Cancer Prevention Research Oral Epithelium as a Surrogate Tissue for Assessing Smoking-Induced Molecular Alterations in the Lungs

The lungs and oral cavity of smokers are exposed to tobacco carcinogens. We hypothesized that tobacco-induced molecular alterations in the oral epithelium are similar to those in the lungs, and thus the oral epithelium may be used as a surrogate tissue for assessing alterations in the lungs. We used methylation-specific PCR to analyze promoter methylation of the p16 and FHIT genes at baseline and 3 months after intervention in 1,774 oral and bronchial brush specimens from 127 smokers enrolled in a randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites / total sites per subject) was analyzed in a blinded fashion. At baseline, promoter methylation in bronchial tissue was present in 23% of samples for p16, 17% for FHIT, and 35% for p16 and FHIT; these percentages were comparable to methylation in oral tissue: 19% (p16), 15% (FHIT), and 31% (p16 and FHIT). Data from both oral and bronchial tissues were available for 125 individuals, in whom the two sites correlated strongly with respect to alterations (P < 0.0001 for both p16 and FHIT). At baseline, the mean bronchial methylation index was far higher in patients with oral tissue methylation (in either of the two genes; 39 patients) than in patients without oral tissue methylation (86 patients): 0.53 ± 0.29 versus 0.27 + 0.26 methylation index (P < 0.0001). Similar correlations occurred at 3 months after intervention. Our results support the potential of oral epithelium as a surrogate tissue for assessing tobacco-induced molecular damage in the lungs and thus have important implications for designing future lung cancer prevention trials and for research into the risk and early detection of lung cancer. Lung cancer is the leading cause of cancer-related death in the United States (1), with a 5-year survival rate of only 15% (2). Intensive research into the therapy and prevention of lung cancer over the past 30 years has not substantially improved control of the disease. Prevention approaches of smoking cessation and chemoprevention focus on chronic tobacco smokers, who are the highest known risk group for lung cancer. Even chronic smokers who quit the habit remain at a substantially higher-than-average lifetime risk of lung cancer, particularly if they quit at age 50 years or older (3). Chemoprevention has been limited by a lack of effective agents and by the logistics of clinical testing in this setting. The lifetime risk of chronic smokers for developing lung cancer is only 8% to 10%, strapping lung cancer chemoprevention trials with the need for thousands of trial subjects who must be treated and followed for up to 10 years to assess efficacy. The molecular revolution in cancer research contains potential solutions to both the efficacy and logistics problems of chemoprevention. Advances in the molecular understanding of multistep lung tumorigenesis are helping discover tolerable molecular-targeted preventive agents and are suggesting molecular markers with the potential to select the highest-risk chronic smokers for reducing the logistics of randomized controlled trials. We and others have identified early genetic and epigenetic alterations in tobacco-exposed lung epithelium, such as chromosomal deletions and promoter methylation of tumor suppressor genes (4–6). These alterations have been proposed as markers of lung cancer risk and as intermediate markers for assessing the effects of novel molecular-targeted chemopreventive agents in lung cancer prevention trials (7). A major limitation on the use of these markers, however, is the anatomic nature of the lungs, which limits access to bronchial epithelium, particularly in relatively healthy smokers. Bronchoscopy to obtain bronchial tissue samples is invasive and expensive and can be done only in a limited number of patients. Sputum is another source of lung tissue for molecular analysis, but few of the many cell types in sputum samples come fromairway epithelium, complicating data interpretation. Authors' Affiliations: Departments of Thoracic/Head and Neck Medical Oncology, Biostatistics and Applied Mathematics, and Pulmonary Medicine, The University of Texas M. D. Anderson Cancer Center; and Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas Received 03/20/2008; revised 03/20/2008; accepted 04/23/2008. Grant support: National Cancer Institute grants CA091844 and CA-16672. Requests for reprints: Li Mao, Department of Thoracic/Head and Neck Medical Oncology, Unit 432, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713792-6363; Fax: 713-792-1220; E-mail: [email protected]. ©2008 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0058 39 Cancer Prev Res 2008;1(1) June 2008 www.aacrjournals.org Cancer Research. on June 19, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Because the entire airway from the oral cavity to the lungs is exposed to tobacco carcinogens in smokers, we hypothesized that these carcinogens induce similar molecular alterations throughout the airway, making the oral epithelium a potential surrogate tissue for assessing tobacco-induced molecular alterations in the lungs. This surrogacy would greatly simplify screening for high lung cancer risk and early detection and would greatly facilitate repeated biomarker analyses of lung cancer chemoprevention in readily accessible oral tissue. To test our hypothesis, we compared the promoter methylation status of two important tumor-suppressor genes involved in early lung carcinogenesis, p16 and FHIT, in bronchial cells with the methylation status of these genes in oral epithelial cells obtained from chronic smokers. Materials and Methods Trial design and subjects Our study cohort came from a prospective placebo-controlled double-blind randomized chemoprevention trial conducted at The University of Texas M.D. Anderson Cancer Center among current and former smokers who had a minimum smoking history of 20 packyears. Current smokers were defined as active smokers or those who had quit smoking less than 12 mo before their registration for the clinical trial; former smokers had quit smoking longer than 12 mo before their registration. Bronchoscopic and buccal brushing were donein participants at baseline and 3 mo after treatment with either celecoxib (200 or 400 mg twice daily) or placebo. Buccal brushing was done at one site, whereas bronchial brushing was done at six predetermined sites: the main carina, the bifurcation of the right upper lobe, the right middle and lower lobes, the left upper lobe, and the anterior bronchus of the left lower lobe, as shown in Fig. 1. The samples were collected after obtaining appropriate Institutional Review Board approval of the protocol and written informed consent from the subjects. Sample processing and DNA extraction Specimens obtained from bronchoscopic and buccal brushing were placed in DMEM (Life Technologies, Inc.) in sterile tubes and stored at 4°C for processing the same day. DNA was extracted by digestion of cells with 10× proteinase K-SDS solution [5 mg/mL proteinase K (Roche Molecular Biochemicals) and 10% SDS (Life Technologies)] at 42°C overnight followed by phenol and chloroform extraction. Fig. 1. A flexible bronchoscope is inserted through the nose or mouth to examine the airways after i.v. sedation (bottom right). Bronchial brushings are taken from six predetermined sites (top right). Oral brushing using a cytologic brush is done (top left) by rubbing the inner side of the left cheek (bottom left). From Visual Art © 2008, The University of Texas, M. D. Anderson Cancer Center; with permission. Cancer Prevention Research 40 Cancer Prev Res 2008;1(1) June 2008 www.aacrjournals.org Cancer Research. on June 19, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Methylation-specific PCR At least 100 ng of sample DNA, mixed with 1 μg of salmon sperm DNA (Life Technologies), were subjected to chemical modification following the protocol of Herman et al. (8). PCR was then conducted with primers specific for either the methylated or unmethylated versions of the p16 and FHIT promoter regions (5, 9). The 12.5-μL total reaction volume contained 25 ng of modified DNA, 3% DMSO, all four deoxynucleoside triphosphates (each at 200 μmol/L), 1.5 mmol/L magnesium chloride, 0.4 μmol/L PCR primers, and 0.625 unit of HotStar Taq DNA polymerase (Qiagen). Negative controls included water to control for DNA contamination and normal tissue DNA was used as a negative control for methylation. DNA from the NCI-H460 lung cancer cell line treated with SssI methylase (New England Biolabs Inc.) was used as a positive control. PCR products were separated on 2% agarose gels and visualized after staining with ethidium bromide. Statistical analysis Methylation status was determined at baseline and 3 mo after intervention with the brush site (both oral and bronchial) and participant (with multiple bronchial brushes) as the units of analysis. When the participant was used as the unit of analysis, that individual was considered to be methylation positive when any of the bronchial brush sites showed promoter methylation. The methylation index was determined for each gene by dividing the number of bronchial brush sites exhibiting promoter methylation by the total number of sites examined in each participant. Statistical analysis was done using χ test or Fisher's exact test for correlation among multiple genes and between methylated gene status and sex. Wilcoxon's rank-sum test was used for testing differences between methylated and unmethylated groups in median age, packs per day, years of smoking, pack-years, and years since having quit. Spearman's rank correlation was applied to estimate the association between smoking pack-years and methylation index. Kruskal-Wallis test was used to compare bronchial brush methylation index modulation among oral brush methylation modulation groups (10). All P values were determined by two-sided tests, and P ≤ 0.05 was considered statistically significant.