Hello, everyone. Welcome to the ASLD webinar titled, Biliary Atresia, Where We Are and Where Do We Go From Here? I'm Krupa Mysore, I'm a pediatric hepatologist and transplant physician at Texas Children's Hospital, Baylor College of Medicine, Houston, Texas. We are really excited to have you here. This webinar was developed by the educational subcommittee of the Pediatric Liver Disorder Special Interest Group of ASLD. We have three outstanding speakers for this webinar. And before I introduce our speakers today, a quick overview of the format of this webinar for all the attendees. Each talk will be for 15 minutes, one after the other in succession. And at the end, we'll have a question and answer session and a panel discussion. Please use the Q&A box to type your questions. And if you can also type in the speaker that your question is addressed to, that would be fantastic. Now, before I introduce, there's also a chat box for general comments and any other queries about the webinar. So the first presenter that I want to introduce is Dr. Kathy Looms. Her presentation is on genomics and biliary atresia. Dr. Looms is a professor of pediatrics and is director of the Pediatric Liver Center at the Children's Hospital of Philadelphia. Right after Dr. Looms, the second presenter for the webinar today is Dr. Sanjeev Harpawat. He will present novel prognosticators for biliary atresia. Dr. Harpawat is associate professor of pediatrics at Texas Children's Hospital, Baylor College of Medicine in Houston, Texas. And our third presenter and our final presentation will be by Dr. Saul Carpin. He will present long-term outcomes of biliary atresia after a COSI operation. Dr. Carpin is professor of pediatrics and division chief of pediatric gastroenterology, pathology and nutrition at Emory University School of Medicine in Atlanta. So without further ado, I would invite Dr. Kathy Looms to give our first presentation on genomics and biliary atresia. Thank you so much, Dr. Mysore. Thank you to the organizers of Pediatric SIG and to the ASLB for sponsoring this webinar on biliary atresia, such an important topic. So thank you very much. Let's see if I can get these slides to move. There we go. So I'll be leading off the talks this morning with an overview of what we know about genomics and biliary atresia. These are my disclosures. So genomics and biliary atresia is a really fascinating topic. And you might wonder since biliary atresia is not really a genetic disease, what there is to know about this. It's not a Mendelian disorder, but we think that the genetics of biliary atresia is complex in that there may be a genetic susceptibility in some individuals. So one model of pathogenesis is that there could be an environmental insult that might come from a toxin or an infection. And then both a developmental susceptibility, so this injury can only occur at a certain time point in early infancy, and then also a genetic susceptibility in certain individuals that allows progression of this injury and a progression of inflammation, fibrosis, and immune response. This is a complicated problem to sort out. And since it's likely multifactorial, it makes the analysis challenging. And some of the genetic susceptibility variants that we may identify can also be inherited from the parents because the parents may not have had the same exposure that the affected infant would have. So this is an overview of some of what I'll talk about today. I'll go through a few genome-wide association studies that have been done to identify genetic susceptibility variants. Some studies on copy number variants, some work on whole exome sequencing, and a little bit about how animal and in vitro models can help us understand what's going on. And then just a word about genetics and clinical outcomes. There are some other models that with evidence for pathogenesis and deliria atresia, such as epigenetics, micro RNA, and somatic mutations, as well as studies of the transcriptome, but we won't have time to discuss those today. So leading off with the genome-wide association studies. So basically a genome-wide association study involves genotyping lots, oh, sorry. Oh, sorry. Lots of different single nucleotide variants across the genome, and then comparing the prevalence of those variants in the disease population, which is BA, versus an ethnically matched control population. So this is a study that was done in 2010, looking at a Chinese cohort of 200 deliria atresia patients. And they found a SNP, or a single nucleotide polymorphism on chromosome 10, that had genome-wide significance with a P value of 6.94 times 10 to the minus nine. And this shows some of the SNPs that are really coming up above the others as being important and significant right here on chromosome 10. This particular SNP that they identified was downstream of two genes, XPN-PEP1, which is involved in the metabolism of inflammatory mediators, and also ADD3, which encodes the protein aducin-3. This particular locus on chromosome 10 has been independently replicated in two different populations. So one in the Caucasian population, in a paper by Sai et al, and also again in the Thai population, in a paper in 2018. There have also been some additional studies trying to sort out whether one of these two genes is important in the BA susceptibility identified at this locus on chromosome 10. And it's been found that the risk allele is associated with reduced expression of the ADD3 gene in the liver. And this was reported in 2013 by Chang et al. And this is an agreement with some zebrafish data that was published by Vivian Tang in 2016. And this figure here on the right shows some staining of the zebrafish liver and the bile ducts in a control on the top. And then in knocking down ADD3A with a morpholino in the middle box here, you can see that there's reduced branching of bile ducts. And then in XPN-PEP1, knocking this gene down, the bile duct pattern appears normal. So this points more towards ADD3 as being a possible candidate gene. Another genome-wide association study was published a few years ago. And this identified a locus on chromosome 2P16.1. And the gene identified here is EF-EMP1. So this was a study done by our group with Nancy Spinner, Marcello Devoto. And this was with children in collaboration with the Children Network, the Childhood Liver Disease Research Network funded by the NIDDK. And I'll be mentioning a few studies that have come from the Children Network, which is a multi-center consortium enrolling many children with biliary atresia. And this really shows the power of a network for studying a rare disease. But this discovery cohort was 450 patients with isolated biliary atresia. And then we had a control cohort of almost 2000 patients with a rare eye disease. These DNA samples were genotyped on the Illumina Omni 2.5 platform, genotyping about 2.5 million SNPs. And the most significant signal was found in an intron of EF-EMP1. And this gene encodes an EGF containing fibulin-like extracellular matrix protein. And here you can see the plot showing the peak SNP here on chromosome two. The allele frequency for this SNP in the biliary atresia population was about 43%, as opposed to 32% in the controls. So looking a little bit further at EF-EMP1 structure and function, EF-EMP1 encodes fibulin-3, which is part of a family of extracellular matrix proteins. We found that this gene was upregulated in both biliary atresia, as well as disease control liver, as compared to non-disease controls. And then in looking at where this gene is expressed within the liver or the protein, the EF-EMP1 is stained in red. And here on the left, it's co-localizing with smooth muscle actin. So both in control and biliary atresia liver, EF-EMP1 is expressed in blood vessels. And then if we look at bile ducts and co-expression with CK19, in controls, EF-EMP1 is not expressed in bile ducts, but in biliary atresia, there is a significant co-expression. So this is an interesting difference in expression pattern. There are a couple of other genome-wide association studies that have been done by Rakesh Sindhi's group in Pittsburgh. And this is one identifying ARF6 as a candidate, biliary atresia susceptibility gene. This study enrolled 63 biliary atresia cases and almost 2000 controls. And they found a significant locus on chromosome 14 in an enhancer region for the gene ARF6. ARF6 is important for EGF receptor signaling and also regulates actin cytoskeletal modeling. They did further studies on this gene in the zebrafish. And this upper panel here shows a PED6 assay. So this is almost like a zebrafish to cytoscan in a normal biliary tree. This fluorescent tracer is taken up by the gallbladder. You can see it here with the arrow and the control. And then that is absent in the ARF6 morpholino knockdown. And over here is a graph showing multiple animals counted. And there's definitely absence of the gallbladder in some of these zebrafish that had knockdown of ARF6, as well as an abnormal branching pattern of the bile ducts. So this seems to be a consistent theme with some of these candidate genes. Also from Dr. Cindy's group, a more recent publication has identified MAN1A2 as a possible candidate gene. In this study cohort, there were 137 biliary atresia cases who had all undergone transplant compared with about 2,500 controls. And they found the top two ranked SNPs were in an intron of MAN1A2. This is a gene that's important for ciliogenesis and laterality determination. They found reduced expression of this protein in biliary atresia liver compared with control. And when they looked at it in zebrafish and knocked it down in the zebrafish, laterality was normal in the control animals and randomized in those that were knocked down for MAN1A2. And then again, the PED6 assay showed absence of the gallbladder in many of the animals with knocked down MAN1A2. So moving on to some copy number variants that have been identified in biliary atresia. This study came out in 2013 and identified GPC1 as a candidate biliary atresia susceptibility gene. So this also in this study SNP genotyping was the way that the copy number variants were detected. 61 BA patients were enrolled in this study and there were heterozygous deletions on chromosome two found in six patients. So almost 10% of this patient cohort compared with 0.08% in the controls and with a P value of 4.4 times 10 to the minus 10. This deleted region on chromosome two included one gene, GPC1, which includes Glipican1 and it is a heparin sulfate proteoglycan regulating hedgehog signaling. So again, could be important for biliary development. This was studied in control and biliary atresia liver and in control liver, the protein seems to be localized to the apical surface of the cholangiocyte whereas it's more diffusely localized in biliary atresia liver. And again, in the zebrafish, we have reduced uptake in the gallbladder and the GPC1 morphins and reduced branching of the bile ducts within the liver. One other small study looking at a copy number variant in a single family. This is a affected father and a daughter and this family had a de novo deletion of FOXA2 which is a forkhead box transcription factor important for endoderm development. The father is affected with situs inversus and polysplenia but does not have biliary atresia and the daughter has biliary atresia and heterotaxy. And interestingly, the daughter also inherited a polymorphism of nodal from her mother which we hypothesize may be important as well in terms of laterality determination and potentially the biliary atresia phenotype in this family. So moving on to some exome sequencing studies that have come out more recently and have also been informative in terms of genetics of biliary atresia. Throughout the last 15 years or so, there have been a number of rare gene variants that have been associated with biliary atresia. There was a study from 2002 identifying some of these genes and some variants in JAG1 which of course is the disease gene for allergial syndrome in biliary atresia cases. These I think are of uncertain significance. These variants that were found in JAG1 are not those seen in allergial syndrome and their functional consequences are not 100% sorted out at this point. In addition, there have been some other variants identified in biliary atresia patients with laterality defects. There were some variants found in CFC1 as well as ZIK3. And then more recently there was a report of some variants in some of the known other disease genes for cholestasis found in biliary atresia patients. And these I think are of uncertain significance as well. We may find out more about these types of variants as we continue to do more genetic testing in our patients. And some of them may actually turn out to be genetic modifiers of disease severity. But I think that story is still unfinished. This is an important study that came out recently in hepatology. This exome sequencing study was led by Saul Karpen for the Children Network and involved whole exome sequencing on 67 patients with biliary atresia splenic malformation syndrome. 58 of those patients were trios. So there were also samples available for the parents and all of these patients were involved, enrolled in the children's studies from multiple centers. The exome sequencing was performed on all of these patients' DNA samples. And then there was a list of just over 2000 candidate gene variants that were involved with ciliary function and also cholestasis. So the analysis involved looking at these variants and identifying deleterious variants in the patients that were in this list of candidate genes. And the one, the best candidate that rose to the top here was PKD101. And this gene is involved in ciliary calcium signaling and laterality. There were bioallelic deleterious variants found in five individuals in this cohort and heterozygous variants in three individuals. And this diagram shows the gene and all of the exons and the variants were pretty much spread across the gene. And then this was also, some staining was done in human liver samples and there was reduced expression of PKD101 in biliary atresia liver. And I know that there are further studies ongoing now in animal models to try and understand the function of PKD101. This is a report of another study that was done with exome sequencing also through the Children Network. And exome sequencing was performed on 101 Caucasian non-Hispanic patients with isolated biliary atresia. So not the splenic malformation patients and three experimental models were considered to identify rare and deleterious variants in this population. The cases were looked at on their own as well as in comparison with non-disease controls. And then there was a separate analysis done on 30 trios to look for either variants that appeared de novo or recessive variants that could have been inherited from both parents. In this analysis, there was no gene or pathway that was overrepresented in the cases as compared with the controls. And in the trio analysis, there were 66 de novo variants in 66 different genes. So no gene that came up more than once in more than one patient. Two of the variants that we thought were especially interesting were those in STIP1 and REV1, which are involved in stress response. And we do have some evidence that these variants may increase susceptibility to injury of the bile ducts by the plant toxin biliatrizone in the zebrafish model. So a few words about animal and in vitro models in studying the genomics of biliary atresia. There are a number of different mouse models that have been reported with abnormal biliary development. One of the earliest ones was INV, which in this model, there are laterality defects as well as abnormal bile ducts, extra hepatic biliary tree in these mice. The other important ones are HNF6 as well as SOX17, which has gallbladder abnormalities as well as abnormalities of the extra hepatic bile ducts. So in some ways, these genetic mouse models can help give us clues about genes that may be important for human biliary atresia. And then in some ways, it goes the other way. So if we find a candidate in patients with biliary atresia, a candidate gene, we can then study it in both zebrafish or mouse or even cell culture models to try and understand its function. The RRV mouse model, which is the rhesus rotavirus model that has been very useful for studies of biliary atresia. There have also been some genetic studies done in this model where people have knocked out key inflammatory mediators in order to show which ones are important for pathogenesis of the disease. And some of these have been found to be quite important, such as interferon gamma. Some in vitro models are now becoming quite important and well-characterized in understanding BA pathogenesis. For example, biliary organoids can be isolated from human liver biopsies. And there's a really nice recent paper from the Bizero Lab here from Hepatology 2021. And this is looking at biliary organoids that were developed from normal controls versus biliary atresia versus disease control. And this is just an example of abnormal staining of aquaporin 1 here in the biliary atresia organoids. And there are multiple examples in this really nice paper that shows some delayed epithelial development as well as barrier function abnormalities in the biliary atresia organoids. Becky Wells Lab has developed a bile duct on a chip model that's also really nice and useful for studies of this disease. The bile ducts on the chip are seeded with biliary cholangiocytes and have really nice apical and basal polarity. And just one word on alpha-1 antitrypsin heterozygosity. Has been found to be more common in patients with chronic liver disease and specifically in BA patients, presence of a non-amylyl can be associated with earlier transplant listing and lower survival with native liver at 24 months. So to conclude, although BA is not a Mendelian disorder, we have multiple studies showing us candidate genetic susceptibility genes that we've been able to understand better through animal and in vitro studies. And we hope that future studies are going to reveal novel therapeutic targets for us and infants with BA. And we know there are some upcoming studies through the Children Network and elsewhere in large cohorts of patients that we hope to identify genetic contributors to both susceptibility and outcome. Thank you. So good morning, good afternoon, good evening to everyone. And I thank the AAA, so the organizers, Dominique Clayton and the Pediatric SIG for inviting me to review this very interesting topic, novel prognosticators for biliary atresia. Here, I have the following disclosures. I'll start with the case that could happen at any day at any of the pediatric hepatology centers anywhere around the world. So an infant presents with high-conjugated bilirubin and follows the common course. Concerning ultrasound, liver biopsy suggesting obstruction, abnormal cholangiogram, and then the caesarean portoenterostomy. This highlights two things. First, as a field, we've done a really good job characterizing the early natural history of biliary atresia to the point that very little surprises us. However, for individual patients, what happens after the caesarean still remains largely unpredictable. Will the patient's caesarean drain bile? Will the patient develop portal hypertension? And perhaps the most important question for physicians and parents alike, will the infant need a liver transplant? So Dr. Mysore, our moderator, recently searched the UNOS database and discovered these alarming numbers. BA is the number one reason for pediatric liver transplant by a long shot. Surprisingly, BA is also the leading indication for any solid organ transplant in pediatrics. So the question of whether or not a patient with BA will eventually need liver transplant is a critical one. I've boxed the early period on this slide because this is what I will discuss in this talk and Dr. Saul Karpen will talk about later periods in the next talk. I have the following objectives. A, to review predictive biomarkers that we can measure before caesarean to identify patients most likely to benefit from the operation. And B, discuss prognostic markers that we can measure shortly after caesarean to identify patients who will have the best outcomes. For this talk, there will be three themes. First, for the sake of clarity, I will discuss these markers in the context of only one outcome, which is transplant-free survival at two years. Second, I will discuss work from investigators from around the world, really highlighting the true global effort to define these biomarkers. Most of what I will share may be familiar to many in the audience, but I hope the discussion identifies opportunities and stimulates ideas for further research. And third, I will focus mainly on laboratory and imaging markers and leave discussions of genetic markers and liver biopsy markers for a later time when more data is available. To start now, what are the predictive markers for bilarial atresia? By definition, a predictive marker is a marker which identifies individuals more likely to benefit from treatment. So in terms of bilarial atresia, we can use these predictive markers to determine which patients who are about to undergo the CASAI operation will have the best or worst outcomes. We can use these predictive markers to counsel families, but on a more controversial note, we can also use these markers to possibly skip the CASAI altogether in some patients and move straight to liver transplant. So the Kaplan-Meier curve shown is from California study of 626 patients with BA who either had the CASAI operation, which is denoted as BED or biliary enteric diversion, or had a primary liver transplant. The curve suggests that overall survival may actually be higher in patients skipping the CASAI and moving straight to liver transplant. The most important predictive marker we know about is age at CASAI operation. Morioh Kasai from Japan developed the procedure shown on the left, and he hypothesized that performing the procedure earlier would have better outcomes. This is because he carefully described the bile ducts at the hilum as seen in his drawings and showed that the ducts were small, thread-like and formed amorphous channels at the hilum. He thought connecting the intestines to these minuscule structures had to happen early before the ducts and channels closed off. Interestingly, U.S. surgeons were arguing the opposite and waiting for infants to grow and the ducts to enlarge before performing the portoenterostomy. CASAI, of course, has been proven right in many retrospective studies around the world, France, Canada, Switzerland, Brazil, the U.S., Taiwan, Japan, Netherlands, and others. This is a seminal French study of 695 infants showing that infants receiving the CASAI before 30 days of life had the best transplant-free survival and those receiving the CASAI after 90 days had the worst outcomes. And the table on the right shows the numbers for two-year transplant-free survival. Again, the message is earlier is better. The exciting thing about age is it is a predictive marker that with careful coordination and attention, we can modify when infants receive the CASAI. Now, saying that, it's important to keep in mind there are some exceptions. For example, in a U.S. study of 55 infants shown on the left, a CASAI before 30 days of life actually led to worse outcomes. And in a U.K. study of 34 infants shown on the right, a CASAI after 100 days still led to decent outcomes. Other than age, the field is still looking for reliable, robust predictive markers. For example, a study from Australia of 113 infants suggested that GGT levels below 200 before the CASAI may predict lower transplant-free survival, as shown in the line in blue on this curve. Another potential source of predictive markers comes from imaging. To date, diagnostic findings on ultrasounds that are consistent with the BA, such as an abnormal gallbladder, the triangular cord sign, increased subcapsular flow, or enlarged tidal lymph nodes, have not been shown to be predictive. One imaging finding, liver stiffness, in theory could be predictive. This is because stiffness before CASAI correlates with age. This is a figure from a key study of 15 patients from Taiwan, and liver stiffness was found to increase steadily with age before CASAI. However, unfortunately, liver stiffness before CASAI does not seem to predict outcomes after the CASAI. In this table, I highlighted liver stiffness measurements before surgery, and as seen on the far right, liver stiffness before surgery does not associate with the risk of liver transplant at one and a half years of life. So moving on, in addition to predictive markers, the next question is, what are the prognostic markers in biliary atresia? And a prognostic marker is defined as a marker which identifies the likelihood of disease progression or a clinical event. In terms of biliary atresia, we can use a prognostic marker to identify which patients will do well or poorly after the CASAI, and we can use these markers to counsel families, anticipate complications, and very importantly, serve as endpoints in clinical trials. The most established and accepted prognostic marker is normalization of total bilirubin, which essentially marks good bioflow after the CASAI. This is nicely shown by two studies from the Children Consortium, which Dr. Looms mentioned before. The left is 104 patients, the right with 137 patients. The Kaplan-Meier curves clearly show that a total bilirubin less than two after CASAI associates with transplant-free survival, whereas higher total bilirubin levels associate with need for transplant. These curves are very convincing, and the question arises, are we done? Do we even need more prognostic markers? Now, I would argue the answer to that question is yes. We do need more prognostic markers because of data that came from these same studies. Despite achieving normal total bilirubin levels after the CASAI, which is the blue line, patients still developed ascites at a rate of 18%, malnutrition measured by mid-upper arm circumference at 35%, splenomegaly at 71%, and transplant death at 18%. So at a minimum, we still need to find markers for those patients who normalized their bilirubin levels after CASAI and may or may not have good outcomes afterwards. To address this problem, investigators in Australia and Canada studied 217 patients and combined total bilirubin and albumin levels three months after CASAI. As shown in the Kaplan-Meier curves, this may be one way to further separate patients into prognostic groups after CASAI. Other investigators in Finland stretched the definition of normal total bilirubin levels and looked at very low total bilirubin levels achieved three or six months after CASAI. This really interesting analysis of 41 patients showed that achieving total bilirubin levels very low, below 0.7, 0.4, or even 0.1, correlated with the best chances of transplant-free survival. In addition to total bilirubin levels, there are other serum markers which could be prognostic. One study of 139 patients from the Children Consortium looked at serum bilacids six months after CASAI in patients who achieved normal bilirubin levels. Bilacids less than or equal to 40 micromoles per liter versus greater than 40 micromoles per liter, significantly associated with transplant-free survival over time. As a small tribute, this study was led by Jim Hybee from Cincinnati Children's, who passed away recently. And I and many others are truly grateful for Dr. Hybee's enormous contributions to our knowledge of BA specifically and to the entire field of pediatric, GI and hepatology generally. Another BA marker, MMP7, has recently gained more attention in numerous studies from around the world. MMP7 is thought to increase when the liver remodels, perhaps during the events leading to fibrosis in BA. In a study from Taiwan of 32 patients, as shown in this figure, MMP7 levels, which were measured six months after CASAI, less than or equal to 10.3 nanograms per milliliter, associated with the best long-term transplant-free survival. And from the same group, liver stiffness appears to be a powerful prognostic marker. This table was shown before in a previous slide, but now I've highlighted the time points after CASAI. As seen on the right, liver stiffness at different time points after CASAI did correlate with the need for, or did associate with the need for transplant at age one and a half years. These results seem to suggest the faster liver stiffness decreases after CASAI, the better the long-term outcomes. Interestingly, liver fibrosis would not be expected to resolve as fast as one or two weeks after CASAI. So an intriguing possibility is that liver stiffness at these time points may represent changes in the liver other than fibrosis. In summary, we have discussed two types of markers in BA that can help us anticipate need for liver transplant by age two years. Predictive markers include age at CASAI and GGT levels before CASAI. Future markers may be genetic changes or liver biopsy findings. And ultimately, we can use these markers to decide which patients may not benefit from the CASAI and instead direct them possibly straight to liver transplant. The prognostic markers include total bilirubin after CASAI with other markers such as serum bilacids, MMP7, and liver stiffness as promising future candidates. I wanna emphasize the importance of developing these prognostic markers. With these prognostic markers in hand, we can change the current paradigm of relying on liver transplant as the primary endpoint in BA therapeutic trials. Trials with liver transplant as an endpoint are expensive and take many years to complete. With robust early prognostic markers, we can use the markers as endpoints and rapidly and efficiently test different therapies with the ultimate goal of quickly finding ways to reduce BA's tremendous transplant burden. Thank you, and I'd be happy to take questions at the end. So thank you so much to the SIG. Thank you, Dr. Mysore. And thanks to Kathy and to Sunny for setting this up. I'm gonna talk about long-term outcomes. And as Sunny has mentioned, he really did a beautiful job talking about ages zero to two. I'm gonna focus primarily from ages two onward. So I wanna mention, I have these disclosures and I've asked there is two companies, Alvareo and Merum, because I'm going to be discussing two studies that are sponsored by these companies for which I'm a consultant. So three topics, outcomes during childhood, adult outcomes. And I also think for this particular audience, adjuvant therapeutic landscape is really quite important in these day and age, as Sunny has pointed out. Definitions, as we mentioned, short-term up to age two and long-term is really 10 and above. Remember, we hand off these patients at age 16, 18 or 21, depending on where you are in the world. People need to know about it. So outcomes during childhood. I wanna give you a single case very quickly. Two-month-old BA, beautiful draining cassi. By age six, however, platelet counts 34,000, slightly high GGT. By age eight, you'll see an MR sign here with a massive humongous spleen. Yet this is a success, right? Look at that small liver, look at that gigantic portal vein. By the next year, platelet count drops to 13,000, high GGT. But guess what? The bilis, the conjugated bilis is zero. So on some measure, you would think this is fine, but obviously on other measures, it's not. Things happen to this child that are not important. And so by the next year, he was transplanted. You can see this massive degree of peribiliary fibrosis on the lower right histology. This is the nature of this disease. If we really try to reformat our thinking over the last 10 years, and some of it that both Kathy and Sunny alluded to is that there really is a developmental component to this. It's just a cholangiopathy, but it's one that is at a time of childhood and a time of life which develops this tremendous degree of fibrosis. I would argue it's the most fibrotic liver disease. So what do we know about outcomes? Well, this is an extension of one of the French studies that Sunny alluded to with 1,300 biliary atresia patients going all the way back to 1986. It's fantastic. And the curves I'm gonna be showing you are just quite straightforward, survival with native liver curves. And the easy numbers, I think, are shown here in the blue boxes. By age two, it's roughly 50-50. By age five, you lost another 10%. You lose another 5% every five years thereafter. So by the time you're at age 20, you're at about 25%. So this really does perfectly highlight, to me at least, the ongoing risk in all SNL patients. Just because you have a successful caci and you drain doesn't mean you avoid all those peri-biliary fibrotic issues. And in fact, you generally do not. And I'll lay this out in the next couple of slides. It's easier to kind of think about this perhaps, and Sunny and I worked on this to make age two the discriminating year, that it really almost looks like two different curves, doesn't it? Your survival native liver really plummets. And then all of a sudden, it just seems to level off. But it doesn't really level off. It continues to go down in both this curve and the previous one. So we think about what are the things that lead to the decision for early transplant? And then what are the things that are really impacting this child's life of SNL from ages two onward? And this is nicely outlined with the paper from Georgie Bezerra and others, where they go over the outcomes from birth with liver transplant, survival with native liver, or death. And I know we have not talked about death yet, but I think people do need to talk about it. And in fact, there is a death rate. And in fact, in those on the right-hand side of this flow chart with a Billy Rubin, greater than two and about three months of age, there's about a 10% death rate. So it's not just, are you gonna get a transplant that's gonna be fine? This is an ongoing battle for those of us in large centers, for disease and for actual death. So by the age of two, which is where I put the arrow here, although there's a nice discriminator of a bilirubin of two at three months post-CASI, the reality is it's not binary because the SNL that you have is not 100% if your bilirubin is less than two, it's 80%. And your SNL, if it's more than two, is not zero. It's actually around 20%. So there is some grayness here and there's an absolute need for more prognostic indicators. And these numbers drop as the years go by. So instead of having an SNL of 80% at two years, it's 40% at 10. And throughout this talk, I'm not gonna talk about the survival post-transplant. So what can we know from age two onward? And I wanna focus on a paper that we and some of my colleagues in Toronto and other centers put together, where we just had a simple question of those with SNL at age two, what happens to them? And are there ways to distinguish who's doing well or who are in those category of kids who are ultimately going to need a transplant? We did a very simple study from ages two to age six, which is look at their GGT levels at age two and look at their platelet count as a pretty decent surrogate for the development of portal hypertension. And in fact, if your GGT was less than 100 at age two, which is on the left-hand side of these bar graphs, the platelet count is over 200 and pretty much stays that way. Why GGT? Well, if you think about it, it's a marker for ongoing biliary irritation damage. So those which had higher GGT, those with higher evidence of cholangiopathy had a lower platelet count at age two. In fact, it was close to 150 and that dropped even on this short-term study over age six. So the point here being GGT is at least a decent harbinger of ongoing cholangiopathy in these children. And in fact, if it's high at age two or so, it's likely that there could be progression of portal hypertension. Well, a better study was done with the Children Network, and this was run by Vena Venkat and the group, where instead of just looking at a couple centers, did all the centers, and this was 240 kids who had their liver at age two and asked with the children database, what happened to these children? And were there some biochemical or clinical markers that could predict their outcome over the next 10 or so years? So these 240 kids were followed for a median only of about three years, but there was a decent number that was followed, obviously up to five to 10 years. And what the statisticians in the group did was put these into different quartiles of whether or not you will survive with your native liver. And what I found interesting with this is that as a group shown here on the left, the cumulative incidence from the age of two onward that you don't have survival without transplant is roughly by the time you're 10 years of age, another 20 or 25%. And what was it about this group that maybe distinguishes those that did have their liver and didn't have problems from those that did not? And in fact, it was these different risk groups. It was just bilirubin, whether or not you had ascites or cholangitis, your albumin level, and your platelet count. But in addition to this transplant, what about sentinel events? GI bleeding, ascites, hepato-pulmonary syndrome? Excuse me. Cumulative incidence here, again, by the age 10 is about 20% of all of these. And for these sentinel events, your platelet count, again, this is at age two only, and GGT together were quite predictive of putting you into the category of whether or not you were gonna keep your liver and not have a sentinel event or have one, as in this lower brown bar here. So platelets and GGT, again, in this particular analysis seem to be helpful. So what about adult outcomes? So a couple studies, and I'll go through these. This, again, this fantastic database from the French group. This is an earlier study than the one I presented, where they looked back to 1968 to 83, because they needed to have patients over the age of 20. And they followed 63 of them. Well, here's their SNL curves on the left. And what I highlighted here was roughly from the age of two with this horizontal red arrow. And I highlight here with the solid red arrow from age 20 onward, where they started with 63 patients. You still see the SNL is not horizontal. It still continues to drop. And what happened to these patients? Well, I'll outline some of the key points here. Nearly everybody's cirrhotic by the time of age 20. Two died of acute liver failure as young adults. 10 of these underwent transplant in their 20s. Two died post-transplant. We're handing off a number of these patients with chronic liver disease. And please recognize that. An Italian study showed roughly the same thing. Here, slightly less SNL rate by age 20 of 18%. They also had an 18% mortality here. And that there was really just a small cohort of survivors with native liver who displayed no clinical manifestations of chronic liver disease. So I can't emphasize this point enough. We can't tell our families that everything's gonna be okay just because we don't have the data. We can't tell our patients that everything's gonna be okay just because the CASI works. We can tell them it's gonna be okay early on, but we absolutely need to continue to follow these patients closely. As they get older, there are concerns and reports of decompensations with pregnancy, reports of hepatocellular carcinoma. And we don't know what happened to this 20% with so-called normal liver function for 25 years. We don't even know, quite frankly, did they really have BA? So this is one of those questions, but something that I think plays wise with following these patients into adulthood. The last series I wanna talk about is a compilation from a number of centers in Europe and in Canada where they compiled five series with a median age of 24 and almost 200 patients after the age of 16. As you can see from the list here, three quarters with cirrhosis, almost 70% with portal hypertension, half with varices, a quarter with variceal bleeds and pruritus, as well as episodes of bacterial cholangitis. But we do not have a mechanism as to how best to follow these patients with EGDs, yes or no, different treatments. So we really are handing off these patients and we're not necessarily handing off a treatment plan. So to summarize this, there are significant complications that arise throughout adulthood and perhaps early interventions or some mechanism to intervene to slow progression would be beneficial for a lifetime. And the last bit I'm gonna talk with is adjuvant therapeutic landscape. In 2021, there are a number of anticholestatic targets and therapies that are being studied. And I'm showing this on the left-hand side of the slide. There are bile acid-based therapeutics, such as FXR activators or IBAD inhibitors for which I'm gonna be spending some time talking about here. There's also people looking at NTCP, the bile acid importer, various bile acids, including colic acid, erso, nor-erso and FGF19 analogs. In addition, on the lower left, non-bile acid-based therapeutics. If fibrosis is the problem, why not antifibrotics? If there is a specific gene involved, why not specific gene correctors? But also listed here are these ideas that perhaps there are parts of the inflammatory pathways that could be addressed rationally in some of these older patients. So what about BA after Casai? So what do we use? We use steroids in many parts of the world. We use antibiotics. And we have some CMV and also some other ones. And so in the interest of time, I think I'm gonna jump right to the two things, one about steroids and then IBAD inhibitors so we have enough time as we wrap up this particular webinar, okay? So first and foremost, this will take five seconds. Kaplan-Meier curve, placebo-controlled study, steroids versus placebo, no difference, no benefit. We do not see which patients with BA could possibly benefit. So what about IBAD inhibitors? Well, IBAD inhibitors are designed to block the uptake of bile acids from the distal intestine. And the concept here is that you take a cholestatic liver, stop filling it with more bile acids, and hopefully it will continue to drain bile acids. There are two studies now ongoing worldwide, and I wanna mention these. One with Odavixivat, the other with Maralixivat. One is a phase three study. They're both double-blind randomized control trials, but the outcomes that they're looking at are quite different. One is a clinical outcome of SNL at age two, the other one is a biochemical outcome. But I highly encourage everybody to seek out these studies because the more we do and know about these patients and with BA, the more we're gonna intervene and perhaps make a difference. So to summarize, early outcomes around 50% SNL, I'm gonna skip over the first part in the interest of time, and I'm sorry about that, but you can have these slides afterwards, I'm happy to share. Outcomes during childhood to age 18, about a 25% survival with native liver rate. There are absolutely ongoing concerns for cholangitis and other ramifications of liver disease, including cancer. Perhaps we can do some modeling with standard labs like GGT and platelets. As an adult, they're nearly ulceratic. So same ongoing concerns. And right now for current therapeutics, ERSO has never been tested, remember. There are two iBAT inhibitor trials, the concept to reduce intrapartic bile acid accretion, highly recommend enrolling, and future opportunities for NORERSO or perhaps antifibrotic therapies. And Krupa, I'm gonna end there so that we have time for questions. Thank you. Thank you so much, Dr. Karpen, and thank you for all the speakers, Dr. Looms, Dr. Harpovitz, and Dr. Karpen for this fantastic webinar. And I personally learned a lot from this, and we do have one question by Dr. Nortovitz. I think I will let Dr. Harpovitz answer this first. The question is, most of the work on prognostication has focused on who will develop long-term complications. What about the reverse? Can we identify individuals with BA who have good outcomes, no splenomegaly, no hypersplenosome, or fibrosis? It is rare, but it does occur. It might alter the intensive management follow-up in the small cohort. I will let Dr. Harpovitz answer, and maybe Dr. Karpen to follow on this question as well. Yeah, thanks, Dr. Mysore. It's a great question, and it is probably the right question, not only to lessen the intensive therapies for a subset of children, but also to look at those children and study them intensely and say what went right so that we can apply them to all the other children. So I think it's important. The first question is, how many are there? You saw the last talk. There might be 62 out of 63 patients that fit that bill, or maybe 20% of the adults fit that bill. It's hard to know exactly which. I can tell you from Jim Hybee's studies of the bile acids that I talked about, that might be actually one marker to do it. Serum bile acids less than 40 in kids who clear their bilirubin not only has the amazing transplant-free survival curve that you saw, but it also has great results with development of sentinel events like ascites and GI bleed. Another set of markers that might fit the bill are the group one and group two in the children's study that was presented at the last talk, which combines total bilirubin, cholangitis, ascites, and a few other parameters. So I definitely agree. These patients are important to study. Again, not only, like I said, to prevent too much therapy on them at the beginning, but also to extract the information from that group. And whatever characteristic is about that group will help us understand how to best treat the disease. I can add a quick follow-up if there's time, Krupa. I like this question, but the problem is it's very hard to prove normality. And so you're right. I think that with more ongoing prospective studies that we can say, here's the lab and clinical data at age two, five, and 10. And guess what? If at age two, you had these gorgeous characteristics that just stayed put, then you can follow them and give those parents a greater sense. My pause comes in reading the adult studies because there just seems to be a much greater accretion of liver disease later on. That's it. Thank you both. I have a question for Dr. Looms. Dr. Looms elegantly summarized the various genetic studies. Seems like there are several candidate genes and the SNPs that you alluded to. Do you consider those as more like a disease modifier? Seems like all of these gene defects, probably it's like all roads lead to delirium atresia. Do you think it is a disease modifier or a disease causative defect? For most of the ones that have been identified in the genome-wide association studies, I think of them more as susceptibility genes that perhaps there is a variant in an individual that if two babies had the same exposure to a virus or a toxin of some sort, that a baby who had one of these genetic markers might be more susceptible to actually developing the disease. I don't think we've looked at them yet in terms of genetic modifiers of outcome, but that's something that we wanna do in future studies. Yeah, the current studies have really looked at susceptibility in the VA versus a normal control populations. Thank you so much for that. And we have a couple more questions. And if it's okay, I'll go ahead and address those. And the first one is Dr. Bezira. He says, congratulations on the super presentations. You showed key advances from careful clinical observations. Would one next step be an attempt to prioritize markers of outcomes perhaps at two years of age that can be formally used as endpoints in clinical trials? I think this is for Dr. Karpin. I can start, but I really am curious what Sonny thinks about this. I think, Georgie, like many things, you're spot on. We really need to have deeper dives into the natural history of this disease in the first couple of months. I actually think that we use two years because we don't know what to do at one year. And that as Sonny has pointed out with the data from Finland, where they actually do liver biopsies on these patients, you can probably predict with low risk and you can probably predict with lower bilirubins earlier that you may have better outcomes. The problem is, again, it's the stability of the outcome. And so at least with my hat on long-term outcomes, I really want to help this kid not just get to her own liver at age two, but to be set so that they're not experiencing ongoing peribiliary fibrosis. Yeah, I'll add in to that. And I would say maybe we challenge ourselves even more. Instead of two years, why don't we say six months after CasI? Can we find markers that are even earlier for the practical reason of trials are expensive, they take time, especially looking for transplant-free survival. And unfortunately, not to be pessimistic, but unfortunately we haven't found that magical cure not to be pessimistic, but unfortunately we haven't found that magical therapy yet. So I predict we will be going through a lot of therapies before we find it. So to pull off a two-year or a plus trial where transplant survival is your outcome for multiple therapies, many of which might not work, seems like a tall task. So I would say if we could even go earlier at six months, if these markers truly exist, as the first question might've alluded to, this provides a mechanism for very, very quick testing of therapeutics, almost like in the cancer world, quick testing of therapeutics. And if one doesn't work, we can drop it quickly and move to the next. Can I just follow up on that a little? I think it's important to mention that we don't know how to go shorter. So the problem with going shorter is that, okay, you stop something at six months and then guess what? It really needed to have a longer outcome before we knew if it worked. So we can certainly go longer first and then chip away. And I think that we're so greedy to have things to test, but it's really time. So I think at the moment, because of that almost dogleg SNL curve that we have up to age two, and then we have a different rate after age two, at the moment, we have to use two as our sort of gold standard and then work towards that and say, how does it work? How can we find something better than that? Thank you so much for that wonderful discussion. We have, I think we have like few seconds for one last question. This might be for Dr. Harpavid or any of the panelists. It might be more experience and style question regarding immediate post-COSI antibiotics. The question is for how long and during cholangitis for how long do you continue the antibiotics treatment for? So this is a question I think all panelists will be, have an opinion on and probably everyone in the audience. It's an international question because I can tell you the amount of antibiotics IV or PO after COSI varies across the world. We tend to do a shorter in the US. I know in places in East Asia, they do it for a long period of time and they have the best outcomes. Is there a relationship? I don't know. The bottom line is it's still up in, I think there's no consensus on this. It's in need of studies. When you look at COSI's old monograph, his prodigies, they all developed all these new techniques that connect the intestine to the liver with the hopes of putting in interfering intestines to prevent cholangitis. But in the end, we have what we have because COSI predicted the best way to prevent cholangitis is to just have good bioflow. And then after that, you know, we deal with the antibiotic problem and how long. So the short of it to me is the answer is unknown. Do the other panelists wanna comment on it? Okay, sounds good. Thank you so much. Thanks everybody for attending the webinar and thank you, Dr. Looms, Dr. Harpavid and Dr. Carpenter for those excellent presentations. And thank you ASLD and the Pediatric SIG for this opportunity for all of us. Thank you. Everyone.

Biliary Atresia

Genomics

Genetic factors

Prognostic markers

Casia operation

Transplant-free survival

Long-term outcomes

Complications

Adjuvant therapeutic landscape

Research