The Status And Clinical Significance Of Serum IGFBP7 Gene Promoter Methylation In Patients With Hepatocellular Carcinoma

PART I Methylation of Serum Insulin-Like GrowthFactor-Binding Protein 7 Promoter in Hepatitis B Virus-Associated Hepatocellular CarcinomaBackgroundChronic hepatitis B virus(HBV)infection is the most common cause of hepatocellular carcinoma(HCC)in China.Since HCC was often diagnosed at an advanced stage,a large proportion of HCC patients missed the best opportunity to receive optimal therapy.Even if in the last decades the management of HCC is improved due to the increased diagnostic capacity,the development of evidence-based staging system and the availability of new effective treatments the patients with HCC still have a very dismal 5-year-survival rate and poor prognosis.Therefore,early diagnosis and treatment is essential for improving the prognosis and survival rate of patients with HCC.So far,the screening and diagnosis of HCC still rely on tumor biomarkers and abdominal imaging technologies.Alpha-fetoprotein(AFP)is a well known biomarker for HCC.However,it has inadequate sensitivity and specificity for detecting HCC.Meanwhile,the surveillance ability of imaging technologies are affected by many factors,which makes the effective and timely diagnosis of HCC at its initial stage difficult.Therefore,noninvasive and effective biomarkers for early screening and diagnosis of HCC are urgently needed.HCC development is a multifactorial process involving alterations of a combination of genetic,epigenetic,and environmental factors.DNA methylation,as one of the most extensively investigated epigenetic DNA modifications,is an important epigenetic regulatory mechanism for gene expression.To date,accumulating evidence has confirmed the important role of aberrant DNA methylation in tumor development and considered the methylation changes of specific genes as a promising biomarker for early detection of various cancers.Similar methylation alterations can also be measured in cell-free DNA(cfDNA)isolated from serum or plasma.Circulating cfDNA is defined as extracellular DNA occurring in serum or plasma and is thought to originate from apoptotic and necrotic cancer cells from the primary tumor site,circulating tumor cells,and distant metastases.As a non-invasive liquid biopsy,detecting of cfDNA might provide valuable predictive information for cancer diagnostics and would avoid the need for tumour tissue biopsies.Methylation status of circulating cfDNA in serum represents one of the most promising biomarker for early detection of HCC.Insulin-like growth factor-binding protein 7(IGFBP7)plays an important role in inhibiting tumor growth,promoting apoptosis of tumor cells and affecting the stability of tumor vessels,and it functions as a tumor suppressor gene in numerous cancers.CpG island methylation of the serum IGFBP7 promoter is recently shown in a variety of human cancers,and participates in the development process of the tumors.Recent research shows that IGFBP7 acts as a tumor suppressor gene in liver cancer.Thus,we hypothesize that IGFBP7 promoter methylation may have very important function in the development and progression of HCC,and can be used as a potential early diagnosis and prognosis biomarker for HCC.ObjectiveIn this study,we aimed to evaluate the potential diagnostic value of serum IGFBP7 promoter methylation in patients with HCC.MethodsThe study included a total of 217 subjects,including 136 HCC patients,46 patients with chronic hepatitis B(CHB)and 35 healthy controls(HCs)from Qilu Hospital of Shandong University,recruited between June 2011 and July 2012.HCC was diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases(AASLD)Practice Guidelines for Management of hepatocellular carcinoma.All the patients with HCC were infected with HBV.According to the 2009 update of the AASLD Practice Guidelines for Management of Chronic Hepatitis B,chronic HBV infection was defined as positive hepatitis B surface antigen(HBsAg)for at least 6 months prior to the beginning of this study.The methylation status of the serum IGFBP7 gene promoter was determined using methylation-specific polymerase chain reaction(MSP).Statistical analysis was performed using SPSS version 16.0(Chicago,IL,USA).Correlation between two quantitative variables was evaluated using the Student’s t or Mann-Whitney U tests.We compared the difference of serum IGFBP7 methylation status between different groups and evaluated the association between serum IGFBP7 methylation status of HCC patients and their clinicopathological parameters using the Chi-square test.Multivariate logistic regression was also performed to identify independent risk factors for IGFBP7 promoter methylation.Diagnostic values of IGFBP7 methylation and serum AFP level were assessed by the area under the receiver operating characteristic curves(AUC).Sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)were calculated.The statistical significance was defined as P<0.05.Results1.We analyzed the methylation status of the serum IGFBP7 promoter in 136 HCC patients,46 CHB patients,and 35 HCs.The frequency of serum IGFBP7 promoter methylation in HCC patients(89/136;65%)was significantly higher than that in CHB patients(P<0.001)and HCs(P<0.001),respectively.2.There was no association between the IGFBP7 promoter methylation status and gender,age,HBeAg,smoking,alcohol,tumor number,tumor size,histological grading,tumor node metastasis(TNM)staging,Child-Turcotte-Pugh(CTP)staging,or serum AFP levels,whereas the frequency of methylated serum IGFBP7 promoters strongly correlated with vascular invasion of HCC(P = 0.010).Furthermore,multivariate logistic regression was also performed to identify independent risk factors for IGFBP7 promoter methylation.However,there was no independent risk factor for IGFBP7 promoter methylation.3.We determined whether or not serum IGFBP7 methylation might be a useful biomarker for the detection of HCC.Methylation of the serum IGFBP7 gene promoter was demonstrated to distinguish HCC from CHB patients with a sensitivity of 65%and a specificity of 83%,and to distinguish HCC patients from HCs with a sensitivity of 65%and a specificity of 86%.The AUC of IGFBP7 methylation in discriminating HCC from CHB was significantly higher than that of serum AFP level(0.740 versus 0.618;P<0.05).4.We compared the diagnostic value of the combination of IGFBP7 methylation and AFP with AFP alone in detecting HCC.When AFP was less than 20 ng/ml,the HCC detection rate was significantly higher in the IGFBP7 methylated group than in the unmethylated group(P<0.001).The frequency of HCC was also significantly higher in the IGFBP7 methylated group than in the unmethylated group(P<0.001)when AFP was>20 ng/ml.143 patients with AFP>20 ng/ml or methylated IGFBP7 were regarded as positive,of whom there were 116 HCC patients(true positives)and 27 CHB patients(false positives),whereas 39 patients with AFP<20 ng/ml and unmethylated IGFBP7 were regarded as negative,of whom there were 19 CHB patients(true negatives)and 20 HCC patients(false negatives).Sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)were 85%,41%,81%,and 49%,respectively.We also demonstrated that the combination of both markers has a higher sensitivity(P<0.001)and NPV(P = 0.033)than AFP alone.There was no significant difference in specificity(P = 0.296)and PPV(P = 0.525)between the two markers and AFP alone.Conclusions1.This study demonstrated that aberrant IGFBP7 gene promoter methylation existed in patients with HCC,which implied that serum IGFBP7 methylation might serve as an effective and non-invasive biomarker for HCC.2.Aberrant IGFBP7 gene promoter methylation was more likely to be detected in HCC patients with vascular invasion compared with those without,which implied that IGFBP7 methylation might be used to predict the vascular invasion of HCC.PART II Serum Insulin-Like Growth Factor-Binding Protein 7 Methylation Predicts a Poor Prognosis in Hepatitis BVirus-Associated Hepatocellular Carcinoma after HepatectomyBackgroundHepatocellular carcinoma(HCC)is the most common type of hepatic malignant tumor worldwide,the most serious complication of long-standing chronic liver disease(CLD)and the third most common cause of cancer-related deaths worldwide with rising incidence.Approximately 600,000 patients die every year due to HCC.The mechanisms of hepatocarcinogenesis include several common functions such as oncogene activation,oxidative stress and tumor suppressor function attenuation.The relationship between oxidative stress and the pathogenesis of HCC has been attracting increasing attention.Oxidative stress is a process whereby the body receives stimulation from harmful endogenous or exogenous factors.Accumulating evidence has shown that oxidative stress plays an important role in the development of hepatocarcinogenesis.Reactive oxygen species(ROS)and/or inadequacy of antioxidants are increased when oxidative stress occurs,which exerts a tumorigenic role to promote both genetic mutation and epigenetic alteration.Aberrant DNA methylation is a common event during the pathogenesis of human cancers and one of the important epigenetic mechanisms in carcinogenesis.Representative alterations of DNA methylation associated with carcinogenesis include focal hypermethylations of CpG islands in the promoter region,leading to the inactivation of certain tumor suppressor genes,and genome-wide hypomethylation,resulting in genomic instability and contributing to cell transformation and progression of cancer.Three known catalytically active DNA methyltransferases(DNMTs)have been identified:DNMT1,DNMT3a and DNMT3b.Many studies have provided evidence that cancer-linked DNA methylation alterations may be used as early indicators of HCC,as well as prognostic markers of cancer progression and response to chemotherapy.Malignant tumors can release significant amounts of DNA into the blood of cancer patients as tumor-derived cell-free DNA(cfDNA)by cell death-associated mechanisms like necrosis and apoptosis.Different genetic and epigenetic analyses have already demonstrated the clinical potential of cfDNA.Coupled with the convenience for obtaining specimen,increasing attention has been paid to circulating cfDNA as potential cancer biomarker for non-invasive cancer diagnostic test.Several molecular alterations found in tumor cells,such as DNA mutations and DNA methylation,are reflected in cfDNA.Recently,A substantial number of studies have suggested DNA methylation of cfDNA as a very promising tumour specific and useable diagnostic marker for early detection,risk assessment,treatment response,and prognosis of HCC with minimal invasion and costeffective.The functions of insulin-like growth factor binding protein 7(IGFBP7)include regulation of cell proliferation and differentiation,induction of apoptosis and cellular senescence.Previous studies have established IGFBP7 as a potential tumor suppressor gene for a variety of cancers including HCC.Our previous study reported that methylation of the serum IGFBP7 gene promoter may serve as a useful noninvasive biomarker for HCC diagnosis.In addition,several studies found a significant association between IGFBP7 and not only apoptosis,but also prognosis.However,the prognostic significance of IGFBP7 methylation in HCC remains unclear.ObjectiveThe main purpose of this study was to investigate the relationship between oxidative stress,DNMTs expression and IGFBP7 methylation,and to evaluate the prognostic value of serum IGFBP7 methylation status in patients with HCC after hepatectomy.MethodsA total of consecutive 155 patients undergoing surgical resection of HCC from Qilu Hospital of Shandong University were enrolled in the present study from January 2010 to January 2013.HCC was diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases(AASLD)Practice Guidelines for Management of hepatocellular carcinoma.All the patients with HCC were infected with hepatitis B virus(HBV).We investigated the methylation status of IGFBP7 promoter by means of methylation-specific polymerase chain reaction(MSP)and DNMTs mRNA levels by means of quantitative real-time polymerase chain reaction(RT-PCR).Then,we used enzyme-linked immunosorbent assay(ELISA)to measure the oxidative stress status,including serum levels of malondialdehyde(MDA),xanthine oxidase(XOD),reduced glutathione hormone(GSH)and glutathione-S-transferases(GST).Statistical analyses of the data were performed with SPSS version 16.0(Chicago,IL,USA).We compared the quantitative variables between groups using the Student’s t or Mann-Whitney U test.Chi-square test was used to compare the categorical variables.Kaplan-Meier method and log-rank test were used as univariate analysis to compare the survival time between groups.Multivariate analysis was performed by Cox proportional hazards models.We investigated the prognostic factors affecting overall survival and early tumor recurrence.The statistical significance was defined as P<0.05.Results1.Methylation of IGFBP7 gene was detected in 67.7%(105/155)of HCC serum.We found that IGFBP7 methylation was associated with vascular invasion,overall survival(OS)and early tumor recurrence(ETR).No other correlations was observed between IGFBP7 promoter methylation status and gender,age,AFP,tumor numbers,tumor size,liver cirrhosis,differentiation and tumor node metastasis(TNM)stage(P>0.05).2.MDA level was significantly higher in IGFBP7 gene promoter methylated group of HCC than unmethylated group(P<0.001).XOD level was significantly higher in IGFBP7 gene promoter methylated group of HCC than unmethylated group(P =0.014).GSH level was significantly lower in IGFBP7 gene promoter methylated group of HCC than unmethylated group(P = 0.009),but no significant difference of GST level could be observed between between IGFBP7 gene promoter methylated group and unmethylated group(P = 0.878).3.The DNMT1 mRNA level was significantly higher in IGFBP7 gene promoter methylated group of HCC than unmethylated group(P = 0.001).The DNMT3a mRNA level was significantly higher in IGFBP7 gene promoter methylated group of HCC than unmethylated group(P = 0.002).However,no significant correlation of DNMT3a mRNA level could be found between IGFBP7 gene promoter methylated group and unmethylated group(P = 0.386).4.Survival signatures consider the date of HCC resection as the starting time and the date of death or the last clinical review before 30 January 2016 as the end point.We then evaluated the prognostic value of the IGFBP7 promoter methylation status in HCC patients.Kaplan-Meier curve analysis revealed that IGFBP7 promoter methylation was significantly correlated with OS(log-rank test,P<0.001).5.By univariate analysis,tumor size,vascular invasion,TNM stage,ETR and IGFBP7 methylation were associated with OS in HCC patients.Multivariate analysis confirmed that TNM stage(P = 0.000),ETR(P = 0.000)and IGFBP7 methylation(P=0.000)were independent prognostic predictors for OS.6.Univariate analysis revealed that tumor size,vascular invasion,TNM stage and IGFBP7 methylation were associated with ETR in HCC patients.Then,multivariate analysis confirmed that TNM stage(P = 0.015)and IGFBP7 methylation(P = 0.008)were independent prognostic predictors for ETR.Conclusions1.Our results indicated that IGFBP7 promoter methylation was associated with oxidative stress and DNMTs expression,supporting the hypothesis that oxidative stress induces DNA methylation by affecting DNMTs.2.IGFBP7 promoter methylation was associated with vascular invasion,overall survival and early tumor recurrence,indicating that it might serve as a potentially independent prognostic factor in patients with HCC after hepatectomy.