Expression of P53 Protein in Premalignancies and

10.5005/jp-journals-10011-1318 Poornima Chandra et al ORIGINAL ARTICLE

Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity Poornima Chandra, PG Agnihotri, S Nagarathna

ABSTRACT Introduction: Expression of p53 gene is one of the common findings in premalignancies and malignancies of the oral cavity. The aim of the study was to know whether P53 protein is overexpressed in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. The aim of the present study was to determine the overexpression of P53 protein in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. Materials and methods: Eighty-five histopathologically diagnosed, formalin-fixed, paraffin embedded tissue samples were divided into study group and control group. The study group was further subdivided into three groups: Group 1 consisted of 25 samples of oral submucous fibrosis; group 2 consisted of 25 samples of oral leukoplakia and group 3 consisted of 25 samples of oral squamous cell carcinoma. The control group consisted of 10 normal oral mucous membrane tissue samples. The samples were subjected to immunohistochemistry using indirect immunoenzyme Labelled StreptAvidin Biotin (LSAB) method. The results were analyzed statistically by Chi-square (χ2) test of significance. Results: 8/25 samples of group 1, 5/25 samples of group 2, 8/25 samples of group 3 and none of the samples of the control group were positive for P53 protein. P53 staining was seen mostly in the basal layer of the samples of group 1 while in the samples of groups 2 and 3 the staining was more diffuse. Conclusion: Among P53 may be considered a tumor marker as a definite number of samples were positive for P53. Keywords: Tumor suppressor gene, Cell Immunohistochemistry, p53 gene, P53 protein.

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How to cite this article: Chandra P, Agnihotri PG, Nagarathna S. Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity. J Indian Aca Oral Med Radiol 2012;24(4):300-305. Source of support: Nil Conflict of interest: None declared

INTRODUCTION In recent years, it is has become clear that malignancy arises in humans because of the accumulation of mutations in two major classes of genes, namely proto-oncogenes and tumor suppressor genes. Proto-oncogenes and tumor suppressor genes play an important role in the control of cell proliferation and the development of the malignant phenotype.1

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Proto-oncogenes are genes involved in the growth regulation of normal cells. Oncogenes, the activated counterparts of proto-oncogenes, stimulate cell growth independently and allow cells to escape the normal controls of the cell proliferation. Thus, oncogenes help in tumor progression. Tumor suppressor genes, on the other hand, are those genes which act to limit the growth of tumors by slowing and halting cell cycle progression.2 Tumor suppressor genes express a product that can suppress the expression or function of other genes involved with cell growth and proliferation. These genes are vulnerable site for critical deoxyribonucleic acid (DNA) damage.3 P53 is a tumor suppressor gene located on the short arm of chromosome 17 at the position 17p13.1.1,4 This gene encodes a nuclear phosphoprotein P53, so termed because it is a protein with a molecular weight of 53 Kilo Daltons (Kd).5 Mutations of the p53 gene are the commonest somatic genetic changes found in human cancer resulting in the alteration of the protein. The loss of normal or wild-type P53 protein is believed to occur in the multistep process of oncogenesis in most human cancers. Very few studies have been conducted in Southern India to compare the expression of P53 protein in oral premalignancies and oral squamous cell carcinoma. This study was intended to determine whether the expression of P53 shows any variation among the South Indian population. The aim of the present study was to determine the overexpression of P53 protein in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. The objectives of the study were as follows: To compare the degree of P53 overexpression in oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma with that of the normal oral mucous membrane. To compare the pattern of expression of P53 protein in oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. To determine whether P53 protein can be considered to be a tumor marker. MATERIALS AND METHODS A total number of 85 histopathologically diagnosed, formalin-fixed, paraffin embedded tissue samples were collected and categorized into study group and control group.

JIAOMR Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity

The study group consisted of 75 samples that were collected from the Department of Oral Pathology. A brief case history of the patients was obtained. The age range of the study group was 20 to 79 years. The study group was further divided into: • Group 1 consisted of 25 samples of oral submucous fibrosis (Table 1) • Group 2 consisted of 25 samples of oral leukoplakia (Table 2) • Group 3 consisted of 25 samples of oral squamous cell carcinoma (Table 3). Control group consisted of 10 normal oral mucous membrane tissue samples obtained from healthy volunteers who visited the Department of Oral Surgery for minor oral surgical procedures. The age range of the control group was 20 to 59 years (Table 4). METHODOLOGY The wax blocks of histopathologically diagnosed lesions of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma were selected. The wax blocks of the normal tissue samples were made. Then, 5 mm of tissue sections were made using a microtome and were placed on silane-coated slides. The method used for detection of the expression of P53 protein was immunohistochemistry. The primary antibody used to stain P53 was mouse monoclonal antibody DO-7. The universal kit was based on indirect

immunoenzyme Labelled StreptAvidin Biotin (LSAB) method (Dako, Chennai, India). INTERPRETATION OF THE P53 STAIN The slides stained for P53 were observed under light microscope with a magnification of 400×. The tissue Table 2: P53 status in group 2 Sl. no.

Age (years)

Sex

P53 status

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

45 55 64 63 76 47 69 50 34 35 53 45 50 45 45 60 52 67 59 22 64 58 39 42 52

M M M M M M M M M M M M M M M M M M M M M F F F M

1+ 1+ 1+ 1+ 1+ – – – – – – – – – – – – – – – – – – – –

Table 3: P53 status in group 3

Table 1: P53 status in group 1 Sl. no.

Age (years)

Sex

P53 status

Sl. no.

Age (years)

Sex

P53 status

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

22 64 30 31 29 24 28 33 29 26 51 25 25 21 30 58 33 21 33 25 32 30 34 23 22

M M M M M M M M M M M M M M M M M M M M F M M M M

1+ 1+ 1+ 2+ 2+ 1+ 2+ 1+ – – – – – – – – – – – – – – – – –

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

35 52 48 52 60 70 70 62 50 72 52 62 38 28 62 59 59 60 52 55 49 58 50 47 50

M M M F M M M M F M M M F F F F M F M M F F F M F

2+ 1+ 1+ 3+ 3+ 1+ 3+ 3+ – – – – – – – – – – – – – – – – –

Journal of Indian Academy of Oral Medicine and Radiology, October-December 2012;24(4):300-305

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PATTERN OF P53 EXPRESSION

samples were thoroughly examined and total number of 100 cells were counted. Then, among the 100 cells, the number of cells which had taken up the stain was counted. A brown precipitate seen within the nucleus confirmed the presence of P53 protein. The P53 positive samples were then evaluated semiquantitatively on a 4-point scale based on the percentage of cells showing P53 staining:6 Negative or – if less than 10% of the cells showed positive staining • 1+ if 10 to 30% cells showed positive staining • 2+ if 30 to 50% cells showed positive staining • 3+ if more than 50% of the cells showed positive staining. The results, thus obtained were analyzed statistically by Chi-square (χ2) test of significance. In this test the p-value of less than 0.05 was accepted as statistically significant.

In all the study groups P53 was seen as a brown stain within the nucleus. But the pattern of expression of the stain was different among the groups as follow: • Group 1: The brown stain of P53 was almost limited to the basal layer (Fig. 2). • Group 2: The brown stain of P53 was seen in the basal and immediate suprabasal layers (Fig. 3). • Group 3: The brown stain of P53 stain was diffuse involving all the layers of the epithelium (Fig. 4). INTENSITY OF P53 STAIN The intensity of P53 stain also varied between different study groups are as follows: • Group 1: Less intense staining for P53 • Group 2: Less intense staining for P53 • Group 3: More intense staining for P53.

RESULTS Comparison of P53 staining between the study group and control group: 8/25 (32%) samples of group 1 (Table 1), 5/ 25 (20%) samples of group 2 (Table 2) and 8/25 (32%) samples of group 3 (Table 3) were positive for P53. None of the samples of the control group were positive for P53 (Table 4). Four samples of group 3 showed 3+ staining for P53 while none of the samples of groups 1 and 2 showed 3+ staining (Fig. 1). The p-value was 0.0845 and thus was not statistically significant (Table 5).

DISCUSSION The p53 gene appears to act as a real tumor suppressor gene since its function is not essential for the viability of the organism but acts to profoundly suppress the rate of development of cancer.7 The normal or wild-type P53 protein has been implicated in the control of cell cycle, DNA repair and synthesis, cell differentiation, genomic plasticity and programmed cell death.8 The major function of P53 protein is cell cycle arrest in G1 phase in response to DNA damage, allowing for DNA repair and the regulation of programmed cell death (apoptosis).9,10 The levels of P53 protein in a cell are dramatically increased (5-60 fold) in response to DNA damage. After DNA damage, the high P53 levels block the progression of cell cycle in the G1 phase, so as to permit DNA repair prior to proceeding with DNA replication. This does not permit the duplication of damaged DNA and minimizes errors in the cell cycle. Thus, the P53 protein acts as a checkpoint control in the cell cycle. Hence, P53 is

Table 4: P53 status in control group Sl. no.

Age (years)

Sex

P53 status

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

30 32 46 30 40 27 21 28 20 55

M M M F M M M M F F

– – – – – – – – – –

Table 5: P53 grading among the control group and the study group Groups

P53 grade Negative

Total

1+

2+

3+

Normal Oral submucous fibrosis Oral leukoplakia Squamous cell carcinoma

10 (100%) 17 (68%) 20 (80%) 17 (68%)

0 (0%) 5 (20%) 5 (20%) 3 (12%)

0 (0%) 3 (12%) 0 (0%) 1 (4%)

0 (0%) 0 (0%) 0 (0%) 4 (16%)

10 25 25 25

Total

64

13

4

4

85

Statistic Chi-square

302

DF 5

Value 4.3265

Prob 0.5034

JIAOMR Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity

rightly called the ‘Guardian of the Genome’ 11,7 and ‘Molecular Policeman’.12 In the present study, 8/25 (32%) samples of group 1, 5/25 (20%) samples of group 2 and 8/25 (32%) samples of group 3 were positive for P53. None of the normal tissue samples were positive for P53 protein. These results are in

Fig. 2: Photomicrograph showing P53 staining of oral submucous fibrosis. Black arrows show the P53 staining

accordance with previous studies.13-15 The study conducted by Saranath et al13 showed that overexpression of P53 protein was seen in 6/22 (27%) samples of leukoplakia and 21/62 (37%) samples of oral cancer. Warnakulasuriya et al14 reported that 2/12 (17%) of the samples of dysplasia and 13/37(35%) samples of oral cancer showed positivity for P53 protein. All normal tissue specimens were negative for mutant P53 expression. Murti et al15 observed that 21/68 (31%) samples of precancerous lesions that did not progress to cancer and 6/22 (27%) samples of precancerous lesions and conditions that progressed to cancer were positive for P53. The previous studies showed that P53 expression in premalignant lesions ranges from 17 to 67% and in oral cancer the expression ranged anywhere between 11 and 75%. 14,16,17 The reasons for this wide range in expressibility of P53 protein have been stated by Rich et al18 Diogene et al,17 Paterson et al19 and GR Ogden.20 The reasons are as follows: 1. Variations in the etiological factors and ethnic background of the patients, with a generally lower prevalence of P53 alterations in oral squamous cell carcinoma in patients from the Indian subcontinent and a higher prevalence in patients from Western societies. 2. Variations in the immunohistochemical technique used also explain some of the differences, with the use of microwave antigen retrieval increasing the proportion of positive results. 3. Tumors may have lost both the alleles of the p53 gene. 4. Tumors may have a level of P53 protein that cannot be detected by immunohistochemistry. Comparing all the study groups, it was observed that none of the samples of groups 1 and 2 showed 3+ staining whereas 4 samples of group 3 showed 3+ staining. This indicates that more number of cells contain mutated or stabilized P53 protein in case of oral squamous cell

Fig. 3: Photomicrograph showing P53 staining of oral leukoplakia. Black arrows show the P53 staining

Fig. 4: Photomicrograph showing P53 staining of oral squamous cell carcinoma. Black arrows show the P53 staining

Fig. 1: P53 grading among the study group


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carcinoma. This supports the hypothesis given by Kaur et al21 that a progressive increase in P53 expression occurs in oral squamous cells as they acquire a more malignant phenotype. The intensity of stain was more in the samples of group 3 when compared to groups 1 and 2. Further there was a difference in the pattern of P53 expression among the study groups. While the P53 staining was almost always limited to the basal layers in the samples of group 1, the P53 staining was more diffuse in the samples of groups 2 and 3. This implies that there is progressive accumulation of P53 protein as the normal cells change to dysplastic lesions and later to invasive carcinoma. Previous researchers have reported similar results that correlate with the results of the present study.22-25 The suprabasal expression of P53 could indicate that a larger part of the epithelium than normal is dividing.4 The inability to detect P53 protein in the normal tissue samples can be attributed to the short half-life of P53 in the normal tissue. The normal or the wild-type P53 has a halflife of 6 to 20 minutes and is present in such minute quantities that it cannot be detected by immunohistochemistry. But when the p53 gene is mutated there is excessive production of altered P53 protein which has a long half-life of 6 hours and thus can be detected by immunohistochemical technique.15,20,26 CONCLUSION Although the comparison of P53 grading between the study group and the control group was not statistically significant, there were definite number of samples of the study group that were positive for P53, while all the samples of the control group were negative. This suggests that P53 may play an important role in oral carcinogenesis. Expression of P53 protein may also help in determining the prognosis as well as play a key role in the gene therapy for the treatment of oral squamous cell carcinoma in future. Thus, P53 may be considered one of the important tumor markers in the diagnosis of premalignancies and squamous cell carcinoma of the oral cavity.13-15 However, long-term follow-up studies with larger sample size would further help to assess the significance of P53 protein in oral carcinogenesis. REFERENCES 1. Levine AJ, Perry ME, Chang A, Silver A, Dittmer D, Wu M, et al. The 1993 Walter Hurbert Lecture: The role of the p53 tumor suppressor gene in tumorigenesis. Br J Cancer 1993;69: 409-16. 2. Gleich LL, Salamone FN. Molecular genetics of head and neck cancer. Cancer Control 2002;5:369-78.

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3. Field JK, Pavelic ZP, Spandidos DA, Stambrook PJ, Jones AS, Gluckman JL. The role of the p53 tumor suppressor gene in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 1993;119:1118-22. 4. Nylander K, Dabelsteen E, Hall PA. The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck. J Oral Pathol Med 2000;29:413-25. 5. Langdon JD, Partridge M. Expression of the tumor suppressor gene p53 in oral cancer. Br J Oral Maxillofac Surg 1992;30: 214-20. 6. Agrawal S, Mathur M, Srivastava A, Ralhan R. Mdm2/p53 coexpression in oral premalignant and malignant lesions: Potential prognostic implications. Oral Oncol 1999;35:209-16. 7. Lane DP. p53 and human cancers. Br Med Bull 1994;50(3): 582-99. 8. Harris CC, Hollstein M. Clinical implications of the p53 tumor suppressor gene. New Engl J Med 1993;329(18):1318-27. 9. Girod SC. Tumor suppressor genes and cell proliferation control in the carcinogenesis of the oral mucosa. Quintessenz Verlags GmbH;221-23. 10. Williams HK. Molecular pathogenesis of oral squamous carcinoma. J Clin Pathol: Mol Pathol 2000;53:165-72. 11. Saranath D. Contemporary issues in oral cancer. Oxford Publisher 2000;137-47. 12. Isselbacher KJ, Braunwald E, Wilson JD, Martin JB, Fauci AS, Kasper DI. Harrison’s principles of internal medicine (13th ed). Vol 2; Mc Graw-Hill, Inc;2:1187-88. 13. Saranath D, Tandle AT, Teni TR, Dedhia PM, Borges AM, Parikh D, et al. p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India. Oral Oncol 1999;35(3): 242-50. 14. Warnakulasuriya KAAS., Johnson NW. Expression of p53 mutant nuclear phosphoprotein in oral carcinoma and potentially malignant oral lesions. J Oral Pathol Med 1992;21: 404-08. 15. Murti PR, Warnakulasuriya KAAS, Johnson NW, Bhonsle RB, Gupta PC, Daftary DK, et al. P53 expression in oral precancer as a marker for malignant potential. J Oral Pathol Med 1998; 27:191-96. 16. Girod AC, Krueger G, Pape HD. p53 and Ki67 expression in preneoplastic and neoplastic lesions of the oral mucosa. Int J Oral Maxillofac Surg 1993;22:285-88. 17. Diogene HR, Tetu B, Moreney R, Fortin A, Monteil RA. p53 overexpression in head and neck squamous cell carcinoma: Review of literature. Oral Oncol Eur J Cancer 1996;32B (3): 143-49. 18. Rich AM, Kerdpon D, Reade PC. p53 expression in oral precancer and cancer. Australian Dent J 1999;44(2):103-05. 19. Paterson C, Everson JW, Prime SS. Molecular changes in oral cancer may reflect etiology and ethnic origin. Oral Oncol Eur J Cancer 1996:32B(3):150-53. 20. Ogden GR, Kiddie RA, Lunny DP, Lane DP. Assessment of p53 protein expression in normal, benign and malignant oral mucosa. J Pathol 1992;166:389-94. 21. Kaur J, Srivastava A, Ralhan R. Overexpression of p53 protein in betel and tobacco-related human oral dysplasia and squamous cell carcinoma in India. Int J Cancer 1994;58:340-45. 22. Chiang CP, Lang MJ, Liu BY, Wang JT, Leu JS, Hahn LJ, et al. Expression of p53 protein in oral submucous fibrosis, oral epithelial hyperkeratosis and oral epithelial dysplasia. J Formos Med Assoc 2000;99:229-34.

JIAOMR Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity 23. Trivedy C, Warnakulasuriya KAAS, Tavassoli M, Steingrimdottir H, Penhallow J, Maher R et al. p53 aberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR-SSCP. J Oral Pathol Med 1998;27:72-77. 24. Kerdporn D, Rich AM, Reade PC. Expression of P53 in oral mucosal hyperplasia, dysplasia and squamous cell carcinoma. Oral Dis 1997;32:86-92. 25. Gopalakrishnan R. Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol 1997;83(4):471-77. 26. Field JK, Spandidos DA, Malliri A, Gosney JR, Yiagnisis M, Stell PM. Elevated P53 expression correlates with the history of heavy smoking in squamous cell carcinoma of the head and neck. Br J Cancer 1991;64:573-77.

ABOUT THE AUTHORS Poornima Chandra (Corresponding Author) Reader, Department of Oral Medicine and Radiology, Rajarajeswari Dental College and Hospital, Bengaluru, Karnataka, India, e-mail: [email protected]

PG Agnihotri Professor and Head, Department of Oral Medicine and Radiology, Career Institute of Dental Sciences and Hospital, Lucknow, Uttar Pradesh, India

S Nagarathna Professor and Head, Department of Community Dentistry, Maharana Pratap College of Dentistry and Research Centre, Gwalior, Madhya Pradesh, India


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