Evaluation of Mirror Image Biopsy for Incidence of Multiple Premalignant and Malignant Lesions in Oral Cancer: A Clinical Study

10.5005/jp-journals-10011-1294 Manjula A Hebbale et al ORIGINAL ARTICLE Evaluation of Mirror Image Biopsy for Incidence of Multiple Premalignant and Malignant Lesions in Oral Cancer: A Clinical Study Manjula A Hebbale, R Krishnappa, Anjana Bagewadi, Vaishali Keluskar, Alka Kale, Rajshekhar Halli ABSTRACT Introduction: To histologically evaluate the mirror image biopsy obtained from the apparently normal looking mucosa at corresponding contralateral anatomical sites to determine the incidence of multiple premalignant and malignant lesions in oral cancer. Materials and methods: Fifty patients with unilateral, single, untreated, histologically confirmed oral squamous cell carcinoma underwent mirror image biopsy from apparently normal looking mucosa at corresponding contralateral anatomical site. The mirror image biopsy specimens were subjected to histopathological examination. Results: The age group of the patients ranged from 42 to 70 years with male to female ratio of 1.2:1. Of 50 patients, 38 (76%) revealed abnormal epithelial changes ranging in the degree from hyperkeratosis (12%), dysplasia (48%) and premalignant changes like leukoplakia, erythroplakia, submucous fibrosis, etc. (16%) in the mirror image sites. The incidence was high in males, with increasing age of the patient and duration of exposure to habits. Conclusion: Mirror image biopsies should be considered as useful diagnostic tool for early detection of premalignant changes so as to prevent further progression of these changes to oral cancer. Keywords: Mirror image biopsy, Multiple primary cancers, Multicentric neoplasia, Field cancerization, Squamous cell carcinoma, Aerodigestive tract. How to cite this article: Hebbale MA, Krishnappa R, Bagewadi A, Keluskar V, Kale A, Halli R. Evaluation of Mirror Image Biopsy for Incidence of Multiple Premalignant and Malignant Lesions in Oral Cancer: A Clinical Study. J Indian Aca Oral Med Radiol 2012;24(3):194-199. Source of support: Nil Conflict of interest: None declared INTRODUCTION Oral cancer is often poorly understood by the society in general and because of the asymptomatic nature, is frequently ignored in its early stages, which has led to the increasing incidence worldwide. Oral cancer is thought to arise from a series of stepwise genetic changes induced by various carcinogens. 1 Theodor Billroth was among the first one to recognize that a single patient can develop simultaneous independent cancers, following which numerous studies documenting multiple primary cancers have appeared in the literature. 2 Only in recent years, because of improvement in early 194 detection at the initial examination, relatively high frequency and clinical significance of multicentric neoplasia has been recognized. 3 The upper aerodigestive tract including the oral cavity is thought to be a preferential site for the occurrence of multiple primary cancers. 4-6 The etiology of multiple primary cancers involving a single organ system appears to be related to the synergistic effects of multiple carcinogens acting on a field of growth resulting in field cancerization. 7 Field cancerization is a concept which suggests that the entire epithelial surface exposed to repeated carcinogenic insults is at risk and that such exposure increases the likelihood of development of multiple, independent premalignant lesions and malignant tumors. 1 With this concept in 1953, Slaughter et al opened the door for modern research into field cancerization. The complete clinical significance of multiple primary neoplasms in the aerodigestive tract is only recently being appreciated. Recognition of such simultaneously presenting cancers profoundly affects the treatment strategy. Modern theories of carcinogenesis suggest that premalignant change may occur in any area of mucous membrane exposed to a carcinogen. Hence, patients with oral cancer are at risk of developing second or multiple primary cancers within the upper aerodigestive tract. 1,8 The reasons for this increased incidence are multifold and the most important fact is that a greater awareness of the phenomenon, which has stimulated a more extensive search for these lesions. The present study was carried out to histologically evaluate the mirror image biopsy obtained from the apparently normal-looking mucosa at corresponding contralateral anatomical sites in patients with unilateral, single, untreated, histologically proven oral squamous cell carcinoma (SCC) to know the incidence of occurrence of multiple premalignant and malignant lesions. MATERIALS AND METHODS The present study included 50 patients with unilateral, single, untreated, histologically confirmed oral squamous cell carcinoma. Exclusion criteria being patients with extensive midline or bilateral tumors, those with metastatic disease, a previously treated oral cancer or patients with JAYPEE

JIAOMR Evaluation of Mirror Image Biopsy for Incidence of Multiple Premalignant and Malignant Lesions in Oral Cancer multiple oral lesions. Ethical committee approval for the study was obtained. With informed consent these patients were subjected for biopsies at two sites. One from the carcinomatous lesion (primary tumor) and other from apparently normal looking mucosa at corresponding contralateral anatomical site (mirror Image site; Figs 1 to 3). Both the biopsy samples were subjected for histopathological examination. Both the specimens were stained with hematoxylin and eosin (H&E). The oral squamous cell carcinomas were graded histologically into well, moderately and poorly differentiated tumors. The mirror image tissue was analyzed histologically for the presence of any abnormal features, ranging from reactive changes to tissue proliferation, hyperkeratosis, cellular atypia, dysplasia, and premalignancy or frank malignancy using standardized pathological criteria. The WHO (1978) definition for oral premalignant lesion a morphologically altered tissue in which cancer is more likely to occur in its apparently normal counterpart was used to determine the premalignancy state of the biopsy specimen. The sum total of physical (visible changes at the cellular level atypia) and morphological alterations are of diagnostic and prognostic relevance and were designated as precancerous changes. The diagnosis of precancer is primarily based on morphology and its grading on histology which is widely practiced to assess the risk of malignant potential of such lesions. Because of inherent discrepancy such lesions may well be designated as potentially malignant. The following statistical analysis was used for the present study: χ 2 2 = (O E) / E O = Observed value, E = Expected value Chi-square test was used to test the significant difference of prevalence rates of different groups (p-value). Fig. 1: Sample case: Extraoral view Fig. 2: Primary tumor site Fig. 3: Mirror image site RESULTS AND OBSERVATIONS The study group consisted of 50 unilateral, single, histologically confirmed oral SCC patients, wherein 54% were males and 46% were females, with male to female ratio being 1.2:1 (Table 1). All the patients had habits of some kind ranging from tobacco chewing, smoking, pan and betel nut chewing either separately or in combination (Table 2). In our study the site of occurrence of primary oral cancer was highest in buccal mucosa (38%), followed by lower alveolus (28%), gingivobuccal sulcus (16%), retromolar trigone (6%), tongue (6%), maxillary tuberosity (4%) and least in the lower lip (2%; Table 3). A total of 79.5% of patients with more than 15 years of exposure to habits had abnormal epithelial changes as compared to 63.6% of patients with less than 15 years of exposure to habits (Table 4). The age group ranged from 42 to 70 years, with the mean age being 52.7 ± 5.08 standard deviation. In the present study, 54% of the subjects were above the age of 50 years Journal of Indian Academy of Oral Medicine and Radiology, July-September 2012;24(3):194-199 195

Manjula A Hebbale et al and 46% below the age of 50 years which shows that oral cancer is a disease of increasing age (Table 5). The epithelial changes at mirror image sites were more in patients having tobacco chewing habit and followed by pan with tobacco chewing habit (Table 6). Our study revealed maximum of histological changes in the mirror image sites as premalignancy and dysplasia in the gingivobuccal sulcus (87.5%), followed by the lower alveolus (64.2%) and buccal mucosa (63.15%) and the other sites such as retromolar trigone, tongue, maxillary tuberosity, lower lip revealing a total of 25% changes (Table 7). Of the 50 primary oral SCC patients, 84% had welldifferentiated SCC whereas only 16% of patients had moderately differentiated squamous cell carcinomas with no patients having poorly differentiated squamous cell carcinoma, which strongly suggests that most of them could be treated successfully (Table 8). The present study revealed 76% abnormal epithelial changes ranging in the degree from hyperkeratosis 12%, dysplasia 48% and premalignant changes 16% in the mirror image sites (Table 1). These abnormal epithelial changes were significantly higher in males (88.9%) as compared to females (60.9%) at the mirror image sites which was statistically significant (p < 0.05). The rate of occurrence of abnormal epithelial changes in the mirror image sites increased with the advancing age, 85% of patients more than 50 years of age revealed abnormal epithelial changes, whereas only 65% of patients under the age of 50 years revealed these changes (Table 5). Only male patients (12%) in the present study revealed hyperkeratosis as abnormal epithelial changes in mirror image sites which can be attributed to the habit of smoking which is not commonly seen in Indian females. Sixteen percent of patients had histologically confirmed oral submucous fibrosis at the contralateral mirror image sites (Table 1). DISCUSSION Oral SCC arises from a series of stepwise genetic changes induced by carcinogens, which subsequently lead to clinical and microscopic changes, summating to form an invasive neoplasm. It has been observed that patients with carcinoma of the oral cavity and oropharynx often develop second malignant or multiple premalignant lesions. This finding led to the theory of field cancerization, which hypothesizes that the entire epithelial surface of the upper aerodigestive tract has an increased risk for the development of malignant and premalignant lesions because of multiple genetic abnormalities in the whole tissue exposed to the same carcinogen. 9 196 Unfortunately, neither the precise nature nor the order of genetic or structural alterations preceding malignant change are characterized at present. 1 Multifocal occurrence of SCC of the upper aerodigestive tract has been studied by many authors and they found that the rate of incidence of second primary tumor on an average was 10 to 35%. 10-12 Two theories have been postulated to explain the high frequency of second primary tumors in the upper aerodigestive tract. The first theory based on the postulation Table 1: Sex-wise distribution Sex Normal oral Hyperkeratosis OSMF Dysplasia mucosa Male (27) 3 6 5 13 Females (23) 9 3 11 Table 2: Habits in relation to sex Habits Male (27) Female (23) Only tobacco chewers (22) 11 11 Only smokers (6) 6 Tobacco chewers + smokers (6) 6 Only pan, betel nut chewers (9) 9 Pan, betel nut + tobacco chewers (7) 4 3 Pan, betel nut + smoking (0) Table 3: Site-wise distribution in relation to sex (primary tumor sites) Site Males (27) Females (23) Buccal mucosa (19) 11 8 Lower alveolus (14) 5 9 GBS (8) 4 4 RMT (3) 3 Tongue (3) 2 1 Maxillary tuberosity (2) 1 1 Lip (1) 1 GBS: Gingivobuccal sulcus; RMT: Retromolar trigone Table 4: Duration of exposure Duration Normal Hyperkeratosis OSMF Dysplasia oral mucosa 5-15 years 4 1 1 5 (group A) (11) 15-30 years 8 5 7 19 (group B) (39) Table 5: Age-wise distribution Age Normal Hyperkeratosis OSMF Dysplasia oral mucosa 50 years (23) 8 3 12 >50 years (27) 4 3 8 12 JAYPEE

JIAOMR Evaluation of Mirror Image Biopsy for Incidence of Multiple Premalignant and Malignant Lesions in Oral Cancer Table 6: Habit-wise distribution Habits Normal oral mucosa Hyperkeratosis OSMF Dysplasia Only tobacco chewers (22) 5 4 2 11 Only smoking (6) 1 1 1 3 Tobacco chewing + smoking (6) 1 1 1 3 Pan + betel nut chewing (9) 4 3 2 Pan + tobacco chewing (7) 1 1 5 Pan + smoking (0) Table 7: Site-wise distribution to epithelial changes Site Normal oral mucosa Hyperkeratosis OSMF Dysplasia Buccal mucosa (19) 4 3 2 10 Lower alveolus (14) 4 1 2 7 Gingivobuccal sulcus (8) 1 3 4 Retromolar trigone (3) 1 1 1 Tongue (3) 1 1 1 Maxillary tuberosity (2) 1 1 Lip (1) 1 Table 8: Histological grading of oral squamous cell carcinoma (primary tumor sites) Histological grading of Male Female Total squamous cell carcinoma Well differentiated 22 20 42 Moderately differentiated 5 3 8 Poorly differentiated of micrometastatic spread which says that micrometastatic spread of premalignant and malignant cells give rise to genetically related second primary tumors. 13 The second theory proposed by Slaughter et al 14 is based on the concept of field cancerization, according to which oral carcinomas, including second primary originate by a process, in which, the whole area of epithelium has been preconditioned by long-term exposure to various carcinogens. Our present study was based on this particular theory of field cancerization. Basically it should be kept in mind that head and neck cancer is not a regional mucosal disease, but is rather a panmucosal disease of the aerodigestive tract. Therefore, the likelihood of developing second primary lesions of the endothelium lining the tract is relatively high. 11 Epstein et al reported that the presence of malignant neoplasms in the upper aerodigestive tract implies an enhanced risk for occurrence of another malignancy in the same anatomic region. 15-18 Ever since the hypothesis of field cancerization in oral SCC by Slaughter et al 14 has been laid, a number of studies have been reported in the literature in this regard. Currently, it has been suggested that premalignant changes may occur, in any area of the mucous membrane exposed to the carcinogens. 1 According to the multifocal theory of neoplasia, there are many agents (e.g. tobacco, alcohol, radiation), which can initiate cancer development. Following this, there is a symptom-free period during which a slow, potentially reversible, cellular changes can transform into preneoplastic and if untreated, to frank malignant changes. 18 The aim of our present study was to quantify the incidence and the type of field change observed in the apparently normal-looking oral mucosa of patients presenting with single primary oral SCC with the help of histological evaluation of mirror image biopsies taken from the contralateral anatomical site. Sankaranarayanan 19 noted that the male to female ratio of oral cancer in India was 2:1. Many recent studies have shown increased incidence of oral cancer in Indian females. In turn data available also shows that, the incidence rates for oral cancer in females are much higher in South India than in other parts where the ratio of male to female is 0.6:1 (South India). 20 Wright and Shear 21 suggested that oral SCC are usually surrounded by areas of leukoplakia or erythroplakia, thus it is reasonable to assume that an altered field extends peripherally from the neoplasm and can demonstrate premalignant and dysplastic features. But such features can also occur at a distant site, which is also exposed to carcinogens. Additional evidence supporting widespread change within clinically normal-looking epithelium of patients with oral cancer has been provided by exfoliative cytological studies. 22 In these studies, smears taken from normal mucosa contralateral to oral cancerous lesion or from tissue adjacent to neoplastic lesions showed reduction in normal cytoplasmic areas and increased nuclear to cytoplasmic ratios. Journal of Indian Academy of Oral Medicine and Radiology, July-September 2012;24(3):194-199 197

Manjula A Hebbale et al Oral submucous fibrosis is found to coexist in the oral cavity of 40% of oral cancer patients and is considered as precancerous. 23,24 Such findings were noted in the present study, 16% of patients had histologically confirmed oral submucous fibrosis at the contralateral mirror image sites. Pindborg reported that patients with oral submucous fibrosis in India have a higher occurrence of carcinoma than those without this disease. 25 Paymaster observed the development of slowly growing SCC in one-third of oral submucous fibrosis patients. 26 Thomson 1 demonstrated that apparently normal-looking mucosa from oral cancer patients may exhibit histological signs of chronic mucosal irritation, dysplasia or premalignancy. The tongue and the floor of mouth showed an increased vulnerability to dysplastic changes in his study. Our study revealed maximum of histological changes of premalignancy and dysplasia in the gingivobuccal sulcus (87.5%), followed by the lower alveolus (64.2%) and buccal mucosa (63.15%) and the other sites such as retromolar trigone, tongue, maxillary tuberosity, lower lip revealing a total of 25% changes. However, this was not statistically significant, but consistent with the study of De Vries et al. 27 Lund 28 and Wynder et al 17 reported that significantly higher number of patients with a primary oral cancer died of a second primary tumor. The second primary tumors, therefore, represent not only a difficult challenge to our understanding of oral carcinogenesis but also, an important clinical problem heavily influencing the survival rate and the quality of the remaining life. It is, therefore, necessary to find both a clinical tool and a reliable histopathological technique to identify high-risk subjects for development of second primary tumor. 10 Routine multiple oral biopsy sampling and cytologic smears, as suggested by few authors, cannot be justified on the basis of practicality nor are they cost-effective. 11,22 Thus, advice on primary prevention by avoidance of risk factors, screening for detection of early lesions by careful clinical examination, should be stressed upon to prevent the occurrence of second primary carcinomas. This will help to diagnose and treat premalignant lesions at the earliest with chemoprevention and thus can prevent the occurrence of frank second or multiple oral cancers. 29 Detailed screening at the initial examination of patients with oral SCC should be done for premalignant changes like oral submucous fibrosis as it may coexist (which was evident in the present study) with oral cancer and may go unnoticed. Thus, patients with single primary oral SCC can be subjected to mirror image biopsy in the presence of multiple risk factors like increasing age, increase in the duration of exposure to carcinogen and the male gender, so as to prevent 198 the occurrence of second primary tumor and even treat the premalignant and dysplastic changes, if present, at the earliest. Conventional oral examination and histological diagnosis are limited by their observational nature. Currently few authors are undertaking further studies of proliferative labeling indices, not only for oral squamous cell carcinomas but also for normal mucosa harvested from cancer patients with the aim of developing such a clinically predictive tool. 2 While the routine use of multiple oral biopsy sampling cannot be justified on the basis of this small sample study, large-sample size and long-term follow-up studies are now required to determine the importance of field change carcinogenesis in modern clinical management. 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