Following histological review (M.-A.B., D.E.) of the initial cohort of 114 cases, 10 had incorrect diagnosis and 7 had tissue processing errors beyond our control. A total of 97 intracranial tumors from 74 patients were included in the present study. Mean and median age at diagnosis was 5.4 and 3.8 years, respectively (range, 8 months to 14.9 years). Patient clinical features are summarized in .
Univariate analysis of clinical and biological factors on 74 primary intracranial ependymomas
Nine of 26 patients (35%) who had complete resection relapsed with recurrent disease. In total, 35 patients (47%) relapsed (mean and median time to first event, 2.5 and 2.2 years, respectively). Twenty-three recurrent tumors from 19 patients were included in the study. Thirty-eight patients (51%) were alive, with mean and median follow-up times of 8.3 and 6.3 years, respectively (range, 2.0–28.9 years), and 36 patients (49%) had died, with mean and median survival times of 3.7 and 2.9 years, respectively (range, 1.2 months to 13.3 years). Five-year OS and EFS were 56% ± 6% and 37% ± 6%, respectively.
Incomplete resection of the primary tumor was significantly associated with a worse OS and EFS (p
= 0.001 and p
0.001, respectively; ). Posterior fossa location was significantly associated with a worse OS and showed a tendency toward a worse EFS (p
= 0.032 and p
= 0.083, respectively; ).
Fig. 1 (A and B) Kaplan-Meier survival analysis of clinical factors for overall survival (OS) and event-free survival (EFS) for resection status. Incomplete resection (indicated by the dashed line) has a significantly less favorable outcome in terms of OS and (more ...)
Adjuvant therapy was administered to 65 of 72 patients. Eighteen patients received radiotherapy alone, 11 had chemotherapy alone, and 36 had a combination of radiotherapy and chemotherapy. Treatment information was unavailable for two patients. Adjuvant chemotherapy (n = 47) did not confer a survival advantage in terms of OS and EFS (p = 0.188 and p = 0.656, respectively). Five-year OS for irradiated patients (n = 54) was 61% ± 7%, which although not significant (p = 0.166), did show a trend toward a more favorable OS compared to nonirradiated patients (n = 18; 5-year OS, 43% ± 12%). Comprehensive radiotherapy data were available for 55 patients. Patients who were electively irradiated as part of primary treatment (n = 29) versus those who did not receive radiotherapy or had delayed radiotherapy (i.e., following the first event; n = 26) had a significantly more favorable EFS (p = 0.039) but not OS (p = 0.47). Other clinical factors, including gender, age <3 years at diagnosis, and histology, were not associated with a worse outcome.
Overall, 10% (range, 9%–15%) of cases were excluded from immunohistochemical analysis due to core dropout, nonviable tumor, or <50% intact cores following processing. Expression levels of the biological factors are summarized in .
Sixty-seven primary tumors (100%) demonstrated Ki-67 staining, with mean and median LIs of 2.9% and 1.0%, respectively (range, <1% to 27%). Tumors were categorized into low (
1%), intermediate (2%–4%), or high (
5%) Ki-67 expression levels. Thirty-five (52%) tumors exhibited low Ki-67 expression, 22 (33%) intermediate, and 10 (15%) high (). Univariate analysis did not show Ki-67 levels to be a contributor to worse OS or EFS when considering low versus high, low versus intermediate/high (OS, p
= 0.132; EFS, p
= 0.792), and low/intermediate versus high (). Stratification of Ki-67 by tumor location also showed no prognostic significance for posterior fossa tumors (OS, p
= 0.184; EFS, p
= 0.547). Data from 82 primary and recurrent tumors showed higher Ki-67 LI values correlated with grade III tumors (p
= 0.02). The mean Ki-67 expression level for grade II and III tumors was 2.2% ± 0.6% and 4.3% ± 0.8%, respectively.
Fig. 2 Positive Ki-67 (A–C) and survivin (D–F) immunostaining was categorized into three expression groups based on the labeling index, as indicated by positive immunostained nuclei: <1%, low (A, D); 2–4%, intermediate (B, E); (more ...)
Sixty-five primary tumors (100%) demonstrated survivin expression, with mean and median LIs of 1.1% and 0.5%, respectively (range, <1% to 6%). Tumors were categorized into low (
1%), intermediate (2%–4%), and high (
5%) survivin expression levels (). Fifty-one tumors (79%) exhibited low expression, 11 intermediate (17%), and 3 high (4%). A strong correlation was observed between survivin and Ki-67 LI values (R2
= 0.753, p
Higher survivin LI also correlated with grade III tumors (p
0.001) but was of no prognostic value when considering low versus high, low versus intermediate/high (OS, p
= 0.110; EFS, p
= 0.360), and low/intermediate versus high (). Stratification of survivin by tumor location also showed no prognostic significance for posterior fossa tumors (OS, p
= 0.363; EFS, p
RTK-I (ERBB) Family
Sixty-two tumors (98%) were negative for EGFR protein expression, and one tumor (2%) was positive (2+ intensity) (). Because EGFR was detected in only one case, further analysis was not carried out.
Fig. 3 RTK-I family expression. (A–C) epidermal growth factor receptor–negative immunostaining was identified in 98% of tumors (A), 2+ intensity positive staining was detected in one tumor (B), and 3+ intensity was achieved in hepatocyte control (more ...)
Three different expression groups were observed for ERBB2: (1) negative staining, (2) cytoplasmic staining including either an epithelioid “dot-like” pattern or diffuse granular staining, and (3) membranous staining of 2+ intensity (). Analysis of ERBB2, when adhering to the FDA scoring criteria, identified 2 positive (2+ membranous staining; 3%) and 64 (97%) negative tumors. Forty-seven corresponding primary tumors screened for ERBB2 with the Dako HercepTest gave good concordance with the results when adhering to the FDA scoring criteria, confirming the two positive cases. When considering cyt-ERBB2 staining, 22 tumors (34%) exhibited immunopositivity with the ERBB2 antibody. Comparatively, with the HercepTest, of 15 representative positive cyt-ERBB2 cases demonstrated with the ERBB2 antibody, 10 (67%) showed similar staining patterns, and 5 (33%) were negative (). Reliability between the two techniques was good (κ = 0.63, p
0.001). The results with the ERBB2 antibody were confirmed by independent analysis (data not shown).
Gene copy number in whole-tissue sections from seven representative cases from the ERBB2 immunohistochemistry was determined by FISH. Despite a small number of tumor cells with a gene copy number of 3 for ERBB2, the two tumors that demonstrated 2+ membranous staining were considered negative by FISH (ratio scores, 1.28 and 1.33). No gene amplification was observed in two ERBB2-negative tumors or in the three cases that exhibited cyt-ERBB2 expression (ratio score range, 1.1–1.37; ).
Univariate analysis on the FDA-scored ERBB2 expression data was not possible in this cohort due to the small number of positive cases (n = 2); however, additional consideration with cyt-ERBB2, demonstrated in 22 cases (34%), did not contribute to a worse outcome compared to negative tumors (OS, p = 0.179; EFS, p = 0.246).
Three groups of cytoplasmic staining were observed for ERBB4 expression, categorized into negative, moderate, and strong. Forty-four (66%) tumors were negative for ERBB4 expression, and 23 (34%) exhibited moderate/strong expression (). Kaplan-Meier survival analysis showed that moderate/strong ERBB4 expression was not a predictor of outcome when compared to negative expression (OS, p = 0.380; EFS, p = 0.458; ). When considering coexpression of cyt-ERBB2 with ERBB4 and/or cases with high Ki-67 levels, no prognostic significance was found.
From 65 primary tumors, 52 (80%) were positive for 44F12 and 13 (20%) were negative. Initially, Fisher exact tests determined no association of 44F12 expression with clinical factors, including tumor location (p = 1.0), histology (p = 0.51), resection status (p = 0.53), and age<3 years at diagnosis (p = 0.341). Kaplan-Meier survival analysis showed that 44F12-positive tumors contributed to both a worse OS and a worse EFS (p = 0.015 and p = 0.016, respectively; , ). Five-year OS and EFS were 92% ± 7% and 77% ± 12%, respectively, for 44F12-negative tumors and 53% ± 8% and 30% ± 7% for 44F12-positive tumors. Stratification of radiotherapy status by 44F12 status was also analyzed for 51 patients. Of the 25 of 51 patients (49%) who received primary radiotherapy, the 5-year EFS for 44F12-positive tumors (n = 21) and 44F12-negative tumors (n = 4) was 28% ± 11% and 100% ± 0%, respectively (p = 0.015; ). All of the patients who received primary radiotherapy with 44F12-negative tumors are alive and event free, with mean and median follow-up times of 13 ± 5.1 and 12.7 years, respectively.
Fig. 4 Immunohistochemical detection of 44F12 (A–E) and nucleolin (F–J). (A and B) Tumors were grouped as 44F12 negative or positive by immunostaining. (C and D) Kaplan-Meier survival analysis identified a poorer overall survival (OS; p = 0.015) (more ...)
Nucleolin protein expression was evident in 64 (100%) primary tumors with a mean LI of 68% (range, 3.0%–92%). Two distinct groups were observed, with 50% LI used as the cutoff to distinguish between high and low expression. Fifty-two cases (81%) demonstrated high expression (mean LI, 75% ± 1.5%), and 12 cases (19%) demonstrated low expression (mean LI, 35% ± 3.6%). Initially, Fisher exact tests determined no association of nucleolin expression with clinical factors, including tumor location (p = 1.0), histology (p = 0.51), resection status (p = 0.53), and age at diagnosis (p = 0.34). Kaplan-Meier survival analysis showed that high nucleolin expression contributed to a worse OS and EFS (p = 0.047 and p = 0.007, respectively; , ). Five-year EFS for high and low nucleolin expression was 31% ± 7% and 74% ± 13%, respectively. Furthermore, 10-year EFS for high and low nucleolin expression was 28% ± 7% and 74% ± 13%, respectively. Of those patients that were 44F12 negative and exhibited low expression of nucleolin (n = 5), all were alive and event-free, with a mean follow-up time of 9.2 years (range, 3.6–21.0 years). We next sought to stratify resection and radiotherapy status by nucleolin expression. Paired data of nucleolin expression and resection status were available for 59 primary tumors. For completely resected primary tumors demonstrating low nucleolin expression (n = 5), the majority of patients (80%) were event-free with mean and median follow-up times of 8.9 ± 4.1 and 5.5 years, respectively (range, 3.6–21.0 years; 5-year EFS, 75% ± 22%). Ten of the 19 completely resected cases with high nucleolin expression (47%) relapsed, with a mean and median times to progression of 4.0 ± 1.3 and 2.7 years, respectively (range, 0.2–13.3 years; 5-year EFS, 56% ± 12%). For incompletely resected primary tumors demonstrating high nucleolin expression (n = 29), the majority of these cases (90%) relapsed, with mean and median times to progression of 2.3 ± 0.6 and 1.7 years, respectively (range, 0.1–12.4 years; 5-year EFS, 17% ± 7%). For incompletely resected primary tumors demonstrating low nucleolin expression (n = 6), the majority of cases (83%) were event-free, with mean and median follow-up times of 6.0 ± 1.7 and 4.4 years, respectively (range, 3.4–12.8 years; 5-year EFS, 83% ± 15%).
Patients who received primary radiotherapy and who had tumors demonstrating high or low nucleolin expression (n = 22) had a 5-year EFS of 23% ± 11% and 70% ± 18%, respectively (p = 0.034; ). Five of seven (71%) irradiated patients demonstrating low nucleolin expression were event-free, with mean and median follow-up times of 7.7 ± 3.3 and 4.4 years, respectively (range, 3.9–21.0 years). By contrast, 12 of 15 (80%) irradiated patients with high nucleolin expression relapsed, with mean and median times to progression of 3.2 ± 0.89 and 2.3 years, respectively (range, 1.1–12.4 years). Additionally, 16 of 22 (73%) nonirradiated patients demonstrating high nucleolin expression relapsed, with mean and median times to progression of 1.2 ± 0.27 and 0.9 years, respectively (range, 0.1–3.1 years). Analysis of nonirradiated patients with low nucleolin expression could not be carried out because the numbers were too small.
Multivariate analysis of clinical and biological covariates (histology, tumor location, resection status, primary radiotherapy, age <3 years at diagnosis, Ki-67 LI, survivin LI, and nucleolin expression) identified incomplete resection (p = 0.001) and high nucleolin expression (p = 0.008) as the only factors to independently predict a worse EFS ().
Cox regression multivariate analysis of clinical and biological factors
Paired data for 17 primary tumors and their first recurrence revealed that mean Ki-67 in primary tumors was 3.3% ± 1.5% compared to 7.0% ± 2.8% in recurrent tumors. A similar pattern was observed for survivin LI, with a mean of 1.0% ± 0.2% for the primary tumors compared to 5.1% ± 3% in recurrent tumors.
Of the 18 patients with 44F12 data on primary and subsequent relapse tumors, only two primary tumors (11%) were negative, and eventually both patients progressed to positive status, showing that ultimately 100% of recurrent tumors were 44F12 positive.
As with primary patients, all recurrent samples (100%) were positive for nucleolin. Among 12 patients with paired data on primary and relapse tumors, two (17%) had low expression in the primary and first recurrence, but in both cases the expression levels subsequently progressed to high, showing again that, ultimately, 100% of recurrent samples expressed high levels of nucleolin.
Telomerase repeat addition processivity assay (TRAP) was performed on 22 representative tissue samples. Positive TRAP products were evident in 19 tumors (86%), indicative of a high level of telomerase repeat addition processivity (). Telomerase activity was evident in all recurrent tumors analyzed by TRAP and in 11 of 14 primary tumors. There was a good agreement between telomerase activity and 44F12 detection (κ = 0.59, p = 0.02). Of six tumors negative for 44F12 detection, three (50%) were positive for telomerase activity. The reverse was never observed; of all telomerase-negative tumors, none were 44F12 positive.
Fig. 5 Telomerase activity and telomere length in ependymoma tissue from a pediatric cohort. (A) Telomere repeat amplification protocol assay indicates variable levels of telomerase activity in pediatric ependymoma patients; 19 of 22 (86%) patients have moderate (more ...)
To determine telomere length alterations, mean telomere restriction fragment (TRF) length was calculated in seven patients with recurrent tumors (data set consisting of 6 primary tumors and 15 recurrences). Mean telomere length ranged from 7.3 to 16.7 kb, with telomere maintenance observed in five of seven patients (71%). Of these five cases, four showed telomere lengthening in relapsed tumors compared to the primary tumor (, ). A moderate increase in mean telomere length was observed in tumors T1–T2 and T9–T12 (7.3–7.6 kb and 9.2–10.3 kb, respectively). In contrast, substantial telomere lengthening was evident in tumors T3–T4 and T19–T20 (9.5–16.7 kb and 7.2–15.9 kb, respectively). Because time to recurrence was 2.9 and 2.8 years, respectively, a rapid telomere lengthening rate is inferred for these latter cases.
Summary of clinical factors and telomere status of recurrent tumors
However, telomere shortening occurred in two of seven recurrent cases (29%); in tumors T5–T8, telomeres shortened from 15.3 to 8.4 kb, and in tumors T16–T19, telomeres shortened from 14.8 to 8.1 kb (, ). Of note, T16–T19 represent tumor tissue from the oldest patient in this cohort, with final recurrence to date occurring at age 22.
We observe variability with respect to the status of the telomere and present evidence for telomere maintenance in the majority of pediatric ependymoma recurrent cases (71%), with telomere shortening evident in a minority.