Patients and specimens
A total of 59 non-mucin producing ICC patients confirmed by pathologically HE staining who received curative surgery from January 2004 to December 2014 (33 from Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China and 26 from University Medical Center Hamburg-Eppendorf, Hamburg, Germany) were enrolled. Two patients lost during the follow-up. Hence, 57 patients, 39 men and 18 women, were eventually included in the study. The mean follow-up duration was 25.7 ± 19.1 months. The tumor stage was determined according to the 2009 UICC TNM classification system [13].
The clinical study was approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiaotong University School of Medicine and the ethics committee of Medical Association of Hamburg. Informed consent was obtained from all participants.
Histology
Liver tissues were fixed in 4% formaldehyde and embedded in paraffin. 4 μm sections was used for hematoxylin–eosin, Sirius red and immunohistochemistry (IHC) staining. The inflammation grades and fibrosis stages of the peri-tumoral tissues were examined by two experienced pathologists according to the Scheuer scoring system [14].
For IHC, the sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 min for antigen unmasking. After cooling, the sections were incubated in peroxidase blocking reagent (Dako) for 1 h and then incubated with the following primary antibodies at 4 °C for overnight: anti-CK19 (Dako, Hamburg, Germany), 1:200; anti-CD133 (R&D Biotechnology, USA), 1:200; anti-TGF-β1 (Santa Cruz Biotechnology, USA), 1:200; anti-p-Smad2 (Santa Cruz Biotechnology, USA), 1:100; and anti-S100A4 (Sigma-Aldrich Biotechnology, Germany), 1:200; anti-E-Cadherin (1:200; Abcam) and anti-Vimentin (1:200; Abcam). Next day, the sections were incubated at room temperature with the secondary antibody and developed with diaminobenzidine for 5 min.
For semiquantitative analysis, IHC scores were calculated as follows: grade 0, < 1% positive cells; grade 1, ≥ 1% and < 25% positive cells; grade 2, ≥ 25% and < 50% positive cells; grade 3, ≥ 50% and < 75% positive cells; and grade 4, ≥ 75% positive cells. Since p-Smad2 is commonly expressed in tumor cells, we also evaluated the intensity of p-Smad2 staining: grades 1–4: (1) weak positive staining: yellow; (2) moderate positive staining: brown; (3) strong positive staining: deep brown; (4) very strong: black. The final immune staining score for p-Smad2 was calculated as positive area * staining intensity.
For double-fluorescence immunostaining for E-Cadherin and Vimentin, the slides were washed with PBS and incubated with anti-E-Cadherin antibody (1:100; Abcam) at 4 °C overnight. Then, the slides were washed with PBS and incubated with anti-Vimentin antibody (1:100; Abcam) at 4 °C overnight. Next, the slides were incubated with secondary antibodies, Alexa 633 IgG and Alexa 488 IgG (Molecular Probes/Invitrogen, Karlsruhe, Germany) for 30 min at room temperature. The samples were mounted using Dako-Cytomation Fluorescence Mounting Medium. The slides were imaged with a confocal microscope (Leica, Heidelberg, Germany).
Statistical analysis
Data were analyzed using the SPSS version 13.0 for Windows (SPSS Inc, Chicago, IL, USA), and are presented as means and standard deviations (± SD). Student’s t-test was used to compare the continuous quantitative data. A two-tailed Wilcoxon signed rank test was used to compare ranked variables. The Kaplan–Meier analysis was applied to evaluate overall and disease-free survival, and different groups were compared with the log-rank test. p < 0.05 was considered to be statistically significant.
