ANPEP/CD13 Expression as a Marker of Lymphovascular Invasion and Survival in Esophageal Adenocarcinoma

Background: The presence of lymphovascular invasion (LVI) in early esophageal adenocarcinoma (EAC) is associated with more aggressive disease. Molecular markers associated with LVI are still largely unknown. Using a combination of transcriptomic analysis and validation experiments, we sought to describe markers for LVI and survival. Methods: We performed NanoString expression profiling using RNA from 60 EAC specimens collected from surgery-only cases between 2000 and 2012. Differentially expressed genes (DEGs) were correlated with pathologic characteristics (T and N status and presence of LVI). Kaplan-Meier and Cox regression analyses were used to correlate gene expression with overall survival. Expression of alanyl aminopeptidase, membrane (ANPEP)/CD13 was validated by immunohistochemistry (IHC) in EAC tissue microarray and in EAC cell lines. Results: We identified >20 up-regulated DEGs in tumor samples containing LVI. Multivariable analysis showed depth of invasion and ANPEP/CD13 expression were independently associated with overall survival, whereas nodal status was not. IHC analysis demonstrated overexpression of the ANPEP/CD13 protein in dysplastic Barrett esophagus and EAC tumors. Kaplan-Meier analysis showed that patients with higher RNA expression and strongly positive ANPEP/CD13 membrane IHC-Histoscore staining have shorter survival (P =.002). Down-regulation of ANPEP/CD13 expression by short hairpin RNA vector reduces colony formation, migration, and invasion of FLO-1 EAC cells. Overexpression of CD13 in SKGT4 EAC cells increases colony formation, motility, and invasion in vitro. Conclusions: Elevated expression of ANPEP/CD13 indicates shorter survival of EAC patients and a more invasive phenotype of cancer cells in vitro. Validation in a larger sample group is required to better understand the clinical significance of ANPEP/CD13 and other candidate genes.