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Table of Contents   
ORIGINAL RESEARCH  
Year : 2022  |  Volume : 33  |  Issue : 3  |  Page : 272-276
Expression of Ki67 as potential biomarker in oral submucous fibrosis: An immunohistochemical study


1 Department of Oral Medicine and Radiology, School of Dental Sciences, Krishna Institute of Medical Sciences (KIMSDU), Karad, Satara, Maharashtra, India
2 Department of Pathology, School of Dental Sciences, Krishna Institute of Medical Sciences (KIMSDU), Karad, Satara, Maharashtra, India
3 Department of Molecular Biology and Genetics, School of Dental Sciences, Krishna Institute of Medical Sciences (KIMSDU), Karad, Satara, Maharashtra, India
4 Department of Periodontology, HKDET'S Dental College, Hospital and Reaserch Institute, Humnadad, Karnataka, India

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Date of Submission07-Jun-2021
Date of Decision06-Sep-2022
Date of Acceptance23-Sep-2022
Date of Web Publication17-Jan-2023
 

   Abstract 


Background: Oral submucous fibrosis (OSMF) is a potentially malignant disorder (PMD) known to transform into oral cancer. One of the important hallmarks of malignant transformation is the uncontrolled growth rate, commonly reflected as increased cell proliferation which can be significantly detected by proliferative markers such as a high Ki-67 index. Aim: The aim of this study is to evaluate the degree and pattern of expression of Ki67 in OSMF, oral squamous cell carcinoma (OSCC) and in normal mucosal (NOM) patients and to correlate the Ki67 expression with clinical and histological grading of OSMF and OSCC patient. Materials and Methods: A prospective cross-sectional study was conducted over a duration of two years. An immunohistochemical study was performed for Ki76 expression on 35 cases of OSMF, 10 cases of OSCC and 10 normal mucosal patients. Statistical Analysis: Data were analysed using SPSS version 21. Chi-squared test was used to analyse the differences between the intensity levels in OSMF, OSCC and NOM. Results: Expression of Ki67 was significantly higher in OSMF than that of NOM samples but less than that of OSCC samples. Expression of Ki67 increased with increasing grade of clinical and histological stages. Conclusion: The study demonstrated a high incidence of Ki67 overexpression in OSMF and OSCC and showed a correlation between clinical and histological grading of OSMF and OSCC. Identification of high-risk oral PMDs and intervention at premalignant stages could constitute one of the key steps in reducing the mortality, morbidity and cost of treatment associated with malignant transformation of these diseases.

Keywords: Immunohistochemistry, Ki67 biomarkers, malignant transformation, oral cancer, OSMF

How to cite this article:
Kamala K A, Kanetkar SR, Datkhile KD, Sankethguddad S. Expression of Ki67 as potential biomarker in oral submucous fibrosis: An immunohistochemical study. Indian J Dent Res 2022;33:272-6

How to cite this URL:
Kamala K A, Kanetkar SR, Datkhile KD, Sankethguddad S. Expression of Ki67 as potential biomarker in oral submucous fibrosis: An immunohistochemical study. Indian J Dent Res [serial online] 2022 [cited 2023 Feb 5];33:272-6. Available from: https://www.ijdr.in/text.asp?2022/33/3/272/367885



   Introduction Top


Oral submucous fibrosis (OSMF) is a potentially malignant disorder (PMD) of the oral cavity seen predominantly in the Indian subcontinent and South East Asia.[1] Many recent epidemiological studies show that there is a marked increase in the prevalence of OSMF.[2] Previous data and intervention studies suggest that areca nut is the main etiological factor for OSMF and is the fourth most commonly used psychoactive substance in the world.[3] Over 90% of oral cancers are oral squamous cell carcinomas (OSCC) which are known to arise from pre-existing PMDs. The reported rate of malignant transformation (MT) in OSMF ranges from 3% to 19%. One of the important hallmarks of MT is the uncontrolled growth rate, commonly reflected as increased cell proliferation.[2]

Ki67 is a proliferative marker identified at the University of Kiel, Germany. The expression of Ki67 occurs in all phases of the cell cycle except G0 phase and early G1 phase. It increases in S phase which correlates with the growth fraction and is indicative of the proliferative capacity of the cell, making it an excellent proliferative marker.[4]

There are a few data on proliferative markers like Ki67 in OSMF as well as its MT at the molecular level. Therefore, this study was undertaken (i) to evaluate the degree and pattern of expression of Ki67 in OSMF, OSCC and in normal patients (ii) to correlate the Ki67 expression with clinical and histological grading of OSMF and OSCC patients and (iii) to show Ki67 biomarker as surrogate and useful in detecting high-risk OSMF patients for MT.


   Materials and Methods Top


Study design and participants

This prospective cross-sectional study was conducted in the Department of Oral Medicine and Radiology (OMR), School of Dental Sciences, KIMSDU, Karad, Satara, Maharashtra, from March 2018 to March 2020. The patients were drawn from the outpatient department (OPD) of OMR using a purposive/subjective sampling technique. The study was approved by the institutional ethical committee, Krishna Institute of Medical Sciences, Deemed to be University (Ref No. KIMSDU/IHC/01/2018), Karad and was in accordance with the 1975 Helsinki declaration and its later amendments.

Thirty-five cases of OSMF who had the habit of chewing areca nut in processed forms and others, and 10 cases of OSCC patients with habits of tobacco and other products, and 10 cases of normal oral mucosa (NOM) without any habits were considered for the study. Normal tissues were obtained from the buccal mucosa of 10 patients during the surgical removal of the third molar which served as negative controls and OSCC patients as positive controls.

The inclusion criteria of the study were individuals with clinically diagnosed OSMF and OSCC with habits. The exclusion criteria were medically compromised patients and patients who did not give a written informed consent. Demographic details were recorded for all the patients, and all the patients were subject to incisional biopsy.

All the biopsied tissue specimens were fixed in 10% neutral buffered formalin for 24–48 hrs and embedded in paraffin wax using standard procedures. The biopsied tissues were cut into 3 μm thickness of tissue sections and haematoxylin and eosin staining was done for histopathological diagnosis for all cases. Additional sequential sections were prepared for immunohistochemical (IHC) study.

Immunohistochemistry protocol

All 35 cases of OSMF were available for high-quality IHC staining. Formalin-fixed paraffin-embedded tissues were sectioned at 3μm thickness and mounted on frosted slides. The sections on frosted slides were deparaffinized 3 times in xylene for 5 minutes each and then rehydrated in different concentrations of ethanol.

The antigen retrieval was carried out in Envision FLEX target retrieval solution, high pH (Dako; K8004) containing Tris-EDTA buffer pH 9 for 30 minutes in an autoclave. After washing with distilled water at 25°C, the slides were incubated with Envision FLEX wash buffer containing Tris-buffered saline solution with Tween 20 pH: 7.6 (Dako: K8007) for 20 minutes, followed by blocking with Envision FLEX peroxidase blocking reagent containing phosphate buffer with 15 mmol/L hydrogen peroxide, sodium azide and detergent (Dako: SM801). After 20 minutes H2O2 blocking, the sections were incubated directly with primary antibodies, Ki67 antigen clone MIB-1, ready to use (Dako Autostrainer/Autostrainer plus, Dako Denmark A/S Produktionsvej 42 DK-2600 Glostrup, Denmark), for 1 hour at room temperature in the humidity chamber.

Thereafter, the sections were washed with wash buffer for 5 minutes followed by treatment with Envision FLEX/HRP goat secondary antibody against rabbit and mouse immunoglobulins coupled with peroxidase molecules (Dako: SM802). After completion of 1-hour incubation with secondary antibody, sections were washed with distilled water for 5 minutes. The sections were stained with Envision FLEX DAB + Chromogen (3, 3' diaminobenzidine tetrahydrochloride) (Dako: DM827) in Envision FLEX substrate buffer containing hydrogen peroxide and preservative (Dako: SM803) till the brownish red colour development. The sections were counter-stained with haematoxylin for 2–3 minutes after washing with distilled water. After drying the slides, slides were dipped into 100% ethanol and thereafter xylene for clearing the sections. The slides were mounted in DPX mountant and observed under a Primovert phase contrast microscope (Carl Zeiss). The immunostaining intensity and percentage of stained cells were reviewed.

Scoring system

All images were captured under 10× and 40× magnification, and the expression of Ki67 protein was carried out based on the intensity of staining. For antibodies staining the nuclei, the labelling index (LI) was calculated, and for this purpose 100 cells were counted and LI was calculated as follows:



The scoring of protein expression was considered negative if <5% of cells stained positive, low if 5%–50% of cells stained positive, intermediate if 50%–75% of cells stained positive and high if >75% of cells stained positive.

Statistical analysis

Data were entered and analysed using Statistical Package for the Social Sciences version 21 (SPSS 21, IBM Corporation, United States). The Chi-squared test and Kruskal-Wallis test were used to analyse the differences between the intensity levels in NOM, OSMF and OSCC. Chi-squared test and Spearman correlation tests were used to see the correlation of Ki67 expression with clinical and histological staging of OSMF and histological grading of OSCC. Differences with a probability value of P ≤ 0.05 were considered statistically significant.


   Results Top


A total of 35 cases of OSMF and 10 cases of each OSCC and NOM were studied for the expression of Ki67 oncoprotein. [Table 1] gives a descriptive analysis of age, education and socioeconomic status of OSMF, OSCC and normal patients. Most of the OSMF patients were in the age group 21–30 years and in OSCC are elderly patients. All patients attained low educational status and belong to low to middle class socioeconomic status. Chi- squared test showed significant (P ≤ 0.05) results with distribution of age groups and socioeconomic status.
Table 1: Demographic data (age, educational and socioeconomic status) of OSMF, OSCC and NOM patients

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Out of the 35 OSMF cases, 12 (34.2%) and 23 (65.7%) showed intermediate and high expression of Ki67, respectively. Out of 10 OSCC cases, 1 (10%) case showed intermediate and the rest 9 (90%) showed high expression. Out of the 10 NOM cases, 1 (10%) case showed low and the rest 9 (90%) cases showed negative expression. The overall expression of Ki67 was highest in OSCC followed by OSMF and was least in NOM patients which were statistically significant (P-0.001). The pairwise difference between OSMF versus OSCC was non-significant whereas OSMF versus NOM and OSCC versus NOM was statistically significant [Table 2].
Table 2: Expression of Ki67 in OSMF, OSCC and NOM patients

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Out of the 35 OSMF cases, 4 (11.42%), 20 (57.14%) and 11 (31.42%) cases were in early, moderate and advanced stages, respectively. When the expression of Ki67 was compared, 10 (28.57%) were in moderate and 25 (71.42%) were in advanced staging with P = 0.0017 and r = 0.600. In histological grading 7 (20%), 19 (54.28%) and 9 (25.71%) cases were in early, moderate and advanced stages of OSMF, respectively. When the expression of Ki67 was compared, 7 (20) and 28 (80) were in moderate and advanced grading with P = <0.001 and r = 0.680. Both Chi-squared test and spearman correlation test showed statistically significant correlation [Table 3].
Table 3: Correlation of Ki67 expression with clinical and histological grading of OSMF Patients

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There were 14 (40%) and 21 (60%) cases of OSCC in moderate and poorly differentiated histological grading, respectively. When the expression of Ki67 was compared, 2 (5.71%), 22 (62.85%) and 11 (31.42%) were in well, moderate and poorly differentiated grades, respectively. Chi-squared test showed statistically significant (P-0317) correlation between the expression of Ki67 histological grading of OSCC and Spearman correlation test was non-significant (r-509) [Table 4].
Table 4: Correlation of Ki67 expression with histological grading of OSCC

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   Discussion Top


The prevalence of OSMF has increased since 2000 (2.42 in 2000 to 6.42/1000/year in 2004). One study recognised that OSCC originating from OSMF is clinically more invasive and also exhibits a higher metastasis and recurrence rate than OSCC not originating from OSMF.[5] Therefore, there has been much focus on investigating biomarkers for the prevention and early detection of their MT.

In the present study, the demographic data showed that a majority of OSMF patients were young (21–30 years) and belonged to lower educational and low socioeconomic status. Distribution of age groups and socioeconomic status in regard to the study and comparison group showed statistically significant (P ≤ 0.05) results. The results of the present study were similar to the studies conducted by Sami et al.,[6] Pratik and Desai,[7] Singla and Khanna,[8] and Yang et al.[9]

McGurg and Craig[10] studied Asian community settled in United Kingdom and they found that most of the OSMF patients were from low or middle-income group. The reason for OSMF cases coming from low socioeconomic group might be due to poor quality of food, low vitamins particularly in iron deficiency and use of more spices and chillies to make the food tasty, coupled with lack of health consciousness.

In a study conducted by Kamala et al.,[11] it was reported that parental education also has an effect over the development of noxious oral habits. It has been seen that higher parental education prevents the development of noxious habits in their offspring. Illiteracy or lower education status encourages the development of noxious habits.

The data for Ki67 in OSMF are very much limited, especially in India. In the present study, the expression of Ki76 was highest in OSCC followed by OSMF and was least in NOM patients, which was statistically significant (P ≤ 0.05). All 35 (100%) cases of OSMF showed expression of Ki67, and a similar percentage was observed in a study by Patel et al. (100%).[12] Raju et al.[13] observed Ki-67 expression in 93.10% of premalignant lesion and 90% of oral cancer. Dragomir et al.,[14] in their study observed that all cases of OSCC expressed Ki-67. Motta et al.,[15] showed 92.75% of cases with Ki-67 expression. Ranganathan and Kavitha[16] assessed the expression of p53, Ki67, Bcl2, Bax in normal epithelium, OSMF, and OSCC by IHC technique. The LI of Ki67 in OSMF patients was significantly higher than that of normal but less than that of OSCC. On the contradictory, Bazarsad et al.,[17] reported that Ki67 and cyclin D1 expressions were found to be reduced in OSMF samples compared with the NOM. The reason could be the proliferating activity of the OSMF largely dependent on the developmental stage.

Our study showed the correlation between Ki76 expression with clinical and histological staging of OSMF and histological grading of OSCC which were statistically significant. Expression of Ki67 increased from early to advanced stages in OSMF and well-differentiated OSCC to differentiated. Similar observation was shown by Iqbal et al.,[18] where the authors correlated Ki67 expression with functional grading. As the functional stage increased, the Ki67 expression also increased in OSMF patients. Ki67 expression was highest in severe functional staging and least in mild functional stage.

Verma et al.,[19] compared the mean Ki-67 expression between normal and OSCC patients. The Ki-67 expression was linearly increased from normal mucosa through various grades of OSCC which was a significant difference (P < 0.001).

Reddy et al.,[20] in their study of IHC to detect expression of both p53 and Ki67 concluded that these biomarkers could be useful in assessing the MT in oral precancerous conditions and may serve as intermediate points for cancer prevention programmes. Higher Ki-67 labelling values may indicate biological malignancy with higher proliferative activity.


   Conclusion Top


Ki67 has been shown to be excellent for the estimation of the growth fraction in both normal and malignant human tissues, and this antibody is now used as the standard for the assessment of cell proliferation as it does not get affected by internal and external factors.

Expression of Ki67 increased from early to advanced stages in OSMF. Thus, this can infer that the level of Ki67 antigen rises as the normal cells changes to dysplastic and later to invasive carcinoma indicating the involvement of Ki67 in MT of OSMF, and these markers can be used as surrogate markers for MT in OSMF.

Furthermore, it has a much shorter half-life, thus producing less residual staining after cells have gone through proliferative stage. The limitation of the present study was the small sample size. However, more studies, with large study samples, long-term follow up and use of other proliferative markers would further help to assess the significance of Ki67 as a prognostic biomarker for MT.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. K A Kamala
Department of Oral Medicine and Radiology, School of Dental Sciences, KIMSDU, Karad - 415110, Satara, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_551_21

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