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British Journal of Oral and Maxillofacial Surgery 48 (2010) 503506

Clinicopathological behaviour of multiple oral dysplastic lesions compared with that of single lesions
O. Hamadah, M.L. Goodson , P.J. Thomson
Oral & MaxilloFacial Surgery, School of Dental Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4BW, United Kingdom Accepted 24 August 2009 Available online 2 October 2009

Abstract Oral precancerous lesions may be solitary or multifocal, the latter being difcult to manage because of extensive eld change. The aim of this study was to characterise differences in clinicopathological features, proliferative labelling indexes for cyclin A, cyclin B1, and Ki67, and clinical outcome 5 years after laser resection in a group of patients presenting with single and multiple oral precancerous lesions. Ninety-six patients with 132 lesions (78 single and 18 multiple) were recruited, and there were no signicant differences between those with single and multiple lesions with respect to age, sex, smoking, or alcohol consumption, although multiple lesions were signicantly more common in smokers who ate little fruit and vegetables (p = 0.02). Clinically, most lesions were leukoplakia, with ulcerated or exophytic lesions appearing singly. There were signicant differences in site, single lesions being most common on the oor of the mouth and the ventrolateral tongue, and multiple lesions preferring the buccal mucosa (p = 0.0002). The most severe dysplasia was seen in single lesions (p = 0.001) with labelling indexes for cyclin A and Ki67 being signicantly higher in these (p = 0.04 and p = 0.01, respectively). Oral squamous cell carcinoma developed in 3/78 single lesions and 4/18 multiple ones. There are distinct differences between single and multiple lesions that have implications for the prophylaxis and management of oral cancer. 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Precancer; Leukoplakia; Single; Multiple

Introduction Dysplastic lesions of the oral mucosa include leukoplakia, erythroplakia, and erythroleukoplakia, and are known to be potentially malignant. The importance of such lesions is their unpredictable rate of malignant transformation, which varies from 0.336%.13 Though clinical appearance, such as non-homogeneous leukoplakia or erythroplakia,4 and anatomical site (notably the oor of the mouth and the ventral tongue) are useful in identifying lesions at high risk of transformation,5 there are no reliable ways of predicting the behaviour of individual lesions or guiding clinical management.

Corresponding author. Tel.: +44 0191 222 8290; fax: +44 0191 222 6137. E-mail address: peter.thomson@ncl.ac.uk (M.L. Goodson).

A particularly difcult group of patients to manage are those with multiple oral precancerous lesions in whom extensive areas of mucosa may show signs of dysplastic change. Modern concepts of carcinogenesis that have evolved from the work of Slaughter et al.,6 have emphasised the existence of molecularly altered preneoplastic elds from which multiple lesions can develop.7 Widespread leukoplakias have been shown to have higher rates of malignant transformation than more localised lesions.8 Multifocal disease has been reported to affect between 3% and 24% of patients with oral precancerous lesions. We have previously reported the use of eld mapping biopsy specimens to characterise multifocal disease histopathologically, which allows targeted laser excision of severely dysplastic areas.9 However, further research is necessary to investigate the risk factors and clinicopathological features that are specic to patients with multifocal lesions.

0266-4356/$ see front matter 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.bjoms.2009.08.027

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Table 1 Sites of oral precancerous lesions according to type (single or multiple). Site of lesion Type of lesion (No. (%)) Single 78 (78 patients) Floor of mouth Lateral tongue Ventral tongue Buccal mucosa Soft palate Buccal sulcus Pillar of fauces Commisure Gingiva Retromolar Dorsum of tongue Total 40 (51) 16 (21) 6 (8) 5 (6) 4 (5) 3 (4) 2 (3) 1 (1) 1 (1) 0 (0) 0 (0) 78 (100) Multiple 54 (18 patients) 12 (22) 7 (13) 5 (9) 15 (28) 6 (11) 3 (6) 2 (4) 2 (4) 0 (0) 1 (2) 1 (2) 54 (100) Total (132 lesions) 52 (39) 23 (17) 11 (8) 20 (15) 10 (7) 6 (5) 4 (3) 3 (2) 1 (<1) 1 (<1) 1 (<1) 132 (100)

We had three aims in this study: rst, to characterise clinicopathological features and clinical outcome for a group of patients with single and multifocal premalignant lesions; secondly, to assess the inuence of risk factors in the development of multiple rather than single premalignant lesions; and thirdly to characterise proliferative activity by quantifying expression of markers of the cell cycle cyclin A, cyclin B1, and Ki67, in patients with multiple and single lesions.

Methods After informed consent and ethics committee approval had been obtained, a group of new, previously untreated patients who presented with either single or multiple oral precancerous lesions to the maxillofacial oncology/dysplasia clinic at Newcastle General Hospital were recruited. Inclusion criteria were: no history of oral cancer, surgical treatment, or radiotherapy. Patients completed an investigator-led questionnaire covering clinical data, smoking and alcohol consumption, and diet. Dysplastic oral precancerous lesions were excised, and the margins vaporised using carbon dioxide laser by a single operator (PJT) following established criteria that have been described previously.11,12 Complete removal of each lesion was conrmed by histopathological examination postoperatively. Patients were followed up for 5 years and clinical outcome documented as: no disease, persistent disease (recurTable 2 Clinical appearance of single and multiple oral precancerous lesions. Clinical appearance Type of lesion (No. (%)) 78 Single (78 patients) Leukoplakia Erythroplakia Erythroleukoplakia Ulcer Exophytic Total 46 (59) 4 (5) 21 (27) 2 (3) 5 (6) 78 (100)

rence at same site), or further disease (presentation at a new site). Excised specimens were examined immunohistochemically and proliferative labelling indexes calculated for cyclin A, B1, and Ki67. Cyclin A is expressed during S phase, cyclin B1 aids cell movement to and from mitosis, and Ki67 (identied throughout the cell cycle) is an overall marker of proliferative activity. By combining these labels, we hoped to construct comprehensive proliferative proles for single and multiple oral precancerous lesions. Immunohistochemical detection of cyclin A, B1, and Ki67 was done on parafn-embedded tissue cut into 4 m sections, using a standard avidinbiotinperoxidase technique as described previously.10 Labelling indexes were quantied using computer-assisted microscopy that superimposed computer images on to the microscope (Zeiss Axiohome TM interactive microscope). Five representative elds of view were dened for each sample and labelling indexes for cyclin A, B1, and Ki67 calculated by counting the number of cells that displayed immunoreactivity among 1000 cells.10 The signicance of differences between single and multiple lesions for both clinicopathological features and risk factors were assessed with the help of the Statistical Package for the Social Sciences (SPSS Inc., Chicago) using the chi-square test. Differences between labelling indexes in the two groups were assessed using the MannWhitney U-test. Two-way analysis of variance (ANOVA) was used to assess the impact of the type of lesion (single or multiple) and his-

54 Multiple (18 patients) 35 (65) 7 (13) 12 (22) 0 0 54 (100)

Total (n = 132) 81 (61) 11 (8) 33 (25) 2(2) 5 (4) 132 (100)

O. Hamadah et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 503506 Table 3 Clinical appearance of oral dysplastic lesions according to type of lesion. Type of lesion Single Multiple Total Mild dysplasia (No. (%)) 3 (4) 18 (96) 21 (100) Moderate dysplasia (No. (%)) 16 (47) 18 (53) 34 (100)

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Severe dysplasia (No. (%)) 21 (84) 4 (16) 25 (100)

tological diagnosis (mild, moderate, or severe dysplasia) on cyclin A, B1, and Ki67.

Results Ninety-six patients were recruited, 78 with single and 18 with multiple oral precancerous lesions. Overall, 132 lesions were excised and analysed. Patients ages ranged from 27 to 91 years (mean 59), with no signicant differences between age, sex, or whether lesions were multiple or single. Histologically conrmed complete removal showed no dysplasia or oral squamous cell carcinoma (SCC) at the excision margins. The results are shown in Tables 14. There were no signicant differences in age or sex. Seventy per cent of patients were smokers at the time of presentation, but there was no association between smoking and the development of either single or multiple lesions (p = 0.26). Three-quarters of patients reported regular alcohol consumption, but there was no association between the developments of single or multiple lesions and the number of units consumed (p = 0.09). Mean alcohol consumption was 27 units/week for patients with single lesions, and 17 for multiple lesions. Seven patients in the study were vegetarians, and 40 ate at least three portions of fresh vegetables or fruit/day. After controlling for smoking, multifocal lesions were most commonly associated with a low intake of fruit and vegetables of less than 3 portions/day (p = 0.01). Proliferative labelling indexes for single lesions were higher than for multiple lesions for all three labels, with cyclin A (p = 0.04) and Ki67 (p = 0.01) being signicantly so (Table 4). Patients were followed up for 5 years. Fifty of 78 (64%) who had presented with single lesions remained disease-free after excision, whereas 25 (32%) developed either persistent disease (recurrence at the same site) or further disease (growth at a new site). These were conrmed histopathologically and further treatment arranged. Follow-up data

for patients with multiple lesions were difcult to classify because of the widespread nature of their mucosal lesions. Seven patients developed oral SCC during follow-up, 3 of the 78 with single lesions and 4 of the18 with multiple lesions. In 6 patients, oral SCC arose at sites distant from their presenting lesions. Differences in malignant transformation between patients with single and multiple lesions was significant (p = 0.02), although development of SCC did not seem to be related to their histopathological diagnosis or labelling indexes. All patients achieved 5-year survival.

Discussion The management of oral precancerous lesions remains controversial, with no guidelines available and a lack of clinically relevant, randomised, controlled trials. We have, however, shown the effectiveness of interventional laser surgery both as a diagnostic and therapeutic tool.11,12 Field change indicating cancer is a serious problem in clinical management in a small, but important, group of patients. Widespread histological changes in the oral mucosa, accompanied by unpredictable and high rates of malignant transformation, compound the difculties. It is clear from our results that there are considerable differences in clinical and pathological variables between single and multiple lesions.9 Leukoplakia remains the commonest clinical appearance for both types, and is the most common presentation of panoral disease. Erythroleukoplakia and ulcerative lesions presented exclusively as single lesions. The presence of large numbers of buccal lesions in multiple disease may reect widespread mucosal instability, although this did not seem to be related to risk factors. There were no differences in age, sex, smoking, and alcohol consumption between the groups with single and multiple lesions. While this may not be surprising given the high incidence of alcohol and tobacco use in the North East of England, it was interesting to note the histopathological differences and varied rates of malignant transformation

Table 4 Mean (SD) cyclin A, cyclin B1, and Ki67 labelling indexes for single and multiple lesions. Labelling index Cyclin A Single Total Basal Suprabasal 13.6 (5.9) 17.3 (9.3) 12.4 (6.4) Multiple 11.3 (4.5) 15.6 (10.2) 10.1 (3.9) Cyclin B1 Single 12.3 (5.7) 16.7 (8.5) 11.2 (6.0) Multiple 10.6 (6.5) 15.8 (11.8) 9.4 (5.7) Ki67 Single 25.4 (10.5) 24.6 (15.9) 25.3 (11.1) Multiple 20.7 (8.6) 26.5 (18.2) 19.1 (8.3)

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between the two groups, which suggests inherent molecular or metabolic differences between the patients with single and multiple lesions. The low intake of fruit and vegetables in smokers with multiple lesions may reect a true difference in dietary habits, but the signicance should be interpreted with caution given the small number of patients with multiple lesions. Severe dysplasia was found mainly in single lesions, while multiple lesions were mainly mildly dysplastic. It is important to identify severe dysplasias in multiple lesions as these are the likely focus of the development of oral SCC. Field mapping techniques provide a pragmatic approach to identication and elimination of these foci.9 To our knowledge this is the rst study to compare immunohistochemical proliferative analyses of single and multiple oral precancerous lesions. The labelling indexes of single lesions were higher than those with multiple lesions, particularly for the higher indexes seen with cyclin A and Ki67. Two-way ANOVA suggested that the differences between single and multiple labelling indexes were related to the grading of dysplasia, with more severe dysplasias having increased proliferative activity. Oral SCC developed in 3 patients with single, and 4 with multiple, lesions. This is despite a higher incidence of severe dysplasias in single lesion disease. However, as single lesions are easier to identify and excise completely, this may explain the lower transformation rate. A higher incidence of carcinoma in multiple lesions probably reects more widespread mucosal instability, so that excision of large areas of mucosa becomes impracticable. Regular and careful clinic review of patients with these lesions remains essential so that we can detect dysplastic disease or oral SCC at the earliest possible stage. There remains a need for objective and reproducible clinical assessment tools to quantify mucosal instability. We have shown that there are distinct clinicopathological differences between patients with single and multiple oral precancerous lesions. Future research should study this in more detail. Multicentre, randomised, controlled longitudinal studies to compare interventional surgery with systemic chemoprophylaxis are now urgently needed, particularly in patients with widespread, multifocal disease.

Acknowledgements We thank colleagues in the Pathology Department and Medical Physics at the Royal Victoria Inrmary, Newcastle upon Tyne, and Epistem Ltd., Incubator Building, University of Manchester, without whom this study would not have been possible.

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