BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) often is a diagnosis determined by exclusion. Distinguishing ICC from other metastatic adenocarcinomas based on histopathologic or immunohistochemical analysis often is difficult and requires an extensive workup. This study aimed to determine whether albumin, whose expression is restricted to the liver, has potential as a biomarker for ICC using a novel and highly sensitive RNA in situ hybridization (ISH) platform.METHODS: Modified branched DNA probes were developed for albumin RNA ISH. The study evaluated 467 patient samples of primary and metastatic lesions.RESULTS: Of the 467 samples evaluated, 83 were ICCs, 42 were hepatocellular carcinomas (HCCs), and 332 were nonhepatic carcinomas including tumors arising from the perihilar region and bile duct, pancreas, stomach, esophagus, colon, breast, ovary, endometrium, kidney, and urinary bladder. Albumin RNA ISH was highly sensitive for cancers of liver origin, staining positive in 82 (99 %) of 83 ICCs and in 42 HCCs (100 %). Perihilar and distal bile duct carcinomas as well as carcinomas arising at other sites tested negative for albumin. Notably, 6 (22 %) of 27 intrahepatic tumors previously diagnosed as carcinomas of undetermined origin tested positive for albumin.CONCLUSIONS: Albumin RNA ISH is a sensitive and highly specific diagnostic tool for distinguishing ICC from metastatic adenocarcinoma to the liver or carcinoma of unknown origin. Albumin RNA ISH could replace the extensive diagnostic workup, leading to timely confirmation of the ICC diagnosis. Additionally, the assay could serve as a guide to distinguish ICC from perihilar adenocarcinoma.