Objectives: Esophageal cancer is the sixth leading cause of cancer death worldwide. The prognosis for esophageal cancer is dismal because most patients are diagnosed at a late stage. Patients can be successfully treated if diagnosed early, but current early detection screening tests are inadequate. Balloon cytology is a simple and inexpensive method of retrieving esophageal cells, but traditional cytologic examination of these cells has poor sensitivity and specificity for detecting precursor lesions. Thus, a biomarker such as DNA hypomethylation, which predisposes cells to chromosomal defects and genetic instability, may be used to improve the early detection of precursor lesions. In this study, we examined whether overall methylation levels of transposable elements Alu and long-interspersed nuclear elements (LINE-1) in the DNA extracted from balloon cytology-collected esophageal cells are associated with precursor lesions of esophageal squamous cell carcinoma (ESCC).
Methods: The Cytology Sampling Study 2, a population-based early detection screening study of ESCC, recruited over 700 healthy subjects from a high-risk population in Linxian, China. The screening study used a mesh-covered balloon to collect esophageal cells. All subjects underwent endoscopy and Lugol's iodine, and precursor lesions were diagnosed by biopsy. Using a nested case-control study design, we examined the association between Alu and LINE-1 methylation levels and esophageal health in 50 esophagitis cases, 38 low-grade dysplasia cases, 55 high-grade dysplasia cases, and 94 matched normal controls. DNA methylation assessment of Alu and LINE-1 repetitive elements was conducted using quantitative PCR pyrosequencing on bisulphate-treated DNA extracted from cells collected by balloon cytology. Receiver operating characteristic (ROC) curves were plotted, and the area under the ROC curve (AUC) was calculated for methylation levels as a diagnostic marker for high-grade dysplasia. Logistic regression models adjusted for age, sex, smoking tobacco, alcohol intake, and hypertension were used to examine the association between Alu and LINE-1 methylation levels and risk of high-grade dysplasia compared with the other diagnoses.
Results: Median Alu methylation levels were comparable among the esophagitis cases (24.5%), low-grade dysplasia cases (24.6%), high-grade dysplasia cases (24.6%), and normal controls (24.6%). Median LINE-1 methylation levels were also comparable among the esophagitis cases (71.8%), low-grade dysplasia cases (72.2%), high-grade dysplasia cases (71.6%), and controls (72.2%). The AUCs for using Alu and LINE-1 methylation levels as a diagnostic marker for high-grade dysplasia were 0.62 and 0.59, respectively. Overall, Alu and LINE-1 methylation levels were not associated with risk of high-grade dysplasia, with odds ratios (95% confidence interval) of 1.1 (0.9-1.4) and 1.1 (0.9-1.3), respectively.
Conclusions: Given that esophageal cancers have very poor prognoses, it is of utmost importance to improve early detection methods. In this study, esophageal cell DNA methylation levels of Alu and LINE-1 were not associated with esophageal precursor lesions, and thus do not appear useful as an early detection marker. Future early detection studies may examine DNA hypomethylation in general or loci-specific methylation levels.
Citation Format: Shih-Wen Lin, Christian C. Abnet, Neal D. Freedman, Lee Moore, Liying Yan, Ann Meyer, Qin-Jing Pan, Mark Roth, Guo-Qing Wang, Wen-Qiang Wei, Ning Lu, Philip R. Taylor, You-Lin Qiao, Sanford M. Dawsey. Measuring Alu and LINE-1 methylation for the early detection of precursor lesions of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C25.