Original Article- A CYTOLOGICAL STUDY OF LEUKOPLASTIC LESIONS IN ORAL CAVITY

Original Article- A CYTOLOGICAL STUDY OF LEUKOPLASTIC LESIONS IN ORAL CAVITY I. GUJRAL*, P. SINGH**, S. SHARMA***, N. GANGANE*** ABSTRACT Oral white lesions that cannot be clinically or pathologically characterized by any specific disease are referred to as leukoplakia. Such lesions are well known for their propensity for malignant transformation to the extent of 10-20%.Exfoliative cytology is a simple and useful screening tool for detection of malignant or dysplastic changes in such lesions. Leukoplastic lesions of the oral cavity are caused by many habits such as tobacco, betel and nut chewing in association with smoking. The cytology similar to gynecological cervical cancer screening by Papanicolau stain was done in the leukoplastic lesions seen in oral cavity. The lesions were more common in the middle age group and also parakeratosis and orthokeratosis was seen. The atypia was only mild and moderate and no severe atypia or malignant cell was observed in the smears. Cytology can play a very important role in identifying the leukoplastic lesions of the oral cavity and can also help in the mass screening programmes for oral leukoplastic lesions especially in developing countries like India, as its cheap, easy to perform and the report can be obtained very fast and also patients undergoing long term observation in whom repeatedly doing cytology will reduce the number of biopsies and enable detection of developing malignant transformation. Key words : Cytology, Leukoplastic lesion, Oral cavity. Introduction : In 1978 a World Health Organization (WHO) group defined oral leukoplakia as : "A white patch or plaque that cannot be characterized clinically or pathologically as any other disease" 1. It is therefore a diagnosis of exclusion from other oral white lesions such as leukokeratosis, infective lesions (candidiasis, syphilitic oral lesion, oral hairy leukoplakia caused by Epstein Barr virus), lichen planus, lupus erythematosus, dyskeratosis congenita, white sponge nevus, submucosal fibrosis and frank carcinomas. 1,2, It is common in adults beyond 40 years of age, and affects 1% of the total population. 3 Leukoplakia is an important premalignant lesion usually associated with smoking and chewing habits. Many surveys have been carried out in India and abroad to demonstrate an association between leukoplakia and smoking habits. As oral cancer presents a *Ex-Resident, **Assistant Professor, *** Professor, Dept of Pathology, MGIMS, Sevagram Corresponding Author : Dr Nitin Gangane, Professor & Head, Dept of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha (MS), India

major health problem in India, 15 to 70%cof all cancers diagnosed are found in oral cavity. There has been found very strong association between smoking, chewing tobacco, betel nut consumption of alcohol or combination with leukoplakia. Leukoplakia, the most frequent precancerous lesion of mouth, was first described in the second half of the last centuary by Hungarian dermatologist, Schwimmer. Initially cytologic studies of the mouth were based on Papanicolaou's gyanecologic cytodiagnosis methods. Cytology is now recognized as an adjunct to biopsy in the diagnosis of carcinoma. Biopsy for premalignant lesions was not always possible especially during mass screening, so cytologic screening was carried out for evaluation of premalignant lesions like leukoplakia. The use of cytologic techniques for evaluation of leukoplakia especially in mass screeing programmes is now practically well established, as it is a very simple, cheap and quick procedure without need of elaborate equipments and helps in mass screening of large population. The present study was carried out with the following aims: 1. To determine the ability of cytological method to determine the type of kertanization in an oral leukoplakia. 2. To correlate the cytological patterns of keratinization with clinical types of leukoplakias. 3. To find out the degree of effectiveness of oral exfoliative cytology as a diagnostic tool for epithelial atypias and thus in the clinical management of oral leukoplakias. Material and methods: Inclusion criteria : The present study consisted of oral cytological study in 250 adults with and without leukoplakic lesions. All the cases were having history of smoking, chewing tobacco, betel nut or the combination of it and drinking alcohol for a desirable time span. The cytological smear was prepared as per the protocol given below - all the patients were asked to rinse their mouth with normal saline 3 times. Dry lesions were moistened with saline. Sterile wooden spatula was used for cytology. The lesions were scraped gently with the spatula and the material obtained was smeared on glass slides. The precaution was taken to avoid smudging of smear. The slides were kept in fixative [mixture of ethanol (80%) and ether (20%)] immediately after spreading for about 30 minutes.

Staining of the smears : The fixed smears were stained by the method of Papanicolaou (Hughs and Dodds 1968). The stained smears were screened for various pathological lesions and classified as 1) Orthokeratotic, 2) Parakeratotic, 3) Combined, as per criteria laid down by Sahiar et al. Similarly smears with atypia were again divided into mild, moderate and severe. Exclusion criteria : Individuals of less than 20 year were not included in the present study. Observations : The present study was carried out in the department of Pathology and consisted of 250 individuals with a definite history of betel nut, tobacco and bidi smoking for duration of more than 5 years. Cytological study was inconclusive for opinion in 21 cases (8.4%) because of scanty material or degenerative changes in the epithelial cells. Opinion was possible on 229 (91.6%) cases only. The maximum number of cases was in the age group of 36-40 years with a mean of 44 years (17.6%) as shown in table 1. A detailed clinical examination of the oral cavity was carried out in each case. The comparison between the clinical lesion and the various cytological features of oral cavity has been depicted in table 2. Showing maximum number of cases with parakeratosis (176) followed by orthokeratosis (31) and combination of both types (22). Table 1 : Incidence of leukoplakia in different age group Age Group in years Number of cases Cases with leukoplakia examined 15-20 - - 21-25 5-26 - 30 37-31 - 35 23 1(4.3%) 36-40 44 1 (2.2%) 41-45 26 4 (15.3%) 46-50 31 12 (38.7%) 51-55 19 6 (31.5%) 56-60 32 7 (21.8%) 61 & above 33 9 (27.2%) Grading of the cytological atypia showed the number of cases with mild atypia were 4, moderate atypia 2. No case showed severe atypia or malignant cells on cytology, as shown in table 3. The effect of different habits prevailing in the study group on the oral mucosa in causing different lesions. It was observed that

combination of habits caused maximum number of clinical lesions such as leukoplakia was 22 (55%) cases, tobacco lime lesion was 22 (55%) cases. Smoking was also an important causative factor in causing oral leukoplakia. 12 (30%) cases of smoking showed oral leukoplakia. Table 2 : Oral cytology in comparison with clinical lesion Clinical impression Orthokeratosis Parakeratosis Combination Unsatisfactory No Lesion 19 116 14 19 Oral Leukoplakia 5 31 4 Tobacco lime 5 20 4 1 lesion Oral submucous 1 3 - - fibrosis Leukoedema - 6-1 Oral submucous 1 - - - fibrosis& Leukoplakia Table 3 : Cytological atypia in oral leukoplakia with grading Cytological Atypia Parakeratotic with atypia Orthokeratotic with atypia Combination with atypia No. of cases Mild Moderate Severe 6 4 2 NIL NIL NIL NIL NIL NIL NIL NIL NIL Table 4: effect of various habits on various clinical lesions Habits No. of lesions Leukoplakia Clinical Lesion Tobbaco & Lime OSMF* Others lesions Betel Nut 16 (9.7%) - - - - Tobacco + 47 (27.9%) 6 (15%) 8 (26.6%) 1 (25%) - Lime Smoking 20 (11.9%) 12 (30%) - - 2 (25%) Pan 13 (7.7%) - - - - Combinations 72 (42.8%) 22 (55%) 22 (73.4) 3 (75%) 6 (75%)

Discussions : Idiopathic leukoplakias are mostly benign lesions occurring in response to chronic irritation. Tobacco in different forms has been described as the most common incriminating factor for such lesions 4,5,6,7. However, other factors, depending on the socio-cultural habits of the patients that lead to chronic irritation of oral and buccal mucosa may also be contributory. It starts as a thin homogeneous grayish white plaque either well defined or blending with homogeneous leukoplakia). Some lesions develop surface irregularities (granular or nodular the surrounding tissue. Patients with idiopathic leukoplakia have the highest risk of developing cancer 8,9,10,11,12. The frequency of dysplastic or malignant change varies from 15.6-39.2% in different studies 9,11,12,13,14. The risk of developing malignancies at lesion sites is 5 times greater in patients with leukoplakia than in patients without them. Frequently oral white patches are noted secondary to some identifiable local irritation. For example, thickened hyperkeratotic lesions are frequently seen over edentulous areas of alveolar ridges (ridge keratosis), chronic tongue chewing (morsicatio linguarum) or chronic cheek chewing (morsicatio buccarum). All such lesions are secondary responses to a chronic irritation leading to compensatory hyperkeratosis of the epithelium developing as a protective phenomenon, similar to the development of a callus over skin of hand or feet. Such lesions do not show any dysplasia and are reversible on eliminating the causative factor. These lesions are better termed as leukokeratosis rather than leukoplakia as agreed by most experts. The usage of the term leukoplakia continues to undergo refinement 7. The findings of the present study are higher as compared to the available literature i.e. 16% of cases. This may be probably due to the selected group chosen for the study with a history of more than 5 years of continuation of habits. Also multiple habits were common in the present study group as compared to others. Oral cancer is the commonest malignancy in Indian males 13. Leukoplakia is the most common precancerous lesion of the oral mucosa 10,11,12,13,14. Its implicating factors, like chewing and smoking of tobacco is widely prevalent in our country. However, few Indian studies have been published on this topic. In the present the adult population was taken into consideration with a definite history of betel nut, tobacco and lime chewing and smoking. Out of total 250 cases, 168 cases (67.2%) cases did not show any clinical lesions. 40 (16%) cases had leukoplakia, 30 (12%) cases had lime lesion, 7 (2.8%) cases had leukoderma and 4 (1.6%) cases had orla submucous fibrosis. In india highest prevelance of leukoplakia was reported by Mehta et al 6 from Bombay (3.5%) in

persons with smoking and chewing habits. Highest prevalence was reported by Atkinson et al 8 from New Guinea (8.1%). The age range was wide showing that no age was exempted whereas several other studies worldwide 4,7,8,9 have shown preponderance of leukoplakia in a later age group beyond 40 years. The discrepancy could be due to the use of tobacco, lime and betel quite prevalent among the younger population in our country. The male to female ratio was found to be 1.16:1. The leukoplakia cases were highest in the 46-50 years of age group. This is in accordance with the findings of Fall et al although it was higher in comparison to other studies by Mehta et al 7. There has been conflicting evidence as to the efficacy of the cytological examination when it was used to diagnose the malignant potentialities of the oral precancerous condition, mainly leukoplakia. In such lesions the presence of thick keratinized layer may lead to a scant exfoliation of atypical cells on the surface and viable cells may be sparse in the smear. The keratinisation pattern of 229 smears was studied as 21 of the smears were unsatisfactory. The smears were categorized into parakeartotic, orthokeratotic or combination. The smear was assessed quantitatively if organophilic,yellowish or eosinophilic nucleated squames were present predominantly. This involved atleast 60% of the cells in the smear and very often they were the only cells present. Similarly, parakeratotic smears were judged by the predominant presence of eosinophilic cells with pyknotic nuclei. The determination of the type of keratinization assumes importance because with parakeratosis the mitotic index rises, indicating proliferative tendency which often leads to epithelial hyperplasia, dysplasia, carcinoma in situ and even carcinoma. Our study showed maximum cases with parakeratosis indicating proliferative tendency in these cases. It was observed that with increase in frequency of habits there was also increase in percentage of combination smears. Parakeratotic tendency indicates a proliferative tendency and therefore in field surveys though oral cytology is not useful in diagnosing orla leukoplakias, it can help in identifying leukoplakias with parakeratosis patterns and leading to those cases which need careful long term follow up. Conclusions : Leukoplakia and its clinical association with the habit of tobacco and betel nut chewing along with smoking manifest itself in various forms, out of which smoking is the most important etiological factor for oral leukoplakias. The determination of the type of keratinisation on cytology assumes significance because it helps in identifying parakeratosis, orthokeratosis and also indicates the proliferative tendency. This study justifies the use of cytology in mass

screening programmes in identifying leukoplakias with parakeratotic patterns and leading to these cases which need careful long term follow up. Competing interests The authors declare that they have no competing interests. Authors' contributions Dr I. Gujral contributed to the literature search, data collection, data analysis, and Dr P. Singh and Dr S Sharma helped in manuscript preparation. Dr N. Gangane gave the cytology impressions. All authors have read and approved of the submitted manuscript. References: 1. Mishra M, Mohanty J, Sengupta S, Tripathy S. Epidemiological and clinicopathological study of oral leukoplakia. Indian J Dermatol Venereol Leprol. 2005 May-Jun;71(3):161-5. 2. Odukoya O, Roberts T, Aroll G. A cytologic study of the effect of Kolanut on the keratinization of the palatal mucosal of Nigerian smokers. Afr Dent J. 1990;4(1-5):1-5. 3. Bancozy J. Exfoliative cytologic changes in oral leukoplakia. J Dent Res 1969;48:17-21. 4. Boyd W. Pathology for surgeons, 7 th edition. Philadelphia Pa. W.B.Saunders company. 1955. 5. Schwimmer E. Szajurges nyelu ny alkahartya nemely ritka koralakjarol. Orv Egyesinet Eukonvyve 48,1887. 6. Sahair E, Daftary K and Mehta S. Cytological and Histological keratinisation studies in leukoplakias of the mouth. J. of Oral Path 1975;4: 19-26. 7. Mehta S, Sanjana K,Shroff C and Doctor H. Incidence of leukoplakia among 'pan (betel leaf) chewers' and 'bidi smokers' 8. Atkinson L, Chester L, Smith G and Ten J. Oral cancer in New Guinea. A study in demography and etiology. Cancer 1964;17:1289-1298. 9. Mehta S and Sanjana K. Relataion of betel nut chewing to periodontal disease. J Am Dent Assoc. 1955;50:581. 10. Pindborg J, Jist O, Renstrup G and Roed B. Studies in oral leukoplakia. A prelimanry report on the period prevalence of malignant transformation in leukoplakia based on a follow up study of 248 patients. J Am Dent Assoc 1968; 78:767-771. 11. Wahi N, Kehar V and Lahiri B. Factors influencing oral and oropharyngeal cancers in India. Br J Cancer 1965;19:642-660. 12. Lahiri and Vijay L. Normal oral smear and the effect of tobacco chewing on the character of buccal epithelial cells. ICMR exfoliative cytology 1971;93-100.

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