|Year : 2020 | Volume
| Issue : 2 | Page : 255-260
Immunohistochemical expression of p63 in oral premalignant disorders and its correlation with oral squamous cell carcinoma
Radhika Manoj Bavle1, K Paremala2, M Soumya1, V Reshma1, M Sudhakara1
1 Department of Oral and Maxillofacial Pathology, Krishnadevaraya College of Dental College and Hospital, Sir MVIT Campus, Bengaluru, Karnataka, India
2 Department of Oral Pathology, Government Dental College and Hospital, Hyderabad, Telangana, India
|Date of Submission||27-Jan-2020|
|Date of Decision||10-Feb-2020|
|Date of Acceptance||22-Feb-2020|
|Date of Web Publication||21-Dec-2020|
Dr. K Paremala
Department of Oral Pathology, Government Dental College and Hospital, Hyderabad - 500 012, Telangana
Source of Support: None, Conflict of Interest: None
Introduction: Oral squamous cell carcinoma (OSCC) represents 95% of all head and neck cancers, and its incidence has increased by 50% in the past decade. Accumulation of genetic alterations (mutations, loss of heterozygosity, loss or gain of chromosomes) is the basis for the progression from a normal cell to a cancer cell. OSCCs are frequently preceded by oral potentially malignant disorders (PMDs). It is difficult to predict the malignant transformation of these PMDs. Thus, early detection of genetic changes in PMDs can facilitate detection of those lesions, which may have potential to progress to malignancy. p63 is a protein coded by p63 gene which is a homolog of p53 gene. It has a critical role in cell cycle regulation and is associated with epithelial tumorigenesis. The present study aims to evaluate the role of p63 in carcinogenesis in PMDs (Leukoplakia, Oral submucous fibrosis and Lichen planus) and OSCCs using immunohistochemistry. Aims and Objectives: The aim is to evaluate the expression of the p63 IHC marker in oral premalignant disorders and to compare its expression in OSCC. Materials and Methods: Tissue sections of PMDs (105 cases) and OSCCs (35 cases) were stained for IHC marker p63. The percentage of positive cells and staining patterns were assessed for all the lesions. Results and Conclusions: Progressive accumulation of p63-immunopositive cells from PMDs to OSCC reflects its major role in the development of OSCC according to a multiple-stage model of carcinogenesis and can be a useful indicator of dysplastic change, thus serving as a biomarker for cancer progression.
Keywords: High risk, oral squamous cell carcinoma, p63, premalignant disorders
|How to cite this article:|
Bavle RM, Paremala K, Soumya M, Reshma V, Sudhakara M. Immunohistochemical expression of p63 in oral premalignant disorders and its correlation with oral squamous cell carcinoma. J Datta Meghe Inst Med Sci Univ 2020;15:255-60
|How to cite this URL:|
Bavle RM, Paremala K, Soumya M, Reshma V, Sudhakara M. Immunohistochemical expression of p63 in oral premalignant disorders and its correlation with oral squamous cell carcinoma. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2021 Jan 16];15:255-60. Available from: http://www.journaldmims.com/text.asp?2020/15/2/255/304252
| Introduction|| |
Oral squamous cell carcinoma (OSCC) is the sixth common cancer in the general population, with variation in incidence rates across different countries. In spite of advances in modern technologies, the 5-year survivalrate of oral cancer patients showed only a meek improvement over the past years.
Oral carcinogenesis is a multistep complex process that involves accumulation of genetic alterations which leads toformationofproteinsthat promote cell growth (byoncogenes), loss of proteins limiting cell proliferation (tumour suppressor genes). Thus any of the molecules involved in these processes can provide markers for early detectionof malignant transformation of potentially malignant disorders (PMDs).
A large proportion of OSCCs are preceded by premalignant disorders such as leukoplakia, erythroplakia and oral submucous fibrosis (OSMF). The prediction of malignant transformation of these oral PMDs is very difficult. Thus, it would be of great help which helps to detect early genetic changes in these lesions, and thus facilitate detection of those lesions, which may have a potential to progress to OSCC.
p63 is a protein coded by p63 gene. It is a member of the P53 tumor suppressor family. The p63 gene shows structural and functional homology with the p53 transcription factor family and is located on chromosome 3q28. It is expressed as three major forms-p63 alpha, p63 beta, and p63 gamma, each of which differ in their c-termini.
p63, a transcriptionfactor plays an important role in the development, differentiation, and morphogenesis of epithelial tissue. The function of p63 in oral epithelium might be for maintaining stem cell function rather than the direct correlation of oncogenesis and malignant transformation. Thus, p63 can have a pivotal role in the neoplastictransformation of the PMDs.
The aim of our present study is to investigate the biologic role of p63 in oral tumorigenesis and its possible role as prognostic marker in oral cancer. Thus, the design of the study is to analyse the expression of immunomarker p63 in PMDs and compare with its expression in OSCC.
| Materials and Methods|| |
The tissue samples for the study included 35 cases of OSCC (Group 1) and 105 cases of PMDs (35 cases each of leukoplakia-Group 2a, OSMF-Group 2b, and oral lichen planus [OLP]-Group 2c). The cases were selected from the Department of Oral and Maxillofacial Pathology, Krishnadevaraya College of Dental Sciences, Bengaluru after reviewing the Hematoxylin and Eosin (H and E) stained sections. The paraffin-embedded blocks of the selected cases were sectioned at 4 μm thickness and stained with IHC marker p63.
The immunohistochemical staining of p63 was performed on sections mounted on Poly-L-Lysine coated slides. The protocol for immunostaining was performed according to the manufacturer's recommendations. The sections were deparaffinized and rehydrated through decreasing concentrations of alcohol. Antigen retrieval was done using EDTA buffer in a pressure cooker. After peroxide blocking, the sections were stained with purified mouse IgG primary monoclonal anti-p63 antibody (prediluted: Biogenex Life Sciences Pvt. Ltd.) followed by incubation with secondary antibody. The sections were treated with Di-amino benzidine chromogen for the visualization. All the sections were viewed under the microscope for the presence of brown-colored end product.
The results were assessed for presence, pattern, and localization of staining. The positive control (prostrate epithelium) and negative control were considered. The grading was performed by two observers independently to prevent interobserver bias.
Ethical approval for this study (KCDS/595/2015-16) was provided by the Ethical Committee of Krishnadevaraya College of Dental College and Hospital on 19/8/2015.
| Observations and Results|| |
All the cases that were positively stained was localized only in the nucleus, i.e., p63 is a nuclear stain. The observations were subjected to statistical analysis.
The location of cells that showed positivity in PMDs is depicted in [Table 1]. Out of 105 cases of PMDs, 63 (60%) cases showed positivity in basal, suprabasal, and spinous cell layers. The positivity was seen till the superficial layers in 33 cases (31%), and only 9 cases (8%) of the PMDs were positive for p63 only in basal and suprabasal layers.
In individual PMDs as in leukoplakia, majority of the cases (52%) showed positivity till the midspinous layers, whereas in OSMF the positivity for p63 was predominantly seen in midspinous and superficial layers. In OLP, the staining was predominantly seen till the midspinous layers, and none of the cases showed positivity only in the basal and suprabasal layers.
For all the PMDs (leukoplakia, OSMF, and OLP) in our study, the low-risk cases had p63 immunopositivity in the basal and suprabasal layers [Figure 1], [Figure 2], [Figure 3] whereas in high-risk cases, the staining was positive till the superficial layers of the involved epithelium [Figure 4], [Figure 5], [Figure 6].
|Figure 1: Photomicrograph showing positive nuclear immunoexpression in the basal and suprabasal layers in low-risk leukoplakia. p63 IHC, ×20|
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|Figure 2: Photomicrograph showing positive nuclear immunoexpression in the basal and suprabasal layers in low-risk oral submucous fibrosis. p63 IHC, ×10|
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|Figure 3: Photomicrograph showing positive nuclear immunoexpression in the basal and suprabasal layers in low-risk lichen planus. p63 IHC, ×20|
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|Figure 4: Photomicrograph showing positive nuclear immunoexpression till the superficial layers in high-risk leukoplakia. p63 IHC, ×20|
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|Figure 5: Photomicrograph showing a positive nuclear immunoexpression till the superficial layers in high-risk oral submucous fibrosis. p63 IHC, ×20|
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|Figure 6: Photomicrograph showing positive nuclear immunoexpression till the superficial layers in high-risk lichen planus. p63 IHC, ×20|
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In OSCC, the positively stained cells in the invaded epithelial islands are more prominent and diffusely distributed in moderately and poorly differentiated lesions. In the well-differentiated OSCCs the positivity was seen in the peripheral part of the islands [Figure 7]a, [Figure 7]b, [Figure 7]c.
|Figure 7: (a) Photomicrograph showing positive nuclear immunoexpression in the peripheral part of the islands in well-differentiated oral squamous cell carcinoma. p63 IHC, ×20. p63 IHC, ×20. (b) Photomicrograph showing positive nuclear immunoexpression in the peripheral part of the islands in moderately differentiated oral squamous cell carcinoma. p63 IHC, ×20. p63 IHC, ×20. (c) Photomicrograph showing positive nuclear immunoexpression in the peripheral part of the islands in poorly differentiated oral squamous cell carcinoma. p63 IHC, ×20. p63 IHC, ×20|
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The average labeling index for OSCC (Group 1) is 82.8. For PMDs (Group 2), the labeling index of 65.15%, 62.3%, and 60.94% was observed in leukoplakia, OSMF, and OLP, respectively [Table 2].
|Table 2: Labeling index of oral squamous cell carcinoma (Group 1) and potentially malignant disorders (Group 2a, b, c)|
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By using Student's t-test for statistical analysis, the P < 0.005 between OSCC and all the MDs [Table 3]. Thus there was statistically significant difference in the labeling index between OSCC and all the PMDs (P < 0.05).
| Discussion|| |
Squamous cell carcinoma of head and neck is the sixth common cancer in the world, the incidence of which is approximately 600,000 cases/year and has a mortality rate of about 50%.
Development of OSCC is a multistep process which requires the accumulation of multiple genetic alterations. It is also influenced by an individual's genetic predisposition and the environmental influence which frequently includes tobacco use, alcohol consumption, and human papillomavirus infection., Understanding the pathogenesis of OSCC in depth is necessary to develop improved therapeutic approaches.
Various and recent advances in relation to oncogenes and tumour-suppressor genes have helped us to study the genetic changes that are occurring at different stages of carcinogenesis that also includes transition from premalignancy to malignancy.
The malignant transformation of leukoplakia varies from 15.6% to 39.2%. This depends on the type of leukoplakia; wherein the speckled variant has more transformation than the homogeneous type. OSMF has a malignant transformation rate of 7%–26%. OLP also has a malignant potential with a transformation rate of 0.4%–3.7%.
The p63 tumor protein is a member of the p53 protein family, which is essential for the development of stratified epithelium. In p53 family of genes, p63 (most ancient member), p53, and p73 are included. Different splicing and promoters produce 6 isoforms ofP63. p63 utilizes an alternative promoter at the 5' end of the gene to allow the expression of two different N-terminal isoforms, one having the N-terminal transactivation domain (TA isoform) and N-terminal truncated isoform (ΔN isoform) that lacks this domain. ΔNP63 is essential for thedevelopment, differentiation, and homeostasis of the oralsquamous epithelium.
Immunohistochemically, p63 is a nuclear stain. In normally stratified squamous epithelium, the expression of p63 is restricted to the basal cell layers, and gradually, its expression downregulates towards the surface of the epithelium, and the superficial layers does not show any positivity.
In the present study, all the PMDs (leukoplakia, OSMF, and OLP) showed an increased labeling index of p63 in the nucleus of immunopositive cells when compared to the normal mucosal epithelium. In leukoplakia, 52% of cases showed positivity for p63 in basal, suprabasal, and in the mid-spinous cell layers; whereas 37% of cases showed positivity up to superficial layers of the epithelium [Table 1]. Morevoer, the average labeling index was 65.15 [Table 2].
In OSMF, 46% and 40% of cases showed p63 positivity till midspinous and superficial cell layers, respectively [Table 1] with a labeling index of 62.3 [Table 2].
In OLP, 83% of cases showed immunopositivity for p63 till the midspinous cell layers and 17% of cases showed positivity up to the superficial cell layers. None of the cases of OLP showed positivity only in the basal and suprabasal cell layers [Table 1]. The mean labeling index was 60.94% [Table 3].
The study is in accordance with Parsa et al. who stated that in dysplasia, the dysplastic keratinocytes present above the basal layers still express p63protein, i.e., shift to a status similar to the embryogenesis producing an anti-differentiation effect, and also maintain the proliferative capacity of dysplastic cells in oral dysplastic mucosa.
In our current study, high-risk cases of all PMDs had the distribution of immunopositive cells extending to the surface layers compared to the low-risk cases. This is in accordance to the study done by Glickman et al., who suggested that the disturbance in the distribution of p63 positive cells in the upper layers in oral epithelial dysplasias varies according to the grade of dysplasia; thus, p63 might play a role in oral tumorigenesis.
Moreover, the architectural disorganization of proliferating cells and stem cells in oral epitheliumcan be a useful index to estimate the grading of epithelial dysplasias if supplemented to histomorphological examinations in H and E staining sections.
Our study is also in accordance to Sinha et al., wherein they proposed the increase in the level of expression and p63 labelling index in dysplastic epithelium when compared to normal epithelium may establish that p63 could be considered as a marker of premalignancy.
In our current study, all the cases of OSCC were positive for p63 staining. The labeling index is more in OSCC (82.85) when compared to all the PMDs and also the normal oral epithelium. These findings of our study are consistent with the studies done by Bortoluzzi et al.
This increase of p63 in OSCCs can be explained by a study done by Oncel et al. wherein they found that in oral tumors, ΔNp63 binds to DNA through its DNA binding domain or it directly interacts with P53/TAP63; and this makes ΔNP63 inhibit cell apoptosis and acts as an oncogene in the occurrence of oral tumors.
According to Nguyen et al., the most abundantTP63 protein product, i.e., ΔNp63 in squamous epithelium promotes renewal of basal keratinocytes by a mechanism which requires downregulation of NOTCH1 and CDKN2A. And also terminal differentiation in squamous epithelium is induced in reaction to genotoxic stress by means of a mechanism that involves p53-dependent transactivation of NOTCH1–an activity antagonized by ΔNp63. Head and neck primary squamous cell carcinoma (HNSCC) involve the transformation of squamous epithelial cell lineage; these findings hypothesized that mutations in the above such genes will disrupt the development/differentiation in precursor cells of malignancy.
The increased p63 expression in OSCC is also supported by a study done by Hibi et al. wherein they found that p63 is a target of amplification and is overexpressed in >80% of HNSCC and also other squamous epithelial malignancies, which suggests the role of p63 as an oncogene.
According to studies done by Lo Muzio et al. and Chen et al., there is preferential overexpression of DNp63a mRNA in cases of HNSCC and epithelial dysplasias.,
Likewise Joseph et al. in his study found that 3q26 region containing the p63 locus is frequently amplified in many tumours and it contains genes for maintaining the immaturity of the tumour cells, which in turn causes disruption of terminal differentiation of cells; thus subsequently preserving their ability to multiply. Thus in neoplastic cells, the DNp63 isoforms can promote growth and survival of neoplastic cells by competing for p53-binding sites and plays an important role in OSCC.
In our study, statistical difference is found between the labeling index of Group 1 (OSCC) and Group 2 lesions (PMDs) [Table 3]. This indicates an increased expression of p63 with increasing grades of PMDs to malignancy thus confirming the role of p63 in carcinogenesis.
| Conclusions|| |
Progressive accumulation of p63-immunopositive cells from PMDs to OSCC may reflect its major role of this protein in the development of OSCC according to a multiple-stage model of carcinogenesis. Our study results indicate that increased p63 expression in PMDs and OSCC may be useful indicators of dysplastic change, serve as a biomarker for cancer progression.
Understanding the histology, premalignant states, and molecular mechanisms of oral carcinogenesis may facilitate the development of novel strategies for the prevention and treatment of oral cancer. In addition, early detection is of critical importance to improve the survival rates of patients with oral cancer.
Advanced and improved knowledge on tumor behavior regarding p63 would be important to assess response to treatment and would lead to novel therapies with higher efficacy and less toxicity.
We are grateful to Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka for providing a grant for this Original research work.
Financial support and sponsorship
This original research work has been funded by Rajiv Gandhi University of Health Sciences, Bangalore.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]