effect of PLK1 inhibition on the proliferation of the medulloblastoma cell lines was more effective compared to the aurora kinases. In conclusion, these data suggest that the inhibition of PLK1 or Aurora kinases may represent a promising approach for novel targeted strategies in the treatment of medulloblastoma. http://www.kidscancerkinome.org Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2588. doi:10.1158/1538-7445.AM2011-2588

Kids Cancer Kinome (KCK) is a European research project investigating aggressive childhood cancers. Focusing on the human protein kinase family nine research centers explore different highly malignant pediatric tumors (medulloblastoma, Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, ALL) to validate novel drug targets and develop novel therapeutic options. Medulloblastomas of the cerebellum represent the most frequent malignant brain tumors of childhood. Previous studies revealed Aurora kinase A (STK15) as independent predictor for an unfavorable outcome of medulloblastoma patients. The Aurora kinases belong to the family of serine/threonine kinases which are involved in the cell cycle by controlling chromatid segregation. It is commonly approved, that defects in the process of cell division are associated with tumorigenesis. Using mRNA expression profiling (Affymetrix) we compared the Aurora kinase mRNA expression of 62 medulloblastoma tumor samples with normal cerebellar tissue (n=9) and an extensive dataset of various other tumor types (n=2028). We discovered a significant overexpression of Aurora kinase mRNA in primary medulloblastoma tumors, as well as 14 medulloblastoma cell lines compared to non-neoplastic tissues (n=685). Kaplan-Meier scans revealed an inverse correlation between overall survival rates and mRNA expression levels of Aurora kinases. To investigate the effects of Aurora kinase inhibition on medulloblastoma cells, we first evaluated the effects of the small molecule inhibitors ZM447439 andVX680 on the proliferation of medulloblastoma cell lines. Incubation of medulloblastoma cells with ZM447439 and VX680 for 72h resulted in a concentration dependent decrease of the proliferative activity as measured by MTS proliferation assays. The GI50 for ZM447439 ranged from 0.2 to 20 µm and for VX680 from 0.02 to 10.0 µm. Cytologically, the cells appeared enlarged and multinucleated after Aurora kinase inhibition. In a second approach, we transduced cell lines with lentiviral particles carrying shRNA targeting Aurora A and B kinase. The specific downregulation of the Aurora kinases with lentiviral shRNA was confirmed by Western blotting and had a negative effect on the viability of the medulloblastoma cells. In conclusion, these data suggest that the inhibition of Aurora kinases may represent a promising approach for novel targeted strategies for the treatment of medulloblastoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-384.