HER2 status in extrahepatic cholangiocarcinoma and gallbladder carcinoma: concordance between immunohistochemistry and chromogenic in situ hybridization in 140 Cases.

BACKGROUND: HER2 represents an actionable target in a subset of biliary tract cancers (BTCs), particularly extrahepatic cholangiocarcinoma (eCCA) and gallbladder carcinoma (GBC). Recent approval of zanidatamab for HER2-positive BTC has further highlighted the clinical relevance of accurate HER2 assessment. METHODS: HER2 status was evaluated in 140 consecutive surgically resected BTCs collected between 2020 and 2025 (87 eCCAs and 53 GBCs) using immunohistochemistry (IHC) and dual chromogenic in situ hybridization (DISH). HER2 expression was scored according to gastric cancer criteria, and HER2 positivity was defined following HERIZON-BTC-01 criteria. Selected cases were additionally analyzed using a combined IHC-DISH approach. RESULTS: HER2 expression was 0 in 72.9% of cases, 1+ in 12.1%, 2+ in 8.6%, and 3+ in 6.4%. HER2 positivity was observed in 9.2% of eCCAs and 9.4% of GBCs. ERBB2 amplification was consistently identified in all IHC 3+ tumors and in a subset of IHC 2+ and, rarely, 1+ cases. The mean ERBB2/CEP17 ratio was significantly higher in HER2 3+ tumors than in amplified HER2 2+/1+ tumors (6.2 vs 3.9; p=0.001). Combined IHC-DISH analysis confirmed concordance between HER2 overexpression and ERBB2 amplification within the same tumor cells. HER2 expression frequently showed intratumoral heterogeneity, often involving less than 50% of tumor cells. CONCLUSIONS: HER2 alterations occur in a subset of eCCA and GBC and show substantial biological heterogeneity, including lower-level amplification and intratumoral heterogeneity, with potential implications for HER2 assessment and patient selection.