|Year : 2014 | Volume
| Issue : 3 | Page : 641-645
Role of Ki-67 labeling index as an adjunct to the histopathological diagnosis and grading of astrocytomas
Mahathi Thotakura, Neelima Tirumalasetti, Ravi Krishna
Department of Pathology, NRI Medical College, Chinakakani, Guntur District, Andhra Pradesh, India
|Date of Web Publication||14-Oct-2014|
Door No.: 1-5-66-2, Mahathi Nursing Home, Ashok Nagar 3rd Lane, Guntur - 522 002, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Aim of the Study: Ki-67 labeling index (LI) is used in the assessment of cell proliferating activity. It has been widely documented for various human tumors, including the brain neoplasms. The aim of the present study is to evaluate Ki-67 as proliferative index in the grading of astrocytomas in order to predict the biological behavior of the tumor and prognosis of patients.
Materials and Methods: In the present study, a total of 105 patients with astrocytomas were included. Ki-67 LI was done on all the astrocytomas and was compared in correlation with World Health Organization histological grading of astrocytomas.
Results: The mean Ki-67 LI in Grade I astrocytomas was 3.36 ± 4.59 standard deviation (SD), 7.05 ± 4.16 SD in Grade II astrocytomas, 28.24 ± 6.23 SD in Grade III astrocytomas and 38.7 ± 7.19 SD in Grade IV astrocytomas. P values were significant between all grades of astrocytomas except between Grade I and Grade II tumors which was 0.5076.
Conclusions: Assessment of astrocytic tumor proliferative potential provides important prognostic information that supplements standard histological grading. Ki-67 LI is the simplest and most reliable methods of all. This study demonstrates that, Ki-67 LI serves as an important prognostic marker in human astrocytomas. Ki-67 LI solely correlates with a grade of the tumor. Average level of Ki-67 LI varies between different grades of astrocytic tumors but some overlap of values does exist.
Keywords: Astrocytoma, immunohistochemistry Ki-67, proliferation markers
|How to cite this article:|
Thotakura M, Tirumalasetti N, Krishna R. Role of Ki-67 labeling index as an adjunct to the histopathological diagnosis and grading of astrocytomas. J Can Res Ther 2014;10:641-5
|How to cite this URL:|
Thotakura M, Tirumalasetti N, Krishna R. Role of Ki-67 labeling index as an adjunct to the histopathological diagnosis and grading of astrocytomas. J Can Res Ther [serial online] 2014 [cited 2019 Oct 16];10:641-5. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/641/139154
| > Introduction|| |
Gliomas constitute a broad class of neuroectodermal tumors believed to originate from sustentacular neuroglial cells.  Astrocytomas form the largest group of gliomas (>75%) and diffusely infiltrating astrocytomas account for more than 60% of all the primary brain tumors. 
The survival time in about one-third of patients is poor, averaging 5 years. The prognosis is unfavorable because a significant number of brain tumors grow infiltratively and diffusely. Therefore they are not amenable to total resection and are prone to recur. They are often located in functionally highly specialized or vital regions. Moreover, therapeutic options are also limited. 
Histological grading is well accepted for evaluation of the prognosis of patients with astrocytomas. However, the morphologic criteria are not always accurate prognostic indicators of individual case.  Hence, there is a need to develop better prognostic markers with the aim of predicting tumor behavior and one such complementary method is an assessment of proliferative index of the tumor. ,
The proliferative index is a potent biologic marker that estimates the growth of neoplasms quantitatively and thus will aid in identifying the prognosis for patients with neoplasms. A variety of methods have been employed to estimate the proliferative index of central nervous system tumors. Of these, one of the most potent methods is the Ki-67 labeling index (Ki-67 LI). The value of Ki-67 LI in the assessment of cell proliferating activity has been widely documented for various human tumors, including the brain neoplasms. 
This study is designed to assess the Ki-67 LI in correlation with the World Health Organization (WHO) histological grades of astrocytomas in order to predict the biological behavior, which may give a direction for using Ki-67 LI as an adjunct to the routine histology in grading astrocytomas precisely.
| > Materials and Methods|| |
A total of 105 patients diagnosed with astrocytomas between the periods of June 2007 and June 2012 were included in the present study.
The specimens were fixed in 10% buffered formalin and were manually processed. Gross features like size, shape, color, consistency, cystic and necrotic changes were noted. The tissues were mostly in fragments/piecemeal. Wherever possible, the specimens were bisected longitudinally and a minimum of four bits each measuring 3-5 mm thickness was taken. After manual processing, sections of 3-5 μ thickness were cut and stained with routine hematoxylin and eosin (H and E). H and E stained sections were graded according to the WHO classification of central nervous system tumors. 
Ki-67 immunostaining was done on all the 105 cases. Poly-L-lysine coated slides were dried overnight at room temperature/placed at 50-60°C in oven for 1 h. Representative blocks of formalin-fixed paraffin-embedded tissues were selected and 4-μm thick paraffin sections were floated onto slides previously coated with poly-L-lysine. The MIB-1 monoclonal antibody (FLEX monoclonal mouse anti-human Ki-67 antigen clone MIB-1) was used as the primary antibody for Ki-67 antigen detection.
Immunohistochemical detection was carried out using a labeled streptavidin - biotin (LSAB) kit (Dako LSAB2 system - horseradish peroxidase [HRP]). The LSAB2 system-HRP is based on modified labeled avidin biotin technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecules. Staining is completed after incubation with substrate chromogen 3-3'- diaminobenzidine for 10 min, which will result in a brown-colored precipitate at the antigen site.
During each batch of staining, appropriate positive and negative controls were used. Sections from lymph node were included as a positive control. Negative control (without adding primary antibody) was included in all the batches. Sections were examined under high power field to observe for the immunoreactivity.
A hot spot (area with the highest density of immunostained nuclei) was selected and adjacent fields were counted to include 1000 nuclei. Distinct nuclear staining of the tumor cells were recorded as positive. Ki-67 LI was recorded as a percentage of positively stained tumor nuclei in 1000 tumor cells.
Vascular components, inflammatory cells, necrotic, degenerated and poorly preserved areas were excluded. The histological grade on H and E was correlated with Ki-67 LI. The results obtained were analyzed for statistical significance.
| > Results|| |
The age groups included in the present study ranged from 0 to 72 years. Majority of the cases were seen in the fourth to fifth decade (constituting 49.52%) with a male preponderance and male: female ratio of 1.84:1.
In the present study, pilocytic astrocytomas were observed in first and second decades. Diffuse fibrillary astrocytomas and anaplastic astrocytomas were most common in the fourth decade. Glioblastoma multiforme (GBM) were more common between fourth and sixth decades.
The most common site of Grade I astrocytomas was cerebellar region (75%), Grade II and Grade IV astrocytomas was fronto-temporal region in 61.9% and 57.9% patients respectively and for Grade III astrocytomas was fronto-parietal region (46.6%).
Most common histological variant of astrocytic tumors in the present study was diffuse fibrillary astrocytoma (35.2%) followed by glioblastoma (34.3%). The distribution of histological variants of astrocytomas is depicted in [Table 1].
Most common histological grade of astrocytic tumors was WHO Grade II tumor (40%) followed by Grade IV (36.19%). Grade I and Grade III tumors constituted 9.52% and 14.29% of cases respectively.
Ki-67 LI increased with the grade of the tumor [Figure 1]. The mean Ki-67 LI with a standard deviation, median and range in different grades of astrocytomas is depicted in [Table 2].
|Table 2: Mean Ki-67 LI with SD, median and range in different grades of astrocytomas |
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|Figure 1: Ki-67 LI in (a) Grade I astrocytoma, (b) Grade II astrocytoma, (c) Grade III astrocytoma, and (d) Grade IV astrocytoma|
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P values were significant between all grades of astrocytic tumors except between Grade I and Grade II tumors which were 0.5076. Rest of the tumors showed P < 0.05. P values on comparison between different grades of astrocytomas are in [Table 3].
|Table 3: P value on comparison between different grades of astrocytomas |
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| > Discussion|| |
Quantitative studies of proliferative potentials in gliomas may provide direct knowledge about the biologic behavior of tumors and will aid in finding an accurate prognosis for patients with gliomas. Although studies show an overall positive correlation between tumor grade and the proliferative indices, the relationship between these markers and outcome remains controversial. ,
The result of the data analysis in the present study, showed male predominance; as in a study by Ganju et al.  Among 105 patients with astrocytic tumors studied, age ranges show significant variations; similar to studies conducted by Whitton and Bloom and Jaskσlsky et al. ,
The distribution of astrocytomas differs by anatomic site between adults and children. 
Burkhard et al.  showed that pilocytic astrocytoma (Grade I astrocytoma) occurred typically in the cerebellum and brainstem in children; similar to the present study.
In the present study, the most common site of Grade II astrocytic tumors was the fronto-temporal region, as in a study conducted by Inskip et al.  Similar to present study, Simpson et al. observed 43% of glioblastomas in the frontal lobe. 
Incidence of histological grades of astrocytomas in various studies is depicted in [Table 4]. , Majority of studies including a study conducted by Zulch.  Observed that the majority of astrocytic tumors were glioblastoma contradicting the present study where diffuse fibrillary astrocytomas were more common.
|Table 4: Incidence of various histological grades of astrocytomas in various studies |
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This particular study was done to evaluate the clinical usefulness of proliferative activity in astrocytomas assessed by Ki-67 LI. Ki-67 LI is one of the most useful markers for evaluating cellular proliferation in various human neoplasms including intracranial tumors. ,
In a study by Montine et al. using the previous monoclonal antibody and frozen sections, the Ki-67 LI was found to be a significant prognostic indicator for the entire group of astrocytomas and was more significantly related to survival than histologic grade. 
But studies by Wakimoto et al. and Rathi et al. demonstrated significant relation between tumor grade and Ki-67 LI. They found a significant difference between low and high grade astrocytomas. ,
In the present study, pilocytic atrocytomas did not show any statistical significance for Ki-67 LI; similar to the study by Tibbetts et al. 
In the present study, on comparing Ki-67 LI between pilocytic astrocytomas and other grades (Grade III and Grade IV) of astrocytomas significant statistical correlation was found. No statistical significance was observed when compared to Grade II tumors; similar observations were made by Ambroise et al. 
Although in pilocytic astrocytoma mitotic activity is generally nil or difficult to demonstrate, Ki-67 LI in some pilocytic astrocytomas is similar to that of Grade II astrocytomas. 
One of the pilocytic astrocytomas in the present study was reported in a 7-year-old male child with supratentorial location (along the optic pathway). The patient came with a complaint of visual disturbance. Magnetic resonance imaging (MRI) scan revealed a well circumscribed, contrast enhancing fusiform mass extending from the optic nerve to optic chiasma. The possible resection in this patient was only 70%. No atypical histological features were reported in the biopsy. Immunohistochemistry showed high Ki-67 LI of 16, whose results were comparable to high grade astrocytomas. This high proliferative index can be attributed to proliferating microglial cells.  The patient was advised chemotherapy after surgical intervention.
Klein and Roggendorf demonstrated that proliferation rates in astrocytomas not only reflect proliferation of tumor cells but also that of microglial cells, especially in pilocytic astrocytomas. So, using this marker to differentiate pilocytic astrocytoma from gliosis should be done with caution and this marker is not reliable for definitive diagnosis. 
All these facts suggest limited role of Ki-67 LI in pilocytic astrocytomas. Similar observations were made by Ambroise et al. 
In the present study, two cases of pilomyxoid astrocytomas were reported in a 6-year-old female child with hypothalamic location and the other in a 10-year-old male child with cerebellar location. Both the patients had non-specific symptoms and MRI scan revealed well circumscribed masses with homogenous contrast enhancement. Ki-67 LI in both the tumors were 2.3 and 6.8 respectively.
In the present study, diffuse astrocytoma (Grade II astrocytoma) showed mean Ki-67 LI of 7.05 with a range of 0.4-18.8.
Majority of the patients in the present study, with diffuse astrocytoma had ill-defined, homogeneous masses of low density without contrast enhancement on computed tomography scan. MRI studies showed hypodensity on T1-weighted and hyperintensity on T2-weighted images. Five cases with diffuse astrocytoma had Ki-67 LI >6. Of which, two of them were above 40 years of age with neurological deficits. One of these patients with neurological deficits progressed to GBM 4 years later. Rest of the three patients with Ki-67 LI >16 and no neurological deficits had disease free survival until date.
Statistically significant P values (<0.0001) were obtained on comparing diffuse astrocytomas with anaplastic astrocytoma and glioblastoma, but not with pilocytic astrocytoma.
Rodrνguez et al. and Hsu et al. identified significant differences when comparing Grade II with Grade III tumors. The Ki-67 proliferation index was lower in Grade II than Grade III astrocytomas (P < 0.0001). , A recent study on cellular proliferation on pilocytic astrocytomas and diffuse astrocytomas points to the value of Ki-67 LI as a predictor of survival and its ability to better differentiate between Grade II and Grade III tumors than does the presence of mitotic figures. 
Most studies showed similar statistically significant differences in Ki-67 LI between high grade (Grade III and Grade IV) and low-grade (Grade II) astrocytomas. 
In the present study, P value on comparing Grade III and Grade IV tumors was 0.004; which is a statistically significant value. Wakimoto et al. and Rathi et al. showed a significant difference of Ki-67 LI between anaplastic astrocytoma and glioblastoma. , While some studies did not show a significant difference between anaplastic astrocytoma and glioblastoma. ,
A different conclusion was reached in a recent retrospective analysis of Ki-67 LI as an independent prognostic marker in a series of 116 GBM patients as in a study conducted by Moskowitz et al. Importantly, this study included only newly diagnosed tumors and Grade IV histology (GBM). The mean Ki-67 LI was 12.5% and varied from 0% to 76.4%. When tumor histology is restricted to GBM, the Ki-67 LI does not have much utility. 
Comparison of mean (range)/Ki-67 LI in various grades of astrocytic tumors according to different studies are shown in [Table 5]. ,,,
|Table 5: Comparison of mean (range)/Ki-67 LI in various grades of astrocytic tumors according to different studies |
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By comparing the Ki-67 LIs reported by other authors, it is evident that wide differences exist among Ki-67 LI values of various studies. Such differences may be explained partially by technical issues with different sensitivity of detection methods like peroxidase anti-peroxidase, avidin, streptavidin, various dilutions of antibodies used and time of incubation, different fixation protocols and most importantly the antigen retrieval methods applied by different workers. Thereby, different laboratories should set up their own range of values for different grades.
Above all, the interobserver counting variability could also be responsible for this wide range. Like some other studies, in the present study the counting was carried out in areas of highest densely labeled nuclei.  Our study emphasizes that Ki-67 LI is not dependent on factors like age and sex but solely dependent on histological grade. Though the average level of Ki-67 LI varies considerably in the different grades of astrocytomas, considerable overlap can be observed between them and Ki-67 LI has certain limitations in cases of pilocytic astrocytoma. Thus, Ki-67 LI should be prudently used in combination of the histopathological features.
| > Conclusions|| |
Though clinical parameters and histopathological grade are important prognostic indicators in astrocytomas, Ki-67 LI can be used as an adjunct to the histopathological diagnosis. It can be of great help in situations where there is lack of correlation between clinical parameters, histological diagnoses and grade of the tumor.
| > Acknowledgment|| |
The authors would like to thank the technical staff and management of NRI Medical College, Chinakakani.
| > References|| |
Kleihues P, Cavenee WK, editors. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Nervous System. 3 rd
ed. Lyon, France: IARC Press; 2000. p. 314.
Gudinaviciene I, Pranys D, Juozaityte E. Impact of morphology and biology on the prognosis of patients with gliomas. Medicina (Kaunas) 2004;40:112-20.
Haberland C. Clinical neuropathology: Text and color atlas. In: Percy C, editor. Tumors of the Central Nervous System. New York: Demos Medical Publishing, LLC; 2007. p. 213.
Hsu CY, Ho DM, Yang CF, Chiang H. Interobserver reproducibility of MIB-1 labeling index in astrocytic tumors using different counting methods. Mod Pathol 2003;16:951-7.
Wakimoto H, Aoyagi M, Nakayama T, Nagashima G, Yamamoto S, Tamaki M, et al
. Prognostic significance of Ki-67 labeling indices obtained using MIB-1 monoclonal antibody in patients with supratentorial astrocytomas. Cancer 1996;77:373-80.
Montine TJ, Vandersteenhoven JJ, Aguzzi A, Boyko OB, Dodge RK, Kerns BJ, et al
. Prognostic significance of Ki-67 proliferation index in supratentorial fibrillary astrocytic neoplasms. Neurosurgery 1994;34:674-8.
Ganju V, Jenkins RB et al
. Prognostic factors in gliomas. A multivariate analysis of clinic pathologic, flow cytometric, cytogenetic and molecular markers. Cancer 1994;74:920-7
Whitton AC, Bloom HJ. Low grade glioma of the cerebral hemispheres in adults: A retrospective analysis of 88 cases. Int J Radiat Oncol Biol Phys 1990;18:783-6.
Jaskólsky D, Zawirski M, Papierz W, Kotwica Z. Mixed gliomas. Their clinical course and results of surgery. Zentralbl Neurochir 1987;48:120-3.
Legler JM, Ries LA, Smith MA, Warren JL, Heineman EF, Kaplan RS, et al
. Cancer surveillance series [corrected]: Brain and other central nervous system cancers: Recent trends in incidence and mortality. J Natl Cancer Inst 1999;91:1382-90.
Burkhard C, Di Patre PL, Schüler D, Schüler G, Yaºargil MG, Yonekawa Y, et al
. A population-based study of the incidence and survival rates in patients with pilocytic astrocytoma. J Neurosurg 2003;98:1170-4.
Inskip PD, Linet MS, Heineman EF. Etiology of brain tumors in adults. Epidemiol Rev 1995;17:382-414.
Simpson JR, Horton J, Scott C, Curran WJ, Rubin P, Fischbach J, et al
. Influence of location and extent of surgical resection on survival of patients with glioblastoma multiforme: Results of three consecutive Radiation Therapy Oncology Group (RTOG) clinical trials. Int J Radiat Oncol Biol Phys 1993;26:239-44.
Wang HC, Ho YS. Clinicopathological evaluation of 78 astrocytomas in Taiwan with emphasis on a simple grading system. J Neurooncol 1992;13:265-76.
Lakhtakia R, Trehan A, Rai R, Mukherjee S. Astrocytomas and prognosis-From morphology to tumor biology. Med J Armed Forces India 2000;56:103-9.
Zulch KJ. Histologic typing of tumours of the central nervous system. In: Zulch KJ, editor. International Histological Classification of Tumours, No. 21. Geneva: World Health Organization; 1979. p. 17-57.
Suri VS, Tatke M, Singh D, Sharma A. Histological spectrum of ependymomas and correlation of p53 and Ki-67 expression with ependymoma grade and subtype. Indian J Cancer 2004;41:66-71.
Roser F, Samii M, Ostertag H, Bellinzona M. The Ki-67 proliferation antigen in meningiomas. Experience in 600 cases. Acta Neurochir (Wien) 2004;146:37-44.
Rathi KR, Radotra BD, Khosla VK. Proliferative index in astrocytic tumours. Indian J Pathol Microbiol 2007;50:754-8.
Tibbetts KM, Emnett RJ, Gao F, Perry A, Gutmann DH, Leonard JR. Histopathologic predictors of pilocytic astrocytoma event-free survival. Acta Neuropathol 2009;117:657-65.
Ambroise MM, Khosla C, Ghosh M, Mallikarjuna VS, Annapurneswari S. Practical value of MIB-1 index in predicting behavior of astrocytomas. Indian J Pathol Microbiol 2011;54:520-5.
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.
Klein R, Roggendorf W. Increased microglia proliferation separates pilocytic astrocytomas from diffuse astrocytomas: A double labeling study. Acta Neuropathol 2001;101:245-8.
Rodríguez-Pereira C, Suárez-Peñaranda JM, Vázquez-Salvado M, Sobrido MJ, Abraldes M, Barros F, et al
. Value of MIB-1 labelling index (LI) in gliomas and its correlation with other prognostic factors. A clinicopathologic study. J Neurosurg Sci 2000;44:203-9.
Hsu DW, Louis DN, Efird JT, Hedley-Whyte ET. Use of MIB-1 (Ki-67) immunoreactivity in differentiating grade II and grade III gliomas. J Neuropathol Exp Neurol 1997;56:857-65.
Moskowitz SI, Jin T, Prayson RA. Role of MIB1 in predicting survival in patients with glioblastomas. J Neurooncol 2006;76:193-200.
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.
Ralte AM, Sharma MC, Karak AK, Mehta VS, Sarkar C. Clinicopathological features, MIB-1 labeling index and apoptotic index in recurrent astrocytic tumors. Pathol Oncol Res 2001;7:267-78.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]