Comprehensive molecular profiling of combined hepatocellular carcinoma and cholangiocarcinoma reveals distinct Notch signaling subgroups with prognostic significance.

Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA) is a rare primary liver carcinoma characterized by dual hepatocytic and cholangiocytic differentiation. Accurate diagnosis remains challenging, and the underlying molecular mechanisms that could inform treatment and prognostic predictions are not fully understood. The Notch signaling pathway has been implicated in the carcinogenesis and tumor biology of cHCC-CCA, yet its role has not been comprehensively investigated. In this study, we analyzed 35 cHCC-CCA, 38 hepatocellular carcinoma (HCC), and 32 intrahepatic cholangiocarcinoma (CCA) samples using immunohistochemistry and RNA sequencing. Compared to HCC and CCA, cHCC-CCA exhibited elevated expression of NOTCH1 and its downstream targets (HES5, ASCL1, and HES1). Based on NOTCH1 and HES5 expression levels, cHCC-CCA tumors were stratified into three subgroups: Group 1 (Notch inactivated; low NOTCH1, variable HES5), Group 2 (Notch-responsive; high NOTCH1 and HES5), and Group 3 (Notch-unresponsive; high NOTCH1, low HES5). Notch-responsive tumors (Group 2) displayed the most aggressive biological characteristics, including higher rates of vascular invasion and significantly poorer progression-free survival. Transcriptomic analyses revealed that the molecular profiles of Group 2 cHCC-CCA resembled those of CCA, further confirming Notch signaling activation and identifying enriched pathways associated with increased tumor invasiveness and poor prognosis. These findings highlight the significant heterogeneity within cHCC-CCA and emphasize the potential of NOTCH1 and HES5 as biomarkers for subgroup classification. Moreover, these subgroups offer actionable insights into targeted therapies, including Notch pathway inhibitors, particularly for Notch-responsive tumors.