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Role of Ki-67 as an Adjunct to Histopathological Diagnosis in the Grading of Astrocytic Tumors
Namita Singh1, Prita Pradhan1*, Ranjana Giri1, Diptiranjan Satapathy2
1Department of Pathology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India-751024. 2Department of Neurosurgery, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India-751024.
Abstract
The Ki-67 Labelling Index (LI) is used as an ancillary tool to assess cell proliferation activity in glioma diagnosis. It is a potent biologic marker that estimates the growth of neoplasm and thus will aid in identifying the prognosis in patients with glioma. To study the expression of Ki-67 in astrocytic tumors and its correlation with clinicopathological parameters. Clinico-radiological details of forty-three cases of glioma were documented. H&E slides were reviewed for histopathological grade and morphology. KI-67 Labelling Index was studied by immunohistochemical analysis at ‘hot-spot’ areas. Cases were segregated into high and low grades taking 4% as a cut-off as per WHO criteria. Statistical analysis was carried out using standard statistical software with a p-value of ≤ 0.05 taken as statistically significant. A total of 43 patients were included comprising 31 males and 12 females with a mean age of 46.14 years. Ki-67 labeling index showed a statistically significant relationship with the advanced age (p=0.01), higher histological grade (p=<0.00001), presence of cellular atypia (p=0.0002), necrosis (p=<0.00001), and microvascular proliferation (p=<0.00001). Ki-67 assessment supplements standard histopathological grading. It is a simple and reliable method. This study demonstrates that it can serve as an important prognostic marker. It can be used as an adjunct to histopathological diagnosis.
Keywords: Astrocytic tumors, Ki-67, Grade, Atypia
Gliomas are common malignancies of the central nervous system(CNS).[1, 2] Scholzer and Gerdes discovered the Ki-67 antigen in the early 1980s, which expresses two protein variants with molecular weights of 345 and 395 kDa.[3] It is present at all active cell cycle phases (G1,S,G2, and M) but absent in resting cells (G0).[4,5] In the later stages of mitosis significant decrease in Ki-67 (during anaphase and telophase) levels takes place.[6] The Ki-67protein (pKi-67) expression has been linked to the proliferation of intrinsic cell components in malignant tumors enabling its use as a measure of tumor aggressiveness.[7,8]
The cell cycle's advancement, changes in cellular distribution, interphase, and process of cell mitosis are all regulated by the expression of Ki-67.[9] Ki-67 is utilized in diffuse astrocytic tumors as a biomarker of tumor cell proliferation that forecasts the biological characteristics of the tumor and the patient's prognosis.[10,11] Low-grade astrocytomas have a considerably different mean Ki-67 LI compared to anaplastic astrocytomas and glioblastoma.[12]
Glial cell proliferation is the foundation of gliomagenesis.[13, 14] It is well established that histological grading can be used to assess a patient prognosis in CNS neoplasm but histological differentiation in glioma may not always be evident, particularly when tiny tissue pieces from stereotactic guided needle biopsies are available.[15, 16] Proliferation indices have thus been utilized for prognostic purposes in both disease progression and patient survival.[17,18] The MIB-1 antibody recognizes the Ki-67 antigen in formalin-treated paraffin-embedded tissue.[19, 20] Classification and prognosis of astrocytomas have been determined using the MIB-1antibody because in order to predict tumor behavior, it is crucial to develop better prognostic markers. [21, 22]
Evaluation of the tumor's proliferative index (Ki-67) is one such complementary method.[23] Among the many useful biological markers available, the proliferative index provides a quantitative measure of tumor proliferation.[24] Several techniques have been used to calculate the
The study was conducted in the Department of Pathology, over two years between September 2020 to August 2022 after approval from the Institute Review Board as well as Institute Ethical Committee, as per the Declaration of Helsinki. Before, the initiation of the study, informed consent was taken from all the participants whose biopsies were considered for the study. Formalin-fixed paraffin-embedded (FFPE) blocks of histopathologically proven cases of glial tumors were included both prospectively and retrospectively from the archives. Inadequate and poorly-fixed tissues were excluded from the study. After taking informed consent, detailed clinical and radiological information was documented.
Consecutive H&E sections slides were reviewed for histomorphological features including-histological type and grade of tumors, degree of cellularity, atypia, mitosis, presence of necrosis, and microvascular proliferation. Grade I and Grade II tumors were referred to as low-grade tumors, while Grade III and Grade IV were tumors referred to as high-grade tumors. The corresponding sections from paraffin block with a viable tumor in highly cellular areas (including necrosis if present) were chosen for IHC.
IHC for Ki-67 was performed on deparaffinized sections taken on albumin-coated slides using two-step indirect methods. Prediluted ready-to-use Ki-67 monoclonal antibody CAT-P-K001-30 (Clone QR015, Berlin, Germany) was used. Placenta tissue was taken as a positive control. For negative control, the same sections were used by omitting the primary antibody step. The observations were taken independently by two pathologists blinded to the clinical details. The discrepant cases were reviewed and evaluated over a multiheaded microscope with an expert for consensus observations.
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 tumor cells was recorded as positive. Ki-67 labelling index was recorded as a ‘percentage’ of positively stained nuclei in 1000 tumor cells. The Ki-67 cut-off value given by WHO for glioma is less than 4% for low-grade gliomas and more than or equal to 5% for high-grade gliomas. We categorically divided Ki-67 expression into low and high expression taking 4% as cut off.
For statistical analysis, data were entered into a Microsoft Excel spreadsheet in 2010. Quantitative data were expressed as range, mean±SD, or median. The categorical variables were analyzed using Pearson’s chi-square test or Fisher’s exact test. Statistical analysis was carried out using Microsoft Excel spreadsheet 2010, GraphPad QuickCalcs ©2021 GraphPad Software, and Social Science Statistics©2021 Jeremy Stangroom. A p-value of ≤ 0.05 was taken as statistically significant.
A total of 43 patients were included with a Mean age of 46.14y with M:F=2.5:1. Tumours most frequently involved the frontal lobe (12/43) with 15low-grade gliomas comprising pilocytic and diffuse astrocytoma (11/43) 25% (Figure 1).
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Figure 1. Showing different grades of glial tumors encountered in the study. a) Grade I-Pilocytic Astrocytoma. (H&E,400X); b) Grade II- Diffuse Astrocytoma.(H&E,100X); c) Grade III- Anaplastic Astrocytoma.(H&E,400X), d) Grade IV-Glioblastoma (H&E, 100X) |
The Ki-67 LI was found to be in 95.3% of cases ranging from 0-80% (Mean: 22.65±24.28%) with strong nuclear positivity. Low Ki-67(<4%) was seen in 14 out of 43 cases (32.6%). The correlation of Ki-67 with the clinicopathological parameters is summarized in Table 1.
Ki-67 LI showed a statistically significant relationship with age, histomorphological grade, presence of cellular atypia, necrosis, and microvascular proliferation (Table 1). Higher Ki-67 LI was associated with higher age of the patient, higher histologic grade, and a greater degree of cellular atypia (Figure 2).
Table 1. Correlation of PD-L1 and Ki-67 with clinicopathological parameters |
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Sl. No. |
Parameters |
Category (n) |
Ki-67 expression |
‘p’- value |
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Low |
High |
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1. |
Age (years) |
<46 (19) |
10 |
09 |
0.01 |
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>46 (24) |
04 |
20 |
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2. |
Cellularity |
Reduced (27) |
13 |
17 |
0.22 |
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Increased (16) |
04 |
12 |
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3. |
Atypia |
Minimal(12) |
09 |
03 |
0.0002 |
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Marked (31) |
05 |
26 |
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4. |
MVP** |
Absence (19) |
13 |
06 |
<0.00001 |
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Noted (24) |
01 |
23 |
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5. |
Necrosis |
Absence (19) |
13 |
06 |
<0.00001 |
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Presence (24) |
01 |
23 |
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6. |
Histologic Grade(WHO) |
Grade I/Grade II (15) |
12 |
03 |
<0.00001 |
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Grade III/Grade IV (28) |
02 |
26 |
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*MVP=Microvascular proliferation.
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