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The term oral cancer encompasses all malignancies that originate in the oral tissues. Squamous cell carcinoma of the oral cavity comprises 90 % to 95 % of oral malignancies (Sunitha and Gabriel 2004).

Among the types of cancers occurring in mankind, the incidence of oral cancer is very high and it is significantly alarming. It is the 6th most frequent cancer of all malignancies occurring in the body (Shin-Ichi et al. 2002, p.327). It is estimated that thousand of people die daily due to oropharygneal malignancies .The incidence rates of oral cancer differ from region to region. The annual age-adjusted incidence rates per 1,00,000 in several European countries vary from 2.0 (UK, south Thames Region) to 9.4 (in France). In the Americans the incidence rate vary from 4.4 (USA) to 13.4 (in Canada). In Asia, it ranges from 1.6 (Japan) to 13.5 (India). In Australia and New Zealand, it varies from 2.6 (New Zealand – Maori) to 7.5 in South Australia (Sunitha and Gabriel 2004). Oral cancer is a major health problem of this subcontinent. In India, oral cancer rate is 10% of the estimated 644600 new cancers that occur in all parts of the body each year (Mehta & Hammer 1993, p.134). The corresponding figure for Sri Lanka and Pakistan are 30% and 33% respectively (Oral Cancer 1998, p. 214).

Bangladesh has a high incidence rate of Oral Cancer. Here, the number of new cancer cases of the whole body is about 200000 per year, of which, oral cancer represents 20% and it is the third leading cancer of this country (Shaheed & Molla 1996, p. 8).

To explore the possible etiological factors of Oral Cancer, various attempts have been made by several investigators .So the literature on the etiology of oral cancer is voluminous. Commonly accepted risk factors include tobacco chewing or smoking and alcohol. In some countries, such as India and the Southeast Asian region, up to 50% of all cancers arise in the oral cavity. In these countries, betel quid consumption plays an important role. However, many cases of oral cancer develop without prior exposure to the previous agents. In these cases, viruses, diet, or genetic predisposition have been hypothesized to play the role (Rodu 2002, p. 511)

In the field of molecular biology there are lots of studies to find out the genetic basis of tumor genesis. It is generally accepted that neoplasm arises from a series of genetic alterations that lead to cellular proliferation and differentiation (Warnakulasuriya and Johnson 1992, p.404). This genetic alteration may be induced by inactivation of tumor suppressor gene. The most common genetic abnormalities, found in different type of human cancers are the mutation of the tumor suppressor gene p53 (Chen, Yang and Li, 1996, p. 204).

The p53 gene is located in the short arm of the chromosome no. 17 at the position of 17 p 13.1 (Warnakulasuriya and Johnson 1992, p. 404). The p53 pathway provides a physiological system for integrating signals from diverse insults and eliciting adaptive cellular responses that include growth arrest in G1 phase of cell cycle and apoptosis (Chang et al. 2000, p.56). Defects in the pathway are prevalent in cancer, most notably being associated with mis-sense mutations in p53 itself (Shahnavaz et al. 2000, p.407).

Inactivation of p53 gene leads to the inability of a cell with DNA damage to induce cell cycle arrest to allow time for DNA repair or the induction of apoptosis (Piattelli et al. 2002, p.532). This leads to the inability of p53 to act as a transcription factor for some DNA repair enzymes and thus to the non-occurrence of downstream events. So the cell reluctantly turns on to a one way street that leads to malignant transformation (Neoplasia 1999, pp.260).

The p53 gene mutation often results in a more stable gene product that is expressed as a tumor marker or biomarker by the tumor cells (Rich., Kerdpon & Reade, 1999) . According to most studies p53 is not detected in normal oral mucosa but it can be demonstrated with immunohistochemical techniques in oral squamous cell carcinoma (Chen, Yang and Li, 1996, p.204). Immunohistochemistry is a much simpler and easier method to detect expression of p53 protein than DNA sequencing (Castle et al. 1999, p.326). It can facilitate the identification of p53 over expression to specific cell types that molecular biological techniques can’t provide (Murti et al 1998). Therefore p53 immunohistochemistry may have a practical, clinical and pathological value (Shahnavaz et al. 2000, p. 407).

To find out the pattern of specific mutation in oral squamous cell carcinoma, different studies have been done to assess the expression pattern of p53 oncoprotein (Cruz et al. 1998, p.99). But in our country no study has so far been done regarding expression pattern of p53 protein in oral precancerous lesions and oral squamous cell carcinoma. The present study was undertaken with the hope of achieving the goal in assessing the expression pattern of p53 protein in our country.


Immunohistochemical analysis of Oral Squamous Cell Carcinoma (OSCC) shows over expression of p53 protein.


General Objectives:

To analyze the expression of p53 protein in Oral Squamous Cell Carcinoma by immunohistochemical method.

Specific Objectives:

a) To assess the p53 protein expression in Oral Squamous Cell Carcinoma.

b) To evaluate the expression pattern (intensity and distribution) of the p53 protein in different grades of Oral Squamous Cell Carcinoma.

c) To correlate the expression of p53 protein with specific risk factors associated with development of Oral Squamous Cell Carcinoma.


What is p53 ?

p 53 is a tumor protein which acts as a transcription factor that regulates the cell cycle and hence functions as a tumor suppressor. P 53 has been described as, ‘the guardian of the genome’. Referring to its role in conserving stability by preventing the genome mutation. It is located on Chromosome 17 p.13.1 (Neoplasia 1999, p.290).

Fig. 3.1 Structural organization of p53 protein (Linda and Carol 1996, p. 1054.)

p53 identification

p53 was identified in 1979 by Arnold Levine, David Lane and Lloyd Old, working at Princeton University, Imperial Cancer Research Fund (UK), and Sloan-Kettering Memorial Hospital, respectively. The p 53 gene was first cloned in 1983 by Moshe Oren (Weizmann Institute).

Its character as a tumor suppressor gene was finally revealed in 1989 by Bert Vogelstein working at Johns Hopkins School of Medicine.


Fig. 3.2 ‘Molecule of the year-1993’ by Science magazine.

In 1993, p53 was voted ‘molecule of the year’ by Science magazine. (Wikimedia Foundation, Inc.)

How does P53 act ?

· p 53 activtes DNA repair protein when DNA has sustained damage.

· It can also hold the cell cycle at the G-1 / S regulation point on DNA damage regulation.

· It can initiate appoptosis , the programed cell death , if the DNA damage proves to be irreversible.

Fig. 3.3 p53 as transcription section: tumor suppressor activity

More than 50% of human cancer contain mutation or deletion of p53 (Neoplasia 1999, p. 290).

Relation between p53 and oral squmous cell carcinoma:

According to the result of the study performed by Jang-Jaer et al. (2005, p.471) activation of P53 play a role of in the process of malignant changes of oral mucosal pre malignant lesions. They also stated that the gradually increasing expression of P53 in normal epithelium , hyperplasia , dysplasia and carcinoma supports the values of this protein as an indicator of OSCC.

The suppressor gene frequently alterd in carcinomas of the upper aerodigestive tract is the p 53 gene. P 53 mutation or over expression has been demonetrated in 43 % to 93 % of cases of oral cells than in any human cancer (Sunitha et al. 2004).

The highly direct correlation between p53 expression and neoangiogenesis suggested that p53 is important in regulation of angiogenesis and both factors may play a critical role in oral carcinogenesis (Abbas et al. 2003, p.197).

Fig. 3.4 The cell cycle (after Ryan and Vousden, Nature, 2002, p.797)

Cruz et al. (2002, p.98) concluded that supra basal p53 immunoexpression has a high positive predictive value for malignant transformation of oral lesion and can be used as a specific marker for lesions that are ‘at risk’ for malignant transformation.

Gonzalez et al. (2001, p. 2889) analised the pattern of P 53 expression and its influence on survival in patients of OSSC. According to their result 57.7% of OSCC express P 53 , with nuclear expression in 52.6% of cases and cytoplasmic expression in 24.4% .

Shahnavaz et al. (2000, p. 417) reported in their study that during oral carcinogenesis p53 gene mutation was seen to occour relatively late and was assosiated with transformation to the invasive phenotype.

Mutation of the p53 tumor suppressor gene are frequent in OSCC of the oral mucosa in patients from Europe , USA & Australia (Alison, Duangporn & Petre 1999, p.103) . The study stated that all the cases of oral mucosa were negative for p53 . The cases of carcinoma had a greater intensity of p53 staining than dysplasia and dysplasia had a greater intensity than hyperplasia.

A follow up study was carried out by Kaur J , Srivastava and Ralhan (1998, p.372) to show the overexpression of p53 protein in 102 of 145 cases that is 70 % of OSCC which included 69 % primary and 76 % recurrent OSCC.

Rowleyh et al.(1997, p.23) examined 90 boipsy specimen and found positive immmunohistochemical detection of the p53, demonstrated by brown nuclear staining in over 80% of mild, moderate, severe dysplastic tissue as well as carcinoma in situ and OSCC.

p53 gene mutation and risk factors of OSCC

Jang-Jaer et al. (2005, p.471) reported that the patient with atrophic form of oral lichan planus and arecaquid chewing habit may have higher risk of malignant transformation. The effect of AQ chewing may be stronger than those of drinking and smoking . These people have a higher risk of contracting oral cancer when compared to those who only smokes and drinks.

Snyder et al. (2004, p.209) provides additional support for a relationship between oral SCC development and exposure to household ETS and may implicate p53 as a potential site for carcinogen-related mutation in this tumor.

The development of oral carcinoma can almost certainly be ascribed to genetic damage and is closely connected with cigarette smoking and alcohol consumption (Abbas et al. 2003, p.1999).

Chang et al. (2002, p.300) suggested that betel quid generates reactive oxygen species that may cause oxidative DNA damage to the surrounding oral tissue which is proved by over expression of p53 protein.

Specimen of 187 OSCC from Taiwanese with various histories of betel quid, tobacco and alcohol use strongly suggests contributive role of tobacco carcinogens to p53 gene mutations (Hsie, Wang and Chen 2001, p.1499).

According to the statement of Chun et al. (2000, p.229) arecanut contains genotoxic chemicals that causes DNA strands breakdown in cultured human buccal epithelial cells and fibroblasts and in turn results in oral carcinoma which is expressed by p53 protein .

The probable differences in the habit of tobacco consumption and ethnic origin may account for the higher freqency of p53 gene mutations observed in the study of Kaur , Srivastava, and Ralhan, (1998, p.370) . They found a direct corelation between the history of tobacco smoking or chewing, pan (betel & arecanut ) and p53 over expression and malignant lesions.

p53 over expression and different grade of OSCC

Wu et al. (2005, p.51) reported that p 53 expression cells were mainly located around the nests in highly differentiated squamous cell carcinoma. Stained cells dispersed throughout the tumor tissues in low differentiated sq. cell carcinoma. P 53 was undetectable in hyperplasia and light dysplasia but was observed in 66.67 % cases of high dysplasia and 72.72 % in OSCC.

Jang-Jaer et al. (2005, p.471) performed a study where they described the distribution and intensity of p53 in different grade of OSCC. In 19 well differentiated OSCC, 6 had less than 5% p53 expression, 6 had 6% to 25%, 5 had 26% to 50% and the rest 2 had 51% to 100% of p53 expression.

In case of moderately differentiated OSCC 4, 3, 3, 1 cases had less than 5%, 6%-25%, 26%- 50% and 51% to 100% p53 expression respectively.

In 8 poorly diffentiated OSCC cases , 2 showed 26% to 50% and 3 had more than 50% p53 protein expression.

The apoptotic pathway assessed by p53 immunostaining was found to be uninformative in the study of Hafian et al.(2004). Analysis of the relationship between immunohistochemical results and clinical and pathological parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate but not with p53 (Hafian et al. 2004, p.749).

On the other hand the findings of the study Hideo et al. (2005, p.602) demonstrated that over expression of Ki-67 antigen and p53 at the deep tumor invasive front of OSCC is associated with histological grade of malignancy.

A study on well diferentiated carcinoma in Japan performed by Shin et al. (2002, p.327) suggested that p53 gene mutations areless frequent in well differentiated TSC. These results indicatethat mutations in the p53 gene may not be strongly involvedin the development of well differentiated TSC.

Famulski et al. (2002, p.203) proved that tumors that stained positive for p53 revealed no statistically significant differences with respect to the stage or grade of the tumor, site and size of the primary tumor, or recurrence rate.

Again Po et al. (2001, p.240) found positive expression of p53 in 45 of 87 cases. There was no correlation of p53 expression with cancer stage, T stage, nodal metastasis, and tumor grade.

In contrast, there was a significant increase in the no. of p53 positive cases among the patients of moderate (56%) and severe (74%) dysplastic lesion in comparison to those having mild dysplasia. In primary oral cancer, a significantly positive association was observed between p53 accumulation and poor histological differentiation of tumors (Kaur, Srivastava, and Ralhan 1998, p.370).

Prognostic and therapeutic value of p53

The two well-defined pathways that are shown to be prominently altered in a variety of cancers are the cell cycle regulatory pathways led by either p53 or Rb genes. The study (Jayasurya et al. 2005, p.1059) was undertaken to find out the pathway that is more altered in oral carcinoma at protein level, with special emphasis on its prognostic significance. p53 expression index showed association with both disease-free survival and overall survival. The study also demonstrated that the Rb pathway proteins are comparatively more important than p53 pathway proteins for the prognostication of oral carcinoma patients.

Sang et al. (2004, p.166) identified clinicopathologic factorsand immunohistochemical biomarkers predicting late cervicalmetastasis in stage I and II invasive squamous cell carcinomaof the tongue.

Molecular factors including p53, cyclinD1, Ki-67, epidermal growth factor receptor, microvessel density(MVD), cyclooxygenase-2 (COX-2), MUC1, laminin-5 g2, E-cadherin,and ß-catenin were selected as candidates. Tumor suppressorgene p53 has been shown to be a useful predictor of regionalrecurrence in stage I tongue carcinoma.

Tumor suppressor gene p53 mutation is the commonest genetic abnormality not only in the OSCC but also in other human cancers. Over expression of p53 is the strongest predictorof poor prognosis in patients with sarcomas of the GI tract (Heriberto 2003, p.192).

In-vitro introduction of the p53 protein to p53-deficient cells has been shown to cause rapid death of cancer cells or prevention of further division. The rationale for developing therapeutics targeting the p 53 protein is that “the most effective way of destroying a network is to attack its most connected nodes”. This can be seen as 50% of cancers have missense point mutations in the p53 gene, these mutations impair its anti-cancer gene inducing effects. Restoring its function would be a major step in curing many cancers.Various strategies have been proposed to restore p53 protein function in cancer cells. A number of groups have found molecules which appear to restore proper tumor suppressor activity of the p53 protein in vitro (Wikimedia, Foundation, Inc.) .


Type of the Study : A cross sectional study.

Place of the Study : Department of Oral and Maxillofacial surgery, Dhaka Dental College & Hospital, Dhaka, Bangladesh

Period of the Study: January 2005 to December 2006.

Study Population : Patients with histologically confirmed oral Squamous Cell Carcinoma who attended at the Department of Oral and Maxillofacial surgery, Dhaka Dental College & Hospital, Dhaka.

z2 pq

Sample Size: n = ———–


n = sample size

p = 0.96 (estimated prevalence of p53 expression among oral sq. cell carcinoma=96%)

q = 1-p = 0.04

z = 1.96 (corresponding value of z of 95% confidence interval)

e = Degree of accuracy (% of relative error) =0.05%

Estimated sample size

(1.96)2x 0.96 x 0.04

n = ———————— = 59 (approx.)



Sampling was done purposively (non randomized), strictly maintaining the inclusion and exclusion criteria. All Patients having suspected oral squamous cell carcinoma were selected on the basis of provisional diagnosis made by history and clinical examination. They were then explained about the objectives of this study and invited to join. Those who gave informed written consent, biopsy materials were collected from the suspected lesions of the oral cavity and the provisional diagnosis was confirmed by histopathological examination. Only the patients with histopathologically confirmed oral squamous cell carcinoma were finally selected for the study.

Standard treatment was provided to all patients who were provisionally selected.

Inclusion Criteria:

Patients with histologically confirmed oral squamous cell carcinoma.

Exclusion Criteria:

a) Patients meeting the inclusion criteria but did not give written informed consent.

b) Previously diagnosed oral squamous cell carcinoma patients those were receiving anticancer therapy.

c) Patients for whom biopsy was contraindicated (Bleeding diathesis, Recent MI etc.)

Data Collection Methods:

Data was collected through a questionnaire, clinical examinations and by laboratory methods.

· Questionnaire

A structured, pre-tested questionnaire was administered to collect relevant information from the patients about the particulars, about ulcer/lesion(s), presence or absence of risk factors and also about the data of the histopathology and immunohistochemistry of p53 protein.

· Laboratory methods

Immunohistochemical methods: Histological slides were prepared from each collected specimen. Then the specimen was stained with monoclonal antibody to p53 according to the standard protocol. Details of the standard protocol is mentioned in the Appendix no. III.

Immunologically stained slides were then examined for expression of p53 protein by light microscope. Staining profile was categorized according to the proportion of the positive or negatively stained cells.


Expression (Jang-Jaer et al. 2005, p.475)

Positive staining : >5% cells are stained.

Negative staining: ?5% cells are stained or not stained at all.

Intensity (Alison, Duangporn & Petre 1999, p.104).

Weak staining : + (6-25% cells take staining)

Moderate staining : ++ (26-50% cells take staining)

Strong staining : +++ (51-100% cells take staining)


Only in basal layer

In all the layers of squamous epithelium

Data from the observations were recorded in pre-designed data sheets.


· Age

· Sex

· Socioeconomic status

· Personal habits (Risk factors)

o Smoking habits

o Chewing habits

· Presence of chronic trauma or precancerous lesions

· Grades of Oral Squamous Cell Carcinoma

· Expression of p53 protein

· Intensity of expression of p53 protein

· Distribution of p53 protein

Data Processing and Analysis:

Data were processed and analyzed using computer software SPSS (statistical package for social sciences) version 11.5. The test statistics used to analyse the data were descriptive statistics, Pearson Chi-square tests. The data presented on categorical scale were expressed as frequency and corresponding percentage, while the quantitative data were presented mean and standard deviation (SD) from the mean. Associations between two variables were justified using Pearson Chi-square tests. For all analyses level of significance was set at 0.05 and p-value <0.05 was considered significant.


5.1 Sociodemographic Data of the Study Subjects

Figure 5.1 shows that 53% of the study subjects were male, while remaining 47% of them were female.

Figure 5.1 – Distribution of the study subject according to sex (N = 59).

Figure 5.2 shows that majority of the study subjects belonged to the age group of 50 to 59 yrs (about 42%) followed by the age group 60 to 69 years (about 31%). The age of the study subjects who fulfilled the inclusion criteria ranged from 30 to 90 years (Mean age ± SD = 58.47± 11.28 years).

Figure 5.2 – Distribution of the study subjects according to their age (N = 59).

Figure 5.3 shows that most of the study subjects were Muslims (about 90%), while remaining 10% of the study subjects were the follower of Hinduism

Figure 5.3: Distribution of the study subject according to their religion (n=59).

Figure 5.4 shows that about 36% of the study subjects were housewives, which also indicates the predominant occupation of the female subjects. About 17% of the study subjects were unemployed. Equal number of respondents (about14%) mentioned agriculture as their main occupation while about 10% of them were day laborers. Other occupations reported by the study subjects were business (about 8%) and fishing (about 2%)

Figure 5.4: Distribution of the study subject according to their current main occupation (occupation for past 12 months) (N = 59).

Figure 5.5 shows that majority of the study subjects (97%) who fulfilled the inclusion criteria were married. About 3% of them are unmarried.

Figure 5.5: Distribution of the study subject according to their marital status (N = 59).

Figure 5.6 show about 46% of the study subjects had primary education (class 1 to 5), while about 22% had secondary school education (6 to SSC). However about 8% and 5% of the study subjects had some college education (post secondary to HSC) and graduate respectively. About 19% of the study subjects had no formal or informal education.

Figure 5.6 : Distribution of the study subjects according to the educational qualification (n=59)

Figure 5.7 shows that about 71% of the study subjects had average monthly family income between 3000 to 6000 Tk, followed by 12% who had average monthly family income ranges between Tk. 6001 to 9000. However the range of average monthly family income of the study subjects varies widely between Tk. 3000 to 20,000 (Mean ± SD = Tk. 6423.73 ± 3944.46).

Figure 5.7: Distribution of the study subjects according to their average monthly family income in Taka for last 12 months (N = 59).

5.2 Clinical Findings

Table 5.1 shows that about 47% of the study subjects reported to have the oral lesion or ulcer for about a year, however about 27% of them had the lesion for about one and half year to two years. Study also revealed that about 14% of study subjects had been suffering from the particular oral lesions or ulcers for less than 1 year. Of the study subjects, about 7% reported to have the lesion for 16 months. However about 5% of them gave history of having lesions or ulcers for two and half years.

Table 5.1: The duration of oral lesion and/or ulcer (in months) (N=59)

Duration of the oral lesions in months n %
3 2 3.39
8 6 10.17
12 28 47.46
16 4 6.78
18 8 13.56
24 8 13.56
30 3 5.08
Total 59 100


Figure 5.8 shows that most (about 98%) of the study subjects had a single oral lesion or ulcer, however approximately 2% had more than one lesions or ulcers in their oral cavity.

Figure 5.8 : The no. of the ulcers or lesions in the oral cavity (n=59)

Figure 5.9 shows that about one third (33%) of the lesions or ulcers located in the buccal mucosa. Beside buccal mucosa the most common site was tongue (29%), followed by alveolar ridge (14%) and buccal sulcus (14%). About 8% and 2% of the lesion among the study subjects were in lip and palate respectively.

Figure 5.9 : The location of ulcers in the oral cavity.

Figure 5.10 shows that when the study subjects were asked whether they could perceive the ulcer(s) growing in size and/ or number about 71% replied yes. However about 29% of them responded that there was no change in size or numbers.

Figure 5.10 : Whether the ulcers rapidly growing in size or numbers.

5.3 Risk Factors

Figure 5.11 shows that about 41% of the study subjects used different types of tobacco products in combination. About 17% and 12% of the study subjects had the habit of using jarda (spiced tobacco) and sada pata (fermented tobacco leaves) respectively. Other smokeless tobacco product commonly used by the study subjects was gul or tobacco dust (about 3%). However about 19% of the respondents also smoked tobacco. Among the study subjects about 8% didn’t not use any kind of tobacco products.

Figure 5.11: Type of tobacco products used by the study subjects (N=59)

Figure 5.12 shows that among the 54 tobacco users 59% used over a very long period of time (>15 yrs). However about 39% of them reported to have using tobacco products over a long period of time (5-15 yrs). Only 2% of the users found to have the habit for a short period of time (<5 yrs).

Figure 5.12 : The duration of using different types of tobacco products (n=54)

Figure 5.13 shows that most of the study subjects had mixed chewing habits (78%), however 7% of them used only betel leaf for chewing. About 15% of the study subjects had no chewing habit.

Figure 5.13 : Whether the study subjects had chewing habit (n=59).

Figure 5.14 shows that among the 50 study subjects who had chewing habits 56% used them for a very long period of time. However about 40% of them reported to have chewing habits over a long period of time. Only 4% of the users found to have the habit for short a period of time.

Figure 5.14 : The duration of chewing habits (n=50)

Figure 5.15 shows that 2% respondent used to consume alcohol and 98% did not.

Figure 5.15 : Habit of consuming alcohol (n=01)


Table 5.2 shows that the only study subject who used to consume alcohol had been taking it for a long period of time.

Table 5.2: Duration of Habit of consuming alcohol (N=01)

The duration of habit of consuming alcohol n %
short duration 0 0.0
long duration 1 100.0
very long duration 0 0.0
Total 1 100

Figure 5.16 shows that the majority of the patients (83%) did not have any history of chronic trauma, while 14% had a history of chronic trauma due to sharp teeth and 3% due to rough surface of denture.

Figure 5.16 : Presence of chronic trauma.

Figure 5.17 shows that among 59 study subjects, 14% (n=9) were suffering from white patches which were diagnosed clinicohistologically as leukoplakia and 2% were suffering from oral submucosal fibrosis.

Figure 5.17 :Presence of premalignant lesions (n=59)

5.4 Laboratory Characteristics of the lesion

Figure 5.18 shows that among the 59 study subjects who were suffering from oral squamous cell carcinoma, 47% (n = 28) were histologically diagnosed as well-differentiated type while moderately differentiated, poorly differentiated and verrucous type covered 32%, 12% and 8% respectively.

Figure 5.18 : Histological type of lesions* (n=59)

* Squamous cell carcinoma


Table 5.3 shows that the immunostaining of the biopsy material of OSCC expressed p53 positively in 88% cases (n = 52), while 12% (n = 7) were negative.

Table-5.3: Expression of p53 among the lesion following immunostaining with monoclonal antibody to p53 (N=59)

Expression of p53 n %
Positive 52 88.14
Negative 7 11.86
Total 59 100.00

* (Note: Staining of >5% cell is considered as positive, ?5% cell – negative)

Figure 5.19 shows the intensity of staining. The frequencies were 52 % ++ (moderate), 42 % +++ (high) & 6% + (low).

Figure 5.19 : Intensity of immune staining of p53 (following staining with monoclonal antibody to p53. n=52)

* (Note: a: +, b: ++, c: +++)

Table 5.4 shows the distribution of p53 expression. Ninety two percent cases expressed intense staining of all the layers, while in 8% cases staining was confined only to the basal layer.

Table-5.4: Distribution of p53 expressing cells following Immunostaining with monoclonal antibody to p53 (N=52)

Distribution of p53 expressing cells Frequency Percent
Only in basal layer 4 7.69
In all layers 48 92.31
Total 52 100

Table 5.5 shows that the total number of well differentiated SCC was 28. Eighty-six percent (n = 24) expressed p53 that corresponded 41% of the total. Fourteen percent (n = 4) did not express p53 which covered 7% of the total. In 19 moderately differentiated OSCC, 95% showed p53 positivity. In 7 poorly differentiated OSCC, 86% (n = 6) expressed p53. In Verrucous SCC, 80% showed positive expression and 20% negative (n=5).

Table- 5.5: Association between histological type and expression of p53 (N=52)

Histological type of squamous cell carcinoma Expression of p53
Positive Negative Total
N n n
% within histological type % within histological type % within histological type
% of total % of total % of total
well differentiated 24 4 28
85.71 14.29 100
40.68 6.78 47.46
moderately differentiated 18 1 19
94.74 5.26 100
30.51 1.69 32.20
poorly differentiated 6 1 7
85.71 14.29 100
10.17 1.69 11.87
verrucous type 4 1 5
80 20 100
6.78 1.69 8.48
Total 52 7 59
88.14 11.86 100
88.14 11.86 100

Pearson Chi-Square Tests: p = 0.728

Table 5.6 shows the relationship between histological grading and immunostaining. Total number of well differentiated SCC was 28. Fifty-four percent (n = 15) of them showed moderate staining (++) and 32% (n = 9) high staining. In moderately differentiated cases, only one case had low staining and also one negative staining, the rest 17 cases either had moderate or high staining. In poorly differentiated OSCC almost 72% cases (n = 5) had high staining. Verrucous type had 40% low, 40% moderate and 20% no staining.

Table- 5.6: Association between histological type and intensity of p53 staining (N=52)

Histological type of

squamous cell carcinoma

Intensity of immune staining of p53 expression
Positive Negative Total
a(+) b (++) c(+++)
N n N N n
% within histological type % within histological type % within histological type % within histological type % within histological type
% of total % of total % of total % of total % of total
well differentiated 0 15 9 4 28
0.00 53.57 32.14 14.29 100.00
0.00 25.42 15.25 6.78 47.46
moderately differentiated 1 9 8 1 19
5.26 47.37 42.11 5.26 100.00
1.69 15.25 13.56 1.69 32.20
poorly differentiated 0 1 5 1 7
0.00 14.29 71.43 14.29 100.00
0.00 1.69 8.47 1.69 11.86
verrucous type 2 2 0 1 5
40.00 40.00 0.00 20.00 100.00
3.39 3.39 0.00 1.69 8.47
Total 3 27 22 7 59
5.08 45.76 37.29 11.86 100.00
5.08 45.76 37.29 11.86 100.00

Pearson Chi-square tests: p = 0.012

Table 5.7 shows that none of the well, moderate or poorly differentiated SCC had expression of p53 protein in basal layer. They were distributed in all layers of the squamous epithelium. On the other hand, the positive verrucous SCCs were distributed only in the basal layer.

Table- 5.7: Association between histological types and the distribution of p53 (N=52)

Histological type of squamous cell carcinoma Distribution of p53 within the lesion
Positive Negative Total

in basal layer


all layers

n n n n

within histological type


within histological type


within histological type


within histological type


of total


of total


of total


of total

well differentiated 0 24 4 28
0.00 85.71 14.29 100.00
0.00 40.68 6.78 47.46
moderately differentiated 0 18 1 19
0.00 94.74 5.26 100.00
0.00 30.51 1.69 32.20
poorly differentiated 0 6 1 7
0.00 85.71 14.29 100.00
0.00 10.17 1.69 11.86
verrucous type 4 0 1 5
80.00 0.00 20.00 100.00
6.78 0.00 1.69 8.47
Total 4 48 7 59
6.78 81.36 11.86 100.00
6.78 81.36 11.86 100.00

Pearson Chi-Square Tests: p = 0.000

Table 5.8 shows the personal habits (tobacco and/or chewing of the respondents). Ninety two percent (n = 54) respondents used tobacco at any form and 85% (n = 50) of the total had chewing habits. On the other hand, only 3 cases did not have any tobacco or chewing habits, which comprises only 5% of the total.

Table 5.8: Tobacco use and chewing habits of the study subjects (N=59

Type of tobacco product used by

study subjects

Chewing habit of the study subjects
Betel leaf only Mixed None Total
n n n n
% within tobacco product use % within tobacco product use % within tobacco product use % within tobacco product use
% of total % of total % of total % of total
Cigarette 1 6 4 11
9.09 54.55 36.36 100.00
1.69 10.17 6.78 18.64
Sada pata 1 6 0 7
14.29 85.71 0.00 100.00
1.69 10.17 0.00 11.86
jarda 2 8 0 10
20.00 80.00 0.00 100.00
3.39 13.56 0.00 16.95
gul 0 1 1 2
0.00 50.00 50.00 100.00
0.00 1.69 1.69 3.39
mixed tobacco products 0 23 1 24
0.00 95.83 4.17 100.00
0.00 38.98 1.69 40.68
None 0 2 3 5
0.00 40.00 60.00 100.00
0.00 3.39 5.08 8.47
Total 4 46 9 59
6.78 77.97 15.25 100.00
6.78 77.97 15.25 100.00

Pearson Chi-Square Tests: p = 0.004


Table 5.9 shows the relation between use of tobacco products and the expression of p53 protein. Fifty four respondents (out of 59) were tobacco user. About 94.4% (n = 51) of them expressed p53 with non expression by 5.6% (n = 3) cases. On the other hand, only one case (20%) was p53 positive among the tobacco non-users.

Table 5.9: Association between tobacco habits and expression of p53 (N=52)

Tobacco product use Expression of p53
Positive Negative None
n n n

within tobacco product use


within tobacco product use


within tobacco product use


of total


of total