Figure 4.

Detection of methylated cytosine by methylation-specific polymerase chain reaction analysis of caudal type homebox transcription factor 2 CpG-island region. Tissue samples were stained with E-cadherin and caudal type homebox transcription factor 2 (Cdx2) to assist cell identification. Cells, either isolated from intestinal metaplasia, intramucosal adenocarcinoma (IMC) or submucosal invasive poorly-differentiated adenocarcinoma (SM-PDA) by laser-assisted microdissection, were subjected to bisulfite conversion and subsequent methylation-specific polymerase chain reaction (MSP). MSP products using primers that specifically amplify only unmethylated DNA are indicated by visible polymerase chain reaction products in line unmethylated pattern (U), while visible polymerase chain reaction products in line methylated pattern (M) indicate those amplified by primers specific for methylated DNA. In intestinal-type metaplasia sections, MSP shows a U pattern, while MSP shows U patterns with partial M patterns in high-grade dysplasia or IMC sections. MSP shows a mostly M pattern in PDA where strong Cdx2 expression is observed (Figure 3D).