| Abstract|| |
Context: Oral cancer is a major health problem worldwide. In cancer, the equilibrium between cell proliferation and apoptosis is disturbed. The defect in the apoptotic pathway allows cells to proliferate with genetic abnormalities. Thus, the apoptotic index (AI) can be used to assess the significance of apoptosis as a proliferative marker in oral epithelial dysplasia. Aims: To assess the apoptotic index in various grades of epithelial dysplasia. Objectives: 1) To calculate the apoptotic index in various grades of oral epithelial dysplasia, 2) To compare the apoptotic index between various grades of oral epithelial dysplasia, 3) To predict the biologic behavior of oral epithelial dysplasia based on an apoptotic index. Settings and Design: Cross-sectional tissue analyzing study. Methods and Materials: This study constituted 30 cases, previously diagnosed with various grades of oral epithelial dysplasia (OED). AI was calculated as the number of apoptotic bodies/cells expressed as a percentage of the total number of cells counted in each case. Statistical Analysis Used: Statistical analysis was carried out using ANOVA test. Results: A statistically significant difference was observed between mild dysplasia and severe dysplasia where P = 0.002. The mean AI was increased progressively with increasing grades of OED. Conclusions: This study demonstrated the clinical significance of apoptosis in assessing disease progression in Oral Potentially Malignant Disorder (OPMD) which may be used as a prognostic indicator in OED. This would, in turn, help in knowing the prognosis of the disease and to develop targeted drug therapy.
Keywords: Apoptosis, apoptotic index, dysplasia, oral squamous cell carcinoma
|How to cite this article:|
Pawar RB, Palaskar SJ, Kalavankar SS. Assessment of apoptotic index in various grades of oral epithelial dysplasia: A cross-sectional study. Indian J Pathol Microbiol 2020;63:534-7
|How to cite this URL:|
Pawar RB, Palaskar SJ, Kalavankar SS. Assessment of apoptotic index in various grades of oral epithelial dysplasia: A cross-sectional study. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 24];63:534-7. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/534/299313
| Introduction|| |
Oral squamous cell carcinomas (OSCC) are amongst the most aggressive of tumors. The 5-year survival rate reported for some parts of the oral cavity is as low as 9%, largely due to late-stage diagnosis of the tumor. In the case of early detection and treatment of OSCC, the survival rate significantly increases from 66% to 85%, It has been previously reported that the diagnosis and management at the “precancerous” stage would further improve survival rates.
OSCC is commonly preceded by a range of tissue and cellular alterations consistent with carcinoma, termed oral epithelial dysplasia (OED). These changes often manifest in a clinical mucosal lesion., Dysplasia, being a histopathological finding, is characterized by loss of polarity of basal cells, basilar hyperplasia, increased nuclear-cytoplasmic ratio, drop-shaped rete ridges, abnormal mitotic figures, bizarre mitoses, hyperchromatic nucleus, loss of cohesion, cellular and nuclear pleomorphism and nuclear hyperchromatism, and so on. Recent clinical advances have shown that 10 to 20% epithelial dysplastic lesions may lead to squamous cell carcinoma. The most accepted classification developed by the World Health Organization divides OED into mild, moderate, severe dysplasia, and carcinoma in situ. Of them, the risk of severe dysplasia to transform into carcinoma is as high as 43%.
Apoptosis is a naturally occurring programmed and targeted cell death and is distinct from necrosis. Apoptosis is an essential phenomenon for the normal growth and development of all organisms. It plays an important role as it also eliminates potentially cancerous and virus-infected cells and maintains balance in the body. Deregulation in the apoptotic pathway can either cause excessive removal or prolonged survival of cells. Researchers have reported that altered apoptotic pathways may lead to malignant transformation thus leading to tumor proliferation. Thus, to study apoptosis; a technique of counting of apoptotic cells or apoptotic bodies has been discussed by various authors. Apoptotic bodies are characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation.,
Our study aimed to assess the apoptotic index (AI) and its significance as a biological marker in various grades of oral epithelial dysplasia.
| Materials and Methods|| |
This study was done on formalin-fixed, paraffin-embedded, and histopathologically diagnosed cases of oral epithelial dysplasia. Ethical approval was obtained from the institutional ethics committee. A total of 30 cases of oral epithelial dysplasia (10-mild, 10-moderate, and 10-severe) were collected from the archives of the Department of Oral Pathology and Microbiology, Sinhgad Dental College and Hospital, Pune. The sample size was calculated using the software Open Epi-Info. These slides were examined using a Leica Research microscope 1000 at 40× magnification. Five fields were selected from respective slides and the number of epithelial cells was counted. Later, the apoptotic bodies were counted and evaluated by using the Leica application suite (LAS 2.0). AI was calculated as several apoptotic bodies/a total number of cells in one field which is expressed in percentage. In this study, all the study samples were observed by two observers. The counting of AI was done independently to reduce observer bias.
Under a light microscope, apoptotic bodies appear as a round oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin [Figure 1], [Figure 2], [Figure 3]. These apoptotic bodies represent morphological changes at various stages of the cell cycle. In OED, apoptotic bodies were located at different locations based on grades of dysplasia but mostly they were present in stratum basale.
|Figure 1: Apoptotic body showing perinuclear halo and pyknotic nucleus in mild epithelial dysplasia (H and E stain, 40×)|
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|Figure 2: Apoptotic body showing intense eosinophilic cytoplasm and fragmented nuclear material in moderate epithelial dysplasia (H and E stain, 40×)|
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|Figure 3: Apoptotic body showing intense eosinophilic cytoplasm, fragmented nuclear material, and perinuclear halo in severe epithelial dysplasia (H and E stain, 40×)|
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| Results|| |
Out of 30 cases, 10 were mild OED, 10 moderate OED, and 10 severe OED. Mean AI was calculated using descriptive statistics. In mild OED, the mean AI was 1.948, moderate OED means AI was 2.622, and severe OED means AI was 3.315. The mean AI was increased progressively with increasing grades of OED. The maximum was reported in severe dysplasia.
Further statistical analysis was carried out using the ANOVA test to compare the mean AI of various grades of OED. On comparing the mean AI, a statistically significant difference was observed between mild dysplasia and severe dysplasia where P = 0.002. The difference between the AI value of mild dysplasia and moderate dysplasia was statistically not significant as P = 0.196. Moreover, there was no statistically significant difference between moderate dysplasia and severe dysplasia P = 0.176. [Table 1] and [Graph 1].
| Discussion|| |
OED is a commonly diagnosed oral lesions in India. In the Indian subcontinent, the incidence rates of OPMDs have ranged from 0.6 to 30.2 per 1000 population. A regional variation in prevalence ranged from 0.2% in Bihar state in the north to 4.9% in Andhra Pradesh state in the east.
OSCC is commonly preceded by a range of tissue and cellular alterations including OED. It has been suggested that alterations in apoptosis accompany the onset of invasion in OPMD. Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. A large number of stimuli can induce apoptosis in a cell. Multiple signaling pathways lead to activation of the apoptosis depending on the triggering factor and the cell type. Apoptosis prevents the development of aneuploidy and other genetic aberrations that are associated with the development and progression of OPMD.
At certain stages during the development of a tumor, the equilibrium between the cell proliferation and its apoptosis is interrupted, resulting in dysregulation of cell proliferation. Thus, a dysfunction in the apoptotic system can lead to a wide variety of diseases including oral cancers.
Kerr et al. had linked apoptosis to the elimination of potentially malignant cells, hyperplasia, and tumor progression. Hence, reduced apoptosis or its resistance plays a vital role in carcinogenesis. There are many ways by which a malignant cell can acquire a reduction in apoptosis or apoptosis resistance. The molecular mechanisms by which evasion of apoptosis occurs are 1) disrupted the balance of pro-apoptotic and anti-apoptotic proteins, 2) reduced caspase function, and 3) impaired death receptor signaling [Figure 4].
|Figure 4: Schematic diagram showing the relation between apoptosis and cancer|
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Apoptotic bodies can be identified on hematoxylin and eosin (Hand E) stained sections under light microscopy. As light microscopy has its limitations, various advanced methods have been developed for the detection of apoptotic cells such as electron microscopy, flow cytometry, gel electrophoresis, immunohistochemistry, in situ end-labeling of fragmented DNA, and so on to overcome the limitations of light microscopy. Although these methods are better for the evaluation of apoptotic cells, these are technique sensitive, expensive, and need standardization.
In this study, the biological and clinical significance of AI was evaluated. The apoptotic cells were observed in the suprabasal and basal regions of early dysplastic lesions but as the severity of the lesion increased the apoptosis becomes more generalized in the epithelium., Our study showed an increased number of apoptotic bodies from mild-to-severe dysplasia suggesting a mechanism whereby apoptosis helps to eliminate cells that have been produced more than the cells that have developed improperly or undergone abnormal mitosis and sustained genetic damage. Analysis of AI showed a progressive increase in apoptosis in parallel with biological aggressiveness, indicates the increased synthetic activity of these proteins during neoplastic progression.
Therefore, it might be argued that the epithelium is attempting to maintain balance by removing cells with damaged genomes using the mechanism of apoptosis. This may be intrinsic “suicide” or be induced by adjacent normal or infiltrating inflammatory cells. Alternatively, another possibility is that the topical factors responsible for dysplastic change may themselves be inducing increased levels of apoptosis.
| Conclusion|| |
This study demonstrated the clinical significance of apoptosis in assessing disease progression in OPMD which may be used as a prognostic indicator in OED. Further studies need to be undertaken with a larger sample size and using advanced methods for the detection of apoptosis to understand the role of apoptosis in the mechanism of carcinogenesis. This would, in turn, help in knowing the prognosis of the disease and to develop targeted drug therapy.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Sciubba JJ. Oral cancer: The importance of early diagnosis and treatment. Am J Clin Dermatol 2001;2:239-51.
Gomez I, Seoane J, Varela-Centelles P, Diz P, Takkouche B. Is diagnostic delay related to advanced-stage oral cancer? A meta analysis. Eur J Oral Sci 2009;117:541-6.
McCullough MJ, Prasad G, Farah CS. Oral mucosal malignancy and potentially malignant lesions: An update on the epidemiology, risk factors, diagnosis and management. Aust Dent J 2010;55(Suppl 1):61-5.
Barnes L, Eveson JW, Reichart P, Sidransky D, editors. World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours. Lyon, France: IARC Press; 2005.
Van Der Waal I. Potentially malignant disorders of the oral and oropharyngeal mucosa; Terminology, classification and present concepts of management. Oral Oncol 2009;45:317-23.
Rajendra R, Shivpathsundaram B. Shafer's Textbook of Oral Pathology 7th
ed. Elesvier Publications; 2011. p. 889.
Macluskey M, Chandrachud LM, Pazouki S, Green M, Chisholm DM, Ogden GR, et al
. Apoptosis, proliferation, and angiogenesis in oral tissues possible relevance to tumour progression. J Pathol 2000;191:368-75.
Nambiar S, Hegde V. Apoptosis in oral epithelial dysplastic lesions and oral squamous cell carcinoma: A prognostic marker. Indian J Pathol Microbiol 2016;59:284-6.
] [Full text]
Kesarwani P, Choudhary A, Gupta R. Apoptosis: A prognostic marker in oral epithelial dysplasia and oral squamous cell carcinoma. Int J Dent Health Concern 2016;1:1-4.
Angela CC. Epithelial pathology. In: Neville BW, Damm DD, Allen CM, Bouquot JE, editors. Oral and Maxillofacial Pathology. 3rd ed. Missouri: SaundersElsevier; 2009. p. 362-425.
Ranganathan K, Kavitha L. Oral epithelial dysplasia: Classifications and clinical relevance in risk assessment of oral potentially malignant disorders. J Oral Maxillofac Pathol 2019;23:19-27.
] [Full text]
Jain M, Kasetty S, Sridhara Jain S, Khan S, Desai A. Apoptosis and its significance in oral diseases: An update. J Oral Dise 2013;1:1-11.
Archana M, Yogesh TL, Kumaraswamy KL. Various methods available for detection of apoptotic cells-A review. Indian J Cancer 2013;50:274.
] [Full text]
Soini Y, Pääkkö P, Lehto VP. Histopathological evaluation of apoptosis in cancer. Am J Pathol 1998;153:1041-53.
Hsu S, Singh B, Schuster G. Induction of apoptosis in oral cancer cells: Agents and mechanisms for potential therapy and prevention. Oral Oncol 2004;40:461-73.
Kaufmann SH, Gores GJ. Apoptosis in cancer: Cause and cure. Bioessays 2000;22:1007-17.
Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239-57.
Santos-García A, Abad-Hernández MM, Fonseca-Sánchez E, Cruz-Hernández JJ, Bullón-Sopelana A. Proteic expression of p53 and cellular proliferation in oral leukoplakias. Med Oral Patol Oral Cir Bucal 2005;10:5-8.
de Vicente JC, Herrero-Zapatero A, Fresno MF, López-Arranz JS. Expression of cyclin D1 and Ki-67 in squamous cell carcinoma of the oral cavity: Clinicopathological and prognostic significance. Oral Oncol 2002;38:301-8.
Kasibhatla S, Tseng B. Why target apoptosis in cancer treatment? Mol Cancer Ther 2003;2:573-80.
Birchall MA, Winterford CM, Allan DJ, Harmon BV. Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: A preliminary study. Eur J Cancer Part B: Oral Oncol 1995;31:380-3.
Rasika B Pawar
44/1 Off Sinhgad Road, Sinhgad Dental College, and Hospital, Pune - 411 041, Maharashtra, India
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]