Introduction
Hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC) regardless of current antiviral therapy with nucleos(t)ide analogs (NUCs). The burden of liver cancer is increasing [
1], and the risk of HBV-related HCC, despite virological and biochemical responses, is still high in the first five years [
2], steadily persists over time [
3], and remains stable, particularly in patients with cirrhosis [
4] or is at least not eliminated [
5]. The HCC risk still exists among those patients outside current treatment criteria or even exists in individuals with HBV that has been functionally cleared [
6,
7]. These above findings suggest that there are some obstinate HCC-driving factors other than viral replication and hepatitis.
HBV integration, oncoprotein hepatitis B x protein, chronic necroinflammation and hepatocellular regeneration, together with chemical carcinogenesis, generally account for hepatocarcinogenesis [
8]. Among these factors, HBV integration has the potential to be one of those obstinate driving factors. It can lead to chromosomal instability, genomic damage and residual expression of viral oncoproteins, which are all known as selective advantages for tumor progression [
9]. Indeed, HBV integration has been found in more than 80% of HBV-related HCC cases, all tumor single cells exhibit the same HBV integration in monoclonal HCC, and integration sites or integration-related genes are believed to be important for hepatocarcinogenesis [
10,
11]. HBV integration events randomly occur as early as in the immunotolerance phase of patients [
12] or 1–3 h post infection in model cells [
13]. Viral integration, thus, has enough time to play roles in narrowing the hepatocyte population by clonal hepatocyte expansion, similar to other HCC contributors [
14]. Clonal hepatocyte expansion, a major risk factor for HCC [
15], often occurs in the later stages of infection such as cirrhosis. It is estimated that at least half of cirrhotic nodules are clonal [
16]. However, clonal hepatocyte expansion also occurs as early as in the liver of patients with so-called immunotolerance [
12,
17]. Therefore, the subsequent fates and evolution of early viral integration in the immune clearance phase (chronic hepatitis) are important parts of hepatocarcinogenesis. Unfortunately, only very few studies have focused on viral integration events in chronic hepatitis B (CHB), and none of them have discussed the subsequent fates and evolution to date.
In this study, HBV integration was successfully detected using a high-throughput viral integration detection (HIVID) assay in liver biopsy samples from 54 CHB patients. The subsequent fates of HBV integrations and clonal hepatocytes in the stage of CHB were mainly influenced by liver damage surrogate indicators, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver inflammation activity grade score. NUC treatment that remitted liver damage was preliminarily found to perhaps reduce viral integration but undoubtedly increase clonal hepatocytes, which may explain why HCC risk cannot be ruled out by NUC treatment.
Discussion
The HBV integration in our CHB patients with hotspots in the region of 1600–1900 bp of the HBV genome and the enrichments to some extent in genes and KEGG pathways are similar to those in most reports [
10,
11,
23], suggesting that our cohort of patients is suited for the study of the subsequent fates and evolution of early viral integration in the immune clearance phase. HBV integration mainly shows dispersed distributions in non-tumor tissues [
10,
11], and the chromosomal distribution of integrated HBV does not change during HBeAg-seroconversion [
23]. However, the HBV integration-related genes in our CHB patients were inclined to gap and tight junctions. These enrichments usually occurred in patients with elevated ALT levels and NUC-untreated patients who usually had significantly higher ALT levels. In contrast, the pathway enrichments may disappear when patients restore to normal ALT levels or receive NUC treatment. These findings suggest that the inventories of HBV integration are dynamically changing with liver damage, and liver damage allows some host genes, especially those genes that are activated for liver repair, to be susceptible to viral integration.
HBV integration events occur in the early stage of HBV infection [
12,
13,
24]; as thus, their fates, compared with integration itself, seem to be even more important to the tumorigenesis of HCC. In this study, we found that HBsAg persistence was favorable and serum HBV DNA load, ALT and AST were unfavorable for the maintenance of viral integration. Compared with ALT and the liver S scores, AST and the G scores had closer correlations with the fate of HBV integration, suggesting that necrosis (liver damage) rather than simple hepatocyte degeneration or fibrosis was actually correlated with the fate of HBV integration. The S scores, however, were differently related with pathways in patients with low and high S scores, suggesting that there is a possibility that advanced fibrosis may be correlated with more dangerous HBV integration to HCC hepatocarcinogenesis. The negative correlations with HBV integration in the number of breakpoint types suggest that liver damage is favorable for the elimination of accumulated integrations, and together with the relationships between transaminase levels and KEGG pathways, these results further suggest that liver damage plays a bidirectional role to increase the chance and to reduce the accumulation of HBV integration. However, the major role of liver damage was to reduce the inventory of HBV integration, perhaps due to integration eliminations surpassing chance increases in CHB. Therefore, the number of breakpoint types was weakly, but significantly negatively correlated with liver damage, and with serum HBV DNA load regardless of its enhancing effect on integration chance [
25]. Therefore, liver damage is the key determining factor of HBV integration fate in CHB.
The evolution of HBV integration may be another important process of hepatocarcinogenesis. The clone sizes of hepatocytes in patients with HBeAg-negative CHB are much larger than those in patients with HBeAg-positive CHB [
23]. When breakpoint frequencies were used as indicators of clonal expansion in this study, a similar phenomenon evidenced by elevated average and maximum frequencies of breakpoints was found in our HBeAg-negative CHB patients. Moreover, some hepatocytes were found to clonally expand to more than 10
3 in scale. The average, maximum and total breakpoint frequencies were all negatively correlated with the liver damage surrogate indicators of ALT, AST and liver inflammation activity grade score, suggesting that liver damage eliminates clonal hepatocytes in CHB. However, many scientists believe that the immune killing of infected hepatocytes is the strongest known pressure point to drive clonal hepatocyte expansion [
14], suggesting that liver damage may also bidirectionally influence clonal hepatocyte expansion but may primarily help to eliminate clonal hepatocytes in CHB.
The underlying mechanism of HCC prevalence despite sustained viral response and remission of liver inflammation during NUC therapy has been a hot topic in recent years [
1‐
5]. In this study, compared with non-NUC-treated patients, treated patients only had a minor decrease in breakpoint types but had significantly higher average and marginally higher maximum frequencies of breakpoints, implying an increase in clonal hepatocytes, though the total frequency was similar because it was minimized by the small decrease in breakpoint types. The remission of liver damage explains the increase in clonal hepatocytes but cannot explain the decrease in breakpoint types since it should increase because of elimination reduction. Therefore, the decrease in breakpoint types may result from persistent HBV replication inhibition that leads to a reduction in the chance of integration and a subsequent decrease in the inventory of viral integration. Together with the significant increase in clonal hepatocytes, the decrease in breakpoint types may explain why NUC treatment reduces but does not rule out the risk of HCC, especially in the later stage when clonal hepatocyte expansion is almost finished, supporting the views of some scientists to conduct antiviral intervention early to reduce genetic damage to a large extent [
12,
14].
Integrated HBV DNA has the potential to express HBsAg, which is unfavorable for a radical cure [
26,
27]. Therefore, knowing how to remove HBV integrations is favorable not only for HCC prevention but also for the radical cure of HBV infection. The significance of liver damage to HBV integration and clonal hepatocyte expansion in this study supports the restoration of host antiviral immunity [
28]. Excitingly, neoadjuvant anti-PD1 therapy significantly decreases HBsAg, especially when there is a flare of ALT, in patients with virally suppressed CHB [
29]. However, persistent inflammation in the liver promotes clonal hepatocyte expansion and increases the risk of HCC [
14,
30]. Therefore, much work still needs to be done before we are able to use host antiviral immunity to prevent HCC. Nonetheless, transient and limited inflammation may benefit CHB patients in either HCC prevention or radical cure of HBV infection.
In conclusion, our study uncovered the characteristics of HBV integration in CHB patients and provided convincing evidence that liver damage increased the chance of HBV integration but might mainly prevent HCC by removing viral integrations and clonal hepatocytes in CHB. NUC treatment may reduce the chance of HBV integration but simultaneously reduce the elimination of viral integration and clonal hepatocytes. These results might have many implications for CHB treatments and HCC prevention in the future.
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