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Study of Ki-67 expression and DNA ploidy in various grades of meningioma

Salem, Mostafaa; Wagih, Mohamedc; Badawy, Manalb

doi: 10.1097/01.XEJ.0000421479.33369.b9
ORIGINAL ARTICLES
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Background Although histological grade is the most relevant prognostic factor in meningiomas, there are some unusual cases in which establishing a diagnosis of high-grade meningioma is extremely difficult. Thus, the search for ancillary techniques may result in improved diagnostic and prognostic accuracy in such cases. In the current study, we aimed to assess the significance of both the immunohistochemical expression of Ki-67 antigen and DNA ploidy status using an image analyzer in various grades of meningioma. Fifty patients (32 women and 18 men) with a diagnosis of meningioma were included in this study. Among the studied samples, there were 38 (76%) benign (grade I), eight (16%) atypical (grade II), and four (8%) anaplastic (grade III) meningiomas.

Results Immunohistochemical study showed that Ki-67 antigen was expressed in all tumors with a low labeling index (LI) (<15%) found in 66% of cases and a high LI (>15%) found in 34% of cases. A statistically significant correlation was found between Ki-67 LI and histological grading and the values increased significantly from grade I to grade III tumors (P=0.001). The mean LIs were 4.3±12.2, 19.6±28.5, and 44.8±23.3% for grade I–III meningiomas, respectively. The result of image analysis of DNA showed that 56% of the studied meningiomas contained only diploid cells, whereas 44% were composed of aneuploid cell populations. Atypical and anaplastic meningiomas showed a significantly higher DNA aneuploidy (P=0.009) and S-phase fraction (P=0.01) compared with benign tumors. Both Ki-6 LI and DNA ploidy status correlated with each other (P=0.007) and allowed a significant differentiation between different grades of meningiomas.

Conclusion We conclude that Ki-67 LI in conjunction with DNA ploidy could be used to support histopathological parameters for accurate diagnosis and grading of meningiomas and appear to be of greatest value in the evaluation of tumors with borderline atypia.

aDepartment of Pathology, Faculty of Medicine, Cairo University

bDepartment of Basic Medical Science (Pathology Researches), National Research Centre, Cairo

cDepartment of Pathology, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt

Correspondence to Mohamed Wagih, MD, Department of Pathology, Faculty of Medicine, Beni Suef University, 62515 Beni Suef, Egypt Tel: +20 100 121 4582; fax: +20 22 575 2449; e-mail: mohamwagih@hotmail.com

Received June 19, 2012

Accepted July 3, 2012

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Introduction

Meningiomas account for about one-third of primary intracranial neoplasms and represent the largest group of benign tumors. The tumor has its origin from arachnoid cells and is most frequently found in middle-aged and elderly patients (Abry et al., 2010). Most meningiomas are benign and slow growing, and are usually curable after complete removal. However, they have an intrinsic trend to recur, and recurrence depends strongly on the tumor grade, subtype, brain infiltration, and extent of resection (Perry et al., 2007).

Although histological grading enables the approximate prediction of the biological behavior of meningiomas, the variables used for this assessment are generally based on qualitative criteria and therefore overshadow objectivity. Furthermore, there are always those cases that do not completely fulfill the histological criteria for atypia, and the mitotic index is border line. Therefore, the need for further quantitative criteria has emerged and this has led to the search for other approaches to identify more aggressive meningiomas; cell kinetic studies have therefore come into focus on the basis of the hypothesis that recurring tumors comprise a higher number of proliferating cells (Terzi et al., 2008).

Cell proliferation is a fundamental process controlled by highly coordinated mechanisms. Measurement of the proliferative activity is important to evaluate the tumor grade and to assess the possibility of recurrence and malignancy (Kayaselçuk et al., 2002). The Ki-67 is a nuclear antigen expressed in the G1, S, G2, and M phases of the cell cycle recognizable by the monoclonal antibody MIB-1 (Mastronardi, 1999). Assessment of the Ki-67-labeling index (LI) is highly recommended in combination with established histopathological features in the evaluation of the malignancy potential of tumors (Eneström et al., 1998).

DNA ploidy has also been shown to be an important factor in the assessment of tumors because it provides valuable information on tumor behavior (Zetterberg and Forsslund, 1991). The DNA content was determined by flow cytometry; however, the advantage of DNA analysis by image cytometry is that it allows the possibility of selecting nuclei to be analyzed, thus avoiding nontumor cells (Berner et al., 1995).

On the basis of this background, the aim of the present work is to investigate the utility of two different indicators of cell proliferation, the Ki-67 LI and DNA ploidy, in the evaluation of various grades of meningioma.

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Materials and methods

The material of this study consisted of 50 paraffin blocks of meningiomas derived from a group of Egyptian patients. All cases were collected from the archives of the Pathology department, Faculty of Medicine, Cairo University, and a private center during the period from March 2011 to August 2011. The cases were classified histologically according to the criteria of the WHO classification system (Perry et al., 2007). This system integrates tumor type and grade into benign meningioma (grade I), atypical meningioma (grade II), and anaplastic meningioma (grade III).

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Ki-67 immunostaining

Immunostaining was carried out on formalin-fixed paraffin-embedded tissue sections using the avidin–biotin immunoperoxidase system for the detection of Ki-67 antigen. The sections were deparaffinized and rehydrated in graded alcohols (95, 85, and then 70%). Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 5 min, followed by a wash for 5 min with PBS. For antigen retrieval, the sections were boiled in a microwave oven in PBS at 100°C three times, 5 min each, and were allowed to cool for 30 min at room temperature and then washed with PBS for 5 min. The sections were incubated with a blocking reagent (normal rabbit serum) for 20 min to suppress nonspecific binding of immunoglobulins. Next, tissue sections were incubated with the MIB-1 mouse antihuman monoclonal antibody (code DF8296; Dako, Glostrup, Denmark) diluted at 1 : 60. The sections were washed twice for 5 min with PBS and incubated for 20 min with anti-mouse biotinylated secondary antibody (Dako). The slides were washed twice for 10 min with PBS and incubated for 20 min in avidin-biotinylated peroxidase complex (Dako). Then, the sections were washed twice for 5 min with PBS and treated with 3,3 diamino benzedine tetrachloride (Dako) as a chromogen and incubated for 3 min at room temperature. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, cleaned, and mounted. The positive control used was a tissue section obtained from a case of non-Hodgkin lymphoma, whereas the negative control was prepared by excluding the primary antibody; PBS was instead used in this step.

Ki-67 immunostaining was evaluated using the CAS 200 Image Analyzer (Becton Dickinson, Cell Analysis System, Elmhurst, Illinios, USA) in combination with the Quantitative Proliferation Index CAS software program (Phoenix Cell Analysis System, Joplin, Missouri, USA). In each case, the analysis was carried out on areas expressing quantitatively the highest number of immunopositive nuclei and 10 microscopic fields at ×400 magnification were measured for each case. The results were expressed as the Ki-67 LI, which is defined as the percentage of positively stained nuclei divided by the total number of counted cells. Tumors were divided into those with a low LI (<15% stained nuclei) and those with a high LI (>15% stained nuclei) (Tallini et al., 1999).

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DNA ploidy analysis

Five to seven micrometer thick sections from the paraffin blocks of the studied cases were deparaffinized and rehydrated. Each staining batch of 10 slides was treated with 5 N hydrochloric acid for 60 min at room temperature and then stained with Fuelgen stain using CAS Quantitative DNA staining (Becton Dickinson, Cell Analysis System), which allows the colored compound in the stained nuclei to be directly proportional to the DNA content within the nucleus and can thus be measured as a quantifiable integrated optical density.

Quantification of nuclear DNA staining of the studied cases was carried out using the CAS 200 Image Analyzer (Becton Dickinson, Cell Analysis System) and the quantitative ploidy analysis software program. Normal rabbit hepatocytes were treated like the tumor tissue and used as a control of diploid cells. Degenerating tumor nuclei, obviously artifactually distorted nuclei, and overlapping nuclei were rejected. For each case, 200 cells were analyzed for nuclear DNA content. DNA histograms were generated and the ploidy of tumor samples was estimated by the DNA index. A sample was defined as DNA diploid if there was a single G0G1 peak and the DNA index had a value between 0.9 and 1.1. Samples with a DNA index of the second peak of more than 1.1 or less than 0.9 were defined as DNA aneuploid. The proliferation index automatically expressed as the percentage of cells engaged in the S-phase of the cell cycle was classified into low (10–20%), moderate (20–30%), and marked (>30%) (Arai et al., 1998).

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Statistical analysis

All numerical data were expressed as mean±SD. Statistical analysis was carried out using the χ2-test, t-test, and analysis of variance test using the SPSS software program, version 11 (SPSS Inc., Chicago, Illinois, USA). P values of less than 0.05 were considered to be significant.

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Results

Among the 50 studied cases, there were 32 (64%) female and 18 (36%) male patients (F : M ratio 1 : 1.8). Their ages ranged from 26 to 79 years, with a mean age of 53.7 years. The cerebral convexities were the predominant localization of the tumor in our studied cases, representing 40% of cases, followed by the parasagittal and paratentorial areas (28%), the skull base (24%), spinal (4%), lateral ventricles (2%), and orbital (2%). There were 38 (76%) benign (WHO grade I), eight (16%) atypical (WHO grade II), and four (8%) anaplastic (WHO grade III) meningiomas.

Positive nuclear immunostaining for Ki-67 was found in all cases. Thirty-three cases (66%) showed a low Ki-67 LI (<15%), whereas 17 cases (34%) showed a high Ki-67 LI (>15%) (Figs 1–5). Our results showed that there was a statistically significant correlation between Ki-67 LI and the WHO meningioma grade (P=0.001). The mean Ki-67 LI was 4.3±12.2 for grade I, 19.6±28.5 for grade II, and 44.8±23.3 for grade III tumors (Table 1).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Fig. 5

Fig. 5

Table 1

Table 1

Twenty-eight cases (56%) in the present study were diploid, whereas 22 cases (44%) showed aneuploid cell populations, of which nine (18%) cases were hypodiploid (DNA index<0.9) and 13 (26%) cases were hyperdiploid (DNA index>1.1) (Fig. 6). The mean DNA index in the studied cases was 0.75±0.21, 0.93±0.53, and 1.32±0.86 in the hypodiploid, diploid, and hyperdiploid cases, respectively. Table 2 shows the DNA ploidy status in different tumor grades. All of the anaplastic meningiomas (WHO grade III) and six of the eight (75%) of the atypical meningiomas (WHO grade II) showed DNA aneuploid populations, whereas of the benign (WHO grade I) meningiomas, only 12 of 38 (31.6%) were DNA aneuploid. This difference was found to be statistically significant (P=0.009). A significant correlation was also found between the WHO tumor grade and the proliferative index [S-phase fraction (SPF)]. The mean percentage of cells in the S-phase was 21.63±7.62 in benign (WHO grade I) meningiomas, 27.36±3.56 in atypical (WHO grade II) meningiomas, and 62.78±12.82 in anaplastic (WHO grade III) meningiomas (P=0.01) (Table 3). In addition, the aneuploid tumors showed significantly higher mean Ki-67 LI and mean SPF values than diploid tumors. The mean Ki-67 LI was 5.7±10.3 in diploid and 26.2±17.6 in aneuploid cases, whereas the mean SPF was 22.3±19.5 in diploid and 33.8±21.7 in aneuploid cases (Table 4).

Fig. 6

Fig. 6

Table 2

Table 2

Table 3

Table 3

Table 4

Table 4

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Discussion

Among neurosurgical tumors, meningiomas are usually considered to be a clinically benign entity. However, the presence of atypical/malignant histopathological features and, sometimes, the localization of the tumor potentiates their risk of recurrence (Louis et al., 2000). Although the WHO classification includes well-defined histological criteria, there are some unusual cases in which establishing a diagnosis of high-grade meningioma is extremely difficult (Bruna et al., 2007). Accordingly, the search for prognostic factors that could be used at diagnosis to identify a subgroup of tumors that are at a high risk of relapse represents a major challenge. Among all disease characteristics, those parameters that are related to the biology of the neoplastic cells such as the presence of genetic abnormalities at the DNA level, as well as the proliferative ability of human tumors, have frequently been shown to contain predictive information for disease outcome. In this sense, previous reports have indicated that the presence of DNA aneuploidy detected in tumors from the central nervous system is frequently associated with malignancy (Tsukazaki et al., 2000; Gardina et al., 2008; Alexiou et al., 2009). Similarly, in neurological tumors, a high proliferative rate has been associated with a worse clinical outcome (Johannessen and Torp, 2006; Yoshida et al., 2010; Habberstad et al., 2011). Despite this, for meningioma tumors, few reports have been published in which the prognostic value of both parameters has been explored.

In the current study, the mean Ki-67 LI was correlated significantly with the histological grade of meningioma. Grade III tumors tended to have a statistically significant higher Ki-67 LI compared with grades II and I tumors. These findings were in agreement with previous studies that have confirmed an increase in the Ki-67 proliferation index within the spectrum of meningiomas and its prognostic significance (Takahashi et al., 2004; Moradi et al., 2008; Shayanfar et al., 2010).

However, in all grades, the observed mean Ki-67 LIs were different from those reported in other studies. This considerable variation from one study to another could be related to several aspects of the immunohistochemical procedure, for instance, the choice of tumor areas to count and the numbers of cells counted to calculate a LI. Tumor heterogeneity with regional differences in cell proliferation is well known; thus, tumor sampling becomes an important source of error as it is crucial to select the block with representative tumor tissue for calculation. As a rule, the area with the most malignant histological appearance is selected for the estimation of proliferative activity. The definition of positive immunostaining is another element of uncertainty, and only distinct nuclear staining should be interpreted as positive. These aspects are the basis of the interobserver variability associated with the determination of proliferative indices (Prayson, 2005).

Between grade I and II/III meningiomas, all studies found a statistically significant difference in the Ki-67/MIB-1 LIs, whereas this was not always the case between grade II and III tumors. For this reason, some authors group the two latter under the entity ‘aggressive meningiomas’. Because of overlap of indices between the malignancy groups, it is difficult to pinpoint a specific LI indicative for a specific tumor grade. Accordingly, a low index does not always indicate a low-grade meningioma as grade II, III tumors may show low proliferative activity as well. In contrast, a high proliferative index in an otherwise benign-looking meningioma should call attention to a more aggressive tumor. Nevertheless, the Ki-67/MIB-1 LI of a particular tumor should be interpreted with caution and in conjunction with established histopathological features of malignancy (Abry et al., 2010).

In the present study, we found no correlation between Ki-67 expression and other demographic and clinical parameters such as age, sex, and tumor location. Baumgartner and Sorenson (1996) have reported a high Ki-67 LI in pediatric meningiomas, with a higher frequency of recurrence. However, Sandberg et al. (2001) have shown that Ki-67 LI in these tumors did not differ significantly from that in adults and added that the more aggressive features of meningiomas in children may be attributable to factors other than the growth rate. Some reports have found a statistically significant higher Ki-67 LI in male patients than in female patients (Matsuno et al., 1996; Wolfsberger et al., 2004), whereas others have not (Roser et al., 2004; Korhonen et al., 2006). In terms of tumor location, most meningiomas arise in intracranial, intraspinal, and orbital locations, and obvious regional differences in proliferative activity have not been found; however, in one study, lower indices in spinal meningiomas were found (Roser et al., 2006).

The assessment of abnormal cellular DNA content can provide clues about the aggressiveness of various tumors and has been shown to be of considerable value in identifying different prognostic patient subsets (Alexiou et al., 2008). In the current study, 44% of the meningioma patients studied showed DNA aneuploidy, with nine (18%) cases showing hypodiploidy and 13 (26%) cases showing hyperdiploidy. This frequency is consistent with that reported by Ironside et al. (1987) (41%). However, different values of DNA aneuploidy have been reported by Arai et al. (1998) (67%) and Maíllo et al. (1999) (14%). The discrepancy in the incidence of aneuploid tumors could be attributed to technical pitfalls because of the use of paraffin-embedded samples or fresh material, lack of approaches used for the exclusion of cell multiplets, analysis of a short series of patients, and/or to the criteria used for the definition of DNA aneuploid populations. In this sense, consensus reports (Shankey et al., 1993; Ormerod et al., 1998) have pointed out that the best results are usually obtained by using either fresh or frozen material. Further studies, on the basis of larger series of meningioma patients in whom a comparative analysis of the results is obtained in fresh and archival material, are required to clarify this question.

Our results showed that DNA ploidy correlated with the tumor histological grading, as there was an increase in the percentage of aneuploid tumors with an increase in tumor grading. This was in full agreement with Spaar et al. (1987) and Butti et al. (1989), who reported a significantly higher rate of aneuploid cell lines in malignant meningiomas using flow cytometry. Also, Cruz-Sanchez et al. (1993) observed that DNA aneuploidy was significantly associated with tumor recurrence. In contrast, Akachi et al. (1991) found no increased DNA aneuploidy in malignant meningiomas, and Nishizaki et al. (1993) showed that evaluation of tumor prognosis on the basis of the presence of aneuploid cell lines yields uncertain estimates. No correlation was found in the present study between DNA ploidy status and the clinical characteristics of the patients. However, Maíllo et al. (1999) reported an association of DNA aneuploidy with older age and tumor location at the cerebral convexity in their studied meningioma cases.

Only a few previous studies have attempted to define hypodiploid and hyperdiploid cell lines in meningiomas. Although Ironside et al. (1987) found six hypodiploid and 10 hyperdiploid cell lines among 16 DNA aneuploid tumors, Cruz-Sanchez et al. (1993) detected 10 hypodiploid and seven hyperdiploid cell lines among 17 aneuploid tumors and found a slightly increased tendency of recurrence in the hyperdiploid tumor group. There is only sparse published evidence on the possible relevance of either hypodiploidy or hyperdiploidy for different biological and therapeutic behaviors of intracranial tumors. Sandberg and Turc-Carel (1987) associated chromosomal hypodiploidy with an aggressive behavior in meningiomas. For glioma cell lines, Shapiro (1989) reported that hypodiploid or near-diploid cell lines become more readily resistant to chemotherapy, whereas hyperdiploid cell lines are more sensitive.

In this study, we also confirmed previous findings of the existence of an association between aggressive meningioma subtypes and a higher proliferative rate of tumor cells as evaluated by the SPF (May et al., 1989; Cruz-Sanchez et al., 1993). SPF was also found to be significantly higher in aneuploid than in diploid tumors, which agrees with that reported by Zellner et al. (1998). Furthermore, from the prognostic point of view, Maíllo et al. (1999) reported that the proportion of S-phase cells is an independent prognostic factor for relapse prediction in meningiomas.

In terms of the relationship between the DNA ploidy status and Ki-67 expression, there was a statistically significant difference in Ki-67 LI between aneuploid and diploid tumors. Aneuploid tumors had a significantly higher mean Ki-67 LI compared with diploid tumors. This was in agreement with Meixensberger et al. (1996), who identified a similar significant correlation between ploidy status and Ki-67 LI in their prospective study of human meningiomas. They added that the nuclear DNA content is an important factor in predicting the risk of recurrence and poor clinical outcome after meningioma surgery.

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Conclusion

The results of this study suggest that Ki-67 LI and DNA ploidy can act as potential supplements in determining the histological grade of meningiomas. Furthermore, they might be beneficial as additional tools in histological borderline cases where one cannot readily differentiate an atypical meningioma from a benign or a malignant one. However, further studies are recommended to correlate Ki-67 expression and DNA ploidy with tumor recurrence, progression, and treatment.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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