THESIS ON ORAL CANCER AND BANGLADESH.
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.
2. OBJECTIVES & AIMS
To analyze the expression of p53 protein in Oral Squamous Cell Carcinoma by immunohistochemical method.
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.
3. LITERATURE REVIEW
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 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).
Fig. 3.2 ‘Molecule of the year-1993’ by Science magazine.
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.
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.
Patients with histologically confirmed oral squamous cell carcinoma.
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.
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.
· 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. OBSERVATIONS & RESULTS
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||%|
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||%|
|very long duration||0||0.0|
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 resence 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||%|
* (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|
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|
|% within histological type||% within histological type||% within histological type|
|% of total||% of total||% of total|
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|
|% 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|
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|
in basal layer
within histological type
within histological type
within histological type
within histological type
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
|Chewing habit of the study subjects|
|Betel leaf only||Mixed||None||Total|
|% within tobacco product use||% within tobacco product use||% within tobacco product use||% within tobacco product use|
|% of total||% of total||% of total||% of total|
|mixed tobacco products||0||23||1||24|
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|
within tobacco product use
within tobacco product use
within tobacco product use
|Tobacco product users||51||3||54|
Pearson Chi-square tests: p = 0.04
Table 5.10 tells about the relationship between the chewing habits and expression of p53 protein. Fifty (85%) respondents had some what chewing habits. Out of that, 49 (98%) positively expressed p53. Among the non chewers (total 9), only 3 (33%) expressed p53 protein.
Table 5.10: Association between chewing habits and expression of p53 (N=52)
|Chewing habit||Expression of p53|
within chewing habit
within chewing habit
within chewing habit
Pearson Chi-square tests: p = 0.04
The development of oral squamous cell carcinoma is generally accepted to be a multi-step process in which alternation in oncogene and tumor suppressor gene, play an important role (Abbas et al. 2003, p. 197).
The aim of this study was to elucidate the outcome of tumor suppressor gene p53 mutation in the pathogenesis of OSCC by immunohistochemistry. Though extensive studies of p53 alteration in oral cancer have been done in western countries and some Asian countries, no study has yet been emerged from Bangladesh. This study would therefore be helpful in providing a Bangladeshi perspective.
This study was performed at Dhaka Dental College Hospital from January 2005 to December 2006, where significant number of OSCC patients attended regularly. Among them 59 study subjects were selected who full filled the inclusion criteria.
According to socio demographic data, 53% of the total study subjects were male. The age group ranges from 30 to 90 years where majority of the study subjects belonged to the age group of 50-59 years. This data corresponds with the information of Langdon et al. (1992, p.214) where they found the incidence of OSCC in higher age group, mostly in 7th decade. About 90% were Muslims rest are Hindus. By occupation, most of the respondents were housewives (36%) followed by farmer (14%). About 17% were unemployed. Forty-six percent of the study subjects took primary education and about 5% were graduates. Nineteen percent study subjects had no formal or informal education. The average monthly family income varies widely between Tk. 3000.00 to Tk. 20,000.00. Most of the study subjects (71%) informed that their family income was between 3000-6000 taka which indicates low socioeconomic status. Taufiqunnessa (1993, p. 7) showed that there was a significant inverse association between the socioeconomic condition of the respondents and the frequency of oral carcinoma i.e. lower the socioeconomic status higher the disease frequency.
On clinical examination of the lesions, we found m the most common site of OSCC was buccal mucosa (34%) followed by tongue (29%). The lowest patient prevalent site was palate (2%). Others are lip, buccal sulcus and alveolar ridges. Similar findings were reported by Hsie et al. (2001, p. 1497) where buccal mucosa followed by tongue was the most occurring site for OSCC. The persistence range of the lesion of our study subjects was 3 months to 30 months where most of the persons gave history of suffering from ulcers for 12 months.
The majority of OSCC (up to 80%) carry p53 mutation indicating that the oral mucosa is one of the more common targets for p53 mutation and suggesting the knockout of p53 normal function is a common/essential step in oral carcinogenesis (Isabel et al. 1998, p.363). Mutation and loss of function of p53 gene can be assessed by molecular biological method and immunohistochemistry and was proved to be an excellent biological marker in several human malignancies (Nakopoulo, Vourlakou & Zervas 1998, p.148). In our study we used the immunohistochemical analysis to detect the p53 protein in OSCC biopsy material. Alison et al. (1999, p. 103) reported that all cases of normal oral mucosa assessed in their study were p53 negative. This is an agreement with most other reports in the literature, regardless of differences in antibodies, immunohistochemical procedure and types of specimen (frozen or paraffin embedded section) used (Warnakulasuriya & Johnson 1992, p. 404). At the earlier stage of our study we evaluated 2 cases of normal epithelium for p53 expression for laboratory standard. Both of them were p53 negative.
Histopathologically 47% of our study specimen were well differentiated, 32% moderately differentiated and 12% poorly differentiated OSCC. Eight percent (8%) specimen were verrucous type. When immunohistochemistry was done on the paraffin block of all those histological type, 52 cases showed positive expression of p53 and 7 cases were negative. In other words 88% showed positive expression and 12% showed negative. All OSCC are not p53 positive according to most of the literatures. Rana et al. (1993, p. 147) found 75% positive cases (24 out of 32 cases). Our series showed 88% positivity which corresponds to the report of Alison, Duangpron & Peter (1999, p. 103) which had 94% positivity .
All the study subjects were classified into 3 major histological groups i.e. well differentiated, moderately differentiated and poorly differentiated. These were counted as grade-I, II & III respectively. Verrucous Squamous cell carcinoma is considered as very well differentiated carcinoma. One of our aim was to find out the pattern of p53 protein in different histological grades. Here the pattern means expression, intensity and distribution of staining of p53 with monoclonal antibody.
We found that 86% (24 out of 28) of the total well differentiated carcinoma were positively stained for p53. Ninety-five percent (18 out of 19) of moderately differentiated carcinoma, 86% (6 out of 7) of poorly differentiated and 80% (4 out of 5) of verrucous carcinoma had positive expression. Famulski et al. (2002, p. 206) & most of the other studies revealed that there was no direct correlation between p53 expression and grading of the tumor. This indicates that the p53 over expressed tumor probably do not have much more aggressive behavior than those without over expression (Yan et al. 1995, p. 138). This information favors the finding of our study regarding this variable. On the contrary Jasbir, Anurag & Ranju (1998, p. 370) tried to show a relationship between these two parameters where well differentiated carcinoma showed 53%, moderately differentiated 77% and poorly differentiated showed 84% positive p53 expression.
When we analyzed the intensity of the staining of p53 and its correlation with histological grading or differentiation, we found the relation statistically significant (p=0.012). The intensity of staining was assessed in the cases by reference to photographic standard in a semi quantitative manner as weak (w), moderate (m) and strong(s), which were also expressed as weak (+), moderate (++) and strong (+++). The result from the present study indicated that p53 abnormalities in the OSCC as detected by the intensity of the immunohistochemical staining increased from verrucous type to poorly differentiated one. There was a very little evidence showing agreement with our study regarding the relation between the intensity pattern and the grading of the OSCC. However the result of our study appeared to be similar to the findings of Hideo et al. (2005, p. 602) where a significant association between intensity of immunostaining of p53 and histological grading of malignancy was found.
It was observed that when immunostaining was done for p53 protein detection, nuclear staining was confined to the suprabasal cells in premalignant lesion (Isabel et al. 1998, p. 360). In SCC, positive p53 staining was found equally in all basal, parabasal and spinous cells layer (Jang Jaer et al. 2005, p. 471). Findings of the distribution of p53 in OSCC, the result of our study correlates with the result of other studies. In our study all types of OSCC other than verrucous type showed distribution of p53 throughout all the layers. But in case of verrucous type the staining was confined only in the basal layer. This association between histological types and the distribution of p53 was tested by chi-square tests. The result was very significant (p = 0.000).
One of the important aims of our study was to find out the relationship between p53 expression and some common specific risk factors (if any) for the development of OSCC. The study conducted by Iqbal, Amzad and Motiur (1995, p.27), reported that in Bangladesh 78% of the patients with OSCC had the habit of tobacco chewing in any form along with lime and betel nut. Tobacco smoking and alcohol drinking were the principal factors associated with p53 expression in oral squamous carcinoma in the west, where as betel quid chewing and smokeless tobacco are important factors in the east. Variable results of p53 expression have been reported and it has been proposed that ethnic difference and variation in the indigenous oral habit may be responsible for the OSCC (Kerdpon D, Sriplung H, Kiethubthew S; 2001, p. 553).
Most of our respondents used to take tobacco in different forms. Forty percent of them took mixed tobacco product i.e. tobacco products in combination. Nineteen percent used smoke tobacco, about 17% and 12% had the habit of using jarda and sada pata respectively. Only 8% subjects did not take any kind of tobacco. Most of the tobacco users (59%) used to take these for a very long duration of time i.e. more than 15 years, 39% for 5-15 years and a few (2%) used these for less than 5 years.
In case of chewing habits (betel leaf, betel nut or mixed), about 78% respondents had mixed chewing habit and 7% had habit of betel leaf only. Fifty-six percent of the chewers gave history of chewing for a very long duration and 40% and 4% gave history of long and short duration respectively. Among all the study subjects only one person gave history of consuming alcohol for a very long duration. We also collected information about presence of chronic trauma and premalignant lesion.
Fifty-four respondents (out of 59) were tobacco users. Almost 95% (n = 51) of them expressed p53 with non expression by 5% (n = 3) cases. On the other hand, only one case (20%) was p53 positive among the tobacco non-users. This was statistically significant (p = 0.04). These findings were also supported by the study of Hsie, Wang & Chen (2001, p. 503) who reported that tobacco carcinogens contributes strongly in the mutation of p53 which is expressed by over expression of p53 protein.
In our series, 50 (85%) respondents had some what chewing habits. Out of that 49 (98%) positively expressed p53. Among the non chewers (total 9), only 3 (33%) expressed p53 protein. This proved a significant relation between them (p=0.004). This correlated with the findings of Leue L et al. (2004, p. 327) where they reported the positive expression of p53 in 8% of betel nut chewers. They also examined that buccal SCC is the most frequently occurring oral cancer in Asians due to the popularity of the betel nut use in this area.
Only 1% gave history of alcohol consumption. It may be due to the fact that majority of the patients were Muslims. But according to several literatures, the relation between p53 protein positivity and alcohol consumption is well established. Hsieh et al. (2001, p.1497) found an increased effect of alcohol on the incidence of p53 mutation in Betel quid users with or without cigarette smoking. They showed a statistically significant (RR = 2.24; 95% CI, 1.21-4.15) effect of alcohol on the incidence of p53 mutations.
Oral cancer of tobacco and betel chewers represent a unique in vivo model to understand the genotoxic effect of tobacco and betel carcinogens on oncogenes and tumor suppressor genes (Shew et al. 2001, p. 113). These findings are supported by many other studies. As use of tobacco products, betel leaf and betel nut are very popular habits of Bangladeshi people, our study would carry a great importance for the diagnostic and prognostic aspects of the oral cancer patients. This type of study was performed by Pande et al. (2002, p. 657) to evaluate the prognostic significance of expression of proteins, involved in cell cycle regulation and loco regional spread of OSCC of habitual betel and tobacco chewers. Multiple logistic regression models showed that p53 over expression may served as adverse prognostic factors for disease free survival of the patients.
So, at the end of the discussion, it can be said that various findings of the present study regarding p53 status in OSCC, there relation with different risk factors are in agreement with many other studies performed in the field of oral cancer.
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 .Immunohistochemistry is a much simpler and easier method to detect the over expression of p53 to specific cell types. This study showed a high prevalence of p53 expression in OSCC specimen from the patients who chewed betel leaf and betel nut with or without tobacco products or smoked cigarettes or both.
It can be evocated that p53 protein level may be a useful indicator of the malignant phenotype of squamous cells and immunohistochemical assessment of p53 may provide clinically relevant informations. Hence our study carries a great importance for the diagnostic and prognostic aspects of the oral cancer patients. Further studies in large scales are needed to support this evidence.
Informed Written Consent Form
I am ………………………………………, aged about ……………years , duly informed about the objectives of the study titled , ‘Expression of p 53 protein in Oral Squamous Cell Carcinoma’.
I have been given the guarantee to the secrecy of my data that would be collected for the study.
I have given the permission to publish my Photographs for publications in any form, if needed, for public interest.
I spontaneously agree to be included in the aforesaid type of study.
Signature / Finger print Of the Patient
ID No. Date of Interview :
Particulars and Socioeconomic status of the patient
1. Name of the Patient : _____________________________________________
2. a. Sex : 1. Male 2. Female
b. Age (in years) :
3. a) Religion :
1. Muslim 2. Hinduism 3. Christianity 4. Buddhism 5. Others
b) Occupation :
1. Agriculture 2. Fishing 3. Businessman 4..Service
5. Laborer 6. Housewife 7. Unemployed 8. Student 9. Others………………
4. Address : ____________________________________________________
5. Marital Status : 1. Married 2. Unmarried
6. Educational Qualification : 1. Illiterate 2 . Primary 3. Secondary
4. Intermediate 5. Graduate
7. Monthly Family Income (Taka) :
Ulcer(s) / lesion(s)
8. How long are you suffering from oral Ulcer(s) / Lesion(s) ? ( Months )
9. How many Ulcer(s) / Lesion(s) are there ?
1. One 2. More than one 3. Don’t know
10. Where the Ulcer(s) / Lesion(s) is / are ?
1. Tongue 2. Palate 3. Buccal mucosa, 4. Lip 5. Buccal sulcus
6. Alveolar ridge 7. Floor of the mouth 8. Mixed 9. Don’t know
11. Are the Ulcer(s) / Lesion(s) rapidly growing in size and numbers ?
1. Yes 2. No
Presence of any Risk Factor
12. Do you have any tobacco habit ?
1. Cigarette 2. Sada pata 3. Jorda 4. Gool 5. Mixed 6. None
13. How long do you have this habit ?
1. Short duration 2. Long 3. Very long duration
14. Do you have any Chewing habit ?
1. Betel leaf 2. Betel Nut 3. Mixed 4 . None
15. How long do you have this habit ?
1. Short duration 2. Long 3. Very long duratio
16. Do you take Alcohol ?
1. Yes 2. No
17. How long do you take this ?
1. Short duration 2. Long 3. Very long duration
18. Presence of any chronic trauma ?
1. Due to Sharp teeth 2. Rough surface of the denture 3. None 4. Others…………….
19. Clinical presence of the premalignant lesion(s) :
1. Oral sub mucous fibrosis 2. Lichen plannus 3. Leukoplakia
4. Erythroplakia 5. None 6.Others…………………………
Data for Biopsy & Expression of p53 protein
20. Date of the Biopsy taken : ………………………………..
21. Histological diagnosis : Squamous Cell Carcinoma
1. Well Differentiated 2. Moderately Differentiated 3. Poorly Differentiated 4. Verrrucous
22. Comments on the Expression of p 53 protein :
1. Positive a. + b. ++ c. +++ 2. Negative
23. Intensity of Immuno-staining with antibody to p 53 :
a. + b. ++ c. +++
24. Distribution of p 53 : 1. Only in basal layer 2. In all layers .
Signature of the Doctor
Case Illustration no. : 1
Thesis case no. : 26
Particulars of the Patients:
1. Hospital registration no.: 1703/21
2. Name : Mr. Monjur Mia
3. Age : 55 years
4. Sex : Male
5. Religion : Muslim
6. Occupation : Farmer
7. Address : Vill. Fulpur
PS & PO. Goforgaon
1. Ulceration on the right angle of the mouth – 16 months
2. Difficulty on taking food – 6 months
3. Occaissional pain – 3 months
There is an exophytic, cauli flower like growth with ulcerated surface over the right angle of the mouth & the lateral one trhird of the lower Lip, measuring about 3cm X 2cm in diameter. The ulcer was non-tender. One hard and non fixed submandibular lymph node was palpable on the right side.
The patient had a very long 16 years history of smoking cigarrette, taking betel leaf with betel nut and jarda (spiced tobacco).
Fig: 9.1 Verrucous carcinoma, Thesis case no. 26
We took the biopsy under L/A on 21.06.2006 and sent the specimen for histopathological examination. According to the result of H/P, it was a case of verrucous carcinoma.
Paraffin block was examined by Immunohistochemical technique for p53 expression with the help of an expertise & setup.
· Comments of expression p53 protein was positive ( More than 5% cells took staining).
· Intensity + means 6-25% epithelial cells show positive expression for p53.
· The intensity of staining + also means the cells were weakly stained.
· Besides these the staining was distributed in the tumor cell nuclei of the basal layer only.
This patient was advised for hospitalization for proper treatment.
Case Illustration no. : 2
Thesis case no. : 51
Particulars of the Patients:
1. Hospital registration no.: 2081/12
2. Name : Mrs. Sushila Rani Baroi
3. Age : 60 years
4. Sex : Female
5. Religion : Hindu
6. Occupation : Houswife
7. Address : Vill. Sohagdol,
PS & PO. Inderhat,
1. Ulceration on right buccal mucosa – 12 months
2. Difficulty on taking food – 3 months
There was an ulcer on the right buccal mucosa measing about 3cmX2cm in diameter with evarted margine and the floor was covered with slough. The ulcer was non-tender. One hard and fixed submandibular lymph node was palpable on right side.
The patient had a long 12 years history of taking betel leaf with betel nut
and jarda (spiced tobacco).
Fig. 9.2 A case of OSCC .Thesis Case no. 51
We took the biopsy under L/A on 22.08.2006 and sent the specimen for histopathological examination. According to the result of H/P, it was a case
of well differentiated squamous cell carcinoma.
Paraffin block was examined by Immunohistochemical technique for p53 expression with the help of an expertise.
· Comments of expression p53 protein was positive.
· Intensity +++ means 51-100% epithelial cells show positive expression for p53.
· The intensity of staining +++ also means the cells were strongly stained.
· Besides these the staining was distributed in the tumor cell nuclei of all the layers.
This patient was advised for hospitalization for proper treatment.
Method for p53 immunostaining
Fixation: 10% formal saline.
Processing: First the tissue is fixed in 10% formal saline for 24 hours. Then the tissue is dehydrated through different concentration of alcohol. Then the tissue is cleared by xylene followed by paraffin impregnation at 56 degree C for one hour. Then preparation of paraffin block. Paraffin wax, with xylene, as the clearing agent.
Sections: Sections are cut by a microtome between 3 and 5 microns in thickness. Mount on poly-L-lysine coated slides. Dry at 37°C for overnight is preferable.
1. Dewax sections in xylene 5 mins.
2. Rinse in a fresh bath of xylene.
3. Rinse in 2 baths of absolute alcohol.
4. Place the slides in pre warm Tris-EDTA (pH 9) solution containing coupling jar. The jar is then placed in a water bath at 98 degree C for 20 minutes.
5. The coupling jar is then kept in room temperature and when the internal temperature become equal to the room temperature, then the slides are taken out of Tris-EDTA solution and ringed in water followed by tris buffer.
1. Incubate the slides in peroxidase block (Dako) for 15 mins.
2. Ringe in tris buffer and drain off excess fluid and wipe off by lint free tissue.
3. Incubate in primary antibody (Dako lot no S-2144) for 30 minutes
4. Ringe in tris buffer and drain off excess fluid and wipe off by lint free tissue.
5. Incubate in labeled polymer for 30 minutes.
Fig.9.3: Positively stained nucleuses of the OSCC cells of this series , low power field .
6. Ringe in tris buffer and drain off excess fluid and wipe off by lint free tissue.
Fig 9.4: Positively stained nucleuses of the OSCC cells of this series , high power field field.
7. Incubate in DAB solution for 10 mins.
8. Wash in running tap water.
9. Counterstain in haematoxylin.
10. Dehydrate, clear. Mount sections in DPX
Nuclei-Brown -Positive for P 53 protein over expression in OSCC.