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High messenger RNA (mRNA) expression of the tropomyosin receptor kinase C gene (TrkC) has been associated with favorable survival in medulloblastoma patients. Untested is whether it plays a role through modulating the response to therapy or whether it might be a surrogate marker for a favorable molecular subgroup. The medulloblastoma-derived cell line DAOY was stably transfected to overexpress TrkC (clone DAOY-TrkC) and compared to a control (clone DAOY-EV, empty vector transfected). Cell viability (MTS assay) was tested after irradiation or incubation with chemotherapeutic drugs. Neuroradiologic response to postoperative chemotherapy or craniospinal irradiation (CSI) of medulloblastoma patients aged 3–21 years with postoperative residual disease treated within the consecutive trials HIT’91/HIT2000 was compared to TrkC mRNA expression in their tumor samples. Five well-characterized independent expression-profiling studies covering together 686 medulloblastoma patients were analyzed for TrkC levels according to the molecular subgroups. Cell viability of DAOY-TrkC compared to DAOY-EV was not different after exposure to increasing doses of irradiation, cisplatin, etoposide, or vincristine. While TrkC mRNA expression tended to be higher in non-responders (n = 5/19) to postoperative CSI (p = 0.03, ratio 15.5, 95% CI 9–267), this was the case in responders (n = 23/43) to chemotherapy (p = 0.04, ratio 6.1, 95% CI 1.1–35), both analyzed with Mann–Whitney U test (not significant after Bonferroni adjustment). The highest TrkC mRNA levels were found in the SHH subgroup across all expression-profiling studies. High TrkC mRNA expression appears to be frequent in the SHH subgroup and seems not to have a major effect on therapy responsiveness in medulloblastoma patients.

Item does not contain fulltext Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Embryonal tumors of the central nervous system represent a heterogeneous group of childhood cancers with an unknown pathogenesis; diagnosis, on the basis of histological appearance alone, is controversial and patients’ response to therapy is difficult to predict. They encompass medulloblastoma, atypical teratoid/rhabdoid tumors and a group of primitive neuroectodermal tumors. All are aggressive tumors with the tendency to disseminate throughout the central nervous system. The large amount of genomic and molecular data generated over the last 5–10 years encourages optimism that new molecular targets will soon improve outcomes. Recent neurobiological studies have uncovered the key role of microRNAs (miRNAs) in embryonal tumors biology and their potential use as biomarkers is increasingly being recognized and investigated. However the successful use of microRNAs as reliable biomarkers for the detection and management of pediatric brain tumors represents a substantial challenge. This review debates the importance of miRNAs in the biology of central nervous systemembryonal tumors focusing on medulloblastoma and atypical teratoid/rhabdoid tumors and highlights the advantages as well as the limitations of their prospective application as biomarkers and candidates for molecular therapeutic targets.

Embryonal tumors of the nervous system are the leading cause of childhood cancer-related morbidity and mortality. Medulloblastoma, supratentorial primitive neuroectodermal tumors, atypical teratoid/rhabdoid tumor and neuroblastoma account for more than 20% of childhood malignancies and typify the current neural embryonal tumor model in pediatric oncology. Mechanisms driving the formation of these tumors point towards impaired differentiation of neuronal and neuron-associated cells during the development of the nervous system as an important factor. The importance of microRNAs (miRNAs) for proper embryonic cell function has been confirmed and their aberrant expressions have been linked to tumor development. The role of miRNAs in controlling essential regulators of key pathways implicated in tumor development makes their use in diagnostics a powerful tool to be used for early detection of cancer, risk assessment and prognosis, as well as for the design of innovative therapeutic strategies. In this review we focus on the significance of miRNAs involved in the biology of embryonal neural tumors, delineate their clinical significance and discuss their potential as a novel therapeutic target.

Background: microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated. Methods: Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinical outcome was assessed, and the impact of microRNA-9 restoration was functionally validated in medulloblastoma cells. Results: microRNA-9 expression is repressed in a large subset of MB samples compared with normal fetal cerebellum. Low microRNA-9 expression correlates significantly with the diagnosis of unfavourable histopathological variants and with poor clinical outcome. microRNA-9 silencing occurs via cancer-specific CpG island hypermethylation. HES1 was identified as a direct target of microRNA-9 in medulloblastoma, and restoration of microRNA-9 was shown to trigger cell cycle arrest, to inhibit clonal growth and to promote medulloblastoma cell differentiation. Conclusions: microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis. Thus, re-expression of microRNA-9 may constitute a novel epigenetic regulation strategy against medulloblastoma.

MB-108. EXPRESSION OF TROPOMYOSIN RECEPTOR KINASE C (TrkC) HAS NO MAJOR IMPACT ON THE RESPONSE TO THERAPY OF MEDULLOBLASTOMA IN VITRO AND IN A CLINICAL PATIENT COHORT Carsten Friedrich1, Tarek Shalaby2, Christoph Oehler3,4, Martin Pruschy5, Burkhardt Seifert6, Monika Warmuth-Metz7, Rolf-Dieter Kortmann8, Stefan Rutkowski9, Michael A. Grotzer2, and Andre O. von Bueren10,11; Division of Pediatric Oncology, Hematology and Hemostaseology, Department of Woman’s and Children’s Health, University Hospital Leipzig, Leipzig, Germany; Department of Oncology, University Children’s Hospital, Zurich, Switzerland; Department of Radiation Oncology, University Hospital Zurich, Zurich, Switzerland; Department of Radiation Oncology, Hospital Graubuenden, Chur, Switzerland; Laboratory for Molecular Radiobiology, Radiation Oncology, University Hospital Zurich, Zurich, Switzerland; Department of Biostatistics; Epidemiology, Biostatistics and Prevention Institute, University of Zurich, Zurich, Switzerland; Department of Neuroradiology, University of Wuerzburg, Wuerzburg, Germany; Department of Radiation Oncology, University of Leipzig, Leipzig, Germany; Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Pediatrics, Division of Pediatric Hematology and Oncology, University Medical Center Goettingen, Goettingen, Germany; Department of Pediatrics and Adolescent Medicine, Division of Pediatric Hematology and Oncology, University Hospital of Geneva, Geneva, Switzerland BACKGROUND: High mRNA expression of the tropomyosin receptor kinase C (TrkC) has been associated with favorable survival in medulloblastoma patients. A not yet tested explanation could be that TrkC mRNA expression modulates the response to therapy. PATIENTS AND METHODS: Medulloblastoma-derived cell line DAOY, stably transfected to overexpress TrkC (clone DAOY-TrkC) and compared to a control (clone DAOY-EV, empty vector transfected), was grown under serum-free conditions (+ TrkC ligand neutrophin-3, NT-3) and cell viability was tested after irradiation or incubation with chemotherapeutic drugs after 72 h using the MTS-assay. Neuroradiologic response topostoperativechemotherapyorcraniospinal irradiation (CSI) of medulloblastoma patients aged 3-21 years with postoperative residual disease treated within the consecutive trials HIT’91/HIT2000 was compared to TrkC mRNA expression in their tumor samples. RESULTS: Cell viability of DAOY-TrkC compared to DAOY-EV was not different after incubation with increasing doses of cisplatin (0-4.8 mg/ml), etoposide (0-1.3 mg/ml), or vincristine (0-20 ng/ml), and irradiation (2, 5, 10 Gy). WhileTrkC mRNAexpression tended tobe higher in non-responders (n 1⁄4 5/19) to postoperative CSI (p 1⁄4 0.03, ratio 15.5, 95% CI 9-267), this was the case in responders (n 1⁄4 23/43) to chemotherapy (p 1⁄4 0.04, ratio 6.1, 95% CI 1.1-35, both analyzed with Mann-Withney U test and not significant after Bonferroni adjustment). CONCLUSIONS: TrkC mRNA expression was not associated with a considerable improvement in response to therapy neither in vitro nor in a MB patient cohort. The study is limited by a relatively small medulloblastoma patient cohort and the availability of only one TrkC mRNA overexpressing medulloblastoma cell line. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.103 #The Author(s) 2016. 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), the most common malignant brain tumour in children, is characterised by a high risk of leptomeningeal dissemination. But little is known about the molecular mechanisms that promote cancer cell migration in MB. Aberrant expression of miR-21 is recognised to be causatively linked to metastasis in a variety of human neoplasms including brain tumours; however its function in MB is still unknown. In this study we investigated the expression level and the role of miR-21 in MB cell migration. miR-21 was found to be up-regulated, compared to normal cerebellum, in 29/29 MB primary samples and 6/6 MB-derived cell lines. Inverse correlation was observed between miR-21 expression and the metastasis suppressor PDCD4, while miR-21 repression increased the release of PDCD4 protein, suggesting negative regulation of PDCD4 by miR-21 in MB cells. Anti-miR-21 decreased protein expression of the tumour cell invasion mediators MAP4K1 and JNK, which are also known to be negatively regulated by PDCD4, and down-regulated integrin protein that is essential for MB leptomeningeal dissemination. Moreover miR-21 knockdown in MB cells increased the expression of two eminent negative modulators of cancer cell migration, E-Cadherin and TIMP2 proteins that are known to be positively regulated by PDCD4. Finally and importantly, suppression of miR-21 decreased the motility of MB cells and reduced their migration across basement membranes in vitro. Together, these compelling data propose miR-21 pathway as a novel mechanism impacting MB cell dissemination and raises the possibility that curability of selected MB may be improved by pharmaceutical strategies directed towards microRNA-21.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma. Mol Cancer Res; 9(7); 925–35. ©2011 AACR.

Purpose: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in medulloblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in medulloblastoma. Experimental Design: The expression pattern and functions of class IA PI3K isoforms were investigated in medulloblastoma tumour samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110α, p110β, or p110δ by means of RNA interference or inhibition with isoform-specific PI3K inhibitors. Results: Overexpression of the catalytic p110α isoform was detected in a panel of primary medulloblastoma samples and cell lines compared with normal brain tissue. Down-regulation of p110α expression by RNA interference impaired the growth of medulloblastoma cells, induced apoptosis, and led to decreased migratory capacity of the cells. This effect was selective, because RNA interference targeting of p110β or p110δ did not result in a comparable impairment of DAOY cell survival. Isoform-specific p110αinhibitors also impaired medulloblastoma cell proliferation and sensitized the cells to chemotherapy. Medulloblastoma cells treated with p110α inhibitors further displayed reduced activation of Akt and the ribosomal protein S6 kinase in response to stimulation with hepatocyte growth factor and insulin-like growth factor-I. Conclusions: Together, our data reveal a novel function of p110α in medulloblastoma growth and survival.

Purpose: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in neuroblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in neuroblastoma.Experimental Design: The expression pattern and functions of class IA PI3K isoforms were investigated in tumor samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110α or p110δ in SH-SY5Y and LA-N-1 cells by means of RNA interference.Results: Overexpression of the catalytic p110δ and regulatory p85α isoforms was detected in a panel of primary neuroblastoma samples and cell lines, compared with normal adrenal gland tissue. Although down-regulation of either p110α or p110δ led to impaired cell growth, reduced expression of p110δ also had a selective effect on the survival of SH-SY5Y cells. Decreased levels of p110δ were found to induce apoptosis and lead to lower expression levels of antiapoptotic Bcl-2 family proteins. SH-SY5Y cells with decreased p110δ levels also displayed reduced activation of ribosomal protein S6 kinase in response to stimulation with epidermal growth factor and insulin-like growth factor-I.Conclusions: Together, our data reveal a novel function of p110δ in neuroblastoma growth and survival.

BACKGROUND: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner. METHODS: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival. FINDINGS: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084). INTERPRETATION: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection. FUNDING: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Central nervous system (CNS) cancer is a devastating illness with unmet therapeutic needs. Establishing biomarkers that have the potential to guide accurate CNS cancer diagnosis or are helpful in predicting disease progression or therapy response is of great interest. Cerebrospinal fluid (CSF) has been extensively targeted for the detection of molecules that might be useful markers for cancer detection. However, so far very few of such markers have found a standardized routine clinical application. This review examines the current scientific knowledge about the biochemical elements in the CSF that have been reported in the literature as brain cancer biomarkers and highlight reasons why the role of most markers is not yet established in the managment of CNS tumors.

Purpose Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known. Methods Four independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses. Results Molecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN_PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN_PFA, a substantial proportion of patients with EPN_PFB can be cured with surgery alone, and patients with relapsed EPN_PFB can often be treated successfully with delayed external-beam irradiation. Conclusion The most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN_PFA and EPN_PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN_PFA, even with adjuvant radiation therapy. Patients with EPN_PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence.

Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in infants. Although the prognosis of ATRT patients is poor, some patients respond favorably to current treatments, suggesting molecular inter-tumor heterogeneity. To investigate this further, we genetically and epigenetically analyzed 192 ATRTs. Three distinct molecular subgroups of ATRTs, associated with differences in demographics, tumor location, and type of SMARCB1 alterations, were identified. Whole-genome DNA and RNA sequencing found no recurrent mutations in addition to SMARCB1 that would explain the differences between subgroups. Whole-genome bisulfite sequencing and H3K27Ac chromatin-immunoprecipitation sequencing of primary tumors, however, revealed clear differences, leading to the identification of subgroup-specific regulatory networks and potential therapeutic targets.

AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class IA PI3K p110α isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.

The MYC family plays essential roles during brain development and their oncogenic deregulation is implicated in the formation of embryonal neural tumors such as medulloblastomas (MB) and neuroblastoma (NB). Amplification of the MYCN is the predominant marker for aggressive NB and correlates with poor prognosis, while c-MYC overexpression is a defining feature of MB subgroups inflected with aggressive biological behavior and increased likelihood of metastasis. Not surprisingly MYC has emerged as an attractive target for pediatric neural cancer therapy. However despite three decades of intensive research in MYC biology and an impressive number of 30,000 publications, inhibition of MYC as therapeutic strategy remains an elusive goal in cancer medicine. This review discusses the potential and challenges of targeting the oncogenic effects of MYC as therapeutic strategy for MYC over-expressing embryonal neural tumors where current therapies are inadequate.

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (

Aim: Medulloblastoma (MB) is the most common malignant brain tumor in children. The crucial role of extracellular-microRNAs (ex-miRNAs) in cancer has been widely recognized; however, their role in MB remains unknown. This study aimed to investigate MB-driven ex-miRNAs. Methods: Microarray analysis was used to disclose the identity and quantity of key miRNAs excreted in culture-medium (CM) of 3 human MB cell lines and cerebrospinal fluid (CSF) of brain tumors (including MB) and leukemia patients. MiRNA expression was validated by quantitative reverse transcription polymerase chain reaction. Results: We have demonstrated that the 3 MB cell lines tested commonly expressed 1,083 miRNAs in their spent CM. Among them, 57 miRNAs were specific to the CM of metastasis-related cell lines which represents the aggressive group 3 and group 4 MB subtypes. A significant number (1,254) of ex-miRNAs were identified in the CSF of a MB patient. Eighty-six of these miRNAs were found to be differentially expressed in this patient's CSF compared with controls. Interestingly, 3 metastasis-associated miRNAs over-represented in CM of metastasis-related MB cell lines were found to be significantly enriched in the CSF of the MB patient. Conclusion: Although more samples are required to fully verify these results, our work provides the first evidence for the presence of a significant amount of miRNAs excreted extracellularly by MB cells and raises the possibility that, in the near future, miRNAs could be probed in CSF of MB patients and serve as novel biological markers.

Medulloblastoma (MB), the most common pediatric malignant brain cancer, typically arises as pathological result of deregulated developmental pathways, including the NOTCH signaling cascade. Unlike the evidence supporting a role for NOTCH receptors in MB development, the pathological functions of NOTCH ligands remain largely unexplored. By examining the expression in large cohorts of MB primary tumors, and in established in vitro MB models, this research study demonstrates that MB cells bear abnormal levels of distinct NOTCH ligands. We explored the potential association between NOTCH ligands and the clinical outcome of MB patients, and investigated the rational of inhibiting NOTCH signaling by targeting specific ligands to ultimately provide therapeutic benefits in MB. The research revealed a significant over-expression of ligand JAG1 in the vast majority of MBs, and proved that JAG1 mediates pro-proliferative signals via activation of NOTCH2 receptor and induction of HES1 expression, thus representing an attractive therapeutic target. Furthermore, we could identify a clinically relevant association between ligand JAG2 and the oncogene MYC, specific for MYC-driven Group 3 MB cases. We describe for the first time a mechanistic link between the oncogene MYC and NOTCH pathway in MB, by identifying JAG2 as MYC target, and by showing that MB cells acquire induced expression of JAG2 through MYC-induced transcriptional activation. Finally, the positive correlation of MYC and JAG2 also with aggressive anaplastic tumors and highly metastatic MB stages suggested that high JAG2 expression may be useful as additional marker to identify aggressive MBs.

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O(6)-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7-92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n = 46; Pearson correlation r (2) = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT'91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not.

Further progress in cancer diagnosis and therapy will be only possible with ongoing translational research. Accordingly, it has become extremely important to collect prospectively tumor samples along with the clinical information in a systematic manner to distribute it to dedicated research groups. Pediatric liver tumors are rare entities and the availability of large series of high-quality tumor samples and their associated clinical data is crucial for biomedical research to thrive in these childhood malignancies. The SIOPEL Liver Tumour Storage Programme includes the centralized collection and storage of tissues under controlled and standardized conditions, a comprehensive inventory, a process to distribute specimens to investigators, and policies to define responsible research.

Background To study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB). Methods We used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging. Results In DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR) vs. 5 non-responders (SD, PD)) or chemotherapy (23 CR/PR vs. 20 SD/PD) was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively). Conclusions c-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex–forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC50, 0.25–0.39 μmol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc–dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors. Mol Cancer Ther; 9(1); 167–79

Myc family genes are often deregulated in embryonal tumors of childhood including medulloblastoma and neuroblastoma and are frequently associated with aggressive, poorly differentiated tumors. The Myc protein is a transcription factor that regulates a variety of cellular processes including cell growth and proliferation, cell cycle progression, differentiation, apoptosis, and cell motility. Potential strategies that either inhibit the proliferation-promoting effect of Myc and/or activate its pro-apoptotic function are presently being explored. In this review, we will give an overview of Myc activation in embryonal tumors and discuss current strategies aimed at targeting Myc for cancer treatment.

BACKGROUND Most recent studies analyzing candidate biological prognostic factors in childhood medulloblastoma (MB) are limited by small patient numbers due to dependence on fresh-frozen tumor material. By contrast, large archives of formalin-fixed, paraffin-embedded MB samples exist from homogeneously treated patients. PATIENTS AND METHODS We have optimized RNA and DNA isolation from formalin-fixed paraffin-embedded MB samples. We then analyzed archived tumor samples from well-documented patients treated within the prospective randomized multicenter trial HIT'91 for DNA amplification of c-myc and N-myc, and mRNA expression of c-myc and trkC. RESULTS TrkC and c-myc mRNA expression were identified as independent prognostic factors by multivariate analysis. Three risk groups were identified: 1) Favorable risk group: All 8 patients (2 metastatic) with elevated trkC and reduced c-myc mRNA expression (compared to levels of human cerebellum) remained relapse-free (7-year EFS 100%). 2) Poor risk group: 10 of 15 patients with metastatic disease and high c-myc and low trkC mRNA expression relapsed (7-year EFS 33%). 3) Intermediate risk group: The 7-year EFS of the remaining 78 patients was 65%. CONCLUSIONS While the collection of fresh-frozen tumor samples is remaining a major challenge in large clinical trials, routinely processed paraffin-embedded tissue samples can be used to quantitate biological prognostic factors on the DNA and RNA level. Upon prospective validation of cut-off levels, this may lead to better risk-based stratification systems for children with medulloblastoma.

Most recent studies analyzing candidate biological prognostic factors (including neurotrophin receptor TrkC and proto-oncogene c-MYC) in childhood primitive neuroectodermal brain tumors (PNET) are limited by small patient numbers due to dependence on fresh-frozen tumor material. In contrast, large archives of formalin-fixed, paraffin-embedded PNET samples exist from homogeneously treated patients. The ability of real-time RT-PCR to assay very small mRNA fragments makes this assay amenable to studies where the RNA is moderately or even highly degraded. We have optimized RNA isolation from archive PNET samples and found that TrkC and c-MYC mRNA measurements significantly correlated with those obtained from matching fresh-frozen tissues. Exploitation of already existing archives of formalin-fixed paraffin-embedded PNET samples may accelerate the building of better stratification systems for PNET patients.

Loss of caspase-8 expression - which has been demonstrated in a subset of Medulloblastoma (MB) - might block important apoptotic signalling pathways and therefore contribute to treatment resistance. In this study, IFN-gamma mediated up-regulation of caspase-8 in human MB cells was found to result in chemosensitization to cisplatin, doxorubicin and etoposide, and sensitisation to radiation. These effects were more prominent in D425 and D341 MB cells (low basal caspase-8 expression) when compared to DAOY MB cells (high basal caspase-8 expression). IFN-gamma mediated chemosensitization and radiosensitization effects were reduced by treatment with the caspase-8 specific inhibitor z-IETD-fmk. Treatment of IFN-gamma resulted in activation of STAT1 in DAOY MB cells and to a lesser extent in D425, but not in D341, indicating that IFN-gamma acts in MB cells through STAT1-dependent and -independent signalling pathways. Taken together, our results demonstrate that IFN-gamma mediated restoration of caspase-8 in MB cells might enhance apoptotic pathways relevant to the response to chemo- and radiotherapy.

The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC50 values ranging from 0.15 to 5 μM. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP-AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combination of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensitized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation. © 2006 Wiley-Liss, Inc.

Neuroblastoma is a tumour derived from primitive cells of the sympathetic nervous system and is the most common extracranial solid tumour in childhood. Unfavourable tumours are characterised not only by structural changes, including 1p deletion and amplification of the MYCN proto-oncogene, but also by high telomerase activity. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. In this study, we examined telomestatin, a G-quadruplex interactive agent, for its ability to inhibit telomere maintenance of neuroblastoma cells. Telomere length was determined by the terminal restriction fragment method, telomerase activity was measured by a quantitative telomeric repeat amplification protocol, and the expression of human telomerase by quantitative real-time polymerase chain reaction (RT-PCR). Short-term treatment with telomestatin resulted in dose-dependent cytotoxicity and induction of apoptosis. Long-term treatment with telomestatin at non-cytotoxic, but still telomerase activity-inhibiting, concentrations resulted in telomere shortening, growth arrest and induction of apoptosis. These results suggest that the effect of telomestatin is dose-dependent and at least 2-fold. Prolonged low-dose treatment with telomestatin limits the cellular lifespan of NB cells through disruption of telomere maintenance.

Primitive neuroectodermal tumors (PNETs), including medulloblastoma (PNET/MB) and supratentorial PNET (sPNET), are the most common malignant brain tumors of childhood. The stabilization of telomere lengths by telomerase activation is an important step in carcinogenesis and cell immortalization. Epigallocatechin gallate (EGCG), the major polyphenol in green tea, is a telomerase inhibitor with antiproliferative and anticarcinogenic effects against different types of cancer. In this study, we used real-time reverse transcriptase-polymerase chain reaction to measure the mRNA expression of the human telomerase reverse transcriptase (hTERT) in 50 primary PNET samples (43 PNET/MB, 7 sPNET), 14 normal human brain samples, and 6 human PNET cell lines. Compared to normal human cerebellum, 38/50 (76%) primary PNET samples hador= 5-fold upregulated hTERT mRNA expression. We then examined PNET cell lines for telomerase activity using a quantitative telomeric repeat amplification protocol (TRAP), and for telomere length using terminal restriction fragment analysis. While a positive correlation between hTERT mRNA expression and telomerase activity was detected in PNET cell lines, no correlation was found between telomerase activity and telomere length. Treatment of PNET cell lines with EGCG resulted in a dose-dependent inhibition of telomerase activity at micromolar levels. Although EGCG displayed strong proliferation inhibitory effects against TRAP-positive PNET cell lines, it had no significant effect against TRAP-negative D425 cells. These results provide evidence for a possible role of telomerase in the pathogenesis of most PNETs and indicate that subsets of PNETs maintain telomere length by alternative mechanisms. Inhibition of telomerase function represents a novel experimental therapeutic strategy in childhood PNETs that warrants further investigation.