Staging Staging describes the extent or severity of a cancer based on the

extent of the original (primary) tumour and the extent of spread in the body. The TNM system is one of the most commonly used staging systems. This system has been accepted by the international union against cancer (UICC) and the American Joint Committee on Cancer (AJCC). The TNM system is based on the extent of the tumour (T), the extent of spread to the lymph nodes (N), and the presence of distant metastasis (M). A number is added to each letter to indicate the size or extent of the tumour and the extent of spread. The staging system used for this study is based on the spread of the tumour through the body, KU-57788 molecular weight and therefore considered summary staging. Many cancer registries, such as the National Cancer Institutes (NCI) surveillance use summary staging. The staging system used for this study is a modified three category staging system and is based on the invasion and spread of the tumour. The tumours are staged in three categories: Stage 0: macroscopically there is only one tumour process in the liver and/or microscopically the tumour is well circumscribed or encapsulated. There are no indications for intrahepatic or extrahepatic metastases; Stage 1: Microscopically the tumour has

spread beyond the original (primary) site to the adjacent tissue and/or vessels or microsatellites can be seen and/or there are macroscopically multiple tumour processes present in the liver; Stage 2: The tumour has spread from the

p38 MAPK inhibitor review primary site to the lymph node and/or other organs (distant metastasis). Immunohistochemistry Immunohistochemistry (IHC) was performed for K19, K7, HepPar-1, and glypican-3 (GPC-3) on all liver tumour samples. Antibody characteristics, manufacturer, source and dilution are provided in Table 1. Slides were air dried (30 min, RT) and deparaffinised. Heat induced antigen retrieval was performed with 10 mM citrate buffer (pH 6.0) or 10 mM Tris with 1 mM EDTA for 10 minutes in a microwave (850 W) with a cool down period for O-methylated flavonoid 10 minutes at RT (Table 3). Antigen retrieval by enzymatic digestion was performed with proteinase K for 15 minutes at room temperature (Table 3). Endogenous GDC-0994 datasheet peroxidase activity was blocked in 0.3% H2O2 (30 min) and background staining was blocked with 10% normal goat serum (30 min). The primary antibodies were diluted in the appropriate buffer and incubated as indicated in Table 3. The Envision system was used for secondary antibody labelling (Dakocytomation, Glostrup, Denmark). The signal was developed in 0.06% 3,3′-diaminobenzidine (DAB) solution (Dakocytomation) for 5 minutes and finally counterstained with Mayer’s hematoxylin (Mayer’s haematoxylin, Klinipath B.V. Duiven, The Netherlands).