Immunopathological And Clinical Factors Associated With The Prognosis Of Primary Hepatic Carcinoma

Primary hepatic carcinoma(PHC)is a highly malignant tumor with poor outcome,which is usually asymptomatic at early stage and diagnosed at late stage.Therefore,the investigation of PHC prognostic relevant factors is crucial to improve the outcome of PHC patients.In this study,we investigated the prognostic associated factors of PHC by analyzing the immunopathological and clinical characteristics in a large sample size to provide the scientific evidence for improving the outcome and survival rate of patients with PHC.Methods:1.Collection of tissue specimens: Tissue specimens were collected from the PHC patients who underwent surgical treatment in Jiangxi Provincial Tumor Hospital,including all-age group or elderly group of patients.All specimens were confirmed as hepatocellular carcinoma(HCC)by pathology.All patients did not receive radiotherapy,chemotherapy and radio frequency ablation before operation.2.The expression of Skp2,p27 and β-catenin in HCC: The expression levels of Skp2 and p27 were measured by immunohistochemical Envision method in cancer tissues from 80 cases of all-age group HCC,and their correlations with clinicopathological characteristics were analyzed.Similarly,the expression levels of Skp2,p27 and β-catenin were measured by immunohistochemical SP method in cancer and corresponding para-cancer tissues from 56 cases of HCC of elderly group,and their correlations with clinicopathological characteristics were also analyzed.3.The expression of PCAF in HCC: The expression levels of protein PCAF were measured in human hepatocyte line LO2 and hematoma cell lines HepG2 and SMMC-7721 by Western blotting and in 40 cases of HCC and corresponding para-carcinoma tissues by RT-PCR and immunohistochemistry.The correlation between the expression of PCAF and clinicopathological characteristics were analyzed.4.Collection of clinical data: The clinical data of 905 PHC patients who hospitalized in Jiangxi Provincial Tumor Hospital from June 1999 to November 2008 with complete follow-up data were collected,including demographic information,etiology data,laboratory test results,imaging findings,treatment and so on.5.Analysis of clinical data: All data were statistically analyzed by using SPSS19.0 statistical software.Measurement data were shown as mean±standard deviation and count data shown as frequency.The prognosis-related factors of PHC were analyzed by univariate analysis.And the survival rates of each factor were compared with logarithmic rank test.The major prognostic factors were selected by using the Cox proportional hazards model.And the survival rates were compared by Kaplan-Meier method.P<0.05 was considered statistically significant.Results:1.The expression of Skp2,p27 and β-catenin in HCC: In all-age group,the positive rate of Skp2 was significantly higher in HCC than that in normal control group(P<0.05),however,the positive rate of p27 was significantly lower in HCC than that in normal control group(P<0.05).The positive rates of Skp2 and p27 in HCC were closely related to histological grade,clinical stage,tumor size and intrahepatic metastasis.The positive rate of Skp2 in HCC was closely related to lymph node metastasis and alpha-fetoprotein(AFP)levels.And the expression of Skp2 was negative correlation with p27 in HCC.In elderly group,the positive rate of Skp2 and β-catenin were significantly higher in HCC than that in normal control group(P<0.001),and p27 was significantly lower in cancer than that in para-cancer tissues(P<0.001).The positive rate of β-catenin in HCC was closely related to intrahepatic metastasis and Edmonson classification(P<0.001).And the positive rate of Skp2 in HCC was closely related to intrahepatic metastasis,Edmonson classification and TNM stage(P<0.001).The positive rate of p27 in HCC was closely related to Edmonson classification and TNM stage(P<0.05).The expression of β-catenin was positively correlation with Skp2 in HCC(r=0.571,P=0.002).And the expression of p27 protein were negatively correlated with β-catenin(r=-0.429,P=0.029)and Skp2(r=-0.667,P=0.001),respectively.2.The expression of PCAF in HCC: The expression of PCAF protein in HepG2 and SMMC-7721 cell lines were significantly higher than that in LO2 cell line(P<0.05).The expression levels of PCAF mRNA and protein in HCC tissues were significantly lower than that in para-cancer tissues(P<0.001).The expression levels of PCAF protein were significantly correlated with intrahepatic metastasis(r=0.413,P=0.037),portal vein invasion(R=0.589,P=0.011)and TNM stage(r=0.513,P=0.029).3.The clinical characteristics of 905 PHC patients:(1)There were 775 cases of male(85.6%)and 130 cases of female(14.4%)in 905 PHC patients,with the male to female ratio of 5.96: 1.00.(2)The highest incidence was in the age of 40-59.(3)441 cases of PHC with right upper abdominal pain as the first clinical symptom,accounting for 53.3%,and 201 cases with upper abdominal pain as the first clinical symptom,accounting for 24.3%.(4)95.1% of patients were infected with HBV,1.7% infected with HCV,and 14.6% were alcohol-related PHC(ADI≥800),1.5% infected with schistosomiasis,and 19.3% were cryptogenic PHC(except the viral,schistosomiasis infected and alcohol-related PHC).(5)496 cases of PHC patients with positive of HBsAg,HBe Ab and HBcAb,accounting for 59.3%,and 751 patients with negative of HBeAg,with the highest rate of 94.3%.(6)Liver function damage: 81.3% PHC patients with abnormally elevated AST;73.4% with abnormally elevated ALT,and the AST level significantly higher than that of ALT.50.7% with abnormally elevated TBIL,92.1% with abnormally elevated GGT,and 52.9% with abnormally elevated ALP.(7)74.4% PHC patients with abnormally elevated AFP,more than half of which with AFP level less than 400ng/ml and about a quarter with AFP negative.The AFP level of HBV infection patients were significant higher than that of HBV negative patients(P<0.01).The AFP levels were significantly different among young,middle-aged and elderly group(P=0.002).(8)57.0% of tumor in the right lobe and 18.7% in the left.Among the tumor type,massive PHC accounted for 40.3%,huge PHC accounted for 32.5%,and nodular type accounted for 15.8%.72.7% with one tumor lesion,14.4% with diffuse type PHC,and 7.1% with two tumor lesions.20.3% with ascites,23% with portal vein tumor thrombus,12% with lymph node metastasis,and 10.5% with distant metastasis.4.Prognosis-related factors analyzed by univariate analysis: A total of 51 factors were significant with PHC prognosis(P<0.05).Categorical data including: history of liver cirrhosis,family history of PHC,physical status score,platelet,total bilirubin,albumin,hepatitis B e antibody,AFP,ascites,classification of portal vein tumor thrombus,tumor thrombus,lymph node metastasis,vascular invasion,intrahepatic metastasis,distant metastasis,Child-Pugh classification,treatment method(simply supporting treatment,giving up treatment,comprehensive treatment,interventional therapy,surgical treatment,radical operation,hepatic artery embolization,hepatic artery perfusion,portal vein pump and alcohol injection),tumor type and tumor edge.Quantitative data included: WBC,NEUT,PLR,DBIL,GLB,AGR,ALT,AST,AST/ ALT,APRI(AST/ALT),ALP,LDH,GGT,Pche,Cr,Na +,CL-,PT,INR,Fbg,the largest diameter of tumor,tumor numbers and the thickness of spleen.5.Prognosis-related factors analyzed by multivariate analysis: The independent prognostic factors of PHC include tumor thrombus,simply supporting treatment,comprehensive treatment,radical operation,WBC,DIBL and LDH levels.Tumor thrombus,simply supporting treatment,and high levels of WBC,DIBL and LDH were related to poorer prognosis,while comprehensive treatment and radical operation were the protective factors.Conclusions:(1)The expressions of Skp2 and p27 in HCC were closely associated with the histopathological parameters related to prognosis and the expression of β-catenin,suggesting that Skp2,P27 and β-catenin have synergistic effects in development and metastasis of HCC and they are potential biomarkers for predicting HCC outcome.(2)The expression of PCAF was significantly lower in hepatoma cell lines than in human normal hepatocyte cell line and in HCC tissues than in para-cancer tissues,and positively correlated with intrahepatic metastasis,portal invasion and high TNM stage,suggesting that PCAF is closely related to the prognosis of HCC.(3)The PHC in this region was more frequent in male than in female;the highest incidence of PHC was at age 40-59 years old;the main cause was hepatitis B virus infection and usually HBeAg negative;right upper abdominal pain was the initial symptom;more than half of patients had AFP levels less than 400ng/ml and one fourth patients was AFP negative.Therefore,males over 40 years who have abdominal discomfort,HBV infection and HBeAg negative should be alert to PHC.(4)Tumor thrombus,simply supporting treatment,comprehensive treatment,radical operation,elevated levels of WBC,DIBL and LDH were independently prognostic factors of PHC,in which comprehensive treatment is a favorable factor,suggesting comprehensive treatment is beneficial to improve the outcome of patients with advanced PHC.