Cancer Prevention Research Promoter Methylation in Cytology Specimens as an Early Detection Marker for Esophageal Squamous Dysplasia and Early Esophageal Squamous Cell Carcinoma

The incidence of esophageal squamous cell carcinoma (ESCC) is very high in northern China. This cancer has a very poor prognosis, mostly because it is usually diagnosed at a late stage. Detection at an earlier stage can dramatically improve prognosis. Microscopic evaluation of esophageal balloon cytology (EBC) specimens has been the most common method for early detection of ESCC, but this technique is limited by low sensitivity and specificity. The use of molecular markers may improve these screening characteristics. This study evaluates whether measurement of gene methylation in EBC specimens may have utility for the detection of esophageal squamous dysplasia and early ESCC. We evaluated the presence of methylation in eight genes shown to be methylated in ESCC in previous studies in EBC specimens from 147 patients with endoscopic biopsy diagnoses ranging from normal mucosa to severe squamous dysplasia. Methylation status was determined using quantitative methylation-specific PCR techniques. The sensitivity and specificity of methylation of each individual gene and of combinations of these genes to detect biopsyproven high-grade (moderate or severe) squamous dysplasia were determined. For individual genes, the sensitivities ranged from 9% to 34% and the specificities ranged from 77% to 99%. Using a panel of four genes (AHRR, p16INK4a, MT1G, and CLDN3) resulted in sensitivity and specificity of 50% and 68%, respectively. This study suggests that evaluation of gene methylation in EBC samples may have utility for early detection of esophageal squamous dysplasia and early ESCC; however, identification of more sensitive methylation markers will be required for development of a clinically useful screening test. Esophageal cancer is the eighth most commonly occurring cancer and the sixth most common cause of cancer death in the world (1). There is wide geographic variation in the incidence of esophageal cancer, with the highest rates occurring in China, South-Central Asia, and Eastern and Southern Africa (1). In these high-risk areas, nearly all cases of esophageal cancer are esophageal squamous cell carcinoma (ESCC). The prognosis for esophageal cancer is poor, with 5-year relative survival rates of ∼10% (2). The main reason for this poor prognosis is that most early cases are asymptomatic, so ESCC is usually diagnosed only at a late stage when the cancer has spread beyond the esophagus and is unresectable. Squamous dysplasia is the precursor lesion of ESCC, and subjects with moderate or severe dysplasia are at high risk (10-fold and 28-fold risk, respectively, compared with subjects with normal mucosa) for developing ESCC (3). When detected, squamous dysplasia and early ESCC are treatable by endoscopic techniques or esophagectomy, and survival can be improved dramatically (4). This highlights the necessity of developing a clinically useful early detection test for squamous dysplasia and early ESCC. Among the techniques currently used for ESCC screening in high-risk regions is esophageal balloon cytology (EBC; ref. 5). In this technique, a deflated mesh-covered balloon is swallowed into the stomach, inflated, and then withdrawn, collecting esophageal mucosal cells, which are stained and read visually like a Pap smear by a cytopathologist. The sensitivity of balloon cytology for detecting squamous dysplasia has been shown to be at ∼50% (5–7), which is insufficient to be effective for population screening. Molecular markers may be able to improve the sensitivity and specificity of this method. The main problems associated with cytologic screening of the esophagus seem to be sampling error in cell collection and in cell review and insensitivity for finding rare abnormal cells among the cells that are reviewed. Blind balloon Authors' Affiliations: Genetics Branch, Center for Cancer Research and Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland; Department of Cancer Epidemiology, Department of Endoscopy, and Department of Pathology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China Received 03/28/2008; revised 05/20/2008; accepted 06/04/2008. Requests for reprints: Mark J. Roth, Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, 6120 Executive Boulevard, EPS/320 MSC 7232, Rockville, MD 20852. Phone: 301-402-8276; Fax: 301-435-8645; E-mail: [email protected]. ©2008 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0061 357 Cancer Prev Res 2008;1(5) October 2008 www.aacrjournals.org Cancer Research. on April 18, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from sampling may miss small mucosal lesions, and even if neoplastic cells from these lesions are successfully retrieved, they are still rare cells in a much larger population of normal cells, and thus they may not appear on the slides that are reviewed or they may be misinterpreted by the slide reader. The evaluation of molecular markers, such as gene methylation, cannot help cell collection, but it may be able to improve the other steps in this screening procedure. Gene methylation refers to abnormal methylation of CpG islands in the promoter regions of genes that are normally unmethylated, and this abnormal methylation results in the silencing of gene expression. DNA methylation assays have been shown to be highly sensitive (they can detect as few as 20 copies of methylated DNA among a large number of normal unmethylated copies; ref. 8). In addition, hypermethylation of some genes has been shown to occur early in esophageal squamous dysplasia and is common in ESCC (observed in up to 80% of cases in some series; refs. 9–11). In this study, we measured the prevalence of methylation in selected genes in balloon cytology specimens collected in a screening study conducted in Linxian, China, a county with some of the highest rates of ESCC in the world (12). The purpose of this study was to evaluate whether the presence of methylation in these genes in EBC samples could identify individuals with high-grade (moderate or severe) esophageal squamous dysplasia, who should be referred for endoscopic examination. Materials and Methods