Expression OF p16INK4a Gene in Premalignant and Malignant Lesions of Oral Cavity

Squamous cell carcinoma is the summits malignant neoplasm of the oral cavity. Tobacco and alcohol is identified as risk factors, but squamous cell carcinoma can occur in patients with no known risk factors. Oral cancer is the sixth most common malignancy and is one of the major causes of cancer morbidity and mortality worldwide. Cancer is caused due to a series of alteration in genetic and epigenetic factors that occur in multiple steps and is influenced by the genetic predisposition of the individual and by exogenous environmental factors. These factors result in a series of molecular alteration, including inactivation of tumor suppressor genes expression of p16 has been proposed as a marker for malignant transformation. The p16 staining was correlated between the control and study groups and p 16 was shown to be increasing expressed in premalignant and less expressed in malignant category and was found to be statistically significant by Fischer’s exact test. This study concluded that p16 was increasingly expressed in premalignant lesions and less expressed in malignant lesion. In the present study 9 of the control cases were p16 negative and one case showed sporadic staining. The study group I showed 1 case of sporadic staining, 6 cases of focal staining and 8 cases of diffuse staining. The study II showed 14 cases of sporadic staining, 6 cases of focal staining and 5 cases of diffuse staining. Hence variations cannot be accurately assessed, but it plays a crucial role in assessing pre-malignant lesions progressing to malignancy. To confirm this, a larger sample study is required. As advances in research have leads to greater understanding of potentially malignant lesions in the oral cavity. Original Research Article Raj and Marylilly; JPRI, 33(22A): 53-62, 2021; Article no.JPRI.66767 54


INTRODUCTION
Oral cancer ranks 6 th among the most common cancer worldwide. It is constitut 5.5% of all malignancies. In India [1], 65,000 cases are diagnosed annually and 500, 000 new oral cancers are recorded worldwide [2]. Etiological tobacco usage and alcohol consumption as a major source of intra-oral carcinogens globally leading to oral & oropharyngeal cancers [3,4]. In India, the increased prevalence of oral cancer seems to be due to high exposure to sunlight due to farming, smoking, alcohol, spicy food, paan -masala and neglect overall oral hygiene [2]. Tobacco usage can also cause two clinically recognized precancerous lesion like leukoplakia and erythrop lakia. In India & several other South-East Asian countries, oral sub-mucous fibrosis associated with areca-nut chewing as precancerous conditions [5].
In India 19% of tobacco consumed in the form of cigarettes and other forms of tobacco usage is as bidi and chewable tobacco. Areca nut is used as naturally crude areca nut, betel quid or paanparag. Other formulations are as supari, guthka (with tobacco) and paan masala [6]. Tobacco has been demonstrated in many states as carcinogenic, teratogenic and genotoxic [4]. Although oral cancers do occur in people who do not use tobacco too. The possibility thus existing is genetic susceptibility and inherent genetic alteration. Genetic susceptibility can be done to chemical carcinogens, oncogenic viruses and radiant cosmic energy [7].
Genetic susceptibility is multistep, complex process that includes initiation, promotion and progression. The most important and decisive event of the chemical carcinogenesis is between presumed carcinogens and macromolecules such as DNA, proteins and lipids [8]. These genetic damages result in tumors by disrupting the normal regulatory pathways that control basic cellular functions. Recently, i t is found that malignancy arises from accumulation of mutation in two major classes of genes, which are protooncogenes and tumour suppressor genes [9]. Tumor suppressor genes stop the growth of tumors by arresting cells in cell cycles [10]. The expressed product suppresses the expression or function of the other genes involved with cell growth and proliferation [11].

Inclusion Criteria
For control group: 10 formalin fixed paraffin embedded samples of oral cavity, histopathologically diagnosed as normal.For study group: 40 formalin fixed paraffin embedded samples which was histopathologically diagnosed as oral leukoplakia an d oral squamous cell carcinoma.

Exclusion Criteria
Oral biopsy specimen who is not received in formalin Biopsy specimen from patients who underwent chemotherapy or radiotherapy. Specimen with histopathological diagnosis other that premalignant and malignant conditions such as Lichen planus, Pemphigus, Pemphigoid etcetera.

RESULTS
The present study was designed to determine the expression of p16 in oral premalignant and malignant lesions. A group of 40 histologically diagnosed formalin fixed, paraffin embedded oral biopsy/ surgical resection tissue samples were collected and were group into control group and study group. The control group consisted of 10 formalin fixed paraffin embedded tissue samples diagnosed as hyperkeratosis/ chronic inflammatory pathology. The study group included histologically diagnosed formalin fixed, paraffin embedded tissue samples oral biopsy/ surgical resection and are sub-divided into  Oral Leukoplakia as Group I  Oral Squamous Cell Carcinoma as Group II Control group: Out of 10 samples, 5 were males and 5 were females.
Study group: In group I: Out of 15 samples, 12 were males and 3 were females. In group II: Out of 25 samples, 18 were males and 7 were females. In the control group, 9 cases were negative for p16 whereas 1 case showed sporadic staining and in the study group I which included leukoplakia had no cases of negative staining, 1 case staining sporadic, 6 cases staining focally and 8 cases staining diffusely. The data are tabulated as Table 3.
In the control group, 9 cases were negative for p16 whereas 1 case showed sporadic staining and in the study group II which included oral squamous cell carcinoma had no cases of negative staining, 14 cases staining sporadic, 6 cases staining focally and 5 cases staining diffusely. The data are tabulated as Table 4.

DISCUSSION
The gene expression of p16 INK4A is prominent in premalignant lesions where it acts as a cell cycle inhibitor by binding to CDK4/6 and prevents its interaction with cyclin D and thereby arresting cell proliferation and arresting carcinogenesis. In my current study of expression of p16 in premalignant and malignant lesions of oral cavity, there is strong expression in premalignant category and weak expression in malignant category [12].
In another study by Angiero F et al. [17] in a study of expression of p16 in progression of epithelial dysplasia of oral cavity in the year 2008. He found in 54 biopsy specimen, 18 specimen were normal mucosa, 25 specimen were dysplastic, and 11 specimen were invasi ve carcinoma. p16 was negative in normal and no dysplasia and was increased in moderate to severe dysplasia. P16 was expressed in 54.5 % in invasive carcinomas which is also contradictory.
All controls were normal oral mucosa showed negative staining. He concluded telling the existence of a subset of malignant lesions staining p16 positive. Azizi et al. [18] [19].
Expression of p16 INK4A is not seen in normal cell cycle. In case of carcinogenesis, p 16 INK4 a is expressed to act as a cell cycle inhibitor by binding to CDK4/ 6 and prevents its interaction with cyclin D and thereby arresting cell proliferation and arresting carcinogenesis. In premalignant lesions, p16INK4A is highly expressed limiting cell cycle and inhibiting the cells to turn malignant and so p16 is found to be high in the premalignant slides. In malignant lesion s, there is loss of p16 activity and hence there is a high turnover of cells without any checkpoint and hence is the reason for malignancy [20][21][22][23][24][25].

CONCLUSION
The current study supports this hypothesis by saying whenever an oral biopsy is taken, expression of p16 can help in categorizing malignant or premalignant and can also help with the prognosis of the condition. p16 may be considered as a tumour marker and can be used in conjunction with routine hematoxylin & eosin slides to help in determining the premalignant/ malignant nature and also the prognostic nature. But our sample study is small. Hence variations cannot be accurately assessed, but it plays a crucia l role in assessing pre-malignant lesions progressing to malignancy. To confirm this, a larger sample study is required.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
Ethical clearance for the study was obtained from the Institutional Human Ethical Committee, Sbmch.