Clinical Significance Of HBV Gene Mutation In The HBV-related Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. The major risk factor for the development of HCC is hepatitis B virus (HBV) infectioa A peculiar aspect of chronic HBV infection is the persistence of HBV genomes in the absence of serum HBs antigen (HBsAg), so called ’occult’ infection. HBV covalently closed circular DNA (cccDNA) is an important intermediate in the life cycle of HBV, from which the HBV pregenomic RNA and all HBV messenger RNA transcripts originate.Part 1. This study aimed to investigate the virological status in liver (both tumor and adjacent non-tumor tissue), the clinical features and the contribution of occult HBV infection (OBI) to postoperative prognosis in HBeAg-negative(-) hepatocellular carcinoma (HCC) patients in China. Using quantitative TaqMan fluorescent real-time PCR assays, HBV covalently closed circular DNA (cccDNA) and total DNA (tDNA) were both quantified in 11 [HBsAg(-)] and 57 [HBsAg-positive(+)] pairs of tumor tissue (TT) and adjacent non-tumor tissue (ANTT) obtained from HBeAg(-) HCC patients who received no antiviral treatment and were negative for anti-HCV before surgical treatment. Of 11 HBsAg(-) patients,36% were with HBsAb(+) HBeAb(+) HBcAb(+). However, only 9% of the HBsAg(-) patients were HBsAb(-) HBeAb(+) HBcAb(+), while this pattern accounted for the majority (93%) in the HBsAg(+) group. TT and ANTT HBV tDNAs in 11 HCC patients with HBsAg (-) and HBeAg (-) were all detectable. HBV cccDNA and tDNA were all lower in the HBsAg(-) group than those in the HBsAg(+) group. Using Kaplan-Meier analysis, patients with OBI were associated with a lower risk of cirrhosis and better overall survival (OS). Thus, the intracellular HBV DNAs, such as HBV cccDNA and tDNA are valuable biological markers for the diagnosis of occult HBV infection in HCC patients. This would assist the clinical implementation of a more personalized therapy for viral re-activation control and improve the survival rate of OBI patients.Part 2. This study aimed to investigate the association between HBV mutations and intrahepatic HBV status and to determine the clinical features and the contribution of HBV mutations to postoperative prognosis for hepatocelluar carcinoma (HCC) patients with HBsAg (+) in China. Using TaqMan fluorescent real-time PCR, HBV covalently closed circular DNA (cccDNA) and total DNA (tDNA) were both quantified in 106 pairs of tumor tissues (TT) and adjacent non-tumor tissues (ANTT) obtained from HCC patients who received no antiviral treatment before surgical treatment. The prevalence of 19 hotspot mutations were evaluated by Sanger sequencing. HBV cccDNA and tDNA were lower in TT than in ANTT. The loads of cccDNA and tDNA in ANTT were associated with serum HBV DNA and HBeAg. Three Pre-S mutants (A2962G and C2964A in Pre-S1 and C105T in Pre-S2) were associated with higher tumor cccDNA levels (P< 0.05), and A2962G/C2964A mutants were associated with higher AFP levels. This would assist to disclose the virological features, to implement a more clinically personalized therapy and to improve the prognosis of HBV-related HCC patients.Part 3. This study aimed to investigate the pre-S variations of HBV by the Next-generation sequencing (NGS), which is the high sensitivity and high throughput new technology, and the association between HBV pre-S variations and hepatocellular carcinoma. We found that HCC and chronic hepatitis B (CHB) patients were infected predominantly both B and C genotype of HBV; The complexity of HBV pre-S gene in HCC patients was obviously higher than that of CHB patients; In tumor, HBV cccDNA levels were associated with lower percentage of HBV gene type A and type B (r=-0.369, p = 0.001; r=-0.274, p= 0.012), but were associated with higher percentage of HBV gene type C (r= 0.338, p= 0.338). However in adjacent non-tumor tissues, there were no significant correlation between HBV cccDNA with HBV A, B and C genotypes; In CHB group, the serum HBV deletion were mainly located in nt 2850-2863 and nt 2-55. In HCC group, HBV deletion of tumor and adjacent non-tumor tissues were mainly located in nt 2850-2863, nt 2889-2951, nt 3051-3078, nt 7-27 and nt 40-54; The serum HBV deletion in HCC patients were mainly located in nt 2850-2864, nt 3152-3197 and nt 40-52. The percentage of pre-S deletion in HCC patients was significantly higher than that of patients with CHB. The applications of the next-generation sequencing on the sequencing of HBV, it is not only beneficial to indicate the real and accurate status of the virus in the body, promote personalized auxiliary diagnostic methods, but also will be helpful to clinical follow-up and prognosis judgement, and is likely to provide the basis for individualized treatment, finally realizes the personalized care.