Expression of p27KIP1 in human gliomas: Relationship between tumor grade, proliferation index, and patient survival☆☆☆

Article Outline

• Abstract
• Materials and methods
  • Tissue samples
  • Western blot analysis
  • Immunohistochemistry
  • p27KIP1 and MIB-1 scoring
  • Statistical analysis
• Results
  • p27KIP1 protein expression in nonneoplastic brain disorders and astrocytic and nonastrocytic brain tumors
  • Relationship between p27KIP1 protein expression, tumor grade, patient survival, and proliferation index
• Discussion
• References
• Copyright

Abstract 

Numerous studies examining the prognostic significance of p27KIP1 expression in human cancer have shown that decreased expression often is an independent prognostic factor associated with worse survival. However, the prognostic value of p27KIP1 expression in gliomas is less well established. To further address this issue, we evaluated the relationship between p27KIP1 protein expression in a series of 50 astrocytomas with clinicopathologic parameters including age, tumor grade, MIB-1 proliferation index, and patient survival using both Western blot analysis and immunohistochemistry. The level of p27KIP1 protein expression in 9 nonneoplastic brain tissue specimens served as a control. Sixteen high-grade astrocytomas were analyzed by Western blot, and 26 high-grade astrocytomas were analyzed by immunohistochemistry for levels of p27KIP1 protein expression. Regardless of the technique used to measure p27KIP1, approximately 50% of the high-grade tumors were low expressors and the other 50% were high expressors. Thus, expression of p27KIP1 was independent of tumor grade. Loss of p27KIP1 expression is often associated with an increase in proliferative activity. We measured the rate of tumor cell proliferation using MIB-1 immunostaining in 16 high-grade astrocytomas to determine whether there was an inverse correlation between p27KIP1 expression and proliferation. No correlation between p27KIP1 expression and MIB-1 labeling index or patient survival was found. Using immunohistochemistry, we noted that the staining pattern of p27KIP1 in glioblastomas was mainly in the pseudopalisading cells that outline areas of necrosis. Because p27KIP1 can be up-regulated by hypoxia, this staining pattern would be consistent with our observation that hypoxia-inducible factor 1α is expressed primarily in pseudopalisading tumor cells around necrotic zones. It has been shown that a high level of p27KIP1 prevents apoptosis in hypoxic cells. Thus, maintenance of high levels of p27KIP1 in gliomas could result from the hypoxic microenvironment present within the tumor. No correlation was found between p27KIP1 expression and any of the clinicopatholgic parameters tested, including patient age and tumor grade, the 2 strongest predictors of survival among glioma patients. HUM PATHOL 34:48-53. Copyright 2003, Elsevier Science (USA). All rights reserved.

Keywords:  p27KIP1, glioma, tumor grade, proliferation index, survival

Abbreviations:  AA , anaplastic astrocytoma, CKI , cyclin-dependent kinase inhibitor, EPEN , ependymoma, GBM , glioblastoma multiforme, GG , ganglioglioma, GSM , gliosarcoma, HIF-1 , hypoxia-inducible factor 1, JPA , juvenile pilocytic astrocytoma, LI , labeling index, LGA , low-grade fibrillary astrocytoma, WHO , World Health Organization

Cyclin-dependent kinase inhibitors (CKIs) govern progression through the cell cycle.¹, ² p27KIP1 is a key CKI protein that controls G₁ phase to S phase transition by inhibiting the activity of G₁ cyclin–cyclin-dependent kinase complexes, thereby negatively regulating progression through the G₁ and S phases of the cell cycle. Increased levels of p27KIP1 are found in quiescent and in contact-inhibited cells, suggesting that p27KIP1 can play a role in maintaining cells in the G₀ phase.², ³ Loss of p27KIP1 expression has been linked to tumor development and/or progression.²

Numerous studies have correlated reduced p27KIP1 expression with poor prognosis in breast,⁴, ⁵ gastric,⁶ prostate,⁷ non–small cell lung,⁸ ovarian,⁹ colorectal,¹⁰, ¹¹, ¹² endocrine,¹³ and head and neck tumors.¹⁴ Proteosome-dependent degradation of p27KIP1 occurs during the cell cycle; enhancement of this activity in some tumors has been correlated with poor prognosis.¹⁵

Previous studies have examined p27KIP1 expression in gliomas and its role as a prognostic factor.¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹, ²², ²³ In some studies reduced p27KIP1 expression showed an association with tumor grade,¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰ whereas other studies found no association.²¹, ²², ²³ The aim of this study was to evaluate the relationship between p27KIP1 protein expression in 50 astrocytomas and clinicopathologic parameters including tumor grade, MIB-1 proliferation index, and patient survival, using both Western blot analysis and immunohistochemistry.

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Materials and methods 

Tissue samples 

A total of 60 brain tumors were evaluated. The tumors were classified according to the World Health Organization (WHO) criteria,²⁴ except that the presence of necrosis was required for a diagnosis of glioblastoma. For Western blot analysis, 24 fresh-frozen samples of astrocytic tumors, including 16 high-grade tumors [10 glioblastoma multiformes (GBMs), 3 gliosarcomas (GSMs), and 3 anaplastic astrocytomas (AAs)] and 8 low-grade tumors [3 low-grade fibrillary astrocytomas (LGAs) and 5 juvenile pilocytic astrocytomas (JPAs)] were assessed for p27KIP1 expression. Ten nonastrocytic tumors were used in this study, including 6 ependymomas (EPENs) and 4 gangliogliomas (GGs). For controls, 9 samples of nonneoplastic cerebral tissue were obtained during neurosurgical procedures to treat intractable epilepsy.

For immunohistochemical staining, paraffin-embedded tumors from 26 patients with the diagnosis of high-grade astrocytoma who underwent surgery at New York University Medical Center between 1979 and 1990 were evaluated. Histopathologic grading of the tumors was reevaluated by 2 neuropathologists (D.Z. and D.C.M.) at the time of the present study. Nine tumors were classified as AA (WHO grade III) and 17 were classified as GBM (WHO grade IV).

Western blot analysis 

Fresh-frozen brain tumor samples were extracted as described previously²⁵ and stored at −80°C until used. Protein concentration was determined by the Bradford assay. Tubulin expression for each sample was used for normalization and to control for equal loading of the samples. Samples (30 μg) were resolved on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose. Proteins were detected using anti-p27 (Transduction Laboratories, Lexington, KY) and anti-tubulin (Sigma, St. Louis, MO) antibodies with horseradish peroxidase-linked secondary antibody (Amersham, Piscataway, NJ). Levels of p27KIP1 and tubulin expression were quantitated for each sample by scanning densitometry using the National Institutes of Health image program.

Immunohistochemistry 

Sections (6 μm thick) were cut from formalin-fixed, paraffin-embedded tissue blocks and mounted on Superfrost/Plus glass slides (Fischer Scientific, Pittsburgh, PA). The sections were deparaffinized in xylene and rehydrated. For antigen retrieval and p27KIP1 detection, the sections were heated in a microwave oven for a total of 30 minutes (3 cycles of 10 minutes each) in 10 mmol/L sodium citrate buffer at pH 6.0. The sections were stained using standard streptavidin-biotin complex immunoperoxidase methods (Histostain-SP kit; Zymed Laboratories, South San Francisco, CA) on a Ventana ES machine (Ventana Medical Systems, Tucson, AZ). The p27KIP1 antibody (Transduction Laboratories) was diluted to 1:400 in PBS containing 1% normal rabbit serum. Peroxidase activity was localized with chromogen 3,3'-diaminobenzidine tetrachloride in 0.5 mmol/L Tris buffer. The slides were counterstained with hematoxylin.

For proliferation studies, we used the mouse monoclonal antibody MIB-1 (Amac, Westbrook, ME) that recognizes the nuclear cell proliferation–related Ki-67 antigen in formalin-fixed, paraffin-embedded tissues diluted to 1:60 in phosphate-buffered saline. MIB-1 labeling indices were measured for 16 of the 26 cases analyzed for p27KIP1 expression. Then 4-μm-thick sections were cut from the paraffin blocks, deparaffinized, rehydrated, and stained by standard methods. For antigen retrieval and MIB-1 detection, the sections were first hydrated and then heated in a microwave for 10 minutes in 10 mmol/L citrate buffer. Lymph nodes were used as positive controls for MIB-1 immunostaining. For negative controls, the immune serum was replaced by phosphate-buffered saline or by nonimmune serum. MIB-1 nuclear staining was quantitated on the SAMBA 4000 Image Analysis System (Immuno Software Program, IPI, Chantilly, VA).

p27KIP1 and MIB-1 scoring 

Sections stained for p27KIP1 were assessed on a multihead microscope, and the percentage of immunostained cells was determined by a consensus estimate of 3 observers. Only a distinct brown nuclear stain was scored as positive. A cutoff value of <5% immunopositive cells was considered negative, and >5% immunopositive cells was considered positive. The positive samples were scored according to the frequency of p27KIP1 immunopositive cells as 5% to 25%, 26% to 50%, 51% to 75%, and >75%, as previously described.⁹ Samples from patients with <50% p27KIP1-positive tumor cells were considered low expressors, whereas those with >50% p27KIP1-positive tumor cells were considered high expressors.⁹

For MIB-1 immunostaining, 10 fields at ×400 magnification were analyzed to count at least 1000 total cells per sample. The labeling index (LI) of MIB-1 was calculated as a percentage by the following equation: LI (%) = (number of cells with positive staining)/(total number of tumor cells counted)×100. Only cells with unequivocal nuclear staining were counted as positively stained. Weak nuclear or cytoplasmic staining was considered a negative finding.

Statistical analysis 

The quantitative data were expressed as mean ± standard deviation (SD). The data comparison was performed using a 2-sided unpaired Student t test. Correlation between the parameters was assessed by Pearson's correlation coefficient. A P value <0.05 was considered significant.

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Results 

p27KIP1 protein expression in nonneoplastic brain disorders and astrocytic and nonastrocytic brain tumors 

p27KIP expression was determined by Western blot analysis for 9 nonneoplastic epileptic brain samples, 24 astrocytic brain tumors, and 10 nonastrocytic brain tumors. Table 1 and Figure 1 summarize the level of p27KIP1 expression relative to tubulin expression for each sample and the clinicopathologic data for tumor grade, patient age, and gender.

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• Fig. 1. 

  Expression of p27KIP1 in nonneoplastic brain and astrocytic and nonastrocytic brain tumors. Western blot analysis showing levels of p27KIP1 expression in 9 nonneoplastic brain specimens, 24 astrocytic brain tumors (10 GBMs, 3 AAs, 3 GSMs, 3 LGAs, and 5 JPAs) and 10 nonastrocytic brain tumors (6 EPENs and 4 GGs). The molecular weight of p27KIP1 is 27 kDa, and that of tubulin is 50 kDa. For immunoblot analysis, 30 μg of extract were loaded per lane, separated on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and blotted with the appropriate antibodies. Blots were stripped and reprobed with an antibody to tubulin to ensure equivalent loading and transfer. The relative ratio of p27KIP1/tubulin in each sample was quantitated by densitometry.

The epileptic brain samples were used as a control to determine the level of p27KIP expression associated with nonneoplastic brain tissue. The average relative ratio for p27KIP1 in the 9 nonneoplastic brain samples was 0.78±0.3. Samples with values of p27KIP1 expression within 2 standard deviations of the levels seen in nonneoplastic brain were considered low expressors (L) of p27KIP1, whereas those with values greater than 2 standard deviations above the control were considered high expressors (H) of p27KIP1. Among the high-grade astrocytomas (GBM, GSM, and AA), 7 of 16 (44%) were low expressors (range, 0.08 to 0.82) and 9 of 16 (56%) were high expressors (range, 1.67 to 7.05) of p27KIP1. Among the low-grade astrocytomas (LGA and JPA), equal numbers (4 of 8) of tumors were low expressors (range, 0.29 to 1.05) or high expressors (range, 2.93 to 6.43) of p27KIP1. An additional group of nonastrocytic EPENs and GGs were evaluated for p27KIP1 expression. In this group, 7 of 10 (70%) were low expressors (range, 0 to 1.38) and 3 of 10 (30%) were high expressors (range, 3.37 to 6.48) of p27KIP1. There was no association between levels of p27KIP1 expression and tumor grade.

Table 1. p27KIP1 protein expression in nonneoplastic brain disorders and astrocytic and nonastrocytic brain tumors

Relationship between p27KIP1 protein expression, tumor grade, patient survival, and proliferation index 

p27KIP1 expression was determined by immunohistochemistry for 26 astrocytomas, consisting of 9 WHO grade III and 17 WHO grade IV tumors. Samples from patients with <50% p27KIP1-positive tumor cells were considered low expressors, whereas those with >50% p27KIP1-positive tumor cells were considered high expressors.⁹ MIB-1 proliferation index was evaluated in 16 of 26 tumors also assessed for levels of p27KIP1 protein expression. Table 2 summarizes the level of p27KIP1 protein expression and the clinicopathologic parameters for patient age, tumor grade, survival, and proliferation index.

Table 2. Relationship between p27KIP1 protein expression, tumor grade, patient survival, and proliferation index

L, lost to follow-up.

Among the 26 samples examined, 50% were low expressors and 50% were high expressors of p27KIP1. The patients' median age was 58 years (range, 28 to 80). P27KIP1 staining did not correlate significantly with tumor grade. A representative tumor stained for p27KIP1 protein expression is shown in Fig 2.

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• Fig. 2. 

  Immunohistochemical analysis of p27KIP1 and Ki-67 antigen by MIB-1 immunostaining in GBMs. A representative GBM stained with hematoxylin and eosin demonstrates pseudopalisading cells (P) around an area of necrosis (N). The same tumor immunostained with a monoclonal p27KIP1 antibody shows a high density of positive tumor nuclei, as well as intense p27KIP1 staining of the nuclei of pseudopalisading cells (P) outlining the focus of necrosis (N). The expression of the proliferation marker Ki-67 was detected with the monoclonal MIB-1 antibody in the same tumor. Immunopositive cells were also seen around necrotic zones (N). (Original magnification ×200.)

The greatest density (labeled nuclei/area) as well as the highest intensity of p27KIP1 staining was seen in the nuclei of pseudopalisading cells that outline the geographic foci of necrosis in glioblastomas. Among the low expressors of p27KIP1, 8 of 13 (62%) were WHO grade IV, compared with 10 of 13 (77%) of the high expressors. The average survival times were 13 months in the low expressor group and 19 months in the high expressor group. MIB-1 LIs varied widely in the 2 groups; average MIB-1 LIs were 15% in the low expressors and 20% in the high expressors. No correlations were found between p27KIP1 protein expression and any of the clinicopathologic parameters tested.

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Discussion 

Numerous studies have examined the prognostic significance of p27KIP1 expression in human cancer and have shown that decreased expression often is an independent prognostic factor associated with poor survival.² Tumors showing loss of p27KIP1 expression have demonstrated increased proteasome-mediated degradation of the p27KIP1 protein associated with increased aggressive growth behavior.¹⁰ However, the prognostic value of p27KIP1 levels in gliomas is less well established. Conflicting data on the expression of p27KIP1 and its role as an independent prognostic factor have been reported. To date, only 2 studies have indicated that reduced expression of p27KIP1 showed an association with survival outcome.¹⁸, ¹⁹ To further address this issue, we evaluated the relationship between p27KIP1 protein expression in a series of 50 astrocytomas with clinicopathologic parameters including age, tumor grade, MIB-1 proliferation index, and patient survival, using both Western blot analysis and immunohistochemistry.

Levels of p27KIP1 protein expression were first assessed in 9 epileptic brain specimens using Western blot analysis to determine the range of p27KIP1 protein expression associated with nonneoplastic brain tissue. This provided a cutoff point that allowed us to group patients with brain tumors as low or high expressors of the p27KIP1 protein. For immunohistochemical analysis of p27KIP1 protein expression, there is a well-established convention for grouping samples demonstrating <50% p27KIP1-positive tumor cells as low expressors and samples demonstrating >50% p27KIP1-positive tumor cells as high expressors.⁹ A total of 16 fresh high-grade astrocytomas were analyzed by Western blotting, and 26 archival paraffin-embedded high-grade astrocytomas (Table 1, Table 2) were analyzed by immunohistochemistry for levels of p27KIP1 protein expression. Regardless of the technique used, approximately 50% of the high-grade tumors were low expressors of p27KIP1 and 50% were high expressors. Thus our data suggest, contrary to several other reports,¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰ that p27KIP1 expression in high-grade astrocytomas is independent of tumor grade and the aggressive growth behavior characteristic of these highly malignant tumors.

P27KIP1 is a CKI that acts as a negative regulator of progression through the G₁ and S phases of the cell cycle. Loss of p27KIP1 expression in tumor cells would be expected to influence the cells' proliferation rates. Thus, many studies examining the role of p27KIP1 as a prognostic marker in human cancers also measure expression of the proliferation marker Ki-67 antigen detected with the monoclonal antibody MIB-1. Some studies of astrocytomas have reported an inverse relationship between p27KIP1 expression and proliferative activity within the same tumors as assessed by Ki-67 immunostaining.¹⁶, ¹⁹, ²² We also measured the rates of tumor cell proliferation using MIB-1 immunostaining in a subset of the high-grade astrocytomas (Table 2) to determine if there was an inverse correlation between p27KIP1 expression and proliferation. We found no correlation between p27KIP1 protein expression and MIB-1 LI. The average MIB-1 LIs were 15% in the low expressors of p27KIP1 and 20% in the high expressors. Our findings are similar to those reported by others. A large study of 130 astrocytomas by Mizumatsu et al,¹⁸ in which it was claimed that p27KIP1 was a prognostic marker related to favorable outcome in astrocytoma patients, reported no correlation between the Ki-67 and p27KIP1 LI parameters. A study by Nakasu et al²² noted 2 staining patterns among the 28 astrocytomas evaluated for both p27KIP1 expression and MIB-1 proliferation index. Although some tumors showed an inverse relationship, a subset of gliomas also showed very high MIB-1 LI, despite a high level of p27KIP1 expression, similar to our results reported herein. Similar findings were described by Fuse et al¹⁹ in a study of 42 gliomas, although the primary data to support this claim, including the LI for p27KIP1 or Ki-67 for the tumors that they studied, were not presented in their report. Taken together, these results suggest that malignant gliomas not only are a heterogeneous group of tumors, but also are tumors that differ from epithelial cancers in the relationship between the level of p27KIP1 expression and the rate of proliferation in the same tumors. Further studies are required to determine the relationship, if any, between p27KIP1 expression and MIB-1 LI in gliomas.

What other factors could influence p27KIP1 expression in gliomas? We would like to advance the notion that unfavorable changes in the microenvironment, such as hypoxia, could result in the up-regulation of p27KIP1 expression in high-grade astrocytomas that we have noted in this study. Using immunohistochemistry, we showed that the greatest density of immunopositive tumor cells and the highest intensity of p27KIP1 staining were seen in perinecrotic pseudopalisading cells of glioblastomas that outline areas of necrosis (Fig 2). This staining pattern of p27KIP1 in the pseudopalisading cells was a constant feature observed in gliomas. To our knowledge, this is the first report of such a finding.¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹, ²², ²³

A recent study of small cell lung cancer also demonstrated high levels of p27KIP1 expression.²⁶ Transfection of p27KIP1 into small cell lung cancer cell lines prevented apoptosis when cell cultures were grown under unfavorable growth conditions, such as nutrient deprivation or hypoxia.²⁷ These results suggest that high expression of p27KIP1 favors cell survival under adverse growth conditions. This would be consistent with our previous observation in gliomas for the expression pattern of hypoxia-inducible factor 1 (HIF-1)α in glioblastomas.²⁸ HIF-1 is a heterodimeric basic helix–loop helix–PAS transcription factor composed of HIF-1α and HIF-1β subunits. The HIF-1α subunit is unique to HIF-1. Its expression increases as cellular O₂ concentration decreases, thus reflecting the level of HIF-1 activity.²⁸ HIF-1α was expressed primarily in tumor cells palisaded around necrotic zones. We also found that HIF-1α expression was significantly greater in high-grade astrocytomas than in low-grade astrocytomas and was associated with the degree of vascularity.²⁸ Thus, a plausible explanation for the maintenance of high levels of p27KIP1 in gliomas is its up-regulation through hypoxia to prevent apoptosis in tumor cells in hypoxic zones throughout the tumor. Several studies have shown that hypoxia up-regulates p27KIP1 in embryonic stem cells, murine embryonic fibroblasts, human ovarian carcinoma cells, and human seminoma cell lines.²⁹, ³⁰, ³¹, ³² One interpretation of our results is that hypoxia also may be associated with the up-regulation of p27KIP1 in gliomas. Further studies are needed to evaluate this hypothesis.

Factors predicting the prognosis in patients with gliomas could offer therapeutic options. The aim of this study, as well as other investigations,¹⁸, ²³ was to examine the utility of p27KIP1 expression in astrocytomas as an independent prognostic marker for patient outcome. In this study, we found no correlation between p27KIP1 protein expression and any of the clinicopathologic parameters tested, including patient age and tumor grade, the 2 strongest predictors of survival in glioma patients.³³, ³⁴, ³⁵, ³⁶ The continuing challenge for molecular neuro-oncologists will be to catalog multiple genetic alterations in a single tumor specimen. Tumor classification based on DNA microarray gene expression profiling has recently been applied to medulloblastomas, another heterogeneous group of tumors.³⁶ Using this approach, the clinical outcome for children with medulloblastomas was highly predictable. In the near future, gliomas, which are very heterogeneous in their histopathology and biology, may benefit from gene-based predictors for clinical outcomes and advance novel therapeutic approaches for the treatment of this malignant disease.

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