Hazel Rogers

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.

An investigation of WNT pathway activation and association with survival in central nervous system primitive neuroectodermal tumours (CNS PNET).

Central nervous system primitive neuroectodermal tumours (CNS PNET) are high-grade, predominantly paediatric, brain tumours. Previously they have been grouped with medulloblastomas owing to their histological similarities. The WNT/β-catenin pathway has been implicated in many tumour types, including medulloblastoma. On pathway activation β-catenin (CTNNB1) translocates to the nucleus, where it induces transcription of target genes. It is commonly upregulated in tumours by mutations in the key pathway components APC and CTNNB1. WNT/β-catenin pathway status was investigated by immunohistochemical analysis of CTNNB1 and the pathway target cyclin D1 (CCND1) in 49 CNS PNETs and 46 medulloblastomas. The mutational status of APC and CTNNB1 (β-catenin) was investigated in 33 CNS PNETs and 22 medulloblastomas. CTNNB1 nuclear localisation was seen in 36% of CNS PNETs and 27% of medulloblastomas. A significant correlation was found between CTNNB1 nuclear localisation and CCND1 levels. Mutations in CTNNB1 were identified in 4% of CNS PNETs and 20% of medulloblastomas. No mutations were identified in APC. A potential link between the level of nuclear staining and a better prognosis was identified in the CNS PNETs, suggesting that the extent of pathway activation is linked to outcome. The results suggest that the WNT/β-catenin pathway plays an important role in the pathogenesis of CNS PNETs. However, activation is not caused by mutations in CTNNB1 or APC in the majority of CNS PNET cases.

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.

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.

Comparative Expression Analysis Reveals Lineage Relationships between Human and Murine Gliomas and a Dominance of Glial Signatures during Tumor Propagation In Vitro

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor.

WNT/ β -catenin pathway activation in Myc immortalised cerebellar progenitor cells inhibits neuronal differentiation and generates tumours resembling medulloblastoma

Background:Medulloblastoma is the most common malignant childhood brain tumour. Aberrant activation of the WNT/β-catenin pathway occurs in approximately 25% of medulloblastomas. However, its role in medulloblastoma pathogenesis is not understood.Methods:We have developed a model of WNT/β-catenin pathway-activated medulloblastoma. Pathway activation was induced in a Myc immortalised cerebellar progenitor cell line through stable expression of Wnt1. In vitro and in vivo analysis was undertaken to understand the effect of pathway activation and identify the potential cell of origin.Results:Tumours that histologically resembled classical medulloblastoma formed in vivo using cells overexpressing Wnt1, but not with the control cell line. Wnt1 overexpression inhibited neuronal differentiation in vitro, suggesting WNT/β-catenin pathway activation prevents cells terminally differentiating, maintaining them in a more ‘stem-like’ state. Analysis of cerebellar progenitor cell markers demonstrated the cell line resembled cells from the cerebellar ventricular zone.Conclusion:We have developed a cell line with the means of orthotopically modelling WNT/β-catenin pathway-activated medulloblastoma. We provide evidence of the role pathway activation is playing in tumour pathogenesis and suggest medulloblastomas can arise from cells other than granule cell progenitors. This cell line is a valuable resource to further understand the role of pathway activation in tumorigenesis and for investigation of targeted therapies.

Histologically-defined central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) display heterogeneous DNA methylation profiles and show relationships to other paediatric brain tumour types

To the editors: Central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) are a group of rare childhood embryonal brain tumours associated with a poor prognosis (approximately 50% overall survival) and defined by a common histology according to the current consensus World Health Organisation (WHO) classification [7]. CNS-PNETs occur supratentorially and are defined by histological features shared with cerebellar PNETs (termed medulloblastomas), however the histological classification of CNS-PNET can be challenging. Individual CNS-PNETs are often reclassified as other paediatric supratentorial tumour groups, including anaplastic astrocytoma, atypical teratoid rhabdoid tumour (ATRT), anaplastic oligodendroglioma and anaplastic ependymoma, following central immunophenotypic and histological review [3, 12]. Initial studies have shown that substantial molecular heterogeneity exists within CNS-PNETs; molecular features characteristic of other cerebral brain tumour types (e.g. IDH1 mutation, CDKN2A deletion) have been detected in subsets, but unifying genomic defects have not yet been reported [4, 8, 10, 11]. The recent definition of embryonal tumours with abundant neuropil and true rosettes (ETANTR) as a discrete tumour entity occurring in very young children within the CNS-PNET group - characterised by focal amplification of 19q13.42 and dismal outcome [5, 6, 9] - suggests the existence of currently unrecognised molecular pathological variants, and a refined understanding of CNS-PNET biology could lead to their improved subclassification and the subsequent development of directed therapies. We and others have recently demonstrated the utility of DNA methylation profiling for the discovery and distinction of clinical and molecular sub-classes of brain tumour types including medulloblastomas, gliomas and ependymomas [13-15]. To investigate the potential of DNA methylation profiles to enhance the molecular classification of CNS-PNETs, we assessed 1505 CpG residues across 807 genes in a series of 29 archival CNS-PNETs using established methods [14], alongside assessment of clinical and molecular characteristics (Figure 1h). All biopsies underwent central neuropathological review according to WHO criteria [7] by a three pathologist panel (TSJ, KR and JL). Tumours representing ETANTRs, CNS-PNETs with significant glial (GFAP) or neuronal (synaptophysin) differentiation, and SMARCB1/INI1-negative tumours (by immunohistochemistry (IHC)), were excluded and not assessed, thus defining a study population of morphologically homogeneous CNS-PNETs for analysis (Figure 1a). Finally, DNA methylation profiles from 136 further paediatric brain tumours were generated contemporaneously and assessed in comparison. These included medulloblastomas of defined molecular subgroup (n=60; 15 representative examples each from the WNT (MBWNT), SHH (MBSHH), Group 3 (MBGroup3) and Group 4 (MBGroup4) [14]), alongside ependymomas (n=61; 45 posterior fossa (16 anaplastic, 29 classic; median age at diagnosis, 2.8 years), 16 supratentorial (9 anaplastic, 7 classic; median age, 6.9 years)) and cerebral high-grade gliomas (pHGG; n=15; 12 glioblastoma multiforme, 3 anaplastic astrocytoma; median age 7.1 years) with histology confirmed by central histological review (by WHO criteria [7]). Figure 1 Consensus clustering of CNS-PNETs with other molecularly and histologically-defined paediatric brain tumours does not identify a discrete CNS-PNET tumour subgroup We first undertook unsupervised clustering of the CNS-PNET tumour group based on their DNA methylation patterns using non-negative matrix factorisation (NMF [2, 14]). Three sub-groups produced the most consistent consensus clustering; the majority of tumours clustered confidently into a single large group (21/29), while the two smaller remaining groups (n≤5) were less well defined (Figure 1b). Next, we sought to compare the DNA methylation patterns observed for CNS-PNETs with those of the seven other paediatric brain tumour groups with available data. Prior to the addition of CNS-PNETs into our analysis, these tumours formed seven groups as expected (Figure 1c,d,f), representing discrete confidently-defined (average silhouette width, 0.82) groups of MBWNT, MBSHH, MBGroup3 and MBGroup4, posterior-fossa ependymomas and pHGG tumours, and a mixed tumour group containing all (n=16) supratentorial ependymomas alongside some posterior fossa ependymomas (n=9) and pHGGs (n=3). Whilst the inclusion of CNS-PNETs in the analysis yielded 8 optimal clusters (Figure 1c,e,f), the overall quality of these clusters was reduced (average silhouette width, 0.69) and CNS-PNETs did not form a single discrete group; indeed, CNS-PNETs clustered into six of the different tumour groups observed (Figure 1e,f), showing closer similarities to the other clinically and molecularly-defined paediatric brain tumour groups investigated than to each other. Finally, we made an initial assessment of relationships between the clustered CNS-PNETs and other clinical and molecular disease features (Figure 1g,h). Although numbers were limited, TP53 nuclear stabilisation was common and detected in most clusters, while TP53 mutation and MYCN amplification were rare. Most notably, both IDH1 mutations were exclusively detected in pHGG-like CNS-PNETs arising in adults [4], although no pHGG-characteristic HIST1H3B or H3F3A hotspot mutations were observed [15]. The single WNT pathway-activating CTNNB1 mutation was detected in a MBWNT-like CNS-PNET tumour. No relationships to clinical or pathological disease features were observed in this cohort (Figure 1h). In summary, our data show that despite a defining histological homogeneity using current diagnostic criteria, CNS-PNETs display highly heterogeneous DNA methylation patterns which are more commonly related to other paediatric brain tumour types than to each other. These initial findings raise important issues in the classification of CNS-PNETs and indicate their current clinical definition and grouping by common ‘PNET’ histology [7], and treatment using uniform therapeutic approaches, does not adequately address their underlying biological and clinical complexity. Moreover, our data suggest the potential of refined molecular sub-classification for the improved diagnosis and discrimination of CNS-PNET molecular variants, and to support molecularly-directed clinical trials across tumour types defined currently by clinical and pathological criteria. Despite the modest resolution of our platform, robust discrimination of recognised non-CNS-PNET tumour groups was achieved, both supporting these conclusions and highlighting the potential benefits of higher-resolution molecular investigations in expanded cohorts to validate and extend our findings. The variable DNA methylation patterns observed in CNS-PNETs are likely to represent complex factors, including cellular and developmental origins and ‘driver’ events in tumourigenesis [1]. The collection of snap-frozen tumour cohorts will now be essential to support comprehensive integrated genomic/epigenomic investigations, and comparison with transcriptomic features [11], which were not tractable in our current archival cohort. Finally, understanding the biological significance of epigenetic events in CNS-PNET and related tumour types could lead to the development of novel and/or targeted approaches for the improved therapy of these tumours.

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. Clin Cancer Res; 19(23); 6450–60. ©2013 AACR.

The role of the WNT/β-catenin pathway in central nervous system primitive neuroectodermal tumours (CNS PNETs)

Background:Central nervous system primitive neuroectodermal tumours (CNS PNETs) are embryonal tumours occurring predominantly in children. Current lack of knowledge regarding their underlying biology hinders development of more effective treatments. We previously identified WNT/β-catenin pathway activation in one-third of CNS PNETs, which was potentially linked to a better prognosis. In this study, we have extended our cohort, achieving a statistically significant correlation with prognosis. We additionally investigated the biological effects of WNT/β-catenin pathway activation in tumour pathogenesis.Methods:A total of 42 primary and 8 recurrent CNS PNETs were analysed for WNT/β-catenin pathway status using β-catenin immunohistochemistry. Genomic copy number and mRNA expression data were analysed to identify a molecular profile linked to WNT/β-catenin pathway activation.Results:Pathway activation was seen in 26% of CNS PNETs and was significantly associated with longer overall survival. Genes displaying a significant difference in expression levels, between tumours with and without WNT/β-catenin pathway activation, included several involved in normal CNS development suggesting aberrant pathway activation may be disrupting this process.Conclusion:We have identified WNT/β-catenin pathway status as a marker, which could potentially be used to stratify disease risk for patients with CNS PNET. Gene expression data suggest pathway activation is disrupting normal differentiation in the CNS.

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.