Molecular Characteristics of Pediatric Ependymomas: A Systematic Review
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The prognosis for patients diagnosed with ependymoma is relatively poor, with a 5-year overall survival rate of 24–75%. Currently, tumors are treated by surgical resection followed by radiotherapy, as resection is the most consistent prognostic marker (up to 80%). Therefore, there is a pressing need to improve our understanding of the biology of these tumors and to develop new therapeutic targets. The present work was a systematic review of the current molecular knowledge of pediatric ependymomas. From January 2000 to December 2017, we carried out a search using “MeSH” (Medical Subject Heading), and “free-text” protocols in the databases Medline/PubMed, SCOPUS, Web of Science, and EMBASE (OVID platform), combining the terms chromosomal alterations, genetic changes, epigenetic changes, and protein expression changes. We selected articles with samples from pediatric patients and chose publications with complete clinical features. Only 33 articles met the criteria for a meta-analysis, suggested by the state of methylation and expression of a characteristic marker of pediatric ependymomas. We found a chromosomal alteration and one gene associated with survival; these are candidates for bad prognosis biomarkers.
KeywordsEpendymoma Molecular characteristic Systematic review Pediatric patients
Ependymoma (EP) arises from the ependymal cells of the fourth cerebral ventricle and the spinal cord. These tumors can develop in both adult and children patients; however, intracranial EP occurs more frequently in children, whereas spinal EPs are more frequent in adults. These tumors are classified by their location as infra and supratentorial EPs .
EP is the third most common pediatric tumor of the central nervous system and the prognosis is relatively poor for patients with this diagnosis, with a 5-year overall survival rate of 24–75%; therefore, EPs are considered a public health problem. These tumors are treated with surgical resection followed by radio- and chemotherapy. Actually, resection is the best prognostic marker (up to 80%) used for clinical diagnostic; therefore, it is necessary to understand the tumorigenesis of EP in order to develop new therapeutic targets [2, 3].
The clinical features of EPs used for prognosis—such as patient age, tumor location, extent of surgical resection, and tumor histopathology grade—are insufficient and have inconsistent results, so it is necessary to come up with strategies to improve treatment and provide an exact prognosis [4, 5]. Several studies have suggested that epigenetic silencing of tumor suppressor genes and expression changes are an important mechanisms of EP pathogenesis in supratentorial and spinal tumors .
In this systematic review, we aimed to determine the molecular characteristics of these tumors that may establish tumor markers.
Search Strategy and Selection Criteria
Included articles were based on an a priori selected set of criteria: articles published in English, complete data of patients, complete text, cross-sectional studies, molecular data studies, and studies carried out totally or partially in pediatric patients (under 18 years of age). If the reviewed articles were based on pediatric and adult patients, it was considered that the samples would be easily identified through the codes or numbering that different authors granted for each of the samples. The patients had complete clinical characteristics that included age, sex, and diagnosis; in addition, the results described were specific for each sample.
The primary outcome was data on changes in chromosomal alterations, methylation status, gene and protein expression reported in pediatric patients, cross-sectional studies, molecular changes associated with the patient’s prognosis, frequency of appearance of molecular changes, and values of hazard ratios and odds ratios.
The exclusion criteria included case-controlled studies, studies without measures of association, and case series. No systematic reviews or meta-analyses on this topic were found.
Three reviewers participated in the review process. Two reviewers completed the initial review, examined the papers, confirmed the inclusion and exclusion criteria, and completed the second stage, extracting all data; a third reviewer independently examined the data to identify any discrepancies between reviewers. Discrepancies in article selection were resolved by discussion among the reviewers. A similar approach was used to determine which of these studies should be included in the meta-analysis. Information about date of publication, country where the study was undertaken, sample size, data relating to participants, a specific illness, age, sex, histopathology diagnosis, and tumor location was acquired from the included articles for full review. Odds ratios (ORs), and rate ratios (RRs), with 95% confidence interval (CI) for each chromosomal alteration, genetic, epigenetic, and protein expression were extracted when they were available.
Thirty-three studies were selected and included for this systematic review. The number of studies, analyzed by condition, varied from two to five. For each study and each characteristic, an event rate and its CI were computed from the reported numbers, considering the reference group. Review Manager 5.3 was used for the analyses [Review Manager (RevMan) [computer program]. Version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014]. Random-effects models were conducted, separating the studies into one analysis for those reporting OR statistics and another for those reporting OR. Heterogeneity was estimated using the I2 statistic . The I2 describes the percentage of variation across studies due to heterogeneity rather than chance alone [6, 7]. As the I2 percentage increases, so too does the proportion of effect size variability that is due to between-study heterogeneity.
In order to calculate bias, the tests described by Begg et al. and Egger et al. [8, 9] were used. Funnel plots were generated with a confidence interval of 95%, according to the random-effects meta-analysis proposed by DerSimonian and Laird ; Tau2 adjusts the standard error and the effects of the intervention of the studies included in the meta-analysis. Finally, the frequency of chromosomal, genetic, and epigenetic alterations was analyzed.
Articles included in the systematic review
Suzuki et al. 2000 
Lamszus et al. 2001 
Hirose et al. 2001 
Ward et al. 2001 
Dyer et al. 2002 
Gilbertson et al. 2002 
Jeuken et al. 2002 
Singh et al. 2002 
Zamecnik et al. 2003 
Waha et al. 2004 
Ammerlaan et al. 2005 
Tabori et al. 2006 
Karakoula et al. 2008 
Methylation and expression
Moronaru et al. 2008 
Pezzolo et al. 2008 
Rand et al. 2008 
Snuderl et al. 2008 
Puget et al. 2009 
Rousseau et al. 2010 
Andreiuolo et al. 2010 
Korshunov et al. 2010 
Alexiou et al. 2011 
Modena et al. 2012 
Hagel et al. 2013 
Moreno et al. 2013 
Barszczyk et al. 2014 
Gupta et al. 2014 
Virág et al. 2014 
Mack et al. 2014 
Li et al. 2015 
Araki et al. 2016 
Chromosomal alteration, protein
Chen et al. 2016 
Gojo et al. 2017 
Methylation and expression
We selected four articles that met the eligibility criteria with 136 samples. It was observed that genes with the most frequent expression changes were hTERT (36%), ERBB (33%), ERBB1 (12%), ERBB2 (12%), ERBB3 (9%), STB (4%), SHC1 (6%), and TPR (9%). We observed that hTERT and ERBB genes are features of pediatric intracranial ependymal tumors, but only hTERT correlated with a bad prognosis for patients with the following statistical values: Tau2 = 1.21, χ2 = 3.31, P = 0.19, I2 = 40%, which indicated that our model was heterogeneous (Fig. 4b) The funnel plot shows symmetric data dispersion.
We selected 14 publications that met the eligibility criteria with protein expression assays, with a total of 697 patients showing different states of protein expression. We reviewed the frequencies of the results, where it was observed that EGFR was found with 22.02% of the cases, with strong expression, and with 18.75% with absence of protein expression; for Cav-1 with 12.35% had a strong expression, and 13.54% had weak staining. With respect to YB1, the expression was weak with a prevalence of 19.94%. EZH2 showed weak or negative staining with a prevalence of 19.35%. NCL showed strong staining in 19.94% and weak staining in 10.86% of the cases (data not shown).
We found 3564 articles that were related to EP, but most referred to the clinical aspects of this neoplasm and some others did not meet the inclusion criteria, such as articles published in English, with complete data of patients, complete text, cross-sectional studies, molecular data studies, and studies carried out totally or partially in pediatric patients (under 18 years of age). Therefore, a total of 33 studies related to EP were included in this work. These papers were related to molecular studies in EP and included complete data of patients and complied with the eligibility criteria. Regarding data analysis, we considered the value of OR and HR; heterogeneity was evaluated with χ2, P value, and I2; the publication biases were evaluated using the funnel plot and Tau2 values [8, 9, 10].
EPs have been classified into subgroups: Posterior Fosa Group A (PFA) and Group B (PFB), each associated with distinct transcriptomic, genetic, epigenetic, and clinical features. Cases of the PFA subgroup were almost exclusively found in young children. The subgroups of supratentorial EP ST-YAP1 and ST-RELA are common in children; the C11orf95-RELA fusions are the main drivers of ST-EPN-RELA subgroup tumors . It is important to determine if other molecular characteristics are involved in tumorigenesis and are candidates for use as prognostic markers.
The following alterations were reported: losses in 1p, 3p11, 3p12, 3p23-p13, 3p24, 3q23-qter, 4q33-qter, 18q22.2, 22, gains distributed along chromosomes 7, 12, 15, cytobands 6q14–q27, 9q13, 9q21–q32, 9q33, 9q34, 10q25.2–q26.3, 10q25.2–q26.3 and 19p13, the loss at 18q22.2. These alterations were significantly associated with patients over 3 years [24, 28]; after data analysis and considering biases analysis, we found that the most frequent and characteristic chromosomal changes of the ependymal tumors were gains at 1q, 9q, and 17q and losses at 1p, 3p, 6q, 13q, and 22q. Furthermore, the regions 6q24.3 and 6q25.2–q25.3 were defined as the regions with the highest number of deletions and could play a role in the pathogenesis and biology of EP .
It was determined that the most important chromosomal alterations, such as the chromosome 22 loss, was that found in 57.5% of the studied EP cases; it was associated with 45% of intracranial EP cases and 82% of spinal EP . This is consistent with the idea that the loss or mutation of NF2 is frequently involved in their development, ranging from 30% to 71% of the patients [12, 15, 18]. Another important chromosomal alteration is a gain at 1q, which is one of the most common regions with a gain in EP [24, 25]. It has therefore been important to investigate the gain at 1q as a potential marker of a poor prognosis in pediatric EPs treated in a standard manner . The cytoband 1q25 is associated with a bad prognosis for patients because of this correlation with lower progression-free survival and overall survival .
Finally, it has been defined that 9q33–34 is the region with the most frequent gains and its occurrence correlated significantly with relapse . We found a greater incidence at relapse compared with the initial diagnosis for a gain at 1q, 9q34, 15q22, and 18q21, and losses at 6q [27, 30].
Regarding the genes reported to have changes in methylation, we found that the most frequent genes were CHIBBY, RAC2, and hTERT, in 8.8%, 9.2%, and 27.8% of the cases, respectively. It is possible that loss of RAC2 function has a greater impact in younger patients, whose central nervous system is still under development. It has been reported in EPs that the CHIBBY promoter has a high frequency of methylation. The downregulation of CHIBBY in cancer cells might provide information regarding the use of CHIBBY as a therapeutic target and prognostic factors have been associated with tumorigenesis [22, 43]. Furthermore, the results suggested that apart from less aggressive molecular subtypes, hTERT promoter hypomethylation might characterize intracranial ependymal tumors with a more favorable outcome . It has even been suggested that hTERT and CHIBBY methylation changes are characteristic of pediatric ependymomas, but they are not yet associated with the patient’s prognosis. Furthermore, the CpG island methylator phenotype in EP proposed by Mack et al.  allows us to stratify these tumors in PFA-CIMP+ and PFB-CIMP−, thus highlighting the distinct epigenetic differences among them.
In relation to gene expression, we found the hTERT, ERBB, ERBB1, ERBB2, ERBB3, STB, SHC1, and TPR genes were overexpressed. In several publications, a series of characteristic and prognostic molecular markers have been suggested, but no consensus on the matter has been reached. However, it has been reported that ERBB1, ERBB2, ERBB3, and ERBB4 are important in the development of EP and participate in cell proliferation . Witt et al.  classify EPs into two transcriptionally defined main subgroups: PFA and PFB; Group A tumors arose in younger patients (median age 2.5 years) whereas Group B tumors occurred predominantly in older patients (median age 20).
Moreover, it has been proposed that inhibition of telomerase enzymes is associated with an increased progression resulting in the lack of proliferation, self-renewal, and tumorigenicity; these findings suggested that the telomerase can be used as prognostic marker and therapeutic target in pediatric EP . By exploring molecular mechanisms, telomerase activity has been described as a characteristic potential biomarker, showing an association of telomerase reactivation with a chromosome 1q gain and RelA fusion . According to our analysis, considering the biases analysis, OR, and HR value, the hTERT and ERBB genes are characteristic in pediatric EP, but only hTERT can be correlated with the prognosis. The hTERT expression can be used to divide the resected tumors into good and bad prognostic groups because the EP lacking telomerase activity are unable to maintain telomeres and proliferative indefinitely, suggesting that less aggressive therapeutic intervention may be offered for children with telomerase-negative tumors [21, 34].
About protein expression, several research groups have proposed different proteins as features of ependymomas and as potential prognostic biomarkers. A previous study suggested an association between the overexpression of PDGFR protein, in tumor and in the tumor endothelia, has been presented that overexpression of PGDFRs could have a good prognostic role in EP . It has been observed that EPs showed at least focal immunopositivity for MDM2, and only some showed immunopositivity for P53. These findings are consistent with previous reports describing ependymomas rarely have P53 gene mutations and that neoplasms with MDM2 amplification typically lack P53 mutations that deregulate the cell cycle . Our findings correlate with Gupta et al. ; the proteins NOTCH-1, TN-C, and Hes-1 showed a significantly higher expression in grade III tumors in comparison to grade II tumors.
The general results suggest that protein expression plays an important role in pediatric ependymomas and that it can be considered as a possible molecular biomarker of prognosis. Chromosomes 1q and 22q are features in pediatric EP, and the gain at 1q25 has a high probability of being used as a prognostic biomarker. When considering the poor prognosis and survival rate of patients, the hTERT gene changes in expression and methylation status may play an important role in the development of pediatric ependymal tumors. Nevertheless, the results should be interpreted with caution. Additional research is needed to assess the true effect of protein expression to identify patients at risk.
We thank the scholarship CVU 404762 granted from CONACYT and IMSS 011-2013 for the development of the student, and the postgrad program of Ciencias Biológicas, from the Universidad Nacional Autónoma de México. We thank the research scholarship of IMSS Foundation A. C. given to Dr. Normand García Hernández. We thank the given support to Dr. Fabio A. Salamanca Gómez from Hospital de Pediatría “Dr. Silvestre Frenk Freund,” IMSS.
All authors contributed to the study conception and design. Conceptualization by N.G.-H. and M.P.-R. Material preparation, data collection, and analysis were performed by M.P.-R., T.J.-C., A.G.-M., and N.G.-H. The first draft of the manuscript was written by M.P.-R. and T.J.-C., and all authors commented on previous versions of the manuscript. Supervision by N.G.-H. All authors read and approved the final manuscript.
We are grateful for the funding provided by the CONACYT Sectoral Funds S0008-2010-1, 142013. We thank the FIS IMSS for the support FIS/IMSS/PROT/949, 2009-785-042 and R-2014-785-094.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interests.
The project was carried out under the authorization of the molecular biomarker search protocol for ependymal tumors in pediatric patients with registration number R-2014-785-094, at the Ethics Committee of National Ethics Commission from the Instituto Mexicano del Seguro Social, IMSS. All data have been kept in strict confidentiality.
The tests were carried out following the rules established in the Helsinki agreement. All participants were duly informed and provided their written consent for each of the procedures.
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