| BackgroundPrimary liver cancer is one of most common malignant tumors in the world, which threatens public health. Hepatocellular carcinoma (HCC) is the most common liver cancer. It is the fifth most common malignant tumor and the third most usual cause of cancer-related death worldwide after pulmonary cancer and gastric cancer. Almost one million people die of HCC every year. The number of patients with liver cancer in China accounted for43.7%of the worldwide incidence. There are about360000new patients every year, and more than300000patients die every year. It is the third most common malignant tumor and the second most common cause of cancer-related death in China.There are no specific symptoms and signs of HCC. Less than50%HCC patients were diagnosed at early stages, while most diagnosis was too late to operate effectively. Diagnosis of HCC mostly depends on CT, ultrasound, MRI and serum a-fetoprotein (AFP), however, the sensitivity and the specificity of AFP were relatively low. Elevated AFP also could be found in parts of patients with liver cirrhosis and patients with chronic hepatitis. Furthermore, AFP was negative in some HCC patients. Recent studies showed AFP variants and Glogi Protein73could be used as new tumor markers of HCC, but they could not be used in general survey because of low specificity and high price. Identification of new high-quality serum markers is of importance, which can be used in general survey. Thus, more patients may be diagnosed at early stages, and it may improve the prognosis of HCC patients.There are several treatment methods for HCC, including surgery, radiotherapy, chemotherapy, and interventional embolization. Surgical resection is still the optimal treatment for HCC. The long-term survival of HCC patients remains dismal due to high postoperative recurrence rate. Identification of tumor markers for tumor recurrence and survival of HCC patients is very important. Firstly, it can be used to evaluate prognosis of HCC patients. Secondly, it will be helpful to select patients at risk for postoperative adjuvant therapy. Thus, it can prolong survival time of HCC patients and improve prognosis. However, there is no ideal tumor marker which can predict prognosis of HCC at present. Pathological staging system is the most common tool to evaluate prognosis, while it's not precise enough. Patients with the same stage HCC are often associated with different results, especially for those at early stages.The chemokine ligand (CCL20), also termed liver-and activation-regulated chemokine (LARC) or macrophage inflammatory protein-3a (MIP-3a), belongs to the family of CC-chemokines. The chemokine receptor6(CCR6) is the unique receptor for CCL20. It is mostly expressed in liver, lung, colon, and skin. The well-known function of CCL20is to recruit immature dendritic cells to inflammatory sites. Recent studies suggested that CCL20was involved in the development of many malignant tumors, including oral squamous-cell carcinoma, colorectal cancer, pancreatic carcinoma, nasopharyngeal carcinoma, and prostate cancer. High expression of CCL20was also found in cell lines of HCC. CCL20was significantly overexpressed in HCC tissue than in peritumoral liver tissue and normal liver tissue. It could promote the growth of HCC cells in vitro, and enhance the migration of HCC cells. Serum CCL20was related to tumor recurrence after radiofrequency therapy. Thus, CCL20may play an important role in the carcinogenesis, invasion, and development of HCC, however, the diagnostic and prognostic value of CCL20remains unclear for HCC.In the present study, we aimed to detect serum CCL20in different populations by enzyme-linked immunosorbent assay (ELISA), to analyze the relationship between serum CCL20and clinicopathological features, and to evaluate the diagnostic significance of CCL20for HCC. Meanwhile, the other aim of this study was to determine expression of CCL20by immunohistochemical staining in both intratumoral tissue and peritumoral liver tissue to evaluate its prognostic value. It may provide a new high-quality tumor marker for HCC and basic evidences for evaluation of prognosis and selection of postoperative patients at risk. Part I Diagnostic value of serum CCL20for hepatocellular carcinomaObjective1. To compare the levels of serum CCL20and AFP in HCC patients, patients with liver cirrhosis, patients with chronic hepatitis, and healthy controls, analyze the relationship between CCL20and AFP in HCC patients, and investigate the short-term change of CCL20after surgery.2. To determine the correlation of CCL20with clinicopathological characteristics of HCC.3. To evaluate the diagnostic value of serum CCL20for HCC.Methods1. From September2011to January2012,78HCC patients,25patients with liver cirrhosis,24patients with chronic hepatitis, and33healthy controls who presented to Qilu Hospital were enrolled in the study. The levels of serum CCL20were detected using ELISA, and AFP levels were measured using electrochemical luminescence immunoassay. The difference among groups was examined by Kruskal-Walis H test, and the differences between two groups were tested by Mann-Whitney U test. The correlation of CCL20with AFP was determined using Spearman analysis. The levels of serum CCL20were detected on postoperative days1,3,5,7and14in HCC patients.2. Clinical data of78HCC patients were collected prospectively and analyzed retrospectively. The relationships between CCL20and clinical features were analyzed by using Kruskal-Walis H test or Mann-Whitney U test.3. The diagnostic value of CCL20, AFP and the combination of CCL20and AFP was evaluated by using the ROC curve. The optimal cutoff points were determined according to Yuden index. The area under the ROC curves was compared by using Z test.Results1. There were significant differences among four groups in serum CCL20and AFP (Both P<0.001). The levels of serum CCL20and AFP were significantly higher in patients with HCC than those in patients with liver cirrhosis, patients with liver cirrhosis and healthy controls (All P<0.050). The levels of serum CCL20and AFP were higher in patients with liver cirrhosis and chronic hepatitis than those in healthy controls (Both P<0.050). There was no obvious difference in the levels of serum CCL20and AFP between patients with liver cirrhosis and patients with chronic hepatitis (P>0.005). The levels of serum CCL20were related with the levels of AFP in HCC patients and those with liver cirrhosis (P=0.005and0.007, respectively) other than those with liver cirrhosis and healthy controls (P=0.519and0.304, respectively). The levels of CCL20in HCC patients were decreased obviously one week after surgery and slowly in the second week, while they were still higher than those in healthy controls (P=0.040).2. The levels of CCL20in HCC patients were related with tumor size (127.8pg/ml vs89.50pg/ml, P=0.007), vascular invasion (134.3pg/ml vs85.70pg/ml, P=0.005), encapsulation (133.4pg/ml vs71.95pg/ml, P<0.001), and differentiation (128.4pg/ml vs85.30pg/ml, P=0.029).3. The area under the ROC curve for CCL20was0.892with an optimal cutoff point of63.50pg/ml, providing a sensitivity of82.1%, a specificity of87.8%, a positive predictive value of86.5%, a negative predictive value of83.7%and an accuracy rate of85.0%. The area for AFP was0.793with an optimal cutoff point of108.1ng/ml, providing a sensitivity of82.1%, a specificity of63.4%, a positive predictive value of68.1%, a negative predictive value of78.8%and an accuracy rate of64.4%. The area for CCL20was significantly larger than that of AFP (P=0.021). The area for combined CCL20and AFP was0.895. There was no significant difference in the area between CCL20and combined CCL20and AFP (P=0.933).Conclusion1. The levels of serum CCL20were increased in HCC patients and were related with the levels of AFP. The levels of CCL20were decreased within two weeks after surgery. CCL20may be related to tumorigenesis of HCC.2. Serum CCL20was associated with biological behaviors. It may play a role in tumorigenesis and development of HCC.3. The diagnostic value of CCL20was higher than that of AFP. It may be a promising tumor marker for HCC. Part â…¡ Prognostic value of CCL20expression in both intratumoral tissue and peritumoral liver tissue for hepatocellular carcinomaObjective1. To compare the expression levels of CCL20in intratumoral tissue and peritumoral liver tissue.2. To investigate the relationships between expression of CCL20in both intratumoral tissue and peritumoral liver tissue and clinicopathological characteristics.3. To analyzed the correlation of expression of CCL20in both intratumoral tissue and peritumoral liver tissue with survival of HCC after curative resection.4. To determine the association of CCL20expression with postoperative early tumor recurrence.Methods1. One-hundred and twenty five HCC patients were consecutively selected at Qilu Hospital between January2005and August2006. Expression of CCL20was detected by immunohistochemical staining. The difference of expression of CCL20in intratumoral tissue and peritumoral liver tissue was compared by using Mann-Whitney U test.2. Clinical data of these patients were reviewed retrospectively. The relationships between CCL20expression and clinicopathological features were analyzed using Chi-square test.3. Follow-up data of these patients were collected retrospectively. The recurrence-free survival rate and overall survival rate were calculated by the Kaplan-Meier method and compared by using log-rank test in univariate analysis. Cox proportional hazard model was used to identify independent factors for survival of HCC patients in multivariate analysis. The ROC curve was used to determine the predictive value of these independent factors.4. The Chi-square test was used to identify factors associated with early recurrence in univariate analysis, and logistic regression was adopted to identify independent factors of early recurrence in multivariate analysis. The ROC curve was used to determine the predictive value of these factors. Results1. CCL20was mostly expressed in cytoplasma both in intratumoral tissue and in peritumoral liver tissue. The expression of CCL20was significantly higher in intratumoral tissue than that in peritumoral liver tissue (P<0.001).2. CCL20expression in intratumoral tissue was associated with tumor size (P=0.002), tumor number (P=0.031), vascular invasion (P=0.003), and tumor differentiation (P=0.024). CCL20expression in peritumoral liver tissue was related to tumor encapsulation (P=0.014) and vascular invasion (P=0.041).3. Univariate analysis showed the recurrence-free survival rate and the overall survival rate were higher in patients with low expression of CCL20in intratumoral tissue than those in patients with high expression of CCL20(61.9%vs21.0%and71.4%vs37.1%, both P<0.001). Multivariate analysis indicated CCL20expression in intratumoral tissue was an independent factor for recurrence-free survival and overall survival (Hazard ratio2.573, P=0.001and Hazard ratio2.930, P=0.001, respectively). CCL20expression in intratumoral tissue could be used to predict tumor recurrence (The area under the ROC curve=0.711). Expression of CCL20in peritumoral liver tissue was not associated with recurrence-free survival (P=0.225) or overall survival (P=0.574) of HCC patients.4. Univariate analysis showed the early recurrence rate was higher in patients with high expression of CCL20in intratumoral tissue than that in patients with low expression of CCL20(48.4%vs9.52%, P<0.001). Multivariate analysis suggested CCL20expression in intratumoral tissue was an independent indicator for early tumor recurrence (Hazard ratio8.760,95%Confidence interval2.952-25.99, P<0.001). ROC curve analysis confirmed CCL20expression in intratumoral tissue could be used to predict early recurrence for HCC (The area under the ROC curve=0.737). Expression of CCL20in peritumoral liver tissue was not associated with early tumor recurrence after curative resection (P=0.062).Conclusion1. CCL20was mostly expressed in cytoplasm, and expression of CCL20was enhanced in intratumoral tissue. It was associated with biological behaviors of HCC, and it may be involved in tumorigenesis, invasion and development of HCC.2. Expression of CCL20in intratumoral tissue was associated with recurrence-free survival, overall survival, and early recurrence. It was an independent prognostic indicator for HCC. It can be used to predict early recurrence after surgery and select patients at risk for postoperative adjuvant therapy. |
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