Welcome to today's webinar, New Insights into the Pathogenesis and Management of Cystic Fibrosis-Related Liver Disease. It's presented by AASOD's Pediatric Liver Disorder Special Interest Group. Today's webinar presenters are Drs. Daniel Leong and Jay Freeman. Dr. Michael Narkowitz will serve as our moderator. Today's webinar will be available on demand on AASOD's online learning platform, Liver Learning. Be sure to connect with us on Instagram, LinkedIn, Facebook, and Twitter. Also tune into our YouTube channel to view our videos. If you're not an AASOD member, today's a great time to join. As a member, you can join as many special interest groups as you would like, listed or a few other member benefits. Be sure to check out the new AASOD Journals app. Access to all four AASOD journals are in one place. Visit aasod.org slash publications to learn more. AASOD has many upcoming meetings covering a variety of topics. Visit aasod slash calendar for a full list of our events. The liver meeting 2019 is November 8th through 12th in Boston, Massachusetts. The deadline for early registration, pricing, and housing is Friday, October 11th. Please visit our website at aasod.org for detailed information. DDW is May 2nd through the 5th, 2020 in Chicago, Illinois. The abstract submitter is now open and closes December 1. Visit www.ddw.org slash abstracts to learn more. You can help support the future leaders in hepatology by giving a donation to the AASOD Foundation. Your donation in any amount is tax deductible. Visit aasodfoundation.org slash donate to learn about ways to give back. You may submit questions in the Q&A box at the top or bottom of your screen. Questions will be answered at the end of the presentation. So now I'll turn it over to the moderator, Dr. Narkewicz. So I would like to welcome everyone to this webinar put on by the Pediatric SIG of the AASOD. I'm going to just take one slide to highlight some of the big issues for cystic fibrosis liver disease. Throughout this webinar, we'll refer to this as CFLD. This is a broad term. It encompasses a variety of disorders and a spectrum of disease across cystic fibrosis. It can include anything from mildly abnormal liver enzymes through advanced liver disease and portal hypertension. As background information, I think it's very clear that the pathogenesis is still very uncertain. There's many hypotheses and Dr. Leung is going to update us a little bit on that. What most individuals who care for liver disease and CF care about is really advanced liver disease. It manifests as portal hypertension. With cirrhosis, which is the most common form in children, however, there's certainly an emerging data suggesting that there's a large component of individuals with CF who have what's referred to as non-cirrhotic portal hypertension. This seems to occur across all ages, but it's certainly much more common in adults. We're seeing a dichotomy in the presentation of this disorder. Both Dr. Leung and Dr. Freeman are going to address issues around these particular points. The agenda today is shown here. I'd like to introduce our first speaker for the meeting. It will be Dr. Dan Leung. Dan is the Director of Hepatology and Liver Transplant Medicine at Texas Children's Hospital. I'm going to let Dan take it away. Great. Thanks, Mike. Over the next 15 or so minutes, we're really going to try to highlight some of the potential mechanisms of injury, which eventually lead to CFLD. I did want to just briefly mention my disclosures as shown there before we get started. My objectives are relatively simple. We will discuss some of the putative mechanisms behind CF-related liver disease, cirrhosis, and portal hypertension. Then we'll close with a brief overview of the emerging CFTR modulators that have recently hit the market and their potential to really modify progression of CFLD risk. For the remainder of my talk, I'm going to primarily center on CF-related cirrhosis. As Mike mentioned, this really is a pediatric phenomenon. In the largest series of over 500 patients with CF with portal hypertension, the overwhelming majority presented by 18 years of age, in fact, with a mean age of diagnosis of 10 years. This does happen early. Second, really, to only respiratory and transplant complications, CF cirrhosis and portal hypertension is the number three cause of death in CF. There are well-known clinical associations, which may include pancreatic insufficiency, having two severe mutations, also known as the class 1 through 3 mutations. There does appear to be a male predominance in patients who develop CF cirrhosis. Mike will touch on research ultrasound patterns as being a risk factor as well. Roughly 20 years or more, most people have thought that CFTR dysfunction within the apical site of the biliary epithelium leads to hepatobiliary disease, and this would make a lot of sense. CFTR is critically important to ultra-chloride and bicarbonate transport. When CFTR is dysfunctional, there's a lack of alkalinization of the bile. This leads to a very acidic bile environment, resulting in peribiliary plugging, eventually fibrosis and cirrhosis. So, it makes sense for us to really consider CFTR dysfunction within the biliary epithelium as a primary cause. I think it's also very important for us to understand how CFTR works in the biliary epithelium. In wild-type CFTR, the protein is made in the nucleus and then appropriately folded in traffic to the cell surface, where its primary function really is chloride secretion. At least within the biliary epithelium, this chloride-bicarbonate exchange is extremely important to result in bicarbonate-rich choleresis. When I mentioned the association of CF cirrhosis with severe CFTR mutations, I'm really referring to the class 1 through 3 mutations. In the class 1 mutation, the protein is not produced at all. In class 2 mutations, the protein is produced but unable to be folded appropriately and thus is not trafficked to the cell surface. And then with class 3 mutations, there's absolutely no function to the CFTR protein. You can see here that class 4 through 6 mutations are much less mild, and its association with CF cirrhosis is significantly less. So, we've discussed CF liver disease as primarily being a hepatobiliary problem. We've talked about CFTR's role in the biliary epithelium, but what about our clinical observations? It's very interesting that most of our patients are either asymptomatic or compensated for years and years. Very few of our patients with CF cirrhosis demonstrate low albumin or synthetic dysfunction. They also don't present with a rapid progression to thrombocytopenia, and very few of them present with ascites until very end-stage, late disease. And so, why is that? Is this truly hepatobiliary cirrhosis? And interestingly, of the many patients that I've taken care of, I have rarely if ever seen a patient with clinical evidence of cholestasis. Rare if any patients present with jaundice, pruritus, and certainly cholangitis is quite rare. So, then we started to take a look at pathologic observations, and when you look at the lungs and the pancreas, you can really appreciate fibrocystic changes. But these fibrocystic changes, when you look at the explants of patients with CF, do not persist. In fact, as you can see here in the cut surface of the liver, there are really no fibrocystic changes in the liver. So, the disease process in the liver appears to be a little bit different. Why might that be? Over the last 10 years, there has been emerging evidence of possibly a presinusoidal vascular component in addition to CFTR abnormalities, and we know this because our Australian and European colleagues have demonstrated that in CF, poor hypertension can actually precede cirrhosis and appears to be out of proportion with the degree of fibrosis. In fact, our Australian colleagues have demonstrated that cirrhosis was present in less than a third of patients with poor hypertension, even when performing dual-pass biopsies. Also very interesting in Europe, where hepatic venous pressure gradients have been obtained, in patients with CF cirrhotics, having esophageal varices, is their pressures are significantly below the threshold of clinically significant poor hypertension, ranging between 4 to 9 millimeters of mercury. And then lastly, Peter Witters from Belgium has demonstrated very nicely that there does appear to be this phenomenon known as obliterated poral venopathy, with dense fibrosis in the poral vein branches of the porotracts in liver biopsies of adult-age patients. And so that leads me to the second possible idiopathogenesis of CF liver disease, and this may involve vascular abnormalities within the endothelium or perhaps a prothrombotic environment in the CF patient who is constantly in a state of inflammation. And so Peter Witters from Belgium first introduced this concept of, is CF liver disease with poor hypertension more of anopathy as opposed to hepatobiliary cirrhosis? This was a really nice case series of young adults. Their median age was 22 years of age. They all had clinical poor hypertension, and in fact, 10 out of 12 had endoscopic evidence of esophageal varices. And when you look closely at these H and E stains, it is very hard to make out any poral veins. Many of the poral branches are either missing or obliterated, as shown in these four panels. And so at Texas Children's, we decided to take a look at the explants in patients with CF who were transplanted for the indication of poor hypertension, and we wanted to correlate whether or not our findings in children was the same as those in adults. What we found very interestingly is that many of these patients did demonstrate a nodular capsular surface, as shown here by the arrows, but you can appreciate that that nodularity is quite lacking in the cut surface of the liver. Moreover, the areas of fibrotic replacement are significantly less than other hepatobiliary diseases such as biliary atresia or primary sclerosing cholangitis. You really don't see these large, broad bands of dense fibrotic tissue as you would expect. And lastly, while there was some cholestasis in a few of our explants, the green discoloration was really limited to areas of thin fibrocypta, again, not large, broad bands of cirrhotic tissue. And interestingly, in patients with poor hypertension who did not necessarily have nodularity on the outer surface did demonstrate, on many occasions, focal nodularity in the central areas near the vessels. So, this idea of a vascular finding really warranted a closer look histopathologically. As a reference point, the poral vein is quite sizable when compared to the hepatic artery and bile duct, but at least in our 17 explants, we found that the poral veins were significantly diminished. If you look at the circumference of the poral vein in comparison to the hepatic artery and bile ducts in patients with CF-related cirrhosis, you can see that they're very equivalent, very, very diminutive. In fact, 65% of our explants in Houston had these really thick coats of smooth muscle surrounding and obliterating these poral veins. And very interestingly, when we conducted reticulin stain, we found that 94% of our explants demonstrated features of nodular regenerative hyperplasia with central lobular sinusoidal dilation rather than true histologic cirrhosis. And so, this really begs the question of if there may be a vascular phenomenon. When we looked at the clinical features of these patients with their explants, we found that very few of them presented with pruritus or jaundice. However, many of them had endoscopically proven esophageal varices even in childhood. Many of them even received variceal ligation. The overwhelming majority of them did demonstrate splenomegaly, and many of them, as you can see, had clinically significant thrombocytopenia as well. So, it certainly makes a lot of sense following Poiseuille's law that if you have diminished poral veins and decreased flow, that this certainly could explain intraepatic presinusoidal hypertension. And when you think about children with CF, they are presented with early infections at an early age that are quite recurrent, can become multidrug resistant. And so, we know that thrombotic predispositions and subsequent vascular damage has been postulated to cause non-cirrhotic poral hypertension and certainly may be relevant in patients with CF. And again, from our explant findings, I think you will agree with me that it's very challenging to find a normal poral vein in each of these poral tracts. There's either significant smooth muscle hypertrophy or even calcifications within the poral veins. So, what might cause non-cirrhotic poral hypertension in CF? We really don't know. That is a work in progress, but it has been suggested that there may be inflammatory spillover directly from an abnormal microbiome into the poral vein. Patients with CF may have an increased risk for microthrombi because their platelets are known to be hyperactive. Or might there be a primary vascular problem? We know that patients with cystic fibrosis have a higher incidence of abnormal vasculitis markers. And so, we've talked about CFTR dysfunction. We've now talked about the potential of a vascular or platelet problem. It's really hard to talk about evolving liver disease without at least mentioning the microbiome. And CFTR certainly plays an important role in the intestinal epithelium as well. It keeps intestinal secretions nice and fluid so that microbes can be removed appropriately. If there is inspissated intestinal secretion leading to an overabundance or an accumulation of these microbes through translocation into the poral vein, it is thought that the canonical stellate cell activation pathways could be triggered as well. So, I am going to talk a little bit about the microbiome and CF liver disease. This is not a new relationship. In fact, in CFTR knockout mice, these mice frequently demonstrate intestinal inflammation associated with the development of liver disease. In several case series of pancreatic insufficiency of patients who have had capsule endoscopy, a majority of them do have visible intestinal inflammatory lesions. And it's been well described that patients with cystic fibrosis have elevated fecal calprotectin. And even patients without CF who have liver disease have also been demonstrated to have elevated fecal calprotectin level. This is a really nice study done by Thomas Flass and Mike Narkowitz in Denver where they compared patients with CF cirrhosis with CF patients with no evidence of liver disease. And after performing eight-hour capsule endoscopies, they had two blinded reviewers score these and a cutoff of greater than or equal to four was considered a severe capsule endoscopy score. And what did they find? They found some differences in those who had CF liver disease cirrhosis. In fact, it was really only the CF cirrhotics who had a severe capsule score of four or higher. Patients with CF cirrhosis also had increased small bowel transit time but did not demonstrate any clear small bowel permeability when compared to those without liver disease. They went on to move to comparing clinical variables with phyla and genre, and they found a trend towards a higher relative abundance of firmicutes and a lower level of bacteriodoides in those with CF-related cirrhosis. And when they conducted their clinical correlations, they did find a positive correlation of firmicutes with the number of red spots visualized and a negative correlation with bacteriodoides and the number of red spots visualized. So their work has certainly justified the question, if not the hypothesis, of differences in the fecal microbiome disrupting the gut and possibly playing a role in the pathogenesis of cirrhosis in CF, which is quite interesting. I did want to just end with a brief overview of some of the emerging CFTR modulators, which is really going to drastically change the landscape of cystic fibrosis. There are now three FDA-approved therapies to either restore or potentiate CFTR function in CF patients, and the primary benefits are really in the areas of lung function, improved weight gain, and a reduction in the number of pulmonary exacerbations compared to placebo. These modulators now exist in the form of combination therapy, and a triple therapy trial is now in process. And so, these CFTR modulators show a lot of promise, but what are they? I'll just briefly introduce the concept of CFTR potentiators. Ivacaftor is the first potentiator to hit the market, and the way that these potentiators work is that it increases the probability through a defective CFTR channel that it's going to be open for a longer duration, hence allowing increased chloride ions to pass through the channel pore. This is going to be critically important for patients with CF liver disease because this allows increased bicarbonate exchange, resulting in alkalization of bile, and perhaps improved choleresis within the bile ducts, decreasing the risk for peribiliary plugging. The FDA has now approved Ivacaftor in children greater than two years of age who have at least one of 23 residual function mutations, G551D being the most common. Another class of CFTR modulators include the CFTR correctors, which are critically important in the appropriate folding of the CFTR protein. That improved folding results in better trafficking of CFTR proteins to the cell surface. And so, given in combination, Lumacaftor and Ivacaftor, for example, the first drug would increase the number of mature proteins at the cell surface, while the second drug, Ivacaftor, will enhance the function of the CFTR protein. The FDA has now approved combination therapy for patients greater than six years of age who have two copies of Delta-508. And for the combination of Tezacaftor, Ivacaftor, and Ivacaftor, these are approved for patients greater than 12 years of age with a single Delta-508 deletion. So these CFTR modulators show tremendous promise, but it should be mentioned that CFTR modulators given in combination are associated with some mild to moderate hepatitis, and there are a few case reports of cholestasis as well. So, it is important to monitor the liver transaminases and the total bilirubin prior to beginning combination therapy, and every three months after in the first year of treatment. Clearly, if there's an abrupt rise in the transaminases or an abnormal bilirubin, these treatments should be held, interrupted before considering restart. I think that these CFTR modulators are really going to demonstrate potential, but it's probably going to take a good 10 years for us to see if CFTR modulators change the natural history of CF liver disease development. So, in summary, I hope I've impressed upon you that the idiopathogenesis of CFLD with poor hypertension is probably multifactorial, certainly may include CFTR dysfunction at the level of the biliary epithelium. I've also introduced the concept of an obliterated poral venopathy, not only seen in adults, but in children as young as 8 years of age. And of course, the intestinal microbiome may certainly alter the risk for the development of CFLD via the stellate cell activation pathways. I hope I've also demonstrated that these new emerging CFTR modulators may really brighten the future of patients with CF. I think it will require at least a decade for us to determine if there's a true decreased risk of CFLD in patients who are taking these modulators, and because of reports of elevated transaminases on combination therapy, treatment should be monitored very closely and regularly. Thank you very much. It's really now my pleasure to introduce Mike Narkowicz. He's a professor of pediatrics at the Children's Hospital of Colorado, and he's going to share some really unique insights on what might predict or modify the risk of CF liver disease. Mike? Thanks, Dan. So here are my disclosures. Today, at the end of this session, I really hope you can all understand the predictors of CF-related liver disease, including genetic, clinical, and biochemical and biomarkers, and discuss how these may reflect on the presentation and progression of liver disease and CF. And the real question is, are there any good biomarkers for cystic fibrosis liver disease? And why do we ask that? Well, biomarkers actually serve many different questions for us. And often the question is, does my patient have CF liver disease? And then many others will ask, how severe is the liver disease? And then in the end run for hepatologists, is this individual patient at risk for complications of advanced liver disease? One key question, particularly the advent of new therapies, is the patient in front of me at risk for developing cystic fibrosis liver disease, particularly advanced liver disease in the future, such that I might intervene or prioritize therapies? So we ask these questions in large part because CF liver disease is relatively silent. It does not cause symptoms or signs until very late in its course. Liver biopsy has limitations. There's diagnostic accuracy issues because of sampling error in this patchy disease. In the Australian study of dual-pass liver biopsies that Dan referred to, there could be up to two stages of fibrosis difference between the biopsies. And over 30% to 40% of those biopsies had different degrees of fibrosis. And it's really hard to know which is which. Is it the worst or the least of the biopsy? And as we know, for certainly following the course of disease, repeated biopsy is not feasible, especially for research in children and in CF with a higher risk because of pulmonary hyperinflation from liver biopsies. We also think that new therapies such as those that Dan described may prevent CF liver disease and potentially could reverse it, but I think have more opportunity to prevent. These include the highly effective CFTR modifiers, including ones that will come on the market probably by the end of this year or early next year, drugs that affect bioproduction and circulation, antifibrotics that might be targeted at any liver disease. So let's start with identifying individuals at risk for advanced liver disease. A lot of work has gone in on to modify your genes and to genotype-phenotype analysis. So as Dan said, CF liver disease is primarily found in those who have two loss of functions mutations. Indeed, this is necessary but is not sufficient for the development of liver disease. In a large GWAS study that was done by the North Carolina group and Mike Knowles in association with the Toronto group, they screened the largest series of CF patients with a portal hypertension and each subject with portal hypertension was individually reviewed by hepatologists and they had 131 controls and they had both an initial study and a replication study. And what this shows is those with advanced liver disease had the presence of a heterozygote state for the alpha-1 antitrypsin Z allele in 9.7% of the subjects while only 2.4% of the controls had this. So the odds ratio, if you had a Z allele of a developing advanced liver disease was about 4.7 in the first cohort and 3.4 in the second cohort. So what we can take from this is that alpha-1 antitrypsin Z heterozygote state puts individuals at increased risk for advanced liver disease. However, I would also note that the majority of the patients who had the Z allele still did not develop advanced liver disease and it only accounted for 9 or 10% of the subjects who had advanced liver disease and this may vary depending on where you are. I think this risk would be different in California with a different population than it might be in other areas of the U.S. or Europe. What about clinical correlates? Well, pancreatic insufficiency, again, is necessary but not sufficient. The male predilection is somewhere between 1.5 and 2 to 1 and prior data suggested that there was a risk with both Meconium milleus and other clinical correlates. However, subsequent studies that were more directed at identifying liver disease have not supported this association. There are some suggestions from the large PUSH study, which is a multicenter CF liver disease network study that includes about 700 subjects and we found in that study that early pseudomonas infection, less than 2 years of age, was interestingly protective for development of ultrasound abnormalities in children and we found a more frequent association of cystic fibrosis diabetes in individuals who had advanced liver disease, had abnormal ultrasound. That was a relatively low risk of about two-fold increased risk. CFLD is also associated with an increased risk for CF-related diabetes and the odds ratio is quite high, about 11, and particularly for CFRD if you have portal hypertension. So what do I take from this? I take that there are no good predictors of CFLD from clinical features when you have a patient in front of you. So we need better biomarkers for that. What about liver enzymes? The CF Foundation guidelines suggest checking liver enzymes every year and we took the opportunity to look back at our newborn screen population. Colorado was one of the first two states to institute newborn screening back in the early 80s and as a result, we have a large cohort of children who've been followed from birth over a long period of time. We looked at almost 300 children who were diagnosed by newborn screening in Colorado and we characterized their liver enzymes when they were well over a period of time. You can see the scatter plot for liver enzymes and from this, you can see that liver enzyme abnormalities are relatively frequent, but we then looked for correlations between early persistent abnormalities and the subsequent risk of being identified with advanced liver disease, meaning cirrhosis and or portal hypertension. And you can see that persistent early abnormalities in children who are less than five years of age, when they are well at their annual visits, demonstrate that all of these enzymes have an increased risk with GGT having about a fourfold increased risk. I point out GGT because in a study from the Netherlands, not in a newborn screen population, but in a case controlled population, they demonstrated that high GGT preceded the diagnosis of advanced liver disease by over two years. And here you can see those who did not develop advanced liver disease and those that did and their GGT two years prior to their diagnosis. And they demonstrated in their small population that a relatively modest GGT cutoff of 21 identified those individuals at risk for advanced liver disease who had CF. Platelet count is another marker that's been looked at and in this study out of Toronto that's been published in abstract form, they demonstrated again in a case controlled manner that those individuals with advanced liver disease, again, meaning portal hypertension and cirrhosis, had declines in platelet counts and a platelet count less than 150 was able to identify two-thirds of the individuals. So it had good specificity when compared to their controls. And you can see the development of low platelets was relatively early in this cohort that had a mean age of diagnosis at about 11 years of age. So platelet count persistently low can be another biomarker. So if we combine some of these indices, can we get more specific and help to identify individuals with advanced liver disease? So APRI, which is the ASD Platelet Ratio Index, has been used in a variety of liver diseases to look at fibrosis and has been studied in CF in that same cohort from Australia with dual-pass liver biopsy. In this study with Dan Leung and the Australian group, they looked at all of the individuals who underwent liver biopsies, mainly dual-pass liver biopsies, and looked at their APRI score. In summary, what they found was that APRI did identify individuals who had some evidence of fibrosis on liver biopsy and that it seemed to be able to separate out those with advanced versus less advanced fibrosis. And this is shown best in the fibrosis stage. And what I would point out here is this seems to be most effective at separating out those with advanced liver disease. So in this case, we're asking the question, how bad is the liver disease in my patient? It appears that APRI is good at identifying individuals with advanced fibrosis who have CF. I would point out that these cutoffs are much lower than you would see in something like hepatitis C, where the cutoffs for advanced fibrosis are more like an APRI of 1.5. And some of this may be reflected by the fact that AST is rarely very significantly elevated. And this may be really a manifestation of the thrombocytopenia that I demonstrated on a previous slide. Well, what about other markers for fibrosis? And elastography is certainly one that comes to mind in identifying CF liver disease. So I'm going to review the data on elastography. So if we look at liver stiffness by vibration-controlled trans and elastography, this has been the most studied technique, also known as FibroScan. And in these data from the Boston group, you can see the results of VCTE in CF subjects. And what they demonstrated is in individuals who they felt clinically and biochemically did not have liver disease. They had fairly normal elastography or liver stiffness. Those with liver disease, slightly higher, but it was really very good at identifying those with portal hypertension. Shown another way on the graph on the right, you can see that those with advanced liver disease really separated out fairly well from all the others. However, those with liver disease that was milder and no liver disease were very difficult to separate out by elastography. So from these data, which are fairly similar to some of the other data in other diseases, elastography, at least VCTE, seems to be able to identify those with advanced liver disease. What's interesting is the cutoffs from a variety of studies are quite varied, anywhere from 6 kilopascals up to 20 kilopascals to identify those individuals with advanced liver disease. I think there's more work to be done in this area, but it is clearly a tool that is making its way into the clinical practice. There are certainly other elastography modalities, depending on what you have at your center. ARFI, there are nine studies in CF, and you can see the various range of the data. But again, ARFI measures elastography somewhat different and looks at velocities, and you can see there's large overlap between CF liver disease and those with no liver disease, and probably better differentiation, much like vibration-controlled transient elastography of advanced liver disease. Supersonic shear wave elastography, there's really no overlap of, there is a lot of overlap of CF liver disease and no liver disease, and again can differentiate advanced liver disease, and MRE elastography. Now this is not as useful in young children because of the need for sedation potentially, however it does have an advantage over all the other tools in that it provides whole liver elastography. I want to be clear that the KPA values of MRE elastography does not equate to the VCTE values, so you can't use the same cutoffs. There's one published case series of four adults with CF. I'm aware of several ongoing studies, one in Cleveland and one with our Cystic Fibrosis Liver Disease Network, and this is an example of what you can see, and I like this example from the MR case series in that it shows the liver with a heat map, and you can see the patchy nature of the fibrosis, in this case of a subject with advanced fibrosis, and in this case of a subject with less advanced fibrosis, and you can see the patchy nature of this, and you get a whole average of the liver, and you can see the ranges. So advanced liver disease here on the MR has a kilopascals of 5.6, which again is very different from VCTE. So the bottom line is elastography appears to be very good at identifying advanced CF liver disease. It does not appear to be useful, though, in predicting who's going to have liver disease, and it has much less utility in those with mild abnormalities of liver stiffness. What about change over time in liver stiffness? So in this very nice study, they followed individuals over time with liver stiffness, and they demonstrated that there were large increases in stiffness over time that may enable the early diagnosis of progressive CF liver disease. What's interesting is overall, there's a gradual trend towards increase in liver stiffness in CF anyways, at a rate of about 0.1 to 0.3 kilopascals per year. So any increase does not identify someone with advanced liver disease, but rapid increase as seen here does. The final biomarker I'm going to talk about is ultrasound. We often do not think about this as a biomarker, but in the CF liver disease network, we've been studying whether a heterogeneous liver ultrasound pattern is associated with the increased risk for advanced liver disease in CF children. So for this study, we screened over 700 subjects who were all pancreatic insufficient with research ultrasound, and they got a grade by three study radiologists. So this is not a single individual radiologist. Heterogeneous subjects were matched one to two by the factors shown there to subjects with a normal ultrasound in CF. And our endpoint is development of nodular ultrasound pattern at four years consistent with cirrhosis. So what did we find? Here are the imaging patterns that our radiologists have used. But what we basically found is if you have a heterogeneous pattern at entry, at the end of four years, you have a ninefold increased risk of developing a nodular liver compared to controls with a normal liver. So you can see 24% of the heterogeneous group developed nodular liver at the end of four years, and only 2% of those with a normal pattern at entry developed this. So it appears that heterogeneous liver on research grade ultrasound can identify individuals with a high risk, but still it's only at 24% risk at two years, at four years, sorry. So in summary, I've shown you that there are genetic modifier studies that have identified the Z allele as a risk factor for advanced liver disease. There are several biochemical markers, and I think the two best markers are probably platelets and GGT to predict whether someone's at risk of advanced liver disease or identify someone with advanced liver disease using APRI. Transcene elastography and other elastography techniques can identify advanced CF liver disease, but are not yet ready for prime time for identifying early liver disease. As I just showed you, ultrasound pattern may also identify a cohort at high risk for the development of advanced liver disease. There are lots of questions that remain. One that keeps me awake a little bit is whether CFTR modulators will alter the development of CF liver disease, but the questions that I think are prominent to our audience today are now that I've identified someone with advanced liver disease, what do I do? And that's going to be the subject of our next talk. So with that, I would like to introduce Jay Freeman. Jay is the Director of Digestive Health for Cystic Fibrosis at Emory University School of Medicine. And Jay, take it away. All right, Mike. Thank you very much. For my part of the session, I'm going to be talking about our experience in shunts and really other interventions in liver transplant in patients with cystic fibrosis-associated liver disease. And I hear from the two or from our other speakers that portal hypertension is mostly what we're talking about with advanced cystic fibrosis liver disease and really synthetic dysfunction is rarely encountered. Here's my disclosures. At the end of this session, I really want to be able to discuss what the experience is and a number of modalities to address portal hypertension and advanced CFLD and really potential considerations that one must think about when talking about either isolated liver or multiple organ transplant in patients with advanced CFLD. So just to be clear, we are really talking about that 5% to maybe 10% of patients with advanced CFL disease. This is a very interesting population in my opinion because not only do they have the same risks of advanced liver disease sequelae as other diseases, there seems to be an overall survival risk by having this condition. This is shown in the Dutch study up here in the upper left and where they looked at patients with cirrhosis related with their cystic fibrosis and compared to controls that were matched on both pulmonary and nutritional status. And as you can see in the six-year follow-up, there was an over 10% decrease in survival amongst those patients that had cirrhosis and only 18% of those were from liver-related deaths. Over two-thirds, their cause of death was still pulmonary in origin. This mirrors data from the CF Registry in the United States in which the CFLD Network reviewed those patients that were diagnosed with cirrhosis and looked at the 10 years following their diagnosis and in the following 10 years, only 7% went on to have an episode of variceal bleeding, just less than 10% required a liver transplant and only 7% experienced a death related to their liver disease. But if we looked at all-cause mortality, then we went all the way up to 40% of patients died, again, mostly from pulmonary disease, not liver disease. Given the impact that CFLD seems to have on global outcomes and the unclear pathophysiology as Dan described earlier, it makes it difficult to know what the ideal treatment is for these patients. In the next few slides, though, we're going to talk about some of the options that have been used and what the success rates and failures, quite frankly, are in some of them. So to begin, we'll talk about splenic embolization in which there's no real good case series or research. We're usually left with case reports such as this 11-year-old girl who at the age of 11 developed hematemesis and over the next three years had progressive splenomegaly to the point that at age 14, the team decided to use a partial splenic embolization of the middle and lower splenic artery. The authors highlighted an improvement in platelets and white blood cells to show the success of the surgery or, I'm sorry, the embolization in the first month. But in further review, the patient spent an entire month in the hospital and suffered post-embolization syndrome including fevers, severe abdominal pain, pneumonia, ascites, and even ongoing GI bleed that required three transfusions. Fortunately, this patient survived and at 14 years, the follow-up was doing well. But given the high rate of complications of partial splenic embolization which had been reported as high as over 30% and the fact that it does not address the underlying cause of the portal hypertension, it's not surprising that it is not seen more frequently in the literature. There is a greater experience though with partial splenectomies. This table is from the experience of the group in Lyon, France in which 19 patients were admitted for 14 days of antibiotics and underwent partial splenectomy on day 8. There was a median follow-up period of 8 years and at the bottom, you can see that four of the 19 patients passed away despite the intervention with an additional three patients undergoing further GI bleeding. Although abdominal pain improved in all of these patients, you can see that lung function was not significantly improved, hepatic insufficiency developed in five patients, and at the time of their last assessment, 13 of the 19 patients still had grade 2 or higher varices. Again, given the modest improvement in clinical condition and the increased risk of surgical approach, again, partial splenectomy has not been used with great frequency in this population. This has turned people to look at total splenectomy as a better choice when dealing with severe splenomegaly. In theory, this would have a better impact on overall clinical care as it's been shown that massive splenomegaly can push on the diaphragm which can cause restriction to the lungs and cause shunting that would ultimately impact pulmonary function. In this review, over 25 years in Melbourne, Australia, they reviewed the 650 patients under their care and found nine patients who underwent total splenectomy. As you can see, the mean age at diagnosis was eight and splenectomy was typically performed at around 14 or 15 years of age. Of these nine patients in which there was approximately a mean follow-up of six years, the indications for their splenectomy are listed below with all patients having at least two indications. Not surprisingly, low platelet count and complications of splenomegaly were the main indications. Amongst these patients, one patient died of hepatic decompensation but none had overwhelming sepsis or other infection issues. Abdominal pain improved in all patients but most importantly was the improvement in lung function. If we look at the graph on the right, in the two years prior to total splenectomy, patients were averaging a decline of FEV1 at 10% per year. By two years after the procedure, this had reversed to a 2% gain in FEV1 after the procedure. This must be interpreted with some caution though as there is transplant data that showed a similar trend for approximately three years after transplant but the benefit to lung health seemed to disappear after that time and we do not have longer data in this cohort to say what long-term benefit was. Of course, total splenectomy is not without its risks. There is, of course, the well-known postoperative pain that has been associated with this surgery as well as the concern for infection with encapsulated organisms after a splenectomy. To address the root cause or the portal hypertension that is causing the splenomegaly, not surprisingly, there's been quite an experience with systemic shunts. I believe any conversation on shunt procedures in CF really needs to start with this landmark paper of 44 French children who were diagnosed with severe portal hypertension but must also be noted that this took in account patients from 1960 to 1996. Of these 44 patients, 12 underwent surgical shunt procedures with or without splenectomy with multiple shunt procedures used as indicated in this middle column. Follow-up for these patients ranged from three weeks all the way up to 15 years and during this time period, you can see that eight of the 12 patients were deceased with one patient alive only after a combined lung-liver transplant. Of these 12 patients, only one showed significant improvement in their lung function at six months and not shown in this cohort is three patients that underwent liver transplant of which two died, comma, one of hepatic artery thrombosis and the other of sepsis. Although this manuscript does not paint a great picture for shunt procedures, again, it must be noted that this was performed before the year 2000 and since that time, surgical approach and tools have gotten better. Hence, I think it's important to look at this most recent publication from the group at Lurie Children's. Their philosophy has been to restrict liver transplant only to those patients who have synthetic dysfunction and those patients who just show signs of portal hypertension are offered a distal splenorenal shunt. In the time period from 2008 to 2017, five patients underwent distal splenorenal shunt with a mean follow-up of just over four and a half years. As you can see, none of these patients went on to require liver transplant and none of them actually required further intervention for their varices. One patient did pass from pulmonary complications at one year but went into the procedure with an FEV1 less than 30% and was deemed not a transplant candidate for other reasons. One patient underwent an asymptomatic stenosis or was found to have an asymptomatic stenosis on routine ultrasound with extrinsic compression of the right, I'm sorry, the left renal vein. This patient felt angioplasty but never had any clinical sequelae of the condition and has just been monitored and has been stable. In addition of outcomes, we'll look at the pulmonary function testing. As you can see, after the shunt, a decrease in the FEV1 but really over a five-year period, this was 2% per year which is probably what we would expect from this population, although the numbers are pretty low to start with. There was a significant improvement in spleen size and although it didn't meet statistical significance, all of the growth parameters improved at least slightly after the procedure. Of course, with shunt procedures, there is the general risk of thrombosis which given our previous talks on possible thrombotic events in CF liver disease gives some pause and of course, there is the risk of hepatic encephalopathy especially in a patient population that has a pretty aggressive diet. In this cohort though, there was no complications reported of hepatic encephalopathy. So if total splenectomy worked some and distal splenorenal shunt had benefit, why not combine the two which is what this cohort in Belgium did. They looked at six pediatric patients who underwent splenectomy with the distal splenorenal shunt in which the indication was massive splenomegaly with impaired nutrition and severe abdominal pain or variceal bleeding. Of these six patients, three had a 10% or greater increase in their weight in the first year and three patients who had been diagnosed with delayed puberty advanced to appropriate pubertal stage. There was no benefit in FEV1% but there was a decrease in overall risk of pulmonary infections. No talk about shunts could be complete without talking about tips. In an initial report of five children from the Czech Republic, there was a decrease in portosystemic pressures from 17 to 10 but as Dan showed earlier, much lower pressures have been associated with significant variceal complications so it's not surprising that over 70 months of follow-up, there were six additional bleeds and then routine ultrasound showed 15 asymptomatic stenosis that required intervention. One patient went to transplant and two died in a relatively short period of time. More recently, the group out of Seattle reported 34 patients who underwent TIPS, three of which had cystic fibrosis. The first was simply a bridge to get to transplant with the second being a palliative procedure for a patient who was deemed not a transplant candidate with the last patient doing well several years after follow-up. Overall, long-term data is lacking but most people have used the TIPS procedure as really more of a palliative procedure or a bridge to transplant. With that, we will bridge to transplant. I feel like the data on transplant is more familiar to most care providers but the one question that does come up frequently is what are the real indications and contraindications for transplant in this population? This is a table that we published earlier this year with strong and moderate indications. We've already talked about portal hypertension at the top but I also think we have to comment on nutrition and poor growth. Often liver disease is blamed for their poor growth but there's a number of reasons why patients with CF may not be growing or have adequate nutrition and deserve a full workup. Coming down lower, we will talk more about CF-related diabetes when we talk about liver or pancreas. And then finally, I think complications related to infection and colonization of multiresistant organisms are often overlooked and definitely deserves the involvement of an ID specialist in the perioperative period to help with managing what can be quite complicated situations. One of the arguments against transplant in this patient population has been the decreased survival. On the left, you can see in the adult population that patients in blue or CF patients have much lower survival compared to other indications, although this is less dramatic in kids. In this table, we have shown that over the last few years that graft survival in CF liver transplant has improved, but it's unclear if this is just from improvement in transplant care or from CF care. I've also been asked ethically, is it appropriate to give a patient a single organ knowing that it's not going to treat their underlying disorder? To answer that question, I think it's important to look at what the graft survival is. This was also published in our earlier publication this year, and if you look at five-year survival, this entire graph goes over about 27 years, but at five years, there's no real difference between liver alone, liver lung, or liver pancreas graft survival. And with statistical analysis completed, you can see there's no difference. So if we move forward with the idea that there's no survival benefit at least between these three transplant approaches, then often the question becomes a time question. In the graph here on the bottom, we can see that the majority of patients who require a liver transplant for CF do so in their teen years, while those who require a lung transplant are typically in their 20s. This begs the question, are you better off giving a liver transplant early in life and then giving a second organ transplant from a different donor later in life, or should we use one of the modalities discussed earlier to bridge until the patient may need a lung transplant in the future? Clearly this is a case where there cannot be one-size-fits-all, and each patient deserves individual consideration. Finally, if we are going to think about doing dual organ, which organ needs to go in first? Typically it has been thought that due to the liver's ability to tolerate prolonged cold ischemic times that the thoracic organ should be placed first. In this review of a Belgian group that did intrathoracic and liver combined transplants, 11 of their 14 underwent this classic approach, but three patients underwent a liver-first approach in which the lungs were kept warm by ex vivo warm perfusion. The thought was the potential benefits of doing a liver-first approach would mean that the diseased lungs absorb the reperfusion injury, you could restore coagulopathy that may decrease transfusions and pulmonary edema in the lungs, and there could be less cold ischemia resulting in less biliary strictures. In this cohort there were four cases of biliary stricture, only one of which was in the liver-first approach. And as always with dual organ, with the liver, there is an immune privilege status that tends to go along with these patients. There was a decreased rate of rejection in all of these patients, although length of stay was not surprisingly longer. Finally, the last consideration is the liver-pancreas consideration. These patients all essentially have pancreatic insufficiency and the link between CF-related diabetes and advanced liver disease has already been covered. In a review of UNOS data through 2014, we saw that the primary indication for transplant for CF in liver was 303 patients and over 4,000 with lung. Amongst those in which liver was the primary indication, approximately 20% were known to have CF-related diabetes, but only 17 really received a liver-pancreas transplant. In lung, it was even worse with over a third of patients being recognized as also having CF-related diabetes, but only three had a dual transplant. Needless to say, those patients that had their pancreas transplanted with them, only two developed post-transplant diabetes with rates much higher than those that did not, suggesting we are underutilizing this resource. Finally, in this study on the right from the group from Indiana and Toronto of eight patients who underwent liver-pancreas, we can see that BMI improved across these patients and none of the eight patients had either exocrine or endocrine insufficiency. With that, if you were expecting me to tell you what the exact decision should be when it comes to advanced liver disease, I'm sorry. I think it is a patient-by-patient conversation, and I think that the long-term impact as either a bridge to transplant or as a long-term solution of shunt procedures requires further study, but given the correct indication and correct patient, liver alone or combined organ transplant should be considered in this population in the context of a multidisciplinary team approach. With that, I'll turn it over to Mike so we can answer some questions. Thank you. Perfect. There are a couple of questions. One that I think went through this was a question about which patients should be reviewed and followed by hepatology formally. So Dan, you want to take that one? Yeah, sure. You know, the patients that I'm most eager to see are not necessarily those with, not necessarily those who already present with a spleen and their pelvis, but really patients with a heterogeneous ultrasound with a gradual drop in platelet count. So even if the platelet count has not dropped below 150, if six months ago it was 250 and it's now 200 and they've had elevated transaminases for more than six months, even with that gradual interval drop in platelet count, those are patients that I would want to more closely monitor, particularly if they're male or if they have two class one through three mutations. Those are the patients that I generally would want to monitor more closely than just performing their annual labs. Those are patients I might recommend to my annual CBC with the full different platelets as well as a liver panel. My question is really how do people monitor those individuals with elevated LFTs, which I think was really a question of what type of evaluation do you recommend in CF patients who have elevated LFTs to either confirm the diagnosis of CF liver disease and what do you exclude typically? Jay, you want to start with that and then we can chime in? Yes. So I think given some of the previous work, so we're talking about the patient that has prolonged elevation liver enzymes, what is the workup? So we will do a chronic hepatitis workup essentially to make sure that we're just not so unlucky that we pick up two conditions. And of course, given what you showed earlier, I'm always curious to see what the alpha one phenotype will be. We typically will get an ultrasound and then I will try to get a fiber scan because we do have a clinical available one, but unless there is dropping platelet values or some of the other markers that you presented nicely, Mike, we typically don't try to use any other advanced imaging modalities and said, will you read the profile? I think the other issue in addition to chronic hepatitis workup comes up in CF. There's a fair amount of drug exposure and looking closely for potential drug toxicity either from antibiotics or other interventions. And in which case I'm going to take one of the questions that was in the Zoom chat. And the question was really about the CFTR directed therapies. The question was, how do you anticipate the effect of these modulators on CF liver disease and are there any concerns that it might worsen liver disease? So the currently existing CFTR modulators, Ivacaftor, Lumacaftor, and Ivacaftor-Tezacaftor really have kind of minimal effect on CFTR function. They definitely improve pulmonary function and reduce pulmonary exacerbations. But if you look at what they do to CFTR function, it's a relatively modest increase. If you compare that to Ivacaftor and what are called the gating mutations where there was a huge gain in CFTR function to about 25 to 30% in the test tube and much bigger gains in body mass index gains and pulmonary function gains, that's what's really referred to as highly effective modulator therapy. So the new therapies that are going to be coming available for those individuals who are both homozygous for F508-Del or have two severe mutations, one of which is F508-Del, those therapies will appear in the test tube and appear in the preliminary studies to be similar to Ivacaftor in function. They've not included a lot of individuals with liver disease. As a matter of fact, liver disease has been an exclusion, particularly advanced liver disease has been an exclusion. So I don't think we know exactly what the effect is going to be on liver disease. There is a warning for Ivacaftor and Lumacaftor for all of the modulator therapies to monitor for acute increases in transaminases, particularly in the first year of therapy, because there have been, in the FDA reports, some rare increases in transaminases above five times the upper limit of normal or five to eight times, and particularly if it includes an abnormal bilirubin, there are recommendations to interrupt therapy. I think that's what we know at this particular point in time. I think one of the key issues is if we could identify individuals at risk for advanced liver disease, could these therapies prevent progression, whether they act on the liver or they act on the gut, and improve gut integrity and reduce gut-liver axis injury? I think that's an open question to be answered. And then there have been a couple of questions about ERSO, what those, how long, and Dan, you want to? Yeah, thanks a lot, Mike. No controversy there at all. I think the lessons that we've learned about ERSO-Diol, at least in patients with CF, is that at least dosed at no more than 20 or 25 mg per kilo per day, that it seems safe. It certainly does not appear to be harmful. There's not been any toxic bile acid accumulation in patients who have been on long-term ERSO-Diol with CF, but obviously, taking a lesson out of the PSC handbook, we're really not comfortable with starting anything higher than 30 mg per kilo. But it does appear to be safe. I think the real question is, what are we treating? Do these children have a history of TPN-related cholestasis or liver disease? Do they have true cholestasis? Have they been a patient with a history of meconium ileus where they may benefit from a finite duration of ERSO-Diol? But starting ERSO-Diol and leaving it on for years and years for a GGT of 35 or 40 or 51 with transaminases in the 50s or 60s, I'm not sure that there's any significant benefit there. And at least the Cochrane reviews have demonstrated that, unfortunately, with the data out there in peer review, there's nothing that has shown a change in clinical outcome just because you've been prescribed ERSO-Diol for three or more years. Yeah. I would just add two things, one on either end of this comment about ERSO. There was just a large study from the French group that looked at ERSO and their prevalence of advanced liver disease. And their conclusion from that study, and it was one of the authors was Dominique Debray, it was that there was not a change in the prevalence of advanced liver disease with aggressive early use of ERSO. And their prevalence has basically stayed the same and similar to what was presented. There is one paper I'm aware of that has demonstrated some improvement in transient elastography with ERSO use. But again, there's a lot, as I pointed out, there's a fair amount of variability in that testing, particularly in narrow windows. So Dan, here's one, I'm sorry, Jay, here's one for you. What's your opinion about preemptive liver transplant in a stable cirrhotic patient with worsening lung function? I guess the short answer would be probably not in this case. So I think for a number of reasons, I briefly mentioned the one paper that showed that there was a modest improvement in lung function after transplant that really disappeared after about two to three years. And so there doesn't seem to be any real long-term benefit to the lungs with a preemptive essentially liver transplant. Additionally, for the data that Dan showed earlier, how you define cirrhotic, whether it's just imaging or otherwise, it may remain stable for years and years and is likely not causing the overall decline in lung function. If you feel like you have advanced portal hypertension and there may be splenomegaly contributing as we talked about in some of my earlier slides, then it may be reasonable to address the spleen and not necessarily go through an entire liver transplant for this indication at least. Good. And then here's a question from one of the transplant surgeons. Would you use EOVS to distinguish FNH? I think it was a focal nodular hyperplasia that was the attempt of nodular versus cirrhosis. I think the answer is this is really nodular regenerative and not focal nodular, so there is not imaging evidence to distinguish this. The recommendations have really been, are evolving and I think in an individual with advanced liver disease and nodularity, this may be now a new, we should go back and biopsy those individuals to look for cirrhosis. And the Europeans recommend transjugular venous, hepatic venous pressure gradients to try and distinguish these individuals. Elastography may individual, may separate them out a little bit. Most individuals with nodular regenerative hyperplasia do not have KPAs by vibration controlled transient elastography above 12 or 13. Many of us who see advanced CF liver disease see values in the 20s, 30s, and even 40s on our advanced liver disease patients. So I think we'll go last question. Dan, I'm going to throw it to you. Any evidence for supporting the use of cyclic antibiotics for small bowel, small intestine bacterial overgrowth in CF? Related to CF liver disease, I'm assuming. And does it help CF liver disease? Yeah. I'm not aware of any well-powered studies that suggest antibiotics modify the risk of liver disease. I agree. I've never seen anything with that. I think some of the studies are provocative about what could that role be and the data from the CFLD network with early pseudomonas being protected for liver disease. Does that mean that early antibiotic therapy might change the microbiome? I think it's all open and no one knows the answer. And I guess there was one other question I thought was provocative, which was, is there any evidence for the use of anticoagulants to prevent liver disease if we think it's a vasculopathy? I'm not aware of any. Dan or Jay, are you aware of any evidence? I'm not aware of any. No, I don't think there is, but it's a very interesting point. You know, whether it's heparinization, whether or not it's aspirin, I think there are people doing investigative work from a metabolomic and proteomic standpoint that may sort of shed some light on this, but right now it's too soon to say. Great. Well, with that, I would like to thank the panelists, Dan and Jay, for their efforts in this webinar. I'd like to thank Dominique for helping organize all of this. Please do complete your evaluations. That would help the AASLD to look forward and provide the education that you're looking forward to. And again, if you have specific questions, all of the panelists are more than willing to have individual conversations if you would like.

advanced liver disease

cystic fibrosis

CFTR dysfunction

fibrosis

cirrhosis

microbiome

biomarkers

treatment options

liver transplantation

CFTR modulator therapies

diagnosis