Ependymal tumors constitute a clinicopathologically heterogeneous group of brain tumors. They vary in regard to their age at first symptom, localization, morphology and prognosis. Genetic data also suggests heterogeneity. We define a newly recognized subset of ependymal tumors, the trisomy 19 ependymoma. Histologically, they are compact lesions characterized by a rich branched capillary network amongst which tumoral cells are regularly distributed. When containing clear cells they are called clear cell ependymoma. Most trisomy 19 ependymomas are supratentorial WHO grade III tumors of the young. Genetically, they are associated with trisomy 19, and frequently with a deletion of 13q21.31-31.2, three copies of 11q13.3-13.4, and/or deletions on chromosome 9. These altered chromosomal regions are indicative of genes and pathways involved in trisomy 19 ependymoma tumorigenesis. Recognition of this genetico-histological entity allows better understanding and dissection of ependymal tumors.

Background

Ependymal tumors include a broad histological and clinical spectrum of lesions presumably derived from ependymal cells contributing to the lining of the cerebral ventricles and the remnants of the central canal of the spinal cord [1]. Their overall incidence is 0.23 cases per 100,000 individuals per year in the United States, with a mean age at diagnosis of 35 years and an overall 5-year survival of 66% [2]. Ependymal tumors represent the seventh most frequent primary brain tumor in adult and the third in children. The last WHO classification comprises WHO grade I myxopapillary ependymoma and subependymoma, WHO grade II ependymoma, and WHO grade III anaplastic ependymoma [1].

In contrast to astrocytic and oligodendroglial tumors, in which molecular alterations associated with tumorigenesis are relatively well established, less is known about molecular changes in ependymal tumors. Monosomy of chromosome 22 and gain of chromosome 7 occur more frequently in spinal cord than in intracranial tumors [3]. In contrast, gain of chromosome 1q, and losses of chromosomes 6q, 9, and 13, are more frequently observed in the latter [3-5]. The involvement of chromosome 9 in ependymal tumors led to study the three 9p21 located tumor suppressor genes, CDKN2A, CDKN2B and p14ARF. Deletions of CDKN2A were found in 25% of the investigated tumors [6]. Promoter methylation of CDKN2A, CDKN2B and p14ARF was detected in 20–30% of tumors, with variations according to clinico-pathological characteristics [7].

Here we have analyzed ependymomas which have lost at least 9p and discovered that most of these tumors share architectural features, reminiscent of clear cell ependymoma. We performed array-CGH on a series of such tumors and observed trisomy 19 to be present in all of them. This copy number change was associated with alterations in chromosomes 9, 11 and/or 13. This data allowed us to define a new genetico-histological ependymal tumor entity, the trisomy 19 ependymoma. Histologically, they are compact with a clear tumor-to-parenchyma interface, presenting a branched capillary network and regularly dispersed tumoral cells. Clinically, most are supratentorial WHO grade III tumors of the young. This data underscores the heterogeneity within ependymal tumors and the need for further genetic dissection, a mandatory step to establish personalized oncological practice.

Results

Chromosome 9 microsatellite analysis of the formalin-fixed and paraffin-embedded ependymal tumor series and association with clinico-pathological parameters

PCR amplicons with interpretable results for chromosome 9 markers used were obtained for 131 of the 149 tumors (89%). Fifty-nine were located in the posterior fossa, 27 in the supratentorial compartment, 17 in the spinal cord, 26 in the conus-cauda-filum, and 2 in an unknown localization. For 9 tumors, most of the 29 tested microsatellites on chromosome 9 were deleted (median 89%, with a range between 78% and 100%, Figure 1: A1-2, A4-5 and A10 and, B1-4). For an additional tumor, deletion was restricted to the p arm (Figure 1: A6) and in a last one, interstitial deletions of both arms were observed (Figure 1: A9). These 11 tumors were classified as having a deletion on chromosome 9 (8,5%). Three of them were located in the posterior fossa (Figure 1: B1-3), and presented classical ependymoma histology. The other 8 were located in the supratentorial compartment (Figure 1: A1-2, A4-6, A9-10 and B4). Seven of them demonstrated a compact architecture, regularly dispersed tumoral cells, chicken-wire vessels (Figure 1: A1-2, A4-6 and A9-10), and sometimes foci of clear cells (Figure 1: A1, A4-5, and A9-10). This prompted us to perform genomic profiling for tumors of such clinico-pathologic characteristics.

Whole genome array-CGH

Two of us (MMR and CG) re-reviewed the histology of the supra-tentorial ependymomas of the formalin-fixed and paraffin-embedded series to identify additional tumors with histology similar to that of the above described 7 tumors, and without chromosome 9 deletion. Five such tumors were identified bringing the total to twelve ependymomas with the histology of interest (marked A1-12, Figure 1). As DNA extracted from archival tumors is suitable for array-CGH [8-11], this technique was applied to 11 of these tumors for which enough DNA was available (Figure 1: A1-9 and A11-12). In addition, 3 ependymomas with classical histology (Figure 1: B1, B3 and B19) were profiled. Two of them presented a monosomy 9 by microsatellite analysis (Figure 1: B1 and B3). Results were obtained for 79% (11/14) of tumors, 9 with the histology of interest and 2 controls (Figure 2 and Figure 1: A1-9, B1 and B19). Genetic profiles divided them into two distinct groups.

The first one comprised of 9 tumors: 3 were recurrent and 6 primary (Figure 2C: A1-9 and Figure 1: A1-9). They all shared trisomy of chromosome 19 (Figure 2A, B, C), which was non-complete in some tumors (tumor A4, lack of telomeric q region amplification: Figure 2B, C). In this group, altogether 118 genetic anomalies were detected with a mean of 13 per tumor. They consisted of 74 gains (64%) and 44 losses (36%). Deletions on chromosome 9 were observed in 7/9 tumors (Figure 2A, B, C and Figure 1), limited to 9p in one tumor (tumor A6: Figure 2B, C and Figure 1), to interstitial deletions in another (tumor A9: Figure 2B, C and Figure 1) and, without 9qter loss in 3 (Figure 2B: A2, A4 and A5). An equally frequent deletion of 30 Mb was located at 13q21.31-13q31.2 (Figure 2A, B, C). Gain of a small region located at 11q13.3-13.4 was observed in 6 tumors (Figure 2A, B, C). Chromosome 17 telomeres were co-amplified in 5/9 tumors, and a sixth one had an amplification restricted to 17q telomere (Figure 2B). Chromosomes 1, 7, and 20 also frequently showed telomeric p or q amplifications. Other whole chromosomal abnormalities, though less frequent, included monosomy 3 (1/9), trisomy 6 (1/9), trisomy 7 (2/9), trisomy 8 (2/9), monosomy 18 (1/9), trisomy 18 (1/9), trisomy 20 (1/9), and trisomy 22 (2/9)).

The second group comprised of two tumors: neither one of which presented the recurrent chromosomal anomalies of the first group, with the exception of monosomy 9 in one tumor (B1). Nine genetic anomalies were observed per tumor consisting of 7 gains (39%) and 11 losses (61%).

Whole genome profiling of frozen ependymal tumors by SNP-arrays

On the series of 24 frozen ependymomas, all presenting classical histological features of ependymoma, we observed only one ependymoma with partial trisomy of chromosome 19. This lesion was located at the spinal cord. It appeared as a compact tumor of low cellularity, devoid of mitosis, vascular proliferation and necrosis. This tumor, like the 23 other frozen ependymomas, did not fulfil clinico-histological aspect of the 9 ependymomas with a trisomy 19. Thus, altogether, 26 controls (24 frozen ependymomas + 2 paraffin-embedded ones, B1 and B19) were analyzed, and only one showed a partial trisomy 19 giving a p value < 0,001 (Fisher’s exact test) for the presence of trisomy 19 in our first group of tumors.

Characterization of trisomy 19 ependymoma

The tumor group defined by the presence of trisomy 19 on array-CGH (A1-9), shared many clinico-pathological features (Figure 1: A1-9). In 8/9 cases, localization was supratentorial, for one it was unknown. The age at diagnosis ranged between 0.4 and 30 years, with a mean at 14 years and a median at 18 years. Histologically, they appeared compact with a well-demarcated brain-to-tumor. They presented a rich network of branched capillaries, reminiscent of the one of 1p/19q-deleted oligodendroglioma and tumoral cells were regularly distributed. In addition, pseudorosettes were always detected, meanwhile sometimes rare Microcalcifications were frequently encountered, and regions of classical ependymoma histology could be observed.

Tumoral cell phenotype varied between tumors and sometimes within the same tumor In 3/9 tumors, most of the cells were ovoid with a clear perinuclear halo In four tumors, the cells were almost devoid of cytoplasm with round nuclei A2-4 and A6, and, Figure. In these tumors, anucleated areas could be seen. They were not always centred on a vessel, imparting an initial impression of a neuroid-derived tumor. In one of these tumors, transition between neuroid to clear cell pattern was observed (Figure 1: A4 and Figure 4I and 4J). Finally, the remaining two tumors were composed of intermediate-to-large, ovoid or fusiform cells, with abundant cytoplasm that appeared palely stained or eosinophilic, suggestive of an oligo-astrocytic.

The immuno-histochemical profile was similar in all trisomy 19 ependymomas. GFAP was always detected, although with intra and inter-tumoral variations. Some tumoral areas could be almost completely negative, while others showed intensely labeled ependymal cells. At least, perivascular positivity of cell end-feet was always found. EMA immunopositivity, appearing as intracytoplasmic dots, was observed focally in all tumors (Immunolabeling for neurofilaments was consistently negative in the tumor, although it was positive in the surrounding normal brain.

All but one ependymal tumor with trisomy 19 presented at least two of the following signs of anaplasia: (1) endothelial cell proliferation, (2) Ki-67 labeling index higher than 10% and (3) frequent mitotic figures Therefore, they were considered as WHO grade III tumors.

Discussion

We recognized a new genetico-histological association within ependymal tumors, the trisomy 19 ependymoma. Most of these tumors are supra-tentorial WHO grade III tumors of the young. Trisomy of chromosome 19 is frequently associated with deletion of 13q21.31-31.2, three copies of 11q13.3-13.4, and/or deletions on chromosome 9. The histological hallmark is a prominent branched capillary network around which tumoral cells are regularly dispersed. Clear cells may be present in these compact lesions, evoking the diagnosis of clear cell ependymoma. Trisomy 19 ependymomas have an immunohistological profile of ependymal tumors: positive for glial fibrillary acidic protein (GFAP) and epithelial membrane antigen (EMA), and negative for neuronal markers.

In the “cancer chromosomes database” [12], we calculated chromosomal trisomy to occur with a mean frequency of 4% among all tumors (n = 50,380), which was comparable for chromosome 19 (4.2%). In brain tumors (n = 1644), trisomy 19 occurred in 6%, but only in about 3% of meningiomas (n = 817) and, interestingly in about 9% of astrocytomas (n = 569) and ependymomas (n = 111). This more frequent observation in glial tumors suggests an ethiopathogenic role within this group of tumors. Our results linked trisomy 19 to a subset of supra-tentorial ependymomas that we could recognise on histological criteria. Furthermore, in the two control series, we observed only one tumor with a partial chromosome 19 trisomy (3,8%), giving a p value < 0,001 for the presence of trisomy 19 in our tumor group of interest. Because trisomy 19 appears non-complete in some of our ependymomas, it has to be looked for using a technique which profiles the entire chromosome 19 for copy number alterations.

The other copy changes are helpful in recognition of trisomy 19 ependymomas. Interstitial deletion of 13q and deletions on chromosome 9 were identified in 7/9 (78%) of the tumors. They were observed associated with trisomy 19 only once and twice respectively in the NCBI database (frequency of 0,9% and 1,8%) [12], and not in our control. Similarly, association between trisomy 19 and amplification of 11q13.3-13.4, which was observed in 6/9 (66%) of our trisomy 19 ependymomas, was rarely reported in the NCBI database (4,5%) and not found in the control series. All of our trisomy 19 ependymomas presented at least one of these associated anomalies (100%) and 7/9 (78%) two of them.

The WHO classification describes clear cell ependymomas as having an “oligodendroglia-like appearance with clear perinuclear haloes” [1]. This definition highlights the importance of two histological features: clear cells and chicken-wire vessels. The combination of our genetic and histological data emphasized the latter. Chicken-wire vessels were constantly observed in ependymal tumors bearing trisomy 19, whereas clear cells were not. Thus clear cell ependymoma are a subgroup of trisomy 19 ependymoma.

Previously, trisomy 19 ependymomas may have been reported as haemangioblastoma, PNET, central neurocytoma, oligodendroglioma or oligo-astrocytoma. Amongst them, haemangioblastoma is the only one with a reticulin rich stroma. Neuronal markers are positive in PNET and central neurocytoma, but negative in trisomy 19 ependymoma. GFAP and EMA, which are negative in neuronal tumors, are positive in ependymal tumors, although this can be focal Because of this, differential diagnosis between trisomy 19 ependymomas and oligodendroglioma/oligoastrocytoma may be difficult. Genetic analysis can help. Deletion of 1p and 19q is restricted to oligodendroglioma and oligo-astrocytoma whereas trisomy 19 suggests trisomy 19 ependymoma.

Rickert and co-workers recently analyzed by classical CGH a series of 13 clear cell ependymomas. They pinpointed monosomy 9 to be always associated with WHO grade III and sometimes with WHO grade II clear cell ependymoma. They defined clear cell ependymomas as comprising of at least 50% of clear cells. In our series of trisomy 19 ependymomas, 3/9 tumors contained similar percentage of clear cells (Figure 1: A1, A5 and A9), one less (Figure 1: A4), and five none (Figure 1: A2-3, A6-8). All 4 trisomy 19 ependymomas with clear cells had an additional chromosome 9 deletion, in agreement with the published series. Amongst the 5 trisomy 19 ependymomas devoid of clear cells, three had a deletion on chromosome 9. Moreover, chromosome 9 deletion was also observed in other subtypes of ependymomas (Figure 1: B1-4). There is a technical difference as well. The total number of reported chromosomal anomalies was 2,7 per tumor in the Rickert analysis, whereas we demonstrated a mean of 13 anomalies per tumor, illustrating the higher resolution of array-CGH. In addition, Rickert’s series did not demonstrate anomalies of chromosome 19, whereas trisomies of this chromosome were observed in a series of clear cell ependymomas analyzed by FISH Presence of high amount of heterochromatin in chromosome 19 is known to induce false positive and negative CGH results, a reason why this chromosome is often difficult to interpret and frequently excluded from classical CGH analysis

Cancer arises from accumulations of genetic changes in pathways involved in cell cycle, cell proliferation, apoptosis, angiogenesis and interaction with extracellular matrix Interestingly, all these biological processes can already be altered by genes located on chromosome 19. The 3 other chromosomic regions involved in trisomy 19 ependymomas could reinforce potential deregulation of these pathways]. Furthermore, epigenetic changes are of importance. Chromosome 19 contains BRG1 (19p13.2), and chromosome 9 BRM (9p24.3). These genes are ATPase subunits of SWI/SNF complex, one of the two enzymes implicated in chromatine remodeling and promotor methylation

Conclusion

We describe a new subgroup of ependymal tumors, the trisomy 19 ependymoma, which bears specific clinico-histological characteristics. This newly identified association is a significant additional argument for considering disparity in tumorigenesis pathways involved in ependymal tumors as suggested by Lukashova and co-authors [46]. Differences have also been illustrated on differential expression of DAL-1 and NF2 between intracranial and spinal cord ependymomas [47], methylation of RASSF1A and TRAIL pathway-related genes in childhood intracranial ependymomas [48], and methylation of 9p21 tumor suppressor genes following clinico-histological parameters of ependymal tumors [7]. Further genetico-histological dissection of tumors is needed for development of targeted oncological practice.

Methods

Tumor samples and DNA extraction

Two independent series of ependymal tumors were analyzed. None of the tumors were simultaneously included in both series. The first one comprised of 24 frozen ependymal tumors and the second of 149 formalin-fixed and paraffin-embedded ones. Both series were retrieved from the archives of 9 neuropathological centers based on their original diagnosis of ependymomas (Institute of Neurology, London, UK; Hospital Roger Salingro, Lille, France; Laboratory of neuropathology, CHU-Caen, France; St. Luc hospital, Université catholique de Louvain, Belgium; La Timone’s Hospital, AP-HM tumor bank, Marseille, France; Hospital Erasme, Université libre de Bruxelles, Belgium; CHU Kremlin-Bicêtre, Paris, France; CHU-Lariboisière, Paris, France; University of Newcastle, Newcastle, UK). Before inclusion in the study, all tumor diagnoses were revised following the latest WHO classification [1]. The study was approved by the ethics committee of the Medical Faculty of Université catholique de Louvain, Brussels, Belgium.

The 24 frozen ependymomas were obtained from 23 patients, 18 corresponding to primary tumors and 6 to recurrent tumors. For one patient, we received both the primary and the recurrent tumor. Age at operation varied between 4 months and 63 years with a mean age of 27.5 years and a mediane age of 33 years. Histologically, the tumors were classified as myxopapillary ependymomas (WHO grade I, n = 2, 8%), ependymomas (WHO grade II, n = 11, 46%) and anaplastic ependymomas (WHO grade III, n = 11, 46%). Thirteen were located in the spinal cord (54%), 4 in the posterior fossa (17%) and 7 in the supratentorial compartment (29%). This series was used for single nucleotide polymorphism (SNP) microarrays analyses.

The 149 formalin-fixed and paraffin-embedded tumors were obtained from 146 patients. In three patients, tumors were collected from 2 consecutive surgical resections. In 17 patients samples were available only from the recurrence. Age at operation varied from 3 months to 80.6 years, with a mean of 29.4 years and a median of 27.4 years. More precisely, 19 patients were aged between 0 and 3 years (13%), 29 between 3 and 15 years (19%), and 100 were older than 15 years (67%). For 1 patient, the age was unknown (<1%). Histologically, the tumors were classified as subependymomas (WHO grade I, n = 15, 10%), myxopapillary ependymomas (WHO grade I, n = 28, 19%), ependymomas (WHO grade II, n = 63, 42%), and anaplastic ependymomas (WHO grade III, n = 43, 29%). Forty-nine were located in the spinal cord (32.9%), 63 in the posterior fossa (42.3%), 33 in the supra-tentorial compartment (22.1%), and 4 in an unknown location (2.7%) This series was used for microsatellite analysis and array-CGH.

For microsatellite analysis, DNA was extracted from formalin-fixed and paraffin-embedded tumors using QIAamp DNA Mini Kit (Qiagen, Westburg, Leusden, Holland) after deparaffinization of tumor shaves with xylene. This was performed following a tissue dissection step if section contained more than 10% of normal brain parenchyma. For 6 patients, enough normal tissue was obtained to be used as control DNA.

For array-CGH, tumoral DNA was extracted from formalin fixed and paraffin embedded tumors using PUREGENE DNA Purification Kit (Gentra Systems, Minneapolis, Minnesota, USA) after deparaffinization of tumoral shaves using xylene. Control DNAs were extracted from blood samples of healthy individuals. Pools of either 5 males or 5 females were constituted. Absence of DNA anomaly in both pools was confirmed by hybridization of male pool against female pool. For SNP chip analysis, DNA was extracted from frozen tumors as described for array-CGH, but without deparaffinization.

Immunohistochemistry

Immunohistochemistry was performed following classical protocols. Briefly, 5 μm sections were deparaffinized using Histosafe (Yvsolab, Beerse, Belgium), rehydrated in propanol and finally water. After blocking endogenous peroxydase by incubating sections for 30 min in 0.3% H₂O₂, antigens were retrieved using a citrate buffer pH 5.7 at 95°C for 95 min. Non-specific binding was inhibited under 10% normal goat serum and 1% bovine serum albumin. The slides were subsequently incubated overnight at 4°C with the following primary antibodies: Glial Fibrillary Acidic Protein (GFAP, rabbit polyclonal obtained from Dako, dilution 1/2000), Epithelial Membrane Antigen (EMA, mouse monoclonal, Neomarkers, dilution 1/200), Neurofilaments 68 and 200 (NF, mouse monoclonal, Sigma and Boehringer, used mixed together at 1/200 and 1/25 dilution), Neuron-specific Nuclear Protein (NeuN, mouse monoclonal, Chemicon, dilution 1/100), and Ki-67 (mouse monoclonal, Dako, dilution 1/100). After a wash with Tris-HCl 0.05 M, pH 7.4, either a biotinylated anti-mouse (Vector, dilution 1/500) or anti-rabbit (Boerhinger, dilution 1/500) secondary antibody was applied on the sections for 30 min at room temperature. The slides were then washed with Tris-buffer and incubated with a streptavidin-peroxydase complex (Roche, dilution 1/100) for 30 min at room temperature. After a wash, the chromogen was revealed using 0.05% DAB (Fluka) in PBS buffer (pH 7.2) with 0,01% H₂O₂ during 10 min. The slides were rinsed with water, counterstained with Mayer’s Hematoxylin and mounted.

Microsatellite analysis

Twenty-nine microsatellites of chromosome 9 were chosen from the Human MapPairsTM Genome-Wide Screening Set 8 (Weber set, Research Genetics) or 9 (Li-Cor, Westburg, the Netherlands), or in the Unified Database for Human Genome Mapping on the basis of their map position [49]. The latter were synthesized by Eurogentec (Belgium) or MWG (Germany). Of the 29 microsatellites, 23 were located on 9p (D9S917, D9S288, D9S1810, D9S2169, D9S2156, located at 9p24; D9S775, D9S921, D9S168, D9S269, D9S254, located at 9p23; D9S285, D9S156, D9S157, D9S925, located at 9p22; D9S162, D9S1749, D9S1748, D9S171, D9S1679, D9S1121, D9S251, D9S1118, and D9S1788, located at 9p21) and 6 were located on 9q (D9S301, D9S1122, D9S922, located at 9q21; D9S930, located at 9q32; D9S934, and D9S1825, located at 9q33).

For amplification of microsatellites with primers of the Weber set 8, one of the two primers was end-labeled with gamma-32P ATP (Amersham-Pharmacia), using T4 polynucleotide kinase (TAKARA). PCRs were performed in a final volume of 10 μl containing 18 ng of template DNA, 0.6 μM of each primer, 1× Biotools buffer, 0.2 mM of each dNTP, and 0.025 U/μL Biotools DNA polymerase (Lab Systems, Belgium). After 5-minutes denaturation at 95°C, 35 cycles were realized with 94°C for 40 seconds, 55°C for 50 seconds and 72°C for 50 seconds, with a final extension of 5 minutes at 72°C. For amplification of the Weber set 9 (Westburg, Holland) and other fluorescent markers modified with IRD 700 or IRD 800 fluorochromes (MWG, Germany), PCRs were carried out as above, except that 36 ng of template DNA and 2 to 5 mM MgCl2 were used. All PCRs were performed in duplicate.

To visualize alleles of microsatellites, amplicons were heat denatured (95°C for 5 minutes) after addition of 10 μl of a denaturing loading buffer. 2.5 μl of the obtained solution was run on gel. For radio-active markers, a 5% polyacrylamide denaturing gel was used. The gels were subsequently vacuum-dried and exposed overnight on Kodak X-OMAT AR films (Amersham-Pharmacia, Belgium). For fluorescent markers, a 6,5% SequaGel XR gel (National Diagnostics) was used on a Li-Cor Gene Readir 4200 DNA Analyzer (Westburg, the Netherlands), operated by E-Seq DNA Sequencing and Analysis software (version 1.0 Westburg, the Netherlands). The analysis was made with Gene ImagIR software (version 4.0, Westburg, the Netherlands).

Interpretation of microsatellite results was performed as previously described [50]. Briefly, the status of chromosome 9, for a given tumor, was only taken into account when more than 70% of the tested microsatellites gave interpretable amplicons. In addition, to be considered monosomic, 90% of the amplicons had to reveal either the presence of a single allele or an important difference in intensity between the two alleles.

Array-CGH

Array-CGH was performed according to manufacturer’s instructions (Spectral Genomics, USA) with few modifications. After DNA extraction and purification with the Qiagen PCR purification kit (Westburg, Holland), the DNAs were labeled with Cy3-dCTP and Cy5-dCTP (Amersham) using the Gibco/BRL Bioprime DNA labeling kit. Labeled test and reference DNA were subsequently mixed in the proportion of 1.2–1.4 μg of Cy3 labeled DNA to 1 μg of Cy5 labeled DNA. Spectral Hybridization buffer I containing cot-1 DNA was added to the mixture. DNAs were precipitated with 5 M NaCl/isopropanol solution, rinsed with 70% ethanol and air-dried. The precipitate was resuspended in water and mixed with the Spectral Hybridization buffer II. Denaturation at 72°C for 10 minutes was followed by incubation of 10 min on ice and subsequently of 30 min at 37°C. Hybridization on the array (HU 1K BAC Array, Spectral Genomics, USA) was performed in a hybridization chamber at 37°C for 16 hours. To increase specificity, each hybridization was also performed by flipping tumor and control fluorochromes. The arrays were rinsed in the 5 subsequent solutions: 2 × SSC, 0.5% SDS for 20 min at 45°C; 2 × SSC, 50% formamide for 20 min at 45°C; 2 × SSC, 0.1 × NP-40 for 20 min at 45°C, 0.2 × SSC for 10 min at 45°C, and a brief rinse in H₂O. The arrays were scanned and analyzed using the Spectralware software (version 2,0).

Single Nucleotide Polymorphism microarrays

The Affymetrix high-density (50 K) oligonucleotide array-based SNP genotyping was performed according to the standard protocol for Affymetrix GeneChip Mapping 100 K arrays (Affymetrix, Inc.). Only tumors with a call rate higher than 94% were included in the analysis. Briefly, 250 ng of genomic DNA was digested by a restriction enzyme (XbaI), which allowed to ligate an adaptor used for PCR primer annealing for the subsequent whole genome amplification. The obtained PCR products were size-restricted by digesting with DNAseI. This fragmented DNA was labeled with a biotinylated nucleotide analogue and hybridised to the microarray. Hybridised fragments of tumoral DNA were revealed using a three step detection system constituted of a first streptavidin-phycoerythrin binding, followed by an anti-streptavidin biotinylated antibody incubation and, lastly, a final streptavidin-phycoerythrin step. After scanning of the array, the SNPs were genotyped by GeneChip DNA Analysis Software (GDAS, version 3.0.2.8; Affymetrix, Inc.). Raw signals (genotype and intensity data of the SNP probes) were exported from the Affymetrix platform and analyzed for copy number alterations using the Copy Number Analyser for GeneChip (CNAG, version 1.0) [51] and dCHIP (version 2005) softwares.

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OBJECTIVE AND IMPORTANCE

 Extraventricular ependymomas account for 50% of supratentorial ependymomas. Some tumors may extend to the gray matter reaching the pial surface, but pure cortical ependymomas are uncommon. Here, we report three patients with supratentorial intracortical ependymoma.

CLINICAL PRESENTATION: We reviewed the clinicopathological findings of all patients operated on for ependymomas at the Bellaria Hospital during an 11-year period and found three lesions described as cortical ependymomas. The three lesions represented 2.5% of all ependymal tumors and 21.4% of supratentorial tumors operated on during the study period. The patient were aged 52, 24, and 11 years (mean, 32.3 yr). One was female. All presented with seizures. On imaging, the lesions were confined to the gray matter, were solid, and demonstrated diffuse enhancement.

INTERVENTION: Gross total resection was achieved in all instances. Two patients were treated with surgery, and one was treated with surgery and postoperative radiotherapy. All tumors were low grade. After a mean follow-up of 92.6 months, no patient had recurrence or leptomeningeal dissemination. Review of preoperative magnetic resonance imaging scans confirmed an intracortical location. Routine sections were reviewed, and additional immunoreactions for epithelial membrane antigen, glial fibrillary acidic protein, synaptophysin, neurofilament proteins, S-100 protein, and Ki-67 and electron microscopy were performed.

CONCLUSION: Cortical ependymomas seem to behave as benign tumors amenable to surgical removal. Local recurrence and leptomeningeal dissemination seem to be unlikely. Postoperative radiotherapy is unnecessary.

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Ependymomas are central nervous system (CNS) tumours that originate from the wall of the ventricular system along the entire craniospinal axis. Although ependymomas from different regions of the CNS are histologically similar, they are clinically) and genetically distinct, suggesting that they represent a collection of different diseases. Intracranial ependymomas often recur at the primary tumour site and less than 60% of children with this disease will survive more than 5 years. Despite this significant clinical burden, we have learned almost nothing new about the biology and treatment of ependymoma during the last 20 years. Indeed, less than 5% of the 14 200 brain tumour research studies published in the last 5 years have investigated ependymoma. Unless we can increase understanding of the biology of ependymoma we are unlikely to reduce the morbidity and mortality associated with this disease.

CANCER STEMS CELLS

Cancer researchers have invested a great deal of effort in characterising the genetic alterations that accumulate in end-stage tumours. Although these studies can provide lists of the genetic events that contribute to the expansion of malignant clones, they do not inform us regarding the chronology or relative importance of each of these alterations in the cancer process. A more comprehensive understanding of cancer that spans the life of the disease from the birth of the first malignant cell to clinical presentation, would be invaluable in the hunt for more effective treatments of cancer. With this in mind, considerable excitement has surrounded the recent discovery of cancer stem cells (CSC). Cancer stem cell make up just a small fraction of the total population of the malignant cells in many solid tumours and leukaemias. However, evidence indicates that these self-renewing and multipotent stem cell-like cells generate all of the phenotypically diverse cells that populate tumours. The discovery of CSC has therefore provided researchers with a practical point of focus for studying the natal cellular and molecular events of tumorigenesis.

The identification of CSC is likely to have important implications for the treatment of cancer. If tumours are derived entirely from CSC, then it would follow that to be curative, cancer treatments should disable or destroy these cells. Indeed, drugs that are designed to kill CSC could prove highly effective treatments of cancer. On the other hand, evidence that CSCs are remarkably similar to normal stem cells predict that such treatments may also possess significant toxicities. For example, brain CSC express the neural stem cell markers Nestin and CD133, whereas acute myeloid leukaemic CSC display the CD34⁺CD38^- immunophenotype of haematopoietic stem cells (HSC). Thus, the development of safe and effective therapies for all cancers is likely to require understanding of the similarities and differences between normal and malignant stem cells in tissues.

RADIAL GLIA IS CANDIDATE CELLS OF ORIGIN OF EPENDYMOMA CSC

Comparative studies of normal and malignant HSC have been facilitated by the availability of robust assays for all stages of haematopoiesis). In contrast, knowledge of the stem and progenitor cell populations of non-haematologic organs is limited. Therefore, identifying the cell of origin of solid tumour CSC and the molecular alterations that transform these cells is less straight forward. Recently, we compared the gene expression profiles of developing tissues and ependymoma subsets to identify populations of cells in the CNS which act as the cells of origin of ependymomas

In an analysis of over 100 ependymomas, we found that tumour subsets exhibit distinct patterns of gene expression and regions of chromosome gain and loss that correlate with the anatomic location of the tumour (supratentorial region, posterior fossa or spine. Gene expression signatures that most discriminated supratentorial, posterior fossa and spinal ependymoma included many genes that are known regulators of neural precursor cells in the corresponding region of the CNS. For example, we found that supratentorial tumours express markedly elevated levels of members of the EPHB-EPHRIN and NOTCH cell signal systems that play key roles in maintaining normal neural stem cells in the cerebral subventricular zone (SVZ). Conversely, spinal ependymomas expressed multiple Homeobox (HOX) family members that coordinate antero-posterior tissue patterning and development of the spine. Therefore, we reasoned that subsets of ependymoma either maintain, or recapitulate the developmental expression profiles of anatomically restricted progenitor cells. To identify these precursor cells in the normal CNS, we used in situ hybridisation and immunofluorescence to map the site of expression of ependymoma signature genes in the developing mouse. These data identified remarkable similarities between the distinct gene expression patterns observed in embryonic radial glia (RG)  that are neural progenitor cells (see below)  and those observed in human ependymomas from the corresponding region of the CNS. The great majority of intracranial tumours in our study arose in children; whereas the spinal tumours were obtained from adult patients. Therefore, our data suggest that RG are a likely source of ependymomas independent of patient age. It is noteworthy that astroglial cells with functional and molecular characteristics of RG persist in the SVZ of the lateral ventricles and possibly the spinal cord, suggesting that some RG give rise to adult neural stem cells. It is possible that these RG-derived stem cells are cells of origin of adult ependymomas. We are conducting extensive genomic and functional studies of stem cell populations in normal and malignant embryonic and adult neural tissues to identify the precise cell of origin of each type of ependymoma.

Importantly, we showed also that self-renewing and multipotent CSC isolated from fresh samples of ependymoma are: bipolar RG-like cells; express the CD133+Nestin+RC2+brain lipid-binding protein (BLBP)+ immunophenotype of RG; and are both required and sufficient to generate tumours in vivo. Our data suggest a new hypothesis for the origin of ependymoma: that RG in different parts of the CNS are predisposed to acquire distinct genetic abnormalities that transform these cells into CSC of supratentorial, posterior fossa and spinal ependymoma.

WHAT ABERRANT PROCESSES TURN RG INTO CSC?

To determine whether ependymoma CSC arise from RG, and how this malignant transformation may occur, it is important to understand the processes that regulate the generation and fate of RG in the CNS. In vertebrates, neural stem cells first appear as a layer of pseudostratified epithelium that lines the neural plate and neural tube before the onset of neurogenesis. These neuroepithelial cells (NEC) are highly polarised along their apicalbasal. Of particular note, the apical cell membrane of NEC contains characteristic transmembrane proteins, for example, prominin-1 (the mouse orthologue of human CD133), and adjacent adherens junctions that are thought to regulate cell proliferation and fate decisions.

As neurogenesis begins, NEC give rise to RG. RG retain the highly polarised features of NEC and express CD133, Nestin and RC2, but they also express astrocyte-specific proteins that include glutamate transporter and BLBP Previously, RG were believed to generate only astroglial cells. However, studies conducted within the last 5 years indicate that RG are mitotically active, multipotent progenitor cells that probably give rise to the majority of neurons, astrocytes, oligodendrocytes and ependymal cells in the brain ().

Neuroepithelial cells and RG each possess the capacity for symmetric and asymmetric cell division. Symmetric division expands the neural stem and progenitor cell pool, as this generates two identical daughter cells that resemble the parent NEC or RG cell. In contrast, asymmetric divisions generate unequal daughter cells: one stem cell and one cell that is fated to differentiate. In normal tissues, stem cell self-renewal  cycles of division that repeatedly generate at least one daughter equivalent to the mother cell  is tightly regulated, and deregulation of this process is emerging as a key event in the development of CSC . Thus, if ependymoma CSC arise from RG, then the factors that coordinate RG cell division might represent targets for mutations that underlie the initiation of this brain tumour.

Aberrant RG cleavage

The apical plasma membrane appears to play an important role in determining the fate of NEC and RG daughter cells (. Daughter cells that inherit the apical membrane retain the proliferative stem cell properties of the parent cell. Thus, it has been suggested that the apical membrane acts as a transducer of pro-proliferative signals from the neural tube to NEC and RG (. Indeed, equal distribution of the apical membrane between the progeny of symmetrically dividing RG, might explain why this division results in identical daughter cells with proliferative progenitor cell properties. Whether RG undergo symmetric or asymmetric division appears to be controlled, at least in part, by certain transcription factors. For example, Emx2 induces symmetric divisions that can lead to expansion of RG cell numbers, whereas Pax6 activates neurogenic, asymmetric division. Interestingly, we found that expression of EMX2, but not PAX6, is markedly upregulated in supratentorial ependymomas compared to ependymomas from other regions of the CNS (37-fold expression difference P<0.0001;. Thus, aberrant and prolonged expression of EMX2 within embyonic cortical RG might contribute to increased symmetric cell division and the formation of CSC of pediatric supratentorial ependymoma. One important caveat against these rather simple models of tumourigenesis, is the capacity of genes to confer context dependent effects on stem cells. For example, studies have shown that Emx2 can operate as a negative regulator of symmetric cell division in adult neural stem cells. Better understanding of the precise cell of origin of ependymomas should help to clarify the role of specific genes in the development of these diseases.

Disruption of RG adherens junctions

Neuroepithelial cell and RG each contain concentrations of adherens junctions that are located immediately beneath the apical plasma membrane . Evidence indicates that disruption of these complexes could also contribute to the transformation of RG. Indeed, deletion of the essential adherens gene E-Catenin from neural progenitor cells in mice results in severe disruption of the apical cell junctions, loss of cell polarity, increased stem cell proliferation and the formation of tumour-like masses in the brain . It remains to be determined whether disruption of adherens complexes, that occurs frequently in human cancers, might contribute to the formation of CSC in the brain
Deregulation of cell signal pathways

Certain cell signal pathways that control RG self-renewal are deregulated in ependymoma. Most notable among these is the NOTCH cell signal pathway. Upregulation of the Notch ligand Jagged1 maintains the self-renewal and multipotency of adult neural stem cells, whereas deletion of Notch1 depletes the neural stem cell fraction Further, retroviral transfer of activated Notch1 into cells lining the SVZ of the forebrain of embryonic mice promotes the formation and maintenance of RG . Interestingly, the Notch cell signal pathway might serve as a link between the symmetry of NEC and RG division and the fate of daughter cells. In this regard, deletion of Lethal Giant Lavae-1 from dividing neural stem cells of mice, prevents the asymmetric localisation the Notch inhibitor Numb to daughter cells, resulting in failure of asymmetric cell divisions and the formation of tumour-like masses within the brain. We found that the signature genes that are most upregulated in supratentorial ependymomas include the NOTCH ligands JAGGED 1 and 2, and the NOTCH signal targets HES1 and HES5. Interestingly, the ERBB2 oncogene that is expressed to high levels in ependymoma, is an important target of Notch signalling in RG. Downregulation of ErbB2 expression or activity in RG causes these cells to differentiate, whereas activation of ErbB2 maintains RG proliferation. Thus, deregulation of NOTCH signalling, either alone, or in concert with other cell signal molecules might promote the formation of ependymoma CSC.

p19^Arf and p16^Ink4a that are encoded by the Ink4aArf locus, are two additional regulators of neural stem cell proliferation. In this regard, Bmi1 promotes the self-renewal of neural stem cells by repressing transcription at the Ink4aarf locus; whereas deletion of Ink4a significantly expands the neural progenitor cell population . We found that concurrent activation of NOTCH cell signalling and deletion of INK4AARF affect the great majority of supratentorial ependymomas. Specifically, using array comparative genomic hybridisation and fluorescence in situ hybridisation, we have shown that INK4AARF is selectively deleted from >90% of tumour cell nuclei of supratentorial ependymomas but is rarely deleted from tumours arising in other regions of the CNS . Thus, cortical RG might be susceptible to transformation into ependymoma CSC by concurrent activation of NOTCH signalling and deletion of INK4AARF.

Further studies will be required to characterise fully the factors that contribute to the formation of ependymoma CSC. These efforts are likely to be assisted by ongoing studies of flies that have identified already tumour suppressor genes that regulate the proliferation of neural stem cells. For example, the Drosophila tumour suppressor protein Brat is normally distributed asymmetrically to one of the daughters of dividing neural stem cells, fating that cell to differentiate, whereas the remaining daughter cell self-renews. In Drosophila brat mutants both daughter cells self-renew, resulting in expansion of the stem cell pool and the formation of larval brain tumours.

EPENDYMOMA CSC AND THE CLINIC

If the CSC hypothesis proves correct and brain tumours, including ependymoma, arise from rare fractions of stem-like cancer cells, then these findings will lead to a paradigm shift in the way we treat CNS tumours. In particular, we should begin to develop classification systems and targeted treatment strategies that focus on the eradication of CSC. This strategy may prove particularly effective in tumours such as ependymoma that include developmentally and molecularly distinct subgroups that are unlikely to respond uniformly to all treatments.

At least two approaches might be adopted in the development of anti-CSC therapies. First, as normal stem cells are protected from environmental insults by both cell intrinsic and extrinsic factors, then CSC might be inherently resistant to conventional chemo- and radiotherapies. Thus, agents that counteract CSC drug resistance mechanisms might prove useful in the treatment of cancer. Second, targeting the pathways that regulate aberrant self-renewal could be used to disable or destroy CSC. Clinical trials of one such class of drugs, inhibitors of -secretase, are currently underway among patients with leukaemia, and plans to trial these agents among children with ependymoma are in advanced stages within the US Pediatric Brain Tumor Consortium. NOTCH signalling is activated following -secretase mediated cleavage of the NOTCH receptor. Thus, inhibitors of -secretase might be effective treatments of supratentorial ependymomas.

The success of anti-CSC therapies will require not only that these drugs kill or disable CSC, but also that they spare normal stem cells. This issue is especially important when considering the treatment of children with brain tumours whose nervous system is still developing. Thus understanding further the origins of CSC in the brain and how best to target these in the clinic will likely require the ongoing collaboration of developmental biologists, cancer biologists and clinicians.

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A new study shows that long-term survivors of childhood leukemia and brain tumors are at increased risk of stroke well after their cancer treatment has ended, and that this risk is higher among those who were treated with a particular type of radiation therapy. The research, conducted as part of the Childhood Cancer Survivor Study (CCSS), will be published online November 6 in the Journal of Clinical Oncology (JCO).

Leukemia and brain tumors together account for 53% of all cancers diagnosed in children younger than 15. Current five-year survival rates are nearly 80% for leukemia and 74% for brain tumors, making the long-term side effects of treatment for these cancers an important area of study. Treatment for both diseases involves therapy that targets the central nervous system; treatment sometimes includes moderate or high-dose radiation therapy to the brain (known as cranial radiotherapy).

The researchers surveyed 4,828 leukemia survivors and 1,871 brain tumor survivors participating in CCSS, as well as a control group of 3,846 of their siblings who had not had cancer, about their history of stroke. Among leukemia survivors, the occurrence of stroke was 0.8% (one in every 125 survivors), compared with 0.2% (one in every 500 survivors) for the control group. The average time from leukemia diagnosis to stroke was 10 years. For brain tumor survivors, the occurrence of stroke was 3.4% (one in 30 survivors), and as high as 6.5% (one in every 15 survivors) for patients who had been treated with both cranial radiotherapy and chemotherapy. Among brain tumor survivors, the average time from cancer diagnosis to stroke following treatment was 14 years.

What Does This Mean for Patients?

It is important for survivors and their doctors to know that the long-term effects of childhood cancer and its treatment can be reduced through careful planning of follow-up screening and care.  Survivors of childhood leukemia or brain tumors should also be aware of their small but increased risk of stroke, and should seek immediate medical attention for any symptoms of stroke (such as temporary weakness on one side of the body.

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Cancer stem cells (CSC) are recently proposed to be the cancer initiating cells responsible for tumorigenesis and contribute to cancer resistance. Advances have been made in identifying and enriching CSC in leukemia and several solid tumors, including breast, brain and lung cancers. These studies suggest that, like normal stem cells, CSCs should be rare, quiescent, and capable of self-renewing and maintaining tumor growth and heterogeneity. Although the concept of CSC originates from that of normal stem cells, CSCs are not necessarily aberrant counterparts of normal stem cells. In fact, they may arise from stem cells or committed progenitors of corresponding tissues, and even cells from other tissues. At the molecular level, the alteration of stem cell self-renewal pathway(s) has been recognized as an essential step for CSC transformation. Better understanding of CSC will no doubt lead to a new era of both basic and clinical cancer research, re-classification of human tumors and development of novel therapeutic strategies specifically targeting CSC.

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“Identifying the cancer stem cell and the evolutionarily conserved genetic programs underlying self-renewal in ERMS will likely lead to new insights into how to destroy these cell types in established malignancies,” explains Dr. Zon.

Rhabdomyosarcoma (RMS) is an aggressive cancer that arises from a primitive skeletal muscle cell called a “rhabdomyoblast”. Depending upon on the histology of the cancerous cells, there are several different subtypes of RMS. Embryonal rhabdomyosarcoma (ERMS) is the most common subtype, usually found in children under 15, in the head and neck region and genitourinary tract.

Dr. Zon and colleagues have developed an animal model to identify and test therapeutic targets of human ERMS. The scientists artificially activated the RAS pathways to induce ERMS in a strain of genetically engineered zebrafish. Some transgenic zebrafish developed visible tumors by 10 days of age.

Through their model, Dr. Zon and colleagues were able to identify both an ERMS tumor-cell-of-origin and a novel genetic signature that underlies ERMS progression in zebrafish and human patients. Cancer stem cells make up only a small fraction of the overall number of cells in a tumor. However, they are capable of giving rise to other cancer cells, and thereby drive tumor growth and metastasis. To prevent recurrence and progression, effective long-term therapies must target the self-renewing population of cancer stem cells.

“The zebrafish is ideally suited for use in targeted chemical genetic approaches to specifically inactivate cancer pathways we have identified in our study. Identifying drugs that inactivate these pathways in the ERMS cancer stem cell may have far reaching implications for treatment of patients with this disease.”

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Myxopapillary ependymomas (MPEs) are low-grade neuroepithelial tumors typically occurring in the conus - cauda equina - filum terminale region. Limited molecular and cytogenetic analysis of MPEs has not demonstrated consistent abnormalities. In an attempt to clarify the chromosomal status of these tumors and identify commonly aberrant regions in the genome we have combined 3 molecular/cyto/genetic methods to study 17 MPEs. Comparative genomic hybridization of 7/17 tumors identified concurrent gain on chromosomes 9 and 18 as the most frequent finding. The majority of the 17 tumors were also studied using microsatellite analysis with marker spanning the whole chromosomes 9 and 18 and interphase-FISH with centromeric probes for both chromosomes. Our combined results were consistent with concurrent gain in both chromosomes 9 and 18 in 11/17 cases, gain of either chromosome 9 or 18 and imbalance in the other chromosome in 3/17 tumors and allelic imbalances of chromosomes 9 or 18 in 3/17 and 1/17 tumors, respectively. Other abnormalities observed included gain of chromosomes 3, 4, 7, 8, 11, 13, 17q, 20, and X and loss of chromosomes 10 and 22. Our findings represent some steps towards understanding the molecular mechanisms involved in the development of MPE.

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Report of Three Cases and Review of the Literature

The clinical, microscopic, immunohistochemical, and ultrastructural features of three carcinoid tumors of the presacral region are reviewed. All tumors occurred in young women and did not involve the rectum. The predominant microscopic pattern was trabecular. The differential diagnosis included paraganglioma and myxopapillary ependymoma. Immunohistochemically, neuroendocrine markers and low molecular weight cytokeratins were expressed in all cases. Neurosecretory granules were identified in the single case studied by electron microscopy. One case was associated with a tailgut cyst (retrorectal cystic hamartoma). Two patients were treated with complete local excision and are free of disease 3 and 4 years after surgery. One case metastasized to both breasts and recurred locally after an incomplete excision. This report expands the already long list of sites where carcinoid tumors can arise. The frequent association of these tumors with tailgut cysts and their histologic similarities to rectal carcinoid tumors suggest that the most likely derivation of presacral carcinoid tumors is from hindgut rests.

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Cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells, which may belong to different lineages. Recent advances in stem cell research have demonstrated the existence of tumour-initiating, cancer stem cells (CSCs) in non-solid and solid tumours. These cells are defined as CSCs because they show functional properties that resemble those of their normal counterpart to a significant extent. This concept applies to CSCs from brain tumours and, particularly, to glioblastoma stem-like cells, which self-renew under clonal conditions and differentiate into neuron- and glia-like cells, and into aberrant cells, with mixed neuronal/astroglia phenotypes. Notably, across serial transplantation into immunodeficient mice, glioblastoma stem-like cells are able to form secondary tumours which are a phenocopy of the human disease. A significant effort is underway to identify both CSC-specific markers and the molecular mechanism that underpin the tumorigenic potential of these cells, for this will have a critical impact on the understanding of the origin of malignant brain tumour and the discovery of new and more specific therapeutic approaches. Lately, the authors have shown that some of the bone morphogenetic proteins can reduce the tumorigenic ability of CSCs in GBMs. This suggests that mechanisms regulating the physiology of normal brain stem cells may be still in place in their cancerous siblings and that this may lead to the development of cures that selectively target the population CSCs found in the patients’ tumour mass.

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For both intracranial and spinal cord primary lesions, as complete a resection as possible is attempted (8). Progression-free survival is improved if radiation therapy is given, with doses of at least 45 Gy being employed (7).

The optimal radiation volume for intracranial primary lesions is controversial. It is reasonable to consider localized radiation for those with low-grade lesions, supratentorial site, complete resections, negative MRI and no evidence of spinal seeding. One may also consider local radiation for high-grade supratentorial lesions, although no definite recommendation can be made. All others should receive craniospinal radiation, with a dose of approximately 35-40 Gy to the craniospinal axis, and a boost of approximately 15-20 Gy to the intracranial primary site and 10 Gy to drop metastases in the spine (12).

For spinal ependymomas, the use of adjuvant post-operative radiation has been advocated after less than total resection of low-grade lesions (9). However, this has recently been questioned, with some suggesting an expectant policy with possible repeat surgery when complete resection is not achieved (10). A randomized trial would be useful in this situation.

There is no indication that chemotherapy is useful in the treatment of primary ependymomas in adults.

1.                    Postoperative radiotherapy of intra-cranial ependymoma in pediatric and adult patients. Shaw EG, Evans RG, Scheithauer BC, Ilstrup DM, Earle JD. Int J Rad Onc Biol Phys 13(10):1457-62, 1987.

2.                    Improved survival in cases of intracranial ependymoma after radiation therapy. Late report and recommendations. Salazar OM, Castro-Vita H, VanHoutte P, Rubin P. J Neurosurgery 59(4):652-9, 1983.

3.                    Ependymomas: results of radiation treatment. Garrett PG, Simpson WJ. Int J Rad Onc Biol Phys 9(8):1121-4, 1983.

4.                    Intracranial ependymoma: long term results of a policy of surgery and radiotherapy. Vanuytsel LJ, Bessell EM, Ashley SE, Bloom HJ, Brada M. Int J Rad Onc Biol Phys 23(2):313-9, 1992.

5.                    Ependymoma: results, prognostic factors and treatment recommendations. McLaughlin MP, Marcus RB Jr., Buatti JM, McCollough WM, Mickle JP, Kedar A, Maria BL. Int J Rad Onc Biol Phys 40(4):845-50, 1998.

6.                    The clinical and prognostic relevance of grading in intracranial ependymomas. Ernestus RI, Schroder R, Stutzer H, Klug N. Br. J. of Neurosurgery 11(5):421-8, 1997.

7.                    Postoperative radiotherapy of spinal and intracranial ependymomas: analysis of prognostic factors. Stuben G. Stuschke M, Kroll M, Havers W, Sack H. Radiotherapy & Oncology 45(1):3-10, 1997.

8.                    Brain Tumor. Part 2 of 2. Black PM. NEJM 324(22):1555-1564, 1991.

9.                    The role of radiotherapy in the management of spinal cord glioma. Shirato H, Kamada T, Hida K, Koyanagi I, Iwasaki Y, Miyasaka K, Abe H. Int J Rad Onc Biol Phys 33(2):323-8, 1995.

10.                 Spinal ependymomas - the value of postoperative radiotherapy for residual disease control. Sgouros S, Malluci CL, Jackowski A. Br. J. of Neurosurgery 10(6):559-66, 1996.

11.                 Practice Guidelines for Brain Tumors - Draft. Krawcczyk, J., Unpublished.

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