Abstract:Aims/Background: Midkine (MK) is a novel heparin-binding growth factor whose gene was identified in embryonal carcinoma cells in the early stages of retinoic acid-induced differentiation. This study investigates the overexpression of MK in intrahepatic cholangiocarcinoma (CC). Methods: Forty-five primary CC specimens from patients (aged 19–81 years, 24 males and 21 females) were examined. Histologically, 17 cases of CC were classified as the well-differentiated type, 19 as moderately-differentiated and 9 as poorly-differentiated. Immunohistochemical analysis was performed using a rat IgG2a monoclonal antibody against the carboxyl terminal region of human MK. Results: We successfully applied this monoclonal antibody against MK to analyze archival paraffin sections. The cancer tissues showed a positive reaction to this antibody, and there was an intense reaction in their cytoplasm. Approximately 40% of individuals with CC (17/45) had tumor cells that expressed MK, and these were classified into the following types: moderately-differentiated type (9/19), well-differentiated type (8/17) and poorly-differentiated type (0/9). In situ hybridization analysis revealed that signals of MK transcripts were found in the cytoplasm of the cancer cells; the distribution and localization of the MK-transcript signals determined by in situ hybridization analysis were similar to those obtained by immunohistochemical analysis. Conclusions: These findings revealed that CC express increased MK at the messenger RNA and protein levels.