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ORIGINAL ARTICLE
Year : 2013  |  Volume : 9  |  Issue : 4  |  Page : 587-591

Oral squamous cell carcinomas in age distinct population: A comparison of p53 immunoexpression


1 Department of Oral and Maxillofacial Pathology, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
2 Department of Oral and Maxillofacial Pathology, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, India
3 Department of Oral and Maxillofacial Pathology, Mahe Institute of Dental Sciences and Hospital, Palloor, Mahe, Puducherry, India
4 Department of Oral and Maxillofacial Pathology, Dental College, Azamgarh, India
5 Department of Oral and Maxillofacial Pathology, Shree Bankey Bihari Dental College and Research Centre, Ghaziabad, India
6 Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Web Publication11-Feb-2014

Correspondence Address:
Akhilesh Chandra
2/29 Rashmi Khand, Sharda Nagar, Lucknow - 226 002, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.126452

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 > Abstract 

Aims: The study aimed to assess the diffused expression of p53 protein in patients with OSCC and its association with age at diagnosis (using 50 years as a cut point).
Study Design: Ten normal oral mucosa and sixty OSCC lesions from age-distinct patient populations were immunohistochemically analyzed for the expression of p53 protein.
Results: In OSCC cases, 31 out of total 60 cases (51.67%) showed positive expression for p53 protein and it was more common in older study group (56.67%) than in younger study group (46.67%) but the difference was not statistically significant. Moreover, there was also no significant difference in the p53 indices (percentage of p53 positive cells) between the two study groups (P = 0.827).
Conclusion: The alteration rates were found to be slightly higher in older patients although not statistically significant.

Keywords: Immunohistochemistry, p53 protein, oral squamous cell carcinoma


How to cite this article:
Chandra A, Singh A, Sebastian BT, Agnihotri A, Bali R, Verma PK. Oral squamous cell carcinomas in age distinct population: A comparison of p53 immunoexpression. J Can Res Ther 2013;9:587-91

How to cite this URL:
Chandra A, Singh A, Sebastian BT, Agnihotri A, Bali R, Verma PK. Oral squamous cell carcinomas in age distinct population: A comparison of p53 immunoexpression. J Can Res Ther [serial online] 2013 [cited 2019 Nov 22];9:587-91. Available from: http://www.cancerjournal.net/text.asp?2013/9/4/587/126452


 > Introduction Top


Oral squamous cell carcinoma (OSCC) is a disease found particularly in low income communities and mainly a problem of older men, 90% being in the >45 year age group who are exposed to the known risk factors of tobacco and/or alcohol (IARC 2004). [1] However, there are changing pattern in both lip and intraoral cancer with a decrease in male incidence of lip cancer over about a 30-year period, but several studies have shown an increase in OSCC, particularly in younger patients, currently attributed to smoking and drinking among young people. [2],[3]

Patients with OSCC have a poor prognosis and, typically, more than one-third of the patients die within 5 years of diagnosis. [4] The disease is largely preventable and early diagnosis greatly increases a patient's chances of survival as the mouth is easily accessible for clinical and self examination. [5]

Mutation in the TP53 gene is the most common genetic change found in OSCC, and it has been seen in 40-50% of the OSCC cases. [6],[7] TP53 is tumour suppressor gene located on the short arm (p) of chromosome 17. The p53 protein, which is encoded by this gene, is one of the proteins that regulate the cell cycle. Once activated, the p53 protein can induce growth arrest as well as cell death. The wild type p53 protein has a very short half life (6-20 min) and, therefore, it is hard to detect in normal tissue. [8] However, it can remain longer in tissue for a certain reasons such as mutation, a defect in the degradation pathway, or by binding to some other protein. Mutation and subsequent inactivation of a tumour suppressor gene causes 'loss of function'. [6] This can damage its DNA binding properties and transcription factor function, thus, inhibiting its normal function in cell cycle control and in cell proliferation. [9] The clinical significance of young age at diagnosis has been the subject of several reports with some series suggesting a worse prognosis. [2],[10] Very few studies of comparison of p53 expression between older and younger OSCC patients exist. [11],[12],[13],[14]

This study was planned, therefore, with the aim to compare and study the diffused expression of p53 protein in OSCC in a group of younger patients (≤50 years) with similar lesions in a group of older patients (>50 years) and to compare the results with that of p53 protein expression in normal oral mucosa. The results obtained will provide idea about the biological behaviour of OSCC in two age groups.


 > Materials and Methods Top


The study comprised of 70 patients, who were divided into three groups: Group I (control group) contained 10 cases with normal oral mucosa; Group II consisted of 30 cases, in which biopsy samples of histologically proven OSCC of patients ≤50 years of age were included (study group-1); Group III had 30 cases, in which biopsy samples of OSCC of patients >50 years of age were included (study group-2).

The biopsy tissues for the control group were obtained from patients who had undergone minor surgical interventions. The samples for study groups-1 and 2 were collected from the formalin-fixed and paraffin-embedded blocks of the histologically proven OSCC as well as from the patients attending the dental hospital during the study period. The paraffin blocks were sectioned on a rotary semiautomatic soft tissue microtome into two tissue sections of 5-μm and 3-μm thickness. The 5-μm thick section was stained with Haematoxylin and Eosin for the diagnosis of lesion, while the 3-μm thick section was taken on poly-L-Lysine-coated glass slides for immunohistochemical (IHC) staining. Heat-mediated antigen retrieval was done in citrate buffer in pressure cooker. Slides were then incubated in 3% hydrogen peroxide to block endogenous peroxidase activity. The p53 (DO-7) mouse monoclonal primary antibody was applied for 60 min, followed by super enhancer for 25 min. [10],[15] For secondary antibody application, the slides were incubated with polymer-HRP reagent (anti-mouse and anti-rabbit IgG labelled with enzyme polymer in PBS) for 30 min. [16] Slides were then incubated in freshly prepared 3,3-Diaminobenzidene Tetrahydrochloride (DAB) solution, and, for counter staining, the slides were dipped once in Mayer's Haematoxylin. [17],[18] When a distinct brown staining was confined to the nuclei, p53 expression was considered positive. Sections of colon carcinoma served as positive control for p53 staining. [19],[20]

Counting procedure

The IHC sections were first analyzed for the expression of p53 at low power (10X). The selected fields were used for the quantitative assessment of the p53 positive cells, however, the epithelium next to the lesional area was not evaluated. A minimum of 500 tumour cells were identified and counted in five different histological fields using a magnification of 40X. [17],[21] An eyepiece grid was used to prevent the overlapping of fields. p53 overexpression was considered for those cases that demonstrated ≥20% positively stained nuclei of tumour cells. [10] Negative result was defined as absence of stained nuclei or immunoexpression under 20% of tumour cells. The p53 indices were prepared to correlate between mean-indices of p53 protein expression and the presence of TP53 gene alterations and calculated as the percentage of positively stained cells among the total cells counted. [10],[22]

In control group (Group I), all subjects expressed negative p53 expression, while, in Group II, majority (53.3%) subjects had negative expression. In Group III, most of the subjects (56.7%) had positive expression. Statistically, there was a significant difference in p53 expression between Group I and study groups (Group II and Group III). However, there was no significant difference between two study groups (P = 0.438) [Table 1] and [Table 2].
Table 1: Comparison of groups according to p53 expression

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Table 2: Multiple comparisons of groups for p53 expression

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The comparison of study groups according to p53 index showed that in both the groups, maximum proportion was observed for 26-50% and 51-75% categories. Statistically, there was no significant difference between two groups (P = 0.817). In addition, the mean of p53 indices in the study groups was 53.79 ± 16.58% in Group II and 52.53 ± 15.19% in Group III. Statistically, there was no significant difference between two groups (P = 0.827) [Table 3] and [Table 4].
Table 3: Comparison of study groups according to p53 index

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Table 4: Mean of p53 indices in the study groups

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 > Discussion Top


OSCC most commonly seen in the middle-aged and older individuals. [13] Squamous cell carcinoma of oral cavity and oropharynx is rare in patients of age ≤50 years, being primarily a disease that occurs in males in their 6 th and 7 th decade. [23] Recently, it has been reviewed that there is nearly five-fold increase in incidence in OSCC patients <40 years of age, many with no known risk factors. [24]

Changing patterns in the incidence of OSCC suggest that a younger population may also be at the risk of developing aggressive disease. This may reflect differences between the carcinogens to which the younger patients are exposed and the carcinogens responsible for malignant progression in the traditional patient population. [2] High frequencies of genetic aberrations in the TP53 gene in OSCC are highly suggestive of a functional role for p53 inactivation in the pathogenesis of this tumours. [2]

All cases of normal oral mucosa (Control Group) assessed in the present study were p53 negative. It might be due to the fact that p53 protein has shorter half-life and low levels in normal tissue and is hard to be detected by IHC. It was consistent with the studies of Langdon, Partridge (1992), Rich et al. (1999), Nylander et al. (2000), and with most other reports in the literature, regardless of differences in antibodies, IHC procedures, and types of specimens (frozen or paraffin-embedded sections) used. [20],[21],[22],[23],[24],[25]

On the contrary, the normal oral mucosa was found to be p53 positive by Piffko et al. (1995), but this was tissue adjacent to malignant epithelium that might represent an early event in oral carcinogenesis. This might also indicate a normally working p53 system activated in genetically stressed cells that could either be restored or pushed towards apoptosis or enter the vicious circle of malignant transformation. [16] In OSCC cases, including both the study groups, we found that 31 out of total 60 cases (51.67%) showed positive expression for p53 protein by IHC analysis. Our findings were in agreement with Kurokawa, who performed a study to evaluate a potential association between the histological grade of malignancy at the deep invasive front and the expression of Ki-67 antigen and p53 protein in OSCC patients and found p53 immunoexpression in 54% of tumours. [26]

Similar results had been described by Chiang, who found positive p53 staining in 47 of 81 (58%) cases of OSCC. [27] Kuttan observed that 13 of the 23 OSCCs (56.5%) demonstrated nuclear p53 staining. [28] In the study conducted by Danieli, they found p53 immunoexpression in 44% of tumours from patients with tongue SCC. [10] Atula observed p53 protein expression in 33.3% tongue SCC samples. [11]

However, other workers found higher frequency of p53 expression, i.e., by Castle, [2] who found positive p53 expression in 78% of cases and Liu [29] found it in 81% of cases. Sa [7] described that 76.8% lesions showed positivity for p53 protein while Panjwani and Sadiq [22] found p53 immunopositivity in 76.8%. Some factors had been considered to influence such discrepancy, such as the methodology employed, type of tumour material and heterogeneity of tumour sites examined. [22] False-positive results apart from TP53 mutation might have occurred because of the various reasons such as p53 upregulation in an attempt to promote DNA repair or apoptosis. Similarly, p53 stabilization and accumulation could be the result of p53 binding to cellular or viral proteins. Type and dilution of primary antibody [16] as well as pretreatment used [18] are also the factors of influence in IHC results. On the other hand, our p53-negative cases, apart from technique failures, could result from biallelic deletion of TP53 gene, very low levels of mutant or wild-type p53, a nonsense mutation or a truncated p53 protein in its N-terminal portion, which would not be recognized by the antibody. [18] Another possible explanation would be an accumulation of MDM2 protein caused by gene amplification, promoting p53 degradation and non-detection by IHC. [22]

In the present study, the p53 positive cases in younger study group were 14 (46.67%), while, in older study group, 17 (56.67%) cases were p53 positive. Therefore, older patients showed more p53 expression than younger patients, but the difference was not statistically significant. Our findings were in accordance with many researchers who found more p53 expression in older patients. Rodrigues [21] studied p53 and Ki-67 IHC expression in 30 patients with laryngeal SCC among patients < 50 years and in patients of ≥50 years. It was found that in younger group, p53 expression was of 50%, while that, in the older group, p53 expression was of 61.5%. [21] Similar results had also been described by Castle [2] who found 82% of older and 75% of younger OSCC stained positive with p53. Regezi [12] described positive p53 staining in 18/36 of the <35-year group (50%) versus 24/36 of the >75-year group (66.6%) in patients of tongue SCC (P = 0.149). Khabir [30] found accumulation of p53 in 81% of the cases for patients >30 years of age, but in only 38% of specimens for younger patients (P = 0.00013) of nasopharyngeal carcinomas (NPCs).

More p53 expression in older patients might be attributed to longer duration of exposure to the carcinogens, decreased immunity, and more advanced stage of disease at presentation. [23]

On the contrary, Danieli [10] found that p53 immunoexpression was more common among younger patients (58%) than in older patients (36%). Siriwardena [13] also found significantly higher number of nuclear aberrations in younger group (P < 0.001).

Moreover, in our study, the p53 index (percentage of p53 positive cells) ranged between 24% and 84% in younger study group [Figure 1], with a mean of 53.79 ± 16.58%, while, in older study group, it ranged between 23% and 80% having a mean of 52.53 ± 15.19% [Figure 2]. Statistically, there was no significant difference in the p53 indices also between the two study groups (P = 0.827).
Figure 1: Photomicrograph of p53 stained section showing positive result in oral squamous cell carcinoma in a 38-year-old patient (×40)

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Figure 2: Photomicrograph of p53 stained section showing positive result in oral squamous cell carcinoma in a 60-year-old patient (×40)

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The p53 protein expression detected by IHC alone does not always reflect the presence of mutant p53 protein and neither does the absence of p53 staining preclude it. [31] Our study was also limited for assessing TP53 mutation by IHC as we did not aim to analyze our samples at gene level. However, IHC technique had assured its importance in some recent studies between mean-indices of p53 protein expression and the presence of TP53 gene alterations. [22] The study conducted by Cruz [31] to identify p53 immunostaining that was predictive of TP53 mutations and had established a cut-off value of 25% of p53 immunostained tumour cells, as indicative of TP53 mutations.


 > Conclusion Top


An abnormal p53 protein is detected in 51.67% of the tumours as in the present study. Furthermore, the alteration rates were found to be slightly higher in older patients. Further researches on longer scale is still needed to elucidate whether OSCC in the younger patients arise due to the same pathogenic mechanism as those in the older patients. Potentiating factors for OSCC including genetic and hereditary factors and diet as well as possible demographic factors need to be investigated. Larger study groups and comprehensive assessment of multiple genes involved in the OSCC pathogenesis are required to get a conclusive result.

 
 > References Top

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