Clinical Significance Of HBV RT/S Gene Variations In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of most common liver cancer. It is estimated that approximately 2 billion people are infected with hepatitis B virus in the world, which3.5-4 billion for chronic hepatitis B carriers, about 600 thousand people died of HBV related diseases each year.According to theannual report of Chinese cancer registry of 2014,the incidence of liver cancer in China were 21.35/10 million, ranking fourth, and the mortality were18.43/10 million, ranking second. HBV infection is the main risk of HCC occurrence, development and recurrence factors as reported in the literature, HBV genome variations are closely related to the occurrence, development and recurrence of HCC.Part1. Sanger sequencing based HBV RT/S mutations and the impact of mutations on prognosis of hepatocellular carcinoma patientsHBV reverse transcriptase(RT) is encoded by polymerase gene in reverse transcriptase region which overlapped with the S gene. The relationship between mutation of HBV RT and the pathobiological features of hepatocellular carcinoma(HCC) has not been clearly elucidated up to now. This study aims to explore the mutations in this region of HBV genome and its clinical implications. HBV total DNA(HBVt DNA) was extracted from 84 pairs of HCC tumor tissue(TT) and corresponding adjacent non-tumor tissue(ANTT). The RT/S regions(nt130-1161) were amplified and sequenced using Sanger method. Meanwhile, the covalently closed circular DNAs(ccc DNAs) and HBVt DNA were quantified by specific real-time PCR, respectively. The relationship between RT mutations and clinical characteristics of the HCC patients were analyzed.Twenty-seven and 29 mutations with occurrence frequency more than 5% in the RT/S region were identified,respectively. Serum HBV DNA correlated well with ANTT HBVt DNA(r=0.419, P<0.001)and ccc DNA(r=0.370, P<0.001). The levels of HBVt DNA in ANTT were significantly higher than in TT(P=0.029). Rt F221 Y variation together with tumor size larger than 8cmwas found to be independent risk factors for postoperative recurrence of HCC, with hazard ratio of1.838(95% CI: 1.069-3.161; P=0.028) and 2.345(95% CI: 1.391-3.953; P=0.001),respectively. Rt F221 Y was also an independent risk factor for poor overall survival(HR=2.557, 95% CI: 1.344-4.866; P=0.004). The surface antigen of s R122 K, s T140 S and s F183 V have obviously difference in TT and ANTT, these three amino acids was all located in MHR(major hydrophilic region), this is so call B cell antigen epitope. The mutation of R122 K in HBV S protein is closely related to tumor recurrence(p<0.001).Conclusions: Rt F221 Y was identified to be a risk factor for poor prognosis, and would be a potential viral marker for predicting HCC prognosis.Part II. Clinical significance of HBV RT gene mutation in HBV related HCC based on the next generation sequencing.By deep sequencing the HBV RT region, we focused on the mutations related to liver disease progression and drug-resistance. The CHB(n=37) and HCC(n=95)patients were included, and more comprehensive and abundant HBV sequence information were obtained by deep sequencing. Comparison of next generation sequencing, Sanger sequencing and reverse hybridization method, we found 5/8 cases were same in HBV genotyping when using three these methods,and another 3 case were B, C mixed infection. The results of resistance mutations were shown that 5/8 samples were consistent, and these sample were basically the same based on two sequencing methods.In detection of drug-resistance mutation; the results were similar by two sequencing methods. The average mutation frequency of the adjacent tissue was higher than that of the tumor tissue. Low frequency of nucleotide analogues(NAs) resistant mutations, such as rt L180 M, A181 T, A202 G and M204I/V, were found in NAs non-treatment na?ve patients. The complexity value of CHB patients in RT region were obliviously higher than that in HCC TT and ANTT both at nucleotide(CHB: 0.653±0.029, HS: 0.624±0.038, ANTT:0.370± 0.057, TT: 0.344±0.058,p<0.001) and amino acid levels(CHB: 0.493±0.017, HS: 0.443±0.021, ANTT: 0.282±0.039, TT: 0.262±0.055, p<0.001). The average amino acid mutation frequency of CHBgroup was significantly higher than that in HCC group(CHB: 0.0672 ±0.0096, HS:0.0553±0.0086, ANTT : 0.0575 ± 0.0218, TT: 0.0563± 0.0215, P = 0.019), and paired comparison displayed that mutation frequency in HCC ANTT group was higher than that in the TT group(0.0482±0.0042 vs 0.0462 ±0.01102, P < 0.001). We also found patients suffering from chronic hepatitis B(CHB) were tend to harboring such mutations: rt S53N(OR=0.895, P = 0.011), rt D134N(OR=0.964, P = 0.008), rt I224V(OR=0.956, P= 0.014),while mutations: I91L(OR=1.066, P=0.025), N121I(OR=1.108, P= 0.045),L269I(OR=1.022, P = 0.030) were independently associated with HCC development. With the help of next generation sequencing, we can evaluate HBV genome variations more objectively and accurately and, get deeper insight into the HBV population dynamics,which would be helpful to guide individualized treatment.Part III. Association between serum HBcr Ag and the intracellular HBV DNAs in patients with hepatocellular carcinoma.We aimed to assess the clinical performance of a newly developed HBV core-related antigen(HBcr Ag) for the detection of ccc DNA and total HBV DNA in tumor tissueand adjacent non-tumor tissue of HCC patients. 95 HBV-related patients who received hepatic resection for HCC were enrolled. Serum HBcr Ag were detected by Chemiluminescence enzyme immunoassay(CLEIA). HBVt DNA and ccc DNA were extracted from tumor tissue and adjacent non-tumor tissue and quantified by PCR Fluorescent probe. The level of HBcr Ag in HBe Ag+/HBe Ab- group, HBe Ag+/HBe Ab+ group and HBe Ag-/HBe Ab+group was 6.41±1.18 lg U/ml、5.80±0.50 lg U/ml、5.16±0.94 U/ml, respectively, and significant difference among these three groups were detected. There was conspicuous correlation between serum viral markers and ccc DNA or total HBV DNA in ANTT:HBcr Ag r=0.359, r=0.440, HBe Ag r=0.412, r=0.417, serum HBV DNA r=0.560, r=0.653,all above correlation analysis p<0.05. Conclusions HBcr Ag can help to distinguish HBe Ag positive and negative HCC patients. Although HBcr Ag had correlation with ccc DNA, totalHBV DNA in HBs Ag positive HCC patients, it did not show a better correlation than other serum markers such as HBe Ag, HBV DNA.In summary, Sanger sequencing and NGS could be used for the study of RT/S gene variation, We can obtain more comprehensive information from NGS because the high throughput, sensitive and accurate characters of NGS. This study has found close relationship between HBV RT/S gene mutation and HCC incidence and prognosis. HBcr Ag can help to distinguish HBe Ag positive and negative HCC patients, similar to HBe Ag and HBV DNA, HBcr Ag has a correlation with HBVt DNA and ccc DNA in ANTT.HBcr Ag is another alternative markers for HBC infection monitoring.