|Year : 2015 | Volume
| Issue : 2 | Page : 471-474
Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study
Shweta Jaitley1, Pankaj Agarwal2, Ramballabh Upadhyay2
1 Department of Oral Pathology and Microbiology, Mahatma Gandhi Dental College and Hospital, Jaipur, Rajasthan, India
2 Department of Oral Pathology and Microbiology, Kanti Devi Dental College and Hospital, Mathura, Uttar Pradesh, India
|Date of Web Publication||7-Jul-2015|
Department of Oral Pathology and Microbiology, Mahatma Gandhi Dental College and Hospital, Sitapura - 302 022, Jaipur, Rajasthan
Source of Support: None, Conflict of Interest: None
The present study was undertaken with an aim of determining the cytological features observed in mucosal smears of oral submucous fibrosis (OSF) patients and comparing them with that of features of normal mucosal cells. The observed features were than analyzed for their reliability in detecting malignant changes in this premalignant condition. Objective of the study was to conduct an oral exfoliative cytology (OEC) study on 30 clinically diagnosed cases of OSF and 30 cases of clinically normal mucosa with no other systemic disease. We observed that all the smears from clinically normal buccal mucosa showed Class I cytology. The exfoliated cells were of normal size and shape with normal staining intensity and normal nuclear characteristics. All the 30 cases of our study group showed features suggestive of benign atypical cytological changes (Class II cytology). In the present study, despite the small number of cases, cytological features consistently observed in all the cases, were indicative of a premalignant change and emphasized a regular follow-up of patients. Early detection of a premalignant oral lesion promises to improve the survival rate of patients suffering from these conditions.
Keywords: Cellular atypia, genotoxic damage, nuclear aberrations, oral exfoliative cytology, premalignant condition
|How to cite this article:|
Jaitley S, Agarwal P, Upadhyay R. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study. J Can Res Ther 2015;11:471-4
|How to cite this URL:|
Jaitley S, Agarwal P, Upadhyay R. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study. J Can Res Ther [serial online] 2015 [cited 2019 Nov 17];11:471-4. Available from: http://www.cancerjournal.net/text.asp?2015/11/2/471/151421
| > Introduction|| |
Oral submucous fibrosis (OSF) is a habit associated chronic insidious disease with a premalignant potential of transformation into oral squamous cell carcinoma. Malignant transformation rate of OSF has been found to be in the range of 7-13%, which is considered to be the highest among all premalignant lesions and conditions.  Squamous cell carcinomas, the most common form of oral cancer, can be preceded by precursor lesions/conditions in the form of leukoplakia, erythroplakia, or OSF. Oral exfoliative cytology (OEC) is a nonaggressive technique that is well accepted by the patient, and is therefore a suitable option for the early diagnosis of oral cancer.  Recently, OEC has reemerged due to improved methods of early diagnosis of oral precancer and cancer. It focuses on the morphological and staining characteristics of the individual cells. However, because of subjective nature of its interpretation and presence of only a small number of abnormal cells identifiable in a smear, the technique requires more skilled cytopathologists. Recent application of quantitative and cytomorphometric techniques has refined the potential role of OEC.  But in the present study, conventional methods of smear collection, staining, and interpretation were followed as the study aimed at evaluation of nuclear features and further elaborates the scope of this simple technique in mass screening of oral cancer in patients with premalignant condition like OSF.
| > Materials and methods|| |
Objective of the study was to conduct an OEC study on 30clinically diagnosed cases of OSF and 30 cases of clinically normal mucosa with no other systemic disease. The patient group comprised of 24 males between 25 and 32 years of age and six females between 27 and 31 years of age. Most of the patients gave a history of areca nut chewing and few of gutka chewing. On clinical examination of buccal mucosa, all the patients showed blanching and stiffness of oral mucosa with limitation of mouth opening. Palpable fibrotic bands were also exhibited in the study group. Normal male patients under 25-35 years age group with no history of habits, no systemic disease, and no clinical features of the disease on examination were selected for the control group. The aim of the study was to evaluate certain specific cytologic features which are indicative of a premalignant condition and compare them with that of normal cytology.
The procedure of smear collection was carried out by a simple conventional technique using clinical examination instruments. Buccal smears were taken from right buccal mucosa of subjects of both study and control group. The slides were then stained with routine hematoxylin and eosin staining procedure and studied under light microscope.
| > Results|| |
All the smears from clinically normal buccal mucosa showed Class I cytology [Figure 1]. The exfoliated cells were of normal size and shape with normal staining intensity and normal nuclear characteristics. All the 30 cases of our study group showed features suggestive of benign atypical cytological changes (Class II cytology). Certain common cytological features observed among all the cases of study group were; cellular and nuclear pleomorphism, irregular cellular outline, perinuclear halo, free nuclei, and both intranuclear and intracytoplasmic vacuolization along with numerous bacterial colonies. In addition to these findings, cells showed inflammatory changes like indented cellular outline suggestive of cells undergoing autolysis. Such cells also showed intracytoplasmic vacuoles and intracytoplasmic bacterial colonies.
|Figure 1: Epithelial cells showing normal cellular and nuclear morphology|
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| > Discussion|| |
It has been proposed that, cytological analysis oral exfoliative smear is a useful early diagnostic method for epithelial atypia. The assessment of cytological atypia was done using criteria described by Ahmed et al.  Based on our observations, the cytological findings were categorized into two broad categories, that is, inflammatory cellular atypia and noninflammatory cellular atypia. The noninflammatory cellular atypia included features like cellular pleomorphism, nuclear pleomorphism, nuclear budding, prominent nucleoli and micronuclei [Figure 2],[Figure 3],[Figure 4] and [Figure 5]. The inflammatory cellular atypia included intracytoplasmic bacterial colonies, inflammatory cells, perinuclear halo, free nuclei [Figure 6], and indented cellular outline indicative of cytolysis. In the present study out of 30 cases, 90% cases showed both inflammatory and non inflammatory cellular atypia (Class 2 cytology). Earlier studies conducted on OEC in premalignant lesions/conditions have concluded that the technique is useful in lesions of leukoplakia and OSF. It has been known to be useful for diagnosing very early malignant change. It was also demonstrated that exfoliative accurately reflects early epithelial dysplasia in the development of the experimental tumor and a high degree of correlation was also obtained.
|Figure 3: Epithelial cells showing nuclear pleomorphism, prominent nucleoli, nuclear budding, and micronuclei|
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|Figure 4: Perinuclear halo seen around the nucleus along with nuclear budding|
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|Figure 5: Cells with prominent and multiple nucleoli along with some mitotic figures|
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|Figure 6: Epithelial cells showing clumping of chromatin within the nucleus and free nuclei|
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Ogden et al., obtained a reduction in the cytoplasmic area ofnormal buccal mucosal cells in patients with malignant disease distant from the oral cavity.  A cytomorphometric study suggests that a decrease in the mean cytoplasmic diameter of exfoliated buccal mucosal cells could serve as an early indicator of dysplastic change, especially in lesions which appear histologically. 
Nuclear aberration in the form of malignancy-associated changes were first reported by Neiburgs et al.  This kind of studies were criticized for the low number of malignancies included and the failure to consider the following effects of anemia within the cancer group. In later studies it was suggested that such diagnosis should be used to help identify patients at increased risk of developing cancer.  Micronuclei may serve as marker for increased cancer risk, since they have been reported to arise in response to DNA damaging agents.  Studies have shown that micronucleus is the name given to the small nucleus that form whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division. They are induced in cells by numerous genotoxic agents that damages the chromosomes. The lagging elements are included in the daughter cells, too; but a considerable proportion is transformed into one or several secondary nuclei, which are, as a rule, much smaller than the principal nucleus and are therefore called micronuclei. , The head and neck region micronuclei are found at increased frequencies from normal mucosa to potentially malignant disorders (PMDs) to carcinoma, suggesting ever increasing chromosomal instability. 
Tolbert et al., , suggested the following criteria to consider an extranuclear body as a micronuclei: Rounded smooth perimeter suggestive of a membrane; less than one-third the diameter of the associated nucleus; staining intensity similar to that of the nucleus; texture similar to that of nucleus; same focal plane as the nucleus; and absence of overlap with the nucleus. In a study, the incidence of micronuclei was observed to be increased on buccal mucosa cells of smokeless tobacco users.  Carcinogenic and mutagenic compounds, including tobacco-specific nitrosamines are known to be present in ST forms and are believed to be responsible for the induction of micronuclei.  Although contamination by the bacteria that are commonly found in the mouth can interfere with micronuclei scoring. These can be differentiated from micronuclei by their characteristic shape, smaller size, color, staining intensity, and their presence upon and between buccal cells on the slide. , Another common similar particles are dye granules, that may sometimes resemble micronuclei but usually have a slightly different refractility and color intensity. ,
Thus, a cytological smear exhibiting many features of noninflammatory cellular atypia in any potent premalignant condition or lesion is indicative of a high risk lesion. This mechanism can be explained in the following way: Areca nut/tobacco chewing causes genotoxic damage to cells leading to release of reactive oxygen species. This would contribute to cellular atypia/dysplasia and further to oral cancer.
The more recent application of quantitative techniques, together with advances in immunocytochemistry, have refined the potential role of cytology in the diagnosis of oral cancer. Many studies have considered the influence of the quantitative analysis of cytomorphology, DNA analysis, and other tumor markers applied to oral exfoliative cytological samples. Ramaesh et al., revealed cytomorphometric changes in the buccal mucosal cells of cigarette smokers and betel nut chewers. The authors concluded that betel chewing with tobacco influences both nuclear diameter and cellular diameter and smoking influences only the nuclear diameter of exfoliated cells.  These studies indicate that oral cytology may provide an important adjunct in the assessment of the patient with a potentially cancerous oral lesion.
| > Conclusion|| |
Although exfoliative cytology cannot take the place of biopsy in making a definite assessment of the nature of a lesion, it is still widely applied in the evaluation of oral lesions. In the present study, despite the small number of cases in the cohort, cytological features consistently observed in all the cases were indicative of a premalignant change and emphasized a regular follow-up of patients. Early detection of a premalignant oral lesion certainly promises to improve the survival rate of patients suffering from these conditions. OEC is an adjunct to biopsy in such cases and makes screening of cancer easy to manage as more number of cases can be screened in less time. However, further studies conducted on a larger study group would establish the role of oral exfoliative cytology in predicting the premalignant potential of oral submucous fibrosis.
| > References|| |
Thilakaratne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol 2006;42:561-8.
Epstein JB, Zhang L, Rosin M. Advances in the diagnosis of oral premalignant and malignant lesions. J Can Dent 2002;68:617-21.
Hegde V. Cytomorphometric analysis of squames from oral premalignant and malignant lesions. J Clin Exp Dent 2011;3:441-4.
Ahmed HG, Idris AM, Ibrahim SO. Study of oral epithelial atypia among Sudanese tobacco users by exfoliative cytology. Anticancer Res 2003;23:1943-9.
Ogden GR, Cowpe JG, Wight AJ. Oral exfoliative cytology: Review of methods of assessment. J Oral Pathol Med 1997;26:201-5.
Neiburgs HE, Herman BE, Reisman H. Buccal cell changes in patients with malignant tumours. Lab Invest 1962;2:80-8.
Burger G, Aubele M, Clasen B, Jutting U, Gais P, Rodenacker K. Malignancy associated changes in squamous epithelium of the head and neck region. Anal Cell Pathol 1994;7:181-93.
Livingston GK, Reed RN, Olson BL, Lockey JE. Induction of nuclear aberration by smokeless tobacco in epithelial cells of human oral mucosa. Environ Mol Mutagen 1990;15:136-44.
Schmid W. The micronucleus test. Mutat Res 1975;31:9-15.
Palve DH, Tupkari JV. Clinico-pathological correlation of micronuclei in oral squamous cell carcinoma by exfoliative cytology. J Oral Maxillofac Pathol 2008;12:2-7.
Casartelli G, Bonatti S, De Ferrari M, Scala M, Mereu P, Margarino G, et al
. Micronucleus frequencies in exfoliated buccal cells in normal mucosa, precancerous lesions and squamous cell carcinoma. Anal Quant Cytol Histol 2000;22:486-92.
Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in Buccal Smears: A field test in snuff users. Am J Epidemiol 1991;134:840-50.
Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmuller S, et al
. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gaps. Mutat Res 2008;659:93-108.
Vellingiri B, Balasubramaniam L, Kuppanan S, Pappusamy M, Raman S, Subramaniam M, et al
. Cytogenetic damage in khaini users of Tamilnadu, Southern India. Braz J Oral Sci 2007;7:1559-62.
Nersesyan A, Kundi M, Atefie K, Hermann RS, Knasmuller S. Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells. Cancer Epidemiol Biomarkers Prev 2006;15:1835-40.
Samanta S, Dey P. Micronucleus and its applications. Diagn Cytopathol 2012;40:84-90.
Ramaesh T, Mendis BR, Ratnatunga N, Thattil RO. The effect of tobacco smoking and of betel chewing with tobacco on the buccal mucosa: A cytomorphometric analysis. J Oral Pathol Med 1999;28:385-8.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]