So it's 11 o'clock. I think we can maybe get started. Good morning, good afternoon, and good evening. Welcome to our 2022 ASLD Hep B-SIG webinar on HPV DNA integration, mechanisms, pathogenesis, and clinical relevance in liver disease progression and functional cure. My name is Kyungmi Chang from University of Pennsylvania, Perlman School of Medicine and the Crescent VA Medical Center in Philadelphia. I'm honored to co-moderate this webinar with my esteemed colleague, Professor Haitao Guo from Hillman Cancer Center at the Pittsburgh School of Medicine. I also want to recognize the ASLD, HPV-SIG basic science subcommittee members, and the HPV-SIG leadership team, of course, ASLD staff, Dominique Clayton and Brian Kirkland, who's helped us organize this program. So live from Pennsylvania, our goal today is to provide all just in a single hour the state-of-the-art insights to HPV DNA integration with associated disease pathogenesis and clinical implications with three outstanding speakers. First speaker is Professor Stefan Wieland from University Hospital Basel, Switzerland, a well-recognized expert in HPV virology and pathogenesis, especially with seminal observations regarding stealth and cunning of HPV and HCV, and more recently, host virus interactions in the liver of HPV-infected patients. Today, he will speak on basic virology of HPV DNA integration with clinical relevance. Second speaker is Professor Irene Ng from University of Hong Kong. Professor Ng has been using cutting-edge genomic transcriptomic and single-cell analyses to conduct elegant studies on cancer pathogenesis, heterogeneity, and tumor microenvironment in HPV-associated HCC. She will speak on HPV DNA integration and hepatocarcinogenesis. The final speaker is Professor Patrick Kennedy from Barts and the London School of Medicine and Dentistry. He has been redefining disease phases and age-related immune changes in HPV patients. Also, as a clinical hepatologist, has been working to optimize therapeutic strategies for chronic hepatitis B. Professor Kennedy will speak on pathogenetic impact of HPV DNA integration and therapeutic implications. So, for all of you in the audience, each speaker will speak one after the other without break, and we will then have about 10 minutes of Q&A for all three speakers, with Professor Guo as the moderator. We invite the audience to upload their questions and comments by the Q&A box at the bottom of your screen. That could be addressed at the end during the Q&A sessions as time allows. So now, without any further ado for the first speaker, Dr. Violand, take it away. Thank you very much, Kyungmi, and thank you to the organizers to give me the opportunity to present our data as this interesting webinar. And as soon as I get my pointer here, well, if I don't get it, I just say, can you see the mouse? I think so. So, as the title here implies, I would like to give a very brief introduction into the very basic virology of HPV DNA integration, and then one aspect of its clinical relevance that we recently observed. And I have nothing to disclose. And so, HPV integration has been studied for several decades, and I would like to direct your attention to some recent reviews, some of them listed here, that have been, that summarize all this work and have been written by the definitive experts in this area. And these reviews are part of a whole series that has been published last year. So, that's the source where you can get all the information. So, HPV integration obviously starts with hepatitis B virus, and here is just a little graphical illustration of the virus. And the most important feature here is that the HPV virus is a partially double-stranded relaxed circular DNA genome, and that contains a number of promoter and enhancer sites that direct the transcription of this genome, and that is actually run by the host RNA polymerase II system and also host transcription factors. So, the most important RNA that is made from that is from this promoter here, and that's the pink labeled RNA here, which is the pre-genomic RNA that then serves for reverse transcription and assures replication of the virus. And from other promoter in this, in the viral genome, there are sub-viral RNAs produced, the major three ones are shown here that produce all the viral envelope proteins and the X-protein that is instrumental in establishing infection. So, and the genome also contains two direct repeat areas, and they are important for replication. And so, how does this replication work and why is that related to integration? So, here we go back to an old, this depiction of the HPV reverse transcription, and as Hayato already realized, this is actually from the HPV, but it serves the purpose. So, we have the pre-genomic RNA on which the viral polymerase is initiating the reverse transcription on the epsilon sequence, and then starts the miracle of HPV reverse transcription, and that is a number of template switches. So, this little piece, which is to the three prime end of the RNA, and then starts synthesizing the minus strand, and at the same time removes the RNA. And so, you end up with the single strand DNA that has a little primer left here. Now, this primer gets relocated again, and second DNA synthesis starts, there is another template switch, and then finally the relaxed circular or cDNA genome is produced. But sometimes that doesn't really work out, and actually at this step here, rather than this switching over here, this primer, it is used directly for second strand DNA synthesis, and that produces then the so-called double strand linear DNA, BSL DNA, and that is what is the important factor for integration. So, how does that then look like? So, we have this double strand linear DNA here, it contains a direct repeat on one end, direct repeat here, and a little bit of it on this end, and this piece has now open ends, and it will be integrated into the host genomic DNA by the non-homologous end joining DNA repair system that actually repairs double stranded breaks in the genome. And typically, this happens at random sites, and it also involves small deletions of the double strand linear DNA. So, you end up with something that looks like that in the host, which has host sequences to the left and to the right, and this BSL DNA in the middle. And as you can see, it still contains all the promoter elements, the polyadenylation site, and it contains the open reading frames for all the viral genes. However, the promoter that drives expression of the bridge genomic RNA is on the three prime end of the construct. So, this RNA encoding these proteins cannot make, cannot be made, and thus they are not expressed. And so, this construct is actually replication deficient. However, the RNAs, the sub viral RNAs driven by these other promoters, they can be initiated and they will be transcribed. However, they are lacking the polyA site over here, because that is now at the five prime end of the integrated DNA. And so, they can typically transcribe into the host and they produce viral host chimeric transcripts. They could also, in some cases, there is a cryptic polyA site here that also could be used. Importantly, however, these two RNAs can produce all the authentic HPS antigen molecules L, M, and S. And the X transcript can also produce X, but that is typically truncated at its three prime end, or it can even occur as a fusion. So, when does that actually happen? And this cartoon actually comes out of one of these reviews I was mentioning before. So, it all starts with the virus that contains double-stranded relaxed circular DNA, enters the cell, delivers the DNA to the nucleus, produces CCCDNA, produces all the viral RNAs, the bridge genomic RNAs, encapsulated, reverse transcribed into viral relaxed circular DNA, secreted as virus, and on the go. But sometimes, as I mentioned, this reverse transcription can produce this double-stranded linear DNA. However, this capsid can also be enveloped and secreted, and it basically looks like the virus from the outside. So, that can enter another cell, the double-stranded linear DNA is delivered to the nucleus, and typically, that is when this gets integrated into the host genome. It can also form a type of CCCDNA, but that's a non-functional molecule at this point. It's also conceivable that this capsid with the double-stranded linear DNA can actually enter the nucleus again and integrate. So, now, with these basics, I would just like to show you an example of the consequences that can have at the clinical level. And this is something we observed recently in our own studies, that there could actually be a big ubiquitous expression of HBs antigen from integrated HPV DNA in some patients. This all started with the observation that it is known that there is a correlation between HBs antigen levels in the serum and viral replication in HPE antigen-positive patients that have high viral loads and really replicate the virus. However, this correlation is lost in the HPE antigen-negative patients, in many cases, that have low level of viral replication, kind of suggesting that TRS antigen and replication are uncoupled from each other. And, in fact, what we observed in some of our patients, that we had a subset of patients with E antigen-negative chronic infection, called EMCI, that presented with more than 90% of HBs antigen-positive hepatocytes in the liver, that's the black bars here, and also had ubiquitous intrapathic HPV RNA expression, that are the red bars here. As an example, you see that down here, S antigen expression and in situ hybridization for HPV RNA. And this, despite the fact that their viral load was below 2,000 units of international units per ml. Now, when we did the more closer analysis of the liver of these patients and looked for CCC DNA and total HPV DNA, we noticed that maximally 3 to 29% of the hepatocytes in these patients, in the liver of these patients, could actually contain at least one CCC DNA molecule. And this, even though that more than 80% of the hepatocytes were actually HPV RNA positive, that makes it unlikely that the CCC DNA can be the transcriptional template for all this RNA. However, when looking at total HPV DNA levels in these livers, we found that they were always around two to four copies per hepatocyte in average. So that makes it very likely that it's actually integrated DNA that is the source for all the RNA and thus the HBs antigen expression. We then also looked at that by RNA-seq and we're specifically looking for the virus host kinetic transcripts or reads because they are an indication that RNA is produced from integration. And so here is just a cartoon of the HPV genome. And here is the DR1 region where we saw before that that is the typical integration site breakpoint where HPV integrates into the genome. And when we looked for transcribed HPV positive strength breakpoints and summed them up, we realized that most of them were right at this DR1 sequence, very much consistent with integration of double-stranded linear DNA and production of chimeric transcripts. And when we just looked at the read coverage of HPV, we saw that there were some reads whenever we were in the region where S transcription was actually expected and the reads completely disappeared right at the junction where it went into the host genome. And so overall, there were more than 87% of the breakpoints mapped into two sequences up to 100 nucleotides upstream of the DR1 region in the genome, right where it should be for integrations of double-stranded linear DNA. We found more than 100 unique virus host chimeric reads in the liver of each patient. So it's a big variety of integration sites. And the total HPV read coverage shows expression of the entire envelope coding region that will be expected from such an integration. So this is just the short version of all of that. And what we also found is that others did that too, is look how distributed, where there is a specific distribution of these integrations. And what we found is that there is no integration bias for this transcriptionally active HPV DNA integration, because that's all based on the chimeric reads. And as you can see on this graph here is that the number of unique integrations per chromosome was not significantly different from the expected frequency. So black bars is the observed number and the gray one is the expected. Also, there was actually integration sites were not shared among the different patients, again, suggesting that the integration is pretty much random in these patients. So in summary, what has been developed over the years and what we found is the double strand linear HPV DNA is the major substrate for HPV DNA integration. Integration occurs already early after infection and throughout chronic infection that has been shown by others. Integrated HPV DNA is replication defective, but the integrated DNA has the potential to express viral proteins such as L, M and S in the native form and truncated or host fusion versions of X. And what we found in our study is that transcriptionally active HPV integration can actually extend to the entire liver and support this HPV envelope expression. Altogether, it seems clear that the integration driven viral protein expression should probably be considered in pathogenesis, clinical diagnosis and treatment of chronic HPV infection, and you will hear more about that in the next talks. Now, questions that are under investigation by many different labs at this point and need to be addressed are the integration events throughout different disease phases of chronic HPV infection. So when does it happen? How much? And so on. What's the relationship of infection parameters like viral loads, host responses, liver disease, etc. on integration frequency in the different disease phases? Impact of the HPV integration on clonal expansion of hepatocytes that has been already studied a lot, but that's a very important subject and we will learn more about that later on. What is the contribution of HPV integration to the serum HPS antigen levels? Is there a direct link or not? And what is the relative expression levels of the different forms of S from integration? Is that always the same? Does it depend on the integration, etc.? There can be mutations in the integrated HPV DNA, especially in the S that has been described, in 3S especially. What's the impact of those? What's the role of HPV integration in ACC development? You will hear more about that in the next presentation. And that's more of a philosophical question here. Is there something like biological resolution of infection even in the presence of integration derived S antigen? And last but not least, what's the impact of integration driven S antigen expression on therapeutic modulation of antiviral immunity if that is made by all the cells? That might be a problem. so with that, I come to the very end and the acknowledgements, and I would specifically thank Marcus Heim, who has started a pipeline to collect biopsies about 20 years ago. And thanks to that, we actually have the opportunity to look into the liver of some patients with chronic infection. And with that, I'm closing here. Thank you very much for your attention. Thank you. Yeah, so I'm Irene Ng. Yeah, I'd like to thank the organizers for inviting me to give this talk. And I'm very pleased to share with you on the topic of HPV DNA integration and hepatocardiogenesis. Sorry, I have nothing to disclose. We know that HPV is very prevalent worldwide. And indeed, there are about 300 million HPV carriers worldwide, particularly in Chinese. And we know that liver cancer is one of the notorious complications of chronic hepatitis B viral infection. And this is highly prevalent in Asia. Even in Western countries, there are fast increasing rates. So it's a deadly cancer. And worldwide, more than 50% of the HCC cases are associated with the chronic hepatitis B infection. So there is this question, how does HPV contribute to HCC pathogenesis? For HPV integration in the context of hepatocardiogenesis, there can be at least three mechanisms that HPV integration in the host genome can induce HCC. Insertion of HPV sequences can lead to abnormal or increased transcription of host genes. HPV insertion can also lead to truncation or alteration of the host gene that can generate novel fusion proteins with altered properties and enhance HCC formation. And HPV integration can disrupt the chromosomal stability. I'm going to show these two in my talk. I will talk on the TERP promoter integration by HPV and also HPV integration generating oncogenic chimera and also the C-terminal truncated HPX. With the advances in next-general sequencing, the patterns of viral inclusions into the host genome can be studied. And here, the recurrent HPV integrations were identified in cancer-related genes. For example, the TERP MLL4 RoC1. And by all means, TERP is the most common gene with HPV integration. About the TERP gene itself, it is the commonest mutation at the TERP promoter region. It is the most common gene mutation in HCC and is reported to be up to 60%. And TERP promoter mutation, you can see here, that it can be present in premalignant lesions. But how about HPV integrations involving the TERP gene? In a recent study to investigate HPV integration in HCC, we found that HPV integration at the TERP promoter region is frequent, amounting to about 35% here also. And on the other hand, TERP promoter mutation itself, without HPV integration into the TERP promoter region, amounts to only 15%, less than that reported elsewhere. Here, you can see that HPV integration, HPV integration at the TERP promoter region and also TERP promoter mutation itself are mutually exclusive here and here. And this suggests that acquisition of either one type of the TERP genomic alteration might be sufficient to result in activation of TERP in HCC. Of note, the HPV integration at the TERP promoter region here is associated with upregulation of the TERP expression in the tumors. And upregulation of TERP expression indicates TERP activation. And TERP activation is well-known to promote tumor progression. And furthermore, we found that here, you can see that the HPV integration at the TERP promoter region is associated with more aggressive tumor behavior. Here with more frequent venous invasion, for example here. And also with a trend of a more advanced tumor stage and also a trend of poorer overall survival. In another of our studies, we employ RNA sequencing on HPV-associated HCC to examine HPV integrations. And we found that HPV integration could result in a generation of a chimeric HPV human transcript. And this human gene is the cyclin A2 gene. So resulting in HPV cyclin A2 chimeric transcript. And we know that cyclin A2 is very important because it regulates cell cycle progression. The generation of this chimeric transcript uses a very novel splicing mechanism. So at the genomic level here, the long HPV gene sequence is integrated into the cyclin A2 gene at the intron two region, between exon two and exon three, integrated in the intron region. So with this novel pre-rmRNA splicing here, a shorter HPV gene sequence located between the exon two and exon three of the cyclin A2 gene is resulted. And the chimeric transcript is able to produce the protein. And this protein, chimeric protein is able to enhance HCC cell growth. So this is one way that the chimeric protein subsequent to HPV integration can result and also enhance HCC progression. And we know that HBX protein is highly carcinogenic as also alerted to by Dr. Steven. And random HPV integration here can lead to truncation of the HPV genome, especially on the X gene locus and especially at the C-terminus of the X gene. And this will then result in a C-terminal truncated HBX protein. In our previous work, we could detect full length HBX integration in slightly more than half of the HPV associated HCC tumors. And then in the remaining about 46% of the tumors, we can detect C-terminal truncated HBX in these remaining cases. And when we compare using clinical pathological correlation and compare the C-terminal truncated HBX with the full length HBX present within the tumors, we found the presence of the C-terminal truncated HBX was significantly associated with more frequent venous invasion here, which is a feature of metastasis. And this indicate a more aggressive tumor behavior. And indeed this C-terminal truncated HBX has functional implications. And this truncated HBX is able to enhance migratory and invasive ability as shown here using migration and invasion as. Furthermore, HCC cells expressing the C-terminal truncated HBX was able to up-regulate matrix metalloprotease. This is MMP10. And MMP10 is known to be involved in breakdown of the extracellular matrix. And then mechanistically, we found that C-terminal truncated HBX was able to up-regulate the C-gene activity, which in turn up-regulates the MMP10 expression and enhance cell invasion. And this is associated with the more venous invasion in human tumors. There have been more subsequent and recent work of other labs, and they report how C-terminal truncated HBX affects other signaling pathway here, as in here, and also promoting HCC tumorogenicity and reprogramming glucose metabolism. So much so for the HBV integration in HCC, I'd like to talk on two studies with regard to HBV-associated HCC. So here, NGS, here nowadays, can identify specific mutations that may be useful for patient stratification and also using single-cell RNA sequencing. It can delineate the immune landscape within the HCC tumors. I'll present two studies. To find biomarkers for stratifying HCC patients for clinical management, we have performed exome sequencing and also targeted DNA sequencing in HBV-associated HCCs. We focused on the mTOR pathways. We found that TSC1 and TSC2 gene mutations, which stands for Tuberous Sclerosis Complex 1 and 2 mutations, which amounts to up to 16%, which is quite frequent in HCC. And these mutations are inactivating or truncating mutations, which means that with such inactivating mutations, the downstream mTOR activity, which is normally suppressed by the TSC1 and 2, will be upregulated. The TSC1 and 2 will not be suppressed, but will be upregulated, resulting in increase in the downstream phospho-S6. And phospho-S6 is known to be able to promote cell proliferation. However, as the name mTOR implies, so this is mammalian target of rapamycin. So mTOR or mTOR activity is sensitive to the action of rapamycin. Here, they are being used in patients for different diseases, such as immunosuppression after liver transplantation. Our results indicate that TSC2 mutations can also be detected in formalin-fixed paraffin-embedded tissue samples from patients' HCC tumors using immunohistochemistry. So patients' HCC with TSC2 mutation here show no immunostaining of TSC in the tumor. And there is upregulation of the downstream phospho-S6 because of taking away the suppression. In contrast, patients' HCC with no TSC2 mutation shows positive staining in the tumor and minimal staining of the phospho-S6, which is downstream. So in addition, when we gave rapamycin to the patients' derived HCC tumor xenografts from patients with mutations of TSC2, and these tumors show a significantly higher sensitivity to rapamycin. So this may have treatment implication. So in summary, TSC1-2 mutation may act as molecular biomarker for HCC stratification for treatment. On another note is that immune tumor microenvironment nowadays is recognized as an important layer contributing to intratumoral heterogeneity. And there are various amounts of immune cell infiltrates within HCCs as shown in these two representative cases. There are a lot of lymphocytes here, immune cells within the tumor, whereas there is just scanty lymphocytes within the tumor. Also nowadays with immunotherapy and particularly the immune checkpoint inhibitor treatment, the immune tumor microenvironment may play an important role in the treatment response. The inhibitory immune checkpoints refer to those that can inhibit the normal function of T cells. The normal functions of T cells are to kill or suppress tumor cells. And the most well-known ones are PD-1 and PD-L1 and L2 immune checkpoint. And immune checkpoint inhibitors are used to block the immune checkpoints so that the T cells can function normally and kill the tumor cells. We have recently performed a single cell RNA sequencing using chromium 10 X on patients HPV associated HCC samples to explore the immunosuppressive landscape and also the interaction between tumor cells and immune cells. We discover that apart from the well-known immune checkpoints here, PD-1, PD-L1, and there are multiple tumor infiltrating immune cells cooperate to build up an immunosuppressive landscape. I'd like to highlight our findings here of the novel involvement of TIGIT and Nectin-2-axis here. TIGIT and Nectin-2-axis. And you can see a very strong interaction between cells in this Nectin and TIGIT axis. And TIGIT is expressed in T cells and Nectin-2 is expressed in tumor cells and other cells. We further perform in vitro and in vivo work to support this finding. First, we isolate mouse splenic T cells, yeah, and co-culture them with mouse HCC cells. And we found that using anti-Nectin-2 neutralizing antibody which can block the TIGIT-Nectin interaction, this could significantly restore the proliferation of both CD4 and CD8 T cells in a co-culturing system. Similarly, we knock out here, we knock out the Nectin-2 and this knock out also significantly restore both CD4 and CD8 cell proliferation in a co-culturing system. I'd like to briefly tell about our in vivo confirmation findings in animal model. We knock out Nectin-2 using hydrodynamic tailing injection model. The knockout might so significantly reduce tumor size here as compared with the wild type and restore the T cell function. All these findings suggest that Nectin-2 in HCC cells suppress T cell activities. And this implicates the therapeutic potential of targeting TIGIT and Nectin-2 access in HCC. And of note, anti-TIGIT antibody is now in clinical trial of a number of solid cancers. So this is a potentially very interesting and probably effective treatment modality. So to summarize here, HBV is a major cause for hepato-carcinogenesis in many areas in the world. And HBV integration in the host genome can result in genetic changes, for example, the TERT gene and also up-regulation of oncogene, for example, by having a kind of a chimeric mRNA and also protein. And genomic analysis has provided new knowledge in tumor microenvironment, which provide potential new targets for intervention. I'd like to acknowledge my team members for their hard work. I also like to thank my collaborators for their valuable input. Thank you very much for your kind attention. Good evening. Good evening, everybody from London. It's a pleasure to be here. I would like to thank Haito and Kuang-Mi for the invitation, along with the organizers, to be part of this excellent webinar. And it's a real pleasure to have just listened to the talks of Stéphane and Irene. So I'm going to build on this and I'm going to focus more on the clinical elements of this. So... These are my disclosures. So first of all, I want to just draw your attention to the global burden that chronic hepatitis B represents. And I don't want to dwell too long on this because Irene's already talked about it. But if you consider the diversity of chronic hepatitis B, the numbers of patients that we have to manage from the East to the African continents, we need to think about how we're going to approach management of chronic hepatitis B better, and then how we're going to deal with the whole concept of HPV integration. One of the key elements of HPV DNA integration to me is shown on the bottom quadrant of this slide here, showing you that in hepatitis B, unlike other etiologies, you can develop HCC from what is essentially a normal liver or where there may just be chronic hepatitis in the absence of significant fibrosis or indeed cirrhosis. So that's a key element to highlight here and may well be some of the main reasons why we need to give greater consideration to HPV DNA integration. This is a slide that I don't want to dwell too much on numbers, but I do think it's important again to highlight the gravity of the situation or the challenge that we face. We know that chronic hepatitis B can result in about 800,000 deaths per year. If we look at the latest data from the Polaris Observatory, we see that the numbers of patients treated, the numbers of patients diagnosed is really far too small. So all of these factors are highlighting the challenges that we face in terms of the management of chronic hepatitis B. But one of the key elements and one of the elements that I stress to my patients when I see them in the clinic is that those patients with chronic hepatitis B have a 10 to 25% lifetime risk for the development of HCC. And this is something that we really need to think about. And this is really should be the springboard for how we maybe change our approach to the management of hepatitis B as we move forward into the coming years. This is a slide I like to use because if very crudely we divide, let's say, potential risk or potential factors driving HCC, we would look at host factors and we'd look at viral and disease factors. And if I just draw your attention to the factors in the entry to chronic hepatitis B and development of HCC, I would argue that if you say age over 40 is a key determinant of development of HCC, my argument would be you should treat these patients earlier. If there's a family history of HCC, I would argue that you treat these patients earlier also. Okay, we cannot do much about gender. We cannot do much about where you're born as we talked about Sub-Saharan Africa or Asia, but we can, again, on the viral and disease factors, we can be more aggressive to prevent the development of cirrhosis. We can treat earlier, we can treat viral loads, we can treat elevated ALTs. So there are still a lot of factors that we can address. And this is what I use this slide to show you to hopefully mitigate against the development of HCC. The importance of timing of HPV treatment is something that I've been debating for a number of years and it's something that I think I'll continue to push in the coming years. And I'm sure it's something that we'll also touch on in the later phase of the webinar. But if you look at HPV integration, I'm conscious that both Stefan and Irene have covered some of this, but just to think, looking at this, the cartoon here, that the CCCDNA, pre-genomic RNA, inflammation fibrosis paradigm is what we describe as the indirect mechanisms associated with hepatic carcinogenesis, while the HPV integration is the direct element of hepatic carcinogenesis that we will focus more on as we go forward. Some of this work stem from previous work that I've done with Bill Mason and Antonio Bertoletti, where we really examined in great detail this immunologically or what was considered immune tolerant phases of chronic hepatitis B. But one of the things that we always argued was that this is not a benign disease phase, so these patients shouldn't be excluded from treatment. And some of our studies have shown that we see the development of clonal hepatocyte expansion, we see HPV integration in these very early stages of disease and in very young people. We also see a preserved virus-specific T-cell response in this so-called immune tolerant phase of disease. And that's quite important because it may suggest to us that there may be a window of opportunity that we can capitalize on by treating patients earlier. And then finally, from a more public health perspective, by treating patients earlier, we know that we can reduce the pool of HPV infection and possibly reduce transmission in young people. For any of you who are clinicians or working in your clinic, I think this slide's quite interesting because if you take young patients, all below the age of 30 in this study, which is published in Gastroenterology, irrespective of what disease phase they were, Janssen positive, immune active, or immune tolerant, Janssen negative immune active disease, that when you biopsy these young patients, we almost always got the same results. So it was a fibrosis stage of one out of six in the ISHAC fibrosis stage. And we looked in more detail, but we saw very little difference again in terms of collagen proportionate area, suggesting to us that again, that these patients labeled immune tolerant weren't in a state of kind of immunological inertia, but that there was some factors driving some degree of disease. Then I move on to, you know, looking more at the immunological markers. So on the left of this slide, as you look at it, we're looking at a surrogate of immune activity. That's PD-1 and CDAT cells. And you can see that in those patients labeled Janssen positive chronic infection from Janssen positive chronic hepatitis. So formerly immune tolerant versus immune active, we're seeing very little difference in that degree of, let's say, immune activity, when you look at this CDA population using the PD-1 positive marker. If you look at CD1 27 low, PD-1 positive CDAT cells, a marker of exhaustion, what you're seeing here on the bottom of this slide is that with age, there is a continuous or progressive exhaustion of those T cells. And then that brings me to the publication of Quang My, the trial paper published in JCI Insights last year, which is a fascinating paper, but really picks up on some important elements here. First of all, there's more work that we need to do to better understand these disease phases and try to disentangle them. But what I found striking about this paper is that, you know, even though we see a lot more immune activity in the immune active phases of the disease, this to me reflects that potentially nonspecific inflammatory infiltrate that we get in the immune active phases of the disease. And that may suggest to us that that window of opportunity where there's a more calmer liver microenvironment to maybe introduce new therapies may be lost. So I think these papers really match each other very nicely in terms of developing some of the ideas as we try to move forward and understanding better what patients we should be treating, how we should be prioritizing, and how they may benefit more. But back to some of our previous work, and we look at a clone lepidocyte expansion. And here I'm showing you these various phases of the disease. And we know that clone lepidocyte expansion is associated risk for development of HTC. We know that the maximum clone sizes are seen in those patients who develop HTC. But let me draw your attention to the top of this slide here showing you patients as young as 15, 17, and 18 in the immune tolerant phase of disease with clone sizes, which are virtually no different to those patients in the immune active phase of disease, suggesting that there's probably an immune pressure driving this with HTC integration and leading to this clone formation. And if we don't do anything about this clone formation, we think these patients are at risk for progressive disease and the complications of chronic hepatitis B, namely HTC development. This really picks up on what Stefan has talked about, and even what Irene has talked about. This is just showing you the frequency of HIV integrations across the various disease phases. And the real take home here is Stefan labeled in his talk is that integration is a random event in the human genome. What these integrations are resulting in, this is where the future work will have to be undertaken to better understand what these integrations are resulting in. Or can we reduce these integrations? And by doing so, can we reduce some of the sequelae of chronic hepatitis B? This brings me to our more recent publication in Gut from last year. This is looking at integration in those patients with the antigen negative chronic hepatitis B, and specifically in those patients formerly labeled as asymptomatic inactive carriers of chronic hepatitis B. Both Stefan and Irene have touched on this, that the surface antigen production that you're seeing in the antigen negative disease is primarily coming from integrated DNA. So it won't come as a surprise to us to see that inpatients below the treatment threshold of 2000 international units have evidence of integration. And in fact, the integration events that we saw in those patients, looking at the whole exome integration where we see more pathogenicity is no different whether you're below 2000 international units and between 2000 and 20,000 international units. So again, maybe challenging some of those dogmas around treating patients above and below thresholds of 2000 international units of virus. But these are really important studies because likewise, they're not showing us the integration is just associated, let's say with the development of HTC, but they're also showing us that some of the cell proliferation, inflammatory responses, immune responses are also affected by integration. I also want to just highlight this important area around occult B infection. And this is important because this is where we have to think about patients where we don't have the presence of surface antigen, but we may have HPV DNA replicating at very low levels. Well, patients with occult B infection are also at risk for development of HTC. And it is most likely, as we're showing you in this very nice review, that the development of HTC in occult B infection is being driven by, or is coming from, again, HPV DNA integration. I use this slide really just to highlight where the field is shifting and how the field is moving. So if you take it and you look at this, I've shown you, in fact, with some of the previous slides, and you look at the bottom panel here, I'm showing you CCCDNA, you treat patients with antivirals, and we expect over a period of time, we get significant reduction in CCCDNA, and we get marked, marked reduction to the point that there may be very, very few copies of CCCDNA remaining. Just above that in the panel is the integrated HPV DNA. And what we don't know is, well, what is the effect of treatment in integrated DNA? Are we able to reduce this? We certainly know that we haven't performed the studies to be able to say that what we see in CCCDNA happens, but we do have the various markers, such as HPV DNA, HPV RNA, correlated antigen, which are telling us about some of these factors about the CCCDNA, about reducing transcriptional activity of CCCDNA. But let me draw your attention to the surface antigen line here while on treatment, that you're seeing it remaining almost flat over the course of treatment. So the question becomes, are we able to, you know, are we able to modulate integration by giving antiviral therapy? And if we are able to modulate that, how do we measure it? What's the readout for it? And what's happening to integration at the same time? So this brings me to the Kim paper published in Gut a few years ago, really to me, a seminal piece of work, showing us that when you looked at untreated immune tolerant patient versus their immune active peers who were treated, well, the untreated immune tolerant patients did far worse. So those patients had a 10-year risk of HCC, which was two and a half times higher than those with immune active disease who were treated. And they had a 10-year risk of death and transplantation, which was again, 3.3 times the risk of those patients labeled immune active and offered treatment. So those patients with potentially more aggressive disease being treated, doing better than the patients with the benign disease untreated. So if we move this forward to where we are with the current standard of care of treatment, if I look at what we are doing at the moment, HPV DNA, we can suppress it, and we probably should be suppressing it probably early the better. Abnormal liver enzymes, we give antiviral therapy. We can normalize liver enzymes. We're reducing, removing the immune mediated liver damage. Fibrosis, the seminal papers have shown we can achieve reversal of fibrosis. But what happens with HPV integration? Do we know? So let's see where the field is now moving. And that brings me to two excellent studies which are ongoing and I expect to see very shortly in publication. And this is, first of all, the CHOW study, which was presented as of last year. As expected, these are studies looking at paired liver biopsies. And in fact, if I show you here, this is baseline, one year of treatment, and then in a very small proportion of patients, biopsies again performed 10 years later. But it won't come as a surprise to you. HPV DNA is suppressed. Interhepatic DNA is almost removed completely. The CCC DNA, likewise. But the really important finding here was the significant reduction in median hepatocyte clone size. And this was a first for us in showing us that you have clonal hepatocyte expansion, you treat with an antiviral, you go and you look, and you're able to see a reduction in that clone size. So this, to me, is certainly progress and is something that I consider, you know, something where we need to look at this in more detail as to where we can actually get to with this. The next study is the HUGH study, also presented as of 2020. I'm bringing you back to some of the earlier talks of Stefan and of Irene. You will see that integrations correlate with baseline markers. So very high levels of HPV DNA, we see more integration. But then as the viral load is reduced, we're seeing maybe fewer integrations. But remember, what is the consequence of integration over that period of time? So this study looks at patients who remain on placebo versus those who are treated with an antiviral. And what I'm showing you here in the middle panel is at three years following treatment, you are seeing the full change in integrations after three years of tenofovir therapy. And similarly, if you look at this, you're also seeing the full changes in integration dysregulated genes after three years of therapy with significant difference in those patients who are treated with antiviral therapy. So in summary here, what we're getting back to is the very, I would say, preliminary data of what antivirals, of what nukes are doing to, let's say the downstream effects of HIV integration to hepatocyte clone size is just beginning to emerge. But as Irene said, what you're seeing is the use of all these gene mapping of HIV integration using these new technologies, such as RNA sequencing. And this RNA sequencing, I think is really gonna give, shed a lot of light on integration, how we manage integration as we move forward. As I stressed earlier, integration frequently involves genes involved in other factors, such as cell proliferation, antiviral responses, inflammatory responses, et cetera, and dysregulated protein coding genes, some of which are cancer related has been touched on in Irene's study. But the take-home here is that nukes can reduce viral genomic perturbation and the downstream effects of integration. So another reason to make that case of treating patients with nukes and earlier. So I would suggest that if we talk about treatment, there's a losing argument against early treatment, and that's based around antiviral resistance. We know that there's virtually no antiviral resistance. I think the issue of safety has well and truly been covered at this stage. I think the issues around cost are being addressed all the time. We have access to generic anticoverts and offerverts at this stage, which means that cost is not a major issue. And then finally, if you believe in the functional cure program, we do not expect that these patients will have a lifelong treatment. So this is not an argument for starting a patient, say, in their second decade or in their early 20s. And I'll finish by these concluding remarks. HPV DNA integration and clonal hepatic expansion are recognized events associated with hepatic carcinogenesis as shown by Stefan and by Irene. I think, you know, bringing back to some of my work, the immune tolerant phase may provide an ideal immunological window of opportunity to treat or to use novel therapies. You know, although this is going against the grain of what we would have thought previously, but these are certainly things that we need to explore. And for this reason, I say there's a compelling case to treat immune tolerant chronic hepatitis B and indeed to include immune tolerant chronic hepatitis B patients in the functional cure program. And finally, I probably go as far as saying as that we should consider treating all patients with chronic hepatitis B with HPV DNA of greater than 2000. It would make it more a simplified approach to the management of patients. And based on the emerging data, I would say there's certainly a good case which can be made for this. So I finish by thanking you for your attention. I thank you again to the organizers and to all my collaborators over the years who've contributed to this work. Thank you. Thank you. And thanks Stefan, Irene and Patrick for the wonderful talks. So now I think we're open to a discussion. And there are several questions in the chat box and also in the QA. The first question is from Dr. Maren Peters. So his question is for studies of new HPV therapies, being able to distinguish a surface antigen from CCC DNA and the integrated DNA is critical to determine functional cure. Are there and will there be assays to distinguish surface antigen from these two sources? Will the assay of truncated HBX be able to distinguish the two sources? So speakers, can you? Well, what I would say from my point of view is that currently I don't really see another option than looking at the liver. And I guess one thing that is being pursued now more is actually long read sequencing where you can sequence the entire integration. And by that you can really distinguish whether you have RNA from CCC DNA or from integration. But I guess none of these techniques at this point would be completely discriminatory because you always have, I mean, it really depends on how much integration you have, how much CCC DNA with an expression you have. So I think that's a tall order. Measuring the X, for all I know, measuring X is just very, very, very difficult. Yes, they are not in the serum, they're only in the liver. And yeah, we don't have a really good antibody. I'd probably come back to Marion's question, which I think is a very good one. And this one that comes up again and again. And in Stefan's list of studies which need to be done, I think that really puts the nail in there in terms of we need to look at this in more detail. It will involve looking at the liver at this stage and maybe the components of surface antigen. Maybe we'll learn more from those as we study them more. But one point maybe worth raising is that, it may go back to a reevaluation of this concept of functional cure. Because if we're trying to achieve surface antigen loss coming from integrated HPV DNA, we may need to go back to determine do we really need to do that if the CCC DNA is silenced and what the implications of that might be. Yeah, it's a good point. Okay, so there's another question from Dr. Bob Gish. His question is, with this profound integration story and the data, that early treatment decreased and the reverse integration numbers. So should we treat all patients who are HPV DNA positive at the time of HPV diagnosis? So this is a very clinical question for this patient. I'll start, I'm sure Kongmi and the others will wanna come in. I mean, I think, yes, I think that the field has moved on. I mean, the evidence is growing. I think we need to remove the barriers to treatment. I think people with chronic infections for years and years, it's not doing any good. If there's a risk of integration leading to development of HCC or other sequelae, we should be intervening early to prevent that. And I certainly, I would say to Bob, yeah, we should be treating patients earlier. We should be reducing the treatment thresholds and that should be the direction of travel. Everybody agrees. I wish we could actually do like a hands up or down. I think of the audience, I bet there's some pretty differing opinions, you know, why, I mean, it's a virus. So I guess my preference is sort of along with Patrick, but I'm also sensitive to the, maybe the cost benefit analysis. I mean, you know, not every person could actually afford the medication and have the right drugs access and so on. So I think it's probably a complex question, but yeah, I would like to suppress that virus. Bob just had a comment. Yeah, he said, let us move the dial to treat all HPV DNA positive patients and make the guidelines very simple to enhance patient and the provider engagement. Okay, so there's another question from Dr. Tim Block. Yeah, he asked, is there any evidence that people who get HCC in the absence of a liver cirrhosis are more likely to have HPV integrations whereby the integration are causing mutations in important host genes? So to the point that most HPV integrations are random, but in people with HCC and no cirrhosis, perhaps they have the meaningful integrations. So maybe Irene, you can answer this. Yeah, I mean, I'd like to try to take this question. I don't think there is many studies that are studying the HPV integration in non-cirrhotic patients. Of course, we group the chronic hepatitis with fibrosis and cirrhosis to one group, right? So if without cirrhosis, probably we group it into, say, the non-tumorous liver showing not much fibrosis or hepatitic activity. But I don't think, I'm afraid that there may not be a lot of studies studying, say, the mode or the degree of HPV integration in the human genome in these non-cirrhotic or non-chronic hep B fibrotic patients. So I think it would be a good study to look into, yeah. If I can follow, Irene, I'm just wondering, what are your thoughts about underlying genetic predispositions to HCC at the kind of a germline level that may further accelerate and exactly give us any angle at screening? Yeah, so I think there have been studies to look into, say, familial cases of HCC. So to see whether there are, say, genetic backgrounds that would predispose to HCC. However, because HCC occurs usually in patients with HPV or HCV background, so it would be difficult to dissect whether, say, in a family of multiple HCCs, whether this is due to HPV integration or infection or due to genetic, say, in the germline, genetic mutations. I think it would be very difficult, yeah. Okay, so- And there are no definite answers, I mean, in the literature. Yeah, let's move to the next question. So from Takeshi Seto. So he understands that double-strand linear HPV DNA is a preferred substrate for integration, which would not produce a core antigen. But however, it's conceivable that the fragmented or partial CCC DNA could also be integrated into host chromosome. He referred to a paper published in Habitology in 2015, indicate that. So the question is, could a core antigen be produced from the integrated HPV DNA? Stefan? Yeah. You have a slide to show it's not expressed. I guess the thing is that everything is possible. Of course, fragments of DNA can be integrated and things like that. I just think that more and more, the evidence is that this is probably a very rare event. But then again, if you look at the entire liver, a rare event can still occur because you have 10 to the 10 hepatocytes you can play with. The other thing is a way core could actually be expressed is, I mean, we consider the HPV integration and what happens inside there. If the integration happens in the close vicinity of a cellular promoter, then it probably can transcribe into the transgene and they can express core. But I just think it turns out, we'll see if more and more of these integrations are studied how frequent those events actually are or whether they are not worth mentioning at that point. But future will tell. That's right. And yeah, here's a question from Dr. Ashwin. Is that early treatment may help prevent a new integration, but is there a sort for how to treat the existing integrated clones? So for those already integrated, how you treat those? And maybe if I can dovetail to the question from someone named Jay, how early in age or after infection does the integration occur? What's the whole... It's kind of, yes, yeah, related. I mean, how early does the integration occur? I mean, in animal studies and in vitro, it happens within hours after the infection. So it can happen right away. So, but I guess how early that really happens in patients, I don't know how to study that really. I think that a lot of that has to be inferred at some point and obviously treating to get rid of integration will be great. I think what Patrick alluded to is that treatment actually can reduce the load of integration over time by clone size. You could imagine if you would establish a good immune response against S antigen, you could get rid of the cells that express the S. On the other hand, if S is expressed in the entire liver, then you might have a problem if the liver disappears in a day. So I don't think there is something that would specifically attack HPV integration at this point. I don't know whether that answer is any helpful. And I'm wondering if I could just interject, Patrick, Maurizio Bonaccini has a comment. I think you might want to address that, I wonder, about the HPV antiviral. In the QA, yeah. Before advocating HPV antiviral for immune tolerant patients, I would like to have an estimate of the number needed to treat for that intervention. I would suspect the number will be very high on any published data on NNT. I mean, it's a great question, but I think look at the number of HCC-related deaths. You know, the question I think is a broader one. Do we accept the status quo? Do we just say we're going to treat based on what we've been doing, which to me doesn't seem like it's been very successful in terms of its approach. And at the same time, if you look at all of the emerging data, your studies, Kongming, studies of our own, studies of others, Stefan's studies looking at integration, where it's leading to, what it potentially can cause. I just think we do need to look at this differently. And I guess that's the point that we're making. But, you know, we're never going to have a study which is going to tell us that these patients started treatment age 15. These patients weren't treated. And 30 years later, there was less or more HCC. I mean, we can't do studies like that. So we got to use different approaches. Like some of the studies we looked at reducing integration events, reducing clone size on therapy after short periods of time. But, you know, the field is changing and we got to accept that and we got to build up the case and the evidence for doing so. Yeah, I'm also struck by the sort of a generalized B and T cell dysregulation that occurs with chronic HPV. So it's really not just antigen specific, which is interesting. Right, right. Yeah, we are at 12.06 and we probably have a last one or two question. And there's a question from Joseph Yaw. So how does a core-related antigen correlate with HPV integration and the chimeric mRNA expression? Should it be used routinely to monitor antiviral therapy? I think this question kind of separate. Yeah. So the core-related antigen seems similar to the question what Stefan just answered about the core protein. It's probably very minor things randomly happen. I would expect it. Right, right. And there's another last question is from Tanner Gruder. So when double-strand linear DNA is integrated, how much of the ends are chewed up by host repair machinery? I think Stefan already answered that it's about 120 from the DR1 to DR2, right? And Stefan, you have any other answer on the file permanent of the integrated copy? Honestly, I don't have that in my head at this point. I mean, people have looked at these junctions and made some estimates about it, but I think that's also something that probably will be more addressed in the future when you do long-read sequencing and you are actually sure you are looking at the specific event. So I can't really answer that, definitely. Yeah, okay. Thanks. I think that we do have a lot of questions in the comments, but the time is limited. Now I think it's time to close up the webinar and thank again for the speakers who give the wonderful talks and the audience to answer a question that we have a stimulating discussion. And now I think that we have a consensus that the integrated HPV DNA is not a bystander in HPV infection. It plays a role in the chronic hepatitis B, pathogenesis, immunology, and cancer development. I think that we need to do more research and develop a new therapeutic approach to target this for a functional cure of hepatitis B, unless we change the definition of functional cure. Okay, so I would also like to thank the ASLD leadership, the HPV Small Interest Group Subcommittee, and the teams like Dominic Clayton, Jessica Jessop, and Brian Kirkland for coordinating the event and the technical support. Yeah, thank you very much for this wonderful seminar. If, Connie, you have anything to add? No, just thank you all for wonderful presentations and great ideas and Q&As. Thank you. Yeah, and the last thing is that this webinar is recorded and will be available online probably within one week. So all the registered attendees will receive an email about the link of the recording. Okay, thank you very much, guys. Happy 2022. Thank you. Bye-bye, thank you. Bye-bye.

HPV DNA integration

hepatitis B virus

HBV infection

liver disease progression

virology

clinical relevance

therapeutic implications

early treatment

hepatocarcinogenesis

replication-defective HBV DNA