Tissue Microarray for Liver Pathology

Initial analysis was performed using normal liver tissue (HuFPT074), HCC tissue (HuCAT081), and iCCA tissue (HuCAT086; Biomax, Inc.). Hematoxylin and eosin (H&E) stains of each tissue were annotated by a pathologist with the tumor, adjacent to the tumor, and fibrotic regions. Subsequently, analysis was performed using a tissue microarray (TMA; #LV2081, Biomax, Inc) that contained 208 cores with 103 cases (duplicated cores per case): consisting of fifty HCC, twenty iCCA, one clear-cell carcinoma cyst, five metastatic HCC (spleen, chest wall, cerebrum, costal bone, and lymph node), two hepatic cyst, eight tissues with cirrhosis and dysplastic nodules, ten hepatitis-infected tissues, two adjacent normal tissues, and six independent normal tissues. The cores were 1.0 mm in diameter and validated by pathology. The validation tissue cohort TMA (DLV753, US Biolab) contained 75 cases with 75 cores: forty-five cases of HCC, twenty-three cases of iCCA, two cases of mixed carcinoma, and five cases of normal liver tissue. Cores were 1.5 mm in diameter and validated by pathology. All tissue samples were formalin-fixed paraffin-embedded (FFPE) cut into a 5-μm-thick section and unstained before analysis. H&E stainings of all tissues and TMAs annotated by a pathologist for small duct and large duct classifications for each core are provided in Supplementary Fig. S1A–C. Clinical information for TMAs is listed in Supplementary Fig. S2A and S2B.