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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 2  |  Page : 164-166
Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study


1 Department of Pathology, JN Medical College, A.M.U., Aligarh, U.P, India
2 Department of ENT, JN Medical College, A.M.U., Aligarh, U.P, India

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   Abstract 

Background: Oral cancers are a major health problem in India. Recently, parameters of cell proliferation and cell death have emerged as important diagnostic and prognostic tools. Aims: The aim was to study apoptosis in premalignant and malignant squamous cell lesions of the oral cavity and to evaluate its prognostic role in oral cancers. Materials and Methods: The study included 175 patients presenting with oral lesions. Evaluation of apoptotic index (AI) (using light microscopy) was performed on hematoxylin and eosin-stained sections. Statistical Analysis Used: Student's t test was performed. Results: The mean AI increased progressively with increasing dysplasia, with the maximum AI in well-differentiated (WD) squamous cell carcinoma, and a fall was noted with progression toward higher grades. The difference between WD SCC and poorly-differentiated SCC was significant (P < 0.05). Cases with lymph node metastasis had significantly (P < 0.05) lower mean AI values. Conclusion: Apoptosis can be fairly accurately assessed using light microscopy. Tumors that exhibit less apoptosis tend to show aggressive behavior and have a greater potential for metastasis.

Keywords: Apoptotic index, dysplasia, oral cavity, squamous cell carcinoma

How to cite this article:
Jain A, Maheshwari V, Alam K, Mehdi G, Sharma S C. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study. Indian J Pathol Microbiol 2009;52:164-6

How to cite this URL:
Jain A, Maheshwari V, Alam K, Mehdi G, Sharma S C. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 May 19];52:164-6. Available from: http://www.ijpmonline.org/text.asp?2009/52/2/164/48907



   Introduction Top


In India, oral cavity cancers are the most common cancers in males and the third most common in females. [1]

Most of the oral cancers are squamous cell carcinomas and the majority are unequivocally associated with tobacco chewing and usually preceded by premalignant lesions that could present as persistent leukoplakia or oral submucous fibrosis.

Researchers agree that an early diagnosis greatly increases the probability of cure, with minimum impairment and deformity. In the last decade, histological methods that reveal parameters of cell proliferation and cell death have become important and might facilitate identification of individuals who are at a high risk of developing carcinoma besides having prognostic significance.

As counting of apoptotic bodies using light microscopy is feasible and the technique has been used and described by several authors, [2],[3] there has been interest in the enumeration of apoptosis in malignant growths as a putative prognostic marker.

The present study was undertaken with the purpose of evaluating the apoptotic index (AI) in squamous cell lesions of the oral cavity.

Evaluation of AI (using light microscopy) was performed to assess the significance of AI as a proliferation marker in premalignant and malignant lesions of the oral cavity.


   Materials and Methods Top


The study was conducted over a period of 2 years and included 175 patients presenting with oral lesions to the Department of ENT and Oromaxillofacial Surgery at Aligarh Muslim University, Aligarh, UP, India.

A clinical workup and relevant investigations were carried out in each case. The excisional/incisional oral biopsy specimens were then processed. All sections were routinely stained with hematoxylin and eosin (H and E) stain. Care was taken to have sections of a uniform thickness (not greater than 5mm).

The H and E sections were examined using high magnification (oil immersion 100 lens). From each section, 10 fields devoid of any preservation or fixation artifact, inflammation and necrosis were selected. Apoptotic cells/bodies in the stroma around the tumors were not counted. In each section, 1000 tumor cells were evaluated for the presence of apoptotic cells and apoptotic bodies and AI was calculated as the number of apoptotic cells and apoptotic bodies, expressed as a percentage of the total number of non-apoptotic tumor cells counted in each case.

Statistical evaluation was carried out using the Student t test, with P < 0.05 being significant.


   Results Top


The apoptotic cells showed certain well-defined features, which included cell shrinkage, condensation and deep eosinophilia of the cytoplasm and pyknotic, round to crescentic or irregular nucleus [Figure 1]. Karyorrhexis was also observed frequently. Apoptotic bodies, which appeared as tiny, round and pyknotic nuclear fragments, were seen scattered among tumor cells and occasionally forming a cluster [Figure 2]. Potential pit falls included the presence of mononuclear inflammatory cells, densely stained mitotic figures, necrotic cells and nuclear debris.

AI was evaluated in 175 cases, including 57 cases of oral dysplasia (mild = 22, moderate = 18, severe = 17), and 118 cases of squamous cell carcinoma (well differentiated [WD] = 58, moderately differentiated [MD] = 40, poorly differentiated [PD] = 20).

In mild to moderate dysplasia, apoptotic bodies were seen in suprabasal layers while in severe dysplasia and SCC, they were randomly distributed [Figure 3].

The mean AI increased progressively from dysplasia (0.52% 0.33) to SCC (0.61% 0.42). However, it was not statistically significant. Similarly, despite an increase in the mean AI with an increasing degree of dysplasia (mild dysplasia = 0.48% 0.33, moderate dysplasia = 0.52% 0.42, severe dysplasia = 0.56% 0.23), no statistical significance was observed on correlation.

The maximum mean AI was observed in WD SCC (0.76% 0.38) and with progression toward higher grades, a fall was noted, the values being 0.60% 0.37 in MD SCC and 0.47% 0.27 in PD SCC. Only the difference between WD SCC and PD SCC was found to be significant ( P < 0.05). The cases with lymph node metastasis had a statistically significant ( P < 0.05) lower mean AI (0.49 0.25) compared with those without metastasis (0.81 0.42) [Table 1].


   Discussion Top


Our study is based on the evaluation of AI in 175 oral premalignant and malignant squamous cells lesions on light microscopy. Apoptotic bodies were counted using 1000 magnification (under oil immersion) and similar to the views of Soini et al. , [2] we observed that a fairly accurate assessment of apoptosis is possible by light microscopy. Mitotic figures were readily identifiable and were morphologically distinct. Apoptotic cells in the stroma around the tumors should be disregarded and areas of necrosis must be avoided. Although necrosis may also commence with condensation of the chromatin, its redistribution into well-defined deposits abutting on the nuclear membrane is not seen. Furthermore, dissolution of the nucleus into discrete membrane-bound fragments does not occur. The cytoplasm and organelles within the cell swell rather than condense. Necrosis also tends to affect contiguous cells and is commonly associated with an inflammatory infiltrate, with the cell debris being removed by macrophages. [4]

Care must be taken to distinguish lymphocytes from apoptotic bodies and not to include neutrophils or other leukocytes in the count.

As light microscopy has its fallacies, most importantly human errors in counting apoptotic bodies, various other advanced and better methods have been developed to study apoptosis, i.e. electron microscopy, flow cytometry, electrophoresis, in situ end labeling of fragmented DNA and terminal deoxynucleotidyl transferase-mediated d-UTP biotin nick end labeling technique (TUNEL). Although it is accepted that electron microscopy is the best way to identify apoptotic cells, this method is not practical in most histological studies of specimens. The TUNEL technique is now the most commonly used method for evaluating apoptosis but it is expensive and its use in developing countries like India is not economically feasible. Moreover, it is a specialized technique and so its setup and standardization is not available at every institute. Keeping this in mind and due to economic constraints, we have used light microscopy in our study.

We observed an increased AI as the nature of the lesion changed from oral dysplasia to SCC. The results are in accordance with Macluskey et al. [5] and Bentz et al. [6] It has been postulated that apoptosis may have a role in preventing the development of aneuploidy and other genetic abnormalities that are commonly associated with cancer cells and progression of neoplasia. [4]

Tumor growth is a summation of mitosis and cell production and cell loss and death. Thus, a high AI in WD SCC as observed by us possibly suggests that tumors that exhibit more apoptosis may be slower growing and therefore may be less biologically aggressive. [4]

Various studies [7] have shown that histological malignancy grade and lymph node metastasis are significant prognostic variables. The relevance of apoptosis and potential for spread and dissemination, however, is unclear. It has been suggested by some workers that cells with a diminished apoptotic response have an increased propensity for metastatic survival, a contention supported by fibroblast transfection experiments. [4] An association of low AI with higher grade and metastatic phenotype, as found in our study, further implies that a low AI suggests a poor prognosis. The ability to predict which tumor cells will survive to grow as metastases may be useful in predicting the outcome.

The beneficial anticancer effects of chemotherapy are predominantly mediated through induction of apoptosis in tumor cells de novo or as a result of chemotherapy-induced damage of cellular metabolic processes or cell cycle control mechanisms. Thus, it is possible that tumors that exhibit apoptosis may be more sensitive to chemotherapy and hence likely to have a better prognosis. [4]


   Conclusion Top


Apoptotic cells can be readily and accurately demonstrated on routine Hand E-stained sections provided there is strict adherence to established criteria for recognition and counting of apoptotic bodies. However, care must be taken to count a sufficient number of cells and account must be taken of tumor heterogeneity.

Oral cancers that exhibit less apoptosis tend to show aggressive behavior and have a greater potential for metastasis.

Although there appear to be valid biological reasons for a relationship between low AI and poor prognosis, it is likely that other factors in tumor progression such as mitotic rate and invasive capability have a confounding influence on tumor behavior than the AI alone.

 
   References Top

1.Park K. Park's textbook of preventive and social medicine. 18 th ed. Jabalpur: M/s Banarsidas Bhaot; 2005.  Back to cited text no. 1    
2.Soini Y, Pδδkkφ P, Lehto VP. Histopathological evaluation of apoptosis in cancer. Am J Pathol 1998;153:1041-53.  Back to cited text no. 2    
3.Symmans WF, Cangiarella JF, Symans PJ, Cohen JM, Yee HT, Bennett G, et al . Apoptotic index from fine needle aspiration cytology as a criterion to predict histological grade of non-Hodgkin's lymphoma. Acta Cytol 2000;44:194-204.  Back to cited text no. 3    
4.Langlois NE, Eremin O, Heys SD. Apoptosis and prognosis in cancer: Rationale and relevance. J R Coll Surg Edinb 2000;45:211-9.   Back to cited text no. 4    
5. Macluskey M, Chandrachud LM, Pazouki S, Green M, Chisholm DM, Ogden GR, et al . Apoptosis, proliferation and angiogenesis in oral tissues, possible relevance to tumor progression. J Pathol 2000;199:368-75.  Back to cited text no. 5    
6.Bentz BG, Chandra R, Haines GK, Robinson AM, Shah P, Radosevich JA. Nitric oxide and apoptosis during human head and neck squamous cell carcinoma development. Am J Otolaryngol 2002;23:4-11.  Back to cited text no. 6    
7.Prieto I, Prieto A, Bravo M, Bascones A. Prognostic factors for cancer of the oral cavity. Quintessence Int 2005;36:711-9.  Back to cited text no. 7    

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Correspondence Address:
Veena Maheshwari
Department of Pathology, JNMC, AMU, Aligarh, UP
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.48907

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]

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