| Background and Aims:Primary liver cancer is the sixth most common cancer in the world, and accounts for the third most frequent cause of cancer-related death. China has a high incidence of liver cancer. More than half of the new cases are confirmed in China every year. Although better detection methods and various treatment options have been used to control HCC, it still shows a high rate of recurrence and metastasis and the prognosis is poor. The main reason is that HCC exhibits significant heterogeneity. Patients with similar clinical feature and diagnosis may have very different prognosis after receiving the same treatment.In recent years, with the progress of cancer stem cell(CSC) research, studies have provided evidences that tumor heterogeneity is closely related with differentiation of cancer stem cells. CSCs are small subpopulation of cancer cells which have the ability of self-renewed and pluripoteney. It is generally believed that the activation of hepatic progenitor cells(HPCs) is closely related with hepatocarcinogenesis. HPCs is considered to be one of the targets for carcinogenesis. Progenitor cells can differentiate into hepatocytes and cholangiocytes. When a progenitor cell on its way to differentiation develops into cancer, this can give rise to tumours with a whole range of phenotypes with varying hepatocellular and cholangiocellular differentiation characteristics. This makes liver cancer has high heterogeneity. The different directions of HPCs differentiation determines the pathological type of liver cancer.The World Health Organization(WHO) classifies primary liver cancer into hepatocellular carcinoma(HCC), intrahepatic cholangiocarcinoma(ICC), and combined hepatocellular-cholangiocarcinoma(CHC). The prognosis of HCC is better than ICC and CHC, which indicates that different pathological subtype affects patients prognosis. Recently, many studies have proved evidences that HCC with stem/progenitor cell immunophenotype had poor prognosis and more aggressive behavior. Now more and more studies classify HCC with molecular characteristics.Hepatocyte nuclear factor-1beta(HNF1β) is a homeobox transcription factor that functions as a homodimer or heterodimer with HNF1α. It is a nucleoprotein. In adult, HNF1β is strongly expressed in biliary epithelium. Hepatocyte nuclear factors play essential roles in the liver-specific expression of many genes during liver development. Hepatic progenitor cells(HPCs) are bipotential cells capable of proliferation and differentiation into both hepatocellular and biliary cell lineages. HNF1β is involved in the hepatobiliary specification of hepatic progenitor cells to cholangiocytes during liver development. Recently, it has been shown that the expression of HNF1β is associated with cancer risk of several tumors. However, the study of HNF1β in liver cancer is still limited. As a specific marker of cells from the biliary compartment, HNF1β is only expressed in cholangiocytes, not in mature hepatocytes in aldults. So we hypothesize that the expression of HNF1β may related to the pathological type and prognosis in patients with primary liver cancer.In the present study, we analyzed the expression of HNF1β in HCC and ICC using immunohistochemistry, and compared the difference between HNF1β expression in the two pathologic type. Then we investigated the relationship between HNF1β expression and prognosis of patients with HCC, also the potential mechanisms.Methods: Patients and histopathologic analysis.We retrospectively investigated 2 cohorts of patients with primary liver cancers, including 183 HCCs and 69 ICCs. All the two cohorts were randomly collected from patients with liver cancer who underwent hepatectomy.All patients were followed regularly after surgery. Immunohistochemistry and evaluation. Immunohistochemical staining was carried out on both tumor and nontumor tissue slides. We examined a proportion of tumor cells that were positive for HNF1β in the tumor cells nuclei. We used the criteria to classify and analyze the clinical data; 0 for < 5%, 1+ for 5% to 25%, 2+ for 25% to 50%, and 3+ for ≧ 50%. For the staining of HPC markers(K7, K19, Ep CAM and OV6), images of positive representative fields were captured under 200 x magnification, and the density of immunostaining was measured using Image-Pro Plus Version 6.2 software. The proliferation rate in DR was evaluated by calculating the proliferating cell nuclear antigen(PCNA) labeling index.Statistical analysis. Statistical analysis was performed using SPSS software version 12.0(SPSS, Chicago, IL). Clinicopathologic characteristics among negative and positive HNF1β expression were compared using χ2 test or Fisher’s exact probability test. The degree of association was determined by Spearman correlation. OS and DFS were calculated by Kaplan-Meier method, and differences in survival rate were compared using Log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. P <0.05 was considered significant.Results: Clinical and pathological features. Of the 183 HCC patients, 169(92.3%) had recurrence. We found that 15 of the 183 patients were diagnosed as HCC primarily at the first surgery while their recurrence changed to ICC in the subsequent surgeries. HNF1β expression levels in different pathologic type of primary tumor. Of the 69 ICC patients, 59 patients(85.5%) showed HNF1β positive staining. While of the 183 HCC patients, 82 patients(44.8%) showed HNF1β positive staining. The level of HNF1β expression was lower in HCC patients than in ICC patients The association of HNF1β expression in primary tumor with different pathologic subtypes of recurrent tumor. In sections of the pathological transition group, all patients showed HNF1β positive staining, with 12 patients in 3+, 1 patient in 2+,and 2 patients in 1+. The level of HNF1β expression was higher in pathological transition patients than in non-pathological transition patients. Association of HNF1β expression in HCC with clinicopathological parameters. High HNF1β expression was significantly associated with poorly histological grade. Univariate and multivariate analyses of prognostic variables in HCC patients. In univariate analysis, factors with P<0.100 were selected for further multivariate Cox proportional hazards analysis. Multivariate analysis indicated that tumor HNF1β expression, tumor K7 expression, tumor size, tumor number, and microvascular invasion were independent prognostic factors for overall survival. Tumor HNF1β expression, TNM stage, and microvascular invasion were independent predictors for disease-free survival. The association of HNF1β expression in HCC with expression of HPCs and biliary markers. The Spearman’s correlation analysis found that the expression of HNF1β in HCC tissue had a positive correlation with expression of K7(r=0.463, P<0.001), K19(r=0.286, P<0.001), Ep CAM(r=0.210, P=0.004), and OV6(r=0.279, P<0.001). HNF1β expression in nontumor tissues and the association with PI-DR and DFS. The Spearman’s correlation analysis found that the expression of HNF1β in nontumor tissue had a positive correlation with PI-DR(r=0.312, P<0.001). Patients with high nontumor HNF1β expression were likely to be with poor DFS.Conclusion: All of the results revealed that:1. Expression of HNF1β is different in HCC and ICC tissue. HNF1β is highly expressed in ICC and only a few of HCC. The level of HNF1β expression was lower in HCC patients than in ICC patients 2. HNF1β expression may be associated with the change of pathological subtype during recurrence. 3. According to the expression of HNF1β, the HCC patients were divided into two subtype. Patients with high HNF1β expression were likely to be with poor prognosis, which was associated with the stem/progenitor cell immunophenotype. HNF1β positive cells retain some progenitor-like characteristics. |
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