Serum HBV RNA and HBcrAg have been shown to reflect intrahepatic cccDNA transcriptional activity and are, therefore, promising candidate biomarkers to better classify disease status and monitor functional cure in chronic hepatitis B (CHB) patients. However, data combining both markers in large real-life cohorts are still limited. During this year's annual meeting of American Association for the Study of Liver Diseases (AASLD2021), Fabien zoulim - Professor of medicine at lyon university, France, published a study entitled "cross-sectional study of serum HBV RNA and HBcrAg in a real-life prospective cohort of 1500 Chronic hepatitis B patients followed in France and Italy" (LP28). During the conference, we invited Prof. Fabien zoulim to share the research further.
Hepatology Digest: What are the characteristics of serum HBV RNA and HBcrAg as new biomarkers of chronic hepatitis B? What is the mechanism of action?
Dr Zoulim: First of all, what we are trying to develop is novel biomarkers that are non-invasive, so they can be tested in the serum of patients, and that reflect the reservoir of the virus in the liver, and mainly trying to reflect the cccDNA in the liver. The first interesting biomarker is viral RNA in serum. We need these biomarkers because we are seeing that quantitative HBsAg is not sufficient to predict the pool of cccDNA, because HBsAg can come either from cccDNA or from integrated HBV DNA. So if we are looking at new treatments or new strategies aimed at curing infection and eliminating cccDNA, then quantitative HBsAg may not be the best marker because it can come from integrated forms. So we need to have biomarkers that really reflect cccDNA. The first one is viral RNA in serum. When we say viral RNA, we really need to mention that it should be the pregenomic RNA in serum, because pregenomic RNA comes mainly from cccDNA and not from integration. This biomarker is very interesting. There are several studies that have shown the correlation between pregenomic RNA and the transcriptional ability of cccDNA in the liver. Now there are novel tests to measure pregenomic RNA. We are seeing the first of many laboratory-developed tests, and now there are even more tests being developed by diagnostic companies, such as Abbott and Roche Diagnostics. We have seen very interesting studies on that, and maybe we can comment later on those results. These biomarkers and the assays are now in development. We have seen many studies at AASLD that have used pregenomic RNA as a marker. The second one is the HBcrAg or core-related antigen in serum. This time, it is an ELISA, because we are measuring the protein, not the RNA. Those tests for RNA are PCR-based assays, but here it is an ELISA for core-related antigen. It reflects the expression of core protein and proteins that are related to core as well as HBsAg, and it is also thought to be reflective of cccDNA because it is mainly expressed by cccDNA and not by integrated forms. There are several studies that have shown a correlation between the HBcrAg in serum and the cccDNA’s ability to transcribe viral RNA. So it is a very interesting test, because it is an easy ELISA test that reflects the reservoir of transcriptionally active cccDNA. Many studies are being performed to show that. The assay is developed by Fujirebio from Japan. It is also being developed here by diagnostic companies. So we are getting these different biomarkers and there are clinical studies ongoing. We can talk about them in the next question.
Hepatology Digest: In conjunction with this study, could you please outline the predictive value of HBV RNA and HBcrAg in the treatment of different populations with chronic hepatitis B?
Dr Zoulim: Since these biomarkers are very important, they have been used in clinical studies. Regarding nucleoside analog therapies, the drugs that we currently have, one of the big questions is whether we can stop NUCs at some point and not do lifelong therapy. In that setting, the question is whether stopping nucleoside analog therapy will allow relapse of viral replication and inflammation, which would be followed by the clearance of HBsAg and an increase in the rate of HBsAg loss. On the other hand, the other scenario is the relapse of viral replication and inflammation, which may just restart the chronic liver disease. So there are two main scenarios. It is important to predict before stopping treatment of the chance or the risk for the patient to either lose HBsAg (which would be very favorable), or to have an active relapse of the disease. Up until very recently, the field was very confused. There were no predictors. There have been several presentations at AASLD looking at the risk of relapse or the chance of achieving HBsAg loss. Again, it is not very easy to have a simple take-home message. The easiest biomarker so far was quantitative HBsAg, because HBV DNA is undetectable by definition, as patients are already on NUCs. It was suggested by different studies again at AASLD that the lower the HBsAg is, the higher the likelihood of losing HBsAg. But the predictive value is not very strong. That is a problem. So now, adding pregenomic RNA and core-related antigen at the time of stopping NUCs, may be of high value. There are several studies from different parts of the world (China, Japan, Western countries) suggesting that combining the three markers could add better predictive value. If patients have low HBsAg (<100), undetectable pregenomic RNA, and low HBcrAg, then the chances of losing HBsAg may be higher. But we will need to address that in a prospective manner, because for most of the studies, the analysis of the biomarkers was done retrospectively. It seems that we are going in that direction - to use these three biomarkers, and finding the best threshold for the different biomarkers together, and building an algorithm to predict. I think that will be very important. For novel therapies, it is also very interesting in clinical trials with novel modes of action, but we need to learn. We have seen very interesting studies with capsid assembly modulators and siRNAs during AASLD, as well as combinations of capsid assembly modulators and siRNAs. We have seen studies of endpoints using HBcrAg and pregenomic RNA in serum, and I think we are learning, together with the new drugs but also with the new biomarkers. It is still a bit early to say how to use these biomarkers with the new drugs, because we have two new things - biomarkers and drugs. But the studies are really interesting, and I am sure it will be very helpful to have these novel biomarkers for monitoring the efficacy of the novel antiviral treatments.
Hepatology Digest: In the process of detecting hepatitis B virus, cccDNA is very critical. So how do HBV RNA and HBcrAg correlate with cccDNA respectively? Is it necessary to add these two biomarkers in the management of hepatitis B in the future?
Dr Zoulim: As I said at the beginning in my response to the first question, it was nicely shown by different investigators independently that pregenomic RNA and HBcrAg are really good biomarkers of cccDNA transcriptional activity. They are very interesting. They are non-invasive, compared to the liver biopsy that is required for cccDNA analysis. That is very good and very important. Now the question will be how are we going to use these biomarkers? Will it be as tools to better classify patients into the different phases of disease - from immune-tolerant to chronic hepatitis to inactive carriers, and so on? That will be one possibility, and we need to do more studies. There were some studies published recently by the NIH group, but we need to build on those studies to see whether these biomarkers can add value for better characterization of patients, and better diagnoses. Will it be a predictive value - a prediction of loss of HBsAg, therefore a prediction of functional cure, before HBsAg is undetectable? That would be extremely important, and we need to have prospective studies where patients lose HBsAg to determine whether pregenomic RNA and HBcrAg perform even better than quantitative HBsAg. We need these prospective studies to see whether these biomarkers will have a strong predictive value that can be used in the clinic. The third application, I think, will be for the monitoring of the new drugs. But then we will need to learn how to use these biomarkers across all the different drugs. Depending on the mode of action of the drugs, the evolution of the biomarker may be different. We need to learn with the clinical development of the new antivirals how to use the biomarkers to see how they may help with the monitoring of patients. I think it is going very well, and we are learning. I think within one or two years, we will know much better how to use these biomarkers. At AASLD, we have seen new drugs in development, like capsid assembly modulators, siRNA, and immune modulators and checkpoint inhibitors. Yesterday, we saw a PD-L1 antibody in a clinical trial from China. All these are very promising. We need to combine all these drugs together to cure the infection, and the use of biomarkers will be extremely important to assist in the development of these drugs, and later on in the future, to manage patients with chronic hepatitis B in the routine clinical setting.
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