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ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 3  |  Page : 143-148

Cytomorphometric analysis of exfoliated buccal cells to evaluate the malignant changes in individuals with tobacco smoking and chewing habits


1 Department of Oral Pathology, Kamineni Institute of Dental Sciences, Hyderabad, Telangana, India
2 Department of Oral Pathology, SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh, India

Date of Web Publication25-Sep-2017

Correspondence Address:
Ravi T Chitturi
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Guntur - 522 509, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-8632.215530

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  Abstract 

Background: Tobacco is one of the leading causes for potentially malignant disorders (PMDs) and oral squamous cell carcinoma (OSCC) which stands 6th among all malignancies in the world. To prevent the high mortality rates and improve the five-year survival rates of patients with OSCC, it is very important to diagnose PMDs early. Exfoliative cytology combined with quantitative analysis such as cytomorphometry can be an important tool for early detection and diagnosis of these lesions.
Aim: The aim of this study was to evaluate the quantitative changes in cytological buccal smears of tobacco smokers and chewers with that of the normal healthy individuals to assess the malignant changes by using cytomorphometric parameters such as cell diameter (CD), nuclear diameter (ND), and nuclear–cytoplasmic ratio (N/C).
Materials and Methods: The study was carried out among 120 individuals. Group I consisted of 40 tobacco smokers, group II consisted of 40 tobacco chewers, and group III consisted of 40 controls who did not have a habit of tobacco smoking and chewing with apparently healthy appearing mucosa. Smears were made for all individuals and stained with Papanicolou technique. CD, ND, and N/C were calculated using an image analysis software after selecting 200 cells from each smear. All the parameters were subjected to statistical analysis using one-way analysis of variance and Scheffes multiple post hoc procedure.
Results: There was a statistically significant reduction in CD and increase in ND and N/C of tobacco chewers and smokers when compared to that of controls. Age and sex did not have any influence on any of the cytomorphometric parameters.
Conclusion: The habit of tobacco smoking and chewing causes quantitative cellular and nuclear changes in the squames of buccal mucosal epithelial cells, which shows a cause–effect relationship between tobacco and quantitative changes in the cells. Therefore, exfoliative cytology with the application of quantitative techniques can be used as a valuable diagnostic tool for detecting PMDs and malignant lesions.

Keywords: Chewing, cytomorphometry, oral squamous cell carcinoma, pap stain, potentially malignant disorders, smoking, tobacco


How to cite this article:
Syamala B, Chitturi RT, Chandrasekhar P, Prakash Chandra K L, Kumar K K, Ramana Reddy B V. Cytomorphometric analysis of exfoliated buccal cells to evaluate the malignant changes in individuals with tobacco smoking and chewing habits. J NTR Univ Health Sci 2017;6:143-8

How to cite this URL:
Syamala B, Chitturi RT, Chandrasekhar P, Prakash Chandra K L, Kumar K K, Ramana Reddy B V. Cytomorphometric analysis of exfoliated buccal cells to evaluate the malignant changes in individuals with tobacco smoking and chewing habits. J NTR Univ Health Sci [serial online] 2017 [cited 2017 Oct 23];6:143-8. Available from: http://www.jdrntruhs.org/text.asp?2017/6/3/143/215530


  Introduction Top


Tobacco remains one of the most important preventable causes of addiction, sickness, and mortality in the world. Tobacco has reached millions of people all over the world, cutting across national and social barriers. Tobacco was introduced to India by Portuguese traders in the late 16th or early 17th century. Since then tobacco use has spread with remarkable rapidity seeping into all sections of the society. Tobacco is widely used in India in various forms such as smoking and chewing. It is one of the most important risk factor for many oral diseases including oral cancer, oral mucosal lesions, and periodontal diseases.[1] Oral cancer is currently a major global health issue. It is currently the 6th most common malignancy in the world. In India, it is the most common malignancy among men and one of the five most common malignancies among women. Despite advances in surgery, radiotherapy, and chemotherapy, the 5-year survival rate of oral squamous cell carcinoma (OSCC) patients has remained approximately 50%. This poor survival rate among oral cancer patients can be attributed to the lack of early detection and treatment. A key approach to solve this problem would be to detect potentially malignant disorders (PMDs) such as leukoplakia, oral submucous fibrosis, eryhtroplakia, and oral lichen planus at their earliest or incipient stage.[2],[3],[4],[5],[6] One of the techniques by which this can be achieved is by using exfoliative cytology.

The normal physiology of the oral epithelium is to renew itself rapidly probably for every 2 weeks. The rationale of oral exfoliative cytology is based on this physiological process by examining the cells that are desquamated or abraded from the surface of the oral mucosa. The superficial epithelial cells show alterations in these cells and can serve as a reliable indicator of dysplastic or neoplastic changes. It is a relatively easy, simple, and noninvasive clinical technique that can be used as a chairside method for detecting PMDs and other mucosal lesions including OSCC.[7],[8],[9],[10] The use of oral exfoliative cytology in the past was limited due to the subjective nature of its interpretations and high false negative results. These limitations are overcome by the introduction of quantitative methods such as image analysis systems, especially in the assessment of cytomorphometric cellular alterations. Morphometry can be used selectively on structures or samples which are difficult to assess accurately such as variation in cell size, nuclear size, and staining intensity. Because nuclear changes are the most important criteria for diagnosing precancerous and cancerous lesions and because no single structural change is diagnostic by itself, a combination of several abnormalities is always necessary. Because quantitative procedures are objective and reproducible, they may be important aids in making a cytopathologic diagnosis.[11]

The aim of this study was to evaluate the quantitative changes in the cytological buccal smears of tobacco smokers and chewers with that of the normal healthy individuals by using cytomorphometric parameters such as cell diameter (CD), nuclear diameter (ND), and nuclear–cytoplasmic ratio (N/C).


  Materials and Methods Top


The study participants were patients attending our institute for routine dental treatment. They were grouped as controls without any habit (Group I), tobacco smokers (Group II), and tobacco chewers (Group III). Each group consisted of 40 individuals. Ethical clearance was obtained from the institutional ethical committee to undertake the study. Detailed information regarding personal details, medical history, and habits (type, duration, and frequency of smoking and chewing and use of alcohol) were recorded for each individual. Tobacco smoking was defined as the consumption of a minimum of 5 cigarettes or bidis per day over a period of 5 years or more. Tobacco chewing was defined as the consumption of 5 quids per day over a period of 5 years or more. Individuals with anemia, diabetes, oral sepsis, alcohol drinking, or history of any recent systemic disease were excluded from the study. Following informed consent, scrapings were obtained from the buccal mucosa.

Initially, the individuals were asked to rinse their mouth with normal saline to remove debris, and the buccal mucosa was cleaned with a swab. Using a gentle scraping motion and exerting little pressure on the buccal mucosa, the smears were made using a wooden spatula. The cells were scrapped from the clinically normal appearing buccal mucosa behind the commissural area, i.e., buccal mucosa just behind the angle of mouth. The scrapings were evenly smeared onto the center of the glass slides. The slides were immediately immersed in 95% isopropyl alcohol to ensure proper fixation for half an hour. Air-drying of the smears was avoided because it leads to alterations in the cellular morphology. All the smears were stained using Papanicolaou technique.

The slides were viewed under Olympus Research BX 51 Penta-head Microscope (Olympus, Japan) and the cells were projected onto the monitor via Jenoptik Digital CCD Camera (Jenoptik, Germany) at 40× magnification and images were captured for morphometric analysis which was done with the image analysis system, Image Pro Express Version 6.0 (Media Cybernetics, USA). For each slide, 200 cells were selected in a step-wise manner, moving the microscope stage from the left to right and then down and across in such a way as to avoid measuring the same cells again. Cells that were clumped together, overlapped, folded, or had distorted cellular and nuclear outline were not considered for the analysis. The images of individual cells were subjected to morphometric analysis. Using the “measurement” mode in the software, the nuclear and cellular diameters were obtained for clearly defined cells by drawing a line across the diameter using the digitized cursor. The mean cellular diameter (CD) and nuclear diameter (ND), and the nuclear–cytoplasmic ratio (N/C) were calculated for all 200 cells in controls [Figure 1], tobacco smokers [Figure 2], and tobacco chewers [Figure 3].
Figure 1: 40×: PAP stained cytological squame from buccal mucosa of normal healthy individual to measure cell diameter (CD) and nuclear diameter (ND)

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Figure 2: 40×: PAP stained cytological squame from buccal mucosa of a tobacco smoker to measure cell diameter (CD) and nuclear diameter (ND)

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Figure 3: 40×: PAP stained cytological squame from buccal mucosa of a tobacco chewer to measure cell diameter (CD) and nuclear diameter (ND)

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Variables were compared between and within the groups using one-way analysis of variance, and the pairwise comparison between the means was done using Scheffes multiple post hoc procedures using the Statistical Package for the Social Sciences software version 17.0 (IBM, USA). P values of < 0.05 were considered significant.


  Results Top


The total number of participants in the present study was 120 (40 in each group). In group I, 50% of the participants were males and 50% were females. In group II, 85% of the participants were males, whereas in group III all the participants were males [Figure 4]. In the age-wise distribution of the participants, the maximum number of tobacco smokers was in the 50–59 year age group (37.5%), whereas in the tobacco chewers, the maximum number of patients was in the 40–49 year age group (27.5%). There was no statistically significant association between the different age groups and habit of tobacco smoking and chewing [Table 1] and [Table 2].
Figure 4: Distribution of samples by gender in the control and study groups

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Table 1: Distribution of Samples by Age Groups and Study Groups

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Table 2: Comparison of Age Groups in Three Groups (Controls, Tobacco Smokers, and Tobacco Chewers) with ND Scores by One-Way Anova Test

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The mean CD observed in controls, tobacco smokers, and chewers wwas 56.70 ± 2.12 μm, 55.59 ± 1.59 μm, and 55.39 ± 2.48 μm, respectively. The pairwise comparison of the three groups done with respect to CD scores by Scheffes multiple post hoc procedure showed that there was a statistically significant decrease in the CD among tobacco chewers when compared with controls [Table 3].
Table 3: Pairwise Comparison of Three Groups (Controls, Tobacco Smokers, and Tobacco Chewers) with Respect to CD Scores by Scheffes Multiple Post hoc Procedure

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The mean ND observed in controls, tobacco smokers, and chewers was 8.86 ± 0.41 μm, 9.22 ± 0.36 μm, and 9.75 ± 0.51 μm, respectively. The pairwise comparison of the three groups done by Scheffes multiple post hoc procedures showed that there was a statistically significant increase in the ND in tobacco chewers and smokers when compared with the controls. In addition, there was a statistically significant increase in the ND of tobacco chewers when compared with the smokers [Table 4].
Table 4: Pairwise Comparison of Three Groups (Controls, Tobacco Smokers, and Tobacco Chewers) with Respect to ND Scores by Scheffes Multiple Post hoc Procedures

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The mean N/C values of controls, tobacco smokers, and chewers were 0.15 ± 0.01, 0.16 ± 0.00, and 0.17 ± 0.01, respectively. The pairwise comparison of the three groups done with respect to N/C scores by Scheffes multiple post hoc procedure showed statistically significantly higher N/C in tobacco chewers and smokers when compared with the control group (0.15) along with a statistically significant increase in N/C in tobacco chewers when compared with the smokers [Table 5].
Table 5: Pairwise Comparison of Three Groups (Controls, Tobacco Smokers, and Tobacco Chewers) with Respect to N/C Scores by Scheffes Multiple Post hoc Procedure

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  Discussion Top


Tobacco use is widespread throughout the world from countries with low economic status to the most affluent industrialized nations. There are numerous explanations for the habit of tobacco, however, the main reason for its ubiquity is the addictive drug nicotine present in all forms of tobacco leaf which is delivered in varying amounts to the user by the various methods of tobacco use. Tobacco-induced mucosal changes have been seen in exfoliated cells. Exfoliative cytology implicates its importance in the field of diagnosis with the principle that any change in the superficial cells can be a reflection of the change occurring in the immediate underlying tissue. Exfoliative cytology is widely advocated as an aid to supplement clinical judgment and as an adjunct to biopsy. Cytomorphometry is the quantitative description of the geometric features of structures in which various parameters such as nuclear size, cell size, N/C ratio, nuclear shape, nuclear discontinuity, optical density, and nuclear texture can be evaluated collectively to confirm the diagnosis accurately.[3],[11] The concept of cellular or nuclear alterations on exposure to varying forms of tobacco can be due to the nature of cellular response to stimuli from the end-products of different types of tobacco usage. These adaptive alterations can be attributed to dysplastic changes in the cell.[12]

In the present study, there was a significant reduction in the mean CD in tobacco chewers, as observed by Ramaesh et al. and Hande et al., however, there was no statistically significant reduction in CD in tobacco smokers.[13],[14] One of the early cellular alterations that occur in a cell turning dysplastic is the reduction in CD, as observed by Cowpe et al., which was later confirmed by Ramaesh et al.[15],[16] The reason why this occurs can be explained by the fact that cells turning dysplastic or malignant tend to proliferate faster, thus diminishing the ability of the cytoplasm and hence a decreased CD.[3] We further observed that this change was far more visible in tobacco chewers than smokers. This could be due to the fact that there is a difference in the carcinogens present in the smoke as well as smokeless forms of tobacco along with the duration an individual is exposed to smokeless forms. In smokeless tobacco, there is an initiation by tobacco-specific nitrosamines. Subsequently, the promotion process in carcinogenesis is due to chemical irritants such as nicotine and mechanical abrasion. If the process continues for a long time, it ensues carcinoma. The major carcinogens present in the smoking forms of tobacco are tobacco-specific N-nitrosamines, aromatic amines, polonium-210, and polycyclic aromatic hydrocarbons. The carcinogens of tobacco and the temperature increase that is caused by smoking could all act together and stimulate changes in the cells of oral mucosa. Further, the time of exposure to these carcinogens is high in smokeless forms. This could be the reason for the observed differences in the change in CD of chewers rather than smokers, and thus it seems that smoking tobacco could be less carcinogenic than chewing tobacco.[1],[17]

We also observed that there was a statistically significant increase in the ND in both smokers and chewers. The increase in ND can be explained by various reasons. It can be due to an increased amount of nuclear content, which is required for the active proliferation of the cell.[12] It could also be due to the gross changes in the number of chromosomes (aneuploidy), which is also an early marker for a malignant change.[15] We observed that this change is seen in both tobacco chewers and smokers. Thus, it can be considered that increase in ND is also an early marker for a dysplastic change, as observed by Ramaesh et al., and a change occurs first in the nuclear size. Later, as the result of changes in the nucleus, changes in the cytoplasm are also observed.[15],[18] As far as the N/C ratio is concerned, we observed that there was a statistically significant increase in the ratio in both tobacco chewers and smokers when compared with the controls. This altered N/C ratio is seen as a result of increased ND and decreased CD. The changed ratio in a squamous epithelium indicates a lesser differentiation (i.e. the basal/suprabasal cells) and the appearance of these cells in the superficial epithelium also indicates a dysplastic change.[19] Thus, it can be hypothesized from our study that usage of tobacco in smoking as well as smokeless forms causes dysplastic changes in the oral epithelium, with smokeless forms causing more changes comparatively.


  Conclusion Top


The reduction in CD and increase in ND and N/C ratio could be the early indicators of a malignant change. The habit of tobacco smoking and chewing causes quantitative cellular and nuclear changes in the squames of buccal mucosal epithelial cells, which shows a cause–effect relationship between tobacco and quantitative alterations in the cells. Therefore, exfoliative cytology with the application of quantitative techniques can be used as a valuable, easy, noninvasive, adjuvant techniques for detecting premalignant and malignant lesions or the lesions with normal appearing mucosa. These can also be used for the mass screening of dysplastic and PMDs. Further studies with increased sample size are required to assess the use of exfoliative cytology and cytomorphometry in the diagnosis of malignancy.

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Conflicts of interest

There are no conflicts of interest.

 
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