Journal of Dr. NTR University of Health Sciences

: 2018  |  Volume : 7  |  Issue : 3  |  Page : 185--189

A quantitative expression of Ki-67 to evaluate malignant transformation rate in potentially malignant disorders

Mani D Chitipothu, Kiran K Kattappagari, Divyasri Godavarthy, Chandrashekar Poosarla, Sridhar R Gountu, Venkata R. R. Baddam 
 Department of Oral Pathology, SIBAR Institute of Dental Sciences, Tekkallapadu, Guntur, Andhra Pradesh, India

Correspondence Address:
Dr. Mani D Chitipothu
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Tekkallapadu, Guntur - 522 006, Andhra Pradesh


Objective: To evaluate the quantitative expression of Ki-67 in oral leukoplakia, oral submucous fibrosis (OSF) and to compare their expression with normal oral epithelium. Materials and Methods: Thirty archival paraffin-embedded blocks were retrieved from the Department of Oral and Maxillofacial Pathology. It includes three groups with 10 specimens each of histopathologically confirmed oral leukoplakia, OSF, and controls, which were taken as standard for comparison. Ki-67 immunostaining was detected and numbers of positive cells out of 1000 were counted in 10 random high-power fields in each case. Statistical analysis was done using one-way analysis of variance test. Results: Mean and standard deviation of oral leukoplakia was found to be higher (398 ± 82) followed by OSF (343 ± 83) and controls (290 ± 72). Pairwise comparison of three groups with respect to expression of total number and percentage of cells was statistically significant between oral leukoplakia and controls (P < 0.05). Conclusion: The results of the present study emphasize that malignant transformation rate was higher for oral leukoplakia compared with OSF and controls. Hence, it serves as a prognostic tool for early detection of malignancy.

How to cite this article:
Chitipothu MD, Kattappagari KK, Godavarthy D, Poosarla C, Gountu SR, Baddam VR. A quantitative expression of Ki-67 to evaluate malignant transformation rate in potentially malignant disorders.J NTR Univ Health Sci 2018;7:185-189

How to cite this URL:
Chitipothu MD, Kattappagari KK, Godavarthy D, Poosarla C, Gountu SR, Baddam VR. A quantitative expression of Ki-67 to evaluate malignant transformation rate in potentially malignant disorders. J NTR Univ Health Sci [serial online] 2018 [cited 2019 Jun 24 ];7:185-189
Available from:

Full Text


Oral cancer is the most common cancer and constitutes a major health problem in developing countries, representing the leading cause of death. A significant proportion of oral squamous cell carcinoma (OSCC) develops from potentially malignant disorders, most commonly oral leukoplakia and oral submucous fibrosis (OSF).[1] Potentially malignant disorders are defined by the World Health Organization (WHO, 2005) as the risk of malignancy being present in a lesion or condition either at the time of initial diagnosis or at a future date.[2] Leukoplakia is a common lesion of oral mucosa, which is defined as a white patch or plaque that cannot be characterized clinically or pathologically as any other disease. Worldwide prevalence of leukoplakia is 0.2–4.9% and overall malignant transformation rates range from 3 to 6%, depending on the type, site, and length of follow-up.[3] Malignant transformation of leukoplakia with dysplastic changes ranges from 5 to 43%.[4] Increased cellular proliferation as a result of changes in proto-oncogene and tumor suppressor genes is believed to drive the development of oral cancers. These genetic changes are an intimate part of neoplastic development and serve as markers for specific changes involved in tumor progression.[5]

OSF is a chronic, progressive, peculiar disease of oral mucosa affecting a large population in Asian countries, especially in the Indian subcontinent.[6] It is a high-risk precancerous condition characterized by changes in the connective tissue fibers of the lamina propria and deeper parts leading to stiffness of mucosa and restricted mouth opening.[7] Malignant transformation rate of the surface epithelium in OSF ranges from 3 to 19%.[6]

Various proliferative markers such as Ki-67, p53, PCNA, AgNOR, CK 13, 17, and Notch genes have been utilized to identify the rate of proliferation progressing to malignancy.[8] Among these, Ki-67 is a human nuclear protein associated with cell proliferation and maximally expressed in cells with G2 and M phases of the cell cycle but absent in resting cells. Hence, it can be widely used in pathology as a proliferation marker to measure growth fraction of cells in premalignant and malignant lesions, along with normal tissues.[9]

This study was designed with an aim to evaluate the immunohistochemical expression of Ki-67 in oral leukoplakia and OSF and to compare their expression with normal oral mucosa in an attempt to identify alterations which could indicate malignant progression.

 Materials and Methods

Source of data

The present study was carried out using 10 formalin-fixed, paraffin-embedded tissue blocks in each category, diagnosed histologically as oral leukoplakia and OSF, retrieved from the archives of Department of Oral Pathology and Microbiology, SIBAR Institute of Dental Sciences, Guntur. As controls, 10 formalin-fixed paraffin-embedded tissue blocks of normal oral mucosa over the impacted third molars were taken from the patients undergoing surgery for impactions after approval from the Institutional Ethics Committee, SIBAR Institute of Dental Sciences.

The study consisted of 30 samples categorized into three groups:

Group I – Oral leukoplakia (n = 10)Group II – Oral submucous fibrosis (n = 10)Group III – Controls (n = 10)

Serial sections of 4 μm were obtained from the archival material. The sections of Groups I, II, and III were first subjected to routine hematoxylin and eosin stain for histopathological conformation of potentially malignant disorders (10×) [Figure 1]. Later, other sections of all three groups were subjected for immunohistochemical analysis using monoclonal Ki-67 antibody (Dako, CA, USA). The technique used was based on the labeled streptavidin–biotin method. After processing of the specimen, sections of 4 μm thickness were cut carefully, using semiautomatic microtome and fixed on the microslides coated with poly-l-lysine. The sections were de-paraffinized by giving two dips in xylene and then rehydrated using absolute ethanol for 3 min. Then, the sections were placed in distilled water bath and not allowed to dry. Antigen retrieval was done by dipping the tissue sections in Tris–ethylenediaminetetraacetic acid buffer and incubated. After cooling, the endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 5 min. The sections were then covered with the primary antibody (monoclonal anti-Ki-67 antibody) and incubated for 1 h at room temperature. Then, the secondary link antibody was added and incubated for 30 min at room temperature. Later, after removing the excess buffer, the substrate chromogen (mix of 1 ml substrate buffer and 1 drop of diaminobenzidine) was added onto the slides and incubated for 5 min at room temperature. Finally, the slides were immersed in a bath of hematoxylin for 2–5 min and washed under tap water for 5 min.{Figure 1}

Positive Ki-67 expression was observed as light brown staining of the nuclear regions in the cells using Olympus research microscope (BX51M, USA) (20×) [Figure 2]. The numbers of positively stained cells were counted on 10 representative areas of the section using ×20 objective in a minimum of 100 cells per field.{Figure 2}

Statistical analysis

The collected data were entered in an Excel sheet and statistical analysis was done using the software Statistical Package for Social Sciences (SPSS) version 20.0. The normal distribution of number of positive cells was done using one-way analysis of variance (ANOVA) test. Comparison of three groups with respect to the number of positive cells, percentage of positive cells, and score was done using one-way ANOVA test. Pairwise comparison of three groups was done using Tukey's multiple post hoc test.


The present study was conducted to assess the immunohistochemical expression of Ki-67 in 10 cases of oral leukoplakia and 10 cases of OSF and to compare their expression with 10 normal oral mucosa specimens. The mean value of number of positive cells in group I (oral leukoplakia) was 398.80 ± 82.19, group II (OSF) was 343.90 ± 83.98, and group III (controls) was 290.90 ± 72.48. Statistical analysis was done using one-way ANOVA test and a statistically significant difference of mean was obtained (P = 0.0194) [Table 1]; [Graph 1]. Pairwise comparison of three groups with respect to the number of positive cells was done using Tukey's multiple post hoc test. A high statistical significance was noticed on comparison of group I and group III (P = 0.0145). However, there was no statistical significance between group I and group II (P = 0.2888) and group II and group III (P = 0.3130) [Table 2].{Table 1}[INLINE:1]{Table 2}


Oral cancer is the sixth most common cancer worldwide and its prevalence in the Indian subcontinent is highest among all cancers in men. More than one million new cases are being detected annually.[10] Several studies have shown that approximately 80% of oral cancers were preceded by oral precancerous lesions or conditions.[11] Theories of carcinogenesis suggest that premalignant change may occur in any area of mucous membrane exposed to carcinogens.[12] Leukoplakia is a clinical term and the presence or absence of dysplastic cells does not alter the clinical diagnosis. However, it has been suggested that leukoplakia should be redefined to be a combined clinical/histological term.[13]

Studies in leukoplakia have shown that expression of Ki-67 was significantly correlated with the histopathological stage of the tumor and the proliferative status of oral lesions.[14] OSF is a premalignant condition that is mainly associated with the habit of chewing betel quid containing areca nut and it is characterized by inflammation, increased deposition of submucosal collagen, formation of fibrotic bands in oral and para-oral tissues, which increasingly limit mouth opening.[15]

Malignant transformation of these disorders into cancer is often associated with changes at the genetic level, which are reflected as altered expression of proteins related to cell cycle, proliferation, and apoptosis.[16] Among these, cell proliferation is an important adjunct to histologically-based tumor classification and has potential relevance as an indicator of treatment response and relapse.[17] The most commonly used immunohistochemical marker to study cell proliferation is Ki-67 antigen, which was first described in 1983 by Johannes Gerdes, suggesting that it can be used as a marker for proliferating cells.[18]

Kushner et al. (1997) performed a study to estimate the patterns of p53 and Ki-67 expression in epithelial dysplasia. They observed that expression of Ki-67 was increased with the severity of epithelial dysplasia in oral leukoplakia.[19] Kurokawa et al. (2003) assessed the expression of p53 protein and Ki-67 antigen in oral leukoplakia with or without epithelial dysplasia. In normal epithelium, the expressions of p53 and Ki-67 are limited to the basal cells or the immediate suprabasal cell layers, while they were widely distributed in leukoplakia, as epithelial dysplasia progressed from mild to moderate to severe.[20] In the present study, the expression of Ki-67 observed in controls was limited to the basal cells only, whereas in oral leukoplakia and OSF, the expression was seen in the basal and suprabasal cells of the epithelium, Which is in accordance with Birajdar SS et al., in 2014.[21]

Ranganathan and Kavitha (2011) assessed the expression of p53, Ki-67, Bcl2, Bax in normal epithelium, OSF, and squamous cell carcinoma. When the intensity of staining was compared within the controls and OSF, it was statistically significant. The authors concluded that they can be used as surrogate markers of malignant transformation.[16] A comparison of all three groups in this study showed statistical significance between the controls and oral leukoplakia (P = 0.0145), whereas it was not statistically significant on comparing the controls with OSF (0.3130) and oral leukoplakia with OSF (0.2888), which is in accordance with the studies mentioned above.


The results of the present study emphasize that expression of Ki-67 was increased in oral leukoplakia when compared with OSF and controls. In oral leukoplakia and OSF, the expression was seen in basal and parabasal layers of epithelium, whereas in normal mucosa it was restricted to only basal layer, suggesting that Ki-67 can serve as a prognostic marker in the early detection of malignant transformation.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Humayun S, Prasad VR. Expression of p53 protein and Ki-67 antigen in oral premalignant lesions and oral squamous cell carcinomas: An immunohistochemistry study. Natl J Maxillofac Surg 2011;2:38-46.
2George A, Sreenivasan BS, Sunil S, Varghese SS, Thomas J, Devi G, et al. Potentially malignant disorders of oral cavity. J Oral Maxillofac Pathol 2011;2:95-100.
3Smitha T, Sharada P, Girish HC. Morphometry of the basal cell layer of oral leukoplakia and oral squamous cell carcinoma using computer aided image analysis. J Oral Maxillofac Pathol 2011;15:26-33.
4Lind PO. Malignant transformation in oral leukoplakia. Scand J Dent Res 1987;95:449-55.
5Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics. CA Cancer J Clin 2000;50:7-33.
6Tilakaratne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: On etiology and pathogenesis. Oral Oncol 2006;42:561-8.
7Sabharwal R, Gupta S, Kapoor K, Puri A, Rajpal K. Oral submucous fibrosis- A review. J Adv Med Dent Sci Res 2013;1:29-37.
8Soares FD. Immunohistochemistry for Ki-67, Bcl2 and p53 in oral leukoplakias differentiates noncancerous from cancerous states. Appl Cancer Res 2013;33:173-9.
9Gonzalez-Moles MA, Ruiz-Avila I, Rodriguez-Archilla A, Martinez-Lara I. Suprabasal expression of Ki-67 antigen as a marker for the presence and severity of oral epithelial dysplasia. Head Neck 2000;22:658-61.
10Reibel J. Prognosis of oral pre-malignant lesions: significance of clinical, histopathological and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:47-62.
11George A, Sreenivasan BS, Sunil S, Soma Susan V, Thomas J, Devi G, et al. Potentially malignant disorders of oral cavity. Oral Maxillofac Pathol J 2011;2:95-100.
12Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium clinical implications of multicentric origin. Cancer 1953;6:963-8.
13Bouquot JE, Speight PM, Farthing PM. Epithelial dysplasia of the oral mucosa-Diagnostic problems and prognostic features. Curr Diagn Pathol 2006;12:11-21.
14Sittel C, Ruiz S, Volling P, Vasnicka HM, Jungehulsing M, Eckel HE. Prognostic significance of Ki-67, PCNA, p53 in cancer of the oropharynx and oral cavity. Oral Oncol 1999;35:583-9.
15Auluck A, Rosin MP, Zhang L, Sumanth KN. Oral submucous fibrosis, a clinically benign but potentially malignant disease: Report of 3 cases and review of literature. J Can Dent Assoc 2008;74:735-40.
16Ranganathan K, Kavitha R. Proliferation and apoptosis markers in oral submucous fibrosis. J Oral Maxillofac Pathol 2011;15:148-53.
17Jones PH. Epithelial stem cells. Bioessays 1997;19:683-90.
18Gerdes J, Schwab U, Lemke H, Stein H. Production of mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983;31:13-20.
19Kushner J, Bradley G, Jordan RC. Patterns of p53 and Ki-67 protein expression in epithelial dysplasia from floor of mouth. J Pathol 1997;183:418-23.
20Kurokawa H, Matsumoto S, Murata T, Yamashita Y, Tomoyose T, Zhang M, et al. T. Immunohistochemical study of syndecan 1 downregulation and the expression of p53 protein or Ki-67 antigen in oral leukoplakia with or without epithelial dysplasia. J Oral Pathol Med 2003;32:513-21.
21Birajdar SS, Radhika MB, Paremala K, Sudhakara M, Soumya M, Gadivan M. Expression of Ki-67 in normal oral epithelium, leukoplakic oral epithelium and oral squamous cell carcinoma. J Oral Maxillofac Pathol 2014;18:169-76.