Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma

Abstract

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

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 EFS compared to complete resection (p = 0.0001 and p ⩽ 0.001, respectively). (C and D) Posterior fossa tumors (indicated by the dashed line) have a significantly less favorable outcome in terms of OS (C; p = 0.032) and a trend toward a poorer EFS (D; p = 0.083) compared with supratentorial tumors. The distribution of tumor location was not significantly associated with resection status (p = 0.569) by Fisher’s exact test.

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); >5%, high (C, F). Original magnification, ×10.

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 tissue (C). (D–L) Tumor negative by immunostaining and with no gene amplification (D, G, and arrow in J; ratio score, 1.1), tumor exhibiting cytoplasmic staining and with no gene amplification (E, H, and arrow in K; ratio score, 1.2), and tumor demonstrating 2+ intensity by immunohistochemistry and occasional cells with ERBB2 copy number of 3, considered negative overall (F, I, and arrow in L; ratio score, 1.33). In J–L, red indicates ERBB2 probe (locus 17q11.2–q12), and green, CEP-17 probe. ERBB2:CEP-17, ratio score >2 considered amplification. (M–O) ERBB4 was absent in 66% of tumors (M), with expression detected in 34% of tumors with either moderate (N) or strong (O) expression.

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) and event-free survival (EFS; p = 0.016) for 44F12 positive tumors, as indicated by the dashed line. (E) Patients who received primary radiotherapy were stratified by 44F12 status. A poorer EFS for 44F12-positive tumors was also observed (p = 0.015), as indicated by the dashed line. The distribution of 44F12-positive and -negative tumors was not significantly associated with tumor location (p = 0.743) or resection status (p = 0.528) by Fisher’s exact test. (F and G) Tumors were grouped into low or high nucleolin expression by immunostaining. (H and I) Kaplan-Meier survival analysis identified a poorer OS (p = 0.047) and EFS (p = 0.007) for tumors with high nucleolin expression, as indicated by the dashed line. J. Patients who received primary radiotherapy were stratified by nucleolin status. A poorer EFS for tumors with high nucleolin expression was also observed (p = 0.034), as indicated by the dashed line. The distribution of high-nucleolin and low-nucleolin tumors was not significantly associated with tumor location (p = 1.000) or resection status (p = 0.745) by Fisher’s exact test.

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 to high levels of telomerase activity, as indicated by the length and intensity of the telomere repeat amplification protocol product ladder, whereas 3 of 22 (14%) patients show an absence of any detectable telomerase activity under the conditions employed. We used 0.1 μg total protein lysate for each reaction. C, CHAPS-buffer–only control. (B) Telomere length in seven recurrent cases, encompassing 19 independent tissue samples, was determined by the TeloTAGGG assay, and mean telomere restriction fragment length was deduced by comparison to a known molecular standard. Telomere length ranged from 7.2 to 16.7 kb across all samples. Telomere lengthening is evident in four of seven cases (57%; patients 1, 2, 4, and 7), with a rapid telomere lengthening rate observed in patients 2 and 7 (see Table 3). Telomere maintenance was observed in patient 5, and telomere shortening was observed in patients 3 and 6. (C) To further validate this phenomenon of telomere lengthening, digested DNA was separated using pulsed-field gel electrophoresis (PFGE). The pattern of telomere dynamics observed using PFGE was consistent, with considerable telomere lengthening confirmed in patients 2 and 7.