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© 2002 American Society for Clinical Oncology Quantitatively Determined Survivin Expression Levels Are of Prognostic Value in Human GliomasByFrom the Departments of Radiation Oncology and Biostatistics and Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, and Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. Address reprint requests to Arnab Chakravarti, MD, Massachusetts General Hospital, Laboratory of Molecular Oncology, 13th St, Bldg 149, Room 7330, Charlestown, MA 02129; email: achakravarti@ partners.org.
PURPOSE: Survivin is a novel antiapoptotic gene that has been recently cloned and characterized. Its expression has been found to be of prognostic significance in several tumor types. This is the first study on the prognostic significance of survivin expression in human gliomas. MATERIALS AND METHODS: We used quantitative Western blot analysis with densitometry to determine survivin protein expression levels in 92 glioma cases for which frozen tissue was available for analysis. Survivin positivity and expression levels were correlated with histopathologic features of the tumors, apoptosis (as measured by cleaved, or activated, caspase 3 levels), and clinical outcome. RESULTS: Survivin expression has clear prognostic value in human gliomas. Patients with detectable survivin expression had significantly shorter overall survival times (P < .0001) compared with those without detectable expression when all glioma patients were considered. Although glioblastoma multiforme (GBM) patients had significantly higher rates of survivin positivity and higher levels of survivin expression (P < .0001) than their non-GBM counterparts, the prognostic value of survivin expression seemed to be independent of histology alone. Survivin-positive GBM patients had significantly shorter overall survival times compared with survivin-negative GBM patients (P < .0001). Likewise, survivin-positive non-GBM patients had shorter survival times compared with survivin-negative non-GBM patients (P = .029). Furthermore, increasing levels of survivin expression significantly correlated with reduced survival times when all glioma patients were considered, and markedly so for GBM patients (P < .0001). Increasing survivin levels significantly correlated with reduced expression of cleaved caspase 3, indicating its association with antiapoptotic activity. CONCLUSION: Survivin positivity and protein expression levels, as determined quantitatively, are of significant prognostic value in human gliomas and seem to be associated with reduced apoptotic capacity of these tumors.
GLIOMAS ARE AMONG the most aggressive of all human malignancies. Patients with the most malignant histopathologic subtype, glioblastoma multiforme (GBM), carry the worst prognosis, with median survival in the 9- to 10-month range, despite aggressive surgery, radiation, and chemotherapy.1-3 The molecular and genetic basis underlying pathogenesis and treatment resistance for these tumors are under active investigation and are becoming better understood. For example, on the genomic level, loss of chromosome 1p has been found to be associated with enhanced resistance to chemotherapeutic agents.4,5 Specific antiapoptotic genes have been recently identified at this locus.6 An important mediator of both tumorigenesis and resistance to treatment involves suppression of apoptosis.7 By extending the lifespan of abnormal cells, accumulation of transforming mutations can occur, thereby promoting development of the malignant phenotype and resistance to conventional cytotoxic agents. Recently, survivin has been cloned and characterized as an important member of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins. Survivin expression has been found to be undetectable in normal adult tissues. However, it has been found to be abundantly expressed in fetal tissues and a wide variety of human malignancies, including melanoma, bladder, lung, breast, hematopoietic, neuroblastoma, prostate, gastric, and pancreatic cancers.8-20 Survivin expression patterns in human gliomas have yet to be reported as of this writing. Our objective in this study was to determine whether survivin expression levels, as determined by quantitative Western analysis, are of prognostic significance in human gliomas.
Tissue Processing Ninety-two glioma specimens have been prospectively collected (at Brigham and Women’s Hospital) under an institutional review board–approved protocol for the purpose of studying treatment resistance markers in gliomas using quantitative gene or protein expression analysis. The histopathologic breakdown of these tumors is given in Table 1. Each specimen has been snap frozen after surgical removal, carefully sectioned, and histopathologically reviewed. Tissue containing greater than 95% tumor was provided for this analysis. Each tumor tissue was homogenized in RIPA buffer (for survivin isolation) and Chaps buffer (for cleaved caspase 3 isolation) using a Polytron homogenizer (Fisher, Pittsburgh, PA) to generate protein lysates for Western blot analysis.
Western Blot Analysis Bradford assays were performed to determine total protein concentrations, which were normalized to 1 µg/µL for all samples. Samples were then prepared in sample buffer and heated to 95°C for 5 minutes. The samples were run on 16% polyacrylamide gels, and 15 µL of protein lysates in sample buffer from each tissue were loaded within each well. Positive controls were included in each gel and represented lysates from HeLa cells, which are known to express survivin. Gels were run at constant current (40 mA) for 3 to 4 hours for maximum separation. Wet transfer was performed for 4 hours at constant voltage (40 V) using polyvinylidene fluoride membrane presoaked in methanol. The membrane was then blocked for 1 hour in 5% milk in 0.2% TBST. The membranes were then washed in 0.2% TBST three times for 15 minutes each. The membranes were then incubated overnight with primary antibody directed against survivin (no. NB500; Novus Biologicals, Littleton, CO) or cleaved caspase 3 (no. 9661; Cell Signaling Technology, Beverly, MA). Subsequently, the membranes were washed in 0.2% TBST three times for 15 minutes each. The membrane was then incubated with secondary antibody (anti-rabbit) for 45 minutes. Chemiluminescent (BioRad) detection was then used to detect survivin expression, which was quantitated using densitometry. The relative expression of each sample was determined by comparing with positive HeLa controls run on the same gel. Representative Western blot is shown in Fig 1.
Patient Data Histopathologic diagnosis was provided by Brigham and Women’s Hospital pathologists. Clinical data was obtained from the Brigham and Women’s Hospital tumor registry and from hospital charts. This data included patient age at diagnosis, treatment type, treatment dates, and clinical and survival outcomes. Patients with glioblastoma and anaplastic astrocytoma were generally managed by surgery (partial resection), postoperative radiation (60 Gy in 2-Gy fractions), and chemotherapy (carmustine). Patients with low-grade gliomas were generally managed by surgery alone, with radiation deferred for progressive disease. The histopathologic breakdown of these tumors is given in Table 1. The mean age of patients at the time of diagnosis was 48 years. Western blots were performed in a blinded manner, without prior knowledge of clinical outcome. Patient follow-ups were updated at the time of manuscript preparation.
Statistical Methods
Prognostic Value of Survivin Expression Survivin expression was detected in most patients, with 59 (64%) of 92 tumors survivin positive. When all glioma patients were considered together, the survival of patients with survivin-negative tumors was significantly higher than that of patients with survivin-positive tumors (Fig 2, P < .0001). The median survival of survivin-negative patients was 64 months, compared with 11 months for survivin-positive patients. It was next examined whether GBM tumors had a higher rate of survivin positivity compared with non-GBM tumors to help, in part, explain these differences. Indeed, survivin expression was more common in patients with GBM (45 [80%] of 56 tumors) compared with patients with non-GBM tumors (14 [39%] of 36 tumors) (P < .0001 by Fisher’s exact test). To better evaluate whether survivin expression is purely useful as a histologic marker or whether it carries additional prognostic value independent of histology, the association of survivin expression with survival was then examined separately for GBM versus non-GBM tumors. The hazard for death in survivin-expressing patients was significantly higher than for survivin-negative patients for patients with both GBM (P < .0001) and non-GBM (P = .029) histologies (Figs 3 and 4), despite relatively limited numbers of survivin-negative GBM cases (11) and both survivin-negative (22) and survivin-positive (14) non-GBM cases. This seems to suggest that survivin expression may carry prognostic significance for patients with both GBM and non-GBM histologies (see multivariate analysis below).
Prognostic Value of Survivin Expression Levels Next it was investigated whether in addition to the presence or absence of survivin, quantitatively determined survivin expression levels had significant association with worse survival for glioma patients. When all gliomas were considered, increasing levels of survivin expression were clearly associated with worse survival (P < .0001). When subdivided by histology, there seemed to be a stronger correlation with reduced survival times for GBM patients (P < .0001) than non-GBM patients (P = .062). Figure 5 shows a scatter plot diagram of this association for uncensored GBM patients (47 of 56 GBM patients). This seems to suggest that survivin expression at the highest levels is associated with a more extreme malignant phenotype of gliomas and may also be associated with increased treatment resistance.
Multivariate Analysis Two multivariate models were fit, one using expression level and the second using detectability of survivin expression. In a model that includes presence or absence of survivin expression, histology (GBM v non-GBM tumors), and age, GBM histology (P = .0081) and survivin expression (P < .0001) were significantly associated with reduced survival times, whereas age was not. Likewise, in a model that includes survivin expression levels, histology, and age, histology was of borderline significance (P = .087) and survivin expression level was significant (P < .0001) whereas age was not.
Association of Survivin Expression Levels With Apoptosis
In this study, the prognostic significance of survivin expression in human gliomas was examined for the first time. It seems that survivin expression patterns indeed may lend additional insight into the molecular pathogenesis of these tumors and may be important prognostic markers. Because long-term cure of glioma patients presently is elusive, such an understanding is fundamental to developing better therapies for these tumors, the most difficult human tumors of all. It is speculated that there are multiple pathways leading to development of GBM tumors. Primary GBMs are thought to arise de novo, that is, in the absence of preexisting lower grade lesions. Secondary GBMs arise from preexisting lower grade lesions.21 Although the molecular pathways leading to the development of GBMs may differ, almost all GBMs are united by certain common neuropathologic and behavioral features. That is, nearly all GBMs tend to possess high invasive potential, high angiogenesis potential, active proliferation, and resistance to conventional cytotoxic treatments. It is also true that lower grade gliomas possess many of the same traits, albeit to a lesser degree. The ability of gliomas to avoid apoptotic death, both spontaneous and treatment-related, may permit the progression to more aggressive phenotypes (eg, the linear progressive model of gliomagenesis) and may explain why gliomas, even at their earliest stages, are so resistant to conventional chemotherapy and radiation. Previous studies suggest that bcl family members may play an important role in inhibiting apoptosis in gliomas, although it is not clear that their expression levels are of clear prognostic value.22,23 The IAP family is known to include several important antiapoptotic family members, including survivin, IAP1, and XIAP, among others.24 These proteins are thought to be direct inhibitors of effector caspases and may, indeed, act additionally downstream to inhibit apoptosis. Unique among IAP proteins, survivin is only normally expressed during embryonic and fetal development and is not present in differentiated adult tissues. It is now clear that survivin expression is common in many human tumors and may carry an important prognostic value.8,10,12-20 A recent analysis of 3.5 million transcriptomes identified survivin among the top four transcripts uniformly upregulated in human cancers but not in normal tissues.25 In this study, survivin was found to be expressed in most gliomas (59 [64%] of 92 tumors). Although its expression in GBM tumors (45 [80%] of 56) was significantly higher (P < .0001) than that in non-GBM tumors (14 [39%] of 36), the observation that it was expressed in some lower grade tumors suggests that it may play a role in enhancing the malignant behavior of these tumors. This was supported by the observations that for the non-GBM tumors, survivin expression was significantly associated with reduced survival times (P = .029), and survivin expression levels as a continuous variable approached significance (P = .062). It is possible that because as many as 70% of lower grade tumors ultimately transform to higher grade tumors, increasing the barriers to apoptosis may enhance this transformation process. Survivin expression may be one of several pathways leading to this selective advantage. For GBM tumors, the patients with survivin-positive tumors had significantly shorter survival times than patients with survivin-negative tumors (P < .0001). Furthermore, for GBM patients, it was revealing that increasing survivin levels correlated with reduced survival times (P < .0001). It seems that some of the survivin-negative GBM tumors behaved much like their less aggressive anaplastic astrocytoma counterparts, with survivals in some patients extending to 3 years and longer. Because there can be some subjectivity in making such histologic distinctions, molecular classification in combination with histologic diagnosis may better predict the aggressiveness of a given tumor over histologic diagnosis alone, especially in cases where such a distinction using standard criteria (eg, World Health Organization 2000) is in question. Because the number of survivin-negative GBM cases was limited in this study (n = 11), future studies, including a larger number of survivin-negative GBM cases, are planned to more definitively address this issue. Furthermore, although it has been suggested that detection of survivin expression by immunohistochemistry (IHC) can be imprecise,17 the prognostic value of survivin expression in gliomas by multiple methods (eg, Western blotting, IHC, RT-PCR, and so on) should be assessed. These results suggest that increasing survivin expression enhances the malignant phenotype of gliomas in some manner. Survivin is unique in that its expression is detected specifically in dividing cells. Survivin competitively interacts with the cdk4/p16 complex, and the resultant cdk4/survivin complex induces cdk2/cyclin E activation for S phase entry. This suggests a close interaction between survivin and cell-cycle progression.26 Linked to this concept, LOH at the INK4A locus (encoding p16 and p14ARF) was found in almost 41% of GBMs, with shorter survival in the group of patients older than 50 years.27 Our in vitro data on corresponding primary glioma cell lines suggest that survivin expression may directly be mediating resistance to chemotherapy and radiation (manuscript in preparation). This may, indeed, be a distinct possibility in the clinical setting, because all GBM patients in this series were treated on identical regimens. The other possibility is that the reduced apoptotic capability of these cells secondary to survivin expression permitted the accumulation of additional mutations, which enhanced the invasive and angiogenic phenotype of these cells. Because nearly 40% of gliomas in this series failed to express survivin, it must be kept in mind that survivin expression is probably one of several mechanisms that glioma cells use to evade apoptosis. The relationship of survivin with these other antiapoptotic pathways must be better understood to better understand the molecular pathogenesis of these tumors and develop more effective therapeutic strategies.
Supported by National Institutes of Health/National Cancer Institute grant nos. KO8CA82163 (A.C.) and RO1CA64402 (N.J.D.) and the Massachusetts General Hospital/Brian Silber Memorial Fund (A.C.).
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
, Follow-up.
