Elsevier

Oral Oncology

Volume 37, Issue 5, July 2001, Pages 461-467
Oral Oncology

Quantitative estimation of PCNA, c-myc, EGFR and TGF-α in oral submucous fibrosis — an immunohistochemical study

https://doi.org/10.1016/S1368-8375(00)00115-9Get rights and content

Abstract

The objectives of the present study were to evaluate the expression of three proliferation markers, viz., epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-α) and proliferating cell nuclear antigen (PCNA) and one genomic marker, c-myc in OSMF. Oral tissues were stained with monoclonal antibodies to PCNA, c-myc, TGF-α and EGFR. The results were analyzed with Photoquant image analysis software. The expression of PCNA, c-myc, TGF-α and EGFR was higher in oral submucous fibrosis (OSMF) than in normal control oral mucosa. The oral epithelium was divided into a proliferative compartment (stratum germinativum) and a differentiated compartment (stratum spinosum). A differential pattern of expression of PCNA and c-myc was observed in OSMF. While the intensity of staining decreased, the percent area of expression of PCNA and c-myc increased in stratum germinativum in OSMF (P<0.05). This suggests that greater proportions of cells exhibit PCNA and c-myc immunoreactivity and are in the proliferative pool in OSMF. TGF-α levels increased in the proliferative layers and EGFR levels increased in the differentiated layers (P<0.05) of the oral epithelium in OSMF. Quantitative measurement of these oncoproteins substantiates the precancerous nature of OSMF and may provide intermediate end-points in prospective chemopreventive trials.

Introduction

Oral submucous fibrosis (OSMF) is a chronic insidious disease affecting any part of the oral cavity and sometimes the pharynx. It is often but not always preceded by vesicle formation and is always associated with juxtaepithelial chronic inflammatory reaction and fibroelastic changes in the lamina propria leading to epithelial atrophy and trismus [1]. Common presenting symptoms are burning sensation in the mouth, intolerance to spicy foods and progressive inability to open the mouth. OSMF is diagnosed clinically based on the palpation of vertical fibrous bands along the buccal mucosa, soft palate and/or the labial mucosa. There is depapillation and restricted movement of the tongue. In advanced cases there is severe trismus and the inelastic mucosa is forced against the teeth leading to ulceration [2]. Recently Haider et al have reported a clinical staging of OSMF based on the relationship between the severity of the disease as measured by mouth-opening and sites of bands in the mouth [3].

The pathogenesis of OSMF is still unclear, although several hypotheses have been proposed [4]. The histological appearance is characterized by atrophic epithelium with loss of rete-ridges, relatively avascular hyalinized collagen in the lamina propria and localized chronic inflammatory cell infiltrates in the subepithelial zone [5], [6]. It has been stated that the atrophic changes in the mucosa predispose to malignant changes in the epithelium. The precancerous nature of OSMF was first postulated by Paymaster and later emphasized by others based on clinical and epidemiological grounds [7], [8]. Histologically epithelial dysplasia, considered a preneoplastic state, is evident in 7–25% of the lesions [9] and the incidence of oral squamous cell carcinoma (SCC) has been estimated as 7.6% in patients followed for a period of 17 years [10]. The precancerous nature of OSMF is further substantiated by the observation of co-existent oral leukoplakia and concomitant finding of oral carcinoma.

Molecular studies have explored a number of genes that are either altered, amplified, dysregulated in expression or deleted in head and neck tumorigenesis. However, specific genetic alterations are not uniformly observed in the majority of tumors, and thus each alone might not be useful as a marker for the tissue at risk [11], [12]. Over the past decade, several studies have attempted to identify specific biomarker/s to predict the malignant potential of a more widely prevalent oral precancerous lesion, oral leukoplakia [13]. However, only few studies have addressed the molecular markers of malignant transformation in OSMF [14], [15], [16]. The objectives of the present study were to evaluate the expression of three proliferation markers, viz., epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-α) and proliferating cell nuclear antigen (PCNA) and one genomic marker c-myc in OSMF.

PCNA, a member of the cyclin family is an auxiliary component of DNA-polymerase-δ and appears in all proliferating cells. Increase in PCNA expression has been observed as tissues progressed from normal epithelium to hyperplasia, dysplasia and head and neck SCC [17], [18]. EGFR is a high affinity plasma membrane receptor that binds to epidermal growth factor and TGF-α initiating a sequence of intracellular events that ultimately lead to the stimulation of cell division. Increased expression of EGFR and TGF-α has been suggested as early markers of head and neck cancer [19], [20]. c-myc gene is a 62kD nuclear protein involved in growth and differentiation of normal cells and acts as an activator of cell division. Amplification of myc oncogene has been reported in oral SCC in Indian patients where OSMF is highly prevalent [11], [21]. In the present report, quantitative immunohistochemical data have been used to measure the expression of PCNA, c-myc, EGFR and TGF-α in OSMF and to evaluate these oncoproteins as markers of malignancy.

Section snippets

Patient population

The cohort included 15 patients with OSMF and 10 patients with oral SCC attending the clinics of Oral Medicine of Ragas Dental College and Hospital, Madras, India. Demographic data, personal habits of tobacco smoking and chewing, betel nut chewing, use of pan masala and alcohol consumption as well as clinical appearance of the lesions were recorded. The clinical diagnosis of OSMF was based on the clinical features of burning sensation in the mouth and the presence of palpable vertical fibrous

Clinical features

The mean age of incidence of OSMF was 28.4 years. With the exception of one, all patients were males. Chewing of commercially available pan masalas (pan parag, manikchand) was the predominant habit. The average period of exposure at the time of diagnosis was 7.2 months. One patient reported symptoms as early as 2 months after exposure. All patients had burning sensation in the mouth and difficulty in opening the mouth. Atrophy of the dorsum of the tongue (57%) and inability to protrude the

Discussion

OSMF is a premalignant condition of the oral cavity found predominantly in native Asians and Asians settled in other countries [1], [6], [8]. In our study there was an overwhelming preponderance of occurrence in males. This is in contrast to most reports of female sex predilection for OSMF. Considerable popularity for the use of commercially available packaged panmasalas amongst young males could explain the discrepancy. This is consistent with the recent reports of increasing incidence of OSMF

Acknowledgements

Ragas Educational Trust as mentioned in Section 2.1. Department of Experimental Medicine and Sophisticated Instrumentation, Tamil Nadu Dr. M.G.R. Medical University, Madras. India for the use of laboratory facilities.

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