SFRP2 Gene Promoter Methylation Status Assists In The Diagnosis Of HBV-associated Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma is a common malignancy worldwide and is the most predominant pathological type of primary hepatic cancer,with the latest statistics showing that it currently ranks sixth in terms of incidence and third in terms of mortality.China is one of the regions with a high incidence of hepatocellular carcinoma,and chronic hepatitis B virus infection is the most important risk factor for the carcinogenesis.One of the major reasons for the high mortality rate of liver cancer is that its early symptoms are lack of specificity,which makes it difficult for early clinical diagnosis.Most patients were diagnosed at end stage and missed the optimal treatment opportunities.Moreover,the most commonly used biomarker for hepatocellular carcinoma,alpha-fetoprotein,has limited sensitivity for early diagnosis.Therefore,there is a deadly need for new molecular biomarkers that can more effectively assist in the early diagnosis of HCC.DNA methylation refers to the addition of methyl groups to DNA,which usually occurs in specific CpG-rich regions of gene promoters,resulting in reduced or silenced gene expression.Aberrant DNA methylation is a common epigenetic mechanism associated with the development of various tumors including hepatocellular carcinoma.Studies have demonstrated the potential clinical value of methylation level as a biomarker for cancer risk assessment and diagnosis.The classical Wnt pathway plays an important role in cell differentiation,proliferation and apoptosis.Secreted frizzled related protein 2(SFRP2),a member of the secreted frizzled-related protein family,is involved in the Wnt signaling pathway as a Wnt antagonist.It has been shown that SFRP2,as an oncogene,is always silenced by promoter hypermethylation in a variety of human malignancies such as colorectal cancer,gastric cancer,esophageal adenocarcinoma,and leukemia.Therefore,it is reasonable to suspect that SFRP2 promoter hypermethylation also occurs in patients with hepatocellular carcinoma and has the potential to serve as a non-invasive candidate biomarker to assist in the diagnosis of hepatocellular carcinoma.This study aimed to investigate the methylation status of the SFRP2 promoter in peripheral blood mononuclear cells(PBMCs)of patients with hepatitis B virus-associated hepatocellular carcinoma and to evaluate its value as a noninvasive molecular biomarker for diagnosis of hepatocellular carcinoma.MethodsThis study included 132 patients with hepatitis B virus-associated hepatocellular carcinoma(HCC),121 patients with chronic hepatitis B(CHB)and 40 healthy controls(HCs)for analysis.SFRP2 promoter methylation levels in peripheral blood mononuclear cells were detected by methylation-specific quantitative PCR(Methylight).The mRNA expression level of SFRP2 was detected by real-time quantitative PCR(RT-qPCR).Data were analyzed using SPSS 22.0 statistical software.Continuous variables were expressed as median(centile 25;centile 75);Categorical variables were expressed as number(%).Mann-Whitney U test and Kruskal-Wallis H test were used for the analysis of differences between groups of continuous variables.the relationship between SFRP2 methylation levels and clinical data was performed using Spearman’s rank correlation test.The receiver operating characteristic curve(ROC)was used to assess the diagnostic value of SFRP2 methylation levels in patients with hepatitis B virus-associated hepatocellular carcinoma.P<0.05 was considered to be statistically significant.Results1.The methylation levels of the SFRP2 promoter in the HCC,CHB and HC groups were expressed as percent of methylated reference(PMR).The methylation levels of SFRP2 were significantly higher in patients with HCC than in patients with CHB(P<0.001)and HCs(P<0.001).No significant difference could be found between CHB group and HC group.Furthermore,considering that PMR of SFRP2 was positively correlated with age in HCC group and the average age of HCC group was significantly higher than CHB and HC groups,a 1:1 propensity score matching(PSM)was applied to rule out the confounding factor with age as independent variable.Matching was conducted pairwise with a maximum caliper width of 0.02.After PSM,152 matched patients were selected(n=76 for both HCC and CHB group)among 253 and there were no significant differences in age between the two groups(P=0.997).Then,Mann-Whitney U test was performed in post-matched groups and SFRP2 methylation level was still significantly higher in patients with HBV-associated HCC than in those with CHB(P<0.001).And the same statistical process was performed in HCC and HC groups.After PSM,64 matched patients were observed(n=32 for both HCC and HC group)and no significant difference could be found in age between the two groups(P=0.930).In post-matched groups,SFRP2 methylation level was significantly higher in patients with HCC than HCs(P<0.001),consistent with the pre-matched results.2.In contrast to methylation levels,we observed that the mRNA level of SFRP2 was significantly lower in HCC group compared with CHB(P<0.001)and HC groups(P=0.016).There was no significant difference between CHB and HC group.The relationship between SFRP2 methylation level and mRNA level was further analyzed using spearman rank correlation analysis and we found that PMR value of SFRP2 was significantly negatively correlated with mRNA expression level(Spearman’s r=0.119,P=0.042).3.In the HCC subgroup,SFRP2 methylation levels were significantly higher in patients>50 years old,female,with negative HBeAg,negative HBV-DNA and poor differentiation compared with the remaining groups(P<0.05).The relationships between SFRP2 methylation level and age,AFP were further analyzed using spearman rank correlation test and we found PMR value of SFRP2 was significantly correlated with the age of HCC patients(Spearman’s r=0.365,P<0.001).But no correlation could be observed between SFRP2 methylation level and AFP(Spearman’s r=-0.144,P=0.099).4.Receiver operating characteristic(ROC)curve was used to estimate the diagnostic value of SFRP2 methylation level,AFP and the combined determination.We observed that PMR value of SFRP2 promoter showed an AUC of 0.735,significantly higher than that of AFP(AUC 0.615,P=0.013).A cut-off point of 34.51%was selected,with a sensitivity of 55.30%,specificity of 81.80%.Then,a model based on binary logistic regression was constructed to evaluate the diagnostic value of SFRP2 methylation level combined with AFP.The combined determination showed an AUC of 0.805 with sensitivity of 69.70%and specificity of 78.50%,markedly increased compared with AFP testing alone(P<0.001).5.Multivariate logistic regression analysis was performed to identify the risk factors for development of HBV-associated HCC.The methylation level of SFRP2 promoter was divided into two subgroups by the optimal cut-off point of 34.51%mentioned above and AFP was classified according to the most commonly used cut-off point of 20 ng/mL.The results showed that age>50 years(P<0.001),male gender(P=0.010),PMR value of SFRP2 promoter>34.51%(P<0.001)and AFP>20ng/mL(P<0.001)were independent risk factors for development of HBV-associated HCC.Conclusion1.SFRP2 promoter methylation levels were significantly higher in patients with chronic hepatitis B-associated hepatocellular carcinoma than in patients with chronic hepatitis B and healthy controls.2.The mRNA expression level was significantly lower in the HBV-associated HCC group compared with CHB and HC groups.In addition,the methylation level of SFRP2 promoter was significantly negatively correlated with mRNA expression level.3.The diagnostic ability of SFRP2 promoter methylation level combined with serum AFP was significantly higher than that of AFP testing alone,suggesting that SFRP2 methylation level in peripheral blood mononuclear cells has the potential to become a non-invasive biomarker to assist in the diagnosis of HBV-associated hepatocellular carcinoma,having potential clinical application prospects.4.SFRP2 methylation levels were significantly increased in poorly differentiated HCC.It was also an independent risk factor for HCC development,suggesting its potential value of surveillance and prognosis for HBV-associated HCC.