Thus, each tissue block (NT and T) was represented by three independent spots in the TMA. IHC experiments were performed using an automated Discovery XT immunostaining device (Ventana Medical System, Tucson, AZ). TMA sections (4 μm thick) were evaluated for the expression of collagen 4, laminin, osteopontin, TGFβ2, and KIAA0101 (Supporting Table 1). Antigens were retrieved from deparaffinized and rehydrated PLX4032 tissues by incubating the slides for 48 minutes at 95°C in CC1 Tris-based buffer (pH 8.0) (laminin, collagen 4, and KIAA0101) or in Ultra CC2 citrate buffer

(pH 6.0) (osteopontin and TGFβ2) (Ventana Medical System). Detection was performed using a streptavidin-biotin-peroxidase kit (OmniMap, Biotin-free DAB Detection Systems, Ventana Medical System). TMA slides were analyzed by two experienced pathologists (B.T., F.L.G.) in a blinded manner. Staining intensity in the stroma was scored as follows: negative (0), selleck kinase inhibitor mild (1), moderate (2), or strong (3). Given that each stromal sample was represented in triplicate, the sum of the three values was performed to obtain a score of with a range of 0 to 9. This score was finally categorized into four groups to optimize the statistical analysis and to take into account extreme values: 0 (score 0-1), 1 (score 2-3), 2 (score 4-7), and 3 (score 8-9). Differences in protein expression (NT fibrous tissue versus T stroma) were evaluated by chi-squared testing. Relationships

between protein expression and clinical parameters were evaluated using the chi-squared or Fisher’s exact probability test for categorical variables and using the analysis of variance for numerical variables. The correlation of the scoring performed by the two pathologists was estimated by a weighted kappa coefficient; disagreements were weighted according to their squared distance from a perfect agreement in the correlation matrix. The Kaplan-Meier method was used to estimate the overall (OS) 上海皓元 and disease-free survival (DFS), and group differences were analyzed with the log-rank test. A trend analysis was also performed. Univariate

and multivariate Cox regression models for the hazards of OS and DFS mortality were used to evaluate the effect of protein expression. Correlation between the different variables was also evaluated in order to identify putative interaction and confounding factors. The most suited Cox model was selected using a stepwise regression, selecting variables based on the Akaike Information Criterion (AIC). P < 0.05 was considered statistically significant. Statistical analysis was performed with R (v. 2.15.1). Relevant biomarkers for ICC prognosis were investigated by the unsupervised gene expression analysis of the stroma in mass-forming type ICC. To increase the robustness of the study, an initial cohort of clinically well-annotated cases of patients with ICC (n = 87) was used to build a testing set and a validating set as described.