Human liver cancer and surrounding tissues in this study were obtained from 80 HCC patients who underwent operations between January 2006 and August 2008 at the Affiliated Hospital of Nantong University, Jiangsu Province, China. The tissues were immediately frozen in liquid nitrogen and kept at -80 °C until required. The patient cohort inclusion and exclusion criteria included: (1) accurate pathologic diagnosis of primary HCC; (2) complete clinicopathologic and follow-up data; and (3) previously untreated, with surgery as the first treatment. Thus, analysis of the data would reflect the actual impact of tumor biology on the clinical outcome. This study was approved by the Ethics Committee permission (No. TDFY2013008) of the Affiliated Hospital of Nantong University, and all patients signed informed consent. The related important clinical information of each patient was collected from their medical records.

The patient characteristics are listed in Table 1. The patient population consisted of 62 males and 18 females, with a median age of 51 years (range: 35-74). The sizes of tumors were 44 cases with > 5 cm and 36 with < 5 cm. α-fetoprotein (AFP) levels were 28 cases with ≥ 400 ng/mL and 52 with < 400 ng/mL. The tumor number was 64 cases with single and 16 with multiple. Tumor differentiation was graded using the Edmondson grading system[18] with 5 well-, 41 middle-, and 34 poor- differentiation. Tumor staging was 36 cases (45%, 36 of 80) at I and II and 44 cases (55%, 44 of 80) at III and IV based on the 6^th edition of the tumor-node-metastasis (TNM) classification of the International Union against Cancer (IUAC). The incidence of hepatitis B surface antigen (HBsAg) was 81.25% (65 of 80) in these HCC tissues, and 63.75% (51 of 80) had a history of liver cirrhosis. All patients had regular follow-up. The follow-up period was defined as the duration from the date of the operation to the date of either death or the last follow-up. The last follow-up was October 2013. Overall survival (OS) was evaluated by the duration between the dates of surgery and death. During the follow-up period, a total of 48 patients died.

Histopathologic examination of the livers with hematoxylin and eosin (HE) staining showed that all cancerous tissues were accurate pathologic diagnosis of HCC according to the World Health Organization criteria. HCC diagnosis was confirmed according to the criteria set by the Chinese National Collaborative Cancer Research Group[18].

All HCC tissues were stained by HE staining and reviewed by two histopathologists. Representative areas free from necrotic and hemorrhagic materials were marked in paraffin blocks. Tissue cores (2.0 mm) were taken from each representative tumor tissue and peritumoral tissue to construct the tissue microarray (TMA) slides.

The TMA slides used for immunohistochemistry (IHC) were deparaffinized, and peroxidase was quenched with methanol and 3% H₂O₂ for 15 min. For antigen retrieval, the sections were boiled under pressure in citrate buffer (pH 6.0) for 3 min. After that, the tissues were incubated for 120 min with primary mouse anti-Wnt3a (ab81614; Abcam, United Kingdom) at 1:50 dilution. Following washing with phosphate buffered saline (PBS), the sections were incubated with horseradish peroxidase conjugated goat anti-mouse IgG (A21010; Abcam, Cambridge, United Kingdom) for 15 min at 1:5000 dilution and washed again with PBS. The sections were incubated in diaminobenzidine solution, counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and cover-slipped. For the negative control reactions, PBS was used instead of the primary antibody.

The results of IHC staining were assessed by two independent pathologists without knowledge of the clinicopathologic features. The percentages for Wnt3a-positive cells were scored as follows: 0 (0%), 1 (1%-33%), 2 (34%-66%), and 3 (67%-100%). Staining intensity was stratified into four categories: 0 (negative), 1 (weakly), 2 (moderate), and 3 (strongly). The sum of the percentage and intensity score was defined as the IHC staining score. According to above criterion, the HCC tissues with Wnt3a expression were divided into two groups: low with 0-2 scores and high 3-6 scores. The higher score for positive percentages and staining intensity was taken as the final score when there was a difference between duplicate tissue scores.

Statistical analysis was carried out by using SPSS software (version 20.0, Armonk, NY, United States). The difference between HCC and para-cancerous groups was analyzed by χ² test. Quantitative variables were analyzed by Mann-Whitney U test (Z). The relationship between Wnt3a expression and clinicopathological factors was analyzed by χ² test. Survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. Factors shown to be of prognostic significance in the univariate models were evaluated in a multivariable Cox regression model. For all tests, the significance level for statistical analysis was set at P < 0.05.

The over-expression of hepatic Wnt3a and its cellular distribution in cancerous or surrounding tissues analyzed by immunohistochemistry with human anti-Wnt3a antibodies are shown in Figure 1. Positive Wnt3a staining, identified as brown particles, was distributed in the cytosol and the membrane of hepatocytes or only a few cell nuclei. The incidence of hepatic Wnt3a expression in cancerous tissues (96.25%, 77 of 80) was significantly higher (χ² = 48.818, P < 0.001) than that in surrounding tissues (46.25%, 37 of 80). Liver Wnt3a staining and its expression frequency in cancerous or surrounding tissues are shown in Table 2. The liver Wnt3a staining scores were significantly different (Z = 8.144, P < 0.001) between cancerous tissues and surrounding tissues, with strong expression (3-6 scores) in 71.25% (57 of 80) of cancerous tissues and low or no expression (0-2 scores) in 86.25% (69 of 80) of surrounding tissues.

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Figure 1 Analysis of hepatic Wnt3a expression and its cellular distribution by immunohistochemistry. The expression of hepatic Wnt3a was analyzed on tissue microarrays by immunohistochemistry with the primary mouse anti-human Wnt3a antibody. The positive Wnt3a expression with brown staining particles was distributed in the cytosol and membrane of hepatocytes and only a few cell nuclei. A and B: Strong straining of Wnt3a in hepatocellular carcinoma (HCC) tissues; C and D: Light staining of Wnt3a in surrounding tissue. A and C: Original magnification × 400; B and D: Original magnification × 400.

The relationship between hepatic Wnt3a expression and clinical staging of HCC is shown in Figure 2. According to the IUAC clinical staging criteria of HCC patients, there were nine cases at staging I (11.25%, 9 of 80), 27 at II (33.75%, 27 of 80), 38 at III (47.5%, 38 of 80), and six at IV (7.5%, 6 of 80) among 80 cancerous tissues. The incidences of high or low Wnt3a expression in HCC tissues were 22.2% (two of nine) or 77.8% (seven of nine) at I staging, 51.9% (14 of 27) or 48.1% (13 of 27) at II staging, 94.7% (36 of 38) or 5.3% (two of 38) at III staging, and 83.3% (five of six) or 16.7% (one of six) at IV staging, respectively. Brown Wnt3a expression was gradually increased in different clinical stages, with very strong staining at the advanced stage.

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Figure 2 Expression of hepatic Wnt3a at different hepatocellular carcinoma stages. A1 and B1: Low or no Wnt3a expression in the para-cancerous tissues. A2-A5 and B2-B5: The brown staining of Wnt3a gradually increases in cancerous tissues from stage I to IV. A1-A5: Original magnification × 200; B1-B5: Original magnification × 400.

The correlation between Wnt3a levels and the clinicopathological parameters of 80 HCC patients is shown in Table 3. High Wnt3a expression was associated with poorly-differentiated grade (P < 0.001), liver cirrhosis (P = 0.004), HBV infection (P < 0.001), and higher TNM stage (P < 0.001). By contrast, there were no significant associations between hepatic Wnt3a expression and other clinicopathological features, such as HCC patients’ gender or age, tumor number, circulating AFP level, and periportal cancer embolus.

As expected, the overexpression of hepatic Wnt3a showed a significant relationship with 5-year survival of the 80 HCC patients (P < 0.001). Meanwhile, certain HCC clinical prognostic factors, such as liver cirrhosis (P = 0.035) and HBV infection (P = 0.029), also showed a statistically significant correlation with 5-year survival rate based on Cox regression univariate analysis (Table 4). All these factors were enrolled in a multivariable analysis. High Wnt3a expression (P < 0.001), liver cirrhosis (P = 0.033), and HBV infection (P = 0.010) were all identified as independent predictive factors for poor HCC outcome. The Kaplan-Meier survival curves (Figure 3) demonstrated that HCC patients with high Wnt3a expression had a significantly shorter survival time compared to those with low or no Wnt3a expression.

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Figure 3 Overall survival curves of 80 hepatocellular carcinoma patients. The Wnt3a expression curves were calculated by the Kaplan-Meier method. Survival curves of 80 hepatocellular carcinoma patients were made according to cancerous tissues expressing a low or high level of Wnt3a (log-rank test, P < 0.001). The green line is the high Wnt3a group; and the blue line is the low or without Wnt3a group.

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide. The oncogene member 3a of Wingless-type MMTV integration site family (Wnt3a) is located on chromosome 17q21 and has been regarded as an activator of the canonical Wnt signaling pathway. Some studies on human Wnt3a have focused primarily on its key role in human malignancy and its high expression in many kinds of tumors. However, little data is available regarding HCC, and the prognostic value of Wnt3a remains to be explored.

Recently, Pan et al found that higher levels of circulating Wnt3a expression (over 800 ng/L) were present in 92.5% HCC patients, and this elevation was significantly related to α-fetoprotein (AFP) level, liver cirrhosis, hepatitis B virus (HBV) infection, poor differentiation, tumor node metastasis, and extra-hepatic metastasis. Wnt3a level in HCC was obviously higher than that in any other group of benign liver diseases, except for 5.67% cirrhosis cases, suggesting that Wnt3a expression was associated with tumor progression and that it may be a novel specific biomarker for diagnosis and differentiation of HCC.

Some studies on human Wnt3a have focused primarily on its key role in human malignancy, and its high expression in many kinds of tumors was correlated with a worse outcome. The present data showed that the clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade, liver cirrhosis, HBV infection, higher tumor-node-metastasis (TNM) stage, and 5-year survival rate, indicating that oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

The authors report for the first time that Wnt3a expression is a novel prognostic marker for HCC. Multiple key factors along the Wnt/β-catenin signaling cascade may also represent potential diagnosis, prognosis, or therapeutic targets. The present findings are promising, and the initial evidence confirmed that Wnt3a is a key molecule in the Wnt/β-catenin pathway and further studies should clarify the molecular mechanisms of the upregulation of oncogenic Wnt3a expression and its important role in hepatocarcinogenesis.

Key molecules in the Wnt/β-catenin signaling pathway have been recognized to play an important role in HCC. These molecules regulate multiple cellular events through the β-catenin-dependent canonical pathway, such as Wnt1, Wnt2, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, and Wnt10b, characterized by the stabilization and subsequent nuclear transport of β-catenin, which result in the activation of transcriptional responses; and the β-catenin-independent non-canonical pathway, including Wnt4, Wnt5a, Wnt5b, Wnt6, ont7a, Wnt7b, and Wnt11, with more diverse and several different signaling modes that regulate cell behavior.

This research study is well presented, and the reviewers enjoyed reading the article, despite its limitations as a retrospective study. The main discovery by the authors will likely generate further interest in the use of Wnt3a as a molecular biomarker in HCC.