Ototoxicity evaluation in medulloblastoma patients treated with involved field boost using intensity-modulated radiation therapy (IMRT): a retrospective review

Background: Ototoxicity is a known side effect of combined radiation therapy and cisplatin chemotherapy for the treatment of medulloblastoma. the delivery of an involved field boost by intensity modulated radiation therapy (IMRT) may reduce the dose to the inner ear when compared with conventional radiotherapy. the dose of cisplatin may also affect the risk of ototoxicity. A retrospective study was performed to evaluate the impact of involved field boost using IMRT and cisplatin dose on the rate of ototoxicity.Methods: Data from 41 medulloblastoma patients treated with IMRT were collected. Overall and disease-free survival rates were calculated by Kaplan-Meier method Hearing function was graded according to toxicity criteria of Pediatric Oncology Group (POG). Doses to inner ear and total cisplatin dose were correlated with hearing function by univariate and multivariate data analysis.Results: After a mean follow-up of 44 months (range: 14 to 72 months), 37 patients remained alive, with two recurrences, both in spine with CSF involvement, resulting in a disease free-survival and overall survival of 85.2% and 90.2%, respectively. Seven patients (17%) experienced POG Grade 3 or 4 toxicity. Cisplatin dose was a significant factor for hearing loss in univariate analysis (p < 0.03). in multivariate analysis, median dose to inner ear was significantly associated with hearing loss (p < 0.01). POG grade 3 and 4 toxicity were uncommon with median doses to the inner ear bellow 42 Gy (p < 0.05) and total cisplatin dose of less than 375 mg/m(2) (p < 0.01).Conclusions: IMRT leads to a low rate of severe ototoxicity. Median radiation dose to auditory apparatus should be kept below 42 Gy. Cisplatin doses should not exceed 375 mg/m(2).

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Using genomics for drug discovery in medulloblastoma

Tese de doutoramento, Medicina (Neurocirurgia), Universidade de Lisboa, Faculdade de Medicina, 2014

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

A five-gene hedgehog signature developed as a patient preselection tool for hedgehog inhibitor therapy in medulloblastoma

Purpose: 

Medulloblastoma Down Under 2013 : a report from the third annual meeting of the International Medulloblastoma Working Group.

Medulloblastoma is curable in approximately 70% of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5-10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organization's classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Children's Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

Patterns of care in adult medulloblastoma: results of an international online survey

The literature on medulloblastoma in adults is generally limited to case reports and retrospective series, and there is no accepted standard of care. The Cooperative Trials Group for Neuro-Oncology (COGNO) sought to determine the range and consistency of clinicians’ approaches to management as a basis for future trials. We aimed to identify current treatment strategies for adult medulloblastoma through an online survey launched at the 2012 Society of Neuro-Oncology meeting and by email invitation. Clinicians who had treated at least one adult patient with medulloblastoma, primitive neuroectodermal tumor (PNET), or pineoblastoma in the preceding year were asked about their most recent patient and invited to discuss their approach to a typical clinical scenario. Between November 2012 and January 2013, 45 clinicians (11 medical oncologists, 8 radiation oncologists, 5 pediatric oncologists, and 21 others) from Australia (24), United States (3), Europe (4) and other countries (14) completed the survey. Responding clinicians had treated 54 cases in the past 12 months. The most common histological type was medulloblastoma (64 %), then PNET (20 %). Most patients were male (68 %), and had high-risk disease (65 %). Complete surgical resection in 56 and 32 % had molecular testing. Radiotherapy was predominantly cranio-spinal (92 %) and given mostly post-resection (80 %). Combination chemotherapy was more common than single-agent chemotherapy. The choice of chemotherapy varied considerably. There is substantial variation in the treatment of adult medulloblastoma, most pronounced in the choice of chemotherapeutic agents, highlighting the need for further collaborative research to guide evidence-based treatment strategies.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4 alpha. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-beta signalling in Group 3, and NF-kappa B signalling in Group 4, suggest future avenues for rational, targeted therapy.

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5 ' NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Cytogenetic findings in pediatric radiation-induced atypical meningioma after treatment of medulloblastoma: case report and review of the literature

Ionizing radiation is the most recognized risk factor for meningioma in pediatric long-term cancer survivors. Information in this rare setting is exceptional. We report the clinical and cytogenetic findings in a radiation-induced atypical meningioma following treatment for desmoplastic medulloblastoma in a child. This is the second study to describe the cytogenetic aspects on radiation-induced meningiomas in children. Chromosome banding analysis revealed a 46, XX, t(1;3)(p22;q12), del(1)(p?)[8]/46, XX[12]. Loss of chromosome 1p as a consequence of irradiation has been proposed to be more important in the development of secondary meningiomas in adults. Deletions in the short arm of chromosome 1 also appear to be a shared feature in both pediatric cases so far analyzed.

Expression analysis of stem cell-related genes reveal OCT4 as a predictor of poor clinical outcome in medulloblastoma

Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P <= 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.