Survivin Expression in Intracranial Ependymomas and Its Correlation With Tumor Cell Proliferation and Patient Outcome

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1 Anatomic Pathology / SURVIVIN EXPRESSION IN EPENDYMOMA Survivin Expression in Intracranial Ependymomas and Its Correlation With Tumor Cell Proliferation and Patient Outcome Matthias Preusser, MD, 1 Stefan Wolfsberger, MD, 2 Thomas Czech, MD, 2 Irene Slavc, MD, 3 Herbert Budka, MD, 1 and Johannes A. Hainfellner, MD 1 Key Words: Apoptosis; Ependymoma; Proliferation; Prognosis; Survivin DOI: /PP2G5GAAFKV82DTG Abstract Survivin expression has been described as a prognostic factor in various tumor types and has been shown to correlate with cytologic anaplasia in ependymoma. We immunohistochemically studied survivin expression and its association with Ki-67 and topoisomerase IIα (TIIα) expression and outcome in 63 patients with intracranial ependymoma. Survivin is expressed in a fraction of nuclei of tumor and endothelial cells including mitotic figures. Survivin indices range from 0.6% to 43.2% and correlate with Ki-67 and TIIα indices. On average, 62.86% of Ki-67 expressing tumor cell nuclei coexpress survivin, whereas 92.2% of survivin-expressing nuclei coexpress Ki-67. High survivin, Ki-67, and TIIα indices univariately correlated with an unfavorable outcome. In multivariate analysis, only the Ki-67 index remained an independent prognostic factor. In ependymoma, survivin is expressed in a subset of proliferating cells. High survivin expression is associated with poor patient outcome. However, the Ki-67 index is a more accurate prognostic marker than the survivin or TIIα index. Survivin is a recently identified protein that acts as an inhibitor of apoptosis by preventing caspase-9 activation within a functional apoptosome. In addition, survivin regulates mitosis 1,2 by stabilizing microtubules in metaphase spindle formation and regulation of the spindle assembly checkpoint. 3 High expression of survivin has been associated with an unfavorable prognosis in a large number of malignant neoplasms 4-13 and also has been described as a marker of favorable prognosis in some tumor types In a recent study, Preusser et al 17 showed that survivin is expressed in proliferating tumor cells in glioblastoma. In ependymoma, loss of nuclear survivin expression was reported to associate with cytologic anaplasia. 18 In a recent study, Wolfsberger et al 19 showed that the Ki- 67 immunolabeling index is an accurate predictor of outcome in patients with intracranial ependymoma. Considering the role of survivin as regulator of mitosis, its expression might be associated with tumor cell proliferation and patient outcome in ependymoma. To test the prognostic accuracy of survivin in relation to other tumor cell proliferation markers, we revisited the ependymoma series of Wolfsberger et al 19 and systematically analyzed survivin expression and its correlation with tumor cell proliferation markers Ki-67 and topoisomerase IIα (TIIα). Furthermore, we analyzed the impact of survivin expression on patient outcome. Materials and Methods Patients In a previous study, Wolfsberger et al 19 analyzed a cohort of 103 patients who underwent the first operation for intracranial Am J Clin Pathol 2005;124: DOI: /PP2G5GAAFKV82DTG 543

2 Preusser et al / SURVIVIN EXPRESSION IN EPENDYMOMA ependymoma between December 1957 and October Of the 103 cases, 40 were excluded from the present study because material was no longer available (5 cases) or owing to methodological difficulties (35 cases; for more detailed information, see Statistical Analysis in the Results section). Patients with survival times less than 30 days (n = 5) were excluded from survival analyses to exclude deaths due to postoperative complications. The patients underwent the first operation between 1957 and 2000 at the University Hospital of Vienna, Vienna, Austria. For statistical comparisons (see Statistical Analysis ), we subdivided our cohort into 3 time-related groups: (1) operation in 1979 or earlier; (2) operation from 1980 through 1990; and (3) operation in 1991 or later. All tumor specimens were reevaluated histologically by a neuropathologist (J.A.H.) and classified as ependymoma (grade II, 44 cases; grade III, 19 cases) according to the current World Health Organization definition. 20,21 In the patient subgroup younger than 2 years old, 8 tumors were infratentorial and 1 was supratentorial. In patients between 2 and 18 years, 16 tumors were infratentorial and 11 were supratentorial. In patients 18 years or older, 8 tumors were infratentorial and 19 were supratentorial. Of the 63 patients, 37 (59%) underwent gross total resection and 25 patients (40%), subtotal tumor resection. In 1 case (2%), no information on the extent of resection was available. For 16 patients (25%), adjuvant chemotherapy and radiotherapy were given; 25 patients (40%) received only radiotherapy and 1 patient (2%) received only chemotherapy. No adjuvant therapy was given to 20 patients (32%), and for 1 patient (2%), insufficient information about adjuvant therapy was available. Immunohistochemical and Immunofluorescent Staining Specimens were fixed routinely in 4% formalin, embedded in paraffin, and cut at a thickness of 3 µm. Sections were deparaffinized in xylene. For antisurvivin immunostaining, sections were incubated for 1 hour with S2367 TargetRetrieval Solution, ph 9.0 (DAKO, Glostrup, Denmark) in a steamer. For anti Ki-67 and anti-tiiα immunostaining, slides were heated in 0.01 mol/l of citrate buffer (ph 6.0) for 30 minutes in a microwave oven at 600 W. We used the following antibodies for immunostaining: antisurvivin FL-142 (polyclonal rabbit, sc-10811, dilution 1:300, overnight incubation, 4 C; Santa Cruz Biotechnology, Santa Cruz, CA), anti Ki-67 (monoclonal mouse, clone MIB-1, dilution 1:50, 25 minutes, room temperature; DAKO), and anti-tiiα (monoclonal mouse, clone JH2.7, dilution 1:20, 60 minutes, room temperature; Neomarkers, Fremont, CA). Detection of immunostaining was performed using the EnVision kit (DAKO), and diaminobenzidine was used as the chromogen. A tissue sample from a brain metastasis of malignant melanoma was used as a positive control sample for antisurvivin immunostaining. Negative control samples included omission of the primary antisurvivin antibody and nonneoplastic central nervous system tissue samples. For Ki-67 survivin double immunolabeling, we used the EnVision kit and diaminobenzidine as the chromogen for survivin. For Ki-67, we used biotinylated antimouse antibody (DAKO) and streptavidin alkaline phosphatase (DAKO) with fast blue (0.5%, minutes) as the chromogen. For Ki-67 survivin nucleic acid and TIIα survivin nucleic acid triple immunofluorescent staining, the fluorescent-labeled secondary antibody for anti Ki-67 and anti- TIIα was Alexa Fluor 488 goat antimouse IgG (dilution 1:200; Molecular Probes, Eugene, OR). For survivin, we used Alexa Fluor 633 goat antimouse IgG (dilution 1:200; Molecular Probes). Nuclei were labeled using the Sytox Orange Nucleic Acid Stain (S-11368, Molecular Probes) according to the manufacturer s instructions. For Ki- 67 TIIα survivin triple immunofluorescent staining, the secondary antibody for TIIα was Alexa Fluor 546 goat antimouse IgG (dilution 1:200; Molecular Probes). Immunofluorescent labeling was evaluated using a Zeiss LSM 510 confocal laser microscope (Carl Zeiss, Vienna, Austria). We used argon 488-nm and helium/neon 546-nm and 633-nm lasers to elicit fluorescence. Evaluation For the assessment of survivin, Ki-67, and TIIα immunolabeling, 500 tumor cell nuclei were evaluated in each specimen in fields showing the highest density of immunopositive nuclei. The fraction of labeled tumor cell nuclei was expressed as a percentage (survivin, Ki-67, or TIIα index). In 5 cases, double staining for survivin and Ki-67 was performed for evaluation of the fraction of survivin-coexpressing nuclei per 500 Ki-67 expressing nuclei and of the fraction of Ki-67 coexpressing nuclei per 500 survivinexpressing nuclei. Statistical Analysis The Spearman coefficient of correlation, χ 2 test, Wilcoxon signed rank test, and Fisher exact test were used as appropriate. Survival probabilities were computed as outlined by Kaplan and Meier. 22 The log-rank test and Cox proportional hazards model were used for univariate and multivariate analyses of overall survival. Overall survival was defined from the day of initial surgery until death of the patient. Death due to a cause other than ependymoma or survival until the end of the observation period were considered as censoring events in statistical survival analysis. The median was defined as the cutoff value between low and high survivin, Ki-67, and TIIα indices. The survivin, Ki-67, and TIIα indices were entered into Cox regression. For all tests, a 2-tailed P value of.05 or less was considered significant. 544 Am J Clin Pathol 2005;124: DOI: /PP2G5GAAFKV82DTG

3 Anatomic Pathology / ORIGINAL ARTICLE Results Immunohistochemical and Immunofluorescent Staining Antisurvivin immunostaining was performed on a total of 98 cases in which anti Ki-67 and anti-tiiα yielded satisfactory staining results. In 35 of 98 cases, survivin could not be visualized properly (no immunoreactive structures including unstained mitotic figures), apparently owing to material or methodological reasons. (These were mainly older blocks; for more details, see the following Statistical Analysis section). These cases were excluded from further analyses. In 63 of 98 cases used for further analysis, survivin expression was prominent in a fraction of nuclei of tumor cells Image 1A and endothelial cells Image 1B throughout the specimen. Antisurvivin labeled all mitotic figures Image 1C, Image 1D, Image 1E, and Image 1F. Faint cytoplasmic expression was seen in a few tumor cells. There was no antisurvivin staining of surrounding nonneoplastic central nervous system tissue. Quantitative results for survivin, Ki-67, and TIIα expression are summarized in Table 1. Triple immunofluorescent staining and confocal microscopy showed that Ki-67, TIIα, and survivin were coexpressed in a fraction of tumor cell nuclei (Image 1C-1F) Image 1G, Image 1H, Image 1I, and Image 1J. Double immunolabeling analysis of 5 cases showed that a mean of 62.9% (range, 43.8%-76.6%) of Ki-67 expressing tumor cell nuclei coexpressed survivin. In contrast, a mean of 92.2% (range, 86.6%-96.8%) of survivin-expressing nuclei coexpressed Ki-67. Statistical comparison of Ki-67 and survivin index levels including all cases showed that the Ki-67 index was consistently higher than the survivin index (P <.0001; Wilcoxon signed rank test). Statistical Analysis The median patient age at surgery was 11.3 years (range, years). The mean survival time was 7.7 years for patients with grade II tumors and 3.2 years for patients with grade III tumors. Of 63 patients, 31 (49%) died of disease, and 32 patients (51%) were lost to follow-up or died of causes other than ependymoma. There was a significantly higher number of cases that had to be excluded for material or methodological reasons (see also earlier text) during the period before 1979 than in the later periods (P =.001; χ 2 test). So survivin immunodetectability seems to be affected by tissue processing factors in earlier times or duration of tissue sample storage. High survivin (P =.003; Fisher exact test), high Ki- 67 (P =.018; Fisher exact test), and high TIIα (P =.028; Fisher exact test) indices are associated significantly with tumor grade III. The survivin index showed significant correlation with the Ki-67 and TIIα indices Figure 1. Statistical comparison of the Ki-67 and survivin index levels showed that the Ki-67 index was consistently higher than the survivin index (P <.0001; Wilcoxon signed rank test). The survivin, Ki-67, and TIIα indices correlated significantly with overall survival in univariate analysis (Table 1, Figure 1). In multivariate analysis, only the Ki-67 index remained an independent prognostic factor (Table 1). There was no statistically significant difference in survival times between patients with supratentorial vs infratentorial tumors (P =.8357; log-rank test), patients in different age groups ( 2 years, 2-18 years, >18 years; P =.3135; log-rank test), or patients treated during different periods (P =.3534; logrank test). Discussion Immunohistochemical analysis of survivin expression in ependymoma was performed in 2 previous studies on small series of cases. 18,23 In 1 study, survivin expression was described in the cytoplasm but not in the nuclei of tumor cells. 23 In contrast, we observed that immunostaining signals are particularly prominent in tumor cell nuclei and barely visible in the cytoplasm. In vitro studies have shown the presence of survivin in the cytoplasm, cell nuclei, and mitotic apparatus. 2 Different antisurvivin antibodies bind more strongly to cytoplasmic survivin and others to nuclear survivin. We used antibody sc (Santa Cruz Biotechnology), which has been proven to specifically detect survivin in brain tissue by immunohistochemical analysis and Western blotting. 18 It has been described to bind predominantly to nuclear survivin. 18 So one explanation for the discrepancies of our results with previous findings in ependymoma might be the use of different antibodies. Another study on pediatric ependymomas and choroid plexus tumors reported that nuclear expression of survivin correlated with morphologic tumor grade. 18 Cases with high tumor grade had a lower fraction of survivin-expressing tumor cells than did low-grade tumors. In contrast, we found in our larger series a significant correlation of a high survivin index with high tumor grade. In our hands, confocal laser scanning microscopy showed colocalization of Ki-67, TIIα, and survivin in tumor cell nuclei including mitotic figures. These findings confirm expression of survivin in proliferating cells. Tumor cell proliferation commonly is assessed by anti Ki-67 immunostaining To assess survivin as a potential alternative tumor cell proliferation marker, we compared the relation of Ki-67 and survivin expression in tumor cell nuclei. We found by double immunolabeling studies and statistical analysis that anti Ki- 67 stains a significantly larger fraction of tumor cells than does antisurvivin. These findings are in line with previous in vitro studies showing that Ki-67 (expression in G 1, S, G 2, and Am J Clin Pathol 2005;124: DOI: /PP2G5GAAFKV82DTG 545

4 Preusser et al / SURVIVIN EXPRESSION IN EPENDYMOMA A B C D E F G H I J Image 1 Antisurvivin labels nuclei of tumor cells (A, antisurvivin, original magnification 600) and endothelial cells (B, antisurvivin, original magnification 200). C, D, E, and F, Topoisomerase IIα and survivin are coexpressed in a mitotic figure (nucleic acids, blue signal; topoisomerase IIα, red signal; survivin, green signal; original magnification 1,000). F is a merged image of C-E. G, H, I, and J, Ki-67, topoisomerase IIα, and survivin are coexpressed in a tumor cell nucleus (Ki-67, blue signal; topoisomerase IIα, red signal; survivin, green signal; original magnification 1,000). J is a merged image of G-I. 546 Am J Clin Pathol 2005;124: DOI: /PP2G5GAAFKV82DTG

5 Anatomic Pathology / ORIGINAL ARTICLE M phases) 31,32 is expressed in a larger segment of the cell cycle than survivin (G 2 and M phases) 2 or TIIα (S, G 2, and M phases). 33 We conclude that anti Ki-67 identifies proliferating tumor cells more sensitively than does antisurvivin. Univariate survival analysis showed significant correlation of Ki-67, TIIα, and survivin indices with patient outcome. With multivariate analysis including all 3 factors, only the Ki- 67 index remained an independent prognostic parameter. So Table 1 Quantitative Results of Survivin, Ki-67, and Topoisomerase IIα Expression and of Univariate and Multivariate Survival Analyses Range (%) P Index Overall Grade II Grade III Median Cutoff (%) No. of Cases Log-Rank Test Cox Regression Ki *.019 * > Topoisomerase IIα *.570 > Survivin *.886 > * Statistically significant. A Cumulative Overall Survival C Low survivin index (n = 30) High survivin index (n = 28) Survival Time (y) B Ki-67 Index Survivin Index Figure 1 A, Kaplan-Meier curve showing significantly better outcome for patients with a low survivin index (P =.0032). B and C, Scatter plots showing significant correlation of the survivin index with the Ki-67 index (B, r = 0.586; P <.0001) and the topoisomerase IIα (TIIα) index (C, r = 0.733; P <.0001). 40 TIIα Index Survivin Index Am J Clin Pathol 2005;124: DOI: /PP2G5GAAFKV82DTG 547

6 Preusser et al / SURVIVIN EXPRESSION IN EPENDYMOMA the Ki-67 index more accurately predicts patient outcome. This finding might be explained by more sensitive identification of proliferating cells by anti Ki-67. Thus, anti Ki-67 should be the first choice as a tumor cell proliferation marker in the clinical setting. The prognostic impact of survivin expression has been analyzed in various tumor types. In most tumor types, high survivin expression correlated with an unfavorable patient outcome, 4-13 whereas in a few tumor types (eg, gastric cancer, breast cancer) it correlated with a favorable outcome To our knowledge, only 1 previous study on mantle cell lymphoma compared the prognostic influence of survivin and Ki In that study, survivin and Ki-67 correlated with patient outcome in univariate analysis. With multivariate analysis, only the Ki-67 index remained an independent predictor of survival. By comparing the impact of survivin and Ki-67 in ependymoma, we obtained analogous results. Thus, the Ki-67 index seems to predict patient outcome more accurately than does the survivin index in different cancer types. Survivin is expressed in ependymoma predominantly in a subset of proliferating tumor cell nuclei. High survivin expression is associated with an unfavorable patient outcome in intracranial ependymoma. However, the Ki-67 index predicts the outcome for patients with ependymoma more accurately than does the survivin index. From the 1 Institute of Neurology, 2 Department of Neurosurgery, and 3 Department of Pediatrics, Medical University of Vienna, Vienna, Austria. The study was performed within the Austrian Neuro- Oncology Network ( Vienna. Address reprint requests to Dr Hainfellner: Institute of Neurology, Medical University of Vienna, AKH 4J, Waehringer Guertel 18-20, POB 48, A-1097 Vienna, Austria. 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