Validation of esophageal squamous cell carcinoma candidate genes from high-throughput transcriptomic studies

In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.