Abstract 193: Notch pathway activation predicts poor prognosis in combined hepatocellular carcinoma-cholangiocarcinoma
Authors: Seokhwi Kim, Chan Mo Yang, Jaehong Lim, Ara Ko, Taebum Lee, Young-Bae Kim, Dakeun Lee
Journal: Cancer Research (2025)

Abstract Background: Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCC) is a distinct type of tumor that differs from both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC). Although the involvement of the Notch signaling pathway in its development is recognized, its role in clinical specimens has not been extensively studied. Methods: We analyzed surgically resected specimens from 38 HCCs, 32 CCCs, and 42 cHCC-CCCs obtained between 2017 and 2023 at Ajou University Hospital. The expression levels of the HCC marker Glypican-3, the CCC markers CK7 and CK19, and the hepatobiliary progenitor cell markers CK19, EpCAM, and CD13 were investigated through immunohistochemistry (IHC). The expression of Notch receptor subtypes (Notch1-4) and the downstream transcription factors Hes5 and Ascl1 were analyzed using qRT-PCR. Additionally, NOTCH1 and Hes5 protein expression levels were evaluated via IHC. Results: Compared to HCC, cHCC-CCC exhibited reduced expression of Glypican-3 (p&amp;lt;0.0001) and increased expression of CK7 (p&amp;lt;0.0001), CK19 (p&amp;lt;0.0001), and EpCAM (p&amp;lt;0.0001). When compared to CCC, cHCC-CCC displayed significantly elevated levels of Glypican-3 (p&amp;lt;0.0001) and CD13 (p&amp;lt;0.0001). Among the four Notch receptor subtypes, the mRNA level of Notch1 was preferentially elevated in cHCC-CCC compared to HCC or CCC. Similarly, the mRNA expression levels of Hes5 and Ascl1 were higher in cHCC-CCC than in HCC (p=0.0920 for Hes5; p=0.5453 for Ascl1) and CCC (p=0.0586 for Hes5; p=0.0759 for Ascl1). Higher NOTCH1 protein expression was observed in cHCC-CCC compared to HCC (mean H-score: 43.9 vs. 24.0, p&amp;lt;0.01) and CCC (mean H-score: 43.9 vs. 29.4, p&amp;lt;0.05). Similarly, Hes5 expression in cHCC-CCC was substantially greater than in HCC (mean H-score: 52.7 vs. 8.6, p&amp;lt;0.001). A scatter plot analysis of NOTCH1 and Hes5 expression levels in cHCC-CCC revealed three distinct subgroups based on their expression profiles. Group 1 (n=28) was characterized by low levels of NOTCH1 expression (Notch-inactivated). Group 2 (n=5) exhibited elevated expression of both NOTCH1 and its downstream target (Notch-responsive). Group 3 (n=8) demonstrated high NOTCH1 expression but very low levels of Hes5 expression (Notch-non-responsive). Survival analysis indicated that Group 1 and Group 3 cHCC-CCCs had overall survival and progression-free survival (PFS) rates similar to those of HCCs. In contrast, Group 2 (Notch-responsive subtype) exhibited a significantly worse PFS (hazard ratio [HR] 7.708 [95% confidence interval (CI) 0.7807-76.11], p=0.0002), which was comparable to the PFS of CCC (HR 5.355 [95% CI 2.376-12.10], p&amp;lt;0.0001). Conclusion: The Notch signaling pathway is differentially expressed in cHCC-CCC. Tumors with activated Notch signaling components are associated with poorer prognostic outcomes within the cHCC-CCC cohort. Citation Format: Seokhwi Kim, Chan Mo Yang, Jaehong Lim, Ara Ko, Taebum Lee, Young-Bae Kim, Dakeun Lee. Notch pathway activation predicts poor prognosis in combined hepatocellular carcinoma-cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 193.