The Effect Of Hepatitis B Virus Mutations And Antiviral-Treatments On Occurrence And Postoperative Prognosis Of Hepatocellular Carcinoma

Chronic infection with hepatitis B virus (HBV) is the leading cause of hepatocellularcarcinoma (HCC) in China. China alone accounts for more than half of the global HCC,and about85%-95%of these cases are attributable to chronic hepatitis B (CHB). HCC isa highly fatal malignancy. Postoperative prognosis is unfavorable and recurrence is majorobstacle to improve postoperative prognosis. Therefore, to search for risk factors of HBV-related HCC, early prediction and intervention of hepatocellular carcinoma, andpostoperative adjuvant therapy should be important for decreasing the incidence and therecurrence as well as prolonging the survivals of HCC patients. However, the relationshipof HBV mutations and HCC occurrence remains controversial. It is unclear whether HBVmutation is the etiology of HCC or just a byproduct in the process of inflammationassociated with cancer transformation, and whether nucleoside analogue is able to preventor delay the occurrence of HBV-related HCC. There is also no conclusion about the role ofantiviral therapy of HCC patients in reducing postoperative recurrence of HCC patientsand prolonging survivals. Therefore, it is necessary to clarify the spectrum ofgenotype-specific HBV mutations at different stages of liver disease progression, and thento validate the mutations in prospective cohort studies. The effect of antiviral therapy in theoccurrence and prognosis of HBV-related HCC should be important for clinical decisionmakings.Objective: To determine the association of HBV mutations in the core promoter regionand preS region of HBV with progressive liver diseases at different stage and to clarify therole of the HBV mutations in hepatocarcinogenesis and the effect of antiviral therapy in theoccurrence and postoperative survival of HBV-related HCC in prospective cohorts.Methods: A total of1763subjects, including952asymptomatic HBsAg carriers (ASCs),327patients with CHB,209patients with LC, and275HCC cases were enrolled in thiscase-control study to screen for HCC-related HBV mutations. Prospective cohort studywith CHB patients was carried out for validation of those HBV mutations and evaluationthe effect of antiviral treatment in HCC progression. The Prospective cohort study withpostoperative HCC patients was carried out to evaluate the effect of antiviral treatment forprognosis of HCC patients with radical surgery. HBV genotypes were determined byMultiplex PCR. The core promoter region and preS region of HBV were sequenced directly or cloned into plasmid, and then sequenced. HBV mutations were detected byMEGA5.0and Bioedit7.0softwar from sequences of the core promoter region and preSregion. The clinical data of patients enrolled in this study were collected with standardquestionnaire. The follow-up methods included telephone surveys, mail surveys, householdsurveys and Cancer Registry queries. The randomized method was block randomization inthe randomized control trial. All statistical tests were two-sided, and performed by theStatistical Program for Social Sciences software (SPSS18.0)Results:1. Screening the HCC-related HBV mutationsA total of1459sequences in core promoter region and1171sequences in HBV preSregion were successfully sequenced.(1)“Hotsopts” in the BCP region and their associations with CHB, LC, and HCCNinteen hotspots were found in this region of HBV genome. Multivariate logisticregression analyses indicated that age, male, abnormal alanine aminotransferase, T1768A,A1762T/G1764A, and A1846T were associated with an increased risk of cirrhosis, whileage, abnormal ALT, HBV DNA (≥104copies/ml), genotype C, C1653T, T1674C/G,T1753V, and A1762T/G1764A were associated with an increased risk of HCC.A1762T/G1764A had a moderate sensitivity and specificity for HCC.(2) Associations of HBV mutations in the preS regions with CHB, LC, and HCCA total of35HBV mutations in the preS region were evaluated including21preS1mutations and14preS2mutations. Multivariate logistic regression analyses indicated thatC2964A, C3116T, and C7A were novel factors associated with an increased risk of HCCcompared to those without HCC, whereas A2964C and T3116C were independentlyassociated with an increased risk of cirrhosis compared to ASCs plus the CHB patients.2. The effect of antiviral therapy and HBV mutations in the occurrence of HBV-relatedHCC in a prospective cohort studyThe cohort enrolled2114CHB patients. The incidence rate of HCC is7.02per1000person-years in antivirus therapy group, and13.02per1000person-years in control group.Antiviral treatment reduced the incidence rate of HCC (P<0.001) compared to the controlgroup. The multivariate Cox analysis indicated that age (<40years old as control,40-59years old group [HR=12.37，95%CI=1.39-4.02];≥60years old group [HR=2.51，95% CI=1.12-5.60]), male (HR=3.96;95%CI=1.81-8.67), and liver cirrhosis ((HR=1.63;95%CI=1.03-2.59) significantly increased the risk of HCC. The HBV mutations in BCP regionsuch as C1653T (HR=1.63;95%CI=1.01-2.63), T1674CG (HR=1.65;95%CI=1.05-2.59)and A1762T/G1764A (HR=2.11;95%CI=1.17-3.81) significantly increased the risk ofHCC. A1762T/G1764A and C3116T in HBV genotype B and C1653T, A1727G/T,T1753V and A3120T in HBV genotype C are both independent risk factors of HCC.Whereas antiviral treatment significantly reduced the incidence of HCC (HR=0.22;95%CI=0.05-0.88).Further analysis found that the NAs antiviral treatment would significantly decreased thethe risk of HCC only in CHB patients taking the NAs for more than60months (P<0.001).Antiviral treatment would promote the change of HCC specific mutations from mutationtype to wild type so that it decreased the the risk of HCC.3. The effect of antiviral therapy on postoperative prognosis of HBV-HCC patientsIn this cohort study, the4-year overall survival (OS) was significantly higher in theantiviral group than in the control group,(59.6%vs.46.6%; P=0.008). The same was truefor the2-year recurrence-free survival (RFS)(44.6%vs.25.5%; P<0.001) and the4-yearRFS (37.2%vs.16.6%; P<0.001). High viral load (≥104copies/mL) was significantlyassociated with shorter OS (P<0.001) and RFS (P=0.004) in the control group whereasantiviral treatment significantly improved OS and RFS. Antiviral treatment significantlyimproved RFS (P<0.001) and OS (P<0.001) in all patients enrolled in the cohort. Themultivariate Cox regression analysis showed that tumor size, incomplete encapsulation,tumor microsatellite, microscopic vascular invasion, tumor differentiation, advancedBarcelona Clinic Liver Cancer (BCLC) stage, abnormal γ-glutamyl transpeptidase, andhigh viral load significantly predicted poor prognosis, whereas the antiviral treatmentsignificantly reduced HCC recurrence (HR=0.55;95%CI=0.44-0.69), and HCC-relateddeath (HR=0.57;95%CI=0.43-0.76).In the RCT, the2-year OS,2-year RFS,4-year OS and4-year RFS group were93.8%,55.6%,86.4%and37.3%in the antiviral treatment, as well as62.2%,19.5%,47.4%and12.1%in the control group (P<0.001, between the corresponding two groups), respectively.Early HCC recurrence (within2years) was higher in the control group than in the antiviralgroup (80.5%vs.44.4%; P<.001). The multivariate Cox regression analysis confirmed thatthe antiviral treatment significantly reduced HCC recurrence (HR=0.48;95% CI=0.32-0.70)，and HCC-related death (HR=0.26;95%CI=0.15-0.50). Compared to thecontrol group, the antiviral treatment significantly reduce early HCC recurrence (HR=0.41;95%CI=0.27-0.62), and improve the6-month liver function (P=0.001). Those withrecovered liver function had a higher2-year RFS than those without (P=0.003). Ct-HBxexpression in adjacent hepatic tissues significantly predicted an unfavorable RFS in theantiviral group (P<0.001).Conclusions:1. C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G in genotype Cwere indicative for the risk of cirrhosis. A1846T and T1674C/G were novel factorsindependently associated with increased risks of cirrhosis and HCC, respectively. C2964A,C3116T, and C7A were novel markers independently associated with an increased risk ofHCC, while A2964C and T3116C were novel markers independently associated with anincreased of cirrhosis. Combined preS1mutations were specific for HCC.2. Antiviral therapy with NAs can reduce the incidence of HCC and prolong the survivalof CHB patients. The mutations of A1762T/G1764A, C1653T, T1674CG, A1727G/T,T1753V, A3120T and C3116T increased the risk of HCC occurrence from CHB patients.3. Although it might not affect the HCC-promoting potential of Ct-HBx, antiviraltreatment with NAs is effective in normalising liver function, decreasing HBV-HCCrecurrence, and improving postoperative survival.