Dr. Bajaj, it is my pleasure to introduce the next speaker in this session, Dr. Renu Dhanasekaran from Stanford University, who will tell us about cirrhosis as a pre-malignant condition, mechanisms, and clinical application. Thank you for the opportunity to talk on this topic today. I'm Renu. I'm assistant professor at Stanford, and my lab conducts basic and translational research in HCC and also clinical focus on liver cancer. So I've divided my talk into five sections. I'll begin with the background on defining multi-step cancer progression in HCC. In the second and third sections, I'll identify some genetic and epigenetic changes that accumulate during cancer initiation in the cirrhotic liver. In the fourth section, keeping in line with the theme of today's session, I'll talk about the role of gut-liver access. And then I'll try to bring it all together in the last section, where we'll talk about clinical implications. So let's get started with some background. If we think of cirrhosis, whether it's from hepatitis B, hepatitis C, MASH, or alcohol use disorder, there is a final common pathway of causing a cirrhotic liver in which there is a vicious cycle of chronic inflammation and regeneration due to cell death. Within this cycle, there are multiple mechanisms that can contribute to cancer initiation. I will highlight four of them. The first is fibrogenesis, the hallmark of cirrhosis. During fibrogenesis, there can be a transformation of fibroblasts to myofibroblasts, which, through multiple biological and even mechanical mechanisms, leads to cancer initiation. The second is the complex multipronged chronic inflammation, where there is an accumulation of pro-tumor immune-evasive cells, including macrophages and Tregs in the liver, which contribute to cancer initiation. Third is the oxidative stress, leading to accumulation of reactive oxygen species. These can lead to DNA damage, mutagenesis, and cancer initiation, predominant mechanism in both alcohol and metabolic dysfunction associated liver disease. And the fourth I would highlight is what we've already heard some about, dysbiosis. Dysbiosis, through multiple mechanisms, including lipopolysaccharides, DAMPs, and short-chain fatty acids, can contribute to both chronic inflammation and oxidative stress. All of these processes happen in a coordinated fashion over a period of years and decades, leading to initiation of cancer in a cirrhotic liver. Now, let's shift and look at what are the specific genetic events that cause HCC in cirrhosis. This is from a recent review we published in Hepatology, where we looked at the genetic events through the lens of hallmarks of cancer. Hallmarks of cancer refers to particular capabilities a cell acquires during transformation to cancer. Now, I will highlight a few of these genetic events and how they contribute. Activation of beta-catenin or MYC pathways or loss of P53, for instance, can lead to sustained cellular proliferation. Mutations in the third promoter, meanwhile, contribute to replicative immortality. Genetic events involving genes like ARID, BAP, or BRD7 can contribute to epigenetic reprogramming, while loss of cell cycle regulators, like P53 and CDKN1A and 2A, can help the cell evade growth suppression. Activation of genes like VEGF1, VEGFA, and NOT1 can contribute to angiogenesis, while multiple genetic events involving genes can lead to dysregulated metabolism. All of these in coordination ultimately culminate in initiation and progression of HCC in cirrhosis. Now, that was at a cellular level. Let's look at the organ level. 80% to 90% of HCCs, as you all know, arises in a cirrhotic background. Around 10% to 20% can be non-cirrhotic backgrounds, especially in hepatitis B or high-risk MASH. But we'll focus on cirrhosis in this context. Once you have cirrhosis, you're set up to develop these pre-neoplastic lesions, which can be called dysplastic nodules. There are two kinds in that. We'll talk more about that, low-grade and high-grade. And eventually, this can progress to early HCC and advanced HCC. There are some controversies about the precise steps involved in this multi-step carcinogenesis. But this overall framework is very, there is some consensus and very useful for clinical application. Now, what is happening during the progression within a cell? If you look at the cirrhotic nodule, all the cells are of relatively similar size and shape and don't have any atopia. Once you develop a low-grade dysplastic nodule, you begin to see some atopia, some changes in the cell's shape and size. By the time you reach high-grade dysplastic nodule, you have increased cell density and also increase in the nuclear cytoplasmic ratio. And cancer is easier to diagnose because you will obviously see small cell change, lots of atopia, and increased nuclear cytoplasmic ratio, and importantly, stromal invasion. While this is happening within a cell, what is happening in the microenvironment? An important change is the vascular change. It's a hallmark of HCC initiation. In cirrhosis, you have portal triads, which have a portal vein paired with a hepatic artery. In low-grade dysplastic nodule, you mostly maintain this. Most of the blood supply to a low-grade nodule is from the portal vein. However, by the time you reach high-grade dysplastic nodule, you begin to see unpaired hepatic arteries that are outside the portal triad. So there's a decrease in portal flow and increase in hepatic arterial flow. And in early HCC, you have angioneogenesis, multiple hepatic arteries, and a switch from a portal venous to a hepatic arterial blood flow. And we'll talk about this when we come to the clinical applications. So that was a section on the background of the multi-step carcinogenesis. In this section, I'll focus on some of the genetic changes we see in HCC. In this very elegant study, they looked at somatic mutations in the cirrhotic liver. Yes, you heard me right, not in HCC, which we know cancer has mutations, but this is just a cirrhotic liver. They micro-dissected 482 areas, each containing 100 to 500 hepatocytes from five normal and nine cirrhotic livers. You see in the graph here in the bottom, the x-axis shows each patient, and the y-axis shows the number of unique events. As you can see, compared to the normal liver, the cirrhotic liver itself had a significantly higher mutation burden. And they also showed that synchronous HCC, which arises in the background of cirrhosis, shows the same mutational signatures as what was seen in the cirrhosis. There's a higher burden as expected, but this shows you that the genetic events and the mutational signatures are already present in cirrhosis, making it a pre-neoplastic condition. In another study, they again sequenced patients with cirrhosis, exome sequencing of 82 samples. And again, they showed that the number and size of mutant clones increased as a function of fibrosis and tissue damage. So you're seeing a progressive accumulation of mutations, even within cirrhosis. They went a step further and asked, what are the functional implications of these mutations? And they performed an in vivo CRISPR-Cas9 genetic screen of 147 genes. What they found was loss of certain genes, like PKD1, KMT2D, and added 1A, promoted clonal expansion. So these mutations were increasing the clonal fitness of these nodules and promoting regeneration, which obviously has a survival advantage in cirrhosis. How about the dysplastic nodules or the pre-cancerous lesions? In this landmark study, they sequenced, you can see the numbers there, several low-grade, high-grade dysplastic nodules and early HCC. They particularly focused on third promoter mutation, which is the most common mutation in HCC. Interestingly, what they found was 6% of low-grade dysplastic nodules already had mutations in third promoter, and 19% of high-grade nodules. And as you progress, you can see it significantly increases in HCC, which is expected. But what is really interesting is these mutations appear to be very early events in cancer initiation. Now contrast this to 10 other cancer genes they looked at, including beta-catenin and p53. They did not see any mutations in these genes in the dysplastic nodules and only in cancer. Based on this, they suggested that somatic third promoter mutations could be an early event in liver carcinogenesis. This is a different study. A larger number of dysplastic nodules were looked at. They isolated 205 dysplastic nodules and sequenced for 30 most commonly mutated cancer genes. And what they found here is even in dysplastic nodules, you would get mutations in added 1A, p53, or beta-catenin, several cancer-specific genes. And they did not find any mutations in third promoter in this study. But overall, you can see that there is a beginning or initiation of these genetic events in dysplastic nodules. Now let's look at some of the epigenetic changes that precede HCC. Here is a study where they did both genome sequencing and methylome sequencing of early and overt HCC, so not dysplastic nodules. And what they see is there's obviously an accumulation of mutations, as you can see in the green and red boxes. But they also found four epigenetic classes, normal-like, global, stem-like, and hypermethylated. And these methylation differences were present both in early and overt HCC, suggesting that there is a cooperation between the methylation and the genomic events for transcriptional pathway activation. In another study, they actually sequenced the dysplastic nodules. And two genes are shown here, APC and RasF1A. And they show a progressive accumulation of methylated regions, and maybe even a predominantly methylated regions in the earlier stages of HCC development. And this, I think, as a heat map, is easy to follow. What is red is hypermethylated. What is blue is hypomethylated of the genes shown on this heat map. And you can see just visually how the chronic liver disease, 10 samples in the middle, epigenetically appear much more closer to the HCC profile than the normal liver. And this study suggested that the epigenetic priming in chronic liver disease could be sculpting the transcriptional landscape and creating an environment that favors the acquisition of genetic alteration. So we'll move on to the fourth section, which will be about the gut-liver axis in HCC initiation. Multiple human studies, elegantly summarized in this review, show that there is an alterations in gut microbiome between patients with cirrhosis and HCC. And the table is listed here just to show you that there are disparate findings from these studies. Some common bacteria are seen, like E. coli, but different studies identify different bacteria, showing you that the study cohort, the patient population, the underlying risk factors, and exposures are extremely important in shaping the dysbiosis. But overall, multiple studies have shown differences in patients with HCC. I really like this recent study, where they were able to do longitudinal sampling in patients with active hepatitis C. They sampled the portal vein, peripheral blood, liver tissue, and fecal microbiome before and after DAA therapy and patient-achieved SVR. So now you have a relatively well-controlled before-after study. And what they found is there was a central role for mitochondria and paroxysome in hepatic metabolic dysregulation. They identified particular bacteria which were enhancing microbial fatty acids in biosynthesis and glycan metabolism. And these changes were reversed after the patient-achieved SVR, showing that they were clearly connected to active hepatitis C infection and chronic inflammation in the liver. Now, to give a broader overview of mechanisms which have been published from experimental studies in mice, bacterial dysbiosis can cause both cell-intrinsic and cell-extrinsic mechanisms. A few of the cell-intrinsic mechanisms have already been mentioned in more detail. To give a bigger, broader overview, lipopolysaccharides or short-chain fatty acid deficiency, and like Dr. Bajaj mentioned, ethanol production by the gut bacteria, or alterations in bile acid composition can all contribute to signaling changes within hepatocytes through either chronic inflammation or reactive oxygen species-induced DNA damage and lead to cancer initiation. On the cell-extrinsic side, activation of the TLR pathway through the PAMs and DAMs or cytokine changes can contribute to alteration in the immune composition of a cirrhotic liver, and experimental studies have shown multiple mechanisms like enrichment of NKT2 cells, TH2 cells, or M2 macrophages, which can all create an immune-invasive background and promote cancer initiation. So let's come to the last section, which will be on the clinical implications, how all the summary of how you have progressive accumulation of genetic mutations, epigenetic changes, and dysbiosis from a stage of normal to cirrhosis to dysplastic nodules and early HCC is contributing to what we do in clinic. So to summarize what I said before, I told you how there's a progressive decrease in portal vein flow and a progressive increase in hepatic arterial flow during transformation from cirrhosis to dysplastic nodules to HCC. This is clearly translated to what we do with imaging. Here I show two phases of a MRI study, arterial and delayed phase, and we all know that we use these LIRADS criteria or different imaging criteria to make a diagnosis of HCC, which directly reflects the principle we just discussed. As a lesion progresses from low-grade to high-grade dysplastic nodule and early HCC, you see progressive arterial phase enhancement, and with the decreasing portal venous flow, you see progressive washout in the delayed phases. Pathologists use very similar principles to make the diagnosis of dysplastic nodule versus HCC. The arrows in the first two show unpaired hepatic arteries or cellular atopia that they look for, and in early HCC, by definition, you will see some stromal invasion. The differentiation between these can be difficult and you need sometimes expert pathologists to make that. The accumulation of genetic events and the mutations are reflected in these stains that we use to diagnose HCC, including CD34, GLUT synthase, and GPC3. The burning question, and coming back to the case, is what do we do when patients have dysplastic nodules that are diagnosed using LIRADS criteria? There are multiple studies and meta-analysis. I'm just giving you a big picture from two recent multicenter studies. The risk for LIRADS 3 to progress to HCC was around 24%, and for LIRADS 4, around 44%. So not all these lesions will progress. Prudent decision-making is required, taking all the factors into consideration. So to come close to the end, if you ask me where is the pre-neoplastic lesion in the cirrhosis liver, I would say it's the entire liver. The pre-neoplastic landscape is primed for emergence of cancer. You can see the multiple nodules within this cross-section, and HCC initiation can occur within any of these. But let's end on a more positive note. These mechanisms and epigenetic changes can lead to earlier detection now that we have newer liquid biopsy tests, and the advances in genetic tests and spatial analysis can potentially lead to improved diagnosis. And last but not the least, all we want to do is prevent cancer, and modulation of the dysbiosis using diet or medications can hopefully prevent cancer initiation in patients with cirrhosis. Thank you. presentations this morning will involve a debate, so I hope you are all ready for a fierce discussion. And it's my great pleasure to introduce Dr. Jacqueline O'Leary from University of Texas Southwestern and Dr. Florence Wong from University of Toronto, who will debate the two sides of the question, should all patients with ascites receive antibiotic prophylaxis. Dr. O'Leary. Thank you so much for this opportunity to present to you today on all patients with ascites should receive prophylactic antibiotics. So hopefully I only need one slide here and say maybe even one word. Guidelines. So by the easel practice guidelines, primary prophylaxis with norfloxacin is indicated, especially in patients with Child-Burk-Turkot-Pugh score greater than or equal to 9, a serum bilirubin greater than or equal to 3, impaired renal function or hyponatremia, and a low total protein in the ascites fluid. Our particular patient does meet criteria by CPT class, serum bilirubin, hyponatremia, and the ascites albumin level is so low we assume the ascites total protein level as well will meet criteria. By ASLD practice guidance, patients with cirrhosis and low total protein can receive primary prophylaxis if they have renal dysfunction or liver dysfunction. And so once again our patient does meet these criteria. But since I have some extra time, we'll go on to talk about the risk of SBP in patients with ascites, which can reach up to 60% in one year in patients with low total protein in their ascites fluid. I think the real question here is not if but with what. And it is imperative that we remember not all antibiotics are the same. For the purposes of today, we are not talking about absorbable fluoroquinolones. They have a high rate of resistance. They breed multi-drug resistant infections and colonization. They increase a patient's risk for C. diff. They have no impact on hepatic encephalopathy. There are side effects and they decrease the diversity of the gut microbiome. Norfloxacin, which is essentially universally available other than the United States, is widely used. Its efficacy, however, has decreased over time, especially in patients colonized with multi-drug resistant infections. And our first speaker definitely did show in the Norfloxacin trial of almost 220 CPTC patients with ascites over a four year period that although not statistically significant, there was a small non-statistically significant decrease in mortality. But importantly, there was a 10% decrease in infection rate. I'm sure my esteemed colleague will highlight a recent publication that I helped co-author from the National VA Dataset in over 7,500 patients who did develop SBP. Some on primary prophylaxis and others not over a 10 year period of time. And in fact, the patients that developed SBP on primary prophylaxis did have a higher rate of antibiotic resistant organisms. However, what won't come out is all of the kitties that weren't up trees, right? So the people who had primary prophylaxis and never developed SBP, we did not study, nor did we study how long these patients were successfully on primary prophylaxis before they developed an episode of SBP. But ultimately, as Dr. Moreau highlighted before, we really need better options. And so I'm going to highlight some data about rifaximin that it should be influential. Rifaximin, in fact, has been studied as primary prophylaxis, although it's not FDA approved for that purpose. And when compared in meta-analysis to utilization of systemic antibiotics to prevent SBP, had a trend for superiority. Another complication that many of our patients with cirrhosis have is small intestinal bacterial overgrowth that's present in 41% of all patients with cirrhosis. This increases to over half if they are decompensated. And SIBO is more common in patients with ascites, those with bacterial translocation, those with a history of SBP, and patients with plasma endotoxin levels are increased in patients with SIBO. And in fact, rifaximin can be utilized to treat this and in meta-analysis, as you see here, does definitely improve SIBO. Rifaximin has also been utilized to improve the immune state of patients with cirrhosis, specifically by improving TNF alpha levels, as well as lowering TLR4 levels. And rifaximin decreases overall systemic inflammation, which does have a negative impact on the brain, as well as we recently heard, on cancer progression. In a recent small study, rifaximin did decrease systemic endotoxemia, impair oralization of the gut, impair the growth of mucin-degrading species, and improve fecal IL-17 levels, which you heard how importantly have an antibacterial response in invading pathobionts. Of course, not to mention its positive impact on hepatic encephalopathy, as well as, even in this small study, risk for overall infections. And of course, we can't forget the brain. So patients with ascites and cirrhosis, if they haven't already been diagnosed with hepatic encephalopathy, have about a 60% risk of minimal hepatic encephalopathy, which may actually be even higher in our mass old patients, which often decompensate with hepatic encephalopathy as their first presenting symptom. Minimal hepatic encephalopathy, that is often missed in clinical practice because of time to perform this testing, is associated with a decreased health-related quality of life. And patients with minimal hepatic encephalopathy, 40% progress to develop overt encephalopathy over the next year. And Rifaximin similarly has an excellent track record of treating and resolving minimal hepatic encephalopathy. This concept was not lost on the company that owns Rifaximin, and so in a small study looking at patients with cirrhosis and ascites, a new formulation of Rifaximin in a placebo-controlled trial did show that it decreased overall risk for all-cause hospitalization and mortality. As a result of that, there are two now worldwide registration trials looking for this specific indication. They are randomized control trials of patients just like our topic. Cirrhosis with ascites, without overt or known hepatic encephalopathy, and the new formulation of Rifaximin is being compared to placebo, looking at the primary outcome of prevention of hospitalization from overt hepatic encephalopathy. But certainly there's great interest in other areas associated with risk for infections, hospitalization, SPP, and of course cost. So our key takeaway points from this are that patients with cirrhosis and ascites should receive prophylactic antibiotics, but non-absorbable antibiotics such as Rifaximin can be used to prevent infections, prevent and treat minimal hepatic encephalopathy, resolve SIBO, remedy dysbiosis, ameliorate bacterial translocation and endotoxemia, and even decrease hospitalizations and mortality. Thank you so much. Good morning, everyone. So my task is to refute everything that Dr O'Leary has told you. So these are my disclosures. And she talked about the standard guidelines, and the guidelines and the guidance statements from the various academic societies do tell us that patients with cirrhosis and ascites will require secondary prophylaxis. It is an established standard of care, otherwise the recurrence of SVP occurs at the rate of 40 to 70% with an associated mortality rate of 50 to 70% per annum. So I don't argue with her about secondary prophylaxis, but we are talking about primary prophylaxis. Primary prophylaxis is actually controversial, and Dr. O'Leary has already mentioned that we should consider it in sick patients with ascites who have got either renal dysfunction as indicated by a serum creatinine of more than 1.2 mg per deciliter, or a BUN of more than 25 mg per deciliter, or a serum sodium of less than 130, or in patients who have got liver dysfunction as indicated by a child poo score of 9, and a serum bilirubin of more than 3. So this was something that some of the ASLD fellows put on the ASLD website looking at the question of primary prophylaxis. As you can see, there are actually a number of studies that have looked at the question of primary prophylaxis. And the only study that included patients with a low acidic albumin with severe liver disease and renal dysfunction did show a significant result of primary prophylaxis improving the outcome in these patients, but then there was no competing event in the analysis. And as you can see with all the other studies, none of them actually included both patients with severe liver disease or severe renal dysfunction. And this was something that even the trainees knew about. So primary prophylaxis for SBP, the evidence is not strong. It is restricted to patients with very advanced cirrhosis, and we should really individualize it based on the estimated risks and benefits and taking into account of the patient characteristics and the limited data on the various antibiotics. One of the reasons why we don't universally give these patients primary prophylaxis is because of the increasing prevalence of resistant organisms. This is some data that I have found amongst European countries showing you that the resistant organisms are rising, although not as high as in some other parts of the world. I was involved in a global study looking at the prevalence of multidrug-resistant organisms. And as you can see in some countries, it can be very high. So why are we giving sick acidic cirrhotic patients more antibiotics? And if we are to look at another global study, and you will hear about this study on Monday morning, and it shows that in USA and Canada, the resistance rate is 21%. And you remember that Dr. O'Leary says a lot about using rifaximin, and rifaximin has been around in US for a long time, being prescribed to a lot of patients, and yet we are unable to prevent this high prevalence of multidrug-resistant organisms. Are we going to throw out more antibiotics to increase the prevalence of multidrug-resistant organisms? The answer is probably no. And you notice that Dr. O'Leary and I are actually on the same page. You notice that she actually leads and I follow, and we talk about the same thing. And so this is a study performed by the North American Consortium for the study of end-stage liver disease, where we looked at the use of primary versus secondary prophylaxis. And so you notice that we actually matched patients, those who were on primary prophylaxis versus those on secondary prophylaxis. And just to show you the results. So patients on primary prophylaxis, more of them were taken into intensive care, more of them developed AKI, and more of them died. So why are we giving these patients primary prophylaxis? I have no issues with secondary prophylaxis. And in fact, the 90-day outcomes with mortality were significantly higher in those who received primary prophylaxis. And so Dr. O'Leary mentioned this in her talk. She followed the first study with a second study in the veteran population. And once again, comparing patients who received primary prophylaxis versus no prophylaxis, sure, these patients were using rifaximin, but there was a significantly increase in antibiotic resistance against the fluoroquinolone and also against CEPTRA. And you'll notice that these patients, more of them had second SVP, more of them stay in hospital longer. So my question is, why give these patients primary prophylaxis? And in addition, more of them require liver transplant within 30 days. There was no change, no difference in their mortality rate. So I hope that I have convinced you, and so I rest my case. Thank you. Thank you. Just a moment. Florence is certainly a giant in intellect and accomplishments, but not in stature. Okay. So back to my points about how all patients with the CITES should receive prophylactic non-absorbable antibiotics. And do remember, most of the data she was highlighting came from the U.S. talking about absorbable antibiotics. So we're going to go back to the guidelines and guidance document and check off all those boxes for our patient meeting all of the criteria. But ultimately, Florence is such a spectacular friend and colleague. It is so wonderful to see all of her accomplishments being acknowledged by the Distinguished Achievement Award by the ASLD, that I felt it was important to award her something of my own for this so that she could remember this forever in perpetuity. Because when we did work together on yet another guideline document where, yes, she is following my lead here as well, we both agreed that rifaximin may prevent complications of cirrhosis other than hepatic encephalopathy, and we were definitely talking about infection. So here we have why all patients with the CITES should be treated with prophylactic non-absorbable antibiotics. Because they have the power to tackle minimal hepatic encephalopathy, potentially the over 50% rate of SIBO in our decompensated patients, cirrhosis, dysbiosis, endotoxemia, and bacterial translocation. And so I'm going to present Florence with some teepee, I mean this trophy, in honor of our lovely debate here. Thank you so much. As you can see, I cannot beat her. Thank you all. This session was fantastic. And we got to the end of the forum. Mike, Mike, Mike. Sorry. So thank you all, and congratulations, Florence, on the award. This has been unexpected and absolutely delightful. So we came to the end of the formal presentations, and it's my pleasure to invite you from the audience to ask questions from our expert panel. There are microphones in each section, so please feel free to come up. And in the meantime, I would like to invite some of the speakers, in case if you have any questions. Yes, so we start out here in the microphone, on my right. Thank you. My question is to Dr. Renu. Would you advise a biopsy for all patients with hepatocellular carcinoma, with so many new developments? Should we be biopsying everyone before treating them? I personally would, just to understand the disease better. But that's not the question here, whether it is clinically indicated. There are definitely advances in sequencing and molecular tests, which I discussed. The question would be, would that information we get from the biopsy change management? In certain cases, yes. For example, indeterminate nodules, which you're repeatedly scanning, and they're slightly growing, and you're keeping an eye on those. I would advocate for a biopsy to make a more definitive diagnosis, so you can move on with the management. In the cases of stage four disease, where you're planning systemic therapy, it's reasonable to biopsy, especially in the setting of clinical trials we do. With more targeted therapies being approved, I think there will definitely be a role when you do systemic therapy. For most other patients, radiologic diagnosis is easy, and this is one of the advantages of HCC over other cancers. There are characteristic findings on imaging. If you have those, I would say there is no indication for biopsy right now. But the field is rapidly changing, and I won't be surprised if five years from now we are biopsying everyone. Thank you. Thank you. So, we have a question here for Jas. HETalk, the gut microbiome creates alcohol. Is auto brewery a contributor? I didn't get the first part. So it's about gut alcohol production. Yeah. So auto brewery, unfortunately or fortunately for who want to start a career on that, is an extremely rare condition. Most of the things are at the usual brewery, which is not in CERN. It could be potentially a cause, but I don't think we have a way to actually figure out how much of a cause it is in these patients. Now in pediatric NAFLD, where it was first discovered, or first demonstrated, it is very clear that those kids were not out drinking, and it was quite obvious that those endogenous alcohols were able to produce this. Now when you have exogenous alcohol and endogenous alcohol, it becomes very hard for us to do that. So the hope is that at some point you get a very honest history from the patient and go with that, with a non-judgmental attitude, so that you can get the best treatment for the patient. But to answer your question right now, the auto brewery syndrome, to my knowledge, is not a significant contributor that we can attribute to its HE treatment right now. Thank you. Thank you. Isabel Campos Varela from Barcelona, Spain. Thank you for the nice presentations. My question is for Dr. O'Leary. In the scenario that you could choose, supposing that you could choose between norfloxacin or rifaximin, which one would you choose for SVP prophylaxis? So for patients in the United States, unfortunately we do not have norfloxacin, but you do. And I have to say that I've been favoring rifaximin just because of the data of multidrug-resistant infections becoming more prevalent and problematic. And so that's what I favor, as well as its other potential positive effects. Thank you. So here is a question from Dr. Serene, which is, I guess, to Richard. Can we stratify cirrhosis patients based on gene profile from PBMCs, liquid biopsies, studies who may develop HCC, especially in ARLD patients? So it's Richard, and then Dr. Dharnashekhar. The acoustics are really bad here. The question is really about the relationship between gene profiling of PBMCs in order to stratify patients who are going to decompensate, and of course, the risk of HCC. So for both of you. May I speak here? Yes. Thank you. It could be interesting, yes, to use gene profiling in PBMCs. I would prefer whole-blood gene profiling, because in whole-blood you capture genes from neutrophils. And if you want to have a broad view of the landscape, even in the context of cancer, because we know, identify subtypes of neutrophils which are very lazy and could be also involved in the development of cancer. Therefore, I would support actively the broad use of gene profiling in whole-blood, yes. For HCC, what do you think? Yeah, I can just add, you're ultimately looking for a needle in the haystack. The needle is there. The question is, can you find it, especially when the denominator is cirrhosis? I think some of the more promising tests are ctDNA-based, because you can amplify the signal using PCR-based techniques. In terms of looking at PBMCs, in our lab, we look at immune profiling of the PBMCs in normal patients, cirrhosis, and HCC. And what we see is striking changes in cirrhosis itself, like you saw our first speaker allude to. So, the overlap between what's happening in cirrhosis and HCCs is so significant that the challenges are higher when you're looking for a signal there. However, I think with the multi-analyte tests that are coming up, we do expect to be able to stratify using peripheral blood. So, yeah, thanks. Thank you. There's a question. This is Ashutosh Barve from University of Louisville, and this is a question for Dr. O'Leary and Dr. Wong. And so, I wanted to be sure if I got the right message from your talks. It sounded like Dr. Wong conceded a point to Dr. O'Leary. Am I to understand that you're saying, you're advocating that we should go over and above the recommendations in the guidelines, whereby you don't wait for the cirrhosis to get bad or more serious before you start antibiotic prophylaxis? Because I heard you say that all cirrhotics with ascites should have prophylaxis. Did I get it right? No. My understanding is that those patients with advanced liver cirrhosis or those who have got renal dysfunction, the evidence is there for them to receive primary prophylaxis. But the evidence is not there for the other patients who don't have advanced liver disease. So, I would wait for more evidence to become available. And I think for the rifaximin trial, I'm the PI for the RED-C study that was mentioned. It's not even close to being completed. That would be the one that would tell us in a vacuum, because none of those people are on current prophylaxis. None of those people are on current rifaximin. If that study shows not only a benefit in HE, but the rest of the infections, as Dr. O'Leary pointed out, that would be the time then to start Rifaximin SSD. Right now, the evidence is bubbling, but it's not reached up to the surface yet with a traditional molecule. Jackie, would you agree? So I think in general, the talk title was to look at all patients with ascites and cirrhosis, not just the patients that were very severe, like the case that was presented. And so I did highlight that there are a lot of reasons that you may choose Rifaximin in those patients, such as if you find minimal hepatic encephalopathy, if you find SIBO, if you find other reasons to do it. But Jazz is correct that at this time, there are two ongoing registration trials looking at using it in everyone. My opinion, not fact, is that I'm hopeful those will pan out to be positive and we will end up doing that. But it is very expensive and not FDA-approved at this time. Thank you. Pere Ginés, Barcelona. I would like to give a word of caution about using Rifaximin in all patients with decompensated cirrhosis, because this has not been proved in randomized controlled trials. And in fact, we have a randomized controlled trial, which was presented at the last year in ISL that was finished about some months ago, which was a double-blind study comparing Rifaximin plus Simvastatin versus placebo or both, and the trial was negative with respect to all clinical endpoints, including the development of bacterial infections of any sign of progression of the disease. So I think that, of course, we need more information about trials, but also we need to be cautious that with respect to Rifaximin, the only thing that has been proved effectively is the prevention of recurrent hepatic encephalopathy. All the other indications, there are some signs that may be positive, but in fact that have not been proved, and our trial was completely negative with respect to the old endpoints. We hope that the trial will be published soon. So that's correct. You know, certainly Rifaximin has a very long track record of safety, which I think is also very beneficial as far as utilizing it in patients with decompensated cirrhosis other than, you know, in respect to other medications. Certainly our point today in debate was to make you all think and to go over the different data that was out there and to give you potentially a glimpse into the future. So everyone is stating exactly what I think we both feel, which is the data has not arrived yet to deliver Rifaximin to patients every day with ascites and just simply ascites and cirrhosis, but that this is an ongoing concept that is in a stage three, phase three clinical trials and it's of interest. So back to Dr. Wong, does what Professor Gines was saying help your case? I think so. He and I agree on a lot of things. We debated before. So this is again a question back to Dr. O'Leary from one of the audience. So in today's day and age, how do you pay for Rifaximin for primary prophylaxis? Is it reimbursed in the VA or? No, that's correct that Rifaximin is very expensive. And as a matter of fact, if a new formulation, by the way, it's going generic. So that's one of the main reasons why the company is actually creating a new formulation. And so it will become less expensive when it becomes generic. However, in certain cases, such as, for example, the case that was presented, I have very strong suspicions given the ammonia level of 92 and the patient's sleep difficulties that in fact he does have encephalopathy and therefore may actually need the medication, which I think would be definitely an indication for therapy. But once again, no, it is not approved for that indication of primary prophylaxis. So how would the doctor in the audience pay for it, pay for the patient to receive it? Or would you use something else instead of Rifaximin? For primary prophylaxis in this patient? So once again, primary prophylaxis, my opinion on primary prophylaxis just for like either using norfloxacin if you were in Europe, which would be the other non-absorbable antibiotic, would be in especially in a higher male patient who was listed for transplant who may receive transplant in a short period of time. And therefore, the risks of developing colonization with multi-drug resistant infections you may overlook in a shorter period of time. You wouldn't necessarily get that higher risk and have less infections going into transplant. That would be different than our current case, which of course would not be a transplant candidate. It would be more of a long-term treatment. And so I'm not using either absorbable antibiotics or the currently available antibiotics for primary prophylaxis in those cases. So actually, sorry, I had a question because we all work in the VA. The VA has almost outlawed primary prophylaxis right now. So on October 18, October 27, we had a call with the National Antibiotic Stewardship Policy Program in which the draft policy that was approved from the Richmond VA, which was then approved by 17 other VAs, was then adopted to discourage primary prophylaxis and do the best to actually do shared decision making for primary prophylaxis using ciprofloxacin in the United States veterans where the E. coli resistance rate is 50% or higher with ciprofloxacin. So it was done on the basis of efficacy, lack thereof, and then why do you put tendon rupture and all the other things that were mentioned. So this is a huge critical need. And I actually applaud the organizers for thinking of this devious topic that no one can actually win, but actually creates a lot of awareness that we need to think about. So I would like to recite a couple of questions that came in through the online system and that is related to cancer as a pre-cancerous, I mean the cirrhosis as a pre-cancerous state. And the question was that, one was that, do some of the epigenetic changes that are associated with cancer resolve or remain after, for example, elimination of hepatitis C infection in the liver? And then the other question was that, in a previous talk during this morning, it was mentioned that hypermethylation in the liver is kind of a sign of aging in the liver. And how does that kind of relate to the pre-cancerous state? Yeah, thank you. So the questions are on the theme of epigenetic changes in cirrhosis and cancer initiation. For the first question of reversibility, the epigenetic changes track closer with the degree of fibrosis, and so do the genetic mutations. They progressively accumulate over cirrhosis. So similar to what we see for other clinical risk factors in cirrhosis, they are connected more to the elimination or the reversibility of fibrosis, which we know is limited, compared to elimination of the offending agent itself. For instance, if you're alcohol cirrhosis, advanced fibrosis, or hep C with advanced fibrosis, even after the offending risk factor is removed, the fact that there were decades' worth of epigenetic changes need not be essentially reversible. However, early changes, which are clearly connected to chronic inflammation, can be reversed when you have elimination of the inflammation-producing agent. And in terms of aging, there's a very clear connection between aging and accumulating epigenetic changes, and that clearly is not reversible. So I think the cooperation of the epigenetic changes in aging are what lead to the initiation of HCC in cirrhosis in older patients, which, all said and done, is still a much more higher risk in older patients than young adults. All right, we have one more question. This is for Richard. So Richard, there is a question from the audience about obeticolic acid and its usage in patients with decompensated cirrhosis, where it is contraindicated. So what do you think? So the question is whether, so as obeticolic acid is contraindicated in decompensated cirrhosis, how can you use it to modulate the gut permeability? Yes, but is it a strict contraindication? I think so. You think so? So you should find an alternative to this. An alternative FXR, do you think? Yes, yes. And I'm sure that the industry has a lot of molecules. The message is FXR, yes, agonist, yes. Thank you. This is an interesting one for Jazz, I think. So what is it about MASH that leads to HE being the central decompensating event? That was one study done by the NASH CRN, a very beautifully done study, and the endpoints for HE in that, which were thoroughly litigated, were grade three, grade two in hepatic encephalopathy. It's not people like this person, who was feeling a little worried, and then they started them on lactulose. I believe there's a lot of overlap between MASH and dementia. There's a lot of overlap between, as your group has shown very nicely, that is a direct correlation with brain function. So ultimately, the brain can only take so many insults before it kind of becomes confused. And ultimately, you end up having an altered mental status for one way or the other. So we believe that this accumulation, and like Dr. Anna Medeal's beautiful lecture, these are also much older, a decade older than the typical hepatitis C and alcohol use disorder patients. So all those things take a toll. All the comorbid conditions, such as diabetes, et cetera, also take a toll. And ultimately, maybe you would need less of the insult to propagate them towards that. So that is my theory. It has not been proven yet. But in my clinical practice, I pay a lot more attention to the alcohol use disorder patients, because they are much more likely to be either treated too much or not treated at all. There's no middle ground in between. So thank you. And here's a question from the audience. My question is about the dosage of Ripaximin. Do everybody use 2 times 55 milligrams? Or I am asking all the panelists, did you try any lower or higher doses? So the approved dose for Ripaximin, the approved indication for Ripaximin is, of course, to prevent overt hepatic encephalopathy. And the approved dose is 550 milligrams orally twice daily. So that's, whenever I use it, that's the dose that I use. Some observation may suggest that lower doses of Ripaximin may also help to prevent hepatic encephalopathy, but I wonder if any other people tried that. Jazz? Sorry, I need Ripaximin. What were you saying? Will lower doses work? So there are certain resource-poor countries where doses of Ripaximin, which are lower because it's extremely expensive, like there's a study published that I'm aware of from Pakistan in which they used instead of the 1,200 milligrams, which is the four, they used 800. And they found similar changes. Now in the U.S., we are kind of hamstrung. We only have one dose that we use. And lower dose is, it's neither here nor there. You know, if you have to give it, you give the dose that is impossible, that has been proven. That was a necessity in that country because it was very expensive and most patients would not afford it. And they did a trial. So unless more trials are done in this fashion, I would advise to stick with the dose that has been approved. Thank you. Thank you. Another question from the audience? Yeah. Javier Fernandez from Barcelona. One question for Rajeev, for Jaswaja. Which do you think is the role of fecal transplantation in refractory hepatic encephalopathy? When do you indicate this fecal transplantation? So FMT right now is still very experimental. And in patients with decompensated cirrhosis that our patient was, it's very, very high risk right now. Because we try to do it in outpatients who are relatively stable. And our upper limit for MELD in the study that we just completed, which is still in follow-up, was 22, which is still very high. The problem with FMT is in our patients with impaired intestinal barriers is clearly, you know, talked by Dr. Moreau and everyone else, is that if someone develops such as an SBP, it's very, very difficult unless you actually go do very deep molecular techniques as to where it came from. And a study that was published from Boston here two years, like three years ago, they were actually able to, they did not screen the donors as well. And the MDROs were actually transmitted. And the patient with the HE was actually hospitalized for that. So it is still an evolving field. Now other things that are made from FMT, such as the products from Rebiota and the others, they have not been studied yet in hepatic encephalopathy. Because those products clearly do not have any of the MDRO potential, et cetera. They're clearly effective in C. difficile. But in patients and at past a certain level of decompensation, there were two reasons potentially not to use FMT. One is the potential for transmitting something. And the second more important thing is those patients get antibiotics daily. So anytime you get antibiotics, you basically kill the inoculum and make things a little harder for that thing to engraft. So right now, it's still in very much an experimental stage. And believe me, I've tried to get some of the manufacturers interested in this. And they do not want to lose their license, because there will be so many serious adverse events that would be impossible to adjudicate in these very, very sick inpatients. So the outpatients with high MEL are your biggest yield to potentially prevent things from happening, rather than treating them once things have happened. Thank you, yes. And a brief question for Florence and Jacqueline. It's a very difficult question that I think has no answer yet. But what do you do with a patient developing an ESBL-producing enterobacteria and SVP caused by this multidrug-resistant bacteria, while on quinolone and prophylaxis and on rifaximin, which is your option? I personally have not used rifaximin for prophylaxis, because my experience with it as a prophylactic agent is limited in Canada, where I am, we either use ciprofloxacin or norfloxacin. And so for someone who's got drug resistance, we tend to cycle the antibiotics, sometimes use a quinolone, a fluoroquinolone, sometimes we use spectra. So if I understand the question correctly, it's what would I do in a patient who's had SVP with an ESBL organism that developed on primary prophylaxis like norfloxacin? Is that the question? Yeah, yeah, yeah, on both, on quinolone prophylaxis and rifaximin. So in general, what I would do in that case, because this is secondary prophylaxis, which no one would argue that they need prophylaxis, is I would look at what the sensitivities were for the ESBL and hope that something that I could use would cover it. For example, I would look at Bactrim, I would look at cefepidoxime, I would look at other oral antibiotics to see if I could switch to something that would cover that. And would you consider fecal transplantation in this setting? So our big problem with fecal transplantation is once again, we get back to cost. So getting access to it because of being able to have it be paid for. So once again, unless I have an FDA-approved indication for fecal transplantation, I don't have access to it. But I think it's an interesting concept. But once again, I think the spread of multidrug-resistant infections, as you've published on, is a huge problem that's only getting bigger. It's the highest in other places other than the U.S., for example, India. But it's everywhere and getting worse. Thank you. Well, thank you. May I just take this opportunity to thank my co-chairperson, Professor Szabo, and all the speakers for this fantastic session, which has explored many, many, many different aspects of immune dysfunction, gut dysbiosis, HCC, which I certainly have learned a lot from. And I'll just, therefore, bring this session to a close. And thank you very much, everybody. Just two announcements to make. The first is just to remind you that the session starts again at 2 o'clock, where we'll be talking about the crosstalk between cardiopulmonary and the renal systems, which is obviously extremely, extremely important. And the second is there are some box lunches outside. Is that right? So please enjoy your lunch. Thank you.

cirrhosis

pre-malignant condition

cancer progression

HCC

genetic changes

epigenetic changes

gut-liver axis

dysbiosis

early detection

antibiotic prophylaxis