| Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which seriously threat the life of human being. China is among the high incidence area of HCC, with an incidence of 20.37 per 100 thousand which is the third highest incidence in common tumors. According to the statistical data collected in 2002, HCC's incidence ranked the sixth in common cancers while its mortality ranked the third; the annual incidence cases of HCC was 626 thousand which showed a 5.7% increase, while the number of death was 598 thousand, among which 82% of the cases appeared in developing country, especially China enjoyed 55%. Including surgical resection, liver transplantation, vascular intervention, ablation technology, radiotherapy, chemotherapy and the great breakthroughs in drug therapy, there are a lot of therapies for HCC in clinic practice, but still surgical resection is considered as the best therapy for HCC's radical operation. Even with radical resection, there are still 60-70% patients who appeared metastasis and recurrence in 5 years after operation, while the percentage of metastasis and recurrence for local treatment is higher. Metastasis and recurrence have become the crux of further improvement on survival rate of HCC patients. As a result, to seek and predicate HCC's metastasis and recurrence tendency, judge prognosis of HCC patients is the key and focus of HCC's research, as well as its emphasis and difficulty.Epithelial cell adhesion/activating molecule (EpCAM; also known as CD326, C017-1A, EGP, EGP40, GA733-2, KSA, Ly74,M1S2, M4S1, MIC18, MK-1, TROP1, hEGP-2), one of the first tumor-associated antigens identified, is encoded by the TACSTD1 gene EpCAM is highly expressed in a large variety of human adenocarcinomas and squamous cell carcinomas. Although the function of EpCAM and its regulatory mechanism are largely unknown, it has been implicated in cell-cell adhesion and may act as an oncogenic signaling protein. Our recent gene expression profiling study indicates that EpCAM may serve as an early biomarker of HCC because its expression is highly elevated in premalignant hepatic tissues and in a subset of HCC. Similar results were observed by others, reiterating the significance of EpCAM in HCC development. In addition, using gene expression profiling and pathway analysis, we also showed that EpCAM-positive HCC displayed a distinct molecular signature with features of hepatic progenitor cells (HPCs), including the presence of known stem/progenitor markers (e.g., c-Kit, CK19, vimentin, CD133, and a-fetoprotein) and the activation of Wnt-hcatenin signaling.1 It seems that there is a close functional link between EpCAM expression and the activation of Wnt-h-catenin signaling. It is known that mature hepatocytes are negative for EpCAM expression, In contrast, the majority of hepatocytes in the embryonic liver show EpCAM expression. Several recent studies indicate that EpCAM may serve as a hepatic stem cell marker.Hepatocyte nuclear factor 4 alpha (HNF4alpha, NR2A1, gene symbol HNF4A) is a highly conserved member of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors1 and an essential gene in early development as well as in the adult HNF4alpha is expressed in the adult liver, kidney, pancreas, intestine, colon and to a lesser degree in the stomach, as well as in the visceral endoderm. HNF4alpha is a nuclear protein that binds DNA exclusively as a homodimer it also exhibits strong homodimerization activity in the absence of DNA and cannot heterodimerize with retinoid X receptor (RXR), unlike other nuclear receptors HNF4alpha activates the expression of hundreds of genes, especially those involved in intermediary metabolism, including glucose, fatty acid, cholesterol and xenobiotic and drug metabolism There are two promoters (P1 and P2) that drive the expression of at least 6 different HNF4alpha splice variants (often referred to as isoforms) that exhibit varying levels of transactivation activity and are expressed in a temporal and spatial-specific fashion. A recent report indicates that HNF4alpha binds a single fatty acid (linoelic acid, LA, C18:2) in a reversible fashion, and that apoHNF4alpha exists in physiological conditions 12. This finding suggests that HNF4alpha may be a potential drug target, as are many of the other NRs. HNF4alpha is at the center of a complex network of transcriptional control involving other HNFs and NRs and has been directly linked to several human diseases including diabetes, hemophilia and hepatitis B viral infections.This research intends to test EpCAM's distribution and expression in human's comm -on tumor tissues and adjacent tissues by using tissue chip technology and immunohistochemical(IHC) method, also analyze the difference of EpCAM's expression in HCC and its adjacent tissues, to seek monitoring method for HCC's metastasis/recurrence and prognosis evaluation by analysis on the relationship between EpCAM and HNF4alpha 's expression in HCC patients and their clinical pathological parameters combined with retrospectively analysis on the relationship between EpCAM and HNF4alpha's expression and disease-free survival and over survival time of HCC patients. All of thoses could provide the preliminary theory basis for the diagnosis, treatment evaluation of HCC and post-operation monitor, prognosis judgment as well as the choice of therapy target.Methods:1. The distribution and expression of EpCAM protein in human common malignant tumors and adjacent tissuesBy using tissue chip technology and immunohistochemical method, the distribution and expression of EpCAM protein were detected in human malignant tumors and their adjacent tissues. Using tissue chip technology, combined with immunohistoche -mistry, the expression of EpCAM protein were detected in human HCC tissues and their adjacent tissues, while the difference of EpCAM's expression level between HCC tissues and their adjacent tissues was analyzed.2. The expression of EpCAM protein in HCC tissues and its clinical significanceThe expression of EpCAM Protein in HCC tissues (86 cases) were detected by IHC method, and the relationships of the expression of EpCAM protein of HCC patients with their clinical-pathologic features were analyzed. Survival analy -sis was used to analyze the relationship of EpCAM protein expression with HCC patients' disease-free survival and over survival time retrospectively. Cox proportional-hazard analysis was used for univariate and multivariate analysis to explore the effect of variables on HCC patients' survival time.3. The expression of HNF4alpha protein in HCC tissues and its clinical significanceThe expression of HNF4alpha Protein in HCC tissues (86 cases) were detected by IHC method, and the relationships of the expression of HNF4alpha protein of HCC patients with their clinical-pathologic features were analyzed. Survival analy-sis was used to analyze the relationship of HNF4alpha protein expression with HCC patients' disease-free survival and over survival time retrospectively. Cox proportional-hazard analysis was used for univariate and multivariate analysis to explore the effect of variables on HCC patients' survival time.4. Correlation analysis of EpCAM protein and HNF4alpha protein in HCC tissues, the relationship of two protein expression with HCC patients'disease-free survival and over survival timeThe expression of EpCAM protein and HNF4alpha protein in HCC tissues (86 cases) were analysis by Spearmen correlation analysis; the relationship of two protein expression with HCC patients'disease-free survival and over survival time5. Statistical methodsThe difference of EpCAM protein's expression rate and intensity between HCC tissues and their adjacent tissues was analyzed by Chi-square test and Wilcoxon rank sum test respectively. And the relationships of the expression of EpCAM and HNF4alpha protein of HCC patients with their clinical-pathologic features were analyzed using Chi-square test or Fisher's exact test. Kaplan-Meier method was used to analyze the relationship of EpCAM and HNF4alpha protein expression with HCC patients' disease-free survival and over survival time retrospectively. Cox proportional-hazard analysis was used for univariate and multivariate analysis to explore the effect of variables on HCC patients' disease-free survival and over survival time. The expression of EpCAM protein and HNF4alpha protein in HCC tissues were analysis by Spearmen correlation analysis.The SPSS 13.0 software was used for all statistical analyses and a P value of≤0.05 was considered significant.Results:1. The distribution and expression of EpCAM protein in human common malignant tumors and adjacent tissuesExpressions of EpCAM protein in colorectal carcinoma, esophageal squamous cell carcinoma, lung squamous cell carcinoma,gastric carcinoma, pancreatic carcinoma, renal carcinoma, cervical carcinoma were strongly,and expressions of EpCAM protein in and several other tumor adjacent tissues were weak. The positive rate of EpCAM protein in HCC tissues was significantly higher than adjacent tissues (P<0.05).2. The expression of EpCAM protein in HCC tissues and its clinical significanceThe expression of EpCAM protein was found in cytoplasm and cell membrane. The positive rate of EpCAM protein was89.4%, while the high-expression rate was 55.9%. The proportion of different levels of EpCAM expression in HCC tissues were as follows:"-" 10.6%(9/86),"+" 32.5%(28/86),"++" 23.4%(21/86),"+++" 32.5%((28/86). The EpCAM protein expression correlated closely with metastasis /recurrence(P<0.05), while not correlated with liver cirrhosis, histological differentiation, TNM staging and gender, age, serum HBsAg, serum AFP, tumor size(P> 0.05). Comparing to the HCC patient with EpCAM protein high-expression risk of metastasis/recurrence was high. The median disease-free survival time of HCC patients who were detected high-expression EpCAM protein was 4.0 months, while the HCC patients with low-expression EpCAM protein was 29.0 months. And the median disease-free survival time of HCC patients with high-expression EpCAM protein was significantly lower than HCC patients with low-expression EpCAM protein (P<0.05).To identify the variables of potential prognostic significance in HCC patients,univaritate and multivariate analysis of each variable was performed in relation to the disease-free survival time. EpCAM protein, serum AFP and TNM staging were found affect the disease-free survival time of HCC patients (P<0.05).To identify the variables of potential prognostic significance in HCC patientS,univaritate analysis of each variable was performed in relation to the over survival time. EpCAM protein, serum AFP, liver cirrhosis,tumor size, metastasis/recurrence were found affect the over survival time of HCC patients (P< 0.05). Above factors were then determined by multivariate Cox proportional hazards model analysis. Multivariate analysis results showed that metastasis/recurrence, serum AFP might play a role in predicting the over survival time in HCC patients(P <0.05)3. The expression of HNF4alpha protein in HCC tissues and its clinical significanceThe expression of HNF4alpha protein was found in nuclear and cytoplasm. The positive rate of HNF4alpha protein was 81.4%, while the high-expression rate was 46.5%. The proportion of different levels of HNF4alpha expression in HCC tissues were as follows:"-"18.6%(16/86),"+" 34.9%(30/86),"++" 31.3%(27/86),"+++" 15.2%((13/86). The HNF4alpha protein expression correlated closely with serum AFP,histological differentiation (P<0.05), while not correlated with serum HBsAg, gender, age, liver cirrhosis, TNM staging, metastasis/recurrence and tumor size(P> 0.05). The median disease-free survival time of HCC patients who were detected high-expression HNF4alpha protein was 22.0 months, while the HCC patients with low-expression HNF4alpha protein was 4.0 months. And the median disease-free survival time of HCC patients with high-expression HNF4alpha protein was significantly longer than HCC patients with low-expression HNF4alpha protein (P<0.05).To identify the variables of potential prognostic significance in HCC patients,univaritate analysis of each variable was performed in relation to the disease-free survival time. HNF4alpha protein, serum AFP, TNM staging were found affect the disease-free survival time of HCC patients (P<0.05). Multivariate analysis results showed thatTNM staging, serum AFP might play a role in predicting the disease-free survival time in HCC patients(P<0.05)To identify the variables of potential prognostic significance in HCC patients,univaritate analysis of each variable was performed in relation to the over survival time. HNF4alpha protein, metastasis/recurrence,liver cirrhosis, serum AFP, tumor size were found affect the over survival time of HCC patients (P<0.05). Above factors were then determined by multivariate Cox proportional hazards model analysis. Multivariate analysis results showed that HNF4alpha protein, metastasis/recurrence might play a role in predicting the over survival time in HCC patients(P<0.05)4. Correlation analysis of EpCAM protein and HNF4alpha protein in HCC tissues, the relationship of two protein expression with HCC patients' disease-free survival and over survival times clinical significanceEpCAM protein expression was negatively associated with HNF4alpha protein r=-0.226, P=0.038). The median disease-free survival time of HCC patients who were detected high-expression EpCAM+ low-expression HNF4alpha protein, high-expression EpCAM+ high-expression HNF4alpha protein, low-expression EpCAM+ low-expression HNF4alpha protein, low-expression EpCAM+ high-expression HNF4alpha protein were 3.5,12.0,8.0,36.0 months respectively. The disease-free survival time of HCC patients who were detected high-expression EpCAM+ low-expression HNF4alpha protein was significantly lower than others HCC patients(P<0.05).The over survival time of HCC patients who were detected high-expression EpCAM+ low-expression HNF4alpha protein was significantly lower than low-expression EpCAM+ high-expression HNF4alpha protein patients(P=0.001). The over survival time of HCC patients who were detected low-expression EpCAM+ high-expression HNF4alpha protein was significantly longer than high-expression EpCAM+ low-expression HNF4alpha protein patients(P=0.048). Conclusions:1. EpCAM protein is highly expressed in HCC and several other tumor tissues, hints that EpCAM may have a very important affect in HCC and epithecium cancer,So the role of EpCAM in the development and progress of tumor are complex, and worth further research.2.EpCAM protein were detected highly expressed in HCC tissue, while low expressed in several other liver disease, such as hepatitis, liver cirrhosis, fatty hepatic and so on. EpCAM may be used as an reference valuable biomarkers on the diagnosis and differential diagnosis of HCC.3. The EpCAM protein expression correlated closely with metastasis/recurrence, and EpCAM protein can affect the prognosis of HCC.Metastasis/recurrence after operation found in HCC patients with high expression of EpCAM protein was significantly increased. Thus, EpCAM may participate in the development and progress of HCC, as well as monitors metastasis/recurrence of HCC patients and predicts prognosis.4. The HNF4alpha protein expression correlated closely with serum AFP, histological differentiation,and HNF4alpha protein can affect the prognosis of HCC.Metastasis/recurrence after operation found in HCC patients with lower expression of HNF4alpha protein was significantly increased. Thus, HNF4alphamay participate in the development and progress of HCC, as well as monitors metastasis /recurrence of HCC patients and predicts prognosis.5. EpCAM protein expression was negatively associated with HNF4alpha protein, the relationship of two protein maybe was competition or inhibition in the development and progress of HCC. The expression of EpCAM protein and HNF4alpha protein were detected at the same time could increase predicts prognosis. |
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