Methylation Of SOX1 And VIM Promoters In Serum As Potential Biomarkers For Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world,with the third highest mortality rate in all malignancies.Early symptoms of HCC are not obvious,due to lack of timely diagnosis.About 60-70%of patients has entered the late stage in the initial discovery.And they lost the surgical resection and other radical treatment opportunities.Therefore,the early diagnosis of HCC is very important.At present,the diagnosis and prognosis of HCC mainly dependent on clinical pathology and imaging examination.Although the clinical pathology is the gold standard for the diagnosis of HCC,it needs surgical resection of specimens or liver puncture specimens,the invasiveness greatly limits its clinical application.Various imaging techniques,including ultrasound?CT and magnetic resonance imaging,are widely used in clinical examinations.However,these imaging techniques also have some limitations.In addition,alpha-fetoprotein(AFP)and other serum proteins are widely used.But there are false positives and false negative deficiencies,and the sensitivity is only 22-60%,which is far from the clinical needs.Therefore,it is urgent to find specific markers to improve the early diagnosis of HCC.Biomarkers are widely used in the diagnosis and prognosis of cancer.Circulating tumor DNA(ctDNA)refers to the small fragments that the genome in tumor cells release into the peripheral blood,it can be used as a tumor biomarker and provides a simple and noninvasive method to diagnose cancer.Epigenetic changes play an important role in the development and progression of human cancers.The most common epigenetic change in the tumor is abnormal methylation of the gene promoter region,which leads to tumor suppressor gene silence.Many studies have confirmed that DNA methylation changes can be used as biomarkers for early diagnosis of cancer.Aims:This study aimed to investigate the diagnostic value of SRY(sex determining region Y)-box 1(SOX1)and Vimentin(VIM)promoter methylation for HCC.Methods:The study included 360 subjects,240 patients with HCC,29 with liver cirrhosis(LC),66 with chronic hepatitis B(CHB)and 25 healthy controls(HCs).Patients with HCC were selected from those diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases(AASLD)Practice Guidelines for Management of Hepatocellular Carcinoma.Patients with LC and CHB were diagnosed according to the 2009 update of AASLD Practice Guidelines for Management of Chronic Hepatitis B.All blood samples were taken from peripheral venous blood in the morning.After the separation of serum,we use the kit to extract serum DNA,according to the instructions.Methylation-specific polymerase chain reaction(MSP)was performed to detect the serum methylation state of SOX1 and VIM promoters in HCC,LC,CHB and HC.Results:1.The percentage of SOX1 promoter methylation was significantly higher in the HCC group(72.08%)than the LC group(17.24%,p<0.001),the CHB group(15.15%,p<0.001)and the HCs(4.00%,p<0.001).The methylation frequency of the VIM promoter was also higher in the HCC group(61.67%)than the LC group(24.14%,p<0.001),the CHB group(13.64%,p<0.001)and the HCs(12.00%,p<0.001).However,there was no significant difference in SOX1 and VIM promoter methylation rates between LC and CHB,CHB and HCs or LC and HCs.2.The SOX1 promoter methylation was related to the tumor number(?2 =6.107,p=0.013),tumor size(x2=7.986,p=0.018)and TNM stage(x2=12.458,p=0.001).The VIM promoter had a higher methylation frequency in patients with portal vein tumor thrombosis(PVTT)(x2=7.528,p=0.006),tumor number(x2=9.997,p=0.002),tumor size(x2=19.451,p=0.001),TNM stage ?2=8.291,p=0.004)and vascular invasion(?2=8.832,p=0.003).Univariate logistic regression analysis suggested that serum SOX1 promoter methylation in HCC patients was associated with multiple cancer nodules(OR=0.208,p<0.001)and TNM staging(?-?)(OR=4.987,p<0.001);VIM promoter methylation was associated with PVTT(OR = 2.36,p<0.001),TNM stage(?-?)(OR=3.978,p<0.001),tumor size(>5cm)(OR=2.340,p=0.037)and vascular invasion(OR=33.6815,p<0.001).3.To discriminate HCC from LC and CHB,the sensitivity is 72.08%for SOX1 and 61.67%for VIM,higher than AFP alone(56.67%,x2=12.436,p<0.001;x2=1.242,p=0.265).When we combined SOX1 and VIM promoter methylation,higher sensitivity(82.50%)but lower specificity(78.95%)for discriminating HCC from CHB and LC was found.Then,we compared the diagnostic value of AFP combined with SOX1 and VIM methylation.When AFP was more than 20 ng/ml or at least one gene of SOX1 and VIM was methylated,the sensitivity,specificity,PPV and NPV were 88.33%,72.63%,89.08%and 71.13%,respectively.When AFP was more than 20 ng/ml and SOX1 and VIM were both methylated,the sensitivity,specificity,PPV,and NPV were 48.33%,97.89%,98.31%and 42.86%.Moreover,the AUC of SOXl and VIM methylation was significantly higher than that of AFP in discriminating HCC from CHB and LC patients(0.805 versus 0.742;p=0.012).Conclusions:1.The methylation of SOX1 and VIM promoter in serum could be detected in HCC patients and the methylation rate was higher than that of LC and CHB group.2.There is a correlation between SOX1 and VIM promoter methylation and clinicopathological parameters in HCC.3.AFP combined with SOX1 and VIM promoter methylation can significantly improve the diagnosis sensitivity of HCC.It can be used as a potential biomarker for the noninvasive detection of HCC and HCC progress.