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ORIGINAL ARTICLE
Year : 2014  |  Volume : 5  |  Issue : 3  |  Page : 149-154

Assessment and comparison of p53 and p63 expression in oral epithelial dysplasia and squamous cell carcinoma


1 Department of Oral Pathology, Faculty of Dentistry, Melaka Manipal Medical College, Manipal, India
2 Department of Oral Pathology, Annoor Dental College, Muvattupuzha, Kerala, India
3 Department of Oral Pathology, Yenepoya Dental College, Mangalore, Karnataka, India

Date of Web Publication14-Aug-2014

Correspondence Address:
Smitha Sammith Shetty
Department of Oral Pathology, Faculty of Dentistry, Melaka Manipal Medical College, Manipal, Karnataka
India
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DOI: 10.4103/0976-433X.138710

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  Abstract 

Aim: p53 and p63 are the important genes associated with oral cancer. The aim of this study was to evaluate and compare the immunohistochemical expression of p53 and p63 in oral epithelial dysplasia and squamous cell carcinoma (SCC). Materials and Methods: Immunohistochemical expression of p53 and p63 was graded in total of 60 archival cases, which included 30 cases of oral epithelial dysplasia (10 cases each of mild, moderate, and severe dysplasia) and 30 cases of oral SCC (10 cases each of well-differentiated, moderately differentiated, and poorly differentiated SCC). Results: Our study showed statistical significant difference on comparison of p53 and p63 expression in oral epithelial dysplasia. In oral SCC cases, p53 and p63 did not show significant correlation in expression. Conclusions: Our study demonstrated a progression in expression of p53 and p63 along the grades of oral epithelial dysplasia to SCC, suggesting their role in stages of carcinogenesis. However, p53 and p63 may have independent role in oral tumorigenesis.

Keywords: p53 genes, p63 genes, immunohistochemistry, mouth, carcinoma, squamous cell


How to cite this article:
Shetty SS, Krishnapillai R, Prabhu S. Assessment and comparison of p53 and p63 expression in oral epithelial dysplasia and squamous cell carcinoma. SRM J Res Dent Sci 2014;5:149-54

How to cite this URL:
Shetty SS, Krishnapillai R, Prabhu S. Assessment and comparison of p53 and p63 expression in oral epithelial dysplasia and squamous cell carcinoma. SRM J Res Dent Sci [serial online] 2014 [cited 2020 Aug 9];5:149-54. Available from: http://www.srmjrds.in/text.asp?2014/5/3/149/138710


  Introduction Top


Oral cancer is the sixth most common cancer worldwide, accounting for approximately 4% of all cancers. Squamous cell carcinoma (SCC) of the oral cavity may comprise up to 50% of all cancers in developing and underdeveloped countries. [1]

Oral SCC is usually preceded clinically by evident precancerous lesions which appear as white (leukoplakia) or red lesions (erythroplakia). At the microscopic level, these lesions show varying degrees of epithelial dysplasia, from mild to severe. Long-term studies have shown that the overall risk of malignant transformation of all grades of epithelial dysplasia is approximately 16%. A higher grade of dysplasia is generally associated with a higher risk of neoplastic transformation. [2]

The development of oral SCC is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient's genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation and viral infection. [3] Recent advances in the field of tumor-suppressor genes and oncogenes have provided a tool for studying the genetic changes occurring at different stages of carcinogenesis, including transition from premalignancy to malignancy. [1]

Among the genes associated with oral cancer, p53 is a well-known tumor-suppressor gene that is believed to serve as a gatekeeper against carcinogenesis. It has its site of action in the nucleus and is thought to be involved in regulating the replication of DNA. Under normal circumstances, the function of p53 protein is to prevent the propagation of genetically damaged cells. [4],[5] Cells with loss of p53 function are speculated to undergo malignant transformation. Abnormal expression of p53 protein has also been reported in 10-50% of oral epithelial dysplasia suggesting the role of p53 in early phases of multistage carcinogenesis. Until date, mutation of the p53 gene is one of the most common events in human cancers, including oral SCC. [2],[5]

The p63 gene, a member of the p53 gene family is transcribed into six protein isoforms, which are divided into two groups, those containing the transcription activation domain (TA isoforms) and those which do not contain transcription activation domain (∆N isoforms). [6] TAp63 isoforms might behave like p53 because they transactivate various p53 downstream targets, induce apoptosis and mediate cell cycle control. However, the ∆Np63 isoforms have been shown to display opposing functions to TAp63 isoforms by acting as oncoproteins. [7] p63 is known to play an essential role in epithelial development and the proliferation of limb and craniofacial structures. It is expressed in multiplying cells, and is thought to be a marker for keratinocyte stem cells. [8] p63 overexpression has been demonstrated in SCC arising in different organs such as larynx, lung, uterine cervix, urinary bladder, head and neck tumors, suggesting its role in oncogenesis of tumors. [9]

Until date, only one study has been document in English literature on assessment and comparison of both p53 and p63 expression in oral SCC and dysplasia. [8] Therefore, this study was performed to assess the biologic role of p53 and p63 in stages of oral tumorigenesis and to evaluate if any of these markers will represent the early changes in transformation to dysplasia or malignancy.


  Materials and methods Top


Case selection

A range of malignant and potentially malignant oral lesions were included in this study. A total of 60 archival cases were considered, which included histologically diagnosed, 30 cases of oral epithelial dysplasia (10 cases each of mild [MD], moderate [MoD] and severe dysplasia [SD]) and 30 cases of oral SCC (10 cases each of well-differentiated SCC [WSCC], moderate differentiated SCC [MSCC] and poorly differentiated SCC [PSCC]). In addition as a control group, 10 cases of normal oral mucosa were selected.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections were cut at 5μ and stained using standard immunohistochemical methodology. Two primary antibodies were used: Anti-p53 (Clone DO-7, Biogenex Life Sciences Limited [CA, USA]) and anti-p63 (Clone 4A4, Biogenex Life Sciences Limited [CA, USA]). Antigen retrieval for p53 and p63 was done using citrate buffer by pressure cooker method. Super sensitive polymer - horseradish peroxidase detection system, (biotin free detection) supplied by Biogenex Life Sciences Limited (CA, USA) was the visualization kit utilized which carried the chromogen diaminobenzidine.

Sections of oral epithelial dysplasia and SCCs were scored, both by qualitative and quantitative method. The presence of brown colored end products at the site of target antigen (nucleus) was considered as positive immunoreactivity for both markers. The intensity of the staining was graded as negative (0), mild but definite positive (1), moderate (2) and intense (3). Quantitative assessment was done using an ocular graticule. To determine the proportion of positively stained cells, each specimen was assessed by counting a total of 400 cells (including negative and positively stained cells). Then, the percentage of positive cells was calculated. Slides were reviewed independently by two of the authors, which showed concordant results.

The percentages of positive cells for p53 and p63 were compared between normal mucosa, epithelial dysplasia and oral SCCs. Statistical analysis was performed using Mann-Whitney U-test and Kruskal-Wallis ANOVA test. The criterion for statistical significance was P < 0.05.


  Results Top


All cases of normal oral mucosa showed negativity with p53 [Figure 1]a, while prominent nuclear staining in basal and suprabasal layer was seen with p63 [Figure 1]b.
Figure 1: In normal mucosa, p53 (a) showed negative expression and p63 (b) showed intense staining in basal and parabasal layer; in MD, p53 (c) showed moderate intensity of staining in basal and parabasal layer and p63 (d) showed Intense staining extending up to middle spinous layer; in MoD, p53 (e) stained with moderate intensity extending up to middle spinous layer and p63 (f) showed intense staining extending up to entire spinous layer; in severe dysplasia, p53 (g) showed moderate intensity of staining extending up to entire spinous layer and p63 (h) showed intense staining in entire thickness of epithelium (×10)

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Oral epithelial dysplasia cases showed prominent nuclear staining of p53 and p63 with distribution in the basal, suprabasal and spinous layer, with gradual increase in extent of expression on progression in grades of dysplasia. Expression of p63 was more extensive and stronger in more layers than that of p53 in each grade of epithelial dysplasia [Figure 1]c-h. p63 immunostaining was observed in all cases, while p53 positive staining was seen only in 9 cases of MD (90%), 6 cases of MoD (60%), and 5 cases of SD (50%). The intensity of p53 staining in epithelial dysplasia ranged from mild (1) to moderate (2), but in case of p63 it ranged from moderate (2) to intense (3). p53 showed 36-73.3% of positively stained cells, while in p63 it ranged from 70.3% to 95.6%.

Oral SCC cases showed prominent nuclear staining with p53 and p63 in varying proportions. The peripheral cells of tumor islands showed prominent staining in WSCC and MSCC, while the inner layer of cells toward the center and keratin pearls lacked staining with both markers[Figure 2]a-d. In PSCC, two patterns of staining were observed; either diffuse and intense [Figure 2]e-f, or faint to negative. p53 showed positivity in all cases of WSCC (100%), in only 8 cases of MSCC (80%) and 4 cases of PSCC (40%), but p63 positivity was seen in all cases of WSCC (100%) and MSCC (100%) while only 7 cases of PSCC (70%). The intensity of p53 staining in SCC ranged from mild (1) to intense (3), and in p63 it ranged from moderate (2) to intense (3). The percentage of positively stained cells ranged from 90% to 99.3% in p53 and in case of p63 it ranged from 89% to 99%.
Figure 2: In well differentiated p53 (a) and p63 (b) showed intense staining in peripheral cells of tumor islands and lack of staining in areas of keratin pearl formation; In moderate squamous cell carcinoma, p53 (c) and p63 (d) showed moderate intensity of staining in peripheral cells of tumor islands and lack of staining in few cells in the center of nests; in poorly differentiated squamous cell carcinoma, p53 (e) and p63 (f) showed diffuse and intense staining (×10)

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[Table 1]a and b show means and standard deviation (Std.Dev) of p53 and p63 expression in oral epithelial dysplasia (MD, MoD, and SD) and SCCs (WSCC, MSCC, and PSCC), respectively.
Table 1: Means and SD values of quantitative expression of p53 and p63 in oral ED (a) and SCC (b)

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  Discussion Top


Tumor-suppressor gene p53 (also known as TP53) is the most commonly identified mutated gene in diverse types of human cancer. Mutation of the p53 tumor-suppressor gene is thought to be an important component of oral carcinogenesis. Wild-type - p53 has a very short half-life of 20 min; it regulates cell proliferation and DNA repair by inhibiting the cell cycle at G1/S phase. Mutant p53 protein has a relatively long half-life, unlike the wild-type, leading to accumulation of altered p53 protein. [10]

p63 is known to play an essential role in epithelial development and maintenance. Mutation of p63 rarely occurs in malignancies, but amplification appears to be responsible for overexpression of p63 protein in many SCCs from several sites, including the head and neck. [8] The definitive role of the p63 gene in tumor formation and progression still remains controversial. [11]

Our attempt to analyze the p53 and p63 expression showed uniform pattern and intensity of expression in all staining batches, which composed of the normal mucosa, oral epithelial dysplasia, and SCC cases, suggesting that the immunohistochemical procedure utilized is standardized and hence the results can be considered reliable.

In our study, all cases of normal oral mucosa showed negativity with p53. This is in accordance with the studies done by Ogden et al., [12] Rich et al. [13] and Kaur et al., [1] suggesting its short half-life and lack of stabilization in normal mucosa. [10] The p63 nuclear staining was confined to the basal and parabasal layer in all cases of normal mucosa in our study. Similar findings have been reported in oral buccal mucosa, skin, and laryngeal epithelia. [14],[15],[16] These observations suggest a relationship between p63 protein and the differentiation of oral stratified squamous epithelia. [17]

Oral epithelial dysplasia cases showed varying proportion of p53 staining with 9 cases being negative. However, statistical significant correlation was found on comparison of p53 expression with combined grades of epithelial dysplasia [Table 2]a. Our findings were similar to the study conducted by Kerdpon et al. [18] Kaur et al. [1] and Sauter et al. [19] An increased level of p53 protein in premalignant oral lesions indicates that altered p53 expression may be an early event in the pathogenesis of oral neoplasia. [1],[20] The concordance between p53 gene mutation and p53 overexpression is not perfect. In our study, 4 cases of moderate and 5 cases of SD did not exhibit p53 expression. However the probable reason for p53 to be undetectable in premalignant and malignant oral lesions might be that the cells with frame shift or nonsense mutations in the coding sequences of the gene result in absence, truncated, or unstable protein. [21],[22]
Table 2: Comparison of quantitative expression of p53 among grades of oral ED (a) and grades of oral SCC (b) by Kruskal-Wallis ANOVA test

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A high frequency of elevated levels of p53 protein in SCCs of the head and neck has been reported. [12],[23] In our study, 22 cases out of 30 cases of oral SCC expressed p53 protein. On statistical analysis, significant correlation was found between p53 expression and combines grades of oral SCC [Table 2]b. Our results are similar to as reported by Kaur et al. [22] The correlation between p53 expression and poor histological differentiation of oral SCCs, suggests that p53 protein level may be a useful indicator of the malignant phenotype of squamous cells. [22] The negativity of p53 seen in few cases may be attributed to various mutations resulting in absence, truncated or unstable protein, or may be due to a gross deletion of gene that abolishes p53 protein production. [20],[22]

On comparison of expression of p53 in oral epithelial dysplasia and oral SCC, a significant difference was noted [Table 3]. This is in accordance with the study by Kaur et al. [1] These results suggest that p53 may be involved in the development of oral cancers through the dysplasia-carcinoma sequence. [1]
Table 3: Pairwise comparison of quantitative expression of p53 in oral ED and SCC by Mann-Whitney U-test

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p63 expression was demonstrated in all samples of epithelial dysplasia. However, no statistical significant correlation was seen on comparison of expression with combined grades of dysplasia [Table 4]a. Our finding was consistent with study by Bortoluzzi et al. [8] suggesting that p63 protein stabilization may be due to amplification of p63, but analyzing expression may be of less value in potentially precancerous lesions.
Table 4: Comparison of quantitative expression of p63 among grades of oral ED (a) and grades of oral SCC (b) using Kruskal-Wallis ANOVA test

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In oral SCC, p63 expression was seen in 27 out of 30 cases. A significant association was seen on comparison of expression with combined grades of oral SCC [Table 4]b. Our results were consistent with the findings by previous authors. [17],[24],[25] suggesting that the p63 expression being tightly linked to cell maturation in normal squamous epithelium; in carcinomas it may also reflect the immaturity of the tumor cell lineage. Thus, p63 can be an additional marker for diagnostic use in oral SCC. [24] The negativity of p63 in 3 cases of PSCC in our study may be due to its lack of expression in completely de-differentiated cancer cells. [24]

The comparison of p63 expression in oral epithelial dysplasia and oral SCC showed significance difference [Table 5]. This was in contrast to the findings reported in an earlier study. [8] However, the progressive accumulation of p63-immunoreactive cells from dysplasia to SCC in our study may reflect the major role of this protein in the development of SCC according to a multiple-stage model of carcinogenesis. [25]
Table 5: Pairwise comparison of quantitative expression of p63 in oral ED and SCC by Mann-Whitney U-test

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Expression of p53 compared with that of p63 in oral epithelial dysplasia showed significant difference [Table 6]a. p63 showed positivity in all cases of moderate and SD, which were negative for p53 expression. Similar findings were reported by Choi et al., [26] suggesting that p53 and p63 may have independent role in the early development of these lesions or there is compensatory upregulation of p63 in response to p53 alteration.

In agreement with de Oliveira et al. [27] and Bortoluzzi et al. [8] our results showed no correlation between p63 and p53 immunostaining in oral SCC [Table 6]b. These results suggest an independent role of p53 and p63 during oral tumorigenesis.

The study documented by Bortoluzzi et al. [8] suggests that p53 and p63 staining pattern nor percentage of stained cells could be used to differentiate grades of oral dysplasia and carcinomas and carry no diagnostic value, but our study revealed contrast results.
Table 6: Comparison of quantitative expression of p53 and p63 in oral ED (a) and SCC (b) using Mann-Whitney U-test

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  Conclusion Top


p53 and p63 expression progressed from oral epithelial dysplasia to SCC suggesting their role in stages of carcinogenesis. The significant difference in p53 expression among grades of epithelial dysplasia, unlike that of p63, suggests that p53 may play a more predictive role in assessing the earliest changes among precancerous lesions compared to that of p63. In oral tumorigenesis, p53 and p63 may be useful indicators of malignant change, but both may have an independent role.


  Acknowledgments Top


The authors are grateful to the SDM college of dental sciences, Dharwad, India for the support during the study and also thankful to all the faculty members for the guidance.

 
  References Top

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27.de Oliveira LR, Ribeiro-Silva A, Zucoloto S. Prognostic impact of p53 and p63 immunoexpression in oral squamous cell carcinoma. J Oral Pathol Med 2007;36:191-7.  Back to cited text no. 27
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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