Malignant Embryonal Brain Tumors Research Articles

Integrated proteomics spotlight the proteasome as a therapeutic vulnerability in embryonal tumors with multilayered rosettes.

Embryonal tumors with multilayered rosettes (ETMR) are rare malignant embryonal brain tumors. The prognosis of ETMR is poor and novel therapeutic approaches are desperately needed. Comprehension of ETMR tumor biology is currently based on only few previous molecular studies, which mainly focused on the analyses of nucleic acids. In this study, we explored integrated ETMR proteomics. Using mass spectrometry, proteome data were acquired from 16 ETMR and the ETMR cell line BT183. Proteome data were integrated with case-matched global DNA methylation data, publicly available transcriptome data, and proteome data of further embryonal and pediatric brain tumors. Proteome-based cluster analyses grouped ETMR samples according to histomorphology, separating neuropil-rich tumors with neuronal signatures from primitive tumors with signatures relating to stemness and chromosome organization. Integrated proteomics showcased that ETMR and BT183 cells harbor proteasome regulatory proteins in abundance, implicating their strong dependency on the proteasome machinery to safeguard proteostasis. Indeed, in vitro assays using BT183 highlighted that ETMR tumor cells are highly vulnerable toward treatment with the CNS penetrant proteasome inhibitor Marizomib. In summary, histomorphology stipulates the proteome signatures of ETMR, and proteasome regulatory proteins are pervasively abundant in these tumors. As validated in vitro, proteasome inhibition poses a promising therapeutic option in ETMR.

MEDB-22. iPSC-derived cerebellar organoid model for hereditary genetic predisposition in SHH-medulloblastoma

Medulloblastoma is one of the most common malignant embryonal brain tumors in children. Medulloblastomas of the Sonic Hedgehog (SHH) group arise from excessive proliferation of granule neuron progenitor (GNP) cells during cerebellar development. Genetic predisposition accounts for nearly 40% of all pediatric SHH-medulloblastomas. Recently, ELP1, a novel predisposition gene, was shown to be germline mutated in 15% of SHH-medulloblastoma patients. ELP1 encodes the scaffolding member of the Elongator complex and is required for efficient translation. Heterozygous mutations in ELP1 have been associated with the neural disorder Familial Dysautonomia, but not cancer. ELP1-associated medulloblastomas frequently harbor somatic PTCH1 co-mutations. It remains unclear how ELP1 affects the GNP lineage during normal cerebellar development and tumorigenesis in pediatric SHH-medulloblastoma patients. To characterize ELP1 mutations in the GNP lineage in vitro, we established a cerebellar organoid model from human induced pluripotent stem cells (iPSCs). We genetically inserted an EGFP reporter downstream of the endogenous GNP-specific ATOH1 locus in control iPSCs and generated cerebellar organoids according to published protocols. Marker gene and protein expression levels confirmed the cerebellar identity of the 3D model. Furthermore, activation of the EGFP reporter in single cells within the organoid highlighted the specification of putative GNPs. Next, we will determine the specific cell state of putative iGNPs and compare to human GNPs identified in our scRNAseq cerebellum atlas. To analyze tumorigenesis through ELP1 loss, we will introduce patient-specific ELP1mutations into ATOH1-EGFP iPSCs. Cerebellar organoids derived from ELP1-, PTCH1-deficient and control iPSCs will serve as models to study GNP proliferation, differentiation, apoptosis and tumor formation. Combining genome editing, in vitro 3D differentiation and functional studies, we will characterize the novel predisposition gene ELP1in GNPs during cerebellar development. In addition, we will determine the interplay of ELP1and PTCH1 co-mutations, predisposing SHH-medulloblastoma formation.

Efficacy of Carboplatin and Isotretinoin in Children With High-risk Medulloblastoma

Brain tumors are the leading cause of disease-related death in children. Medulloblastoma is the most common malignant embryonal brain tumor, and strategies to increase survival are needed. To evaluate therapy intensification with carboplatin as a radiosensitizer and isotretinoin as a proapoptotic agent in children with high-risk medulloblastoma in a randomized clinical trial and, with a correlative biology study, facilitate planned subgroup analysis according to World Health Organization consensus molecular subgroups of medulloblastoma. A randomized clinical phase 3 trial was conducted from March 2007 to September 2018. Analysis was completed in September 2020. Patients aged 3 to 21 years with newly diagnosed high-risk medulloblastoma from Children's Oncology Group institutions within the US, Canada, Australia, and New Zealand were included. High-risk features included metastasis, residual disease, or diffuse anaplasia. Patients were randomized to receive 36-Gy craniospinal radiation therapy and weekly vincristine with or without daily carboplatin followed by 6 cycles of maintenance chemotherapy with cisplatin, cyclophosphamide, and vincristine with or without 12 cycles of isotretinoin during and following maintenance. The primary clinical trial end point was event-free survival, using the log-rank test to compare arms. The primary biology study end point was molecular subgroup classification by DNA methylation array. Of 294 patients with medulloblastoma, 261 were evaluable after central radiologic and pathologic review; median age, 8.6 years (range, 3.3-21.2); 183 (70%) male; 189 (72%) with metastatic disease; 58 (22%) with diffuse anaplasia; and 14 (5%) with greater than 1.5-cm2 residual disease. For all participants, the 5-year event-free survival was 62.9% (95% CI, 55.6%-70.2%) and overall survival was 73.4% (95% CI, 66.7%-80.1%). Isotretinoin randomization was closed early owing to futility. Five-year event-free survival was 66.4% (95% CI, 56.4%-76.4%) with carboplatin vs 59.2% (95% CI, 48.8%-69.6%) without carboplatin (P = .11), with the effect exclusively observed in group 3 subgroup patients: 73.2% (95% CI, 56.9%-89.5%) with carboplatin vs 53.7% (95% CI, 35.3%-72.1%) without (P = .047). Five-year overall survival differed by molecular subgroup (P = .006): WNT pathway activated, 100% (95% CI, 100%-100%); SHH pathway activated, 53.6% (95% CI, 33.0%-74.2%); group 3, 73.7% (95% CI, 61.9%-85.5%); and group 4, 76.9% (95% CI, 67.3%-86.5%). In this randomized clinical trial, therapy intensification with carboplatin improved event-free survival by 19% at 5 years for children with high-risk group 3 medulloblastoma. These findings further support the value of an integrated clinical and molecular risk stratification for medulloblastoma. ClinicalTrials.gov Identifier: NCT00392327.

MBRS-29. PROSPECTIVE MOLECULAR PROFILING IN PEDIATRIC MEDULLOBLASTOMA PATIENTS ENROLLED ON THE “HEAD START 4” PROTOCOL

Medulloblastoma is the most common malignant embryonal brain tumor in children with only modest improvements in outcomes achieved over the last 20 years. The implementation of irradiation-avoiding strategies, including trials by the “Head Start” consortium, have demonstrated improved cure rates along with enhanced quality of life. Simultaneously, the classification of medulloblastomas has undergone a dramatic shift as molecular testing has made it possible to divide these tumors into distinctive subtypes. Currently, the WHO recognizes four medulloblastoma molecular subgroups; however it remains unclear how patients within these subgroups respond to modern irradiation-avoiding therapies. This study aims to demonstrate the feasibility of prospective molecular profiling in medulloblastoma patients enrolled on the “Head Start 4” trial. Whole-exome sequencing (SureSelect Human All Exon V6+COSMIC) and DNA methylation (Illumina EPIC Array) profiling were performed on 10 paired tumor/blood samples and 4 tumor samples, respectively. High-quality mutational and copy number data were produced for each of the 10 subjects demonstrating well-described gene mutations (SUFU) and chromosomal losses (9q and 10q). Four subjects had methylation profiling which successfully separated them into the WHO subgroups (two SHH and two Group 3). These data showed the feasibility of prospective high-dimensional mutational and DNA methylation analysis using “Head Start 4” patients. Future work will focus on finalizing these profiling efforts, enabling the development of models that predict response to irradiation-avoiding treatment and, in general, a better understanding of the molecular mechanisms underlying treatment resistance and tumor progression, leading to more personalized approaches to treating children with medulloblastoma.

Abstract PR04: Locoregionally administered B7H3-targeting CAR T cells mediate potent antitumor effects in atypical teratoid/rhabdoid tumor

Abstract Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant embryonal brain tumors, arising in very young children, and are associated with inactivation of SMARCB1. The prognosis is extremely poor. Hence new treatments are urgently needed. We show that 100% of ATRT express B7-H3 and residual SMARCA4 SWI/SNF activity drives B7-H3 expression. Further, we demonstrate that prenatal brain expresses high levels of B7-H3, but expression is absent on pediatric brain, suggesting that the cell of origin may conserve high B7-H3 expression. We recently created a 4-1BB second-generation CAR targeting B7-H3. To date, there are little data on the most efficacious route of administration of CAR T cells in malignant brain tumors. The goal of this study was to test the antitumor activity of the B7-H3 CAR in highly aggressive ATRT xenograft models and compare intracavitary (IT), intraventricular (ICV), and intravenous (IV) administration in terms of efficacy, toxicity, and persistence of CAR T cells. Locoregionally (IT and ICV) administered B7H3.BB.z-CAR T cells mediate potent antitumor effects against cerebral ATRT xenografts, with more rapid kinetics, greater potency, and diminished systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. ICV-administered CAR T cells also demonstrate robust trafficking from the CNS into the periphery, and following clearance of ATRT xenografts, ICV or IV administered B7H3.BB.z-CAR T cells mediate antigen specific protection from tumor rechallenge in the CNS or in the flank. In summary, B7H3 is a promising immune target for this largely incurable pediatric tumor. We demonstrate that CNS delivered CAR T cells cure ATRT xenografts and efficiently traffic from the CNS into the periphery and can mediate long-term immune protection from disease recurrence in the CNS or in peripheral tissues. This abstract is also being presented as Poster A12. Citation Format: Johanna Theruvath, Elena Sotillo, Claus Moritz Graef, Sabine Heitzeneder, Louai Labanieh, Robbie Majzner, Crystal Mackall. Locoregionally administered B7H3-targeting CAR T cells mediate potent antitumor effects in atypical teratoid/rhabdoid tumor [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 PR04.

Changes in glomerular filtration rate and clinical course after sequential doses of carboplatin in children with embryonal brain tumors undergoing autologous stem cell transplantation

BackgroundTreatment for malignant embryonal brain tumors in young children usually employs cycles of standardly dosed cisplatinum followed by high-dose carboplatinum-containing conditioning with single or tandem autologous stem cell rescue (HDC-ASCR). High-dose carboplatin is potentially nephrotoxic, and additive platinum exposure may acutely impact renal function. Aiming to determine if decrease in renal function during conditioning assessed prior to each carboplatin dose was associated with acute increases in creatinine, requirement for dialysis or transplant-related mortality (TRM). This was a retrospective study of consecutive patients with medulloblastoma (n = 15) / atypical teratoid/rhabdoid tumor (AT/RT, n = 5) receiving HDC-ASCR. Fifteen patients underwent 1 HDC-ASCR (carboplatin × 3 doses/ etoposide/ thiotepa) and 5 patients underwent at least 1 of 3 planned tandem HDC-ASCR (carboplatin × 2 doses/ thiotepa). Renal function was assessed by daily creatinine and nuclear medicine glomerular filtration rate (GFR)/ creatinine clearance before each carboplatin dose.ResultsIn this cohort of 20 patients, 3 had doses of carboplatin omitted due to decreases in GFR: 1 did not develop nephrotoxicity, 1 experienced nephrotoxicity without need for dialysis, and 1 required dialysis temporarily but recovered renal function. Two patients did not have GFR changes but developed post-ASCR renal failure requiring dialysis and TRM.ConclusionDaily assessment of renal function by GFR, prior each dose of carboplatin during HDC-ASCR, will help in protecting the kidney in heavily treated population of oncology/HSCT patients. Although the study had a small number of patients which is a major limitation of the study, but it points to a serious transplant-related morbidity and mortality. So, larger scale studies are needed to clarify the best approach to carboplatin dosing to insure the optimal balance between efficacy and toxicity.

Molecular Classification and Management of Rare Pediatric Embryonal Brain Tumors.

Malignant embryonal brain tumors (EBTs) of childhood span a wide clinical spectrum but can share remarkably similar morphologic features. This overlap presents significant diagnostic challenges, particularly for tumor entities that are rarely encountered in clinical practice and for which diagnostic criteria were poorly defined. This review will provide an update on the evolving characterization and treatment of rare EBTs. Rapid advances in genomic tools have led to the discovery of robust molecular markers, and identification of novel tumor types and subtypes for almost all major categories of pediatric brain tumors. These developments have had significant impact on improving the diagnostic classification of the rare EBTs, particularly for tumors with newly recognized C19MC alterations, central nervous system primitive neuroectodermal tumors (CNS-PNET), and pineoblastoma (PB). These important developments in the clinical and molecular understanding of rare EBTs are paving the way for novel therapeutic strategies and improved clinical management.

MEDU-34. INTELLECTUAL OUTCOME IN CHILDREN WITH SENSORINEURAL HEARING LOSS FOLLOWING TREATMENT FOR EMBRYONAL BRAIN TUMORS.

Abstract Neurological side effects associated with childhood brain tumors and their treatments contribute to long term neurocognitive morbidity. The aims of this study were to identify the incidence of sensorineural hearing loss (SNHL) in a large sample of children treated for malignant embryonal brain tumors, and to evaluate the effect of SNHL on intellectual functioning following the completion of treatment. We conducted a single center review of 119 prospectively followed patients treated for malignant embryonal brain tumors at the Hospital for Sick Children, between 1996–2015, to evaluate the impact of significant SNHL (Chang > 2b) on intellectual function. Hearing and intellectual function were assessed post-treatment, at comparable time points (median ages: hearing assessment 13.5y (+4.5); intellectual function 12.8y (+ 4.1)). The median interval since treatment was 5.9 y + 3.7. Significant SNHL was identified in half the patients (48.7%, n = 58/119), of which 53% required a hearing aid (31/58). We identified a subset of patients (n = 61) who had assessments of intellectual function. In these patients, intellectual function was significantly affected by severe SNHL, even after controlling for the effect of craniospinal radiation (severe SNHL 22.4 Gy + 13.3, no/mild hearing loss 20.4 Gy +12.8) and boost dose. Severe hearing loss resulted in lower overall IQ (severe SNHL: 72.4 + 16.6; no/mild hearing loss: 92.0 + 20.5) p<0.001 and in significantly lower verbal comprehension (severe SNHL: 78.7 + 15.9; no/mild hearing loss: 94.7 + 13.8) p<0.001, and working memory (severe SNHL: 78.2+ 17.6; no/mild hearing loss: 94.8 + 16.4) p<0.001. Hearing loss is a much more significant complication in children with embryonal tumors than previously estimated. We show the profound impact of hearing loss on intellectual deficit in children. Our results have major implications on future trial design of children being treated with intensive protocols.

Neurocognitive outcome in children with sensorineural hearing loss after treatment of malignant embryonal brain tumors.

2029 Background: Neurological side effects associated with childhood brain tumors and their treatments contribute to long term neurocognitive morbidity. The aims of this study were to identify the incidence of sensorineural hearing loss (SNHL) in a large sample of children treated for malignant brain tumors, and to evaluate the potential relationship between SNHL and intellectual functioning following the completion of treatment. Methods: We conducted a prospective follow-up study at a single center with review of 119 patients treated for embryonal brain tumors at the Hospital for Sick Children, between 1996-2015, to analyze the impact of significant SNHL (Chang &gt; 2b) on intellectual function. Hearing was assessed post-treatment (median age: 13.5y (+4.5)) and the median age for neurocognitive testing was 12.8y (+ 4.1). The median interval from time of diagnosis was 5.8y (+ 3.7). Results: Severe SNHL was identified in half the patients (50.4%, n = 60/119). We identified a subset of patients (n = 61) who had assessments of intellectual function. In this cohort, intellectual function was significantly poorer in the group with severe SNHL, even after controlling for the effect of craniospinal radiation (severe SNHL 22.4 Gy + 13.3, no or mild hearing loss 20.4 Gy +12.8) and boost dose and volume. Children experiencing severe SNHL had lower overall IQ (severe SNHL 72.4 + 16.6; no/mild hearing loss 92.0 + 20.5) p &lt; 0.001 and in significantly lower verbal comprehension (severe SNHL 78.7 + 15.9; no/mild hearing loss 94.7 + 13.8) p &lt; 0.001, and working memory (severe SNHL 78.2+ 17.6; no/mild hearing loss 94.8 + 16.4) p &lt; 0.001, scores. Conclusions: Hearing loss is a much more significant complication in children with embryonal brain tumors than previously estimated. We show the profound impact of hearing loss on intellectual deficit in children. Namely, patients with severe SNHL have difficulty using and understanding verbal language, and they have a reduced ability to concentrate and manipulate information in short-term memory. Our results have implications on future trial designs and follow-up of children treated for embryonal brain tumors.

Overexpression of TEAD4 in atypical teratoid/rhabdoid tumor: New insight to the pathophysiology of an aggressive brain tumor.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal brain tumor that occurs mainly in early childhood. Although most of the tumors are characterized by inactivating mutations of the tumor suppressor gene, SMARCB1, the biological basis of its tumorigenesis and aggressiveness is still unknown. We performed high-throughput copy number variation analysis of primary cell lines generated from primary and relapsed tumors from one of our patients to identify new genes involved in AT/RT biology. The expression of the identified gene was validated in 29 AT/RT samples by gene expression profiling, quantitative real-time polymerase chain reaction, and immunohistochemistry (IHC). Furthermore, we investigated the function of this gene by mutating it in rhabdoid tumor cells. TEAD4 amplification was detected in the primary cell lines and its overexpression was confirmed at mRNA and protein levels in an independent cohort of AT/RT samples. TEAD4's co-activator, YAP1, and the downstream targets, MYC and CCND1, were also found to be upregulated in AT/RT when compared to medulloblastoma. IHC showed TEAD4 and YAP1 overexpression in all samples. Cell proliferation and migration were significantly reduced in TEAD4-mutated cells. We report the overexpression of TEAD4 in AT/RT, which is a key component of Hippo pathway. Recent reports revealed that dysregulation of the Hippo pathway is implicated in tumorigenesis and poor prognosis of several human cancers. Our results suggest that TEAD4 plays a role in the pathophysiology of AT/RT, which represents a new insight into the biology of this aggressive tumor.

Abstract LB-179: Integrated (epi)genomic analyses identify subgroup-specific therapeutic targets in CNS rhabdoid tumors

Abstract 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 &amp; 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.

TMOD-01BCOR IS A NOVEL TUMOR SUPPRESSOR GENE IN SHH-DRIVEN MEDULLOBLASTOMA

Sonic hedgehog (SHH) medulloblastoma is the most well characterized molecular subgroup of this malignant embryonal brain tumor, accounting for 1/3rd of all medulloblastoma diagnoses. Germline/somatic alterations affecting genes in the SHH pathway occur in most cases, resulting in constitutively aberrant activation of the SHH signaling cascade. Pharmacological inhibitors of SHH signaling have shown encouraging albeit transient efficacy in treating patients with SHH subgroup disease, with most patients developing resistance to conventional inhibitors. Therefore, understanding the genetic events cooperating with aberrant SHH pathway activation in medulloblastoma is of paramount importance for the development of rational, more effective combination therapies that improve cure rates for this patient subgroup. Next-generation sequencing studies have identified the BCL6 corepressor, BCOR, as among the most frequently mutated genes in SHH subgroup patients, with the majority of observed variants inferred to result in BCOR loss-of-function. Somatic mutations and homozygous deletions of BCOR are restricted to SHH subgroup patients, suggesting that deregulation of this gene plays a critical role in potentiating SHH subgroup malignancy. To validate BCOR as a novel tumor suppressor in SHH medulloblastoma, we generated Ptch1 + /−; Atoh1-Cre; Bcorfl/fl/Bcorfl/y mice to specifically inactivate Bcor in cerebellar granule neuron progenitors in vivo, presumed cells-of-origin for this medulloblastoma subgroup. Excitingly, Ptch1 + /−; Atoh1-Cre; Bcorfl/fl/Bcorfl/y mice rapidly develop ∼100% penetrant (n = 16/16) medulloblastomas (median latency = 74 days), compared to ∼36% of mice (n = 4/11) that develop medulloblastoma (average latency = 224 days) due to loss of Ptch1 + /− alone. Histologically, tumors resulting from the Bcor null genotype are highly similar to prototypical Ptch1 + /− medulloblastomas, despite exhibiting considerable gene expression differences as determined by Affymetrix expression profiling. Studies aimed at elucidating the molecular mechanism(s) associated with the development of Bcor-deficient SHH medulloblastoma are ongoing and will be essential for extrapolation of these findings into improved treatment options for SHH medulloblastoma patients in the future.

ME-16 * IS AQUAPORIN4 (AQP4) INVOLVED IN ADULT HUMAN MEDULLOBLASTOMA DISSEMINATION OR IN A BENEFICIAL BARRIER FORMATION?

Medulloblastoma (MDB) is a highly malignant embryonal brain tumor located in the cerebellum with tendency to leptomeningeal dissemination, common in children, rare in adults. Recent genetic analysis suggested classification into four subgroups according to gene expression profiles, including the Wingless signaling pathway-activated group (WNT group), the Sonic Hedgehog signaling pathway-activated group (SHH group), group 3and 4, with distinct molecular, clinicopathological, and prognostic characteristics. AQP4 is the main water channel in the normal central nervous system (CNS) responsible for salt and water balance maintaining. When the blood-brain barrier become disrupted, as in gliomas, water moves from the vasculature into the extracellular space in an AQP4-independent manner, down a hydrostatic gradient to form a vasogenic edema. Moreover, AQPs have a role promoting tumor progression, cell migration and metastasis. Aim of our study was to evaluate for the first time AQP4 expression in adult MDB to focus its role in edema, tumor invasion and dissemination. We analyzed by immunohistochemistry AQP4 expression in 40 adult MDBs, also investigated for GAB1, s-catenin, filamin-A, NPR3, according to molecular subgroups. Desmoplastic histology and Sonic hedgehog (SHH) pathway activation were more common feature, with GAB-1 and filamin-A expression. Only one case showed nuclear staining for s-catenin. None expressed NPR3. In classical MDB, AQP4 staining was localized in glial endfoot surrounding tumor vessels. This AQP4 distribution was lost in the desmoplastic type, where the nodular areas were negatives, surrounded by glial elements AQP4 intensely stained. In the molecular layer of the cerebellar cortex in the tumor proximity in classic type MDB it has been observed migrating tumor cells along glial processes AQP4 stained. This finding was mainly observed in patients with early dissemination. These observations could suggest a role for AQP4 in adult MDB, not only in brain tumor edema resolution, but also in leptomeningeal dissemination.

Genomic and transcriptomic analyses match medulloblastoma mouse models to their human counterparts.

Medulloblastoma is a malignant embryonal brain tumor with highly variable outcome. In order to study the biology of this tumor and to perform preclinical treatment studies, a lot of effort has been put into the generation of appropriate mouse models. The usage of these models, however, has become debatable with the advances in human medulloblastoma subgrouping. This study brings together multiple relevant mouse models and matches genetic alterations and gene expression data of 140 murine tumors with 423 human medulloblastomas in a global way. Using AGDEX analysis and k-means clustering, we show that the Blbp-cre::Ctnnb1(ex3)(Fl/+)Trp53 (Fl/Fl) mouse model fits well to human WNT medulloblastoma, and that, among various Myc- or Mycn-based mouse medulloblastomas, tumors in Glt1-tTA::TRE-MYCN/Luc mice proved to be most specific for human group 3 medulloblastoma. None of the analyzed models displayed a significant match to group 4 tumors. Intriguingly, mice with Ptch1 or Smo mutations selectively modeled SHH medulloblastomas of adulthood, although such mutations occur in all human age groups. We therefore suggest that the infantile or adult gene expression pattern of SHH MBs are not solely determined by specific mutations. This is supported by the observation that human medulloblastomas with PTCH1 mutations displayed more similarities to PTCH1 wild-type tumors of the same age group than to PTCH1-mutated tumors of the other age group. Together, we provide novel insights into previously unrecognized specificity of distinct models and suggest these findings as a solid basis to choose the appropriate model for preclinical studies on medulloblastoma.

Combinatorial effects of Resveratrol, 5-aza-2'deoxycytidin, and irradiation on clonogenic survival and stem cell population in human Medulloblastoma cell lines

Recently, the efficiency of anticancer therapeutics is increasingly measured by their potential to eliminate a subpopulation of tumor cells, the so called tumorigenic stem cells (TSC). Also in medulloblastoma (MB), the most malignant embryonal brain tumor, such TSC were identified by expression of CD133 or CD15. In previous studies we could show a radioadditive effect of the demethylating agent 5-aza-2'deoxycytidin (5-aza-dC) on the clonogenic survival of human MB cells. Now we aim to clarify whether 5-aza-dC and irradiation (IR) have similar effects on the TSC subpopulation. In addition we applied resveratrol (RV), to see if combination of above treatments with this differentiation inducer may further reduce the clonogenic potential of MB cells.

Genome-wide molecular characterization of central nervous system primitive neuroectodermal tumor and pineoblastoma

Central nervous system primitive neuroectodermal tumor (CNS PNET) and pineoblastoma are highly malignant embryonal brain tumors with poor prognoses. Current therapies are based on the treatment of pediatric medulloblastoma, even though these tumors are distinct at both the anatomical and molecular level. CNS PNET and pineoblastoma have a worse clinical outcome than medulloblastoma; thus, improved therapies based on an understanding of the underlying biology of CNS PNET and pineoblastoma are needed. To this end, we characterized the genomic alterations of 36 pediatric CNS PNETs and 8 pineoblastomas using Affymetrix single nucleotide polymorphism arrays. Overall, the majority of CNS PNETs contained a greater degree of genomic imbalance than pineoblastomas, with gain of 19p (8 [27.6%] of 29), 2p (7 [24.1%] of 29), and 1q (6[20.7%] of 29) common events in primary CNS PNETs. Novel gene copy number alterations were identified and corroborated by Genomic Identification of Significant Targets In Cancer (GISTIC) analysis: gain of PCDHGA3, 5q31.3 in 62.1% of primary CNS PNETs and all primary pineoblastomas and FAM129A, 1q25 in 55.2% of primary CNS PNETs and 50% of primary pineoblastomas. Comparison of our GISTIC data with publically available data for medulloblastoma confirmed these CNS PNET-specific copy number alterations. With use of the collection of 5 primary and recurrent CNS PNET pairs, we found that gain of 2p21 was maintained at relapse in 80% of cases. Novel gene copy number losses included OR4C12, 11p11.12 in 48.2% of primary CNS PNETs and 50% of primary pineoblastomas. Loss of CDKN2A/B (9p21.3) was identified in 14% of primary CNS PNETs and was significantly associated with older age among children (P = .05). CADPS, 3p14.2 was lost in 27.6% of primary CNS PNETs and was associated with poor prognosis (P = .043). This genome-wide analysis revealed the marked molecular heterogeneity of CNS PNETs and enabled the identification of novel genes and clinical associations potentially involved in the pathogenesis of these tumors.

Frequency, Risk‐Factors and Survival of Children With Atypical Teratoid Rhabdoid Tumors (AT/RT) of the CNS Diagnosed between 1988 and 2004, and Registered to the German HIT Database

To analyze the frequency, prognostic factors, and outcome of children with atypical teratoid/rhabdoid tumors (AT/RT), a rare and highly malignant embryonal brain tumor. Clinical data of patients diagnosed between 1988 and 2004 with AT/RT who were registered to the German HIT trial center, were correlated with outcome. Patient numbers for AT/RT were compared to numbers for primitive neuroectodermal tumors and medulloblastomas (PNET/MB) registered to the population-based HIT trials. We identified 56 patients with the centrally confirmed histopathological diagnosis of AT/RT with a median age of 1.2 years (range, 0.1-14.0 years). The AT/RT:PNET/MB ratio was 1:12.2 for all children, and 1:1.5 for children younger than 1 year at diagnosis. Three-year overall survival (OS) and event-free survival (EFS) for all patients were 22% and 13%, respectively. Eight patients (14%) are considered long-term event-free survivors (follow-up 1.4-10.6 years). By univariable analyses, younger age, metastatic disease, infratentorial location, and less than complete remission at the end of chemotherapy were identified as negative influencing factors for OS. By multivariable analyses, younger age (OS, EFS) and metastatic disease (OS) were identified as independent risk factors. The incidence of AT/RT in children below 1 year is higher than previously reported. A subset of patients with favorable clinical risk factors profits from intensive multimodal treatment. Prospective clinical and biological studies are needed to further define prognostic factors and optimize therapy.

Upregulation of SOX2, NOTCH1, and ID1 in supratentorial primitive neuroectodermal tumors: a distinct differentiation pattern from that of medulloblastomas

Supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma are highly malignant embryonal brain tumors. They share morphological similarities, but differ in their differentiation patterns and global gene expression. The authors compared the expression of specific genes involved in neuroglial differentiation in supratentorial PNETs and medulloblastomas to define the distinct characters of these tumors. The mRNA expression of 8 genes (SOX2, NOTCH1, ID1, ASCL-1, NEUROD1, NEUROG1, NEUROG2, and NRG1) was evaluated in 25 embryonal tumors (12 supratentorial PNETs and 13 medulloblastomas) by quantitative real-time polymerase chain reaction. The expression levels of the transcripts of these genes were compared between the tumor groups. Activation of the JAK/STAT3 pathway was assessed by immunoblotting. Relative expression levels of STAT3 and phosphorylated STAT3 proteins were compared. Supratentorial PNETs expressed significantly higher levels of SOX2, NOTCH1, ID1, and ASCL-1 transcripts, whereas the transcription of proneural basic helix-loop-helix factors, NEUROD1, NEUROG1 (significantly), and NEUROG2 (not significantly) was upregulated in medulloblastomas. The proportion of phosphorylated STAT3alpha relative to STAT3alpha was significantly greater in supratentorial PNETs than in medulloblastomas, indicating activation of the JAK/STAT3 pathway in supratentorial PNETs. These results indicate that supratentorial PNET predominantly has glial features and medulloblastoma largely follows a neuronal differentiation pattern. These divergent differentiation patterns may be related to the location and origin of each tumor.

Molecular Risk Stratification of Medulloblastoma Patients Based on Immunohistochemical Analysis of MYC, LDHB, and CCNB1 Expression

Medulloblastoma is the most common malignant embryonal brain tumor in children. The current clinical risk stratification to select treatment modalities is not optimal because it does not identify the standard-risk patients with resistant disease or the unknown number of high-risk patients who might be overtreated with current protocols. The aim of this study is to improve the risk stratification of medulloblastoma patients by using the expression of multiple prognostic markers in combination with current clinical parameters. Candidate prognostic markers were selected from literature or from medulloblastoma expression data. Selected genes were immunohistochemically analyzed for their prognostic value using medulloblastoma tissue arrays containing 124 well-characterized patient samples. Protein expression analyses showed that the combined expression of three genes was able to predict survival in medulloblastoma patients. Low MYC expression identified medulloblastoma patients with a very good outcome. In contrast, concomitant expression of LDHB and CCNB1 characterized patients with a very poor outcome. Multivariate analyses showed that both expression of MYC and the LDHB/CCNB1 gene signature were strong prognostic markers independent of the clinical parameters metastasis and residual disease. Combined analysis of clinical and molecular markers enabled greater resolution of disease risk than clinical factors alone. A molecular risk stratification model for medulloblastoma patients is proposed based on the signature of MYC, LDHB, and CCNB1 expression. Combined with clinical variables, the model may provide a more accurate basis for targeting therapy in children with this disease.