Immunohistochemical Analysis of Ezrin Expression in Oral Premalignant Lesions

Research Article

J Dent & Oral Disord. 2020; 6(3): 1134.

Immunohistochemical Analysis of Ezrin Expression in Oral Premalignant Lesions

Temelie-Olinici D1, Cotrutz CE1*, Hritcu-Condurache OM1*, Dimitriu DC1, Sin IA2 and Gheuca-Solovastru L3

¹Department of Morpho-Functional Sciences II, “Grigore T. Popa” University of Medicine and Pharmacy Iasi, Romania

²Department of Cell and Molecular Biology, University of Medicine and Pharmacy Targu Mures, Romania

³Department of Dermatology, “Grigore T. Popa” University of Medicine and Pharmacy Iasi, Romania

*Corresponding author: Carmen Elena Cotrutz, Department of Morpho-Functional Sciences II “Grigore T. Popa” University of Medicine and Pharmacy, Romania; Oana Mihaela Hritcu-Condurache, Department of Morpho-Functional Sciences II “Grigore T. Popa” University of Medicine and Pharmacy, Romania

Received: April 27, 2020; Accepted: May 15, 2020; Published: May 22, 2020


Objectives: Across the world, the incidence and prevalence of oral premalignant lesions have increased significantly. These lesions are the precursors to squamous cell carcinoma, one of the most aggressive types of cancer to be diagnosed over the past 30 years. Their phenotypical and molecular variability justifies a sitch from histopathological to molecular diagnosis in order to identify and describe the risk of malignancy. Ezrin, a molecule the ERM protein family (Ezrin-Radixin-Moezin), is one of the latest identified “hot spots” of tumor metastasis mechanisms.

Materials and Methods: Oral mucosal fragments were collected from fortythree clinically and histopathological diagnosed with oral premalignant lesions such as leukoplakia, erosive oral lichen planus and erosive actinic cheilitis have been processed through the immunohistochemical technique using Ezrin Polyclonal Antibody (Cell Signaling TechnologyR - BioZyme antibody). The immunohistochemical samples were examined using the Olympus BX40 photonic microscope with Olympus E330 photo camera attached.

Results: The results reveal an ezrin overexpression in the oral premalignant lesions analyzed, compared with the normal mucosa, indicative of an increased risk of malignant transformation.

Conclusion: Concurrently, our study highlights the importance of further research into the identification of specific markers allowing for targeted and individualized therapeutic strategies.

Keywords: Oral premalignant lesions; Ezrin expression; Squamous cell carcinomas; Malignant transformation; Immunohistochemical study


Physiopathological processes in the oral mucosa are highly complex and impact the health of the entire body. The oral mucosa is where premalignant and malignant lesions may easily occur, due to its poor resistance to chemical, mechanical and traumatic irritations [1].

Oral lesions precursor to squamous cell carcinomas are becoming more and more common worldwide. These carcinomas are extremely aggressive in their complications both in situ as well as elsewhere in the body. Statistically, this also translates into a level of morbidity and mortality increasing with over 5% every year [2,3].

In contrast to other types of epithelial premalignant and malignant lesions, the absence of clinical manifestations which characterize most lesions occurring in the oral mucosa makes early diagnosis difficult and thus contributes to poorer prognosis and survival rates [4].

Most frequently, the development of oral squamous cell carcinoma is a multi-step process during which epithelial cells transform into preneoplastic cells and then tumor cells, gradually undergoing multiple morphological and molecular changes. Consequently, in over 50% of cases, this type of carcinoma occurs in premalignant lesions such as leukoplakia, erythro-plakia/erythroleukoplakia, oral submucous fibrosis, and oral lichen planus. Tobacco keratosis, leukoedema, leukoderma, discoid lupus erythematosus, and bullous epidermolysis are less common [5].

The rate of these lesions’ malignant transformation is approximately 17% over a period of 7 years following diagnosis. The highest is in the case of heterogenous erythroplakia and erythroleukoplakia with dysplastic changes. However, some studies have found that 16-62% of leukoplakia lesions feature modifications indicative of squamous cell carcinoma at the time of diagnosis [1,6,7].

The clinical diagnosis is in most cases difficult because the subjective and objective signs are not specific and do not adequately reflect the degree to which epithelial and connective tissues are affected. In addition, patients do not seek dermatological and/or dental consultations until in advanced stages, which is unfortunate [8,9].

Although some researchers consider anatomopathological examination as the gold standard in evaluating the potential for malignant transformation, this method comes with its own limitations. For one, risk assessment is done mainly based on the presence/absence of dysplasia as suggested by certain structural and cytological changes [1,10-12].

Therefore, as far as epithelial dysplasia is concerned, histopathological diagnosis is to a large extent subjective and depends greatly on the anatomopathologist’s level of expertise. There may be cases in which carcinomas develop freely in dysplastic lesions after having gone unidentified/undiagnosed in previous biopsies. The situation is further complicated by the fact that not all such lesions turn malignant and some may even regress. Also, the prognosis of premalignant and malignant lesions cannot be established with precision based only on their histopathology [7].

These current limitations justify the need for the identification of markers sensitive and specific enough for premalignant lesions precursor to oral squamous cell carcinoma, so that these lesions may be accurately and timely diagnosed, and their potential for malignant transformation adequately assessed. To this end, the study of molecular changes specific to oral premalignant lesions as early as their initial stages of development is an important step forward.

Recently, the ERM molecules (Ezrin-Radixin-Moesin) have been studied and evaluated in certain types of cancers. Ezrin has been found to be involved in pathways which regulate cell survival, proliferation, and migration, as well as in the regulation of cell-cell and cell-matrix adhesion. The suppression of these three proteins is also known now as a determining factor in cellular coupling disorganization [13-15].

Ezrin or cytovillin is a protein present in the plasmalemma of the apical pole of epithelial cells and it achieves the connection between the cytoskeleton and cellular membranes. It binds the F-actin to the cellular membrane, after phosphorylation, and is thus essential to many fundamental cellular processes. It also activates GTPases and RhoA leading to cytoskeletal remodeling, an important process in cell motility, proliferation, differentiation and migration [16-18].

In keratinocytes, the overexpression of E-cadherin and β-catenin may be associated with ezrin suppression, while ezrin expression correlates positively with the ki-67 index, a marker of tumor proliferation and severity [18-20].

A number of studies underline ezrin’s role in tumor development and progression, thus suggesting its applicability as a biomarker useful in establishing the appropriate therapeutic approach in the case of head and neck carcinomas [21].

Some research has also found that ezrin expression is associated with the aggressive phenotype of prostate adenocarcinoma, breast cancer, astrocytoma, uveal melanoma, and soft tissue sarcoma. In such cases, on one hand, elevated levels of ezrin were detected in secondary tumor cells compared to primary ones, suggesting that ezrin plays an important role in metastasis, and, on the other hand, certain therapies were seen to influence the expression of this molecule [14].

By taking all of the above into consideration, and noticing the scarcity of published studies regarding ezrin expression in skin carcinomas (with only one study on skin premalignant lesions and none about oral ones), we sought to conduct this immunohistochemical analysis of the molecule in oral premalignant lesions such as leukoplakia, erosive oral lichen planus and erosive actinic cheilitis.

Material and Method

For the purposes of this research, samples of oral mucosa were collected from 43 patients by means of incisional/excisional biopsy with a scalpel blade. The patients were all non-smoker, non-drinker women aged between 45-60, diagnosed clinically and histopathologically with leukoplakia (28 cases, of which 20 with lightmoderate dysplasia and 8 with moderate to severe dysplasia), erosive actinic cheilitis (10 cases), and erosive oral lichen planus (5 cases). Concurrently, oral mucosa normal in appearance was sampled from the same patients with the same technique, making up the control samples.

Prior to the surgical intervention, each patient gave her informed consent in accordance with the legislation in place. The anatomopathological diagnosis was given at the Anatomical Pathology and Morgue Service of the Railways Clinical Hospital Iasi, and at the Anatomical Pathology Laboratory of the Emergency Military Hospital “Dr. I. Czihac” Iasi. Some of the tissue samples were processed using the paraffin embedding technique, then sectioned and colored with hematoxylin and eosin in order to be diagnosed histopathologically.

Another batch was processed immunohistochemically using the NovoLinkTMMax Polymer Detection System kit produced by Biosystems Newcastle Ltd and Ezrin Polyclonal Antibody (Cell Signaling TechnologyR -BioZyme) as follows: fixation in neutral formaldehyde, dehydration in ethanol in incremental concentrations, clearing with xylol, embedding in paraffin, sectioning and placement on electrostatically charged slides, deparaffinization with xylene, ethanol hydration, tap water cleaning, application of Ezrin Polyclonal Antibody diluted 1/100 μl, washing with deionized water, endogenous peroxidase block with Peroxidase Block for 5 minutes, washing with PBS for 2x5 minutes, 30-min PostPrimary Block incubation, washing in PBS for 2x5 minutes, 30-min NovoLink Polymer incubation, washing in PBS for 2x5 minutes, application of DAB solution (50 μl -1ml), washing, countercoloring with hematoxylin, washing, dehydration, clarifying, mounting on slide and then examination with Olympus BX40 microscope and photography with Olympus E330 camera at the Discipline of Cellular and Molecular Biology, “Grigore T. Popa” University of Medicine and Pharmacy Iasi, Romania.


The analysis of oral mucosa samples processed via the standard technique for hematoxylin eosin coloration revealed signs indicative of the following diagnoses: leukoplakia with mild-moderate dysplasia (hyperplastic epithelial tissue with moderate, hyper and parakeratosis as well as marked acanthosis , the widening of the stratum granulosum and rare typical mycoses in the stratum basale, stroma with edema, vascular ectasia and polymorphous inflammatory infiltrate, as shown in Figure 2a; leukoplakia with moderate--severe dysplasia (epithelium with hyperkeratosis, marked hypergranulosis, moderate and focal acanthosis, focal acantholysis of stratum basale, stroma with edema, vascular ectasia and polymorphous inflammatory infiltrate, as shown in Figure 2b; erosive oral lichen planus (hydropic degeneration of stratum basale, acanthosis with formation of sharp papillary buds, discontinuous hypertrophy of stratum granulosum, hyperkeratosis, subepidermal blisters, band-like lymphohistiocytic inflammatory infiltrate at dermal level, Civatte bodies, as shown in Figure 2c; erosive actinic cheilitis (erythema, edema, ulceration, hyperkeratosis, and mild acanthosis, lymphohistiocytic infiltrate perivascularly and around the glandular ducts, as shown in Figure 2d.

The results of the immunohistochemical analysis aiming to identify ezrin expression, a little-studied molecule in the context of skin premalignant lesions and so far overlooked with regard to oral premalignant lesions, revealed a series of modifications in the case of tissue samples collected from premalignant lesions compared to those taken from normal oral mucosa.

Notably, the examination of normal oral mucosa samples processed via the above mentioned immunohistochemical technique indicated moderate positive perimembranous immunoreactivity to ezrin and lack of immunoreactivity to it cytoplasmically (Figures 1, 3).