ORIGINAL ARTICLE

Cytological Study of Exfoliative Buccal Mucosal Cells of Qat Chewers in Yemen

Hussain Gad Elkarim Ahmed, B.Sc., M.Sc., Ph.D.,1* Ammar Saleh Abdullah Omer, and Shadan Atif Abd algaffar, Honor B.Sc.3
2 B.Sc., M.Sc.,

Prevalence of oral cancer is relatively increasing in Yemen in recent years, which is ascribed to Qat with other carcinogens. Since the establishment of a simple and reliable method for the detection of oral cancerous and precancerous lesions is essential, exfoliative cytology (EC) was applied to a case-control study to appraise the presence and severity of oral epithelial atypia in 300 subjects (150 were Qat chewers and 150 were non-Qat users) without prior knowledge of the subjects Qat exposure. Ten patients with oral squamous-cell carcinoma (OSCC) were included as internal controls. Cytological atypia and hyperkeratosis were demonstrated in six and 24 subjects, respectively, and could not be ascertained in the remaining 294 and 276 correspondingly. All of the 30 (six atypia and 24 hyperkeratosis) were among the 150 Qat chewers. Cytological atypia was found in all the 10 control cases with OSCCs (eight with severe degree and two with mild atypia). For the Qat among those with atypia and hyperkeratosis, the adjusted OR and the 95% condence level were found to be 1.6 (0.87.2) and 3 (10.121.9), respectively. In view of these ndings, we suggest the use of EC for detection and assessment of oral cytological atypia. Qat use contributes to the occurrence of cytological atypia that might develop to oral precancerous or cancerous lesions. Diagn. Cytopathol. 2010;00:000000. ' 2010 Wiley-Liss, Inc. Key Words: Qat; cytology atypia oral

Earlier detection and follow-up of oral precancerous lesions are important to predict their potency to turn into malignancy.1 The primary risk factors for development of
1 Department of Histopathology and Cytology, Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan 2 Department of Histopathology, Hajja Hospital, Yemen 3 Department of Histopathology and Cytology, Medical Laboratory Science, University of Khartoum, Khartoum, Sudan *Correspondence to: Hussain Gad Elkarim Ahmed, B.Sc., M.Sc., Ph.D., Department of Histopathology and Cytology, Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan. E-mail: hussaingad1972@yahoo.com Received 7 March 2010; Accepted 4 May 2010 DOI 10.1002/dc.21462 Published online in Wiley Online Library (wileyonlinelibrary.com).

these lesions or oral squamous-cell carcinomas (OSCCs) are considered to be similar worldwide.1,2 Habits of tobacco use and alcohol consumption have been strongly attributed to the development of these lesions.1,2 However, in Yemen, alcohol consumption is uncommon, while the use of tobacco in form of smoking is mildly common and female male ratio is 1:1.4.3,4 Qat, is a herbal product consisting of leaves and shoots, has been in use in Yemen for centuries and its usage is widespread.5,6 Qat is usually chewed at special social gathering, but it is also used frequently during work.7 It was estimated that 61.1% of the adults in Yemen were current Qat users.8 High frequencies of oral cancers were reported from countries that have high rates of Qat usage.9 Squamouscell carcinoma (SCC) was the most frequent oral cancer (84%), and the tongue (42%), gingiva (23%), and buccal mucosa (20%) were the most common sites. The high relative frequency of oral SCC may be related to the habits of chewing tobacco and Qat.10 There are three main alkaloids present in Qat leaves: cathinone, cathine, and ephedrine.11 The potentiality of Qat to induce cancerous changes has been investigated in vitro using human cell line.12,13 Qat extract, cathinone, and cathine were found to induce cell death by apoptosis, in addition to structural chromosomal changes, such as those seen in cells in the development of cancer.14 In addition, the use of Qat has also been shown to produce a variety of oral mucosal changes, such as acanthosis, hyperkeratosis, and epithelial dysplasia.8 The use of exfoliative cytology (EC) for diagnosis or screening of oral mucosal lesions has received a considerable attention,15,16 some of the studies that have evaluated the role of EC in comparison with biopsy in this respect, have reported similar results, albeit other studies have reported conicting results.16 Exfoliate cytology was found reliable in the diagnosis of oral hairy leukoplakia17
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2010 WILEY-LISS, INC.

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AHMED ET AL.

and during follow-up of patients with a history of head and neck cancers.18 EC, although it has some disadvantages that require continued renements, remains the simple and useful method in oral cancer screening programs in remote areas lacking advanced diagnostic facilities. In Yemen, the high prevalence of Qat use, which might be the major risk for development of oral cancer, advocates the need for simple reliable and cost-effective methods for screening people at high risk. It was, therefore, the objective of the present study to detect the oral epithelial atypia (that might develop into oral precancerous or cancerous lesions) among Yemen Qat chewers.

Sample Size
The sample size was calculated using a software known as the survey system, available at http:/www.surveystem. com.sscalc.htm. The system inertly relies on the equation: n z2pq/d2 [n sample size; z the standard normal deviate, usually set at 1.96, which corresponds to the level of the 95% condence level (CI); p the proportion to the target population, i.e., percentage of the studied group, which is 0.11 in this study; and q 1.0 p].

Sample Collecting and Processing

Cytological materials of exfoliative cells were collected from Qat application site (buccal mucosa) (n 150) and buccal mucosa of non-Qat users (n 150). Using a at wooden-tongue spatula, cytological materials were collected from white mucosal surfaces (in some cases) and normal-appearing mucosa of the remaining study subjects. The surface epithelium was scraped and the collected cells were immediately smeared on cleaned coated glass slide, xed in 95% ethanol for 15 min, and then transferred to the laboratory.

Materials and Methods

As measurement of the promotion of a screening procedure for oral premalignant and malignant lesions, this retrospective cohort study investigated the practicability of using exfoliative cytological to detect and assess presence and degree of oral epithelial atypia among Qat users from Yemen. In this study, the effects of Qat chewing were assessed on oral mucosa by cytology. The study subjects were Qat chewers (ascertained as cases) or non-Qat using (ascertained as controls) volunteers living in the city of Hajja, Yemen. All cases or controls were apparently healthy. All the study subjects were males and were 13 years of age or older and current Qat user for at least 3 years with a frequency of more than two times per day. Oral cancer patients, persons with bad oral hygiene, tobacco users, and alcohol consumers were excluded from the study. Information regarding the subjects personal characteristics and Qat habits were obtained.

Sample Processing
The smears were stained using the Papanicolaou staining method.19 Assessment of the Results. To increase the reliability and reproducibility, strict quality control measures were applied. We included 10 smears from patients with histopathologically diagnosed oral cancer to serve as positive control. To asses the quality of staining, the smears were examined under low (103) power using a light microscope. All smears showed fair staining quality with blue nuclei, pink/orange cytoplasm of keratinizing squamous epithelium, and blue/green staining of the cytoplasm of the nonkeratinizing squamous cells. Cytological smears were labeled in such a way that the examiner was blind to the Qat habit of each subject.

Selection Criteria
Cases or controls were selected from 600 registered individuals. Cases or controls were given serial numbers from 1 to 600, then the participants were selected by the addition of a constant number that gave equal chance for each one of the registered population to be selected. This was achieved by the use of a random method, in which the sample interval is determined by dividing the sample size on the study group. The sample interval in this study was found to be 4, and then the number of the rst participant to be included in the sample was chosen randomly by blind picking of one of the four pieces of papers numbered 1 to 4. The number picked was 4; thereafter, every 4th number was added, starting from participant number 4, to be selected as the study subjects. Consequently, the numbers of the selected patients were: 4, 8, 12, and so on, up to the selection of 150 cases or controls.

Cytological Assessment of the Epithelial Atypia

For interexamination variations in scoring, two examiners performed the assessment twice and independently. Epithelial atypia was assessed using the criteria described earlier.20,21 In brief, the presence of two or more of the following features indicated the presence of epithelial atypia: nuclear enlargement associated with increased nuclear cytoplasmic ratio, hyperchromatism, chromatin clumping with moderately prominent nucleolation and irregular nuclear borders, bi or multinucleation, increased keratinization and scantiness of the cytoplasm, and variations in size and/or shape of the cells and nuclei. Each feature was assessed by comparison with each of the photographs shown elsewhere.22,23 The degree of epithelial atypia was assigned by adopting criteria described elsewhere.21,24 The presence of two

Ethical Consent
Each participant was asked to sign a written ethical consent during the interview before the specimen was taken. 2
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Table I. Showing the Distribution of Study Subjects by Abnormal Cytological Findings Qat chewers Cytological ndings Normal Atypia Keratosis Inammatory inltrate Bacterial infection Benign degenerative changes Total No. 51 6 24 53 8 8 150 % 34 4 16 35.4 5.3 5.3 100 Non-Qat chewers No. 131 0 0 14 5 0 100 % 87.3 0 0 9.3 3.3 0 100 Total 182 6 24 67 13 8 300

or more of the following features were consistent with atypia: nuclear enlargement associated with increase nuclear cytoplasmic ratio, hyperchromatism, chromatin clumping with moderately prominent nucleoli, irregular nuclear membranes and bi or multinucleation, scant cytoplasm, and variation in size and/or shape of the cells and nuclei. The percentages used below were calculated by counting normal and atypical cells in ve microscopic elds using 403 objective. The mean of the count was then calculated for both types of cells. The two means were added together, and the total mean value of cells counted per eld was generated. If the atypical cells were 310%, this was considered slight and given a score value as follows: for 36% atypical cells, the score assigned was (+) and for 710% the score was (++). More than 10 atypical cells were consistent with marked atypia. The marked changes were scored as follows: 1015% was (+) for mild cytological atypia, 1620% was (++) for moderate cytological atypia, and 21% was (+++) for severe cytological atypia. Furthermore, hyperkeratosis and inammatory inltrates were categorized into mild, moderate, and severe adopting similar scoring of that described for the atypia. Subjects with cytological atypia were further referred for biopsies.

Fig. 1. Photomicrograph 1: Buccal smear showing excessive keratinization and nucleated epithelial cells (Papanicolaou, 3400). Photomicrograph 2: Buccal smear showing mild epithelial atypia with inammatory inltrate and red blood cells (Papanicolaou, 3400). [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Data Analysis
Statistical Package for Social Sciences (version 10) was used for analysis and to perform Pearson v2 test for statistical signicance (P value). The 95% CI and CIs were used.

Results
The distribution of cases (Qat chewers) and controls (nonQat chewers) by age was relatively similar. The mean age of the study population was 47 years with a range of 1382 years. The entire study group consisted of males. The distribution of the cases and the controls by abnormal ndings is shown in Table I. White lesions were signicantly more prevalent in the Qat chewers (13%) compared with the nonchewing individuals (0%) (P < 0.001). People with atypical cytological changes among Qat chewers were six (4%) and 24 (16%) with hyperkeratosis, and none of the controls showed cytological atypia or hyperkeratosis, as

indicated in Figure 1, photomicrographs 1 and 2. The adjusted OR and the 95% CI were found to be 1.6 (0.8 7.2) and 3 (10.121.9), respectively. As all of the cytological atypias were mild degree, subsequent biopsies were only done for three individuals with white mucosal surfaces. Histopathologic examination revealed leukoplakia in two cases and the remaining case was found with epithelial dysplasia. By examining the severity of cytological atypia and hyperkeratosis among the cases, we found that all cytological atypias were mild; hence, for the hyperkeratosis, there were seven mild, eight moderate, and nine severe categories of hyperkeratosis. In regard to the duration and frequency of Qat use and cytological abnormal ndings, there was no statistical signicant relationship. In the specimens from the patients with OSSCs, eight (80%) showed a severe degree of epithelial atypia with very dense inammatory inltrate and the remaining two showed amid category. High proportions of atypia and hyperkeratosis were found among older age ranges. Of the six atypical changes, three were detected in age range 4660, two were in 3145, and only one in age more than 60 years old. A high hyperkeratosis was found in the age ranges 4660, 1630, 3145, and 60+ years, constituted 10 (42%), 7 (29%), 5 (21%), and 2 (8%), respectively. Inammatory inltrate was demonstrated in 53 (35.3%) of the cases and 14 (9.3%) of the controls. Cytological features of bacterial infection were observed in eight (5.3%) of the cases and ve (3.3%) of the controls.

Discussion
SCC is the most common malignancy of the oral cavity worldwide. Most of oral SCCs are strongly associated with exposure to carcinogens such as those present in tobacco, alcohol, or other substances. For the early detection of
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premalignant and malignant lesions in high-risk groups, simple, reliable, and cost-effective screening methods are urgently needed. In this study, the occurrence of cytological atypia was investigated in smears obtained from Qat chewers and non-Qat chewers. This is the rst report from Yemen to use cytological method for the assessment of smears obtained from Qat chewers. We found cytological atypia and hyperkeratosis in smears from six (4%) and 24 (16%) of the cases, respectively. None of these changes was demonstrated in the control group. These ndings suggest that Qat chewing might be associated with a risk for occurrence of oral cytological atypia, which can be indicated in oral EC. The ndings may further suggest that Qat components might stimulate the epithelial cells to undergo atypical cellular morphological changes, which may lead to oral precancerous and eventual cancerous lesions as suggested by others.25,26 Our results also revealed a signicant correlation between Qat use and hyperkeratosis in buccal mucosa. Nevertheless, some studies reported the absence of association between Qat use and occurrence of precancerous and cancerous lesions.27,28 However, these studies investigated the presence of oral cancerous and precancerous by clinical methods; hence, we applied cytological methods. These ndings indicate the apparent role of Qat components in inducing varying degrees of hyperkeratosis, which supported other previous reports.8,29 When examining the relationship between age and epithelial atypia or hyperkeratosis in epithelial atypia, 66.6% of epithelial atypia was found among the age range 46+, although the number of study subjects in this group represents only 26.6% of the study group. However, hyperkeratosis was found among 50% at this age range (46+) years. Thus, cytological atypia and hyperkeratosis appeared to increase with increase of age. These ndings are in agreement with other investigations that attributed the age-increased effect to extent of duration of exposure.8 However, one of the limitations of this study was the variation in age between cases and controls, whilst most of the cases were at older age; many controls were at younger age. Although, the habit of Qat chewing started to decline during the recent years among new generation, the vast majority of older Yemeni are heavier Qat consumers. Nevertheless, our ndings found no relationship between mucosal changes and duration or frequency of Qat use. This might be attributed to the fact that all cases were currently heavy users for more than 3 years and regardless to the frequency, they use to store Qat for prolonged period during the day. However, this requires further evaluation among subjects with oral lesions. In this study, we applied cytological method for the assessment of oral epithelial atypia. EC has been used in screening and early diagnosis of oral cancers,30,31 in characterization of normal, malignant, and premalignant oral 4
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mucosal lesions.32,33 It is known that cells available for cytological smears are taken from the epithelial surface, while most of the atypical cells are located beneath the epithelium surface, thus bringing into question the use of oral EC for the diagnosis of precancerous lesions. In our study, and to examine variations between individuals in assessment of cytological atypia, we included smears from 10 patients histopathologically diagnosed as OSCCs to serve as internal control. Thereafter, a strong correlation between cytological atypia and OSCCs was proved. On the other hand, the evaluation of epithelial atypia varies in many studies.34,35 Therefore, to increase the reliability and reproducibility and to reduce the interference in the morphology, two independent investigators have examined the smears using a more specic scoring system.24 Inammatory inltrate was identied in 35.3% of the cases compared with 9.3% of the controls. We think that this variation evidenced the irritation role of Qat to stimulate the inammatory changes; such ndings were previously reported.36 Although the presence of bacteria in the smears does not make a diagnosis of infection but our ndings of the cytological evidences of bacterial changes can be attributed to the fact that, the oral cavity is a traditional site for many commensal bacteria. Though, the variation between cases and controls, in this respect is mild, but we think that the aggregation of bacteria was due to the presence of the dead post irritated epithelial cells. Although, oral fungal infection associated with Qat use was reported,37 but no previous study reported bacterial infection in the oral mucosa. In conclusion, the ndings of this study indicate that Qat chewing might be considered as a risk factor in the occurrance of cytological atypia and hyperkeratosis in the buccal mucosa, which are frequently seen in premalignant and malignant oral lesions. Oral EC can be used for the detection of cytological atypia that is associated with oral precancerous and cancerous lesions. Duration of exposure to Qat requires further assessment. Oral EC might be appropriate within an oral screening program, particularly for those exposed to habits of Qat chewing.

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