[2] The patients with CoCC, HCC or classical CHC were more likely to have suffered from hepatitis virus B or C infection and liver cirrhosis than the patients with CCC (P < 0.001). The tissue samples were fixed in a 10% formalin solution and embedded in paraffin for histological diagnosis and immunohistochemistry. We used the new WHO classification for the histological diagnosis of CoCC, CCC, HCC and classical CHC.[3] The diagnosis of CCC and the CCC components within classical CHC was

confirmed with Alcian blue histochemical staining for mucin and immunostaining for cytokeratin (CK)7 and epithelial membrane antigen (EMA); HCC and the HCC components of classical CHC were diagnosed using immunostaining for α-fetoprotein (AFP), hepatocyte paraffin 1 and MK-8669 in vivo arginase in addition to histological findings. This study was approved by the Research Ethics Review Board of Teikyo University selleck screening library School of Medicine (no. 08–159). Formalin-fixed paraffin-embedded tissue sections, cut at a thickness of 3 μm, were deparaffinized with xylene and rehydrated with

graded ethanol. Immunohistochemistry was performed using monoclonal and polyclonal antibodies with antigen-retrieval methods, as listed in Table S1, and performed using Dako Autostainer Link 48 (Dako, Glostrup, Denmark). Endogenous peroxidase was quenched with 3% H2O2 in distilled water for 5 min. The slides were incubated with primary antibodies for 30 min at room temperature, and the sections were then stained by a detection method using EnVision FLEX (Dako) according to

the manufacturer’s protocol and counterstained with hematoxylin. Immunostaining for the integrins β6, β4 and α3, fibronectin and laminin was defined as positive when more than 10% of positively stained cells or areas were observed. Moreover, the intensity of integrin β6, β4 and α3 staining was semiquantitatively scored as 0 (negative), 1 (weak), 2 (moderate) Lck or 3 (strong). The percentage of cells staining positively for integrins β6, β4 and α3 was also semiquantitatively scored into five categories: 0 (<10%), 1 (11–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). To perform the statistical analysis, the level of positive staining was evaluated by a final score, which was calculated by multiplying the scores of staining intensity by the scores for the percentage of positive cells. Based on the final score for integrin β6, β4 and α3 staining, the cases were grouped as negative for a score of 0, low for 1–2 and high for 3–12. The predominant pattern of positive staining for the integrins and ECM proteins in the hepatic tumors was classified as a cytoplasmic pattern, cell membrane pattern, basal lamina pattern or stromal pattern.