Marc Remke

Dissecting the genomic complexity underlying medulloblastoma

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour–normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.

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

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 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.

Delineation of two clinically and molecularly distinct subgroups of posterior fossa ependymoma.

Despite the histological similarity of ependymomas from throughout the neuroaxis, the disease likely comprises multiple independent entities, each with a distinct molecular pathogenesis. Transcriptional profiling of two large independent cohorts of ependymoma reveals the existence of two demographically, transcriptionally, genetically, and clinically distinct groups of posterior fossa (PF) ependymomas. Group A patients are younger, have laterally located tumors with a balanced genome, and are much more likely to exhibit recurrence, metastasis at recurrence, and death compared with Group B patients. Identification and optimization of immunohistochemical (IHC) markers for PF ependymoma subgroups allowed validation of our findings on a third independent cohort, using a human ependymoma tissue microarray, and provides a tool for prospective prognostication and stratification of PF ependymoma patients.

Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma

Separation of pilocytic astrocytoma from diffuse astrocytomas frequently poses problems mostly related to small sample size. Precise classification and grading are essential due to different therapeutic strategies prompted by diagnoses of pilocytic astrocytoma WHO grade I, diffuse astrocytomas WHO grade II or anaplastic astrocytoma WHO grade III. Recently, genomic aberrations with a high specificity for distinct glioma entities have been described. Pilocytic astrocytomas carry a duplication at chromosome band 7q34 containing a BRAF–KIAA1549 gene fusion in the majority of cases. IDH1 mutations are observed very frequently in adult astrocytomas and IDH2 mutations have been reported in some astrocytomas. We examined a series of 120 astrocytomas including 70 pilocytic astrocytomas WHO grade I and 50 diffuse astrocytomas WHO grade II for both, BRAF–KIAA1549 fusion with a newly developed FISH assay and mutations in IDH1 and IDH2 by direct sequencing. Pilocytic astrocytomas contained the BRAF fusion in 49 cases (70%) but neither IDH1 nor IDH2 mutations. Astrocytomas WHO grade II exhibited IDH1 mutations in 38 cases (76%) but neither IDH2 mutations nor BRAF fusions. Thus, combined molecular analysis of BRAF and IDH1 is a sensitive and highly specific approach to separate pilocytic astrocytoma from diffuse astrocytoma.

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

Precursor T-cell acute 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 expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-beta or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-beta and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

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

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 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.

Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors

Anti-CD47 antibody is effective for treating malignant pediatric brain tumors without detectable toxicity in patient-derived xenograft models. Brain tumors, meet macrophages A protein called CD47 is often expressed on the surface of tumor cells, where it serves as a “don’t eat me” signal that blocks macrophages from attacking the tumor. To overcome this signal and allow the macrophages to “eat” tumor cells, Gholamin et al. engineered a humanized antibody that blocks CD47 signaling. The researchers tested the efficacy of this antibody in patient-derived xenograft models of a variety of pediatric brain tumors. The treatment was successful at inhibiting CD47, killing tumor cells, and prolonging the animals’ survival, all without toxic effects on normal tissues. Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

Treatment developments and the unfolding of the quality of life discussion in childhood medulloblastoma: a review

PurposeTo describe how the quality of life (QOL) discussion in childhood medulloblastoma (MB) relates to treatment developments, survival and sequelae from 1920 to 2014.MethodsArticles containing “childhood medulloblastoma” and “quality of life” were identified in PubMed. Those containing phrases pertaining to psychological, emotional, behavioral or social adjustment in the title, abstract or keywords were selected. Inclusion of relevant older publications was assured by cross-checking references.Results1920–1930s: suction, electro-surgery, kilovolt (KV) irradiation. Survival = months. Focus on operative mortality, symptoms and survival. 1940s: radiotherapy improved. 1950s: chemotherapy and intubation. Survival = years. Opinions oscillated between optimism/awareness of physical sequelae of radiotherapy. 1960s: magnified vision, ventriculo-peritoneal (VP) shunts, megavolt (MV) irradiation. Long-term survival shifted the attention towards neurological problems, disability and carcinogenesis of radiotherapy. 1970s: CT, microscope, bipolar coagulation, shunt filters, neuroanesthesia, chemotherapy trials and staging studies. Operative mortality decreased and many patients (re)entered school; emphasis on neuropsychological sequelae, IQ and academic performance. 1980s: magnetic resonance imaging (MRI), Cavitron ultrasonic aspiration (CUSA), laser surgery, hyper-fractionated radiotherapy (HFRT). Cerebellar mutism, psychological and social issues. 1990s: pediatric neurosurgery, proton beams, stem cell rescue. Reflections on QOL as such. 21st century: molecular genetics. Premature aging, patterns of decline, risk- and resilience factors.DiscussionQOL is a critical outcome measure. Focus depends on survival and sequelae, determined after years of follow-up. Detailed measurements are limited by time, money and human resources, and self-reporting questionnaires represent a crude measure limited by subjectivity. Therapeutic improvements raise the question of QOL versus cure. QOL is a potential primary research endpoint; multicenter international studies are needed, as are web-based tools that work across cultures.

MB3W1 is an orthotopic xenograft model for anaplastic medulloblastoma displaying cancer stem cell- and Group 3-properties.

BackgroundMedulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.MethodsWe established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.ResultsBesides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.ConclusionsThis model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.

Novel oncogene amplifications in tumors from a family with Li–Fraumeni syndrome

Li–Fraumeni syndrome (LFS) represents an inherited tumor syndrome that is typically caused by germline mutations of the tumor suppressor gene TP53. TP53 dysfunction secondarily disturbs the genetic integrity of the cell. Here, we report a family with LFS harboring a germline TP53 mutation (R248W) located in the functional domain of the protein that binds to the minor groove of the DNA. In this family, tumors of the central nervous system were diagnosed as primary malignancies in all carriers of the mutation. The index patient developed an anaplastic medulloblastoma with unusual genomic profile exhibiting six distinct high‐level genomic amplifications, two of them targeting the MYCN and GLI2 genes, respectively. In an extrarenal rhabdoid tumor from the same patient, we found a novel high‐level amplification of the MYC oncogene. The father of this patient was diagnosed with myxopapillary ependymoma (WHO °I), whereas a brother died from an early relapse of a choroid plexus carcinoma. The analysis of this LFS familiy thus revealed novel oncogene amplifications as different second hits that are likely to also play a role in the pathogenesis of their sporadic counterparts.