CHB patients with rtA181T‐mutated HBV infection are associated with higher risk hepatocellular carcinoma due to increases in mutation rates of tumour suppressor genes

The HBV rtA181T mutation is associated with an increased risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study aimed to evaluate the mechanism by which rtA181T mutation increases the risk of HCC. We enrolled 470 CHB patients with rtA181T and rtA181V mutation in this study; 68 (22.15%) of the 307 patients with rtA181T mutation and 22 (13.5%) of the 163 patients with rtA181V mutation developed HCC (p < .05). The median follow‐up periods were 8.148 and 8.055 years (p > .05). Serum HBV DNA and HBsAg levels in rtA181T‐positive patients were similar to that in rtA181V‐positive patients. However, the serum HBeAg levels in the rtA181T‐positive patients were significantly higher than that in rtA181V‐positive patients. In situ hybridization experiments showed that the HBV cccDNA and HBV RNA levels were significantly higher in the liver cancer tissues of patients with the rtA181T mutation compared to that in the tissues of patients with the rtA181V mutation. The percentage of anti‐tumour hot‐gene site mutations was significantly higher in the rtA181T‐positive HCC liver tissue compared to that in the rtA181T‐negative HCC liver tissue (7.65% and 4.3%, p < .05). This is the first study to use a large cohort and a follow‐up of more than 5 years (average 8 years) to confirm that the rtA181T mutation increased the risk of HCC, and that it could be related to the increase in the mutation rate of hotspots of tumour suppressor genes (CTNNB1, TP53, NRAS and PIK3CA).

470 CHB patients with rtA181T and rtA181V mutation in this study; 68 (22.15%)   of the 307 patients with rtA181T mutation and 22 (13.5%) of the 163 patients with rtA181V mutation developed HCC (p < .05).The median follow-up periods were 8.148 and 8.055 years (p > .05).Serum HBV DNA and HBsAg levels in rtA181T-positive patients were similar to that in rtA181V-positive patients.However, the serum HBeAg levels in the rtA181T-positive patients were significantly higher than that in rtA181Vpositive patients.In situ hybridization experiments showed that the HBV cccDNA and HBV RNA levels were significantly higher in the liver cancer tissues of patients with the rtA181T mutation compared to that in the tissues of patients with the rtA181V mutation.The percentage of anti-tumour hot-gene site mutations was significantly higher in the rtA181T-positive HCC liver tissue compared to that in the rtA181Tnegative HCC liver tissue (7.65% and 4.3%, p < .05).This is the first study to use a large cohort and a follow-up of more than 5 years (average 8 years) to confirm that the rtA181T mutation increased the risk of HCC, and that it could be related to the increase in the mutation rate of hotspots of tumour suppressor genes (CTNNB1, TP53, NRAS and PIK3CA).

K E Y W O R D S
HBsAg, hepatitis B virus, ROS, rtA181T mutation, rtA181V mutation critical for reducing the incidence and mortality associated with HCC. 4,12,13veral nucleos(t)ide analogs (NA) treatments have been developed for CHB; the strategies for treating chronic HBV includes pegylated interferon (Peg IFN) or NA such as adefovir dipivoxil (ADV), lamivudine(LAM) and entecavir (ETV). 6,8,14,15However, treatments with early anti-HBV drugs such as LAM and ADV encounter high drug resistance; rtA181T and rtA181V mutations in HBV are mainly caused by LAM and ADV treatments. 168][19] The rtA181T mutation led to a switch in the HBV S gene sequence at the particular locus from TGG to TAG; the resulting stop codon causes a loss of 55 amino acids in the S antigen.This prematurely terminates the HBsAg translation resulting in a truncated pre-S/S proteins, which in turn inhibits the secretion of HBsAg.Reports suggest that rtA181T mutation is associated with HCC 17,[20][21][22] ; however, the underlying mechanism by which this mutation contributes to HCC remains unclear.This could be attributed to the insufficient evidence from research and clinical studies.
Oxidative stress refers to the production of highly active molecules, such as reactive oxygen species (ROS), when the body is subjected to various harmful stimuli.Cellular responses such as proliferation, differentiation and apoptosis are regulated by the ROS generated by the cells. 23Study has revealed that HBV infection enhances ROS production and causes oxidative stress in host cells. 24[27][28] In this study, we used rtA181T and rtA181V resistant specimens obtained from 2009 to 2013 to conduct in-depth research.
We analysed the clinical variables, virological parameters, and the rate of HCC occurrence.HBV DNA and RNA levels in rtA181T and rtA181V mutant HCC liver tissues were evaluated using in situ hybridization.Next generation sequencing was used to analyse the difference in anti-tumour gene' hot site mutation ratio between rtA181T-and no-rtA181T mutated liver tissue of HCC patients, to confirm our hypothesis that a higher rtA181T mutations in the infected liver cells increases the intracellular ROS and induces the anti-tumour hot gene site mutation ratio, contributing to HCC.

| Patients
A total of 470 patients with chronic HBV infection were enrolled in this study; all of them underwent drug resistance testing (direct sequencing) at You An Hospital, Capital Medical University, China, from March 2009 to December 2013.Based on our previous study, 29 we enrolled patients tested separately for rtA181T or rtA181V mutations.Among them, 307 were infected with HBV harbouring the rtA181T mutation, and 163 were carriers of rtA181V-mutated HBV.All patients completed a follow-up period of more than 5 years.They were characterized as HBsAg and/or HBV DNA positive for over 6 months and were diagnosed with HCC was diagnosed according to the following criteria: echoguided liver biopsy, fine needle aspiration cytology and high alphafetoprotein (AFP) levels (>200 ng/mL) accompanied by at least one dynamic imaging study or one dynamic imaging study plus angiography (if alpha-fetoprotein <200 ng/mL). 30

| Analysis of virological markers
Biochemical and serological markers including HBeAg status, HBsAg status, levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), AFP and HBV DNA in the patients harbouring HBV with rtA181T or rtA181V were routinely detected in the Central Clinical Laboratory of You An Hospital, Capital Medical University, China.Sex, age and time of HCC development were recorded (Table 1).

| Tissue in situ hybridization
Fresh liver cancer tissues from patients with the HBV rtA181T (two cases) or rtA181V mutation (two cases) were obtained from the Department of Liver Transplantation Surgery, You An Hospital, Capital Medical University, China.Informed consent was obtained from the patient or family members.
The HBV cccDNA and RNA in situ hybridization assays were performed as described in the Tables S1 and S2.

| Total DNA preparation and next generation sequencing
Total DNA was extracted from the rtA181T and rtA181V mutant HCC samples using the DNA Mini Kit I (Magen).The quality and concentration of the libraries were confirmed using agarose gel electrophoresis, Qubit 3.0, Agilent 2100 Bioanalyzer system.The NGS was performed at Magen Company on an Illumina HiSeq | 953 platform.The site mutations of the hot genes were valuated (Table S3).

| Statistical analysis
Data were analysed using GraphPad Prism version 9.00.Continuous variables are expressed as mean ± standard deviation or median (interquartile-range [IQR]), as appropriate, categorical variables are presented as a number (percentage).Qualitative and quantitative differences between the two subgroups were analysed using the chi-squared test for categorical parameters and Student's t-test or Mann-Whitney's test for continuous parameters.The qualitative and quantitative differences between three or more subgroups were analysed using the chi-squared test for categorical parameters and one-way ANOVA or the Kruskal-Wallis test for continuous parameters.p < .05 was considered statistically significant.

| HBV replicates more in patients with a rtA181T mutation
The clinical characteristics and serological markers of patients with rtA181T and rtA181V mutations are shown in Table 1.Compared to rtA181V-positive patients, rtA181T-positive patients had significantly higher HBeAg levels(p = .003).However, the ALT, AST, total bilirubin (TBIL), serum HBV DNA and HBsAg levels did not differ significantly (Figure 1).HBeAg does not participate in HBV virus assembly; however, it is often considered a marker of HBV replication and infection because it is a secretory protein obtained through the processing precursor protein.The levels of serum HBV DNA and HBsAg in rtA181T-positive patients were similar to that in rtA181Vpositive patients.However, the level of HBeAg in rtA181T-positive patients was higher, suggesting that the rtA181T mutation could affect the secretion of HBV.

| The incidence of HCC was higher in rtA181T-positive group
The rtA181T mutation is associated with the development of HCC. 31 However, there are limitations in both the observation time and number of patients.In this study, follow-up was sustained for no less than 5 years for patients carrying rtA181T or rtA181V mutations.

| RtA181T mutated HBV infected cells have higher levels of cccDNA and RNA
The high-level of serum HBeAg in patients with the rtA181T mutation suggests that there could be more cccDNA and RNA in liver cells.To verify this hypothesis, we designed a prokaryotic DNA expression vector for the single-stranded region of HBV and used the T7 promoter to obtain a digoxigenin probe that cannot hybridize with mature HBV DNA.Full-length HBV DNA was used as a template to obtain an HBV RNA hybridization probe using a random labeling kit.Liver tissue specimens from patients with rtA181T and rtA181V mutations who underwent surgical liver transplantation were used for in situ hybridization.Positive hybridizations for cccDNA and RNA were observed only in tissues infected with rtA181T mutated HBV (Figure 3A,B); this supports the hypothesis that HBV rtA181T particles cannot be released and accumulate in liver cells.*Indicated the significant difference.

| Mutation rate of hotspots of tumour suppressor genes in liver tissues infected with rtA181T-mutated HBV is significantly higher
HBV infection can induce an increase in the level of intracellular ROS, 32,33 which is one of the most critical independent factors causing gene point mutations. 34RtA181T mutation causes the intracellular accumulation of HBV; therefore, we suspected that the rtA181T mutation is associated with an increased mutation rate in tumour suppressor genes.To verify this, we used NGS and identified 87 hotspot mutations among the nine cancer-related genes (BRAF, CTNNB1, ERBB2, FBXW7, HNF1A, KRAS, NRAS, PIK3CA and TP53) associated with liver cancer (Table S3).The results from 10 patients showed that 27 hotspot sites from the CTNNB1, TP53, NRAS, and PIK3CA genes were significantly different [rtA181T(+) vs. rtA181T(−): 7.65% vs. 4.3% (p < .01)](Figure 4A,B).This suggests that infection with rtA181T-mutated HBV can significantly increase the mutation rate of hotspots of tumour suppressor genes in HCC tissues; this could be associated with the incidence of HCC.

| DISCUSS ION
Nucleos(t)ide analogs is a key treatment strategy for the controlling the disease progression in CHB.The most common potent drugs for treating CHB and liver cirrhosis are ETV and TDF 35,36 ; However, LAM and ADV were used as anti-viral drugs earlier.
7][38] The rtA181T mutation could be related to an increased incidence of HCC; however, a large cohort and long-term follow-up were needed to confirm this.Therefore, we evaluated a large cohort that completed more than 5 years of follow-up.The rtA181T mutation was closely associated with a high incidence of HCC in patients with anti-viral drug-resistance; this is consistent with earlier results. 20In addition, the cumulative incidence of HCC was 22.15% in the rtA181T group and 13.5% in the rtA181V group, the follow-up period was 8.148 and 8.055 years, respectively.Considering the large number of people infected with HBV harbouring the rtA181T mutation in China, it is critical to explore In situ identification of HBV cccDNA and RNA.We designed a DNA prokaryotic expression vector for the single-stranded region of HBV and used the T7 promoter to obtain the digoxigenin probe that does not hybridize to mature HBV DNA.The full-length HBV DNA was used as a template for obtaining the HBV RNA hybridization probe with random labeling kit.The liver tissue specimens of rtA 181 T and rtA181 V patients from surgical liver transplantation were used for in situ hybridization.the mechanism by which the rtA181T mutation increases the incidence of HCC.To obtain accountable evidence and explore the relationship between HCC and the rtA181T mutation, 307 rtA181T-mutated HBV-infected patients who completed followup for more than 5 years were enrolled in this study.We tested the serological characteristics of the viruses.At the same time, we observed the accumulated intracellular levels of HBV cc-cDNA/RNA and increased mutation rates of several key tumour suppressor genes in the liver of patients, suggesting a strong association between the rtA181T mutation and an increased risk of HCC.
Generally, HBV DNA and HBeAg are considered markers of HBV replication and indicative of anti-viral therapy, 17 because HBV DNA and HBeAg increase with active HBV replication.Theoretically, the rtA181T mutation reduces HBV DNA levels.However, this study and earlier reports 16 show that the level of HBV DNA in patients with rtA181T was similar to that in rtA181V patients.However, the level of HBeAg in the rtA181T group was significantly higher, suggesting the influence of de-functionalisation of the viral secretion of rtA181T-mutated HBV.
We have reason to believe that rtA181T-mutant HBV replication level is active at the intracellular level because the serum level of HBV DNA is similar between rtA181T and rtA181V groups; however, the HBeAg level is significantly higher in the rtA181T group (p = .0032).In vitro studies have confirmed that rtA181 T mutations can cause defects in HBV secretion, resulting in their retention within the cell. 17,39We directly used the HCC tissues of patients with rtA181T-mutant HBV for situ hybridization; we confirmed that rtA181T-mutant HBV was significantly higher in HCC tissues compared the rtA181V-mutant virus.This is consistent with the results of the in situ hybridization we performed at the cellular level. 40Intracellular HBV infection is closely related to ROS levels.HBsAg, HBcAg and HBx are viral proteins involved in ROS formation. 32,41,42mbined with the results of in situ hybridization, we believe that the accumulation of mutated HBV in cells increases intracellular ROS levels.ROS is recognized as the main cause of single-base mutations in DNA. 32 verify that a high-level of intracellular rtA181T-mutated HBV is associated with hotspot mutations in tumour suppressor genes, we enrolled HCC patients with or without rtA181T-HBV infection.Hotspot mutations in TP53, CTNNB1, NRAS and PIK3CA were detected using NGS; these results were similar to that in an earlier report. 43In addition, the gene mutation rate in the rtA181T-positive group was significantly higher than that in the rtA181T-negative group.Tp53, a tumour-suppressor protein, plays a critical role in responding to cellular stress and inhibiting malignant development. 44Loss of p53 function contributes to the development of most cancers including HCC. 45 The rate of TP53 mutations was higher in the rtA181T group compared to that in the rtA181T-negative group.Combined with the results of in situ hybridization, we believe that the higher incidence of cancer in the rtA181T group could be attributed to the combined effect of TP53 gene mutations.

| CON CLUS ION
To the best of our knowledge, this is the first large cohort study with a long follow-up of more than 5 years (average 8 years) confirming that the rtA181T mutation increased the risk of HCC; the secretion defect of HBsAg and HBV DNA and increased HBV particles in infected cells could be involved in the increased incidence of HCC.
Mechanistically, these HBV elements cause high mutation rates in CHB. Patients co-infected with hepatitis A, C, or D viruses or human immunodeficiency virus (HIV) were excluded.This study was approved by the Ethics committee of the Beijing You An Hospital.The inclusion criteria are shown in Figure S1.We also investigated the NAs use of the patients at baseline, and the detail of NAs information were showed in Figure S2.Afterwards, therapy was adapted, most patients added ETV on the basis of the original medication, or switched to TDF.

F I G U R E 1 F I G U R E 2
ALT, AST, HBV DNA, HBsAg and HBeAg serum levels in rtA181V and rtA181T groupss.Serum levels of (A) ALT, (B) AST, (C) TBil, (D) HBV DNA, (E) HBsAg and (F) HBeAg in patients with rt A181T and rt A181V mutations.**p < .01.The cumulative incidence of HCC.| 955 NING et al.LAM and ADV in China.However, drug resistance is often observed during the anti-viral treatments with LAM and ADV.

4
(A) cccDNA Hybrization, (B) HBV RNA Hybrization.The heatmap of DNA mutaitons in liver tissue of patients with rtA181T mutation versus without rtA181T mutation.(A) The specific location of the mutation.(B) Gene of the mutation.
the tumour suppressor genes by inducing ROS.The results confirmed that the rtA181T mutation increased the risk of HCC by increasing the mutation rates of hotspots of tumour suppressor genes, such as CTNNB1, TP53, NRAS and PIK3CA could be HBV-associated HCC.AUTH O R CO NTR I B UTI O N SQiqi Ning, Tongwang Yang designed the study and analysed the clinical data.Qiqi Ning, Tongwang Yang and Xianghua Guo involved in drafting the manuscript and revising it critically for important intellectual content.Yanxiang Huang, Mengcheng Liu, Pengxiang Yang, Yuanyue Guan, Ning Liu, Yang Wang interpreted and analysed the clinical and laboratory data.Dexi Chen interpreted and analysed the pathological data.All the authors have read and approved the final manuscript.
Demographics and Clinical Characteristics of CHB Patients with rtA181T and rtA181V mutation.
a Mann-Whitney test.b Fisher's exact test.