Evaluation Of Prognostic Markers For Tumor Recurrence And Survival Of Patients With Early-stage Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is a common malignancy worldwide.Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients.Like other cancers,the progression and metastasis of HCC is a multifactorial and multistage pathogenesis.The metastasis of HCC also depends on multiple interactions of metastatic cells with favorable host microenviroment.Although many molecular markers have identified and shown potential prognostic values for HCC patients,so far,no any biomarker has been used to clinical routine practice. Investigating the tumor itself as well as the favorable host microenviroment maybe useful for understanding the mechanisms of HCC metastasis and prognosis prediction. In this study,based on the previous findings,we select the invsion-related molecules including osteopontin(OPN) and CK19,and inflammatory/immune-related cytokines such as IFNG and IL-2,and also tumor-infiltrated lymphocytes to validate their predictive value in early-stage HCC.Meanwhile,we study the correlation of their balance to HCC prognosis in order to understand metastasis of HCC in a new way.PartⅠEvaluation of the prognostic capabilities of traditional staging systems of hepatocellular carcinomaObjective The aim of this study was to compare the ability of different staging systems(American Joint Committee on Cancer/International Union Against Cancer [AJCC/UICC],China classification system[CS],Japanese TNM,United Network for Organ Sharing[UNOS],Cancer of the Liver Italian Program[CLIP],Japan Integrated Staging[JIS],and Barcelona Clinic Liver Cancer[BCLC]) to predict survival and recurrence after liver resection for hepatocellular carcinoma(HCC).Methods Five hundred and fifteen consecutive patients who underwent liver resection for HCC were identified between March 2002 and March 2003.End points were overall survival(OS) and recurrence-free survival(RFS).Sequential stage-wise discrimination of each system was evaluated using Cox regression. Results Four-year overall survival rates were 58%;recurrence-free survival rates were 49%.In the univariate analyses of various clinical variables in the cohort,tumor number,tumor size,vascular invasion,Edmondson-Steiner grade,alpha-fetoprotein (AFP),and several staging systems(TNM,CLIP,BCLC,LCSGJ,JIS,UNOS-TNM and CS) were revealed to be significant predictors for survival;tumor number,tumor size,vascular invasion,AFP and several staging systems(TNM,CLIP,BCLC, LCSGJ,JIS,UNOS-TNM,and CS) were significant predictors for tumor recurrence after surgery.The multivariate Cox regression model for survival showed that Edmondson-Steiner grade,JIS stage and UNOS-TNM were main prognostic factors in HCC.However,the multivariate Cox regression model for recurrence revealed that AJCC/UICC and BCLC were independent predictors of recurrence.Conclusions The Edmondson-Steiner grade,JIS and UNOS TNM staging system can provides the discrimination of survival in patients undergoing liver resection for HCC. The AJCC/UICC and BCLC were independent predictors of recurrence for HCC.PartⅡThe prognostic significance of preoperative plasma levels of osteopontin in patients with early-stage hepatocellular carcinomaObjective to evaluate the prognostic value of preoperative plasma osteopontin(OPN) levels in patients with HBV-positive early-stage hepatocellular carcinoma(HCC).Methods Plasma OPN levels were detected by ELISA in 68 patients with HBV-positive early-stage HCC.Results The median plasma OPN level of patients was 82.51 ng/ml(range 63.15-110.45 ng/ml).Plasma OPN levels in patients with tumor size≥5 cm in diameter were significantly higher than that of patients with tumor size＜5 cm in diameter(104.76 ng/ml versus 75.16 ng/ml,P=0.003).100 ng/ml as cutoff can divide the patients into two groups:the higher plasma OPN group and the lower plasma OPN group.The higher plasma OPN group recurrence rate was significantly higher than the lower plasma OPN group(52.2%versus 24.4%,P=0.022).Meanwhile 20 ng/ml as cutoff can divide the patients into two groups:the higher serum AFP group and the lower serum AFP group.The two groups recurrence rate was 45.5%and 12.5% respectively,P=0.006.A higher plasma OPN level was a leading independent prognostic factor for both overall survival(OS) and recurrence-free survival(RFS) in univariate and multivariate Cox models.Conclusions The preoperative plasma OPN level and serum AFP level in patients with HBV-positive early stage hepatocellular carcinoma can be used as a predictive marker for early stage HCC recurrence and prognosis.PartⅢThe prognostic significance of CD8+ lymphocytic infiltration in patients with early-stage hepatocellular carcinomaObjective To evaluate the prognostic value of CD8+ lymphocytic infiltration,CK19 and MVD in patients with HBV-positive early-stage hepatocellular carcinoma(HCC).Methods The clinical data of 141 patients with HBV-positive early-stage hepatocellular carcinoma were collected.Immunohistochemistry was used to detect the CD8+ lymphocytic infiltration,CK19 and MVD in intratumoral and peritumoral tissues.Results CK19 and CD34 labelled MVD failed to show correlation with prognosis. CD8+ lymphocytes in peritumoral tissues failed to show correlation with the clinicopathological factors but CD8+ lymphocytes in intratumoral tissues showed adverse correlation with size of tumor(r=-0.227).Eighteen per 200×field was defined as the cutoff,CD8+ lymphocytes in intratumoral areas other than in peritumoral areas was significantly associated with HCC recurrence rates.CD8+ lymphocytes in intratumoral areas was significantly associated with 4-year overall survival(OS) and relapse-free survival(RFS).The OS and RFS of patients with CD8+ lymphocytic infiltration both were 100%,that without CD8+ lymphocytic infiltration were 71 and 74%respectively.300 ng/ml was defined as the cutoff,the level of serum alpha fetoprotein(AFP) was significantly associated HCC recurrence rates.The RFS of patients with CD8+ lymphocytic infiltration and negative AFP was superior to patients without CD8+ lymphocytic infiltration and positive AFP.In multivariate Cox models,CD8+ lymphocytic infiltration in intratumoral areas was independent prognosis factor for RFS.Conclusions CD8+ lymphocytic infiltration in intratumoral areas and serum AFP level in patients with early stage of HCC can be used as a predictive marker for early stage of HCC. PartⅣThe Prognostic Significance of APC,IFNG mRNA Levels in Patients With Early- Stage Hepatocellular CarcinomaObjective The aim of this study was to evaluate clinical significance of several biomarkers associated with metastasis and microenvironment in patients with hepatocellular carcinoma who underwent liver resection.Methods One hundred and twelve patients with HCC were evaluated for the expression of adenomatous polyposis coli(APC),osteopontin(OPN),urokinase-type plasminogen activator(uPA),matrix metalloproteinases(MMP2),C-reactive protein (CRP),macrophage colony stimulating factors(CSF1),γ-interferon(IFNG), interleukin-2(IL-2) by real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR).The association of intratumoral expression of these genes of patients with tumor recurrence and survival was analyzed.Results The clinical outcome did not significantly differ in high expressive group and low expressive group of MMP2,IL-2,CSF1,CRP,uPA and OPN.Patients with high levels of IFN-gamma and APC expression had significantly longer overall survival. The 2-year overall survival was 34.4%and 71.2%for patients with low and high IFN-gamma expression,respectively(P=0.011),and the 2-year overall survival was 31.8%and 77.9%for patients with low and high APC expression,respectively(P =0.004).Application of multivariate Cox regression analysis showed APC expression to be an independent prognostic factor for overall survival.Conclusions Elevated APC and IFN-gamma expression correlates with improved clinical outcome in patients with hepatocellular carcinoma.Conclusions1.AJCC/UICCTNM and BCLC staging systems can divide HCC into subsets that behave differently from each other,are independent risk factors for recurrence of HCC.Traditional staging system can only predict approximate outcome as they merely reflect the likely biological behavior of the HCC based on clinicopathological factors.2.Preoperative plasma osteopontin(OPN) level,intratumoral CD8+ lymphocytes and APC mRNA level can act as biomarkers to subdivide early-stage HCC into subgroups that have different outcomes. 3.The patients with CD8+ lymphocytic infiltration obviously in intratumoral areas and lower serum AFP level have better recurrence-free survival.Similarly,patients with high level of APC and high level of IFNG in tumor have better overall survival.The potential application of this work1.AJCC TNM and BCLC staging system are independent risk factors for recurrence of HCC.And they are important for prognosis prediction and therapy decision-making.2.Preoperative plasma OPN levels,intratumoral CD8+ lymphocytes and APC mRNA levels can act as biomarkers to subdivide early-stage HCC into subgroups that may improve the selection of patients for adjuvant therapy.3.Prognosis of patients with HCC depends on characteristics of tumor cells and host microenvironment.This viewpoint has important value for improvement of new treatment strategies and investigation of prognosis prediction for HCC.Originalities of this work1.This is the first report investigating the association between the preoperative plasma osteopontin(OPN) levels,intratumoral CD8+ lymphocytes and APC mRNA levels and early-stage HCC.2.For the first time,this study suggested that prognosis of patients with early-stage HCC depends on characteristics of tumor cells and host microenvironment.It should offer additional clues for treatment of early-stage HCC and prognosis prediction..