Clinical Cancer Research 13, 3868, July 1, 2007. doi: 10.1158/1078-0432.CCR-06-2730
© 2007 American Association for Cancer Research
Imaging, Diagnosis, Prognosis |
Prognostic Significance of the Immunohistochemical Staining of Cleaved Caspase-3, an Activated Form of Caspase-3, in Gliomas
Tatsuya Kobayashi1,2,
Junya Masumoto1,
Tsuyoshi Tada2,
Tetsuo Nomiyama3,
Kazuhiro Hongo2 and
Jun Nakayama1
Authors' Affiliations: Departments of 1 Pathology, 2 Neurosurgery, and 3 Preventive Medicine, Shinshu University School of Medicine, Matsumoto, Japan
Requests for reprints: Junya Masumoto, Department of Pathology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Phone: 81-263-37-3395; Fax: 81-263-37-2581; E-mail: masumoto{at}sch.md.shinshu-u.ac.jp.
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Abstract
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Purpose: Gliomas are common tumors of the central nervous system, and the majority of patients with gliomas have a poor prognosis. The prediction of prognosis is very important in selecting treatment. In the present study, we retrospectively examined the immunohistochemical staining of cleaved caspase-3 (CC3), an activated form of caspase-3 that acts as a lethal protease at the most distal stage of the apoptosis pathway, in gliomas, and the correlation between the prognosis of patients and caspase-3 activation to find useful prognostic indicators.
Experimental Design: Immunohistochemical staining of CC3 was done in 65 patients with gliomas. The percentage of CC3 staining-positive cells was defined as the CC3 immunoreactivity score (IRS). Survival analysis between CC3 IRS of glioma patients and survival time was carried out using the Kaplan-Meier method with the log-rank test and the Cox proportional hazards regression model.
Results: CC3 IRS was statistically analyzed to designate the best provisional cutoff point, and when detected in >10% of glioma cells, it was considered positive. The Kaplan-Meier method with the log-rank test revealed that patients with CC3 IRS-positive tumors had significantly greater survival than those with CC3 IRS-negative tumors among three grades, 2, 3, and 4 (P = 0.0061), and within grade 3 of anaplastic astrocytoma (P = 0.0458). After adjustment for known clinical prognostic factors, such as age, WHO grade, and performance status, the hazard ratio for CC3 IRS-positive was 0.39 with 95% confidence interval between 0.19 and 0.85 (P = 0.0187). Within high grades, including grades 3 and 4, the hazard ratio was 0.40 with 95% confidence interval between 0.20 and 0.86 (P = 0.0192).
Conclusions: CC3 IRS could be useful as a good prognostic indicator for glioma patients.
Glioma is the most common form of primary brain tumors. The prognosis of patients with glioma depends largely on the histopathologic grading. The survival of patients with lower grade glioma is usually longer than with higher grade glioma (1, 2). Prediction of the prognosis of patients with glioma is very important in selecting and evaluating the effectiveness of the treatment. The histologic classification of gliomas has been designed to identify groups of patients with different prognoses; however, the grading system of light microscopic examination remains controversial due to the lack of direct knowledge about their biological behavior (3–7).
Recent studies have indicated that apoptosis occurs not only in normal tissues but also in malignant tumors and plays an important role both in the pathogenesis of tumors and their biological behavior. A family member of cysteine proteases called caspase plays critical roles in the process of apoptosis (8–10). In particular, caspase-3 plays a crucial role in the most distal effector pathway of apoptosis (11–14). Actually, caspase-3 expression was detected in several human malignant tumors, such as neuroblastomas (15), malignant lymphomas (16), esophageal carcinomas (17), and non–small cell lung cancers (18), and its expression was reported to be inversely related to malignant potential; however, the correlation between caspase-3 activation and gliomas remains uncharacterized.
In the present study, we examined the prognostic significance of caspase-3 activation in gliomas using anti–cleaved caspase-3 (CC3)–specific antibody, which recognizes the activated form of caspase-3, by immunohistochemistry and survival analyses with both the Kaplan-Meier method (19) with the log-rank test and the Cox proportional hazards regression model (20) to propose a favorable prognostic indicator in gliomas.
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Materials and Methods
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Patient samples. The analysis of human tissues was approved by the Human Research Ethical Committee of Shinshu University. Sixty-five patients with gliomas, except for brain stem tumors, treated at the Department of Neurosurgery, Shinshu University Hospital, between December 1988 and December 2003, were studied retrospectively (Table 1
). The tumors were diagnosed histopathologically by pathologists according to the WHO classification, and clinicopathologic characteristics are summarized in Table 1. Formalin-fixed, paraffin-embedded materials were used for routine staining with H&E, as well as for staining by immunohistochemical techniques. The patients were followed up until death or 36 months from the diagnostic surgery. Clinical data were retrieved from patient records.
Immunohistochemistry. In all cases, immunohistochemical reactions were done. Serial sections were reacted with CC3-specific antibody diluted 1:200 (Cell Signaling), which recognizes large fragments (17/19 kilodaltons) of activated caspase-3 (21). Three-micrometer-thick tissue sections were deparaffinized in xylene and hydrated in ethanol, and endogenous peroxidase activity was blocked by soaking in absolute methanol containing 0.3% H2O2 for 30 min. Before immunostaining, antigen retrieval was carried out by incubating tissue sections in a microwave in 10 mmol/L Tris-HCl buffer (pH 8.0) containing 1 mmol/L EDTA. Sections were blocked with 1% normal goat serum in 50 mmol/L TBS (pH 7.6) and incubated with the diluted primary antibodies at 4°C overnight. After washing in TBS, slides were incubated with EnVision+ (DakoCytomation) for 60 min. 3,3'-Diaminobenzidine was used as the chromogen. These sections were counterstained with hematoxylin.
Evaluation of immunostaining. We assessed the immunostaining of CC3. Even in low-grade glioma, >500 cells were observed in the same field under x400 magnification. We counted 500 cells from each specimen under x400 magnification in the best-stained tumor area of each section. CC3 immunoreactivity score (IRS) was defined as the percentage of stained glioma cells.
Statistical analysis. Statistical analyses were done using JMP 6 software (SAS Institute, Inc.). Differences between the mean CC3 IRS for WHO histologic grades were compared using ANOVA with multiple comparisons. Survival analysis at 36 months of glioma patients based on CC3 IRS was carried out using the Kaplan-Meier method with the log-rank test. Adjusted analysis of the effect of CC3 IRS on survival in each glioma grade was carried out by multivariate data analysis using the Cox proportional hazards regression model. The following variables were considered for statistical analysis: patient age, sex, WHO histologic grade, total resection or not, preoperative Eastern Cooperative Oncology Group (ECOG)-performance status (PS), postoperative ECOG-PS, adjuvant chemotherapy, radiation, and CC3 IRS. The reference point was the date of diagnostic surgery. The end point was length of survival to death or 36 months from the diagnostic surgery. The Cox proportional hazards regression model was used to estimate the strength of each variable for survival. The stepwise procedure was adopted to select independent prognostic variables in multivariate analyses. Differences were considered significant when P values of <0.05 were obtained.
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Results
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Histology and clinical information. Three cases of WHO grade 1 were pilocytic astrocytomas (pA); 16 cases of grade 2 included 11 diffuse astrocytomas (A), 2 ependymomas (E), and 3 oligoastrocytomas (OA); 25 cases of grade 3 included 22 anaplastic astrocytomas (AA) and 3 anaplastic oligodendrogliomas (AO); and 21 cases of grade 4 were glioblastomas (GB; Table 1). The mean patient age ± SD was 8.3 ± 6.5 years old in grade 1, 36.6 ± 17.2 years old in grade 2, 43.7 ± 18.2 years old in grade 3, and 52.5 ± 20.5 years old in grade 4. Seventeen cases were located in the frontal lobe, 19 cases were temporal lobe, 8 cases were parietal lobe, 16 cases were basal ganglia, and 5 cases were cerebellar. After tumor removal, 54 patients received postoperative radiotherapy: the total dose of irradiation ranged between 50 and 60 Gy. The other 3 cases of grade 2, 2 cases of grade 3, and 3 cases of grade 4 did not receive irradiation because of the decision of neurosurgeons and/or radiologists or reluctance of the patients and their relations. Forty-nine cases of astrocytic tumors, abbreviated as pA, OA, A, AA, and GB indicated by (+) in Table 1, received adjuvant chemotherapy with IFN-ß using ranimustine [6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-
-D-glucopyranoside], which was administered i.v. only once every 6 to 8 weeks at a dose of 1 to 2 mg/kg of body weight. Three cases of oligodendroglial tumors, abbreviated as AO indicated (+) in Table 1, received adjuvant chemotherapy using procarbazine, nimustine hydrochloride [1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride] with additional vincristine. Procarbazine (100 mg) was used internally once a day for 14 days. Nimustine was administered i.v. only once at a dose of 80 mg/mm2 body surface area. Vincristine was administered i.v. only once at a dose of 1.4 mg/mm2 body surface area. Three cycles of chemotherapy were given. The other cases indicated by (–) in Table 1 received no chemotherapy or discontinued chemotherapy because of myelosuppression or deterioration of their general condition. Both case 1, which was finally diagnosed as pilocytic astrocytoma, and case 19, which finally diagnosed as ependymoma, were initially diagnosed as anaplastic astrocytoma and received chemotherapy using ranimustine. Case 11, which was finally diagnosed as ependymoma, was initially diagnosed as medulloblastoma and received chemotherapy using ranimustine. Complete follow-up was obtained except for five patients indicated by asterisks that were missing clinical data (Table 1).
Staining of CC3 in gliomas. Clear immunohistochemical staining for CC3 was obtained using CC3-specific monoclonal antibody (Fig. 1, arrowheads
). CC3 IRS was statistically analyzed to designate the best provisional cutoff point. We determined the cutoff point of 10% for CC3 IRS using JMP 6 software to obtain a lower P value comparing survival between CC3 IRS-positive patients and CC3 IRS-negative patients. According to the cutoff point of the CC3 IRS, 78.5% were CC3 IRS positive and 21.5% were CC3 IRS negative. Precisely, 51 cases were CC3 IRS positive of 65 cases and 14 cases were negative of 65 cases (Table 1). In grade 1, all 3 cases were pilocytic astrocytoma and CC3 IRS positive. In grade 2, 15 cases were CC3 IRS positive but 1 case was CC3 IRS negative. In grade 3, 17 cases were CC3 IRS positive and 8 cases were CC3 IRS negative. In grade 4, 16 cases were CC3 IRS positive and 5 cases were CC3 IRS negative (Table 1). Representative figures of both CC3 IRS-positive gliomas and CC3 IRS-negative gliomas are indicated (Fig. 1). Summary of the clinical characteristics of the 65 patients in this study is shown in Table 2
.
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Fig. 1. Immunohistochemical staining of CC3 in gliomas. Representative of CC3 IRS-positive glioma and CC3 IRS-negative glioma in each grade. Specimens were stained with H&E (Aa-Fa) or immunostained with CC3-specific pAb (Ab-Fb). Arrowheads, stained glioma cells. CC3 IRS was defined as the percentage (%) of stained glioma cells. IRS > 10% was considered positive. CC3 IRS-positive glioma: pilocytic astrocytoma (WHO grade 1, case 1 in Table 1; A), diffuse astrocytoma (WHO grade 2, case 6 in Table 1; B), anaplastic astrocytoma (WHO grade 3, case 29 in Table 1; C), and glioblastoma (WHO grade 4, case 45 in Table 1; E). CC3 IRS-negative glioma: anaplastic astrocytoma (WHO grade 3, case 40 in Table 1; D) and glioblastoma (WHO grade 4, case 62 in Table 1; F). Bar, 100 µm. It was noted that there was no CC3 IRS-negative grade 1 glioma in our cases and only one CC3 IRS-negative grade 2 glioma was ependymoma.
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CC3 IRS and its association with pathologic grading in gliomas. CC3 IRSs of gliomas with grades 1, 2, 3, and 4 were 68.2% (mean) ± 7.1% (SD), 41.5% ± 20.1%, 30.7% ± 25.4%, and 32.6% ± 21.4%, respectively (Fig. 2
). CC3 IRS decreased with the increasing grade of gliomas (Fig. 2). Significant differences were seen in CC3 IRS between grades 1 and 2 (P = 0.0253), grades 1 and 3 (P = 0.0343), and grades 1 and 4 (P = 0.0129) with Mann-Whitney's U test, respectively (Fig. 2); however, there were no significant differences of CC3 IRS among grades 2, 3, and 4 by multiple comparisons with Kruskal-Wallis test (P = 0.2791; Fig. 2).
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Fig. 2. CC3 IRS and its association with pathologic grading in gliomas. *, significant differences were seen in CC3 IRS between grades 1 and 2 (P = 0.0253), grades 1 and 3 (P = 0.0343), and grades 1 and 4 (P = 0.0129) with Mann-Whitney's U test, respectively. There were no significant differences of CC3 IRS among grades 2, 3, and 4 by multiple comparisons with Kruskal-Wallis test (P = 0.2791).
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Correlation between CC3 IRS in gliomas and patient survival. Kaplan-Meier survival analyses with log-rank test among grades 2, 3, and 4 or within the same diagnoses of grade 3 anaplastic astrocytoma or grade 4 glioblastoma for CC3 IRS-positive glioma patients and CC3 IRS-negative glioma patients are shown (Fig. 3A-C
). Kaplan-Meier survival analysis among three WHO grades, including grades 2, 3, and 4, revealed that CC3 IRS-positive patients had a significantly more favorable survival than CC3 IRS-negative patients with the log-rank test (P = 0.0061; Fig. 3A). Within the same grade 3 anaplastic astrocytoma, a statistical significance between CC3 IRS-positive and CC3 IRS-negative glioma patients was obtained for survival with the log-rank test (P = 0.0458; Fig. 3B); however, within the same grade 4 glioblastoma, there was no statistical significance between CC3 IRS-positive and CC3 IRS-negative glioma (P = 0.6727; Fig. 3C). We attempted multivariate survival analysis using the Cox proportional hazards regression model comparing CC3 IRS and independent statistically significant prognostic factors, such as age, WHO histologic grade, ECOG-PS before surgery, and ECOG-PS after surgery versus patient survival in 62 patients within three WHO histologic grades 2, 3, and 4 (Table 3
). The adjusted hazard ratio for CC3 IRS positive versus CC3 IRS negative was 0.39 with 95% confidence interval (95% CI) of 0.19 to 0.85 (P = 0.0187; Table 3). We also attempted multivariate analysis in 46 patients within high-grade glioma, including grades 3 and 4 (Table 4
). In this group, age, ECOG-PS after surgery and radiation were found to be significant prognostic variables via the stepwise procedure. The adjusted hazard ratio for CC3 IRS positive versus CC3 IRS negative was 0.40 with a 95% CI of 0.20 to 0.86 (P = 0.0192; Table 4). In 22 patients with WHO histologic grade 3 of anaplastic astrocytoma, ECOG-PS after surgery and radiation were found to be significant prognostic variables via the stepwise procedure. The adjusted hazard ratio for CC3 IRS positive versus CC3 IRS negative was 0.17 with a 95% CI of 0.05 to 0.57 (P = 0.0046; Table 5
). In 21 patients with WHO grade 4 of glioblastoma, total resection and chemotherapy were found to be significant prognostic variables via the stepwise procedure. The adjusted hazard ratio for CC3 IRS positive versus CC3 IRS negative was 1.36 with a 95% CI of 0.43 to 5.02 (P = 0.6095; Table 6
).
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Fig. 3. Three-year survival in 65 patients with gliomas in relation to CC3 IRS. A, Kaplan-Meier survival curve among three WHO grades, grades 2, 3, and 4, between CC3 IRS-positive and CC3 IRS-negative glioma patients with log-rank test (P = 0.0061). B, Kaplan-Meier survival curve within WHO grade 3 of anaplastic astrocytoma between CC3 IRS-positive and CC3 IRS-negative glioma patients with log-rank test (P = 0.0458). C, Kaplan-Meier survival curve within WHO grade 4 of glioblastoma between CC3 IRS-positive and CC3 IRS-negative glioma patients with log-rank test (P = 0.6727).
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Discussion
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Assessment of prognosis is very important in selecting treatment and also for patients and their families. Accordingly, accurate histologic diagnosis is fundamentally important from both clinical and experimental perspectives. The prognosis of glioma patients has usually been predicted dependent on the histopathologic diagnosis (1, 2).
However, a wide range of survival periods of patients with anaplastic astrocytoma was reported and that may reflect mixed results from inappropriate diagnosis, particularly oligodendroglial tumors and pilocytic astrocytomas (22). Diagnostic accuracy is affected by several factors, such as biopsy size, regional heterogeneity, and the interpretation of histologic criteria used to classify and grade gliomas. An inappropriate diagnosis may result in inadequate therapy for a high-grade glioma or, worse, harmfully aggressive therapy for a low-grade glioma. Furthermore, we and others had a chance to encounter long-term survivors (Table 1; refs. 23–26). Actually, almost the same populations of both long-term and short-term survivors existed among our cases, especially in anaplastic astrocytoma (Table 1). How do we recognize long-term survivors and which marker should we use?
In this study, we got a clear immunohistochemical staining using CC3-specific antibody (21) in glioma (Fig. 1) and propose CC3 IRS as an attractive prognosis indicator (Figs. 2 and 3; Tables 2-6). Thus far, to our knowledge, there has been no available reference about the prognostic significance of CC3 IRS in gliomas by immunohistochemistry using CC3-specific antibody and survival analyses.
Caspase-3 has been reported to play a crucial role in apoptosis (11–14, 27–29), and the evaluation of CC3 should provide a generally useful tool to identify apoptotic cells in archival material, expanding the range of experimental and disease settings in which apoptosis can be accurately evaluated (21). Apoptosis and cell proliferation are opposing processes in tumor growth, and tumor progression is thought to be determined by the balance between apoptosis and cell proliferation. Thus, the lack of caspase-3 activation leading to decreased apoptosis is thought to give an advantage to tumor growth. Kondo et al. (30) showed that the retroviral transfer of caspase-3 induces apoptosis in malignant glioma cells in vitro. Furthermore, treatment of mouse tumors with retrovirus containing caspase-3 significantly inhibited tumor growth through the induction of apoptosis. Consistent with this, low-grade glioma, including grades 1 and 2, exhibits high CC3 IRS (Figs. 1 and 2). Although CC3 IRS does not correlate with glioma grading in multiple comparisons (Fig. 2), survival analyses using the Kaplan-Meier method with log-rank test and the Cox proportional hazard regression model revealed that CC3 IRS was a significant prognostic factor in predicting survival in gliomas (Fig. 3; Tables 2-6). Notably, within high grades, including grades 3 and 4, after adjustment for age, postoperative PS, and postoperative irradiation, the association between CC3 IRS positive and survival is statistically significant (Table 4); therefore, CC3 IRS-positive glioma patients have a better survival rate than CC3 IRS-negative glioma patients. In grade 3, anaplastic astrocytoma, both the Kaplan-Meier method with log-rank test and the Cox proportional hazard regression model revealed that CC3 IRS-positive patients have much better survival rate than CC3 IRS-negative patients (Fig. 3B; Table 5). We speculate that the anaplastic astrocytomas have various biological potential, and CC3 IRS may be one of their indicators. Thus, CC3 IRS can be a candidate for prognostic indicator especially in anaplastic astrocytoma (Fig. 3B; Table 5). In our cases, there is no statistic significance between survival and CC3 IRS in grade 4, glioblastoma probably because of our restricted cases (Fig. 3C; Table 6).
Although further clinical confirmation on a large scale might be needed, our study propose that CC3 IRS could be a simple and useful tool for predicting survival in glioma patients in addition to known prognostic factors.
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Footnotes
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Grant support: Grants-in-Aid for Scientific Research C-18590524 (J. Masumoto) and B-18390113 (J. Nakayama) from the Japan Society for the Promotion of Science and Hokuto Foundation grant (J. Masumoto).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11/16/06;
revised 3/28/07;
accepted 4/ 5/07.
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Abstract
Materials and Methods
