Analysis of Ki-67 Expression in Oral Premalignant Lesions and Normal Oral Mucosa: An Immunohistochemical Study.

AIM: This study aimed to evaluate the Ki-67 expression in oral premalignant lesions and normal oral mucosa.
MATERIALS AND METHODS: The cases were selected on the basis of the details obtained from the patients. A total of 45 specimens were divided into three groups: Group 1 (normal mucosa), Group 2 (clinically and histologically diagnosed as oral lichen planus), and Group 3 (clinically and histologically diagnosed as leukoplakia). Specimens were prepared and the slides for Ki-67 were observed under light microscope with a magnification of ×400. The tissue samples were thoroughly examined, and the pattern of expression was analyzed semiquantitatively by counting the number of positive cells.
RESULTS: The mean positive cell count of normal mucosa was 23.20 ± 2.89, of oral lichen planus was 42.82 ± 2.65, and of leukoplakia was 82.14 ± 3.10. There was a statistically significant difference of expression observed between the groups (P < 0.001). On multiple comparisons using Tukey post hoc test, a statistical difference was found between all the three groups.
CONCLUSION: Ki-67 is an easily applicable marker of cell proliferation whose expression correlates well with the disease progression.

INTRODUCTION

One of the major health problems in developing countries is oral cancer and it represents the leading cause of death. Squamous carcinomas represent about 3% of human cancers and more than 90% of malignant tumors at oral location are being diagnosed worldwide each year in more than 350,000 new cases.[1]

The cell is the basic, living, structural, and functional unit of the body. Cell proliferation is a biological process of vital importance to all living organisms and is fundamental to both embryonic and postembryonic existence.[2] The control on this important biological process is thought to be lost in cancer, and many studies have reported that abnormal cell proliferation appears to be a precursor and may be a predictor of tumorigenesis.[3]

Molecular biological markers have been suggested to play an important role in the diagnosis and prognostic evaluation of oral potentially malignant disorders. Markers of proliferation, epithelial differentiation, and genomic markers could potentially be a good prospective for improving the prognostic evaluation of precursors of oral cancer.[4]

The most common immunohistochemical cell proliferation marker is Ki-67 antigen. It is a nuclear protein expressed in the G2 and M phases of actively dividing cells. Its nuclear expression during a defined period of the cell cycle represents an advantage in its use as a biological marker of mitotic activity. Ki-67-positive cells are often correlated with the clinical course of the disease. It provides significant information about the degree of aggressiveness and prognosis of the disease.[5] Hence, this study was conducted to evaluate expression of Ki-67 in oral premalignant lesion and normal oral mucosa.

MATERIALS AND METHODS

This study was conducted in the Department of Oral Pathology, Educare Institute of Dental Sciences, Kerala, India. Histopathology of paraffin-embedded section of specimens was conducted using hematoxylin and eosin (H&E) stain. Histologically proven oral lichen planus (OLP), leukoplakia, and normal mucosa were selected for the study.

The cases were selected on the basis of the details obtained from the patients. A total of 45 specimens were divided into three groups:

Group 1: normal mucosa;

Group 2: clinically and histologically diagnosed as OLP; and

Group 3: clinically and histologically diagnosed as leukoplakia.

Thick sections (4 μm) were cut from 10% formalin-fixed tissue, which were then deparaffinized in two changes of xylene for 15 minutes each. Dexylinization was performed by immersing the slides in two changes of absolute alcohol for 1 minute each. Sections were alcoholized by immersing the slides in 90% and 70% alcohol for 1 minute each and then washed for 10 and 5 minutes each with tap water and distilled water, respectively.

Antigen retrieval was carried out by placing the sections in citrate buffer and then keeping in a microwave oven for 10 minutes. Borosil jar was then cooled for 20 minutes in the sink with water. Sections were then rinsed with distilled water for 5 minutes and were then washed with two changes of tris buffer solution (TBS) for 5 minutes each. To block the endogenous peroxidase enzyme activity, the sections were treated with peroxidase block for 10–15 minutes, and then again washed with three changes of TBS for 5 minutes each. Sections were then treated with power block for 15 minutes to block nonspecific reaction with other antigens. Sections were then drained and covered with primary antibody against Ki-67 with dilution of 1:100 for 1 hour to identify tumor markers by antigen–antibody reactions and again washed with TBS as described earlier. To enhance the reaction between primary and secondary antibodies, sections were then treated with superenhancer for 30 minutes, and again washed with TBS. Enzymes were labeled by treating the sections with supersensitive poly-HRP Secondary Antibody and washed with TBS. Chromogen was then added to the sections for 5 minutes to give color to the antigens and sections were again washed with TBS. Sections were then washed with tap water for 5 minutes and were counterstained with hematoxylin for 1 minute and washed with tap water, dried, cleared in xylene, and mounted with dibutylphthalate polystyrene xylene.[6]

Interpretation of Ki-67

The slides for Ki-67 were observed under light microscope with a magnification of ×400. The tissue samples were thoroughly examined, and the pattern of expression was analyzed semiquantitatively by counting the number of positive cells.

Statistical analysis

Data analyses were performed using SPSS software, version 21, for Windows. One-way analysis of variance was used. P value was appreciated as statistically significant only when it was under 0.05.

RESULTS

Comparison of Ki-67 with normal mucosa, OLP, and leukoplakia is shown in Table 1. The mean positive cell count of normal mucosa was 23.20 ± 2.89, OLP was 42.82 ± 2.65, and leukoplakia was 82.14 ± 3.10. And there was a statistically significant difference of expression observed between the groups (P < 0.001).

Table 1

Comparison of Ki-67 with normal mucosa, oral lichen planus, and leukoplakia

Group	Mean ± SD	Standard Error	F	P value
Group 1 (normal mucosa)	23.20 ± 2.89	0.1218	48.546	0.001
Group 2 (oral lichen planus)	42.82 ± 2.65	0.2145
Group 3 (leukoplakia)	82.14 ± 3.10	0.1007

Table 2 reveals the multiple comparisons of normal mucosa, OLP, and leukoplakia using Tukey post hoc test. There was a statistical difference found between all the three groups.

Table 2

Multiple comparisons of normal mucosa, oral lichen planus, and leukoplakia using Tukey post hoc test

Group	Compared with	Mean difference	Significance
Normal mucosa	Oral lichen planus	−10.62	0.02
	Leukoplakia	−58.94	0.001
Oral lichen planus	Normal mucosa	10.62	0.02
	Leukoplakia	−39.32	0.001
Leukoplakia	Normal mucosa	58.94	0.001
	Oral lichen planus	39.32	0.001

DISCUSSION

Ki-67 is a perichromosomally located, cell-cycle-related antigen. Its expression is strictly restricted to dividing cells. Therefore, it is regarded as one of the most potent proliferation markers in the field of molecular pathology.[4] In normal oral mucosa, Ki-67-positive cells are detected dispersedly within the parabasal layer, that is, the two cell strata above basal layer. However, its expression beyond that within the suprabasal layer indicates epithelial dysplasia.[7]

Cell proliferation, a vital biological process, is an important adjunct to histologically based tumor classification and has potential relevance as an indicator of treatment response and relapse. Many studies have reported that abnormal cell proliferation appears to be a precursor and may be a predictor of tumorigenesis.[3]

The monoclonal antibody Ki-67 was first described in 1983 by Johannes Gerdes et al., who suggested that it might be used as a marker for proliferating cells.[8] Immunostaining with antibodies to Ki-67 antigen is well established as a quick and efficient method for evaluating growth fractions of various tumor types because of its distinctive reaction patterns that exclusively involve the proliferating cells.[9]

The results of this study are similar to those of the study conducted by García-Pola Vallejo et al.[10], which stated that an immunohistochemistry study performed on 10 specimens of OLP, oral leukoplakia, dysplasia, and normal mucosa compared Ki-67 expression and found that all the tissue analyzed showed some grade of proliferation in the basal layer. Results were statistically significant among mean of the positive nuclei per millimeter of length in normal mucosa and oral leukoplakia and between normal mucosa, OLP, and oral leukoplakia with dysplasia. Previous studies have compared lichen planus, but in this study we have tried to compare the expression of Ki-67 in OLP, leukoplakia, and normal mucosa and found that expression was significantly higher in leukoplakia than in OLP, which is a chronic immunologic mucocutaneous disease. The possible malignant transformation of OLP is the subject of an ongoing and controversial discussion in the literature. The main criticism of studies on this subject relates to the lack of sufficient data to support the initial diagnosis of OLP in cases that finally developed into squamous cell carcinoma.[11]

The most important factor indicating whether a lesion is progressing toward malignancy is the presence and intensity of dysplasia. Therefore, it is crucial to find markers of these changes.

Cellular proliferation in OLP is either similar to or greater than that in normal oral mucosa.[12] In Neppelberg’s study,[13] the intensity of Ki-67 staining in the epithelium was positive (+) in 77.3% of OLP samples and was (++) in 18.2% of OLP samples, which was significantly higher than that in normal mucosa (P = 0.022). The results obtained in the aforementioned study were similar to those of our study. Zargaran et al.[14] found that Ki-67 expressions in OLP and epithelial dysplasia were similar and reported significantly higher levels in oral cellular carcinoma. This study showed that, in OLP, the risk of turning into malignancy is similar to the risk in epithelial dysplasia.

CONCLUSION

In conclusion, Ki-67 is an easily applicable marker of cell proliferation whose expression correlates well with the disease progression. Therefore, it is a marker of malignant transformation and carcinogenesis in oral premalignant lesions, and in future it may serve as a prognostic tool in the early detection of malignancy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.