Progression Of Ependymomas Research Articles

BIOM-50. GENETIC PREDISPOSITION TO LONGER TELOMERE LENGTH AND RISK OF CHILDHOOD, ADOLESCENT AND ADULT-ONSET EPENDYMOMA

Abstract INTRODUCTION Ependymoma is the third most common brain tumor in children, with well-described molecular characterization but poorly understood underlying germline risk factors. Telomerase reactivation in somatic cells has been linked to ependymoma progression, recurrence, and survival, and has been implicated as an important prognostic marker and potential therapeutic target. METHODS To investigate whether inherited predisposition to longer telomere length influences ependymoma risk, we utilized case-control data from three studies: 1) a population-based pediatric and adolescent ependymoma case-control sample from California (153 cases, 696 controls), 2) a hospital-based pediatric posterior fossa type A ependymoma (EPN-PF-A) case-control study from Toronto’s Hospital for Sick Children and the Children’s Hospital of Philadelphia (83 cases, 332 controls), and 3) a multicenter adult-onset ependymoma case-control dataset nested within the Glioma International Case-Control Consortium (GICC) (103 cases, 3287 controls). We investigated the individual effect of telomere-length associated SNPs on ependymoma risk, as well as the combined effect of these SNPs through polygenic score and Mendelian randomization analyses. RESULTS We observed an association between genetic predisposition to longer LTL and increased risk of adolescent-onset (P= 3.97x10-3) and adult-onset (P =0.042) ependymoma, but not ependymoma diagnosed in children < 12 years old (P=0.21), or among the pediatric EPN-PF-A sample (P=0.59). Comparing ependymoma patients ages 12–19 to those under 12 years of age demonstrated that age significantly modified the association between longer telomere length and ependymoma risk (P=0.021). CONCLUSIONS These findings complement emerging literature suggesting that dysregulated telomere maintenance is important for ependymoma pathogenesis and that longer telomere length is a risk factor for various neoplasms of the peripheral and central nervous system.

Abstract 3002: Identification of frequently altered protein networks in ependymomas

Abstract Ependymomas are glial tumors that originate from the walls of the cerebral ventricles or from the spinal canal. The five-year survival for intracranial ependymomas in children is approximately 65%. Ependymomas exhibit a wide range of DNA copy number alterations that vary greatly in size and location. We hypothesize that an underlying protein network exists that is consistently compromised by such DNA copy number alterations in ependymomas. In this study, we analyzed an array-comparative genomic hybridization dataset generated by Johnson and colleagues [Nature 466(7306):632-6, 2010] to determine whether a protein interaction network could be identified that might be consistently functionally compromised in pediatric ependymomas. This cohort of 203 primary ependymomas consisted of 106 posterior fossa, 56 supratentorial, and 41 spinal ependymomas. The genes within each altered region were used to perform network analyses with GeneGo and Ingenuity to uncover protein networks that were altered in a significant number of tumors. For calculation of statistical significance, 100,000 samplings of randomly selected regions - equal in gene number to the regions of gain or loss - were performed. Indeed, some protein networks were found to be over-represented in the structural alterations exhibited by this cohort (p<0.05). These results warrant future investigations to demonstrate the role of the identified networks in the development and progression of ependymomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3002. doi:1538-7445.AM2012-3002

Candidate Genes on Chromosome 9q33-34 Involved in the Progression of Childhood Ependymomas

The molecular pathogenesis of pediatric ependymoma remains unclear. Our study was designed to identify genetic changes implicated in ependymoma progression. We characterized 59 ependymoma samples (33 at diagnosis and 26 at relapse) using array-comparative genomic hybridization (aCGH). Specific chromosomal imbalances were confirmed by fluorescent in situ hybridization, and candidate genes were assessed by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry, sequencing, and in vitro functional studies. aCGH analysis revealed a significant increase in genomic imbalances on relapse compared with diagnosis, such as gain of 9qter and 1q (54% v 21% and 12% v 0%, respectively) and loss of 6q (27% v 6%). Supervised tumor classification showed that gain of 9qter was associated with tumor recurrence, age older than 3 years, and posterior fossa location. Using a candidate-gene strategy, we found an overexpression of two potential oncogenes at the locus 9qter: Tenascin-C and Notch1. Moreover, Notch pathway analysis (qPCR) revealed overexpression of Notch ligands, receptors, and target genes (Hes-1, Hey2, and c-Myc), and downregulation of Notch repressor Fbxw7. We confirmed by immunohistochemistry the overexpression of Tenascin-C and Hes-1. We detected Notch1 missense mutations in 8.3% of the tumors (only in the posterior fossa location and in case of 9q33-34 gain). Furthermore, inhibition of Notch pathway with a gamma-secretase inhibitor impaired the growth of ependymoma stem cell cultures. The activation of the Notch pathway and Tenascin-C seem to be important events in ependymoma progression and may represent future targets for therapy. We report, to our knowledge for the first time, recurrent oncogenic mutations in pediatric posterior fossa ependymomas.

Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma

Pediatric ependymomas are enigmatic tumors, and their clinical management remains one of the more difficult in pediatric oncology. The identification of biological correlates of outcome and therapeutic targets remains a significant challenge in this disease. We therefore analyzed a panel of potential biological markers to determine optimal prognostic markers. We constructed a tissue microarray from 97 intracranial tumors from 74 patients (WHO grade II-III) and analyzed the candidate markers nucleolin, telomerase catalytic subunit (hTERT; antibody clone 44F12), survivin, Ki-67, and members of the receptor tyrosine kinase I (RTK-I) family by immunohistochemistry. Telomerase activity was determined using the in vitro-based telomere repeat amplification protocol assay, and telomere length was measured using the telomere restriction fragment assay. Primary tumors with low versus high nucleolin protein expression had a 5-year event-free survival of 74%+/-13% and 31%+/-7%, respectively. Multivariate analysis identified low nucleolin expression to be independently associated with a more favorable prognosis (hazard ratio=6.25; 95% confidence interval, 1.6-24.2; p=0.008). Ki-67 and survivin correlated with histological grade but not with outcome. Immunohistochemical detection of the RTK-I family did not correlate with grade or outcome. Telomerase activity was evident in 19 of 22 primary tumors, with telomere lengthening and/or maintenance occurring in five of seven recurrent cases. Low nucleolin expression was the single most important biological predictor of outcome in pediatric intracranial ependymoma. Furthermore, telomerase reactivation and maintenance of telomeric repeats appear necessary for childhood ependymoma progression. These findings require corroboration in a clinical trial setting.

Identification of novel candidate genes for ependymoma progression in chromosomal regions over-represented at recurrence and detected by CGH array

2008 Background: The biology and genetics of ependymomas remain poorly understood. As opposed to other pediatric tumors, few recurrent chromosomal abnormalities or relevant pathways have been identified. Method: In order to gain new insight into the molecular mechanisms involved in ependymoma progression, we performed BAC array-based comparative genomic hybridization (aCGH) in a series of 59 tumors at diagnosis and at relapse (42 posterior fossa and 17 supratentorial). Specific chromosomal imbalances were confirmed by FISH. Expression of candidate genes was evaluated by RT-PCR and immunohistochemistry. Results: As compared to CGH (Cancer Genet Cytogenet 2002), aCGH detected more abnormalities since 18/33 samples at diagnosis and 22/26 at recurrence showed chromosomal imbalances. Three distinct patterns were observed : no imbalance (15 at diagnosis, 4 at relapse), large and numerous imbalances (7 at diagnosis, 12 at relapse) and small and rare imbalances (11 at diagnosis, 10 at relapse). At diagnosis, absence of chromosomal imbalance was correlated with young age (p=0.034) and shorter progression-free survival (p=0.014). At relapse, patients with large and numerous imbalances had a shorter survival (p=0.026). Specific chromosomal imbalances were significantly more frequent at recurrence: 9 qter gain (54% vs 21%), 1q gain (12% vs 0%) and 6q loss (27% vs 6%). Samples were available both at diagnosis and relapse for 16 patients. Whole genome changes were towards increase of genomic abnormalities (n=10) but also simplification (n=6), the former being associated with a higher risk of death from disease (Fisher exact, p=0.03). A candidate gene strategy was focussed on the frequently gained 9qter locus. Significant overexpression compared to normal brain was identified for Tenascin-C (median 35 fold, max 220 fold, p<0.0001) and Notch1 (median 6 fold, max 45 fold, p<0.0001). Notch pathway analysis by RT-PCR revealed potential regulatory loops between JAG1/2 or DLL1/3 and Notch1/2. Conclusion: aCGH pattern may identify subgroups of ependymomas with respect to prognosis. Notch pathway and Tenascin-C are important regulatory genes in ependymoma progression and may represent interesting new targets for therapy. No significant financial relationships to disclose.

9p21 and 13q14 dosages in ependymomas. A clinicopathologic study of 101 cases

Ependymomas are glial neoplasms whose clinical behavior is difficult to predict based on histology alone. Recently, a comparative genomic hybridization study identified frequent chromosome 9p and 13q losses in anaplastic ependymomas, suggesting that p16 and RB alterations may be involved in tumor progression. In order to test this hypothesis further, 101 myxopapillary, conventional, and anaplastic ependymomas (51 spinal and 50 intracranial tumors) were tested for RB and p16 deletions using fluorescence in situ hybridization. Clinical follow-up, ranging from 2 to 198 months (median 46 months), was obtained in 90 cases (91%). RB and p16 deletions were seen in 22 of 92 (24%) and 22 of 89 (25%) informative cases, respectively. Polysomies were more frequent in the grade I and II spinal tumors, consistent with prior reports of increased aneuploidy in such cases. No significant genetic associations were seen with tumor grade, recurrence, or death, suggesting that 9p and 13q deletions do not play a prominent role in the malignant progression of ependymomas, as has been implicated in other glioma subtypes.

9p21 and 13q14 dosages in ependymomas. A clinicopathologic study of 101 cases

Ependymomas are glial neoplasms whose clinical behavior is difficult to predict based on histology alone. Recently, a comparative genomic hybridization study identified frequent chromosome 9p and 13q losses in anaplastic ependymomas, suggesting that p16 and RB alterations may be involved in tumor progression. In order to test this hypothesis further, 101 myxopapillary, conventional, and anaplastic ependymomas (51 spinal and 50 intracranial tumors) were tested for RB and p16 deletions using fluorescence in situ hybridization. Clinical follow-up, ranging from 2 to 198 months (median 46 months), was obtained in 90 cases (91%). RB and p16 deletions were seen in 22 of 92 (24%) and 22 of 89 (25%) informative cases, respectively. Polysomies were more frequent in the grade I and II spinal tumors, consistent with prior reports of increased aneuploidy in such cases. No significant genetic associations were seen with tumor grade, recurrence, or death, suggesting that 9p and 13q deletions do not play a prominent role in the malignant progression of ependymomas, as has been implicated in other glioma subtypes.

Gene Expression Patterns in Ependymomas Correlate with Tumor Location, Grade, and Patient Age

To elucidate the molecular events responsible for tumorigenesis and progression of ependymomas, we analyzed molecular alterations on the gene expression level in a series of newly diagnosed ependymal neoplasms (n = 39). To this aim, tumor RNA was hybridized to microarrays comprising 2600 different genes with relevance to mitosis, cell-cycle control, oncogenesis, or apoptosis. For CLU, IGF-2, and RAF-1, which are apparent candidate genes because they had been previously described to be involved in tumorigenesis of other human malignancies, we found a high expression on the mRNA as well as the protein level. We identified gene expression signatures for the differentiation of tumors with respect to location, grade, and patient age. Spinal ependymomas were characterized by high-expression levels of HOXB5, PLA2G, and CDKN2A and tumors in young patients (< or =16 years of age) by high-expression levels of LDHB and STAM. Notably, we were able to classify supratentorial grade II and III tumors with 100% accuracy, whereas this did not apply for infratentorial ependymomas. The similar gene expression patterns of grade II and III infratentorial malignancies suggest that grade III tumors may develop through a secondary multistep transformation process involving genes that are related to cell proliferation (LDHA, cyclin B, MAT2A) or tumor suppression (PTEN). In summary, our results provide new insight in the biochemical pathways particularly intriguing in the pathomechanism of ependymomas and suggest that this entity comprises molecularly distinct diseases.

Molecular cytogenetic studies of pediatric ependymomas.

Cytogenetic and molecular studies of ependymomas have previously demonstrated deletions of chromosomes 17 and 22 as frequent abnormalities, implicating inactivation of tumor suppressor genes in the pathogenesis of these tumors. In the present study, we analyzed 22 childhood ependymomas by standard cytogenetic analysis, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR)-based microsatellite analysis of chromosomes 17 and 22. Microsatellite analysis of chromosome 6 was performed to identify submicroscopic deletions implicated by the cytogenetic studies. Among the 22 cases, we demonstrated loss of chromosome 22 in 2 patients, deletion of chromosome 17 in 2 patients, and rearrangements or deletions of chromosome 6 in 5 patients. These data do not suggest that loss of a gene on chromosome 17p plays a primary role in the initiation of pediatric ependymomas. This is in contrast to what has been reported for pediatric CNS primitive neuroectodermal tumors and malignant astrocytomas, in which deletion of 17p is regarded as a primary event. Furthermore, loss of chromosome 22 may define a subset of ependymomas more commonly seen in adults. Cytogenetic studies in this series, however, suggest that a region on the long arm of chromosome may be involved in the development and/or progression of ependymomas in children.

Infrequency of p53 gene mutations in ependymomas

Ependymomas, which comprise 5% of central nervous system tumors, have not been extensively characterized genetically. The p53 tumor suppressor gene is frequently mutated in human cancer, and is important in the pathogenesis of other central nervous system (CNS) tumors. Chromosomal DNA corresponding to the p53 tumor suppressor gene was amplified by the polymerase chain reaction (PCR) from 31 archival ependymoma specimens. DNA was screened for the presence of p53 mutations by single strand conformational polymorphism (SSCP) analysis; samples with altered mobility were further tested for the presence of mutation by direct DNA sequence analysis. Of the 31 ependymomas tested, one contained a detectable DNA sequence change in the p53 gene. Sequencing revealed a silent mutation in exon 6, at codon 213, which represents a known p53 sequence polymorphism. These finding suggest that in contrast to many other human cancers, p53 mutation is not important in the pathogenesis or progression of ependymomas.

Chromosomes in the genesis and progression of ependymomas

Cytogenetic analysis was performed on cultures of primary ependymal tumors with different degrees of malignancy (I–IV) obtained from four patients, none of whom had received therapy before karyotypic evaluation. The most common abnormalities were monosomy 17 and 22 in four cases and losses of sex chromosomes in three cases. Structural rearrangements of chromosome 2 were a finding for all cases and involved loss of material at 2q32–34. Other structural chromosome abnormalities detected involved chromosomes 4, 6, 10, 11, 12, and X. We also reviewed data on 22 cases previously reported.

Chromosomes in the genesis and progression of ependymomas

Chromosomes in the genesis and progression of ependymomas