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Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-[3-(3-methyl-1,2,4-triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The incidence and biology of IDH1/2 mutations in pediatric gliomas are unclear. Notably, current treatment approaches by pediatric and adult providers vary significantly. We describe the frequency and clinical outcomes of IDH1/2-mutant gliomas in pediatrics., Methods: We performed a multi-institutional analysis of the frequency of pediatric IDH1/2-mutant gliomas, identified by next-generation sequencing (NGS). In parallel, we retrospectively reviewed pediatric IDH1/2-mutant gliomas, analyzing clinico-genomic features, treatment approaches, and outcomes., Results: Incidence: Among 851 patients with pediatric glioma who underwent NGS, we identified 78 with IDH1/2 mutations. Among patients 0-9 and 10-21 years old, 2/378 (0.5%) and 76/473 (16.1%) had IDH1/2-mutant tumors, respectively. Frequency of IDH mutations was similar between low-grade glioma (52/570, 9.1%) and high-grade glioma (25/277, 9.0%). Four tumors were graded as intermediate histologically, with one IDH1 mutation. Outcome: Seventy-six patients with IDH1/2-mutant glioma had outcome data available. Eighty-four percent of patients with low-grade glioma (LGG) were managed observantly without additional therapy. For low-grade astrocytoma, 5-year progression-free survival (PFS) was 42.9% (95%CI:20.3-63.8) and, despite excellent short-term overall survival (OS), numerous disease-related deaths after year 10 were reported. Patients with high-grade astrocytoma had a 5-year PFS/OS of 36.8% (95%CI:8.8-66.4) and 84% (95%CI:50.1-95.6), respectively. Patients with oligodendroglioma had excellent OS., Conclusions: A subset of pediatric gliomas is driven by IDH1/2 mutations, with a higher rate among adolescents. The majority of patients underwent upfront observant management without adjuvant therapy. Findings suggest that the natural history of pediatric IDH1/2-mutant glioma may be similar to that of adults, though additional studies are needed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The prognosis for patients with pediatric high-grade glioma (pHGG) is poor despite aggressive multimodal therapy. Objective responses to targeted therapy with BRAF inhibitors have been reported in some patients with recurrent BRAF-mutant pHGG but are rarely sustained., Methods: We performed a retrospective, multi-institutional review of patients with BRAF-mutant pHGG treated with off-label BRAF +/- MEK inhibitors as part of their initial therapy., Results: Nineteen patients were identified, with a median age of 11.7 years (range, 2.3-21.4). Histologic diagnoses included HGG (n = 6), glioblastoma (n = 3), anaplastic ganglioglioma (n = 4), diffuse midline glioma (n = 3), high-grade neuroepithelial tumor (n = 1), anaplastic astrocytoma (n = 1), and anaplastic astroblastoma (n = 1). Recurrent concomitant oncogenic alterations included CDKN2A/B loss, H3 K27M, as well as mutations in ATRX, EGFR, and TERT. Eight patients received BRAF inhibitor monotherapy. Eleven patients received combination therapy with BRAF and MEK inhibitors. Most patients tolerated long-term treatment well with no grade 4-5 toxicities. Objective and durable imaging responses were seen in the majority of patients with measurable disease. At a median follow-up of 2.3 years (range, 0.3-6.5), three-year progression-free and overall survival for the cohort were 65% and 82%, respectively, and superior to a historical control cohort of BRAF-mutant pHGG patients treated with conventional therapies., Conclusions: Upfront targeted therapy for patients with BRAF-mutant pHGG is feasible and effective, with superior clinical outcomes compared to historical data. This promising treatment paradigm is currently being evaluated prospectively in the Children's Oncology Group ACNS1723 clinical trial., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer disorder caused by a germline mutation in the VHL tumor suppressor gene. Loss of the wild-type allele results in VHL deficiency and the potential formation of cerebellar hemangioblastomas, which resemble embryonic hemangioblast proliferation and differentiation processes. Multiple, microscopic, VHL-deficient precursors, termed developmentally arrested structural elements (DASEs), consistently involve the cerebellar molecular layer in VHL patients, indicating the tumor site of origin. Unlike hemangioblastomas, however, cerebellar DASEs do not express brachyury, a mesodermal marker for hemangioblasts. In this study, neuronal progenitors occupying the molecular layer were investigated as tumor cells of origin. By immunohistochemistry, cerebellar DASEs and hemangioblastomas lacked immunoreactivity with antibody ZIC1 (Zic family member 1), a granule cell progenitor marker with concordance from oligonucleotide RNA expression array analyses. Rather, cerebellar DASEs and hemangioblastomas were immunoreactive with antibody PAX2 (paired box 2), a marker of basket/stellate cell progenitors. VHL cerebellar cortices also revealed PAX2-positive cells in Purkinje and molecular layers, resembling the histological and molecular development of basket/stellate cells in postnatal non-VHL mouse and human cerebella. These data suggest that VHL deficiency can result in the developmental arrest of basket/stellate cells in the human cerebellum and that these PAX2-positive, initiated cells await another insult or signal to form DASEs and eventually, tumors., (Published by Oxford University Press on behalf of American Association of Neuropathologists, Inc. 2022.)

Background: ONC201, a dopamine receptor D2 (DRD2) antagonist and caseinolytic protease P (ClpP) agonist, has induced durable tumor regressions in adults with recurrent H3 K27M-mutant glioma. We report results from the first phase I pediatric clinical trial of ONC201., Methods: This open-label, multi-center clinical trial (NCT03416530) of ONC201 for pediatric H3 K27M-mutant diffuse midline glioma (DMG) or diffuse intrinsic pontine glioma (DIPG) employed a dose-escalation and dose-expansion design. The primary endpoint was the recommended phase II dose (RP2D). A standard 3 + 3 dose escalation design was implemented. The target dose was the previously established adult RP2D (625 mg), scaled by body weight. Twenty-two pediatric patients with DMG/DIPG were treated following radiation; prior lines of systemic therapy in addition to radiation were permitted providing sufficient time had elapsed prior to study treatment., Results: The RP2D of orally administered ONC201 in this pediatric population was determined to be the adult RP2D (625 mg), scaled by body weight; no dose-limiting toxicities (DLT) occurred. The most frequent treatment-emergent Grade 1-2 AEs were headache, nausea, vomiting, dizziness and increase in alanine aminotransferase. Pharmacokinetics were determined following the first dose: T 1/2 , 8.4 h; T max , 2.1 h; C max , 2.3 µg/mL; AUC 0-tlast , 16.4 hµg/mL. Median duration of treatment was 20.6 weeks (range 5.1-129). Five (22.7%) patients, all of whom initiated ONC201 following radiation and prior to recurrence, were alive at 2 years from diagnosis., Conclusions: The adult 625 mg weekly RP2D of ONC201 scaled by body weight was well tolerated. Further investigation of ONC201 for DMG/DIPG is warranted., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Embryonal tumors of the CNS are the most common malignant tumors occurring in the first years of life. This study evaluated the feasibility and safety of incorporating novel non-cytotoxic therapy with vorinostat and isotretinoin to an intensive cytotoxic chemotherapy backbone., Methods: PBTC-026 was a prospective multi-institutional clinical trial for children <48 months of age with newly diagnosed embryonal tumors of the CNS. Treatment included three 21-day cycles of induction therapy with vorinostat and isotretinoin, cisplatin, vincristine, cyclophosphamide, and etoposide; three 28-day cycles of consolidation therapy with carboplatin and thiotepa followed by stem cell rescue; and twelve 28-day cycles of maintenance therapy with vorinostat and isotretinoin. Patients with M0 medulloblastoma (MB) received focal radiation following consolidation therapy. Molecular classification was by DNA methylation array., Results: Thirty-one patients with median age of 26 months (range 6-46) received treatment on study; 19 (61%) were male. Diagnosis was MB in 20 and supratentorial CNS embryonal tumor in 11. 24/31 patients completed induction therapy within a pre-specified feasibility window of 98 days. Five-year progression-free survival (PFS) and overall survival (OS) for all 31 patients were 55 ± 15 and 61 ± 13, respectively. Five-year PFS was 42 ± 13 for group 3 MB (n = 12); 80 ± 25 for SHH MB (n = 5); 33 ± 19 for embryonal tumor with multilayered rosettes (ETMR, n = 6)., Conclusion: It was safe and feasible to incorporate vorinostat and isotretinoin into an intensive chemotherapy regimen. Further study to define efficacy in this high-risk group of patients is warranted., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Medulloblastoma (MB) is the most common malignant brain tumor in children with standard of care consisting of surgery, radiation, and chemotherapy. Recent molecular profiling led to the identification of four molecularly distinct MB subgroups - Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Despite genomic MB characterization and subsequent tumor stratification, clinical treatment paradigms are still largely driven by histology, degree of surgical resection, and presence or absence of metastasis rather than molecular profile. Patients usually undergo resection of their tumor followed by craniospinal radiation (CSI) and a 6 month to one-year multi-agent chemotherapeutic regimen. While there is clearly a need for development of targeted agents specific to the molecular alterations of each patient, targeting proteins responsible for DNA damage repair could have a broader impact regardless of molecular subgrouping. DNA damage response (DDR) protein inhibitors have recently emerged as targeted agents with potent activity as monotherapy or in combination in different cancers. Here we discuss the molecular underpinnings of genomic instability in MB and potential avenues for exploitation through DNA damage response inhibition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McSwain, Parwani, Shahab, Hambardzumyan, MacDonald, Spangle and Kenney.)

Sonic hedgehog (Shh)-driven medulloblastoma (Shh MB) cells are dependent on constitutive Shh signaling, but targeted treatment of Shh MB has been ineffective due to drug resistance. The purpose of this study was to address the critical role of signal transducer and activator of transcription 3 (STAT3) in Shh signaling and drug resistance in Shh MB cells. Herein, we show that STAT3 is required for Smoothened (Smo)-dependent Shh signaling and, in turn, is reciprocally regulated by Shh signaling, and demonstrate that STAT3 activity is critical for expression of HCK proto-oncogene, Src family tyrosine kinase (Hck) in Shh MB. We also demonstrate that maintained STAT3 activity suppresses p21 expression and promotes colony formation of Shh MB cells, whereas dual treatment with inhibitors of both Smo and STAT3 results in marked synergistic killing and overcomes drug resistance in vitro of Smo antagonist-resistant Shh MB cells. Finally, STAT3 inhibitor treatment significantly prevents in vivo tumor formation in genetically engineered Shh MB mice. Collectively, we show that STAT3 is necessary to maintain Shh signaling and thus is a potential therapeutic target to treat Shh MB and overcome anti-Smo drug resistance., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

RNA-based therapies offer unique advantages for treating brain tumors. However, tumor penetrance and uptake are hampered by RNA therapeutic size, charge, and need to be "packaged" in large carriers to improve bioavailability. Here, we have examined delivery of siRNA, packaged in 50-nm cationic lipid-polymer hybrid nanoparticles (LPHs:siRNA), combined with microbubble-enhanced focused ultrasound (MB-FUS) in pediatric and adult preclinical brain tumor models. Using single-cell image analysis, we show that MB-FUS in combination with LPHs:siRNA leads to more than 10-fold improvement in siRNA delivery into brain tumor microenvironments of the two models. MB-FUS delivery of Smoothened ( SMO ) targeting siRNAs reduces SMO protein production and markedly increases tumor cell death in the SMO -activated medulloblastoma model. Moreover, our analysis reveals that MB-FUS and nanoparticle properties can be optimized to maximize delivery in the brain tumor microenvironment, thereby serving as a platform for developing next-generation tunable delivery systems for RNA-based therapy in brain tumors., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The sonic hedgehog subtype of medulloblastoma (SHH MB) is associated with treatment failure and poor outcome. Current strategies utilizing whole brain radiation therapy result in deleterious off-target effects on the normal developing childhood brain. Most conventional chemotherapies remain limited by ineffective blood-brain barrier (BBB) penetrance. These challenges signify an unmet need for drug carriers that can cross the BBB and deliver drugs to targeted sites with high drug-loading efficiency and long-term stability. We herein leverage the enhanced stability and targeting ability of engineered high-density lipoprotein-mimetic nanoparticles (eHNPs) to cross the BBB and deliver a SHH inhibitor effectively to the cancer stem-like cell population in SHH MB. Our microfluidic technology enabled highly reproducible production of multicomponent eHNPs incorporated with apolipoprotein A1, anti-CD15, and a SHH inhibitor (LDE225). We demonstrate the dual-targeted delivery and enhanced therapeutic effect of eHNP-A1-CD15-LDE225 via scavenger receptor class B type 1 (SR-B1) and CD15 on brain SHH MB cells in vitro, ex vivo, and in vivo. Moreover, we show that eHNP-A1 not only serves as a stable drug carrier, but also has a therapeutic effect itself through SR-B1-mediated intracellular cholesterol depletion in SHH MB cells. Through the facilitated and targeted cellular uptake of drugs and direct therapeutic role of this engineered biomimetic nanocarrier in SHH MB, our multifunctional nanoparticle provides intriguing therapeutic promise as an effective and potent nanomedicine for the treatment of SHH MB., Competing Interests: Competing interest statement: In compliance with the institutional guidelines of the Georgia Institute of Technology, Y.K. discloses his financial interest in Mepsgen and Mepsgenlab, two biotechnology companies developing microengineered physiological systems and biomimetic nanoparticles for medical applications. Mepsgen and Mepsgenlab did not support the aforementioned research, and currently these companies have no rights to any technology or intellectual property developed as part of this research.

Challenges in drug development of neurological diseases remain mainly ascribed to the blood-brain barrier (BBB). Despite the valuable contribution of animal models to drug discovery, it remains difficult to conduct mechanistic studies on the barrier function and interactions with drugs at molecular and cellular levels. Here we present a microphysiological platform that recapitulates the key structure and function of the human BBB and enables 3D mapping of nanoparticle distributions in the vascular and perivascular regions. We demonstrate on-chip mimicry of the BBB structure and function by cellular interactions, key gene expressions, low permeability, and 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4 (AQP4) distribution. Moreover, our model precisely captures 3D nanoparticle distributions at cellular levels and demonstrates the distinct cellular uptakes and BBB penetrations through receptor-mediated transcytosis. Our BBB platform may present a complementary in vitro model to animal models for prescreening drug candidates for the treatment of neurological diseases.

Purpose: Medulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics. Histopathological similarities confound rapid diagnoses of these two tumor types. We have conducted a pilot feasibility study analyzing the molecular profile of archived frozen human tumor specimens using mass spectrometry imaging (MSI) to identify potential biomarkers capable of classifying and distinguishing between medulloblastoma and pineoblastoma., Methods: We performed matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry imaging on eight medulloblastoma biopsy specimens and three pineoblastoma biopsy specimens. Multivariate statistical analyses were performed on the MSI dataset to generate classifiers that distinguish the two tumor types. Lastly, the molecules that were discriminative of tumor type were queried against the Lipid Maps database and identified., Results: In this pilot study we show that medulloblastoma and pineoblastoma can be discriminated using molecular profiles determined by MSI. The highest-ranking discriminating classifiers of medulloblastoma and pineoblastoma were glycerophosphoglycerols and sphingolipids, respectively., Conclusion: We demonstrate proof-of-concept that medulloblastoma and pineoblastoma can be rapidly distinguished by using MSI lipid profiles. We identified biomarker candidates capable of distinguishing these two histopathologically similar tumor types. This work expands the current molecular knowledge of medulloblastoma and pineoblastoma by characterizing their lipidomic profiles, which may be useful for developing novel diagnostic, prognostic and therapeutic strategies.

Background: Diagnosis of diffuse intrinsic pontine glioma (DIPG) has relied on imaging studies, since the appearance is pathognomonic, and surgical risk was felt to be high and unlikely to affect therapy. The DIPG Biology and Treatment Study (DIPG-BATS) reported here incorporated a surgical biopsy at presentation and stratified subjects to receive FDA-approved agents chosen on the basis of specific biologic targets., Methods: Subjects were eligible for the trial if the clinical features and imaging appearance of a newly diagnosed tumor were consistent with a DIPG. Surgical biopsies were performed after enrollment and prior to definitive treatment. All subjects were treated with conventional external beam radiotherapy with bevacizumab, and then stratified to receive bevacizumab with erlotinib or temozolomide, both agents, or neither agent, based on O6-methylguanine-DNA methyltransferase status and epidermal growth factor receptor expression. Whole-genome sequencing and RNA sequencing were performed but not used for treatment assignment., Results: Fifty-three patients were enrolled at 23 institutions, and 50 underwent biopsy. The median age was 6.4 years, with 24 male and 29 female subjects. Surgical biopsies were performed with a specified technique and no deaths were attributed to the procedure. Two subjects experienced grade 3 toxicities during the procedure (apnea, n = 1; hypertension, n = 1). One subject experienced a neurologic deficit (left hemiparesis) that did not fully recover. Of the 50 tumors biopsied, 46 provided sufficient tissue to perform the study assays (92%, two-stage exact binomial 90% CI: 83%-97%)., Conclusions: Surgical biopsy of DIPGs is technically feasible, associated with acceptable risks, and can provide biologic data that can inform treatment decisions.

Background: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines., Methods: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MB WNT ), SHH (MB SHH ), group 3 (MB Group3 ), and group 4 (MB Group4 ). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma., Findings: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MB SHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MB SHH subgroup). Patients with germline APC mutations developed MB WNT and accounted for most (five [71%] of seven) cases of MB WNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MB SHH . Germline TP53 mutations presented only in childhood patients in the MB SHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MB SHH , MB Group3 , and MB Group4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes., Interpretation: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MB WNT and MB SHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics., Funding: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.

Medulloblastomas and sPNETs remain highly problematic tumors to treat. Prognosis has improved over the past two decades, but many children who survive treatment have significant long-term sequelae. The improvements in outcome have been due to advances in surgical techniques, the wider use of chemotherapy, and the more judicious use of radiotherapy. For further improvements,the recent impressive discoveries concerning molecular mechanisms of embryonal tumor origin, development,and growth will need to be translated into molecularly based, risk-adapted therapy.

Background: High-dose chemotherapy with autologous stem cell rescue (HDC/SCR) has produced responses and prolonged survival for some children with recurrent brain tumors, but is associated with considerable morbidity and mortality. A Phase I trial of two cycles of HDC/SCR for recurrent brain tumors in children was performed to determine the maximum tolerated doses for a novel regimen., Procedures: Two cycles of HDC/SCR were given. Cycle 1 included thiotepa and carmustine given on days -5, -4, and -3. Four to six weeks later, patients received cycle 2 which included thiotepa and carboplatin given on days -5, -4, and -3. Autologous peripheral blood stem cells (PBSC) were infused on day 0 of each cycle., Results: Thirty-two patients were treated and 25 patients received both cycles of HDC/SCR. Common toxicities included mucositis, emesis, diarrhea, anorexia, and pancytopenia. Eight of 32 (25%) assessable children died from regimen-related toxicity. Pulmonary failure occurred in seven patients. Seven patients had grade 3-4 neurotoxicity. The 3-year event-free survival (EFS) was 25%., Conclusions: We determined the maximum tolerated regimen to be thiotepa 600 mg/m(2) and carmustine 300 mg/m(2) followed by thiotepa 600 mg/m(2) and carboplatin 1,200 mg/m(2) . Pulmonary toxicity was considerable. The toxic death rate was similar to other trials of HDC/SCR for children with recurrent brain tumors performed during the same time period. The regimen resulted in prolonged time to progression for a significant number of patients and long-term survival for some patients with recurrent medulloblastoma and rhabdoid tumor., (Copyright © 2010 Wiley-Liss, Inc.)

Background: Pilocytic Astrocytomas (PAs) are common low-grade central nervous system malignancies for which few recurrent and specific genetic alterations have been identified. In an effort to better understand the molecular biology underlying the pathogenesis of these pediatric brain tumors, we performed higher-order transcriptional network analysis of a large gene expression dataset to identify gene regulatory pathways that are specific to this tumor type, relative to other, more aggressive glial or histologically distinct brain tumours., Methods: RNA derived from frozen human PA tumours was subjected to microarray-based gene expression profiling, using Affymetrix U133Plus2 GeneChip microarrays. This data set was compared to similar data sets previously generated from non-malignant human brain tissue and other brain tumour types, after appropriate normalization., Results: In this study, we examined gene expression in 66 PA tumors compared to 15 non-malignant cortical brain tissues, and identified 792 genes that demonstrated consistent differential expression between independent sets of PA and non-malignant specimens. From this entire 792 gene set, we used the previously described PAP tool to assemble a core transcriptional regulatory network composed of 6 transcription factor genes (TFs) and 24 target genes, for a total of 55 interactions. A similar analysis of oligodendroglioma and glioblastoma multiforme (GBM) gene expression data sets identified distinct, but overlapping, networks. Most importantly, comparison of each of the brain tumor type-specific networks revealed a network unique to PA that included repressed expression of ONECUT2, a gene frequently methylated in other tumor types, and 13 other uniquely predicted TF-gene interactions., Conclusions: These results suggest specific transcriptional pathways that may operate to create the unique molecular phenotype of PA and thus opportunities for corresponding targeted therapeutic intervention. Moreover, this study also demonstrates how integration of gene expression data with TF-gene and TF-TF interaction data is a powerful approach to generating testable hypotheses to better understand cell-type specific genetic programs relevant to cancer.

Little is known about the molecular characteristics of pediatric brainstem gliomas (BSG), which continue to have a dismal prognosis. Targeted molecular strategies are limited due to rarity of biopsy BSG specimen coupled with obstacles associated with the analyses of formalin-fixed paraffin-embedded (FFPE) autopsies. The objective of this study was to develop methodologies to successfully identify the proteome profile from these archived FFPE specimens. Peptides were extracted from both tumor and adjacent normal FFPE brainstem specimen and quantified using (18) O proteolytic labeling strategy and LC-MS/MS analysis. The ingenuity pathway analysis software was used to elucidate interactions amongst differentially expressed proteins. We identified 188 proteins of which 54 (29%) were found up-regulated (≥1.5-fold) in BSG compared to normal sections. Of these, 15 (28%) proteins have previously been reported as potential biomarkers for supratentorial malignant gliomas, while the rest appear to be exclusive to pediatric BSG. Because the majority of differentially expressed proteins are unique to BSG, we conclude that pediatric BSG is distinct from supratentorial gliomas. To the best of our knowledge, this is the first proteome profile of pediatric BSG, which may facilitate discovery of novel therapeutic targets for early diagnostics and improving prognostics., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A phase II study was conducted to assess the efficacy of administering daily thalidomide concomitantly with radiation and continuing for up to 1 year following radiation in children with brain stem gliomas (BSG) or glioblastoma multiforme (GBM). Secondary objectives were to obtain preliminary evidence of biologic activity of thalidomide and to evaluate toxicities from chronic administration of thalidomide in this population. Thirteen patients (2-14 years old) with newly diagnosed BSG (12 patients) or GBM (one patient) were enrolled between July 1999 and June 2000. All patients received focal radiotherapy to a total dose of 5,580 cGy. Thalidomide was administered once daily beginning on the first day of radiation and continued for 12 months or until the patient came off study. The starting dose was 12 mg/kg (rounded down to the nearest 50 mg) and was increased by 20% weekly, if tolerated, to 24 mg/kg or 1,000 mg (whichever was lower). Advanced imaging techniques and urine and serum analysis for anti-angiogenic markers were performed in some patients in an attempt to correlate changes with clinical effect of therapy. No patients completed the planned 12 months of thalidomide therapy and all have since died of disease progression. The median duration of therapy was 5 months (range 2-11 months). Nine patients came off study for progressive disease (PD), three patients due to toxicity and one patient withdrew consent. Several patients on this study required more extended courses of high dose steroids than would have been otherwise expected for this population due to significant peritumoral edema and necrosis. No consistent pattern emerged from the biologic correlative studies from 11 patients. However, advanced imaging with techniques such as MR spectroscopy, MR perfusion and 18-fluorodeoxyglucose positron emission tomography (FDG-PET) were helpful in distinguishing growing tumor from treatment effect and necrosis in some patients. The median time to progression (TTP) was 5 months (range 2-11 months) and the median time to death (TTD) was 9 months (range 5-17 months). In this small patient sample adding thalidomide to radiation did not improve TTP or TTD from historical controls, however, toxicity appeared to be increased.

Intense angiogenesis proliferation, a histopathological hallmark distinguishing malignant from benign astrocytoma, is vital for tumor progression. Thus, identifying and targeting specific pathways that promote malignant astrocytoma-induced angiogenesis could have substantial therapeutic benefit. Expression profiling of 13 childhood astrocytomas to determine the expression pattern of 133 angiogenesis-related genes revealed that 44 (33%) genes were differentially expressed (17 were overexpressed, and 27 were underexpressed) between malignant high-grade astrocytomas (HGAs) and benign low-grade astrocytomas. Hierarchical clustering and principal components analysis using only the 133 angiogenesis-related genes distinguished HGA from low-grade astrocytoma in 100% of the samples analyzed, as did unsupervised analyses using the entire set of 9198 expressed genes represented on the array, indicating that the angiogenesis-related genes were reliable markers of pathological grade. A striking new finding was significant overexpression of hypoxia-inducible transcription factor (HIF)-2alpha as well as high-level expression of FK506-binding protein (FKBP) 12 by HGA. Furthermore, 9 of 21 (43%) genes overexpressed by HGA were HIF/FKBP-associated genes. This group included the epidermal growth factor receptor (EGFR), which promotes HIF synthesis, as well as insulin-like growth factor-binding protein 2 (IGFBP2), a target gene of HIF activity. Differential protein expression of HIF-2alpha was validated in an independent group of 16 astrocytomas (P = 0.02). We conclude that the EGFR/FKBP12/HIF-2alpha pathway is important in childhood HGA and represents a potential new therapeutic target.