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.

Homozygous loss of ADAM3A revealed by genome-wide analysis of pediatric high-grade glioma and diffuse intrinsic pontine gliomas.

Overall, pediatric high-grade glioma (pHGG) has a poor prognosis, in part due to the lack of understanding of the underlying biology. High-resolution 244 K oligo array comparative genomic hybridization (CGH) was used to analyze DNA from 38 formalin-fixed paraffin-embedded predominantly pretreatment pHGG samples, including 13 diffuse intrinsic pontine gliomas (DIPGs). The patterns of gains and losses were distinct from those seen in HGG arising in adults. In particular, we found 1q gain in up to 27% of our cohort compared with 9% reported in adults. A total of 13% had a balanced genetic profile with no large-scale copy number alterations. Homozygous loss at 8p12 was seen in 6 of 38 (16%) cases of pHGG. This novel deletion, which includes the ADAM3A gene, was confirmed by quantitative real-time PCR (qPCR). Loss of CDKN2A/CDKN2B in 4 of 38 (10%) samples by oligo array CGH was confirmed by fluorescent in situ hybridization on tissue microarrays and was restricted to supratentorial tumors. Only ∼50% of supratentorial tumors were positive for CDKN2B expression by immunohistochemistry (IHC), while ∼75% of infratentorial tumors were positive for CDKN2B expression (P = 0.03). Amplification of the 4q11-13 region was detected in 8% of cases and included PDGFRA and KIT, and subsequent qPCR analysis was consistent with the amplification of PDGFRA. MYCN amplification was seen in 5% of samples being significantly associated with anaplastic astrocytomas (P= 0.03). Overall, DIPG shared similar spectrum of changes to supratentorial HGG with some notable differences, including high-frequency loss of 17p and 14q and lack of CDKN2A/CDKN2B deletion. Informative genetic data providing insight into the underlying biology and potential therapeutic possibilities can be generated from archival tissue and typically small biopsies from DIPG. Our findings highlight the importance of obtaining pretreatment samples.

Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease.

PURPOSE: To define copy number alterations and gene expression signatures underlying pediatric high-grade glioma (HGG). PATIENTS AND METHODS: We conducted a high-resolution analysis of genomic imbalances in 78 de novo pediatric HGGs, including seven diffuse intrinsic pontine gliomas, and 10 HGGs arising in children who received cranial irradiation for a previous cancer using single nucleotide polymorphism microarray analysis. Gene expression was analyzed with gene expression microarrays for 53 tumors. Results were compared with publicly available data from adult tumors. RESULTS: Significant differences in copy number alterations distinguish childhood and adult glioblastoma. PDGFRA was the predominant target of focal amplification in childhood HGG, including diffuse intrinsic pontine gliomas, and gene expression analyses supported an important role for deregulated PDGFRalpha signaling in pediatric HGG. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Pediatric and adult glioblastomas were clearly distinguished by frequent gain of chromosome 1q (30% v 9%, respectively) and lower frequency of chromosome 7 gain (13% v 74%, respectively) and 10q loss (35% v 80%, respectively). PDGFRA amplification and 1q gain occurred at significantly higher frequency in irradiation-induced tumors, suggesting that these are initiating events in childhood gliomagenesis. A subset of pediatric HGGs showed minimal copy number changes. CONCLUSION: Integrated molecular profiling showed substantial differences in the molecular features underlying pediatric and adult HGG, indicating that findings in adult tumors cannot be simply extrapolated to younger patients. PDGFRalpha may be a useful target for pediatric HGG, including diffuse pontine gliomas.

The biological, clinical and prognostic implications of p53 transcriptional pathways in breast cancers.

We hypothesized that the functional status of p53 transcriptional pathways, rather than p53 protein expression alone, could accurately discriminate between low- and high-risk breast carcinoma (BC) and inform about individuals' tumour biological behaviour. To test this, we studied a well-characterized series of 990 BCs with long-term follow-up, immunohistochemically profiled for p53, its main regulators and downstream genes. Results were validated in an independent series of patients (n = 245) uniformly treated with adjuvant anthracycline-based chemotherapy. Eleven p53 transcriptional phenotypes were identified with just two main clinical outcomes. (a) Low risk/good prognosis group (active/partially inactive p53 pathways), defined as p53(+/-)/MDM4(+)/MDM2(+/-)/Bcl2(+/-)/p21(+/-), p53(-)/MDM4(-)/MDM2(+)/Bcl2(+)/p21(+/-) and p53(+/-)/MDM4(-)/MMD2(-)/Bcl2(+)/p21(+/-). These tumours had favourable clinicopathological characteristics, including ER(+) and long survival after systemic adjuvant-therapy (AT). (b) High risk/poor prognosis group (completely inactive p53 pathways), defined as p53(+/-)/MDM4(-) MDM2(-)/Bcl2(-)/p21(-), p53(-)/MDM4(-) MDM2(+)/Bcl2(-)/p21(-) and p53(+/-)/MDM4(-)/MDM2(-)/Bcl2(-)/p21(+). These tumours were characterized by aggressive clinicopathological characteristics and showed shortened survival when treated with AT. Completely inactive p53 pathways but intact p21 axis p53(+/-)/MDM4(-)/MDM2(-)/Bcl2(-)/p21(+) had the worst prognosis, particularly patients who received AT. Multivariate Cox regression models, including validated prognostic factors for both test and validation series, revealed that the functional status of p53 transcriptional pathways was an independent prognosticator for BC-specific survival (HR 2.64 and 4.5, p < 0.001, respectively) and disease-free survival (HR 1.93 and 2.5, p < 0.001, respectively). In conclusion, p53 functional status determined by assessment of p53 regulatory and downstream targets provides independent prognostic value and may help determine more adequate therapeutic regimens for specific subgroups of breast cancer patients.