Background: Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluatedEGFRmutation profile as well as the presence of mutations in hotspots ofKRASandBRAFin ESCC patients.Methods: We performed RT-qPCR, immunohistochemistry and Fluorescentin situhybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis.Results: Our results showed an increasedEGFRmRNA expression in tumors compared to surrounding tissue (p T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations ofEGFR,KRASandBRAFas well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC.Conclusion: HER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC.

Esophageal squamous cell carcinoma (ESCC) is one of the 10 most incident cancer types in the world, and it is mainly associated with tobacco and alcohol consumption. ESCC mortality rates stand very close to its incidence, which is a direct consequence of a late diagnosis and an inefficient treatment. Although this scenery is quite alarming, the major molecular alterations that drive this carcinogenesis process remain unclear. We have previously shown through the first ESCC methylome analysis that TFF1 promoter is frequently hypermethylated in ESCC. Here, to evaluate TFF1 methylation as a potential biomarker of early ESCC diagnosis, we investigated the status of TFF1 promoter methylation and its expression in ESSC and histologically normal tumor surrounding tissue of ESCC patients in comparison to healthy esophagus of non-cancer individuals. Analysis of TFF1 promoter methylation, and gene and protein expression in 65 ESCC patients and 88 controls revealed that TFF1 methylation levels were already increased in histologically normal tumor surrounding tissue of ESCC patients when compared to healthy esophagus of non-cancer individuals. This increase in DNA methylation was followed by the reduction of TFF1 mRNA expression. Interestingly, TFF1 expression was capable of distinguishing tumor surrounding normal tissue from normal mucosa of healthy individuals with 92% accuracy. In addition, TFF1 protein was undetectable both in tumor and surrounding mucosa by immunohistochemistry, while submucosa glands of the healthy esophagus showed positive staining. Furthermore, treatment of TE-1 and TE-13 ESCC cell lines with decitabine led to a reduction of promoter methylation and consequent upregulation of TFF1 gene and protein expression. Finally, using TCGA data we showed that TFF1 loss is observed in ESCC, but not in esophageal adenocarcinoma, highlighting the different molecular mechanisms involved in the development of each histological subtype of esophageal cancer. This study shows that TFF1 expression is silenced in early phases of ESCC development, which seems to be mediated at least in part by promoter hypermethylation, and provides the basis for the use of TFF1 expression as a potential biomarker for early ESCC detection.