Predictive Value Of Single Nucleotide Polymorphism And DNA Methylation In Tumor Recurrence Of Patients With Hepatocellular Carcinoma After Liver Transplantation

Hepatocellular carcinoma(HCC)is the most common primary malignant tumor in liver,which ranks fifth in overall frequency,and around 372,000 new cases of HCC are diagnosed annually.China has one of the highest prevalent areas of HCC,mainly because of chronic hepatitis B carders accounting for more than 10%of its population.The prognosis of patients with HCC is generally poor,even treated with combined therapy based on surgery,including resection,radiotherapy,chemotherapy, targeted therapy and liver transplantation(LT).LT has been accepted as a potentially curative option for patients with small HCC.However,long-term survival(at 5 years) remains 47-61%due to the high incidence of recurrence.Similar to other therapy option,recurrence of HCC after transplantation remains one of the most prevalent causes of poor long-term survival.Given the growing organs shortage and the risk of aggressive recurrence,selection of candidates for LT is a crucial factor and still a debate.Efforts are focused on identifying tumor characteristics to improve predictive power of recurrence and to establish optimal recipient selection criteria that can define a subset of HCC patients standing to benefit from LT.Conventional prognostic markers for tumor recurrence are clinicopathological characteristics such as tumor size,tumor number,histopathologic grading,vascular invasion etc. Especially,vascular invasion is regarded as a sole independent risk factor for poor prognosis.However,there are still no specific biomarkers,especially pre-transplant molecular markers,to reliably predict recurrence after LT.With the improvement of recognition for tumor recurrence and metastasis,as well as great advancement on genomics,it is gradually accepted that carcinogenesis and progress of HCC is a course involving multiple genetic alteration and modulation. Recently,numerous studies have focused on the genetic alteration or modulation in a wide range of human cancers.As an important type of genetic alteration,single nucleotide polymorphisms(SNPs)of genes have been investigated in many diseases, such as cancer,inflammation and immunologic disease.On the other hand,CpG island hypermethylation at the promoter region of many tumor suppressor genes have been shown to be associated with regeneration,carcinogenesis,and tumor progression.However,no study has examined the predictive value of SNPs and DNA methylation in tumor recurrence of HCCs after transplantation.In this study, we investigated 16 SNPs of 10 genes and hypermethylation of XAF1 gene in association with the risk of HCC recurrence after LT,and determined whether some polymorphisms or hypermethylation of XAF1 could provide further discriminative genetic power over current recipient selective systems for HCC patients undergoing LT. PartⅠ.Predictive value of single nucleotide polymorphism(SNP)in tumor recurrence of patients with hepatocellular carcinoma after liver transplantationAim:To evaluate the association between SNPs of genes and the risk of HCC recurrence after LT.Methods:Genomic DNA of 99 HCC patients undergoing LT was extracted from peripheral blood lymphocytes.A total of 16 SNPs of 10 genes,including MDR1 (C1236T,G2677T/A,C3435T),MMP-2(-1306 C/T),MMP-9(-1562 C/T),IL-1α(-889 C/T),IL-1β(-31 T/C,-511 C/T),IL-6(-572G/C),IL-8(-251T/A),IL-10(-1082A/G,-819T/C,-592A/C),TNF-α(-308G/A)and TGF-β1(-509T/C, +869C/T),were enrolled in this study.Genotyping was performed rising polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP).The association between polymorphisms and clinicalpathological parameters,as well as 3-years recurrence-free survival after LT was assessed.Results:To make our description laconic,we labeled patients carrying at least one variant A allele(AA,GA,or AT)as 2677A carrier group,and TT,GT,or GG as 2677A non-carrier one in this study.The association between recurrence-free and 2677A carrier was significant(P=0.019).In 2677A carrier group and 2677A non-carrier group,the patients with recurrence were 34.5%(10/29)and 55.7% (39/70),respectively.The 3-year recurrence-free survival rate was 46.2%in 2677A carrier group compared with 22%in 2677A non-carrier group.The median recurrence-free survival for 2677A carrier group was significantly longer than that for 2677A non-carrier group(44.2 vs.10.5 months,P=0.015).In addition,the association between MMP-2 -1306 C/T polymorphisms and recurrence was significant(P=0.029).Recurrence of HCC was identified in 42 of 73(57.5%) patients with CC homozygous,and 6 of 20(30%)patients with CT heterozygous. The cumulative 3-year recurrence-free survival in patients with CC homozygous was 35.5%,compared to that of 64.4%in patients with CT heterozygous.The mean recurrence-free survival for heterozygous CT patients was significantly longer than that for homozygous CC patients by Kaplan-Meier estimates(30.4 versus 19.3 months,P=0.019).In spite of the low frequency of IL-10-1082 GA genotype,we observed that GA genotype was related to the increased risk of tumor recurrence(P =0.024).In IL-10 -1082 GA genotype and AA genotype,the patients with recurrence were 88.9%(8/9)and 49.4%(40/81),respectively.The cumulative 3-year recurrence-free survival in patients with AA homozygous was 37.1%, compared to that of 0%in patients with GA heterozygous.The mean recurrence-free survival for heterozygous AA patients was significantly longer than that for homozygous GA patients(23.5versus 5.7months,P=0.010).In the relationship between above three polymorphisms and clinicopathological data,there were no statistical significance in the distribution of selected clinicopathological parameters as to age,gender,portal vein tumor thrombi(PVTT),preoperative alpha-fetoprotein (AFP)level,or histopathologic grading,tumor size and tumor number after LT between different genotypes(P＞0.05).Meanwhile,there were no significant association between other polymorphisms of genes and tumor recurrence after LT. Univariate analysis revealed that the clinicopathological parameters could provide significant predictive values for recurrence including PVTT,preoperative AFP level, histopathologic grading,tumor size,tumor number,as well as above three SNPs including MDR1 2677A carrier,MMP-2 C-1306T and IL-10 A-1082G.The multivariate analysis revealed that preoperative AFP level,tumor size,and MDR1 2677A carrier status were independent factors for predicting recurrence-free survival (HR:2.447;95%CI:1.123-5.329;P=0.024). Conclusions:Our results illustrated that the MDR1 2677A carrier,MMP-2 C-1306 T and IL-10 A-1082G polymorphisms were associated with the reduced risk of tumor recurrence after LT.Especially,MDR1 2677A carrier status may serve as a potential biomarker for tumor recurrence after LT.PartⅡ.Predictive value of DNA methylation in tumor recurrence of patients with hepatocellular carcinoma after liver transplantationAim:To investigate the methylation status and expression level of XAF1 in HCCs treated with LT,and to evaluate potential predictive value for tumor recurrence.Methods:The XAF1 mRNA expression in three liver cancer cell lines (SMMC-7721,HepG2 and Hep3B)was detected by RT-PCR before and after a DNA methyltransferase(DNMT)inhibitor,5-aza-2'-deoxycytidine(5-aza-dC) treatment.Meanwhile,methylation status of XAF1 in three cell lines and HCC tissue samples from 65 cases treated with LT was measured by methylation-specific polymerase chain reaction(MSP).Immunohistochemistry was also used to assess the XAF1 protein expression in matched 49 HCC tissue samples.We then analyzed the predictive value of expression and methylation of XAF1 in tumor recurrence of HCC after LT.Furthermore,the relationship between methylation status and expression patterns of XAF1 in HCC was analyzed.Results:RT-PCR showed that the mRNA level of XAF1 was very low in HepG2 and Hep3B,while undetectable in SMMC-7721.MSP indicated that XAF1 gene was completed or partially methylated.However,5-aza-dc reactivated XAF1 mRNA expression in SMMC-7721 and upregulated the mRNA level of XAF1 in HepG2 and Hep3B,suggesting that DNA methylation plays a causal role in the transcriptional regulation of XAF1 in HCC.In 65 HCC tissue samples,the methylation frequency of XAF1 was 43 of 65(66.1%).We proved the relationship between methylation status and expression patterns of XAF1 in HCC tissues.In the 30 cases of XAF1 low expression group,27(90.0%)cases displayed hypermethylation in the XAF1 promoter region,whereas 4 of 19 cases(21.1%)in the unchanged expression group (P＜0.001).In the relationship between methylation and the clinicopathological data, we found that XAF1 methylation was more frequent in HCC,with AFP＞400μg/L than those with AFP≤400μg/L(P=0.009).Concerning the correlation between XAF1 expression and the clinicopathological data,our results showed that XAF1 expression positively correlated with PVTT(P=0.018),preoperative AFP level(P= 0.003)and tumor size(P=0.017).The methylated rates of XAF1 in patients with recurrence and without recurrence were 71.1%(27/38)and 59.3%(16/27), respectively.And there were no statistically significant differences between the methylation status and the risk of postoperative recurrence(P=0.426).However,the unchanged expression rates of XAF1 in patients with recurrence of HCC and without recurrence of HCC was 21.4%(6/28)and 61.9%(13/21),respectively.The association between XAF1 unchanged expression and recurrence-free was significant(P=0.007).Recurrence of HCC was identified in 6 of 19(31.6%) patients with XAF1 unchanged expression,and 22 of 30(73.3%)patients with XAF1 low expression.The cumulative 3-year recurrence-free survival in patients with unchanged expression is 61.8%,compared to that of 18.5%in patients with low expression.The mean recurrence-free survival for XAF1 unchanged expression patients was significantly longer than that for XAF1 low expression patients(27.9 versus 12.6 months,P=0.001).Multivariate analysis revealed that XAF1 expression level might be a novel independent factor for predicting recurrence-free survival (HR:4.219;95%CI:1.689-10.526;P=0.002).Conclusions:Promoter hypermethylation is one critical,but not sole mechanism for the gene silencing of XAF1 in HCC.The protein level of XAF1 rather than methylation status of XAF1 gene may be a potential biomarker for tumor recurrence after LT.