Supratentorial ependymomas in children: Analysis of nine cases.

BACKGROUND: Intracranial ependymomas are the third most common primary brain tumor in children. In the present study, we set out to investigate the expression of p-53, p-27, bcl-2, epidermal growth factor receptor (EGFR) and of neuronal markers in pediatric supratentorial ependymomas, in correlation with Ki-67/MIB-1 proliferation index and prognosis.
MATERIALS AND METHODS: Nine children with supratentorial ependymomas that were treated surgically in our institute over the last seven years were identified and included in the study. The extent of resection was classified as gross total and subtotal, and was determined by MRI scans. The ependymal tumors were classified according to WHO classification.
RESULTS: Headache and seizures were the most common presenting symptoms and papilledema the most common sign. In seven cases, gross total excision was performed, and in two cases, the resection was subtotal. All ependymomas were anaplastic. Ki-67/MIB-1 was detected in 20-40% of the nuclei in all tumors. There was also increased expression of p-53, bcl-2, p-27, and EGFR. There was expression of neuronal markers in three cases. After a mean follow-up period of 32.1 months (range 16-74 months), eight children were alive. Five children suffered from tumor recurrence.
CONCLUSIONS: Complete surgical excision should be the goal of surgery. The prognostic role of Ki-67, p-53, p-27, bcl-2, EGFR, and neuronal markers expression needs to be determined in multi-institutional studies due to tumor's rarity.

Introduction

Intracranial ependymomas are the third most common primary brain tumor in children and are usually located in the posterior fossa.[12] Supratentorial ependymomas are rare.[2] Recent data suggested that these tumors may have different biological and clinical behaviors according to their location.[3] To date, many studies group together infratentorial and supratentorial ependymomas, whereas some studies of supratentorial ependymomas frequently include adult population. Besides that, data on the expression of proliferation and apoptosis regulators in supratentorial ependymomas are scarce.[4] In the present study, we retrospectively studied supratentorial ependymomas in children in conjugation with Ki-67, p-53, p-27, bcl-2, EGFR, and neuronal markers expression.

Materials and Methods

We retrospectively studied supratentorial ependymomas that were treated surgically in our institute over the last seven years. The extent of resection was classified as gross total and subtotal and was determined by comparing MRI scans obtained before surgery with those after resection. The ependymal tumors were classified according to World Health Organization. Progression-free survival (PFS) was defined as the time from the initial surgery to the date of evidence of tumor progression confirmed radiologically. Overall survival (OS) was calculated as the time from surgery to death or as the time to the last follow-up appointment of the surviving patients. The study was approved from the institutional review board.

Immunohistochemical analysis

Four-micrometer-thick, formalin-fixed, paraffin-embedded tissue sections were immunostained using the streptavidin-biotin-horseradish peroxidase (HRP) method (Supersensitive Multilink Kit QD0005L; Biogenex, San Ramon, CA, USA). Deparaffinized sections were rehydrated through graded series of alcohol and then microwaved in 0.1 mol/l sodium citrate buffer solution (pH 6.0) for 3 × 5 min at 450 W to unmask antigen epitopes. After treatment with 3% hydrogen peroxide for 5 min to block endogenous peroxidase, the sections were subsequently incubated with the primary antibodies [Ki-67/MIB-1 (clone MIB-1, dilution 1/50, DAKO, Denmark), p-53 (clone DO-7, dilution 1/50, DAKO, Denmark), bcl-2 (clone 100/D5, dilution 1/50, Novocastra/Leica, UK), p-27 (clone 1B4, dilution 1/20, Novocastra/Leica, UK), EGFR (clone epidermal growth factor receptor 25)], Synaptophysin (clone SY38, Monosan, Sanbio), Neurofilaments [clone 2F11 (70 + 200 kda), Monosan, Sanbio], Neu-N (clone A60, Chemicon, Millipore), and b-tubulin III [clone TU-20, serotec) for 50 min at room temperature. Afterward, the sections were incubated with ready-to-use biotin-labeled secondary antibody and streptavidin peroxidase for 20 min each. Tissues were then stained with 0.05% 3,3-diaminobenzidine tetrahydrochloride diluted in DAB substrate and then counterstained with hematoxylin, dehydrated and mounted. Tris buffer solution (pH 7.6) was used for rinsing the sections between incubation steps and the dilutions of primary antibodies. The immunohistochemical expression of Ki-67, p-53, bcl-2, p-27, and EGFR were evaluated by two independent experienced neuropathologists. Results were expressed as the percentage of positive tumor cells out of the total number of counted cells (approximately 3,000 counted cells) in the highest density of stained areas. All cells with staining of any intensity were considered positive, irrespectively of staining intensity. Any discrepancy between the two physicians was solved by consensus.

Results

Nine children (3 males, 6 females, mean age 6.9 years, range 10 months to 13 years) were identified and included in the study. In eight cases, the tumor was lobar without relation to the ventricular system, and in one case, the tumor was intraventricular. Headache and seizures were the most common presenting symptoms and papilledema the most common sign. The mean symptom duration was 8.3 weeks. Table 1 summarizes patients′ presenting symptoms, tumor localization, size, histology, time to recurrence, and follow-up. In seven cases, gross total excision was performed, and in two cases, the resection was subtotal [Figure 1]. Postoperative in one case, there was mild left-sided hemiparesis that gradually resolved and simple partial seizures that were medically controlled. All ependymomas were anaplastic, and all patients received local radiotherapy and adjuvant chemotherapy, apart from one child under the age of three that received chemotherapy alone. Ki-67/MIB-1 was detected heterogeneously in 20–40% of the nuclei in all tumors. P-53 protein was also detected in all ependymomas (range 20–30%), showing a parallel with Ki-67 expression. Bcl-2 protein was also detected in all tumors (range 5–50%). There was expression of p-27 and EGFR in all stained cases. There was expression of neuronal markers in three cases. Table 2 summarizes patients′ immunohistochemical data. After a mean follow-up period of 32.1 months (range 16–74 months), eight children were alive. The deceased patient died because of respiratory infection. Five children suffered from tumor recurrence. Three local relapses were treated by surgery with chemotherapy and two with stereotactic radiotherapy. The mean time for recurrence from the original operation was 23.4 months. All five children achieved disease remission.

Table 1

Detailed patients’ data

Figure 1

(a) Coronal gadolinium-enhanced T1-weighted MRI revealing an heterogenous mass. (b) Axial T2-weighted MRI showing that the tumor is largely heterogeneous with hypointense areas representing calcifications and hyperintense areas representing necrosis. (c) Preoperative and postoperative (d) Sagittal gadolinium-enhanced T1-weighted MRI revealing gross total excision

Table 2

Immunohistochemical expression of Ki-67, p53, p27, bcl-2, EGFR, and neuronal markers

Discussion

Ependymomas constitute 6–12% of all pediatric brain tumors and are the third most common brain tumor after astrocytomas and medulloblastomas.[12] They are usually located in the posterior fossa. Supratentorial ependymomas are less frequent and are usually not related to the ventricular system. They are frequently located adjacent to the cortical surface similar to our case and usually present due to symptoms of raised intracranial pressure.[4] Recent evidence suggested that supratentorial and infratentorial ependymomas have different behavior. Andreiuolo et al. found a significant overexpression of neuronal markers in supratentorial compared with infratentorial ependymomas. A strong neurofilament light polypeptide 70 expression in supratentorial ependymoma was correlated with better progression-free survival.[3] In the present study, neuronal markers were present in 3/9 ependymomas, only one of which recurred. In another study, genetic aberrations were found significantly more often in supratentorial tumors than in tumors with infratentorial location.[5]

In general, patients with supratentorial ependymomas have a better survival rate than patients with posterior fossa ependymomas, because gross total resection is more commonly achieved.[6] The 5-year survival rate for complete resection ranges from 86% to 66%, whereas for subtotal resection is 75% to 11%.[78] Nevertheless, in the present study, there were five recurrences within a mean period of 23.4 months. Patients younger than three years have also a significantly worse outcome than older children. Besides that, duration of symptoms before diagnosis has been suggested to have a prognostic value. Tumors with more indolent course tend to have a better prognosis. Pollack et al. reported that patients with a duration of symptoms before diagnosis of less than 1 month had a worse outcome.[9] This was not verified in our study. Radiotherapy is usually reserved for incomplete excised or anaplastic tumors similar to our case.[1011] Venkatramani et al. recently reported that surgery alone may be an acceptable treatment option in completely resected supratentorial ependymoma. Nevertheless, this study included only 10 patients, and complete excision was achieved in six patients.[6] Kumar et al. studied 16 cases of supratentorial anaplastic ependymoma and found that gross total excision and postoperative irradiation are not effective in preventing early recurrences.[10]

The prognostic significance of ependymoma grade is dubious. Thus, a better understanding of cell-intrinsic mechanisms and pathways involved in tumor growth may lead to better tumor classification and prognosis assessment. In a previous study, we found high Ki-67, p-27, and EGFR expression in infratentorial ependymomas.[2] P-27 is a member of the cip/kip family of cyclin-dependent kinase (CDK) inhibitors that negatively control the cell cycle progression. High p-27 expression suggested that p-27 may be rendered non-functional through interaction with other cell cycle regulator proteins. Furthermore, EGFR has a crucial role in tumor proliferation and spread by promoting cell cycle. High EGFR expression has been associated with adverse prognosis.[12] EGFR may also serve as a therapeutic target. In this study, there was increased EGFR expression in nearly all stained cases. P-53 and bcl-2 expression has been also correlated with tumor′s aggressiveness.[13] In our study, due to the limited number of cases, no correlation with patient′s prognosis could be made.

To conclude, ependymomas should be included in the differential diagnosis of large heterogenous supratentorial lesions. Complete surgical excision should be the goal of surgery. Nevertheless, even after complete excision, close follow-up is mandatory. The prognostic role of Ki-67, p-53, p-27, bcl-2, and EGFR needs to be determined in multi-institutional studies due to tumor′s rarity.