High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma

Eighty four patients with HCC who had undergone surgical resections at Gunma University Hospital between 1996 and 2014 were included in the study. The ages of the patients ranged from 48 to 89 years old. The tumor stage was classified according to the 6th Japanese tumor-node-metastasis (TNM) classification of Liver Cancer Study Group of Japan. Written informed consent for the collection of specimens was obtained from all participating patients with HCC, and the study protocol was approved by the local Ethics Committee.

Clinical formalin-fixed, paraffin-embedded (FFPE) samples were stored in the archives of the Clinical Department of Pathology, Gunma University Hospital. After reviewing the H&E-stained slides, two representative tumor area were marked on FFPE tissue blocks. These tumor areas were extracted as tissue core by using a cylinder. The diameter of tissue core was 2.0 mm. A manual arraying instrument (Beecher Instruments, Silver Spring, MD, USA) was used to assemble the paraffin blocks into TMAs, as previously described [19].

A 4-μm section was cut from paraffin blocks of samples. Each mounted sections were deparaffinized, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity. The sections were then heated in boiled 10 mM citrate buffer (pH 6.0) at 98 °C for 30 min. Nonspecific binding sites were blocked by incubating with 0.25% Casein/1% BSA for 30 min at room temperature. Anti-FOXC2 primary monoclonal antibody (Abnova, Taipei, Taiwan) was used at a dilution of 1:100 at 4 °C overnight, as previously described [12]. The sections were washed in PBS, and the primary antibody was visualized using the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan) according to the instruction manual. The chromogen 3,3-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) was applied as a 0.02% solution containing 0.005% H₂O₂ in 50 mM Tris-HCl buffer (pH 7.6). The sections were lightly counterstained with Mayer’s haematoxylin and mounted. Negative controls were established by omitting the primary antibody. Other IHC was performed using the following primary anti-bodies: anti-E-cadherin (36; Ventana Medical Systems, Tucson, AZ, USA), anti-N-cadherin (6G11, Dako, Glostrup, Dermark), anti-Vimentin (V9, Dako), anti-ZEB1 (D91854, Atlas Antibodies, Stockholm, Sweden) and anti-Ki67 (30–9, Ventana).

The immunohistochemical FOXC2 expression was evaluated independently by two observers. A staining was primarily cytoplasmic in positive cases. The intensity of FOXC2 staining was scored as 0, 1+, 2+, and 3+. Grade 0, 1+, and 2+ staining was considered to be negative for FOXC2 expression, while grades 3 was considered to be positive. E-cadherin was evaluated by proportion score and intensity score. The proportion score are as follows: Score 0: < 10%; Score 1: 10–40%; and Score 2: > 40%. The intensity score are follows: Score 0: negative; Score 1: weak; Score 2: intermediate; and Score 3: strong. Finally, the two scores are combined, and a total score of 3 or more is regarded as positive. The expression of N-cadherin were defined as negative if membrane staining was detected in < 20% of tumor cells. Then we defined Cadherin switch as the samples showing both E-cadherin negative and N-cadherin positive. The expression of Vimentin in the cytoplasm of > 1% of tumor cells was defined as positive, regardless of staining intensity. The expression of ZEB1 in the nuclei of > 1% of tumor cells was defined as positive. The Ki67 labeling index was evaluated as the percentage of positive tumor cell nuclei based of > 500 tumor cells, regardless of staining intensity. The percentage > 1% was considered as high Ki67 labeling index. The IHC slides were evaluated by two independent pathologists in a blind manner.

We used EZR (Saitama Medical Center Jichi Medical University; http://​www.​jichi.​ac.​jp/​saitama-sct/​SaitamaHP.​files/​statmed.​html) for statistical analysis. The χ2 test and Fisher’s exact test were used to evaluate associations between clinicopathologic characteristics and FOXC2 expression intensities. To calculate and analyze overall survival and disease-free survival, we used the Kaplan–Meier method and the Log-rank test. Independent prognostic factors were tested by Univariate and multivariate analyses, which were based on the Cox proportional hazards regression model. A statistically significant p-value was considered to be less than 0.05.

The expression of FOXC2 was evaluated in 84 HCC samples by immunohistochemistry. Cytoplasmic staining of FOXC2 was primarily observed (Fig. 1a). In total, 26 of 84 HCC samples (31%) were considered high for FOXC2 expression, whereas 58 HCC samples (69%) were considered low for FOXC2 expression.

Correlations between FOXC2 expression in HCC samples and patient age, gender, liver cirrhosis, T classification, differentiation, tumor size, pattern of tumor growth, Fc-inf, IM, Vv, Vp, Va, serum AFP level, serum PIVKAII level, and the Ki-67 labeling index are shown in Table 1. High FOXC2 expression was correlated with liver cirrhosis (P = 0.0296), poor differentiation (P = 0.0302), high serum AFP levels (P = 0.00078), and the Ki-67 labeling index (P = 0.0348).

We examined the relationship between FOXC2 expression and IHC staining of the EMT-related markers E-cadherin, N-cadherin, vimentin, and ZEB1. High FOXC2 expression had a strong association with the induction of the Cadherin switch and a high expression of vimentin (Table 2; P = 0.0414 and P = 0.0273) (Fig. 1).

Disease-free survival rates (DFS) and the overall survival rates (OS) of patients with HCC are shown in Fig. 2. Patients with high FOXC2 expression had significantly poorer prognoses than patients with low expression in both DFS (P = 0.0022) and OS (P = 0.031). For OS, FOXC2 expression was a prognostic factor for poor survival in a univariate analysis (Table 3; RR 2.14, 95% CI 1.05–4.34, P = 0.035). Additionally, the pattern of tumor growth, T classification, and portal vein invasion were also prognostic factors in the univariate analysis. In the multivariate analysis, FOXC2 expression was an independent prognostic factor for poor survival (Table 3; RR 2.21, 95% CI 1.06–4.57, P = 0.033).