Esophageal squamous cell carcinoma is a common disease in many countries, and, because of late diagnosis, it has a very poor prognosis. Early detection of ESCC and its precursor lesion, squamous dysplasia, should improve cure rates and reduce mortality, but there is currently no accurate and affordable way to screen the large numbers of asymptomatic people who are at high risk for this disease. The most common current screening technique in high-risk populations, esophageal balloon cytology (EBC), has only a 50% sensitivity for detecting squamous dysplasia or early ESCC, and only a 5% sensitivity for detecting high-grade dysplasia or early ESCC (7
). The current study is part of an effort to identify molecular markers that may improve this sensitivity.
There are three main problems with EBC for the detection of early esophageal neoplasia. Blind balloon sampling may miss small mucosal lesions, so the sample that is collected may not include diagnostic abnormal cells (sampling error #1). Also, because visual examination of cell preparations is time-consuming, current methods for reading EBC samples evaluate only a minority of the collected cells, so rare abnormal cells may have been collected but may not be evaluated (sampling error #2). Finally, rare abnormal cells that are evaluated on the cytology preparations may be missed or misinterpreted by those reading the slides (reading error). Molecular markers, including gene methylation, cannot help incomplete cell sampling (sampling error #1), but may be able to reduce the other two sources of screening error.
In this study we evaluated promoter methylation in 8 genes in EBC specimens from 147 asymptomatic high-risk Chinese adults, and we tested the ability of this methylation to detect individuals with high-grade (moderate or severe) esophageal squamous dysplasia. For most genes, methylation was more common in the EBC samples of patients with worse disease, consistent with previous findings in esophageal tissue specimens (9
). Methylation of individual genes had sensitivities and specificities ranging from 9–34% and 77–99%, respectively, for identifying patients with high-grade squamous dysplasia. A panel of four genes (AHRR
, and MTIG
) had a sensitivity to 50% and a specificity of 65%. While these figures are no better than the sensitivity and specificity of traditional visual evaluation of EBC specimens (6
), this is only the first evaluation of balloon samples for methylation in a few candidate genes, so these figures probably represent minimum values for this technique.
Our findings of gene methylation in balloon cytology samples from subjects believed to have a normal esophagus after endoscopy with Lugol’s iodine staining is not surprising in light of previous studies that found methylation in normal esophageal mucosa from patients without cancer (16
). In addition, Guo et al (10
) evaluated methylation of eight genes in tissues with a spectrum of histologic diagnoses and found that most examples of squamous dysplasia, including low-grade dysplasia, had at least one methylated gene. This suggests that promoter methylation may be an early event in carcinogenesis. Thus, given the cross-sectional nature of our study, it is unclear if the methylation-positive samples in subjects with normal endoscopic and histologic findings identified field effects associated with occult early neoplasia or if they were non-specific findings unrelated to carcinogenesis. A prospective analysis would be required to distinguish these two possibilities.
Our study had several strengths, including using the gold-standard exam, endoscopy with Lugol’s iodine staining and biopsy, to define the disease status in all subjects. Also, we used a very sensitive and validated method to detect gene methylation in the balloon cytology samples. Our study also had some limitations, including testing only a moderate number of EBC samples and examining only a limited panel of genes. Future studies should use high-throughput methods to test larger numbers of clinical samples and genes.
In summary, this study suggests that measuring gene methylation in balloon cytology specimens may have promise as a primary screening technique for squamous dysplasia and early ESCC in high-risk regions. However, identification of more sensitive panels of methylation markers will be required, and sampling error in EBC cell collection will need to be minimized. Prospective studies evaluating multiple genes that have a high prevalence of methylation in ESCC should also shed light on the clinical usefulness of methylation markers in the early detection of ESCC.