Cancer Prevention Research Screening for Oral Precancer with Noninvasive Genetic Cytology Jantine F. Bremmer, 1,4 A. Peggy Graveland, 2 Arjen Brink, 1,2 Boudewijn J.M. Braakhuis, 2 Dirk J. Kuik, 3 C. René Leemans, 2 Elisabeth Bloemena, 1,4 Isaäc van der Waal, 1,4 and Ruud H. Brakenhoff 2 Abstract Oral squamous cell carcinomas develop in precancerous fields consisting of genetically altered mucosal epithelial cells. These precancerous fields may appear as clinically visible lesions, in particular, oral leukoplakia, but the large majority remains clinically undetectable. Theaimofthisstudywastoassessthepotentialvalueofanoninvasivescreeningapproach to detect precancerous fields. As a first step, we developed a suitable assay and investigat- ed25leukoplakiapatientsand20noncancercontrolsubjects.Exfoliatedcellswereremoved by a brush from multiple small areas of the oral mucosa, including the leukoplakia. Brushed samples were investigated for allelic imbalance (AI) at chromosomes 3p, 9p, 11q, and 17p using microsatellite markers known to show frequent alterations in oral precancer. AI was absent in all (137) of the samples of the 20 control subjects, yielding a specificity of 100%.AIwasdetectedinexfoliatedcellsamplesof40%(10of25)oftheleukoplakialesions studied. Genetic changes were also found outside the leukoplakia lesions. Most frequent was AI at 9p (9 of 10). The noninvasive assay was validated against the biopsy results of the leukoplakia lesions yielding an estimate of sensitivity of 78% (7 of 9) and a positive predictive value of 100% (7 of 7). Altogether, these results show the feasibility of a nonin- vasive genetic screening approach for the detection and monitoring of oral precancer. This assay could therefore contribute to the secondary prevention of oral squamous cell carci- noma.Theassayalsoshowspromiseforthedetectionofprecancerouschangesthatarenot macroscopically visible. Early diagnosis of oral squamous cell carcinoma may have a major effect on survival and quality of life. It is well-known that the majority of oral squamous cell carcinomas, if not all, develop in precancerous fields characterized by specific genet- ic alterations (1–3). Clinically, oral precancerous lesions may appear as a white or red lesion (leukoplakia or erythroplakia, respectively). The malignant potential of these lesions is as- sessed by histopathology and mainly based on the presence and the degree of dysplasia in biopsy material, graded as mild, moderate, and severe (4). As histology is still the gold standard, microscopic examination of mucosal biopsies might, in theory, be exploited for early diagnosis of precancerous fields even when these are not visible. However, histopatho- logic grading has limited value to predict the malignant po- tential in individual cases (5). In addition, histopathologic grading requires taking a biopsy, and to monitor the progres- sion of a lesion, repeated biopsies need to be taken, which is a large burden for the patient. Furthermore, histopathologic grading may largely depend on the precise location of the bi- opsy, given the heterogeneity of some lesions. Finally, to iden- tify precancerous fields that are not visible, more or less random biopsies need to be taken, which is too invasive as a screening approach. Hence, screening and monitoring oral precancer by histopathologic examination of tissue biopsies does not seem to be feasible, except for the visible lesions. Notwithstanding, a noninvasive genetic screening assay might be of large value for populations at high risk for devel- oping oral cancer such as treated oral cancer patients, leuko- plakia patients, genetically predisposed subjects such as Fanconi anemia patients, and individuals frequently exposed to environmental carcinogens. Oral cancers are frequently sur- rounded by nonvisible precancerous changes in the oral mu- cosa that are often not completely resected causing secondary tumors. Detection and monitoring of such nonvisible precan- cerous fields by histology would require multiple biopsies sur- rounding the treated area, and a noninvasive screening tool would be a much more attractive alternative. Such an assay would also be of relevance for leukoplakia patients, as it has been shown that these patients can develop oral squamous cell carcinomas outside the visible lesion (6). Therefore, it seems important to screen and monitor leukoplakia patients not only for precancerous changes in the visible lesion(s), but throughout the whole oral cavity. Authors' Affiliations: Departments of 1 Oral and Maxillofacial Surgery and Oral Pathology, 2 Otolaryngology/Head-Neck Surgery, and 3 Clinical Epidemiology and Biostatistics, VU University Medical Center, and 4 ACTA, Amsterdam, the Netherlands Received 07/01/2008; revised 09/24/2008; accepted 12/02/2008; published OnlineFirst 01/27/2009. Requests for reprints: Ruud H. Brakenhoff, Department of Otolaryngology/ Head-Neck Surgery, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-2044-40953; Fax: 31-2044-43688; E-mail: rh.brakenhoff@vumc.nl. ©2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0128 Cancer Prevention Research 128 Cancer Prev Res 2009;2(2) February 2009 www.aacrjournals.org Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/2/2/128/2336168/128.pdf by guest on 13 June 2022