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Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 520-525
Practical value of MIB-1 index in predicting behavior of astrocytomas

1 Department of Histopathology, Apollo Specialty Hospital, Chennai, India
2 Department of Histopathology, Father Muller Medical College, Mangalore, India

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Date of Web Publication20-Sep-2011


Background : The MIB-1 labeling index (LI) has proved to be useful in assigning grading and prognosis to astrocytomas. The purpose of our study was to analyze the utility of MIB-1 LI in differentiating astrocytomas of varying grades and the possible relationships of MIB-1 LI with clinical parameters like age and sex. We also wanted to study the prognostic role of MIB-1 index in predicting behavior of astrocytomas. Materials and Methods : Our study included 145 patients with astrocytic tumors of varying grades. Immunolabeling for all patients was done using MIB-1 antibody. Survival data could be obtained for 64 patients. A Mann-Whitney U test was used to test the difference in MIB-1 LI between different histological grades. The univariate analysis was done by the Kaplan-Meier method, and the multivariate analysis for survival was performed using the Cox proportional hazard model. Results : Significant differences were noted in mean MIB-1 LI of high-grade and low-grade diffuse astrocytomas. MIB-1 LI did not vary significantly with age and sex. Univariate analysis showed favorable prognostic factors for low histopathological grade, young patient age and low MIB-1 LI; however, multivariate analysis showed that only histopathological grade had independent prognostic significance. Conclusions : Our study proves that MIB-1 LI is not dependent on factors like age and sex and is solely dependent on histological grade. Though the average level of MIB-1 LI varies considerably in the different grades of astrocytomas, considerable overlap can be observed between them. MIB-1 LI is a very useful adjunct to the histopathological diagnosis and can be of great help in situations where the clinical and radiological findings do not correlate with histological diagnosis.

Keywords: Astrocytomas, Ki-67, MIB-1 LI, prognostic value

How to cite this article:
Ambroise M M, Khosla C, Ghosh M, Mallikarjuna V S, Annapurneswari S. Practical value of MIB-1 index in predicting behavior of astrocytomas. Indian J Pathol Microbiol 2011;54:520-5

How to cite this URL:
Ambroise M M, Khosla C, Ghosh M, Mallikarjuna V S, Annapurneswari S. Practical value of MIB-1 index in predicting behavior of astrocytomas. Indian J Pathol Microbiol [serial online] 2011 [cited 2021 Jan 22];54:520-5. Available from: https://www.ijpmonline.org/text.asp?2011/54/3/520/85085

   Introduction Top

Astrocytic tumors constitute a wide range of neoplasms that differ in their location, age distribution, growth potential, extent of invasiveness, morphological features and tendency for progression. A progressively poorer prognosis is associated with increasingly undifferentiated histological features of the tumors across this range. Though histopathological features largely help in the determination of prognosis, histological differentiation may not be clear in some cases, especially when only small fragments of tissue from stereotactically guided needle biopsies are available. Studies have employed a wide range of parameters - from tumor suppressor genes to proliferation indices - for predicting clinical outcome and survival.

Many studies have focused on the proliferative activity in these tumors, especially Ki- 67/MIB-1 labeling index. The Ki-67 antigen is expressed during all active phases of the cell cycle (G1 , S, G 2 and M phases) but absent in the resting phase. The MIB-1 antibody recognizes the Ki-67 antigen in both formalin-fixed and paraffin-embedded tissue. [1],[2] The MIB-1 antibody has been used in assigning grading and prognosis to astrocytomas. The purpose of our study was to study and establish the means and ranges of MIB-1 labeling index in astrocytomas and to asses its utility in differentiating astrocytomas of varying grades. We also wanted to analyze the possible relationships of MIB-1 labeling index with clinical parameters like age and sex and to study its role as a prognostic indicator.

   Materials and Methods Top

This study included 145 patients with astrocytic tumors of varying grades diagnosed from January 2005 to June 2007. All the cases were reviewed appropriately and graded using the World Health Organization (WHO) criteria published in 2007. [3] The cases which were originally graded using the St. Anne/Mayo grading system at the time of diagnosis were also classified as per the WHO guidelines. St. Anne/Mayo grades 2 to 4 closely correspond to WHO grades II to IV. In the St Anne/Mayo grading scheme, a single mitotic figure is sufficient to warrant a diagnosis of anaplastic or grade 3 astrocytoma. Since this rule is questionable for large resection specimens, the WHO classification takes the sample size also into consideration.

All patients with scanty/predominantly necrotic material were not included in our study. Our study included 23 patients of pilocytic astrocytomas, WHO grade I; 34 patients of diffuse astrocytomas, WHO grade II (including 4 patients of gemistocytic astrocytomas); 21 patients of anaplastic astrocytomas, WHO grade III; and 67 patients of glioblastoma, WHO grade IV. The mean ages of patients with the four grades I, II, III and IV were 12.96, 36.79, 41.52 and 49.55 years, respectively. Immunolabeling was done using MIB-1 antibody (MIB-1 mouse monoclonal antibody from Novocastra Laboratories Ltd., Newcastle upon Tyne, UK, in a dilution of 1:100).

Representative blocks of formalin-fixed paraffin-embedded tissue of these patients were selected, and 4-mm thick paraffin sections were floated onto slides previously coated with poly-L-lysine. Antigen retrieval was done by boiling in a pressure cooker using sodium citrate buffer (0.01 M; pH, 6.0). Immunostain visualization was achieved with the standard streptavidin-biotin peroxidase technique. The slides were stained with 3, 3'-diaminobenzidine, counterstained with hematoxylin and mounted. During each batch of staining, appropriate positive and negative controls were used. Tonsil was used as positive control, and a negative control slide in which the primary antibody was excluded was used for each batch of slides.

Immunostaining was evaluated in the fields consisting of regions of the tumor having the greatest number of immunoreactive cells as assessed qualitatively at low-power examination. Where an uneven distribution of immunohistochemical labeling was evident, fields from the area of maximal labeling were chosen for counting. In glioblastomas, areas of sections free from necrosis or capillary endothelial proliferation were selected. The MIB-1 labeling index (LI) was the number of MIB-1-labeled tumor nuclei expressed as a percentage of the total number of tumor nuclei counted. A total of at least 1,000 tumor nuclei were counted in each case.

Of the 145 patients for whom MIB-1 estimation was performed , survival information could be obtained for 64 patients. The survival of these patients was assessed on the basis of the follow-up information obtained from the clinical records and by telephonic calls to patients or families. The only statistical endpoint was length of survival. Follow-up duration ranged from 33 to 63 months. A Mann-Whitney U test was used to test the difference in MIB-1 LI between different histological grades. The univariate analysis was done by generating probability curves according to the Kaplan-Meier method and comparing them using the log rank test. Multivariate analysis for survival was performed using the Cox proportional hazard model. In all cases, only P values less than .05 were considered significant. PASW (Predictive Analytics Software) statistics18 software was used for statistical analysis.

The study was approved by the ethics committee, and a waiver of obtaining informed consent was also granted for this retrospective study.

   Results Top

The results with regard to MIB-1 LI in astrocytomas of varying grades are summarized in [Table 1]. The difference in the mean MIB-1 LI between pilocytic astrocytomas and diffuse astrocytomas was not significant statistically [Table 2]. However, the mean MIB-1 LI of pilocytic astrocytomas was significantly lower when compared to that of anaplastic astrocytomas (P value, .016) and glioblastomas (GBMs) (P value, <.0001). The mean MIB-1 LI of diffuse astrocytomas was also significantly lower when compared to that of anaplastic astrocytomas (P value, .004) and GBMs (P value, <.0001). The difference in the mean LI of anaplastic astrocytomas when compared with that of GBMs was also significant statistically (P value, .010). The mean MIB-1 LI of gemistocytic astrocytomas was 3.82 (range, 1.1-5.1). Majority of the pilocytic astrocytomas and diffuse astrocytomas had LI less than 7.5 (Contast [Figure 1] with [Figure 2]); however, considerable overlap was noted in MIB-1 LI across all tumor grades. MIB-1 LI did not vary significantly with respect to age and sex (detailed calculations not shown).
Table 1: Summary of MIB-1 LI in various grades of astrocytomas

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Table 2: Frequency distribution of MIB-1 LI in astrocytomas of various grades

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Figure 1: Photomicrograph showing few MIB-1-labeled nuclei in a case of diff use astrocytoma (MIB-1, x400)

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Figure 2: Photomicrograph illustrating a glioblastoma with numerous MIB-1-labeled nuclei (MIB-1, x400)

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Among the 64 patients for whom survival data was obtained, 15 tumors were grade I, 19 tumors were grade II (including 2 gemistocytic astrocytomas), 9 tumors were grade III and 21 tumors were grade IV. All the patients of pilocytic astrocytomas had survived. Survival analysis was used for the other three grades only.

In astrocytomas, overall, univariate analysis showed favorable prognostic factors for low histopathological grade, young patient age and low MIB-1 LI [Table 3],[Figure 3].The factors which were significant in the univariate analysis were also analyzed by multivariate analysis.
Table 3: Univariate analysis of prognostic factors

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Figure 3: Kaplan-Meier survival curves for tumors with MIB-1 LI ≤5.0 and MIB-1 LI >5.0. A statistically significant diff erence was noted between the two groups (P value.,010)

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Histopathological grade had independent prognostic significance in multivariate analysis also. MIB-1 labeling index and age lost their significance [Table 4].
Table 4: Multivariate analysis of prognostic factors

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Survival analysis of variables was also done for different histological grades of astrocytomas. We could not identify any significant prognostic cut-off levels for MIB-1 labeling index in all the three grades of astrocytomas. Survival with respect to age was not significant in grade II astrocytomas. Though survival was better in younger individuals in anaplastic astrocytomas, the results were not statistically significant. In GBMs, the survival was significantly better in the age group of 20-45 years.

The behavior of 2 gemistocytic astrocytomas was apparently different when compared to that of the rest of the grade II astrocytomas. Both these tumors occurred in males aged 26 and 32 with survival period of 29 and 30 months, respectively, and one of them showed progression. Their MIB-1 labeling indices were 4.8 and 5.1, respectively.

In cases of GBMs, the duration of symptoms did not affect survival significantly. Though the survival of GBM cases with midline shift was lesser compared to GBM cases without midline shift, statistical significance was not evident.

   Discussion Top

Pilocytic astrocytomas have distinct clinical, pathological and prognostic characteristics when compared to diffuse astrocytomas. They are generally well circumscribed and have an excellent long-term prognosis. However, in our study, the MIB-1 labeling index was remarkably high in a few patients, and the mean LI did not significantly differ when compared to that of patients with diffuse astrocytomas. Two patients of pilocytic astrocytomas had LI in the range comparable with that seen in high-grade astrocytomas. Both these patients did not show any atypical histopathological features. The radiological features were also suggestive of pilocytic astrocytoma. One of them had a recurrence, but both the patients are alive, with a follow-up period of more than 3 years. The behavior of both these patients was not different from that of the rest of the pilocytic astrocytomas. Relatively few studies have analyzed MIB-1 labeling index in pilocytic astrocytomas compared to the other three grades. The mean MIB-1 index in pilocytic astrocytomas varies significantly between studies and ranges from 0.44 to 4.8 [Table 5]. [4],[5],[6],[7],[8],[9],[10]
Table 5: Mean [range] of MIB-1 labeling index in astrocytomas of varying grades - A survey of previous studies

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Some studies showed a very low LI for pilocytic astrocytomas and found significant difference in the distribution of MIB-1 LI between pilocytic and diffuse infiltrating astrocytomas. [5],[7] However, other studies have varied results. In an analysis of astrocytomas in children and young adults, no significant difference in LI was found between pilocytic and diffuse infiltrating astrocytomas. One of their pilocytic astrocytoma patients had a high LI (18.6), comparable with LI seen in high-grade astrocytomas and detailed clinical and pathological examinations could not provide any reason for this. [4]

Another study evaluated MIB-1 LI in cerebellar juvenile pilocytic astrocytoma, and the LI ranged from 0.6% to 12%. The mean LI in classical pilocytic astrocytoma did not differ significantly when compared to LI in cases of pilocytic astrocytoma with atypical/anaplastic appearance and in cases showing high amount of neovascularization. [6] This study also did not reveal any significant prognostic role for MIB-1 labeling index in pilocytic astrocytomas. A recent study could not find any significant association between MIB-1 labeling index and event-free survival.[11] A double-labeling study of pilocytic astrocytomas and diffuse astrocytomas using MIB-1 as a proliferation marker and CD68 as microglial marker showed different proliferative activities at different grades of malignancy, with the highest rates of proliferating microglia seen in pilocytic astrocytomas. Thus high MIB-1 LI values in some cases of pilocytic astrocytomas which otherwise behaved in a benign fashion could have probably resulted from proliferating microglial cells. [12] All these facts suggest a limited role for MIB-1 labeling index in determining the diagnosis and prognosis in cases of pilocytic astrocytomas.

Most of the studies have found significant differences in MIB-1 LI between high- and low-grade diffuse astrocytomas. [5],[13],[14],[15] Though some studies have found significant difference in MIB- 1 LI between anaplastic astrocytomas and glioblastomas,[13],[15] others could not find a significant difference between them. [14],[16] However, the average level of MIB-1 LI is found to vary considerably in the different tumor categories when we analyze [Table 5]. For example, the mean LI for GBM varies from 9.1 to as high as 46. Another problem in the diagnostic value of MIB-1 LI is considerable overlap across tumor grades. The LI for high-grade astrocytomas can significantly overlap with that for low-grade astrocytomas. All these facts were evident in our study also.

Johannessen and Torp [17] analyzed 16 studies comprising a total value of MIB-1 LI of 915 patients and found an average value of MIB-1 LI for diffuse astrocytomas, anaplastic astrocytomas and glioblastomas to be approximately 3, 12, and 16, respectively. An analysis of [Table 2] in our study reveals that all diffuse astrocytomas had an MIB-1 LI less than 10%, and in fact more than 90% of diffuse and pilocytic astrocytomas had MIB-1 LI values less than 7.5%. However, it is evident from our study and other earlier studies that a low MIB-1 LI does not rule out high-grade astrocytomas.

Some studies have found that MIB-1 LI is dependent on age, [10],[14],[16] whereas others have not found a similar association. [5],[6],[9] Our study proved that MIB-1 LI is not dependent on factors like age and sex but is solely dependent on histological grade.

Regarding prognostic indicators for astrocytomas, the histopathological grade still appears to be the best guide to prognosis. Age to a certain extent also affects prognosis. The present study reveals that survival of patients with non-pilocytic astrocytomas showing an MIB-1 labeling index of more than 5% was lesser than that of patients with MIB-1 labeling index of less than 5%. However, the significance of this was lost when adjusted for other factors. Though some studies claim that increased MIB-1 LI is an independent prognostic variable, [18],[19],[5] others find it significant only in univariate analysis. [20],[21],[16]

Studies have also differed on identifying a cut-off value for distinguishing between astrocytomas with good and poor prognosis. In their study, Jaros et al. [18] found that a value of 5% is useful for this purpose, whereas Ellison et al. [21] have suggested a value of 2%. Another study found that 3% is useful as a cut-off point in differentiating with regard to the survival times. [19] This variation could be due to inter-laboratory variation in MIB-1 estimation.

In an exclusive study of grade II astrocytomas, MIB-1 proliferation index was found to be an independent prognostic factor. [22] But Hilton et al. [23] could not establish any significant prognostic role for MIB-1 index in fibrillary astrocytomas. Gemistocytic astrocytoma though classified as a grade II tumor generally shows an aggressive behavior and is characterized by a low proliferative activity. In an extensive study on gemistocytic astrocytomas, majority of the patients were young adults, with a marked male preponderance and with a mean MIB-1 LI of 3.7%. [24]

Similarly a clinicopathologic study of 85 similarly treated patients with anaplastic astrocytic tumors revealed that only age was a significant prognostic marker, and proliferation markers were not prognostically significant when age was taken into account. [25] The results of univariate survival analysis in another study revealed that both the progression-free survival and overall survival were significantly reduced in patients with anaplastic astrocytomas with Ki-67 LI >5%. However, this significance was lost in multivariate analysis. [26]

Studies which investigated the role of immunohistochemical markers in glioblastomas did not reveal any prognostic role for MIB-1 labeling index.[27],[28] Another extensive study on glioblastomas also proved that MIB-1 estimation does not provide additional independent information for predicting survival or response to radiation. [29]

Many factors are responsible for such a variation in MIB-1 LI between studies. The MIB-1 LI can be influenced by the fixative used [28] ; immunohistochemical procedures, especially antigen retrieval [13] ; and interpretation of the immunostaining. A low MIB-1 LI value in high-grade astrocytoma could also result from faulty tissue sampling and tumor heterogeneity. Antigen retrieval can be better with hydrated autoclave treatment than with microwave treatment and can result in a higher MIB-1 LI. [13] This could possibly result from more successful denaturation of formalin-fixed antigens. Computer-assisted methods for quantitation of LI seem to underestimate LI (up to 30%) compared to manual methods. [5],[6] Inter-observer variability can also affect the MIB-1 LI estimation. [30]

All these facts have to be remembered while planning the treatment strategy using the MIB-1 LI. Absolute and cut-off values of MIB-1 LIs cannot be reliably used between different laboratories. However, standardization of staining protocols and counting procedures and also clinicopathological correlation are essential while interpreting the MIB-1 LI.

In conclusion, our study emphasizes that MIB-1 LI is not dependent on factors like age and sex and is solely dependent on histological grade. Though the average level of MIB-1 LI varies considerably in the different grades of astrocytomas, considerable overlap can be observed between them; and MIB-1 LI has certain limitations in cases of pilocytic astrocytomas. Thus MIB-1 LI should be used prudently in combination with histopathological features. Though clinical parameters and histopathological grade are important prognostic indicators in astrocytomas, MIB-1 LI is a very useful adjunct to the histopathological diagnosis and can be of great help in situations where there is lack of correlation between histological diagnosis and clinical parameters.

   Acknowledgment Top

We are very thankful to our Immunohistochemistry technicians Mr. Isac Immanuel T., Mrs. Saraswathi K. and Mrs. Kokila M. for all their hard work and technical support for this study.

   References Top

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4.Matsumoto T, Fujii T, Yabe M, Oka K, Hoshi T, Sato K. MIB-1 and p53 immunocytochemistry for differentiating pilocytic astrocytomas and astrocytomas from anaplastic astrocytomas and glioblastomas in children and young adults. Histopathology 1998;33:446-52.  Back to cited text no. 4
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8.Tihan T, Davis R, Elowitz E, DiCostanzo D, Moll U. Practical Value of Ki-67 and p53 Labeling Indexes in Stereotactic Biopsies of Diffuse and Pilocytic Astrocytomas. Arch Pathol Lab Med 2000;124:108-13.  Back to cited text no. 8
9.Bowers DC, Gargan L, Kapur P, Reisch JS, Mulne AF, Shapiro KN, et al. Study of the MIB-1 Labeling Index as a Predictor of Tumor Progression in Pilocytic Astrocytomas in Children and Adolescents. J Clin Oncol 2003;21:2968-73.  Back to cited text no. 9
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Correspondence Address:
M Moses Ambroise
Plot No. 92, Veeramamunivar Street, Radhakrishna Nagar, Thattanchavady Post, Pondicherry - 605 009
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.85085

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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