The patient cohort inclusion and exclusion criteria included (a) accurate pathologic diagnosis of HCC, (b) complete clinicopathologic and follow-up data, (c) no anticancer treatment

prior to curative liver resection, and (d) complete formalin-fixed, paraffin-embedded tissues. The histopathological diagnosis was determined according to the World Health Organization criteria. Tumor differentiation was graded using the Edmondson grading system [23]. Tumor staging was based on the 6th edition of the tumor-node-metastasis (TNM) classification of the International Union Against Cancer. Most patients (82.4%) had a hepatitis B virus background, and only two patients had hepatitis C virus. Almost all patients (316 of 318 for the training cohort and 325 of

328 for the validation cohort) were in the Child-Pugh A classification. The clinicopathologic characteristics GDC-0449 purchase of the two cohorts are summarized in Additional file 2: Table S1. Ethical approval was obtained from the Zhongshan Hospital Research Ethics Committee, and written informed consent was obtained from each patient. Follow-up and postoperative treatment The follow-up data were summarized at the end of December 2011, with a median observation time of 52.2 months. The follow-up procedures were described in our previous study [23, 24]. Postsurgical patient AZD2014 surveillance was undertaken as previously described [23, 25]. OS was defined as the interval between the dates of surgery and death. TTR was defined as the interval between the dates of surgery

and the dates of any diagnosed recurrence (intrahepatic recurrence and extrahepatic metastasis). For surviving patients, the data were censored at the date of death or last follow-up. Tissue microarray and immunohistochemistry Tissue microarray (TMA) was conducted as previously described [26–28]. Briefly, all samples from the HCC patients were reviewed by three histopathologists and representative Sclareol areas located away from necrotic and hemorrhagic materials were premarked in the paraffin blocks. Two core biopsies (1 mm in diameter) were taken from each representative tumor tissue and peritumoral tissue to construct the TMA slides. Consecutive sections measuring 4 μm were placed on 3-aminopropyltriethoxysilane-coated slides (Shanghai Biochip Co Ltd, Shanghai, People’s Republic of China). Immunohistochemistry of the paraffin sections was performed using a two-step protocol (Novolink Polymer Detection System, Novocastra) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized and then rehydrated; after heat-induced antigen retrieval, endogenous peroxidases were blocked for 5 min using 0.