A retrospective analysis of recurrent pediatric ependymoma reveals extremely poor survival and ineffectiveness of current treatments across central nervous system locations and molecular subgroups.

BACKGROUND: Relapse occurs in 50% of pediatric ependymoma cases and has poor prognosis. Few studies have investigated the clinical progress of relapsed disease, and treatment lacks a standardized approach. METHODS AND MATERIALS: We analyzed 302 pediatric ependymoma cases. Tumor, demographic, and treatment variables were investigated for association with relapse risk, time to recurrence, and survival after relapse. DNA methylation profiling was performed for 135/302 cases, and predominant subgroups were EPN_PFA (n = 95) and EPN_RELA (n = 24). Chromosome 1q status was ascertained for 185/302 cases by fluorescent in-situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and DNA methylation profiles. RESULTS: Sixty-two percent of cases relapsed, with a median of two recurrences with no difference between posterior fossa and supratentorial locations (66% vs 55% relapse rate). One hundred seventeen (38%) cases relapsed within two years and five (2%) beyond 10 years. The late relapses were clinically heterogeneous. Tumor grade and treatment affected risk and time to relapse variably across subgroups. After relapse, surgery and irradiation delayed disease progression with a minimal impact on survival across the entire cohort. In the EPN_PFA and EPN_RELA groups, 1q gain was independently associated with relapse risk (subhazard ratio [SHR] 4.307, P = 0.027 and SHR 1.982, P = 0.010, respectively) while EPN_PFA had increased relapse risk compared with EPN_RELA (SHR = 0.394, P = 0.018). CONCLUSIONS: Recurrent pediatric ependymoma is an aggressive disease with poor outcomes, for which current treatments are inadequate. We report that chromosome 1q gain increases relapse risk in common molecular subgroups in children but a deeper understanding of the underlying biology at relapse and novel therapeutic approaches are urgently needed.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

The therapeutic potential of targeting the PI3K pathway in pediatric brain tumors.

Central nervous system tumors are the most common cancer type in children and the leading cause of cancer related deaths. There is therefore a need to develop novel treatments. Large scale profiling studies have begun to identify alterations that could be targeted therapeutically, including the phosphoinositide 3-kinase (PI3K) signaling pathway, which is one of the most commonly activated pathways in cancer with many inhibitors under clinical development. PI3K signaling has been shown to be aberrantly activated in many pediatric CNS neoplasms. Pre-clinical analysis supports a role for PI3K signaling in the control of tumor growth, survival and migration as well as enhancing the cytotoxic effects of current treatments. Based on this evidence agents targeting PI3K signaling have begun to be tested in clinical trials of pediatric cancer patients. Overall, targeting the PI3K pathway presents as a promising strategy for the treatment of pediatric CNS tumors. In this review we examine the genetic alterations found in the PI3K pathway in pediatric CNS tumors and the pathological role it plays, as well as summarizing the current pre-clinical and clinical data supporting the use of PI3K pathway inhibitors for the treatment of these tumors.

PI3K pathway activation provides a novel therapeutic target for pediatric ependymoma and is an independent marker of progression-free survival.

PURPOSE: Currently, there are few effective adjuvant therapies for pediatric ependymoma outside confocal radiation, and prognosis remains poor. The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in cancer. PI3Ks transduce signals from growth factors and cytokines, resulting in the phosphorylation and activation of AKT, which in turn induces changes in cell growth, proliferation, and apoptosis. EXPERIMENTAL DESIGN: PI3K pathway status was analyzed in ependymoma using gene expression data and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein expression and the mutation status of PI3K catalytic subunit α-isoform gene (PIK3CA) was investigated. RESULTS: Genes in the pathway displayed significantly higher expression in supratentorial than in posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway activation, was seen in 72% of tumors (n = 169) and P-AKT expression was found to be an independent marker of a poorer progression-free survival. A significant association between PI3K pathway activation and cell proliferation was identified, suggesting that pathway activation was influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations were identified in PIK3CA. CONCLUSIONS: Our results suggest that the PI3K pathway could act as a biomarker, not only identifying patients with a worse prognosis but also those that could be treated with therapies targeted against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

Loss of INI1 protein expression defines a subgroup of aggressive central nervous system primitive neuroectodermal tumors.

Pediatric embryonal brain tumors can be difficult to classify. Atypical teratoid rhabdoid tumors (ATRT) contain rhabdoid cells, while primitive neuroectodermal tumors (PNETs) are composed of "small round blue cells." Loss of INI1 is a common event in ATRT; therefore, we investigated if the loss of INI1 protein expression was also observed in central nervous system (CNS) PNET and pineoblastoma. A histological review of 42 CNS PNETs and six pineoblastomas was performed. INI1 expression was assessed by immunohistochemistry. Sequencing was performed on the mutational hotspots of INI1. INI1-immunonegative tumors were further investigated using fluorescence in situ hybridization. Epithelial membrane antigen (EMA) protein expression was assessed in six CNS PNETs to further define the phenotype. Five CNS PNETs without rhabdoid cell morphology were immuno-negative for both INI1 and EMA. Of these primary CNS PNET patients, three died <11 months postdiagnosis, which was dissimilar to the INI1-immunopositive primary CNS PNETs where 18/24 (75%) patients were alive 1 year postdiagnosis. We have identified a small subgroup of CNS PNETs which lack INI1 protein expression, but have no evidence of rhabdoid cell morphology. INI1 protein loss may occur through mechanisms other than gene deletion. INI1 immunohistochemistry should be performed for all CNS PNET cases.

Markers of survival and metastatic potential in childhood CNS primitive neuro-ectodermal brain tumours: an integrative genomic analysis.

BACKGROUND: Childhood CNS primitive neuro-ectodermal brain tumours (PNETs) are very aggressive brain tumours for which the molecular features and best treatment approaches are unknown. We assessed a large cohort of these rare tumours to identify molecular markers to enhance clinical management of this disease. METHODS: We obtained 142 primary hemispheric CNS PNET samples from 20 institutions in nine countries and examined transcriptional profiles for a subset of 51 samples and copy number profiles for a subset of 77 samples. We used clustering, gene, and pathway enrichment analyses to identify tumour subgroups and group-specific molecular markers, and applied immunohistochemical and gene-expression analyses to validate and assess the clinical significance of the subgroup markers. FINDINGS: We identified three molecular subgroups of CNS PNETs that were distinguished by primitive neural (group 1), oligoneural (group 2), and mesenchymal lineage (group 3) gene-expression signatures with differential expression of cell-lineage markers LIN28 and OLIG2. Patients with group 1 tumours were most often female (male:female ratio 0·61 for group 1 vs 1·25 for group 2 and 1·63 for group 3; p=0·043 [group 1 vs groups 2 and 3]), youngest (median age at diagnosis 2·9 years [95% CI 2·4-5·2] for group 1 vs 7·9 years [6·0-9·7] for group 2 and 5·9 years [4·9-7·8] for group 3; p=0·005), and had poorest survival (median survival 0·8 years [95% CI 0·5-1·2] in group 1, 1·8 years [1·4-2·3] in group 2 and 4·3 years [0·8-7·8] in group 3; p=0·019). Patients with group 3 tumours had the highest incidence of metastases at diagnosis (no distant metastasis:metastasis ratio 0·90 for group 3 vs 2·80 for group 1 and 5·67 for group 2; p=0·037). INTERPRETATION: LIN28 and OLIG2 are promising diagnostic and prognostic molecular markers for CNS PNET that warrant further assessment in prospective clinical trials. FUNDING: Canadian Institute of Health Research, Brainchild/SickKids Foundation, and the Samantha Dickson Brain Tumour Trust.

Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death.

Epigenetic alterations, including methylation, have been shown to be an important mechanism of gene silencing in cancer. Ependymoma has been well characterized at the DNA copy number and mRNA expression levels. However little is known about DNA methylation changes. To gain a more global view of the methylation profile of ependymoma we conducted an array-based analysis. Our data demonstrated tumors to segregate according to their location in the CNS, which was associated with a difference in the global level of methylation. Supratentorial and spinal tumors displayed significantly more hypermethylated genes than posterior fossa tumors, similar to the 'CpG island methylator phenotype' (CIMP) identified in glioma and colon carcinoma. This hypermethylated profile was associated with an increase in expression of genes encoding for proteins involved in methylating DNA, suggesting an underlying mechanism. An integrated analysis of methylation and mRNA expression array data allowed us to identify methylation-induced expression changes. Most notably genes involved in the control of cell growth and death and the immune system were identified, including members of the JNK pathway and PPARG. In conclusion, we have generated a global view of the methylation profile of ependymoma. The data suggests epigenetic silencing of tumor suppressor genes is an important mechanism in the pathogenesis of supratentorial and spinal, but not posterior fossa ependymomas. Hypermethylation correlated with a decrease in expression of a number of tumor suppressor genes and pathways that could be playing an important role in tumor pathogenesis.

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

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