NAFLD ENDPOINTS CONFERENCE

Transcription

1 AASLD/EASL NAFLD ENDPOINTS CONFERENCE June 29 30, 2018 The Westin Alexandria Alexandria, VA Program Chairs: Arun J. Sanyal, MBBS, MD, FAASLD Mary E. McCarthy Rinella, MD, FAASLD Quentin M. Anstee, MBBS, PhD, FRCP Frank Tacke, MD, PhD aasld.org/endpoints #Endpoints18

2 Schedule-at-a-Glance and Meeting Locations Wi-Fi Network: Westin Meeting Rooms Wi-Fi Password: AASLD18 Thursday, June 28, PM 7 PM Registration Inventors Foyer Friday, June 29, :30 AM 5:15 PM Registration Inventors Foyer 7 AM 8 AM Breakfast Foyer ABCD 8 AM 6:15 PM General Session Edison Ballroom 10:10 AM 10:30 AM Break Foyer ABCD 12:15 PM 1:30 PM Lunch Foyer ABCD 3:20 PM 3:40 PM Break Foyer ABCD 5:30 PM 5:45 PM Break Foyer ABCD 6:15 PM 7:15 PM Poster Reception Foyer ABCD Saturday, June 30, :30 AM Noon Registration Inventors Foyer 7 AM 8 AM Breakfast Foyer ABCD 8 AM Noon General Session Edison Ballroom 10:10 AM 10:20 AM Break Foyer ABCD Notice Information may not be recorded, photographed, copied, photocopied, transferred to electronic format, and reproduced or distributed without the written permission of the presenter and AASLD.

3 TABLE OF CONTENTS General Information...1 Disclosures...2 Conference Agenda...8 Speaker Summaries...11 Poster Abstract Summaries FDA Perspective on NASH and Trial Endpoints Erica Lyons, MD Regulatory Update from EMA: Challenges of Clinical Trials in NASH Kolbeinn Guðmundsson, MD Lessons Learned from the Non-Cirrhotic Space Mary E. McCarthy Rinella, MD, FAASLD. 17 Lessons Learned from Cirrhosis Due to NASH Arun J. Sanyal, MBBS, MD, FAASLD...19 Lessons Learned from Prior Pediatric Trials in NAFLD Joel E. Lavine, PhD, FAASLD Variations in Disease Natural History: Implications for Placebo Arm Performance in Clinical Trials Manal F. Abdelmalek, MD, MPH, FAASLD 23 Practical Application of Lifestyle Management in Clinical Trials: Achieving Parity Across Sites Shira Zelber-Sagi, PhD.. 26 Geographic and Ethnic Variations in Disease Phenotype: Separate Trials or Stratified Analysis? Vincent Wong, MD, MBChB Natural Course of NAFLD in Pediatric Populations Jeffrey Schwimmer, MD...30 Histological Subtyping and Advances in Histologic Assessment- Is the NAS Sufficient? Valerie Paradis, MD, PhD Metabolomics, Sub-phenotyping, Microbiota and Other Modalities to Phenotype NASH Michael H. Trauner, MD, FAASLD Phenotyping and Subtyping the Pediatric Population Stavra A. Xanthakos, MD, MS.. 37 Elastography and Imaging for Staging NAFLD Richard Ehman, MD...40 Clinical Events: Liver-Related Outcomes Quentin M. Anstee, MBBS, PhD, FRCP.43

4 Extra-hepatic Outcomes Michelle T. Long, MD Pediatrics Perspective on Outcomes and Clinical Events Valerio Nobili, MD.. 47 Patient-related Outcomes Jörn M. Schattenberg, MD. 49 Detection and Management of DILI in NASH/NAFLD Subjects in Drug Development Naga P. Chalasani, MD, FAASLD 51 Novel Markers and Technologies to Assess Liver Fibrosis and Remodeling Detlef Schuppan, MD, PhD 54 Which Emerging Signals from Preclinical Models are Most Likely to have Clinical Relevance? Frank Tacke, MD, PhD 56 Defining the Spectrum of Responses to Treatment Brent A. Neuschwander-Tetri, MD, FAASLD...58 What Defines a Clinically Meaningful Response with Respect to Histological Assessment in Pre-cirrhotic NASH? David E. Kleiner, MD, PhD. 60 Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Pre-cirrhotic NASH Elisabetta Bugianesi, MD, PhD.. 62 Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Cirrhosis Due to NASH Mohammad Shadab Siddiqui, MD. 66 Current Clinical Trial Landscape in Early Development and Prognosis for 2025 Vlad Ratziu, MD 69 Current Clinical Trial Landscape in Late Development (phase 3) and Prognosis for 2025 Stephen A. Harrison, MD, FAASLD.. 71 Current Clinical Trial Landscape in Pediatrics and Prognosis for 2025 Miriam B. Vos, MD, MSPH.73

5 2018 AASLD/EASL NAFLD Endpoints Conference June 29-30, 2018 Westin Alexandria Alexandria, VA Program Chairs: Arun J. Sanyal, MBBS, MD, FAASLD, Mary E. McCarthy Rinella, MD, FAASLD, Quentin M. Anstee, MBBS, PhD, FRCP and Frank Tacke, MD, PhD Learning Objectives Upon completion of this activity, learners will be able to: Understand how lessons learned from completed NASH trials may influence future clinical trial design and endpoints Identify potential approaches to phenotyping adult and pediatric patients with NASH Discuss available methods to study clinical outcomes and their limitations in the context of NASH clinical trials Understand the strengths and limitations of currently available biomarkers to stage disease and assess dynamic change Continuing Medical Education (CME) and Maintenance of Certification (MOC) Continuing Medical Education (CME) credits and Maintenance of Certification (MOC) points will not be offered for this conference. Disclosures This live educational activity has been planned in accordance with AASLD and ACCME Standards of Commercial Support by members of the Endpoints conference faculty and the AASLD Governing Board. AASLD is an accredited CME provider and requires that its faculty and planning committees disclose all financial relationships, including those of their spouse or partner, with a commercial interest within the past 12 months. A commercial interest is defined as any entity producing, marketing, re-selling, or distributing health care goods or services consumed by, or used on, patients. All conflicts of interest are resolved prior to participation. Although no continuing medical education credits (CME) will be offered for this course, AASLD still requires completion of the disclosure form for our records. Statement on Off-Label and Investigational Use: Speakers are advised to make a reasonable effort to identify any discussion of off-label or investigative use or application of a product or device during their presentation. Financial disclosures will appear at the beginning of each session.

6 Faculty Disclosures Manal F. Abdelmalek, MD, MPH, FAASLD Grants/Research Support: Intercept, Gilead, Allergan, Genfit, BMS, NGM, Madrigal, Shire, TaiwanJ, Immuron, Conatus, Target NASH, Progenity, Galactin, Excelanz, Enanta; Consulting: TaiwanJ Pharma, NGM Pharmaceuticals, BMS Pharma, Pfizer, Allergan, Medimmune, Lexicon Speakers Bureau: Alexion Quentin M. Anstee, MBBS, PhD, FRCP Grant/Research Support: Abbvie, Allergan/Tobira, Astra Zenica, GlaxoSmithKline, Vertex, Novartis Pharma AG Advisory Committees and/or Review Panels: Newcastle University, Abbott Laboratories, Acuitas Medical, Allergan/Tobira, E3Bio, Eli Lilly & Company Ltd., Galmed, Genfit SA, Gilead, Grunthal, Imperial Innovations, Intercept Pharma Europe Ltd., Inventiva, Janssen, Kenes, Madrigal, MedImmune, NewGene, NGMBio, Novartis, Pfizer Ltd., Raptor Pharma Elisabetta Bugianesi, MD Advisory Committees and/or Review Panels: Pfizer, Intercept Naga P. Chalasani, MD, FAASLD Consulting: Abbvie, Lilly, DS Biopharma, Tobira, NuSirt, Madrigal, Cempra, Shire, Allergan Grant/Research Support: Intercept, Gilead, Galectin Richard Ehman, MD Company: Resoundant Inc. Kolbeinn Guðmundsson, MD Nothing to disclose Stephen A. Harrison, MD, FAASLD Advisory Committees and/or Review Panels: Alimentary Pharmacology and Therapeutics - Associate Editor Speaking and Teaching: Abbvie, Alexion Consulting: NGM Bio, Perspectum, Echosens, Prometheus, Cirius Therapeutics, HistoIndex, Madrigal, Genfit, Intercept, Civi Biopharma, Cymabay, Novo Nordisk, Pfizer, Prometheus, Second Genome, CLDF, Corcept Grant/Research Support: Genfit, NGM, Madrigal, Gilead, Cymabay, Conatus, Intercept, Pfizer, Novo Nordisk, Cirius, Taiiwan J, Immuron, Promtheus, Capulus David Kleiner, MD, PhD Nothing to disclose 2

7 Joel E. Lavine, MD, PhD, FAASLD Advisory Committees or Review Panels: Janssen Consulting: Merck, Pfizer, Viking, Novartis, BASF, Gilead, Pippin Michelle T. Long, MD Grant/Research Support: Echosens Erica Lyons, MD Nothing to disclose Mary E. McCarthy Rinella, MD, FAASLD Advisory Committees and/or Review Panels: Abbvie, Intercept, Enanta, Gilead, NGM, NUSirt Pharma, Novartis Consulting: NGM, Shire, Fibrogen, Gilead, Intercept, Novartis, Nusirt Philip N. Newsome, MD, PhD Advisory Committees and/or Review Panels: Novo Nordisk, EASL Governing Board Grants/Research Support: Pharmaxis, Boehringer Ingelheim Brent A. Neuschwander-Tetri, MD, FAASLD Advisory Committees and/or Review Panels: Nimbus Therapeutics, Bristol Myers Squibb, Boehringer-Ingelheim, Enanta, Novartis, Pfizer, Allergan, MedImmune/AstraZeneca, CoSynance, Tobira, Karos, Afimmune, Arrowhead, Reset, Gilead, Abide, Cymabay, Shionogi, Innovo, Indalo, ph Pharma, Lexicon, NGM, Madrigal Valerio Nobili, MD Nothing to disclose Valerie Paradis, MD, PhD Nothing to disclose Vlad Ratziu, MD Advisory Committees and/or Review Panels: GalMed, Boehringer-Ingelheim, Intercept, Genfit, Enterome, Enanta, Gilead, NGM, Allergan, Madrigal, Novo-Nordisk Arun J. Sanyal, MBBS, MD, FAASLD Advisory Committees or Review Panels: Abbott, Bristol Myers, Exhalenz, Genfit, Gilead, Ikaria, Novartis, Pfizer; Tiziana, Conatus, OWL, Echosens, Immuron Consulting: Echosens, Enanta, Exhalenz, Genentech, Hemoshear, Immuron, Islet Sciences, JD Pharma, Merck, Nimbus, Salix, Takeda, Zafgen, Amarin, Vivelyx, Malinckrodt, Ardelyx, Indalo, Zydus Grant/Research Support: Genentech, Gilead, Ikaria, Intercept, Novartis, Salix, Takeda, Tobira, Novo Nordisk, Boehringer Ingelhiem, Octeta, Shire, Bristol Myers, Merck, Galectin, Malinckrodt, Salix, Immuron Independent Contractor: Elsevier, UpToDate Management Position: Sanyal Biotechnology 3

8 Jorn M. Schattenberg, MD Advisory Committees and/or Review Panels: Gilead Sciences, AstraZeneca, Intercept Pharaceuticals Consulting: Genfit Detlef Schuppan, MD, PhD Nothing to disclose Jeffrey Schwimmer, MD Grants/Research Support: Galmed Pharmaceuticals Mohammad Shadab Siddiqui, MD Nothing to disclose Frank Tacke, MD, PhD Grants/Research Support: Noxxon, Tobira, Galapagos Consulting: Tobira, Galapagos, Boehringer, Intercept Honoraria for CME Activities: Gilead, AbbVie, BMS, MSD, Falk, Intercept Michael H. Trauner, MD, FAASLD Grants/Research Support: Albireo, Falk, Gilead, Intercept, Phenex, Takeda Miriam B. Vos, MD, MSPH Consulting: Shire, Bristol Myers Squibb, Boehringer Ingelheim, Intercept Vincent Wong, MD, MBChB Advisory Committees and/or Review Panels: AbbVie, Allergan, Center for Outcomes Research in Liver Diseases, Gilead Sciences, Janssen, Perspectum Diagnostics, Pfizer, TARGET-NASH Honoraria for CME Activities: Bristol-Myers Squibb, Echosens, Gilead Sciences, Merck Stavra Xanthakos, MD Grants/Research Support: Target PharmaSolutions Shira Zelber-Sagi, PhD Nothing to disclose 4

9 Planner Disclosures Jorge A. Bezerra, MD, FAASLD (Governing Board) Grant/Research Support: Gilead, Molecular Genetics Laboratory of Cincinnati Children's Hospital Medical Center, Shyer Kimberly Ann Brown, MD, FAASLD (Governing Board) Board Membership: ABIM SEP, AST Finance Committee, CLD Journal, CLDF Advisory Committee or Review Panel: AbbVie, Bristol-Myers Squibb, Gilead, Merck Grant/Research Support: AbbVie, Gilead, Hyperion, Janssen, Simply Speaking CME Other: Medscape CME content development for HCV Raymond Chung, MD, FAASLD (Governing Board) Grant/Research Support: AbbVie, Boehringer Ingelheim, Gilead Laurie DeLeve, MD, PhD, FAASLD (Governing Board) Consulting: Medivation, Jazz Pharmaceuticals, Seattle Genetics, Daiichi Sankyo Michael W. Fried, MD, FAASLD (Governing Board) Consulting: TARGET PharmaSolutions, AbbVie, Bristol-Myers Squibb, Gilead, Merck Stock Options: TARGET PharmaSolutions Grant/Research Support: AbbVie, Bristol-Myers Squibb, Gilead, Merck, NIH Julie Heimbach, MD (Governing Board) Nothing to disclose, no conflict of interest John Lake, MD, FAASLD (Governing Board) Grants/Research Support: Ocera, Eisai, Beckman Coulter Consulting: Novartis, SRTR Anna S. Lok, MD, FAASLD (Governing Board) Grants/Research Support: BMS, Gilead, Target Pharma Royalties: UpToDate Bruce Luxon, MD, PhD, FAASLD (Governing Board) Board Membership: ACG Research Committee Consulting: National Hemophilia Foundation Speakers Bureau: AbbVie, Merck Lopa Mishra, MD, FAASLD (Governing Board) Consulting: Lilly Advisory Committee or Review Panel: Mayo Clinic SPORE, CDMRP Integration Panel, NIH Chair & CTE member, AASLD PG Course Grant/Research Support: NIH Other: Cernetics, Founder 5

10 Ronald J. Sokol, MD, FAASLD (Governing Board) Advisory Committee or Review Panels: Alexion, Alnylam, Ikaria, Lumena/Shire, Retrophin Grant/Research Support: Lumena/Shire, Mead Johnson Nutrition 6

11 AASLD Staff Disclosures Melissa Boagey Nothing to disclose Julie Deal Nothing to disclose Steven Echard, IOM, CAE, CEO Nothing to disclose Stephanie Graham Nothing to disclose Sharon Grant Nothing to disclose Janeil Klett Nothing to disclose Melissa Morrison Nothing to disclose Denise Seise Nothing to disclose Anne Wrobel Nothing to disclose 7

12 UConference Agenda Friday, June 29, 2018 Session I: Current Landscape and Lessons Learned from Recently Completed Trials Moderators: Arun J. Sanyal, MBBS, MD, FAASLD and Frank Tacke, MD, PhD 8 am 8:10 am Welcome, Introduction, Goals of Conference 8:10 am 8:30 am FDA Perspective on NASH and Trial Endpoints Erica Lyons, MD 8:30 am 8:50 am Regulatory Update from EMA: Challenges of Clinical Trials in NASH Kolbeinn Guðmundsson, MD 8:50 am 9:10 am Lessons Learned from the Non-Cirrhotic Space Mary E. McCarthy Rinella, MD, FAASLD 9:10 am 9:30 am Lessons Learned from Cirrhosis Due to NASH Arun J. Sanyal, MBBS, MD, FAASLD 9:30 am 9:50 am Lessons Learned from Prior Pediatric Trials in NAFLD Joel E. Lavine, PhD, FAASLD 9:50 am 10:10 am Panel Discussion Chair: Frank Tacke, MD, PhD 10:10 am 10:30 am Break Session II: Placebo Arm and Lifestyle Management in Clinical Trials Moderators: Mary E. McCarthy Rinella, MD, FAASLD and Joel E. Lavine, PhD, FAASLD 10:30 am 10:50 am Variations in Disease Natural History: Implications for Placebo Arm Performance in Clinical Trials Manal F. Abdelmalek, MD, MPH, FAASLD 10:50 am 11:10 am Practical Application of Lifestyle Management in Clinical Trials: Achieving Parity Across Sites Shira Zelber-Sagi, PhD 11:10 am 11:30 am Geographic and Ethnic Variations in Disease Phenotype: Separate Trials or Stratified Analysis? Vincent Wong, MD, MBChB 11:30 am 11:50 am Natural Course of NAFLD in Pediatric Populations Jeffrey Schwimmer, MD 11:50 am 12:15 pm Panel Discussion Chair: Mary E. McCarthy Rinella, MD, FAASLD 12:15 pm 1:30 pm Lunch Session III: Segmenting the Population Phenotyping for Clinical Trials Moderators: Quentin M. Anstee, MBBS, PhD, FRCP and Stavra Xanthakos, MD 1:30 pm 1:50 pm Histological Subtyping and Advances in Histologic Assessment- Is the NAS Sufficient? Valerie Paradis, MD, PhD 1:50 pm 2:10 pm Metabolomics, Sub-phenotyping, Microbiota and Other Modalities to Phenotype NASH Michael H. Trauner, MD, FAASLD 2:10 pm 2:30 pm Phenotyping and Subtyping the Pediatric Population Stavra A. Xanthakos, MD, MS 2:30 pm 2:50 pm Elastography and Imaging for Staging NAFLD Richard Ehman, MD 8

13 2:50 pm 3:20 pm Panel Discussion Chair: Quentin M. Anstee, MBBS, PhD, FRCP 3:20 pm 3:40 pm Break Session IV: Clinical Events and Patient-Related Outcomes Moderators: Vlad Ratziu, MD and Miriam B. Vos, MD, MSPH 3:40 pm 3:55 pm Clinical Events: Liver-Related Outcomes Quentin M. Anstee, MBBS, PhD, FRCP 3:55 pm 4:10 pm Extra-hepatic Outcomes Michelle T. Long, MD 4:10 pm 4:25 pm Pediatrics Perspective on Outcomes and Clinical Events Valerio Nobili, MD 4:25 pm 4:40 pm Patient-related Outcomes Jörn M. Schattenberg, MD 4:40 pm 5 pm Detection and Management of DILI in NASH/NAFLD Subjects in Drug Development Naga P. Chalasani, MD, FAASLD 5 pm 5:30 pm Panel Discussion Chair: Miriam B. Vos, MD, MSPH 5:30 pm 5:45 pm Break 5:45 pm 6:15 pm Novel Markers and Technologies to Assess Liver Fibrosis and Remodeling Detlef Schuppan, MD, PhD 6:15 pm 7:15 pm Poster Reception Saturday, June 30, 2018 Session V: Endpoints Defining Response to Treatment Moderators: Stephen A. Harrison, MD, FAASLD and Philip N. Newsome, MD, PhD 8 am 8:20 am Which Emerging Signals from Preclinical Models are Most Likely to have Clinical Relevance? Frank Tacke, MD, PhD 8:20 am 8:40 am Defining the Spectrum of Responses to Treatment Brent A. Neuschwander-Tetri, MD, FAASLD 8:40 am 9 am What Defines a Clinically Meaningful Response with Respect to Histological Assessment in Pre-cirrhotic NASH? David Kleiner, MD, PhD 9 am 9:20 am Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Pre-cirrhotic NASH Elisabetta Bugianesi, MD, PhD 9:20 am 9:40 am Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Cirrhosis Due to NASH Mohammad Shadab Siddiqui, MD 9:40 am 10 am Panel Discussion Chair: Philip N. Newsome, MD, PhD 10 am 10:20 am Break 9

14 Session VI: What Will the NAFLD Landscape Look Like In 2025? Moderators: Quentin M. Anstee, MBBS, PhD, FRCP and Brent A. Neuschwander-Tetri, MD, FAASLD 10:20 am 10:40 am Current Clinical Trial Landscape in Early Development and Prognosis for 2025 Vlad Ratziu, MD 10:40 am 11 am Current Clinical Trial Landscape in Late Development (phase 3) and Prognosis for 2025 Stephen A. Harrison, MD, FAASLD 11 am 11:20 am Current Clinical Trial Landscape in Pediatrics and Prognosis for 2025 Miriam B. Vos, MD, MSPH 11:20 11:40 am Panel Discussion Chair: Brent A. Neuschwander-Tetri, MD, FAASLD 11:40 an Noon NAFLD Debrief Arun J. Sanyal, MBBS, MD, FAASLD 10

15 SPEAKER SUMMARIES 11

16 Erica Lyons, MD Center for Drug Evaluation and Research Food and Drug Administration Silver Spring, MD FDA Perspective on NASH and Trial Endpoints The development landscape for pharmacologic therapies for nonalcoholic steatohepatitis (NASH) is active and promising. The focus of this presentation is to provide an overview of the current regulatory considerations for clinical trials designed for investigational therapies for NASH through discussion of FDA regulatory pathways, population selection for drug development for NASH, trial design and endpoints for pre-cirrhotic NASH, and pediatric NASH development considerations. Splitting nonalcoholic fatty liver disease into three successive stages (NAFL, pre-cirrhotic NASH, and NASH with cirrhosis) provides a conceptual framework to identify areas of potential drug development. NAFL can exist for many years and may not progress to pre-cirrhotic NASH. Of the histologic features of NASH, fibrosis is considered the strongest independent predictor of adverse clinical outcomes, including liver-related death. Given the significant prognostic differences between NAFL and NASH with fibrosis, and the absence of clear clinical, biochemical, or histological criteria that can identify patients with NAFL who are at risk for progression to NASH, the FDA encourages drug developers to focus development on the area of greatest need and potential health impact, (i.e., pre-cirrhotic NASH with liver fibrosis and NASH with cirrhosis). Efficacy endpoints for late phase trials considered by the FDA as reasonably likely to predict clinical benefit for accelerated approval include: resolution of steatohepatitis on overall histopathological reading and no worsening of liver fibrosis on NASH/CRN Brunt/Kleiner fibrosis score. Resolution of steatohepatitis is defined as an overall histopathologic interpretation of, absent fatty liver disease or isolated or simple steatosis without steatohepatitis (and a NAS score of 0-1 for inflammation, 0 for ballooning and 0-3 for steatosis); improvement in liver fibrosis 1 stage (NASH/CRN fibrosis score) and no worsening of steatohepatitis (defined as no increase in NAS for ballooning, inflammation, or steatosis); or both resolution of steatohepatitis and improvement in fibrosis (as defined above). 12

17 Since the data to support that change in NASH histopathology is predictive of clinical benefit are limited, phase 4 trials that confirm clinical benefit or delay of disease progression are needed. This may be demonstrated through change in progression to cirrhosis on histopathology, reduction in hepatic decompensation events as adjudicated by a committee of experts, difference in change in MELD score from less than or equal to 12 to greater than 15, liver transplant, or reduction in all-cause mortality. There are several additional considerations specific to pediatric drug development for NASH. These include the need for demonstration of the prospect of direct benefit both for enrollment of minors and related procedures such as liver biopsy, lack of comprehensive NASH natural history information in pediatric patients, slower disease progression, and less frequent demonstration of the classic pathophysiologic findings observed in adult NASH. The latter consideration creates challenges in the use of a scoring system similar to that utilized in adults for grading histology as a surrogate endpoint. Potential novel therapies should be studied in pediatric patients once there is sufficient information about dosing, safety, and efficacy in adults to inform the risk/benefit analysis. References 1. Sayiner, M., et al., Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in the United States and the Rest of the World. Clin Liver Dis, (2): p Angulo, P., Clinical trials: Trial design in NASH--realities and challenges. Nat Rev Gastroenterol Hepatol, (8): p Hagstrom, H., et al., Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD. J Hepatol, (6): p Younossi, Z.M., et al., Pathologic criteria for nonalcoholic steatohepatitis: interprotocol agreement and ability to predict liver-related mortality. Hepatology, (6): p Patel, Y.A., et al., Baseline Parameters in Clinical Trials for Nonalcoholic Steatohepatitis: Recommendations From the Liver Forum. Gastroenterology, (3): p e7. 6. Sanyal, A.J., et al., Endpoints and clinical trial design for nonalcoholic steatohepatitis. Hepatology, (1): p Schwimmer, J.B., et al., Prevalence of fatty liver in children and adolescents. Pediatrics, (4): p

18 Kolbeinn Guðmundsson, MD Icelandic Medicines Agency Reykjavík Ísland Iceland Regulatory Update from EMA: Challenges of Clinical Trials in NASH Introduction: There is a clear unmet medical need of pharmaceutical treatment options in NASH. Whereas the number of development programs and potential future drug candidates has increased during recent years the current regulatory experience (or rather lack therof) reveals the need for further guidance.in order to address and to avoid potential pitfalls in drug development. The EMA has published a concept paper on the need for the development of a reflection paper on regulatory requirements for the development of medicinal products for chronic noninfectious liver diseases (PBC, PSC, NASH) EMA/CHMP/197320/2017. The draft Reflection Paper was initially planned to be published for public consultation 2nd quarter 2018 but now anticipated in the 3-4th quarter. The main feature of chronic liver disease, and NASH is their slow progression across several, if not dozens, of years. The slow progression of these diseases constitutes a major challenge for drug development essentially with regard to a balanced choice of patient populations, clinically relevant endpoints and duration of observation periods. Symptoms are usually unspecific or at least non-predictive for the long-term outcome of the disease. Because NASH is currently a diagnosis which is mainly based on liver biopsy with histology, and because non-invasive methods of determining the stage and grade of liver disease have not been fully validated, endpoints in NASH have for the time being to rely largely on histology. With the currently ongoing development programmes, as well as additional validation data, it may, however, be possible (and desirable) to supplement and finally substitute with non-invasive methods (imaging and/or biomarkers) both the diagnosis of the disease, as well as the follow-up in clinical trials (including the final outcome such as death, liver transplantation, and cirrhotic disease with decompensation). EMA offers Qualification of novel methodologies for medicine development through a scientific advice procedure. Early use of this procedure encouraged for novel methodologies. A joint format for parallel FDA/EMA submission is available and generally divides between biomarker and COA (clinical outcomes assessment). Study Design/Endpoints: The natural history of NASH is assumed to end with the manifestation of cirrhosis in the liver, and the subsequent development of portal hypertension and its sequelae, and decompensation of liver function, which ultimately results in liver associated death, or liver transplantation. Because NASH is also associated with a multitude of risk factors for cardiovascular disease (hypertension, obesity, hyperlipidaemia, and type 2 diabetes), a relevant proportion of patients will also be prone to causes of death other than liver related ones, mainly cardiovascular. The potential need for cardiovascular outcome data in the development of new NASH treatments needs special discussion. New obesity treatments potentially claiming a benefit in NASH could also complicate this scenario. The natural endpoint in clinical trials for NASH would be the combination of all-cause mortality, liver transplantation, and the manifestation of decompensation (variceal bleeding, ascites, encephalopathy etc.). The time to manifestation of these events, however, is 14

19 currently largely unknown, and it is unclear whether a reasonably sized trial can be conducted in patients with the earlier stages of disease, such as fibrosis stage 2 and 3 based on these hard endpoints. It is therefore necessary to open up to endpoints which can be accepted as a proof of efficacy and that would be regarded to reflect the increase in the risk of disease progression (to the events described) in a substantial manner. The unmet medical need in NASH, may thus trigger the need for an earlier evaluation of efficacy, with an overall shorter duration of clinical trials, until the final results can be presented for marketing authorisation application. A regulatory strategy has been advocated to present such data as interim evaluations of ongoing clinical trials and to present the final study results at later timepoints. The acceptance of such strategies is considered to be a case-by-case decision, and will (among other factors) largely depend on the demonstration of an ongoing unmet medical need at the time of filing of such data. At this point in time, it is appropriate to display only the endpoints that are currently considered suitable to demonstrate such likelihood of correlation to hard outcomes. This interim endpoint considered acceptable consists of two composite endpoints to be evaluated at the individual patient level: 1. The resolution of NASH with the presence of any grade of steatosis, no ballooning, and only minimal (grade 1) lobular inflammation and at the same time no worsening of the stage of fibrosis. 2. The improvement of fibrosis by 1 stage without any worsening of NASH (no worsening of ballooning and lobular inflammation, a 1 grade change in steatosis may be acceptable). These two composites should be evaluated in co-primary fashion, meaning that both will have to independently demonstrate a statistically significant and clinically relevant difference to placebo. It is obvious that in liver disease where cirrhosis has already been manifested (Stage 4), the use of the above mentioned final composite is not possible. The natural endpoint for patients with already existing cirrhotic liver disease at inclusion would therefore consist of the composite of all-cause death and liver decompensation events. Patient population: There is a need for the identification of the most suitable patient population, balancing unmet medical needs, the mechanism of action of drug candidates, and the disease severity with regard to grade of inflammation and stage of fibrosis development.the requirement for long-term observation also raises questions on the balance between timely availability of new compounds, the choice of appropriate licensing strategies and the conduct or continuation of clinical studies post-approval with the associated problems of patient adherence and ethics as well as the regulatory need for (repeated) reassessment. Proposed and accepted inclusion criteria mainly refer to a population with active NASH, defined as a NAS score of at least 4. Patients with relevant fibrosis (stages 2 and 3) should be the main main focus of developments. Patients with stage 1 fibrosis can be in- or excluded;inclusion of stage I patients might be sensible if restricted to those with high inflammatory activity/nas score. An acceptable strategy identified has been to limit their inclusion to a certain extent (e.g %). Exclusion of stage 1 population will have impact on labelling. Exclusion of stage 4 fibrosis patients is considered acceptable (reversal of cirrhosis difficult/impossible?). Pediatrics: NASH is a frequent disease with increasing prevalence and must be regarded to be a consequences of the obesity epidemic. An additional issue is therefore the increasing pediatric disease burden. In line with the pediatric regulation, studies in children wil be required albeit with different emphasis on patient population and endpoints. 15

20 Regulatory: New accelerated regulatory path that could be of value: PRIME or PRIority Medicines The development of promising new medicines to address unmet medical needs. This has been offered by EMA for just over one year. References 1. Concept paper on the need for the development of a reflection paper on regulatory requirements for the development of medicinal products for chronic non-infectious liver diseases (PBC, PSC, NASH). EMA/CHMP/197320/ Reflection paper on assessment of CV risk of medicinal products for the treatment of cardiovascular and metabolic disease (EMA/CHMP/50549/2015; draft) 3. Qualification of novel methodologies for drug development: guidance to applicants: uideline/2009/10/wc pdf 4. EMA/CHMP/57760/2015, Rev. 1 Committee for Medicinal Products for Human Use Enhanced early dialogue to facilitate accelerated assessment of PRIority Medicines (PRIME) 5. European Association for the Study of the Liver (EASL) & European Association for the Study of Diabetes (EASD) & European Association for the Study of Obesity (EASO): EASL EASD EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. Diabetologia (2016) 59: Rasi G and S Bonini: Innovative medicines: new regulatory procedures for the third millennium. Expert Opinion on Biological Therapy: 2015: 15: Rotman Y and AJ Sanyal: Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut 2017; 66:

21 Mary E. McCarthy Rinella, MD, FAASLD Northwestern University, Feinberg School of Medicine Chicago, IL Lessons Learned from the Non-Cirrhotic Space Finding a cure for NASH has proven to be difficult, although, for perspective, HCV took >20 years of drug discovery to reach high levels of efficacy and tolerability. NASH is, by comparison, a much more complex disease than HCV and may take longer to reach the magnitude of efficacy patients enjoyed by those with HCV. NASH is also a manifestation of chronic metabolic abnormalities, less amenable to cure than viral infections. Not only is the pathophysiology of NASH multifaceted and heterogeneous, it may be dynamic within and across individuals. The scale of the burden of liver disease due to NASH has led to a proportionate interest in therapeutic drug development that has produced a brace of late phase studies. The results of several phase 2 trials have been published and merit detailed consideration for future therapeutic development. By far the biggest unmet need is a biomarker that can accurately identify individuals with key histological features of NASH and that correlate with histologic change (steatohepatitis and fibrosis) over time. While it is critical to pair the mechanism of action of the compound to the efficacy measure in earlier phase trials, all trials that have demonstrated histological response in NASH have had concomitant decreases in ALT and liver fat. MRI-PDFF, a highly sensitive and precise measure of hepatic fat content that is consistent across devices, has been invaluable in drug development. Thus, for a drug to move into more advanced stages of development, it should first demonstrate that it improves ALT and reduces hepatic fat. The currently accepted thresholds of >/=5% absolute decrease and 30% relative reduction in PDFF measured hepatic fat fraction appear to correlate with clinically significant histological, but may require further validation. Theoretically, hepatic de-lipidation may not be a prerequisite signal of efficacy of therapeutic agents that are relatively specifically antifibrotic in mechanism. There are, currently, no predominantly antifibrotic agents in late stage development, nor have published phase 2 data demonstrated sufficient potency. While several wet and radiologic biomarkers are currently being evaluated as surrogates of histologic and clinical endpoints in clinical trials, all biomarkers require correlation with histological and/or clinical endpoints that are recognized as meaningful by regulatory authorities, at least in the medium term. The field s reliance on histology is problematic for many reasons, including sampling variability and dynamic changes that occur within individuals for both steatohepatitis and fibrosis. The current NASH CRN fibrosis scoring system is unable to make a linear assessment of fibrosis, as it is constrained by a categorical score within which the amount of collagen deposition can vary substantially. Alternative indices of hepatic fibrosis that may be more reproducible and linearly dynamic, e.g. collagen surface area, are being explored. It is difficult to compare reported efficacy of therapeutic agents across phase 2 studies, in part because primary endpoints have varied or been inconsistently defined (in the case of NASH resolution). Regardless of how it is defined, demonstrating resolution of NASH, which we believe to be the driver of disease progression has proven a high bar that has been met in statistical terms compared to placebo in several trials, but of modest clinical impact as it occurs in a minority of patients. 17

22 Although fibrosis and its extent drive clinical outcomes, NASH is the driver of fibrogenesis. Several studies have now demonstrated that when NASH improves, fibrosis follows, at least to some degree. Thus, addressing the lipid and other metabolic drivers of disease that drive steatohepatitis seem likely to result in later improvements in fibrosis. Unlike in trials of HCV, where placebo response rates were close to nil, the placebo response in NASH trials is sizable (on average 20%, but as high as 42% in a recent trial, depending on the primary endpoint). Understanding and the variability in placebo response, which is likely related to many factors and appears, to some extent, to be independent of weight loss, is essential to distinguishing comparative efficacy of therapeutic agents. We have also learned that NASH can improve quickly with highly potent therapeutic agents. Even fibrosis can improve potentially in as short as 12 weeks. Though exciting and provocative it remains unclear if rapid hepatic de-lipidation, for example, is necessary, or even good. Future trials will need to strike a balance between rapidity of efficacy and side effects. Improving fibrosis in an isolated manner has proved challenging. Identifying an efficacious antifibrotic has been compounded by inherent difficulties in reproducibly quantifying modest changes in fibrosis, in part related to the lack of linearity of collagen content with NASH CRN fibrosis stage. Currently, a 1 stage improvement in fibrosis is considered significant in clinical trials, but this can be difficult to measure. More quantitative techniques such as collagen proportionate area may prove useful in assessing the response to antifibrotic drugs in the NASH space. Adding to these challenges is the lack of understanding of longitudinal changes in hepatic fibro-inflammatory induced liver stiffness, as measured by elastography, in the short time frame of current clinical trials despite high accuracy in identifying those with advanced fibrosis or excluding advanced disease. It is likely that hepatic collagen content varies at a rate that is distinct from the fluid hepatic lipid compartment. Given the biologic complexity of NASH and its heterogeneity, a strong argument can be made that combination therapy will yield the highest efficacy. Redundancy in inflammatory and metabolic pathways is emerging as a challenging but predictable challenge in translating preclinical to clinical success. 18

23 Arun J. Sanyal, MBBS, MD, FAASLD Virginia Commonwealth University Richmond, VA Lessons Learned From Studies In Cirrhosis Due To Nash There is an urgent unmet need for effective therapeutics for cirrhosis due to NASH. The goals of therapy include: (1) reversal of cirrhosis and (2) prevention of downstream complications of cirrhosis. Therapeutic considerations need to include both treatment of the cirrhotic state and the underlying disease. Together, these should meet the evidentiary burden of demonstrating meaningful clinical benefit or a surrogate thereof. Several trials have been conducted in subjects with cirrhosis due to NASH. These have focused primarily on the treatment of compensated cirrhosis. Once decompensated cirrhosis develops, multiple pathophysiological processes driving terminal complications of cirrhosis such as sepsis and multi-organ failure come in to play reducing the biological plausibility that a focused NASH-specific therapy will reverse the decompensated state unless it is extremely effective in a manner analogous to antiviral therapy for HBV-associated decompensated cirrhosis. It is also noteworthy that disease activity decreases with progression in to NASH cirrhosis which is significantly different from viral hepatitis or alcohol-associated cirrhosis. Some of the key areas where completed clinical trials have contributed to our understanding include: (1) Natural History: The placebo arms of the rigorously performed anti-lysl oxidase antibody have provided critically important information on the natural course of bridging fibrosis and cirrhosis due to NASH. These indicate that cirrhosis can reverse spontaneously although there is controversy about whether this represents sampling or observer variability. The concomitant decrease in ELF score and other markers of fibrogenesis and improved outcomes of those with stable or improved fibrosis over those with fibrosis worsening or increase in collagen content indicate that these are likely to be real findings. Also, the concept of clinically significant portal hypertension being defined by a HVPG > 10 mm Hg has been questioned given numerous instances of clinical portal hypertensive complications developing at lower HVPG. (2) Case subsegmentation: Given the variable natural course of advanced fibrosis due to NASH, there is an urgent need to better define subpopulations with a more homogeneous natural course for inclusion in to clinical trials. Machine learned approaches indicate that an AST < 30 IU/l defines those most likely to improve spontaneously and can correctly identify over 2/3rds of such individuals. A phase 2B trial of Galectin for NASH cirrhosis also identified a subpopulation of those with mild portal hypertension who were more responsive to Galectin. (3) rates of development of hard endpoints: Approximately 20% of subjects included in to the lysl oxidase antibody trial developed a clinical complication of cirrhosis within 2 years. Increasing fibrosis and the baseline ELF score and increases in ELF score predicted clinical outcomes. Ongoing challenges in this space include: (1) so far, no therapy has shown the ability to reverse NASH cirrhosis. Future trials will need to demonstrate decreased disease progression with less proportions of actively treated subjects reaching a MELD score of 15 or higher or actual complications of cirrhosis. For trials pursuing a subpart H with a histological endpoint of reduction of fibrosis stage to bridging fibrosis or lower stages, long term data demonstrating reduced clinical events compared to placebo arms will be needed. It is also an open question whether a purely anti-fibrotic approach will reduce the underlying liver injury and tissue remodeling that sets up hepatocellular carcinoma in NASH. Further, it is not known if disease activity will flare if fibrosis stage is reduced and if anti-fibrotic therapies will need to be combined with therapeutics designed to reduce disease activity for optimal benefit. There is also a need for better subsegmentation of the decompensated cirrhosis stage of NASH to define those 19

24 where disease activity is a driver of outcomes versus those where fibrosis primarily drives portal hypertension and portal hypertensive complications. 20

25 Joel E. Lavine, MD, PhD, FAASLD Columbia College of Physicians and Surgeons New York, NY Lessons Learned from Prior Pediatric Trials in NAFLD Concomitant with the rise in obesity prevalence in children throughout the world, there has been a linear recognition of associated fatty liver disease. Although the prevalence does not approach that found in adults, estimates for pediatric NAFLD from autopsy studies of children dying from unnatural death range from 5-10% of the pediatric population. Risk factors associated with NAFLD in children include obesity, native American Hispanic ancestry, male gender, adolescence, family concordance, and certain metabolic syndrome co-morbidities. In children, there is a pediatric sub-type of histologic features found in about a third; this sub-type generally demonstrates more pronounced hepatic steatosis, increased portal predominance of inflammation and fibrosis, minimal or no lobular inflammation, little or no ballooning degeneration, and lack of perisinusoidal fibrosis. Nonetheless, this pattern is associated with increased likelihood of bridging portal fibrosis and is more common in 8-12 year old boys than in older children and girls of the same age. Given the young age at which this frequent chronic liver disease presents, there is unique opportunity to recognize the genetic factors and environmental provocations that affect this most vulnerable population. Further, few of them have ever ingested ethanol, making the phenotypic manifestations of pure consequence. Since many of these children are unaware of their condition, as routine clinical assessment of serum aminotransferases is not evidence-supported as yet, they may move into adulthood with years of undetected evolving injury that may make them more prone to adverse consequences from alcohol, drugs, or superimposed viral infections. In the past 7 years, two major multi-center RCT have been undertaken by the NIDDK NASH CRN to assess whether any therapeutic intervention(s) could be of benefit in 8-17 year old children. Both of these trials are the only multi-center RCT where pre-specified histologic endpoints were either primary or secondary outcomes. One trial, termed TONIC (For Treatment of NAFLD in Children), assessed natural vitamin E 800 IU/d versus metformin versus placebo for 96 weeks. The other, termed CyNCh (for DR-Cysteamine for NAFLD in Children) assessed dose-based oral cysteamine vs placebo for 52 weeks). As intent-to-treat trials, the follow-up for both were excellent, with more than 85% of subjects in each completing the end of trial research biopsies. All IRBs at each of 8-10 clinical sites approved the protocols including the end of treatment research-only biopsy, MRI, and blood draws. No serious events occurred. From these studies, a number of lessons were learned. This will be the content of my talk. References 1. Chalasani N, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology, (1): Fernandes D et al. Pediatric nonalcoholic fatty liver disease in New York City: An autopsy study. J. Pediatr In press. 3. Fleet S et al. Current concepts in pediatric nonalcoholic fatty liver disease. Gastro Clinics of North America :

26 4. Lavine JE. The intersection of nonalcoholic fatty liver disease and obesity. Science Translational Medicine (323): 323rv1 5. Lavine JE, et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: The TONIC randomized controlled trial. JAMA (16): Patton HM et al. Association between metabolic syndrome and liver histology among children with nonalcoholic fatty liver disease. American Journal of Gastroenterology 2010; 105(9): Patton HM et al. Clinical correlates of histopathology in pediatric nonalcoholic steatohepatitis (NASH). Gastroenterology : Schwimmer JB et al. Prevalence of fatty liver in children and adolescents. Pediatrics Oct;118(4): Schwimmer J et al. In children with nonalcoholic fatty liver disease, cysteamine bitartrate delayed release improves liver enzymes but does not reduce disease activity scores Gastroenterology. 151(6):

27 Manal F. Abdelmalek, MD, MPH, FAASLD Duke University Durham, NC Variations in Disease Natural History: Implications for Placebo Arm Performance in Clinical Trials In the past 20 years, we have gained a better understanding of the natural history of NAFLD and NASH. As obesity rates and its associated comorbid conditions such as diabetes, dyslipidemia and metabolic syndrome have increased worldwide, NAFLD and NASH have become a burden to Western society and to the world. Although simple steatosis subtype of NAFLD generally carries a good prognosis from a liver standpoint and is considered to be nonprogressive or slowly progressive, the same is not true of NASH. NASH can lead to progressive hepatic fibrosis and cirrhosis and its complications. Nevertheless, a limited amount of highquality prospective data on the progression of NAFLD exists, particularly in the primary-care setting, where routine biochemical indices do not accurately reflect disease activity or progression. Paired liver biopsy studies from tertiary care cohorts provide valuable information however are limited in their generalizability due to selection bias. Nonetheless, it is now recognized that variations in disease natural history exists independent of pharmacologic or surgical interventions Several paired liver biopsies suggest that one third of patients with NAFL and NASH have progressive fibrosis and 20% will have some regression over an average follow-up between 2.2 and 13.8 years (1, 2). The rate of fibrosis progression is characteristically slow with a recent meta-analysis determining an average progression of one stage to take 7.7 years. Nevertheless, the rate of progression is twice as high in NASH subjects and a sub-group of both NASH and NAFL patients may progress rapidly from no fibrosis to advanced fibrosis over an average six-year period. In contrast to fibrosis progression over time, features of steatosis, inflammation and ballooning tend to reduce and is paralleled by reductions in aminotransaminase levels. Clinical factors that influence variations in natural history include ethnicity, diabetes, increased age, post-menopausal status, and polymorphisms in the PNPLA3 and TM6SF2 genes. Nevertheless, a limited amount of high-quality prospective data on the progression of NAFLD exists, particularly in the primary-care setting, where routine biochemical indices do not accurately reflect disease activity or progression. There is a paucity of data in the literature regarding the true resolution of NAFLD. This is further confounded by lack of long-term followup and sparse biopsy data on large population-based studies. However, recent longitudinal paired biopsy studies as well as therapeutic trials lend insight that natural variations in disease activity exist. Variations in natural history of NAFLD may be a reflection of alternations in life-style modification. In patients with NAFLD, only 12% of patients lost between 5-7% of their initial weight and 8.5% lost between 7-10%, even during clinical trials, but that even modest weight loss may improve NAFLD and NASH (3). Even minimal dietary changes, dietary advice, and/or frequent meetings or clinical visits with health care advisors (as would occur with longitudinal studies) may contribute, in a manner that has yet to be qualified and quantified, to the variability in the natural history of NAFLD and NASH. Evidence that only a minority of patients with NAFLD 23

28 progress to NASH with advanced fibrosis suggests that disease progression is likely to depend on the complex interplay between environmental factors and genetic predisposition. Variation in natural history of NASH is notable in the placebo arm performance in clinical studies. In the PIVENS and FLINT study, the placebo response for the primary histologic outcome of 2 point improvement in NAS with no worsening of fibrosis was 19% and 21% respectively (4,5). In a study evaluating ethyl-eicosapentanoic acid (EPA-E) on histologic features of NASH, similar proportions of subjects met the primary end point of NAS 3, without worsening of fibrosis; or a decrease in NAS by 2 without worsening of fibrosis at 1 year (40%, 37%, and 35.9% for placebo, low-dosage, and high-dosage EPA-E, respectively) thereby rendering a negative study (6). In a phase 2 study evaluating the elafibranor for NASH, placebo response rate for resolution of NASH (disappearance of ballooned hepatocytes and disappearance or minimal persistence of lobular inflammation) was 12% (6). Further, variations in disease natural history have been associated with a reported fibrosis regression by 1 stage with no worsening of NASH at 1- and 2-years was 10% and 17% respectively in placebo arm performance of Cenicriviroc phase 2 NASH study (7,8). In a recent early phase 2 study evaluating role of JKB-121, a TLR-4 antagonist, a plausible target for a biological pathway in NASH pathogenesis, a > 30% relative reduction was noted in 42% of patients on placebo with no further improvement in JKB-121 treatment groups. This improvement was associated with improvement in ALT, both of which were noted to occur independent of significant change in weight (9). The variations in natural history of NASH will render challenges in translating compounds into early phase clinical studies, particularly in the absence of sensitive and widely validated biomarkers that accurately define histologic disease activity. Efforts to better understand the natural variations in NASH disease activity as well as the environmental, dietary or behavioral factor(s) that contribute to such variation is an unmet need. The placebo arm performance in early phase clinical trials for NASH will have implications on increased sample size, study costs and need to better define the factors contributing variation in natural history when assessing response to pharmacologic interventions. References 1. Calzadilla BL, Adams LA.The Natural Course of Non-Alcoholic Fatty Liver Disease. Int J Mol Sci May 20;17(5). 2. Marengo A, Jouness RI, Bugianesi E. Progression and Natural History of Nonalcoholic Fatty Liver Disease in Adults. Clin Liver Dis May;20(2): Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, et al. Weight Loss Through Lifestyle Modification Significantly Reduces Features of Nonalcoholic Steatohepatitis. Gastroenterology Aug;149(2): Sanyal AJ, Chalasani N, Kowdley KV, et al; NASH CRN. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med May 6;362(18): Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al; NASH CRN. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomized, placebo-controlled trial. Lancet Mar 14;385(9972): Sanyal AJ, Abdelmalek MF, Suzuki A, et al; EPE-A Study Group. No significant effects of ethyl-eicosapentanoic acid on histologic features of nonalcoholic steatohepatitis in a phase 2 trial. Gastroenterology Aug; 147(2): Friedman SL, Ratziu V, Harrison SA, et al. A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology May;67(5):

29 8. Ratziu V, et al. Cenicriviroc (CVC), a CCR2 and CCR5 inhibitor, in patients with NASH: Phase 2b CENTAUR study Year 2 analysis Oral Presentation ILC Paris, France, Abstract # Diehl AM, et al. JKB-121, a TLR-4 receptor antagonist: Phase 2 trial for the treatment of NASH Oral Presentation. ILC 2018, Paris, France, Abstract #

30 Shira Zelber-Sagi, PhD Tel-Aviv Medical Center & School of Public Health, University of Haifa Tel-Aviv Israel Practical Application of Lifestyle Management in Clinical Trials Achieving Parity Across Sites While in clinical practice, a successful diet is a desirable goal, in NAFLD clinical trials, it is often looked at as an obstacle. Very few diseases can improve substantially by diet as NAFLD and NASH, making lifestyle an important determinant in the design and interpretation of NAFLD RCTs. Lifestyle intervention is useful across the entire spectrum of NAFLD patients. A comprehensive lifestyle modification based on reduced energy intake and increased physical activity during 6-12 months induces improvement in liver enzymes, improved liver fat, assessed by proton magnetic resonance spectroscopy (1, 2), and reduced steatosis, necroinflammation and fibrosis detected in paired liver biopsies (3-5). Most studies conclude that at least 7-10% weight loss is required to induce improvement in NAFLD activity score (NAS) (5-7). However, lifestyle changes that produce even modest, sustained weight loss of about 5% of initial body weight can reduce steatosis (1), liver enzymes (8) and metabolic parameters, sometimes used as secondary endpoints in NAFLD clinical trials. Accordingly, the EASL EASD EASO Clinical Practice Guidelines state that in overweight/obese NAFLD, a 7 10% weight loss is the target of most lifestyle interventions. Despite these promising results, weight loss is a major clinical challenge for patients and clinicians. In the study by Vilar-Gomez et al (5), just 10% of patients (29/293) reached a 10% weight loss and more than 70% of the cohort (208/293) did not lose 5% of initial body weight. Physical activity change, by itself, may be of concern in RCTs. Although exercise has a significant and clinically meaningful effect on liver lipid (20-30% relative reduction), its effects are modest in comparison to weight reduction (9). Dietary composition is also related with NAFLD. Saturated fat and added sugar, mainly fructose, are the most evidenced based harmful dietary components. On the other hand, the Mediterranean diet pattern seems to have hepatic protective properties (10). The specific implications of lifestyle in NAFLD pharmacological RCTs need to be elaborated in several ways: 1. Confounding effect of lifestyle (imbalance between arms). Change in BMI/ abdominal fat/ lifestyle parameters during the trial may distribute differently between treatment arms and impact the hepatic outcomes. This problem is assumed to be minimized by randomization (similar proportion of people motivated for diet in each arm) but may still lead to significant confounding. 2. Weight reduction and/or dietary composition change and/or increased physical activity may make the added value of medication negligible (even if balanced between arms). Steatosis is the major concern since it is most affected by even minor changes in weight or dietary composition, but fibrosis is less easily affected and patients with fibrosis are at the highest need for pharmacotherapy. For these reasons, there doesn t seem to be a place for comprehensive lifestyle intervention in NAFLD clinical trials. In addition, patients with mild disease and with successful lifestyle modification are not the target population for pharmacological therapy. The inclusion of patients who failed dieting (those with recent weight reduction >5% of initial body weight defined not eligible) actually mimics real life. Still, a uniform lifestyle treatment must be ensured within the 26

31 study to reduce confounding, and across different trials to enable fair comparisons. Moreover, providing no lifestyle advice brings-up some ethical questions, especially in long-term trials. It seems wise that the lifestyle advice will include: general instructions for diet and moderate physical activity, refraining dramatic lifestyle changes and the avoidance of food supplements (e.g. omega-3, vitamin E). Since spontaneous improvement in lifestyle during trial may occur due to increased clinical monitoring and motivation, it is needed to document anthropometric parameters (weight, waist circumference, visceral adipose tissue if possible). Monitoring physical activity and dietary intake changes is recommended. While physical activity change can be relatively easily evaluated, dietary intake is harder to monitor since it is time consuming and encompasses a significant report bias. The dietary factors which deserve attention are: fructose or sugar, soft drinks, saturated fat and other types of fat and the Mediterranean diet components. The Mediterranean dietary pattern score (or other healthy eating index) can also be assessed and compared between different time points across the trial. References 1. Lazo M, Solga SF, Horska A, Bonekamp S, Diehl AM, Brancati FL, et al. Effect of a 12- month intensive lifestyle intervention on hepatic steatosis in adults with type 2 diabetes. Diabetes Care. 2010;33(10): Patel NS, Doycheva I, Peterson MR, Hooker J, Kisselva T, Schnabl B, et al. Effect of weight loss on magnetic resonance imaging estimation of liver fat and volume in patients with nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol. 2015;13(3):561-8 e1. 3. Harrison SA, Fecht W, Brunt EM, Neuschwander-Tetri BA. Orlistat for overweight subjects with nonalcoholic steatohepatitis: A randomized, prospective trial. Hepatology. 2009;49(1): Eckard C, Cole R, Lockwood J, Torres DM, Williams CD, Shaw JC, et al. Prospective histopathologic evaluation of lifestyle modification in nonalcoholic fatty liver disease: a randomized trial. Therap Adv Gastroenterol. 2013;6(4): Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres-Gonzalez A, Gra-Oramas B, Gonzalez-Fabian L, et al. Weight Loss Through Lifestyle Modification Significantly Reduces Features of Nonalcoholic Steatohepatitis. Gastroenterology. 2015;149(2): e5; quiz e Promrat K, Kleiner DE, Niemeier HM, Jackvony E, Kearns M, Wands JR, et al. Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology. 2010;51(1): Musso G, Cassader M, Rosina F, Gambino R. Impact of current treatments on liver disease, glucose metabolism and cardiovascular risk in non-alcoholic fatty liver disease (NAFLD): a systematic review and meta-analysis of randomised trials. Diabetologia. 2012;55(4): Suzuki A, Lindor K, St Saver J, Lymp J, Mendes F, Muto A, et al. Effect of changes on body weight and lifestyle in nonalcoholic fatty liver disease. J Hepatol. 2005;43(6): Thoma C, Day CP, Trenell MI. Lifestyle interventions for the treatment of non-alcoholic fatty liver disease in adults: a systematic review. J Hepatol. 2012;56(1): Romero-Gomez M, Zelber-Sagi S, Trenell M. Treatment of NAFLD with diet, physical activity and exercise. J Hepatol. 2017;67(4):

32 Vincent Wong, MD, MBChB The Chinese University of Hong Kong Hong Kong China Geographic and Ethnic Variations in Disease Phenotype: Separate Trials or Stratified Analysis? Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, affecting at least 25% of the global adult population. Although a recent meta-analysis shows that the prevalence of NAFLD is similarly high in America, Europe and Asia, the heterogeneity in the methods of diagnosis and time period of the included studies should be noted (1). More recent studies suggest a higher prevalence of NAFLD in North America than other parts of the world. In the United States, Hispanics are at highest risk of having NAFLD, whereas African-Americans have the lowest risk (2). Likewise, although NAFLD is also found in 15-30% of the Asian population, they have less severe histology and a lower incidence of hepatocellular carcinoma and cirrhotic complications (3, 4). A number of reasons may explain the difference in NAFLD prevalence and severity in different geographical regions and ethnicities. For one thing, the heritability of NAFLD is well established, and there is substantial difference in the distribution of gene polymorphisms across ethnic groups. For instance, the minor allele frequency of PNPLA3 rs (patatin-like phospholipase domain-containing protein 3) is highest in Mexicans and Hispanics and lowest in African Americans (5). Interestingly, this gene polymorphism is more common in East Asians than Caucasians, which may explain the relatively high prevalence of NAFLD in East Asians despite a lower metabolic burden (6). Asians also develop NAFLD and metabolic complications at a lower body mass index, thus explaining the condition of non-obese NAFLD in this population (7). It is also important to recognize that different regions and ethnic groups have different dietary habits, physical activity levels and socioeconomic environments, all interacting to shape the local epidemiology and disease burden. If we agree that geographic and ethnic variations would affect the prevalence and severity of NAFLD, how should this knowledge affect drug development? Above all, race and ethnicity are recommended as essential components to be reported in phase III trials, and preferably in proof-of-concept and phase II trials as well (8). Subgroup analysis for different ethnic groups would be informative. Nonetheless, almost all past and ongoing trials are underpowered for subgroup analysis, and the issue of multiplicity is well recognized. Findings from subgroup analysis should be considered as hypothesis generating. If a trial shows signal of differential response or a compound has variable pharmacokinetics across ethnic groups, the findings should be confirmed in a separate trial. References 1. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016;64: Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, et al. Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 2005;41:

33 3. Kawamura Y, Arase Y, Ikeda K, Seko Y, Imai N, Hosaka T, et al. Large-scale long-term follow-up study of Japanese patients with non-alcoholic Fatty liver disease for the onset of hepatocellular carcinoma. Am J Gastroenterol 2012;107: Leung JC, Loong TC, Wei JL, Wong GL, Chan AW, Choi PC, et al. Histological severity and clinical outcomes of nonalcoholic fatty liver disease in nonobese patients. Hepatology 2017;65: Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 2018;15: Shen J, Wong GL, Chan HL, Chan HY, Yeung DK, Chan RS, et al. PNPLA3 gene polymorphism accounts for fatty liver in community subjects without metabolic syndrome. Aliment Pharmacol Ther 2014;39: Fan JG, Kim SU, Wong VW. New trends on obesity and NAFLD in Asia. J Hepatol 2017;67: Patel YA, Imperial JC, Muir AJ, Anstee QM, DeBrota D, Dimick-Santos L, et al. Baseline Parameters in Clinical Trials for Nonalcoholic Steatohepatitis: Recommendations From the Liver Forum. Gastroenterology 2017;153: e

34 Jeffrey Schwimmer, MD UC San Diego and Rady Children s Hospital San Diego, CA jschwimmer@ucsd.edu Natural Course of NAFLD in Pediatric Populations The available data on the natural history of NAFLD in children are limited. Outcomes of interest include disease progression, development of co-morbidities, liver transplantation, and mortality. Factors that influence the ability to assess these outcomes include challenges in understanding when NAFLD begins in children, how the diagnosis is made, follow-up care, and limited published data. Natural history is the usual course of a disease, especially in the absence of treatment. It is unsettled if this means those who go undiagnosed and thus have no opportunity for intervention, those who are diagnosed and receive only standard of care lifestyle counseling, or those who are diagnosed and participate in the placebo arms of a clinical trial. There is no reason to assume that these three scenarios are equivalent. The most common scenario may be those children who have NAFLD but are undiagnosed and thus do not receive an intervention. Data supports that overweight and obesity in childhood and adolescence are associated with greater odds of NAFLD, cirrhosis, liver cancer, and liverrelated death in adulthood. Liver transplantation for pediatric NASH has been reported in both children and young adults. Children who have a histologic diagnosis of NAFLD are retained for follow-up care at twice the rate of children with noninvasive diagnosis of NAFLD. Follow-up liver biopsy in clinical care has only been reported in small series. With standard of care treatment over 2 to 3 years, fibrosis improves in 20%, is stable in 40%, and progresses in 40%. There are additional data from clinical trials. In those enrolled in the placebo arm, fibrosis improved in 28% over one year and 33% over two years. Over the course of 1 year of standard of care, 20% of children with NAFLD will have hypertension and rates in higher for girls than boys. Quality of life improves more in the context of clinical trials then with standard of care alone. In studies that have followed children with diagnosed with NAFLD into adulthood the mortality rate has been unexpectedly high with death in about 7%. References 1. Cioffi CE, Welsh JA, Cleeton RL, Caltharp SA, Romero R, Wulkan ML, Konomi JV, Frediani JK, Vos MB. Natural History of NAFLD Diagnosed in Childhood: A Single-Center Study. Children ;4(5). PMID: DeVore S, Kohli R, Lake K, Nicholas L, Dietrich K, Balistreri WF, Xanthakos SA. 3. A multidisciplinary clinical program is effective in stabilizing BMI and reducing transaminase levels in pediatric patients with NAFLD. J Pediatr Gastroenterol Nutr. 2013;57(1): PMID: Feldstein AE, Charatcharoenwitthaya P, Treeprasertsuk S, Benson JT, Enders FB, Angulo P. The natural history of non-alcoholic fatty liver disease in children: a follow-up study for up to 20 years. Gut. 2009;58(11): PMID:

35 5. Goyal NP, Schwimmer JB. The Progression and Natural History of Pediatric Nonalcoholic Fatty Liver Disease. Clin Liver Dis. 2016;20(2): PMID: Kerkar N, D'Urso C, Van Nostrand K, Kochin I, Gault A, Suchy F, Miloh T, Arnon R, Chu J, Annunziato R. Psychosocial outcomes for children with nonalcoholic fatty liver disease over time and compared with obese controls. J Pediatr Gastroenterol Nutr. 2013;56(1): PMID: Lavine JE, Schwimmer JB, Van Natta ML, Molleston J, Murray KF, Rosenthal P, al., for the NASH CRN. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: The TONIC randomized controlled trial. JAMA (16): PMID Schwimmer JB, Zepeda A, Newton KP, Xanthakos SA, Behling C, Hallinan EK, Donithan M, Tonascia J; Nonalcoholic Steatohepatitis Clinical Research Network. Longitudinal assessment of high blood pressure in children with nonalcoholic fatty liver disease. PLoS One. 2014; 9(11):e PMID: Schwimmer JB, Lavine JE, Wilson LA, Neuschwander-Tetri BA, Xanthakos S, Kohli R, et al. for the NASH CRN. In children with nonalcoholic fatty liver disease, cysteamine bitartrate delayed release improves liver enzymes but does not reduce disease activity scores Gastroenterology. 151(6): PMID:

36 Valérie Paradis, MD, PhD Beaujon Hospital, Clichy, & INSERM Clichy France Histological Subtyping and Advances in Histologic Assessment: Is the NAS Sufficient? Liver histology: the reference standard in NAFLD Non Alcoholic Fatty Liver Disease (NAFLD), the hepatic manifestation of metabolic syndrome and insulin resistance, encompasses a wide spectrum of histological lesions, ranging from steatosis (NAFL) to steato-hepatitis (NASH) that combines inflammation, hepatocellular damages and fibrosis [1]. So far, liver histology is considered as the reference standard providing both diagnosis and staging of NAFLD. Indeed, in spite of the well-known limitations of the liver biopsy (invasive assessment subjected to potential sampling variability and operator dependence), it offers a global assessment of all elementary lesions and their respective extent in a first blink. Beyond diagnosis and staging, the pathological diagnosis of NASH is predictive of metabolic syndrome abnormalities, as well as all individual features of NASH are independently associated with insulin resistance [2]. In addition, histological diagnosis of NASH is associated with increased risk of cirrhosis and liver-related mortality [3,4]. Among the different morphological features, fibrosis stage is the strongest predictor of liver- and cardiovascular-related mortality [5]. Scoring systems in NAFLD In line with chronic liver diseases, scoring systems, based on a semi-quantitative assessment of elementary morphological features, including steatosis, inflammation and ballooning, have been developed in the context of NAFLD. The NASH Clinical Research Network (NASH-CRN) proposed the NAFLD Activity Score (NAS) and Fibrosis score [6,7]. The NAS, ranging from 0 to 8, is based on unweighted scores of steatosis (from 0 to 3), lobular inflammation (from 0 to 3), and ballooning (from 0 to 2). Although NAS is not a diagnostic scoring system and does not replace the pathologist s diagnostic assessment in patients with NAFLD, it is the most widely used for defining and quantifying disease activity in observational and therapeutic clinical trials [7]. Accordingly, a NAS score > 5 is associated with a diagnosis of definite NASH while a NAS < 3 rules out a diagnosis of NASH. It is of note that the NAS score is poorly correlated with factors of metabolic syndrome and is not associated with advanced clinical outcomes, either progression of fibrosis or risk of mortality [8,9]. As NAFLD is a dynamic chronic liver disease with a continuous spectrum in which ballooning and lobular inflammation, but not steatosis, define activity for the diagnosis of NASH, the European Fatty Liver Inhibition of Progression (FLIP) consortium has proposed the SAF score ([10]). The SAF score is a histological algorithm assessing semi-quantitatively and separately steatosis (S), Activity (A), which takes into account ballooning and inflammation (), and fibrosis (F). Diagnosis of NASH requires presence of steatosis and both ballooning and inflammation at any degree. Accordingly, SAF may identify categories of NAFLD severity into mild disease (A<2 and F<2) and significant disease (A>2 and F>2). While validation of the SAF score in clinical trials is awaiting, intra- and inter-observer reproducibility is very good. Liver histology as an endpoint in clinical trials Liver histology is used as a surrogate endpoint for evaluating clinical benefit in therapeutic trials for NASH. Resolution of NASH without worsening fibrosis and reduction of the NAS 32

37 score without worsening fibrosis have been initially recommended as the main histological endpoints. Reduction of NAS score was defined by at least a 2-point improvement (with >1 point from each of 2 of the components of NAS: steatosis, lobular inflammation ballooning). While resolution of NASH is probably a better validated endpoint, its definition needs to be carefully stated. As fibrosis is a powerful predictor of long-term outcome in patients with NAFLD, fibrosis staging is a key step in the evaluation of liver histology. To date, the fibrosis staging system currently used in this context is derived from the CRN (). Although fibrosis regression may be achieved in clinical trials, the use of an expanded semiquantitative fibrosis staging system (from a 4- to a 6-scale) as well as a quantitative morphometry approach will be more sensitive and may help to identify patients with fibrosis regression. References 1. Nascimbeni F, Ballestri S, Machado MV, Mantovani A, Cortez-Pinto H, Targher G, et al. Clinical relevance of liver histopathology and different histological classifications of NASH in adults. Expert Rev Gastroenterol Hepatol 2018;12: doi: / Ballestri S, Zona S, Targher G, Romagnoli D, Baldelli E, Nascimbeni F, et al. Nonalcoholic fatty liver disease is associated with an almost twofold increased risk of incident type 2 diabetes and metabolic syndrome. Evidence from a systematic review and meta-analysis. J Gastroenterol Hepatol 2016;31: doi: /jgh Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116: Younossi ZM, Stepanova M, Rafiq N, Makhlouf H, Younoszai Z, Agrawal R, et al. Pathologic criteria for nonalcoholic steatohepatitis: interprotocol agreement and ability to predict liver-related mortality. Hepatol Baltim Md 2011;53: doi: /hep Dulai PS, Singh S, Patel J, Soni M, Prokop LJ, Younossi Z, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatol Baltim Md 2017;65: doi: /hep Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94: doi: /j x. 7. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatol Baltim Md 2005;41: doi: /hep Brunt EM, Kleiner DE, Wilson LA, Belt P, Neuschwander-Tetri BA, NASH Clinical Research Network (CRN). Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatol Baltim Md 2011;53: doi: /hep Ekstedt M, Franzén LE, Mathiesen UL, Kechagias S. Low clinical relevance of the nonalcoholic fatty liver disease activity score (NAS) in predicting fibrosis progression. Scand J Gastroenterol 2012;47: doi: / Bedossa P, FLIP Pathology Consortium. Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease. Hepatol Baltim Md 2014;60: doi: /hep

38 Michael H. Trauner, MD FAASLD Medical University of Vienna Wien Austria Metabolomics, Sub-phenotyping, Microbiota and Other Modalities to Phenotype NASH NASH is a rather heterogenous disorder and many current clinical trials designs therefore focus on certain subgroups with varying disease activity and stage (fibrosis) (1). Metabolic co-morbidity (T2DM, central obsesity, dyslipidemia, arterial hypertension), advanced age and sex (postmeopausal women) are predictive of the progression / severity of NASH and fibrosis, and many ongoing clinical trials therefore stratify for presence of T2DM. Other endocrine factors such as thyroid function and vitamin D status may also be involved. Additional phenotyping tools are urgently needed to further segment and stratify the NASH population in clinical trials. As such, genetic markers, alterations of gut microbiota (dysbiosis) and metabolic markers (often reflecting alterations of lipid metabolism genes and microbiota) correlate with severity of NASH and may help to better subphenotype patients. Various genetic variants have been linked with NASH susceptibility/severity (e.g. PNPLA3, TM6SF2, MBOAT7 GCKR) or protection against NASH (e.g. CES1, HSD17B13) (2). Although the function of PNPLA3 is still controversial, some authors have suggested to distinguish PNPLA3-NAFLD/NASH from metabolic (non-pnpla3-associated) NAFLD/NASH which also show different hepatic lipid composition. As such, a saturated, ceramide-enriched liver lipidome is found in 'metabolic NAFLD' but not in 'PNPLA3 NAFLD' where the increase in liver fat is mainly due to polyunsaturated triacylglycerols (3). The PNPLA3 I148M variant has been linked to response to lifestyle and less established - pharmacological interventions, but genetic variants have so far not been rigorously used to stratify patients in clinical trials (4). Specific plasma biomarkers may be associated with disease activity and severity of fibrosis in NASH and are potentially valuable tools for stratification of patients as well as identification of targets for therapeutic interventions. Since lipotoxicity is a key determinant in the progression of NASH, changes in serum/hepatic lipid composition may reflect disease severity (5-7). A stepwise increase in lipoxygenase metabolites 5-hydroxyeicosatetraenoic acid (5-HETE), 8-HETE, and 15-HETE was found in the progression from NAFL to NASH. Notably, 11-HETE, a nonenzymatic oxidation product of arachidonic acid, was increased only in NASH (6). Recently, a score based on mass spectrometry (glutamate, isoleucine, glycine, lysophosphatidylcholine, phosphoethanolamine), AST, fasting insulin, and PNPLA3 genotype has been suggested to determine the risk of NASH (8). Increased activated plasminogen activator inhibitor 1 was strongly associated with NASH, while biomarkers associated with significant fibrosis included higher levels of pro-inflammatory cytokines/adipokines (e.g. IL-8, MCP-1, TNF-a, resistin) and lower levels of insulin-like growth factor 2 (9). In addition to plasma metabolites, CAP, MRI-PDFF and MRS may help to evaluate hepatic lipid content and composition in the context of clinical studies (1). Bile acids (BAs) are important metabolic integrators and play an important role in controlling intestinal microbiota and maintaining intestinal integrity. Conversely, microbiota metabolize primary into secondary BAs. The presence and severity of NASH and fibrosis is associated with specific changes in circulating BAs (10). While serum BA levels are eleated with increasing severity of NAFLD/NASH, BA signaling is impaired as reflected by reduced levels of FXR and FGF19 (11). However, other studies have reported that BA alterations in obese patients may reflect insulin resistance rather than presence of NASH (12). Interestingly, the 34

39 changes in serum and fecal BA composition in NASH may be an indicator altered metabolic function of intestinal microbita reflecting the degree of dysbiosis in NASH (13). NAFLD/NASH severity associates with gut dysbiosis and a shift in the metabolic function of the gut microbiota (14). Several studies suggest differences in gut microbiota composition between NAFL and NASH (15-17). As such, associations of Bacteroides with NASH and Ruminococcus with significant fibrosis have been reported (18) Another study identified a set of 37 bacterial species that distinguish mild/moderate NAFLD from advanced fibrosis (19). The gut-microbiome derived metabolite (3-(4-hydroxyphenyl)lactate) has been linked to hepatic steatosis and fibrosis (20). Notably, gut microbiota and their metabolites may experimentally not only promote but also protect against hepatic fibrosis. While low amounts of (exogenous) alcohol may (paradoxcially) protect agaist NAFLD/NASH (e.g. by improving insulin resistance) (21), recent studies suggest that a NASH-specific microbiome has increased abundance of endogenous alcohol-producing bacteria (17). Alcohol metabolites in urine and plasma may help to identify patients with otherwise underrecognized alcohol consumption (21). In summary, deep patient phenotyping applying genetic, metabolomic, microbiome and other (e.g.circulating mirnas) markers may help to better segement and stratify patients with NASH in clincal routine and for clinical trials. References 1. Konerman MA, Jones JC, Harrison SA. Pharmacotherapy for NASH: Current and emerging. J Hepatol Feb;68(2): Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. J Hepatol Feb;68(2): Luukkonen PK, Zhou Y, Sädevirta S, Leivonen M, Arola J, Orešič M, Hyötyläinen T, Yki- Järvinen H. Hepatic ceramides dissociate steatosis and insulin resistance in patients with non-alcoholic fatty liver disease. J Hepatol May;64(5): Sevastianova K, Kotronen A, Gastaldelli A, Perttilä J, Hakkarainen A, Lundbom J, Suojanen L, Orho-Melander M, Lundbom N, Ferrannini E, Rissanen A, Olkkonen VM, Yki-Järvinen H. Genetic variation in PNPLA3 (adiponutrin) confers sensitivity to weight loss-induced decrease in liver fat in humans. Am J Clin Nutr Jul;94(1): Puri P, Baillie RA, Wiest MM, Mirshahi F, Choudhury J, Cheung O, Sargeant C, Contos MJ, Sanyal AJ. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology Oct;46(4): Puri P, Wiest MM, Cheung O, Mirshahi F, Sargeant C, Min HK, Contos MJ, Sterling RK, Fuchs M, Zhou H, Watkins SM, Sanyal AJ. The plasma lipidomic signature of nonalcoholic steatohepatitis. Hepatology Dec;50(6): Siddiqui MS, Fuchs M, Idowu MO, Luketic VA, Boyett S, Sargeant C, Stravitz RT, Puri P, Matherly S, Sterling RK, Contos M, Sanyal AJ. Severity of nonalcoholic fatty liver disease and progression to cirrhosis are associated with atherogenic lipoprotein profile. Clin Gastroenterol Hepatol May;13(5): Zhou Y, Orešič M, Leivonen M, Gopalacharyulu P, Hyysalo J, Arola J, Verrijken A, Francque S, Van Gaal L, Hyötyläinen T, Yki-Järvinen H. Noninvasive Detection of Nonalcoholic Steatohepatitis Using Clinical Markers and Circulating Levels of Lipids and Metabolites. Clin Gastroenterol Hepatol Oct;14(10): Ajmera V, Perito ER, Bass NM, Terrault NA, Yates KP, Gill R, Loomba R, Diehl AM, Aouizerat BE; NASH Clinical Research Network. Novel plasma biomarkers associated with liver disease severity in adults with nonalcoholic fatty liver disease. Hepatology Jan;65(1): Puri P, Daita K, Joyce A, Mirshahi F, Santhekadur PK, Cazanave S, Luketic VA, Siddiqui MS, Boyett S, Min HK, Kumar DP, Kohli R, Zhou H, Hylemon PB, Contos MJ, Idowu M, Sanyal AJ. The presence and severity of nonalcoholic steatohepatitis is 35

40 associated with specific changes in circulating bile acids. Hepatology Jul 11. [Epub ahead of print] 11. Nobili V, Alisi A, Mosca A, Della Corte C, Veraldi S, De Vito R, De Stefanis C, D'Oria V, Jahnel J, Zohrer E, Scorletti E, Byrne CD. Hepatic farnesoid X receptor protein level and circulating fibroblast growth factor 19 concentration in children with NAFLD. Liver Int. 2018;38(2): Legry V, Francque S, Haas JT, Verrijken A, Caron S, Chávez-Talavera O, Vallez E, Vonghia L, Dirinck E, Verhaegen A, Kouach M, Lestavel S, Lefebvre P, Van Gaal L, Tailleux A, Paumelle R, Staels B. Bile Acid Alterations Are Associated With Insulin Resistance, but Not With NASH, in Obese Subjects. J Clin Endocrinol Metab Oct 1;102(10): Mouzaki M, Wang AY, Bandsma R, Comelli EM, Arendt BM, Zhang L, Fung S, Fischer SE, McGilvray IG, Allard JP. Bile Acids and Dysbiosis in Non-Alcoholic Fatty Liver Disease. PLoS One May 20;11(5):e Wieland A, Frank DN, Harnke B, Bambha K. Systematic review: microbial dysbiosis and nonalcoholic fatty liver disease. Aliment Pharmacol Ther Nov;42(9): Mouzaki M, Comelli EM, Arendt BM, Bonengel J, Fung SK, Fischer SE, McGilvray ID, Allard JP. Intestinal microbiota in patients with nonalcoholic fatty liver disease. Hepatology Jul;58(1): Wong VW, Tse CH, Lam TT, Wong GL, Chim AM, Chu WC, Yeung DK, Law PT, Kwan HS, Yu J, Sung JJ, Chan HL. Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis--a longitudinal study. PLoS One Apr 25;8(4):e Zhu L, Baker SS, Gill C, Liu W, Alkhouri R, Baker RD, Gill SR. Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology Feb;57(2): Boursier J, Mueller O, Barret M, Machado M, Fizanne L, Araujo-Perez F, Guy CD, Seed PC, Rawls JF, David LA, Hunault G, Oberti F, Calès P, Diehl AM. The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology Mar;63(3): Loomba R, Seguritan V, Li W, Long T, Klitgord N, Bhatt A, Dulai PS, Caussy C, Bettencourt R, Highlander SK, Jones MB, Sirlin CB, Schnabl B, Brinkac L, Schork N, Chen CH, Brenner DA, Biggs W, Yooseph S, Venter JC, Nelson KE. Gut Microbiome- Based Metagenomic Signature for Non-invasive Detection of Advanced Fibrosis in Human Nonalcoholic Fatty Liver Disease. Cell Metab May 2;25(5): Caussy C, Hsu C, Lo MT, Liu A, Bettencourt R, Ajmera VH, Bassirian S, Hooker J, Sy E, Richards L, Schork N, Schnabl B, Brenner DA, Sirlin CB, Chen CH, Loomba R; Genetics of NAFLD in Twins Consortium. Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD. Hepatology Mar 23. [Epub ahead of print] 21. Hagström H, Nasr P, Ekstedt M, Kechagias S, Önnerhag K, Nilsson E, Rorsman F, Sheikhi R, Marschall HU, Hultcrantz R, Stål P. Low to moderate lifetime alcohol consumption is associated with less advanced stages of fibrosis in non-alcoholic fatty liver disease. Scand J Gastroenterol Feb;52(2):

41 Stavra A. Xanthakos, MD, MS Cincinnati Children s Hospital Medical Center University of Cincinnati College of Medicine Cincinnati, OH stavra.xanthakos@cchmc.org Phenotyping and Subtyping the Pediatric Population While pediatric NAFLD shares features seen in adult-onset NAFLD, it also has distinct histological and epidemiological characteristics. A smaller proportion of children have NAFLD, approximately 10% in the United States, compared to the 24-32% estimated rates in adults. The earlier onset of disease may reflect the interaction of genetic traits with the heightened pediatric susceptibility to a variety of environmental risk factors for NAFLD. Though highly heritable, NAFLD is not a monogenic condition with a well-defined pathogenic pathway. Various genetic polymorphisms have been linked to prevalence and severity of disease in both children and adults, but a recent study has also identified novel polymorphisms associated with histological disease severity in children with NAFLD, not previously described in adults. Further, a number of environmental factors, including early life prenatal exposures, nutritional and microbial factors, and potentially chemical exposures, may uniquely contribute to pathogenesis in children. These complex gene-environment interactions may in part account for the ongoing challenge in identifying one highly and broadly efficacious pharmaceutical target for NAFLD. An increased recognition of phenotypes and subtypes within pediatric NAFLD may ultimately help illuminate reasons for variable outcomes and response to interventions, opening the pathway to more personalized and effective treatments. The most well-known distinction in pediatric NAFLD is the unique portal and periportal zone 1 pattern of steatosis, inflammation and/or fibrosis more commonly found in children. This pattern appears to be influenced significantly by age, as it is most common among younger children. More typical adult histological features of zone 3 centered injury with more prominent ballooning degeneration, lobular inflammation and perisinusoidal fibrosis are more likely to emerge among adolescents. It is unclear whether the pediatric portal-predominant pattern of disease will carry different long-term risks or outcomes, or whether it may respond differently to treatment. In a recent clinical trial of cysteamine bitartrate in children with NAFLD, subgroup analyses indicated that the pediatric zone 1 pattern was more prevalent among responders to cysteamine compared to placebo. Finally, while bridging fibrosis has been reported in about 15-20% of pediatric cohorts, cirrhosis is very rare compared to adult cohorts This may in part reflect shorter duration of disease, as older age has been associated with severity of fibrosis in adults. Conversely, some pediatric studies have identified higher severity of fibrosis among younger age children compared to adolescents. Biological sex is also associated with the prevalence of NAFLD. Most studies show a male predominance in both children and adults with NAFLD, up until menopause in women, likely related to the influence of sex hormones in the disease pathogenesis. Underscoring the role of sex hormones, women with polycystic ovarian syndrome (PCOS) and hyperandrogenism (classical phenotype) have a higher prevalence of NAFLD compared to women with PCOS and no hyperandrogenism. It is unclear whether the lower prevalence of the portal predominant NASH pattern in adolescence is related to sex hormone changes during and after puberty. As in adults, pediatric NAFLD prevalence also varies by racial and ethnic groups. The highest prevalence of hepatic steatosis has been identified in children of Hispanic ethnicity, Caucasian and Asian race in multi-ethnic cohorts. While children of African heritage have the lowest rates 37

42 of NAFLD in these studies, NAFLD and NASH can occur in all races and ethnicities. These racial and ethnic disparities may be linked to variation in genetic risk factors but require further investigation. Comorbidity burden is also variable among pediatric patients with NAFLD pointing to heterogeneity in pathogenesis of NAFLD. While obesity, insulin resistance or impaired glycemic control, and cardiovascular disease risk factors are highly prevalent among children with NAFLD, these risk factors can be variably present. Prediabetes (23.4%) and diabetes (6.5%) among children with NAFLD is associated with greater odds of having NASH, but are not a prerequisite for developing NAFLD or NASH. For example, a proportion of children with NAFLD are likely to have familial heterozygous hypobetalipoproteinemia marked by very low total cholesterol and LDL cholesterol levels, but generally normal insulin resistance. Obstructive sleep apnea has also been associated with NASH in several pediatric studies, independent of BMI and standard metabolic risk factors. While comorbidity burden varies considerably, meeting criteria for the metabolic syndrome does appear to be associated with greater histological severity among children with NAFLD. Finally, the degree of overweight and obesity can vary significantly among pediatric cohorts with NAFLD. For example, mean BMI in many pediatric studies in the United States ranges from 33 to 34 kg/m2, whereas mean BMIs in European or Asian studies in children of similar ages are often lower (BMI kg/m 2 ). Lean NAFLD may also be more common among Asian cohorts. Pediatric bariatric surgery cohorts have the highest BMI ranges (mean kg/m 2 ) as expected. Increasing BMI is sometimes identified as a risk factor for increased severity of disease in pediatric studies, but the severity of NAFLD among children of varying BMI category (lean, overweight, obese, severely obese) has not been well characterized in children. The wide range in weight that can be seen among children with NAFLD must also be considered in clinical trials when determining drug dosing strategies as it may impact pharmacokinetics and even adherence. Due to lack of long term prospective natural history studies in children as they age into adulthood, the impact of histological and biological heterogeneity on liver and CVD-related outcomes among children with NAFLD remains unknown. In addition, robust phenotyping of these characteristics in pediatric clinical trials is critical to discern if histological phenotypes and clinically or genetically definable sub-types impact efficacy of the interventions in children. Ultimately, treatment selection targeted to specific biological phenotypes within NAFLD, rather than a one-size-fits-all approach, may lead to better health and quality of life outcomes for both children and adults with NAFLD. References 1. Alqahtani A. et al. Features of nonalcoholic steatohepatitis in severely obese children and 2. adolescents undergoing sleeve gastrectomy SOARD 2017;l13: Della Corte C et al. Fatty liver and insulin resistance in children with hypobetalipoproteinemia: the importance of etiology. Clinical Endocrinology 2013;79: Kleiner DE and Makhlouf HR. Histology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults and children. Clin Liver Disease 2016:20: Newton KP et al. Prevalence of Prediabetes and Type 2 Diabetes in Children with Nonalcoholic Fatty Liver Disease. JAMA Pediatr Oct 3;170(10):e Patton HM. Association between metabolic syndrome and liver histology among children with nonalcoholic fatty liver disease. American Journal of Gastroenterology 2010; 105(9):

43 7. Santoro N. et al: A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology 2010; 52: Schwimmer JB et al. Prevalence of fatty liver in children and adolescents. Pediatrics Oct;118(4): Suzuki et al. Association between puberty and features of nonalcoholic fatty liver disease. Clinical Gastroenterology and Hepatology 2012;10: Wattacheril J et al. Genome-wide associations related to hepatic histology in nonalcoholic fatty liver disease in Hispanic Boys. J Pediatr 2017;190: Xanthakos et al. High prevalence of nonalcoholic fatty liver disease in adolescents undergoing bariatric surgery. Gastroenterology 2015;149:

44 Richard L. Ehman, MD Mayo Clinic Rochester, MN Elastography and Imaging for Staging NAFLD Introduction The traditional role of imaging in patients with chronic liver disease has been to assess anatomic changes that might indicate the presence of cirrhosis and to detect complications of advanced liver disease such as varices and hepatic malignancy. The advent of quantitative imaging technologies that can noninvasively estimate important histologic changes in the liver has markedly expanded the role of imaging in patients with NAFLD and other diseases. Hepatic Steatosis MRI-based methods for quantitatively estimating hepatic proton density fat fraction (PDFF) have been available for several decades. Advanced versions of this technology are now available for all major MRI systems, providing reliable measurements of PDFF with an imaging time of less than 1 minute (1). Ultrasound (US)-based methods for estimating hepatic fat fraction (based on the relationship been fat content and attenuation) have also been developed, exemplified by the CAP parameter provided by transient elastography (TE) systems. Both MRI and US technologies have been shown to be useful in clinical practice. However, most studies comparing these techniques have concluded that MRI-based PDFF measurements have higher diagnostic performance than US-based estimates (2,3). Hepatic Fibrosis Liver fibrosis usually causes little or no anatomic change in the liver until advanced disease is present. Therefore, conventional anatomic imaging technologies like CT, US, and MRI are unreliable for assessing this condition. Many quantitative biomarkers accessed by US, CT, and MRI have been explored as potential indicators of hepatic fibrosis (4,5). Among these, MRIbased proton diffusion metrics, MRI-based T1 relaxation time mapping, and US-based and MRIbased elastography have received most attention. Publications have demonstrated a significant relationship between apparent diffusion coefficient (ADC) and fibrosis stage but multiple comparative studies have indicated that the diagnostic performance is limited compared with elastography (6). Quantiative assessment of T1 relaxation time in the liver has been advocated as a method for diagnosing liver fibrosis (7). However, recent studies have shown that T1 mapping is not a reliable method for assessing liver fibrosis (8). Fibrosis is well known to substantially increase the mechanical stiffness of liver tissue. Liver stiffness progressively increases with the fibrosis stage such that a cirrhotic liver can be as much ten times harder than normal. This observation has provided motivation for developing technologies for quantitatively assessing the stiffness of liver tissue. Conventional US and MR imaging techniques do not provide such information. Methods developed over the last two decades for quantitatively assessing tissue mechanical properties are collectively known as shear wave elastography techniques. They are based on the principle that the speed of a propagating mechanical shear wave in a medium is governed by the stiffness of the medium. Ultrasound-based shear wave elastography technologies use shear waves generated by a surface vibrator (Transient Elastography) or by an acoustic radiation force impulse (ARFI) generated by focused ultrasound (Point Shear Wave Elastography and 2D Shear Wave Elastography). The speed of the resulting propagating shear waves is measured with 1D or 2D speckle-tracking ultrasonography. The performance of US-based elastography has been most 40

45 extensively tested for assessing fibrosis. The AUROC for TE in diagnosing stage F3 and higher fibrosis has been reported to be in the range of The Point and 2D shear wave elastography technologies were developed more recently than TE, so the validating literature is smaller but growing. The trend in these reports is that these newer techniques probably perform slightly better than TE. Advantages of US-based elastography include the fact that TE is widely-available. US-based elastography equipment is portable, exams are quick, and exam cost should be low. Disadvantages include technical failures and unreliable results due to obesity and ascites and potential sampling error, reducing diagnostic performance. Magnetic resonance elastography (MRE) is a technology that is now widely available (over 1000 sites worldwide). Shear waves are generated in the liver with a source of vibrations placed against the body wall. The resulting shear waves are imaged with a special MRI sequence and the resulting data are automatically processed to generate quantitative cross-sectional images, showing tissue stiffness. Imaging time is short: about 1 minute. A pooled meta-analysis of 12 published studies found that the sensitivity, specificity, and AUROC for diagnosing stage F3 and higher fibrosis are 85%, 85%, and.093, respectively (9). Several studies have compared MRE with US-based elastography in assessing hepatic fibrosis and most have demonstrated that MRE has higher diagnostic performance and lower technical failure rates (2,3,10,11). Advantages of MRE include the large volume of liver evaluated with the technique, uniform implementation across MRI systems, low operator dependence, ability to obtain reliable measurements in the presence of obesity and ascities, and high test-retest repeatability. There is emerging consensus that MRE has the closest correlation to liver biopsy of all non-invasive techniques. Disadvantages include higher exam cost, failures due to claustrophobia, and lack of portability. NASH, NAS-Score, Inflammation Multiple studies have shown that the onset of NASH is associated with increased liver stiffness. While many patients with elevated liver stiffness have fibrosis, the presence of necroinflammatory changes alone, without fibrosis, is also associated with modest elevation of liver stiffness. The extent to which stiffness alone, or in conjunction with other biomarkers such as MRE-derived damping ratio or T1 mapping can reliably identify NASH prior to the onset of fibrosis, or quantify inflammation, is still being explored (12). Recent reports have also suggested the possibility of using multiparametric MRI/MRE to non-invasively predict NAS- Score in NAFLD patients, using artificial intelligence technology (13). References 1. Yokoo T, Serai SD, Pirasteh A, Bashir MR, Hamilton G, Hernando D, Hu HH, Hetterich H, Kühn JP, Kukuk GM, Loomba R, Middleton MS, Obuchowski NA, Song JS, Tang A, Wu X, Reeder SB, Sirlin CB; RSNA-QIBA PDFF Biomarker Committee. Linearity, Bias, and Precision of Hepatic Proton Density Fat Fraction Measurements by Using MR Imaging: A Meta-Analysis. Radiology Feb;286(2): doi: /radiol Epub 2017 Sep Imajo K, Kessoku T, Honda Y, Tomeno W, Ogawa Y, Mawatari H, Fujita K, Yoneda M, Taguri M, Hyogo H, Sumida Y, Ono M, Eguchi Y, Inoue T, Yamanaka T, Wada K, Saito S, Nakajima A. Magnetic resonance imaging more accurately classifies steatosis and fibrosis in patients with nonalcoholic fatty liver disease than transient elastography. Gastroenterology. 2016;150: Park CC, Nguyen P, Hernandez C, Bettencourt R, Ramirez K, Fortney L, Hooker J, Sy E, Savides MT, Alquiraish MH, Valasek MA, Rizo E, Richards L, Brenner D, Sirlin CB, Loomba R. Magnetic resonance elastography vs transient elastography in detection of fibrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease. Gastroenterology. 2017;152:

46 4. Petitclerc L, Sebastiani G, Gilbert G, Cloutier G, Tang A. Liver fibrosis: Review of current imaging and MRI quantification techniques. J Magn Reson Imaging May;45(5): doi: /jmri Epub 2016 Dec Venkatesh SK, Ehman RL. Magnetic resonance elastography of liver. Magn Reson Imaging Clin N Am 2014; 22: Wang QB, Zhu H, Liu HL, Zhang B. Performance of magnetic resonance elastography and diffusion-weighted imaging for the staging of hepatic fibrosis: A meta-analysis. Hepatology. 2012;56(1): Banerjee R, Pavlides M, Tunnicliffe EM, Piechnik SK, Sarania N, Philips R, Collier JD, Booth JC, Schneider JE, Wang LM, Delaney DW, Fleming KA, Robson MD, Barnes E, Neubauer S. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol Jan;60(1): doi: /j.jhep Epub 2013 Sep Eddowes PJ, McDonald N, Davies N, Semple SIK, Kendall TJ, Hodson J, Newsome PN, Flintham RB, Wesolowski R, Blake L, Duarte RV, Kelly CJ, Herlihy AH, Kelly MD, Olliff SP, Hübscher SG, Fallowfield JA, Hirschfield GM. Utility and cost evaluation of multiparametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease. Aliment Pharmacol Ther Mar;47(5): doi: /apt Epub 2017 Dec Singh S, Venkatesh SK., Wang Z, et al. Diagnostic performance of magnetic resonance elastography in staging liver fibrosis: a systematic review and meta-analysis of individual participant data Clin Gastroenterol Hepatol Mar;13(3): e6. doi: /j.cgh Epub 2014 Nov Cui J, Heba E, Hernandez C, Haufe W, Hooker J, Andre MP, Valasek M, Aryafar H, Sirlin CB, Loomba R. Magnetic resonance elastography is superior to acoustic radiation force impulse for the diagnosis of fibrosis in patients with biopsy-proven nonalcoholic fatty liver disease: a prospective study. Hepatology. 2016;63: Chen J, Yin M, Talwalkar JA, Oudry J, Glaser KJ, Smyrk TC, Miette V, Sandrin L, Ehman RL. Diagnostic performance of MR elastography and vibration-controlled transient elastography in the detection of hepatic fibrosis in patients with severe to morbid obesity. Radiology. 2017;283(2): Yin M, Glaser KJ, Manduca A, Mounajjed T, Malhi H, Simonetto DA, Wang R, Yang L, Mao SA, Glorioso JM, Elgilani FM, Ward CJ, Harris PC, Nyberg SL, Shah VH, Ehman RL. Distinguishing between Hepatic Inflammation and Fibrosis with MR Elastography. Radiology Sep;284(3): doi: /radiol Epub 2017 Jan Allen AM, et al. Novel multiparametric magnetic resonance elastography (MRE) protocol accurately predicts NAS score for NASH diagnosis. DOI: 42

47 Quentin M. Anstee PhD, FRCP Institute of Cellular Medicine Newcastle University Newcastle-Upon-Tyne United Kingdom Clinical Events: Liver-Related Outcomes Non-Alcoholic Fatty Liver Disease (NAFLD) is the most frequent cause of persistently elevated liver enzymes in North America and Europe and has rapidly become a common cause of end-stage liver disease. Indeed, data from United States adult UNOS database indicates that the number of adults with NAFLD awaiting liver transplant has almost tripled since 2004 (1). In addition, the presence of NAFLD is an independent risk factor for development of cardiovascular disease and stroke (2). However, an important paradox exists: whilst the majority of individuals that exhibit features of the metabolic syndrome develop steatosis, explaining why NAFLD is highly prevalent in the general population, only a minority will progress to advanced liver disease characterized by hepatic fibrosis, cirrhosis or hepatocellular carcinoma (HCC) and experience liver related morbidity (2). There are limited prospective data evaluating the progression of NAFLD to cirrhosis, hepatic decompensation or hepatocellular carcinoma (HCC). Based on available evidence from multiple-biopsy studies(3, 4) and longitudinal cohort studies(5-7), it appears that that a highly dynamic bi-directional transit between the states of NAFL and NASH occurs within the liver. Subsequent fibrosis progression in most NAFLD patients is generally slow, taking approximately 8-years to progress from stage F0 to stage F1. There is however a subgroup of NAFLD patients that are rapid progressors who can progress 3-4 stages within 2-6 years (4). In general, the risk of progression to cirrhosis and hepatic decompensation is low, with an estimated incidence of 3.1% for these end-points during a mean 7.6 year follow-up (8). However, the prevalence of NAFLD-cirrhosis may be under-estimated, as levels of steatosis often fall as disease progresses to cirrhosis leading to misclassification as cryptogenic cirrhosis (9). NAFLD patients exhibit increased mortality. In the US based PREHLIN study, deaths caused by complications of cirrhosis, HCC, or liver transplantation occurred in 18 of 193 patients (9.3%) (7). Similarly, in a Scandinavian study with a mean 26.4 years follow-up, a hazard ratio 1.29 (95%CI , P=0.020) was reported, due to an increased risk of cardiovascular disease, hepatocellular carcinoma and cirrhosis compared with the background population (5). Development and progression of hepatic fibrosis have consistently been demonstrated to be the strongest predictors of disease outcomes in NAFLD(5-7). This is well described in the metaanalysis of longitudinal data conducted by Dulai et al where, in a combined cohort of 1,495 NAFLD cases, liver-related mortality increased exponentially with each increase in the stage of fibrosis: stage 1, 1.41 (95%CI ); stage 2, 9.57 ( ); stage 3, ( ); and stage 4, ( ) (6). Once cirrhosis has developed, the risk of developing a major complication of portal hypertension is 17%, 23%, and 52% at one, three, and 10 years, respectively (10) and survival rates thereafter decline rapidly following hepatic decompensation events(11). References 1. Wong RJ, Aguilar M, Cheung R, Perumpail RB, Harrison SA, Younossi ZM, Ahmed A. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology 2015;148:

48 2. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 2013;10: McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications for prognosis and clinical management. J Hepatol 2015;62: Singh S, Allen AM, Wang Z, Prokop LJ, Murad MH, Loomba R. Fibrosis Progression in Nonalcoholic Fatty Liver vs Nonalcoholic Steatohepatitis: A Systematic Review and Meta-analysis of Paired-Biopsy Studies. Clin Gastroenterol Hepatol 2015;13: e Ekstedt M, Hagstrom H, Nasr P, Fredrikson M, Stal P, Kechagias S, Hultcrantz R. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology 2015;61: Dulai PS, Singh S, Patel J, Soni M, Prokop LJ, Younossi Z, Sebastiani G, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology 2017;65: Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, Mills PR, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 2015;149: e Dam-Larsen S, Becker U, Franzmann MB, Larsen K, Christoffersen P, Bendtsen F. Final results of a long-term, clinical follow-up in fatty liver patients. Scand J Gastroenterol 2009;44: Caldwell SH, Oelsner DH, Iezzoni JC, Hespenheide EE, Battle EH, Driscoll CJ. Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease. Hepatology 1999;29: Singal AK, Guturu P, Hmoud B, Kuo YF, Salameh H, Wiesner RH. Evolving frequency and outcomes of liver transplantation based on etiology of liver disease. Transplantation 2013;95: Agopian VG, Kaldas FM, Hong JC, Whittaker M, Holt C, Rana A, Zarrinpar A, et al. Liver transplantation for nonalcoholic steatohepatitis: the new epidemic. Ann Surg 2012;256:

49 Michelle T. Long, MD Boston University School of Medicine Boston, MA Extra-hepatic Outcomes Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity, dyslipidemia, type 2 diabetes mellitus, and increased cardiovascular morbidity and mortality 1, 2. NAFLD is a multisystem disease with many potential extra-hepatic manifestations 3, 4. A bi-directional relationship exists between NAFLD and cardiometabolic disease, where NAFLD is a risk factor for incident cardiovascular disease and cardiovascular disease risk factors increase the incidence of NAFLD 5. The potential mechanism which drive metabolic syndrome and cardiovascular disease may also cause NAFLD and NAFLD may contribute to cardiovascular disease risk by both propagating and exacerbating these disease mechanisms creating a vicious cycle 6. As such, it is possible that treatments for NAFLD and non-alcoholic steatohepatitis (NASH) may have secondary effects on comorbid diseases. When designing clinical trials, it important to capture information related to comorbid diseases in the form of secondary endpoints. The choice of secondary endpoint relies both on patient factors and therapy factors. Patient selection is important so investigators can be deliberate about including or excluding patients with certain comorbidities. It is critical to assess how well any underlying conditions are under control prior to enrolling in a clinical trial 7. The mechanism of action of the investigational drug should be consider so that any potential secondary or off-target effects can be anticipated and study subjects can be monitored appropriately to ensure safety. The goals of extra-hepatic secondary endpoints in NASH studies are to ensure patient safety and to demonstrate a neutral (at least) or beneficial (ideally) impact on important related conditions 8. There are numerous examples of potential secondary endpoints which should be considered in NASH trial design. Though we are most interested in hard patient outcomes, it is often not practical to have hard endpoints in early- or mid-stage clinical trials because of the low incidence of events and the large number of patients required to study these endpoints. As such, we rely on surrogate endpoints to suggest safety and test for possible secondary or offtarget effects. Depending on the patient population, a number of potential secondary endpoints should be considered. In general, measuring blood pressure, lipid parameters and body weight should be considered for all studies. During the presentation we will discuss other potential surrogate markers to consider and how surrogates endpoints should be selected. We will discuss limitations of surrogate endpoints, including their poor correlation with clinical outcomes and reasons why this may occur. We will discuss lessons learned from drug development in type 2 diabetes and cardiovascular disease. Finally, we discuss use of proteomics to assist in predicting off target effects of therapies. References 1. Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 2015;149: e Targher G, Day CP, Bonora E. Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N Engl J Med 2010;363: Armstrong MJ, Adams LA, Canbay A, et al. Extrahepatic complications of nonalcoholic fatty liver disease. Hepatology 2014;59:

50 4. VanWagner LB, Rinella ME. Extrahepatic Manifestations of Nonalcoholic Fatty Liver Disease. Curr Hepatol Rep 2016;15: Ma J, Hwang SJ, Pedley A, et al. Bi-directional analysis between fatty liver and cardiovascular disease risk factors. J Hepatol 2017;66: Francque SM, van der Graaff D, Kwanten WJ. Non-alcoholic fatty liver disease and cardiovascular risk: Pathophysiological mechanisms and implications. J Hepatol 2016;65: Sanyal AJ, Brunt EM, Kleiner DE, et al. Endpoints and clinical trial design for nonalcoholic steatohepatitis. Hepatology 2011;54: Sanyal AJ, Friedman SL, McCullough AJ, et al. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop. Hepatology 2015;61:

51 Valerio Nobili, MD Bambino Gesù Children s Hospital IRCCS Rome Italy nobili66@yahoo.it Pediatrics Perspective on Outcomes and Clinical Events Nonalcoholic steatohepatitis, a progressive form of nonalcoholic fatty liver disease (NAFLD), is one of the most common hepatic diseases in children who present with particular risk factors including obesity, sedentary lifestyle, and/or a predisposing genetic background. The worldwide prevalence of NAFLD in children is a worrying phenomenon because this disease is closely associated with the development of both cirrhosis and cardiometabolic syndrome in adulthood. Several studies have demonstrated a prevalence of 3 to 10% in general pediatric populations, which increases up to 60 to 70% in individuals with metabolic comorbidities. However, NAFLD prevalence varies widely depending on geographical area and diagnostic methods used. To date, the etiopathogenesis of primary NAFLD in children is unknown. Understanding the pathogenetic mechanisms provides the basis to characterize early predictors of the disease and noninvasive diagnostic tools and to design novel specific treatments and possible management strategies. There is much heterogeneity in investigations of NAFLD in children,particularly in regard to the use of biopsy, and establishing biomarkers that correlate with histology progression and clinical outcomes remains a major target. The treatment of pediatric NAFLD can be divided into conservative, medical, and surgical approaches. The goals are multidimensional: improve the metabolic health of children to reduce their long-term cardiovascular risk and reduce liver-related clinical events, presumably by targeting fibrosis. Weight loss is the core therapy for pediatric NAFLD and for all children with obesity. This may be achieved by dietary modification and/or physical activity. A variety of diets have been used in randomized and nonrandomized clinical trials. There are no approved pharmacological therapies of pediatric NAFLD. The main groups of agents that have been tested to date are antioxidants, metformin, polyunsaturated fatty acids (PUFAs), probiotics, and vitamin D. However The American Association for the Study of Liver Diseases (AASLD) guidance recommends vitamin E as the only therapy that is potentially efficacious in pediatric NAFLD. Further investigation into the pathogenesis of pediatric NAFLD is needed to complement translational studies, with an aim to develop novel therapeutic strategies. There are only a few agents in trials in children, and drug development is progressing at a much slower rate than in adults. To date despite major advances in understanding, research in pediatric NAFLD has not yet translated into patient benefit. It is an important condition with unmet scientific and clinical needs that requires urgent attention to slow the future epidemic of end-stage liver disease in adults. References 1. Nobili V, Svegliati-Baroni G, Alisi A, Miele L, Valenti L, Vajro P. A 360-degree overview of paediatric NAFLD: recent insights. J Hepatol 2013;58(06): Mencin AA, Lavine JE. Nonalcoholic fatty liver disease in children. Curr Opin Clin Nutr Metab Care 2011;14(02):

52 3. Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67(01): Vajro P, Mandato C, Franzese A, et al. Vitamin E treatment in pediatric obesity-related liver disease: a randomized study. J Pediatr Gastroenterol Nutr 2004;38(01): BoyrazM,PirgonÖ,DündarB,ÇekmezF,HatipoğluN.Long-term treatment with n-3 polyunsaturated fatty acids as a monotherapy in children with nonalcoholic fatty liver disease. J Clin Res Pediatr Endocrinol 2015;7(02): Goss A. ACarbohydrate-Restricted Diet to Reverse Fatty Liver in Adolescents with Obesity. Birmingham, AL: University of Alabama at Birmingham; Alisi A, Bedogni G, Baviera G, et al. Randomised clinical trial: the beneficial effects of VSL#3 in obese children with non-alcoholic steatohepatitis. AlimentPharmacolTher2014;39(11):

53 Jörn M. Schattenberg, MD University Medical Center Mainz Germany Patient-related Outcomes Assessing subjective well-being is an important aspect in everyday patient care. As a matter of fact, patient reported quality of life (QOL) has likely a strong influence on a physician s decision to order further diagnostic testing and initiate or change a therapeutic measure. The attempt to standardize patient reported outcomes (PRO) in the setting of a clinical trial or beyond this during regulatory approval, is hampered by the difficulties to objectively measure PRO. QOL is influenced by many dimensions and represents the sum of positive and negative subjective aspects. Additionally, different QOL domains are not uniformly weighted by everyone. While health is clearly among the most important domains, other modulators including culture, values, and spirituality aspects can also have a strong impact. Health-related quality of life (HRQL) more specifically refers to the aspects that influence QOL through physical or mental health. At an individual level HRQL summarizes physical and mental health perceptions including health risks, global functional status, social support, and socioeconomic status as defined by the CDC (1). Three most commonly used generic HRQL instruments include the Medical Outcomes Study Short Form-36 (SF-36), the Nottingham Health Profile (NHP), and the Sickness Impact Profile (SIP). HRQL assessment has become an important component of public health surveillance and are generally considered a valid indicator of unmet needs and intervention outcomes (1). Three major domains that are assessed include physical, psychological, and social functioning (2). Importantly, HRQL is based on self-assessed health status and as such does not require expensive resources and can be a powerful predictor of outcome. In the context of chronic liver disease, different generic instruments have been developed (3). These vary greatly from the more general health-assessment questionnaires, considering that liver-specific symptoms are often subtle and related to GI-complaints or fatigue. The most widely used liver disease-specific measure is the chronic liver disease questionnaire (CLDQ), which comprises 29 questions in six domains with domain-specific in addition to an overall score (4). The liver disease quality of life (LDQOL) measure uses SF- 36v2 as a generic core and adds 12 liver-specific scales comprising of a total of 75 questions. A shorter version, SF-LDQOL, has also been developed with 36 liver-specific questions split into nine scales (5). Considering the good utility of the CLDQ, most data in patients with chronic liver disease has been generated using this tool. Both gender and the severity of liver disease have been linked to the HRQL (6). Importantly, the degree of liver injury appears to influence the degree of HRQL impairment independently of the underlying liver disease (7). In HCV, this has translated to improvement in CDLQ scores following HCV cure, even independently of the severity of fibrosis (8). In the context of NAFLD, there is only sparse data available, but the CLDQ has been suggested as a valid instrument (9). In a recent report at the AASLD Liver Meeting in 2017, a correlation of improvement in hepatic fibrosis in patients with NASH and F2-F3 at baseline was associated with improved HRQL (10). Thus further assessment and refinement of HRQL tools in the context of NASH is warranted. The lecture will address evidence on the use of HRQL questionnaires in patients with NASH looking at both strength and weaknesses. 49

54 References 1. CDC. Measuring healthy days: Population assessment of health-related quality of life. Atlanta, Georgia; Gutteling JJ, de Man RA, Busschbach JJ, Darlington AS. Overview of research on healthrelated quality of life in patients with chronic liver disease. Neth J Med 2007;65: FDA. Guidance for Industry Patient-Reported Outcome Measures: Use in Medical Product Development to Support Labeling Claims Silver Spring; 2009; p Younossi ZM, Guyatt G, Kiwi M, Boparai N, King D. Development of a disease specific questionnaire to measure health related quality of life in patients with chronic liver disease. Gut 1999;45: Kanwal F, Spiegel BM, Hays RD, Durazo F, Han SB, Saab S, Bolus R, et al. Prospective validation of the short form liver disease quality of life instrument. Aliment Pharmacol Ther 2008;28: Afendy A, Kallman JB, Stepanova M, Younoszai Z, Aquino RD, Bianchi G, Marchesini G, et al. Predictors of health-related quality of life in patients with chronic liver disease. Aliment Pharmacol Ther 2009;30: Alt Y, Grimm A, Schlegel L, Grambihler A, Kittner JM, Wiltink J, Galle PR, et al. The Impact of Liver Cell Injury on Health-Related Quality of Life in Patients with Chronic Liver Disease. PLoS One 2016;11:e Younossi ZM, Stepanova M, Afdhal N, Kowdley KV, Zeuzem S, Henry L, Hunt SL, et al. Improvement of health-related quality of life and work productivity in chronic hepatitis C patients with early and advanced fibrosis treated with ledipasvir and sofosbuvir. J Hepatol 2015;63: Chawla KS, Talwalkar JA, Keach JC, Malinchoc M, Lindor KD, Jorgensen R. Reliability and validity of the Chronic Liver Disease Questionnaire (CLDQ) in adults with nonalcoholic steatohepatitis (NASH). BMJ Open Gastroenterol 2016;3:e Younossi Z, Stepanova M, Goodman ZD, Lawitz E, Charlton MR, Loomba R, Myers RP, et al. Improvements of Hepatic Fibrosis and Hepatic Collagen Deposition in Non- Alcoholic Steatohepatitis (NASH) Treated with Selonsertib are Associated with Improvement of Patient-Reported Outcomes (PROs). In: AASLD San Francisco; p. 1170A. 50

55 Naga P. Chalasani, MD, FAASLD Indiana University Medical Center Indianapolis, IN Detection and Management of DILI in NASH/NAFLD Subjects in Drug Development Nonalcoholic fatty liver disease (NAFLD) and its severe sub-phenotype, nonalcoholic steatohepatitis (NASH), have become highly prevalent conditions and they impact clinical trials in two ways. First, many metabolic and endocrine conditions such as dyslipidemia, diabetes, hypertension, gout, hypothyroidism, hypogonadism and hypopituitarism have concomitant NAFLD which may be clinically silent, i.e., may not exhibit elevated serum aminotransferases. Clinical trials investigating therapies for these metabolic and endocrine conditions may enroll patients with NAFLD without actually realizing such a phenomenon. For example, more than 50% of patients participating in type 2 diabetes clinical trials may have underlying NAFLD. Second, NASH has become an important focus for drug development and more than 100 phase1-3 clinical trials are currently registered at Clinicaltrials.gov. How to detect underlying NAFLD and NASH in patients participating in non-nafld/nash clinical trials? NAFLD is diagnosed based on clinical, laboratory and imaging criteria whereas liver biopsy is required for establishing a diagnosis of NASH. Detailed work up for identifying those with underlying bona fide NAFLD would likely be quite difficult practically. Therefore, one might consider serum alanine aminotransferase (ALT) levels as a surrogate for NAFLD but the caveat is that ALT levels can be normal in NAFLD and even in NASH. On a study by study basis, one might consider liver ultrasound, transient elastography or Fatty Liver Index for identifying participants with underlying with NAFLD. Noninvasive tools liver stiffness measured by Fibroscan or FIB4, APRI, or NFS may identify NAFLD with advanced fibrosis. How to detect DILI in patients with NAFLD/NASH who are participating in clinical trials? It has recently been suggested that multiples of baseline of ALT rather than multiples of ULN as a threshold for suspecting DILI. The FDA 2009 guidance has recommended using ALT threshold values of >2x baseline in patients with elevated liver enzymes at enrolment; however, others have suggested an increase of >3x baseline and >5x baseline as more appropriate for a hepatic safety signal. In a recent commentary (Gastro 2016; 151: ), the following criteria were proposed for suspecting DILI in patients with underlying NAFLD or NASH: Those with normal baseline ALT: ALT > 5 x ULN or Total Bilirubin > 2 ULN (if baseline TB is normal, or doubling of direct bilirubin if Gilbert s syndrome) Those with elevated baseline ALT: ALT > 5 x baseline or >300 U/L (whichever occurs first) or Total Bilirubin > 2 ULN (if baseline TB is normal, or doubling of direct bilirubin if Gilbert s syndrome) How to manage suspected DILI in patients with NAFLD/NASH participating in clinical trials? There is no clear-cut guidance from regulators in terms of discontinuation rules for suspected DILI events in clinical trials. Recently suggested algorithm (Gastro 2016; 151: ) for monitoring and management of suspected DILI in phase 2-3 NASH studies in patients with normal and elevated baseline ALT is as follows: 51

56 Treatment emergent ALT Normal baseline: ALT>5x ULN Elevated baseline: ALT> 5x baseline or >300 U/L (whichever occurs first) Normal baseline: ALT>8x ULN Elevated baseline: ALT> 8x baseline or >500 U/L (whichever occurs first) Normal baseline: ALT >5 ULN Elevated baseline: ALT> 5x baseline or >300 U/L (whichever occurs first) Normal baseline: ALT >5 ULN Elevated baseline: ALT> 5x baseline or >300 U/L (whichever occurs first) Treatment emergent Total Bilirubin Normal Patients with Gilbert s syndrome: No change in baseline TBL Normal Patients with Gilbert s syndrome: No change in baseline TBL TBL>2x ULN Patients with Gilbert s syndrome: Doubling of direct bilirubin Normal or elevated Liver symptoms None None None Severe fatigue, nausea, vomiting, right upper quadrant pain Action Repeat ALT, AST, ALP, TBL, in 2-5 days Follow-up for symptoms. Interrupt study drug. Initiate close monitoring and workup for competing etiologies. Study drug can be restarted only if another etiology is identified and liver enzymes return to baseline. Interrupt study drug. Initiate close monitoring and workup for competing etiologies. Study drug can be restarted only if another etiology is identified and liver enzymes return to baseline. Interrupt study drug. Initiate close monitoring and workup for competing etiologies. Study drug can be restarted only if another etiology is identified and liver enzymes return to baseline. *Baseline is the average of two screening ALT measurements. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBL, total bilirubin, ALP-alkaline phosphatase; ULN, upper limit of normal Paying Attention to Autoimmune Hepatitis As more patients are participating in longer duration NASH trials, we are starting to see newly diagnosed AIH emerging during NASH clinical trials. This speaker is aware of few cases of suspected AIH emerging during NASH clinical trials. Some have presented with elevations in ALT whereas others have shown histological evidence on end-of-treatment biopsy. Autoantibodies (ANA and ASMA) are present in a sizable proportion of individuals with NAFLD and NASH and NASH clinical trials enroll a sizable proportion of women who are at risk for AIH. 52

57 Thus, it is plausible that NASH clinical trials population may be risk to exhibit de novo AIH during clinical trials, without necessarily being related to the test compound. Therefore, the clinical trial protocol should carefully consider baseline autoantibody titers, immunoglobulin levels and liver histology to ascertain that there isn t co-existing (perhaps low grade) AIH at baseline and also developing criteria for promptly identifying incident (suspected AIH) during the clinical trial. References 1. Chalasani N, Regev A. Drug induced liver injury in patients with preexisting chronic liver disease in drug development How to identify and manage? Gastroenterology : Vuppalanchi R, Gould RJ, Wilson LA, Unalp-Arida A, Cummings OW, Chalasani N, Kowdley KV, NASH CRN. Relationship between serum autoantibodies and liver histology in patients with NAFLD: results from the Nonalcoholic Steatohepatitis Clinical Research Network. Hepatol Int 2012; 6: Disclosure: This syllabus is an updated version of material that was submitted to the 2017 FAD/AASLD Workshop on DILI 53

58 Detlef Schuppan, MD, PhD University of Mainz Mainz Germany Novel Markers and Technologies to Assess Liver Fibrosis and Remodeling In contrast to e.g. untreated viral hepatitis C which shows a fairly constant rate of fibrosis progression, NASH is characterized by difficult-to-predict phases of fibrosis progression and regression. With advanced fibrosis stage 3-4, the risk of further progression to hard endpoints increases steeply, while the potential of fibrosis regression decreases. Therefore, therapies that prevent further progression or induce regression of advanced fibrosis have becomes a first priority. The development of noninvasive tools that permit the detection of advanced fibrosis with a high predictive value has made significant progress, including imaging modalities like ultrasound & MR elastography, and serum marker combinations like FIB-4, Fibrometer and the enhanced liver fibrosis (ELF) test. Predictive values for F3-4 vs F0-2 are satisfying for elastography, but insufficient in view of the huge numbers of NAFLD patients in the US and worldwide that would need to be screened. This applies even more to the current serum marker panels. Therefore, there is an urgent need for the development and validation of cheap, highly sensitive and specific technologies, preferably serum marker to screen-select patients in need of (antifibrotic>anti-inflammatory) treatment, possibly followed by confirmatory imaging. At least equally important are noninvasive biomarkers for the quantification of the dynamics of liver fibrogenesis, i.e., de novo formation, and especially fibrolysis, i.e., removal of excess fibrotic tissue. Direct fibrosis markers, those related to remodeling of the fibrotic matrix, such as the ELF-panel that consists of P3NP, TIMP-1 and HA, will mainly reflect matrix turnover and possibly fibrogenesis rather than static fibrosis, but most correlations of ELF-levels have been made with liver biopsy stage. Recently, novel serum markers have been developed that appear to more truly reflect fibrogenesis and even predict antifibrotic therapeutic response, as evidenced in well defined clinical studies with patients that display fast vs slow progression, or are treated with indirect antifibrotic agents, such as antivirals for hepatitis B and C, resulting in fibrosis regression. A plausible fibrogenesis marker is Pro-C3, a small peptide epitope that is specific for the aminopropeptide of procollagen type III that is mainly released from the procollagen before collagen fibrils can be deposited. Another novel marker is Pro-C6, the carboxyterminal propeptide of the α3-chain of procollagen type VI, which also serves as a prodiabetic and fibrogenic adipokine (endotrophin). Other novel markers derive from cell surface molecules involved in the processing of the ECM or from matricellular molecules, with markers that either reflect fibrogenesis or fibrolysis. Their combinations may permit a better assessment of the matrix being deposited or removed, especially in patients with advanced fibrosis. The retrospective and prospective validation of these markers in large follow-up cohorts of well defined NAFLD patients, including patients within biopsy controlled clinical trials with novel agents, combined with refined imaging, is currently under way within the EU EPOS and LIMUS and the US NIMBLE initiatives. There is also promise in testing circulating (exosomal) micro-rna, as markers of inflammation and fibrosis, but a more thorough standardization and validation is required. Moreover, evolving multi-omics approaches that encompass genetic, epigenetic, circulating micro-rna, metabolomic and proteomic signatures may allow a personalized and exact classification of patients as to disease stage, activity and progression. 54

59 Apart from the serum biomarkers and elastography, there is also progress in quantitative imaging of both fibrosis and fibrogenesis, using PET- or MR-ligands that either bind to fibrillar collagen or to surface receptors of cells that drive liver fibrogenesis, such as activated myofibroblasts/hepatic stellate cells and fibrogenic cholangiocytes. Since these methods are ontarget and quantitative, they could serve as gold standard especially for further validation of the serum markers. These recent developments will likely permit shorter and smaller proof-of-concept trials. Moreover, they will facilitate personalized antifibrotic therapies, employing an individualized dose titration and use of drug combinations for improved efficacy and reduced side effects. References 1. Baues M, Dasgupta A, Ehling J, et al. Fibrosis imaging: Current concepts and future directions. Adv Drug Deliv Rev 2017;121: Caussy C, Hsu C, Lo MT, et al. Genetics of NAFLD in Twins Consortium. Novel link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD. Hepatology 2018 Mar 23 [Epub ahead of print] 3. Dulai PS, Sirlin CB, Loomba R. MRI and MRE for non-invasive quantitative assessment of hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical practice. J Hepatol 2016;65: Karsdal MA, Henriksen K, Nielsen MJ, et al. Fibrogenesis assessed by serological type III collagen formation identifies patients with progressive liver fibrosis and responders to a potential antifibrotic therapy. Am J Physiol Gastrointest Liver Physiol 2016;311:G1009- G Karsdal MA, Henriksen K, Genovese F, et al. Serum endotrophin identifies optimal responders to PPARγ agonists in type 2 diabetes. Diabetologia 2017;60: Pirola CJ, Sookoian S. Multiomics biomarkers for the prediction of nonalcoholic fatty liver disease severity. World J Gastroenterol 2018;24: Schuppan D, Surabattula R, Wang XY. Determinants of fibrosis progression and regression in NASH. J Hepatol 2018;68: Schuppan D, Ashfaq-Khan M, Yang AT, Kim YO. Liver fibrosis: Direct antifibrotic agents and targeted therapies. Matrix Biol Apr 12 [pubmed ahead of print]. 9. Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepatol 2018;68: Yang J, Li C, Zhang L, Wang X. Extracellular Vesicles as Carriers of Non-coding RNAs in Liver Diseases. Front Pharmacol 2018;9:

60 Frank Tacke, MD, PhD University Hospital Aachen Aachen Germany Which Emerging Signals from Preclinical Models are Most Likely to have Clinical Relevance? Nonalcoholic fatty liver disease (NAFLD) has an increasing prevalence worldwide, and no specific pharmacotherapy is currently approved for NAFLD. Simple steatosis and nonalcoholic steatohepatitis (NASH) can progress to liver fibrosis. Fibrosis might progress to cirrhosis and predisposes to hepatocellular carcinoma (HCC). Moreover, patients with NAFLD are characterized by typical concomitant extrahepatic diseases such as obesity, insulin resistance, type 2 diabetes, metabolic syndrome, cardiovascular diseases and others (1). The lack of specific pharmacotherapy and the increasing medical burden of the disease prompted intense research, both from academia as well as industry, to better understand NAFLD pathogenesis and to identify the most promising therapeutic targets. Ideally, researchers would like to use a NAFLD model that is fast, reproducible, reflects all features of human biology and responds to dietary or pharmacological interventions similar to human patients. To this end, several innovative in vitro models (e.g., liver slices, biochips, organoids) as well as in vivo rodent models (genetic, chemical and/or dietary) have been developed and validated. A core challenge is that none of the current models fully reflects all aspects of human disease. Nonetheless, distinct facets of human NAFLD can be well recapitulated in mouse models, including steatosis, inflammation or fibrosis. There is solid evidence from a large body of experimental studies that signals from preclinical models are to a certain extent able to recapitulate pathogenic mechanisms from human NAFLD and may predict the efficacy of investigational drugs (2, 3). Due to the close association between fibrosis and clinical endpoints in longitudinal studies in humans (4), development of fibrosis is desired in preclinical models. Classical liver fibrosis models like repetitive carbon tetrachloride (CCl4) injections or feeding a methionine and choline-deficient (MCD) diet do not have features of the metabolic syndrome (5), while standard high fat or Western diet feeding does not induce rapid fibrogenesis in mice. Newer dietary formulations or combined interventions (e.g., genetic plus dietary, chemical plus dietary) partially overcome this limitation (2). Nonetheless, there is emerging evidence that not a single magic model might provide best insight into efficacy of new interventions, but the rational use of models that reflect best the pathogenic aspect, which is targeted by a new compound (6). For instance, the anti-fibrotic effects of the chemokine receptor CCR2/CCR5 inhibitor cenicriviroc in patients (7) could be predicted from standard fibrosis models in mice (8), which have a strong pathogenic involvement of CCR2-dependent monocyte recruitment (9). Similar findings can be obtained for the apoptosis signal-regulating kinase 1 (ASK1) inhibitor selonsertib, which showed antifibrotic signals in patients (10). The following principles might help to predict the clinical relevance of emerging signals from preclinical models: The signal should be consistent in more than one experimental model. Ideally, the signal should be also recapitulated in different labs and conditions (e.g., microbiota, mouse strains etc.). The experimental model should well reflect the pathogenic mechanism that is targeted by a drug, and this specific mechanism has been validated in human disease before. 56

61 The beneficial signal is not associated with adverse outcome in regression models (e.g., switching mice from a pathogenic diet to normal chow to induce disease regression). In human disease, progression and regression pathways may occur simultaneously or intermittently. References 1. Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, George J, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 2018;15: Cole BK, Feaver RE, Wamhoff BR, Dash A. Non-alcoholic fatty liver disease (NAFLD) models in drug discovery. Expert Opin Drug Discov 2018;13: Santhekadur PK, Kumar DP, Sanyal AJ. Preclinical models of non-alcoholic fatty liver disease. J Hepatol 2018;68: Dulai PS, Singh S, Patel J, Soni M, Prokop LJ, Younossi Z, Sebastiani G, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology 2017;65: Weiskirchen R, Tacke F. Liver Fibrosis: From Pathogenesis to Novel Therapies. Dig Dis 2016;34: Tacke F. Targeting hepatic macrophages to treat liver diseases. J Hepatol 2017;66: Friedman SL, Ratziu V, Harrison SA, Abdelmalek MF, Aithal GP, Caballeria J, Francque S, et al. A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology 2018;67: Krenkel O, Puengel T, Govaere O, Abdallah AT, Mossanen JC, Kohlhepp M, Liepelt A, et al. Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis. Hepatology 2018;67: Krenkel O, Tacke F. Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol 2017;17: Loomba R, Lawitz E, Mantry PS, Jayakumar S, Caldwell SH, Arnold H, Diehl AM, et al. The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: A randomized, phase 2 trial. Hepatology 2018;67:

62 Brent A. Neuschwander-Tetri, MD, FAASLD Saint Louis University St. Louis, MO Defining the Spectrum of Response to Treatment In routine clinical practice, we often think of our patients as being somewhere on a onedimensional spectrum of NAFLD from mild steatosis with no fibrosis to very active NASH with stage 4 fibrosis. However, in NASH as in other types of chronic liver disease, an accepted conceptual framework separates the severity of ongoing inflammation and injury from the consequence of this disease activity which is fibrosis. 1 Using this separation, the spectrum of NAFLD severity can be visualized in terms of the spectrum of severity of steatohepatitis on one axis and the spectrum of fibrosis severity on separate axis of a plot. Additionally, just as any biopsy can be placed on this plot, the response to therapy can be shown as well. An effective anti-nash drug would reduce NASH severity and would likely indirectly improve fibrosis. By comparison, an effective purely antifibrotic drug (e.g., a drug that the alters the stellate cell response to fibrogenic stimuli) would decrease fibrosis without an impact on the hepatocellular injury and inflammatory response that drives the fibrogenesis. Pretreatment (1) and post-treatment (2) liver biopsies from two hypothetical patients. One is treated with a hypothetical effective anti- NASH drug that results in resolution of NASH (but persistent NAFL) and also has secondary improvement in fibrosis. The second patient receives a hypothetical effective anti-fibrotic agent that improves fibrosis with changing the severity of the underlying NAFLD. The challenge for treatment trials is to identify the best histologic features and biomarkers that reflect NAFLD severity and fibrosis severity (i.e., the axis labels of this plot). Currently, histological assessment of inflammation and hepatocyte ballooning are key histologic assessments of NAFLD severity and the pathologist s staging from 0 (none) to 4 (cirrhosis) using the Brunt/Kleiner staging system or 0 to 6 using the Ishak staging system are often used to assess fibrosis severity. Image analysis of liver biopsy collagen staining is also being evaluated. In the future, the axis labels of this type of plot may evolve to be numerical scores rather than these currently used relatively broad categorical variables. For treat treatment trials, validation of alternative measures will need to link changes along either axis with changes in hard outcomes. 58

63 References 1. Brunt EM, Janney CJ, Di Bisceglie AM, et al. Non-alcoholic steatohepatitis: A proposal for grading and staging the histologic lesions. American Journal of Gastroenterology 1999;94:

64 David E. Kleiner, MD, PhD National Cancer Institute Bethesda, MD What Defines a Clinically Meaningful Response with Respect to Histological Assessment in Pre-cirrhotic NASH? When a pathologist assesses a liver biopsy from a patient with nonalcoholic fatty liver disease (NAFLD) there are several pieces of information in the report that are relevant to a patient s disease management and prognosis. These include the disease diagnosis and evaluation of the severity of the various disease features. In adults with NAFLD, the diagnosis can be divided broadly into two categories: steatohepatitis (NASH) and fatty liver disease which does not meet criteria for NASH. This latter category is often termed nonalcoholic fatty liver (NAFL) to distinguish it from the broader category of NAFLD. The liver biopsy may also show evidence of other liver diseases either alongside or instead of fatty liver disease. However, if there is only evidence of NAFLD, the pathologist will also assess the individual histological features of NAFLD, principally steatosis, ballooning injury, inflammation (portal and lobular) and fibrosis. Other features, such as ductular reaction, may also be significant, but are less well studied. Standard methods exist for the semi-quantitative assessment of these features and these scores are typically used in clinical trials to assess changes in disease in populations under study. Histological changes in liver disease are often grouped as stage or grade. The stage of a disease is a measure of how far the disease has progressed from a normal, non-disease state to organ or system failure. Fibrosis usually serves as the histological surrogate of stage, because cirrhosis is recognized as a point at which the patient is at high risk of liver-related complications and lesser fibrosis stages have prognostic significance. In contrast, the grade of a disease is a measure of how fast the disease is progressing at a particular point in time. Various histological features (or combinations) have been assessed as surrogates of grade, including steatosis, inflammation and ballooning. Of these, the features that have shown the most promise are ballooning and portal inflammation. Evaluating changes of stage and grade have become important tools to measure response to an intervention in NAFLD. The FDA defines a Clinically Meaningful Response to therapy as one in which the patient shows improvement in how they feel, function or survive. NAFLD evolves from beginning to end over decades and liver function is not usually significantly affected until patients reach the stage of cirrhosis. Thus, in patients with pre-cirrhotic NAFLD, the goal of therapy has usually been focused on preventing patients from becoming cirrhotic. In cross-sectional studies of untreated NAFLD that have examined disease progression to clinically important endpoints (death or liver transplantation), fibrosis (disease stage) is frequently identified as either the most powerful or the sole histological predictor of outcome. Because of such studies, attention is often focused on disease stage to the exclusion of other histological findings. This attitude represents a huge oversimplification based on natural history studies unperturbed by effective intervention. If an intervention reduces disease grade (or activity) to zero, fibrosis would not progress and cirrhosis would never develop. Reduction in grade may also lead to a reduction in fibrosis as has been demonstrated in post-therapy biopsies of patients with chronic viral hepatitis. 60

65 Based on these considerations, a typical histological endpoint in NAFLD considers elements of stage and grade typically focusing on reducing grade while observing no worsening of stage. The NASH Clinical Research Network developed a scoring system that uses a composite score that sums the semi-quantitative scores for steatosis, ballooning and lobular inflammation (the NAFLD Activity Score or NAS) as a measure of grade. A 2-point improvement in this 8-point score has been judged to be a useful measure of overall disease improvement in response to interventions. If grade improves while fibrosis does not worsen, then the supposition is that the patient s overall prognosis has been improved at least to the degree that we have reset their disease to a less severe form. Many questions remain: How quickly will the disease recur if the intervention is stopped? Is a patient with a particular stage and grade of NAFLD following therapy at the same point in the natural history curve as an untreated patient at that same stage and grade? Is it meaningful (or possible) to affect stage without affecting grade? In an intervention affects only some aspect of grade, such as steatosis, without affecting other aspects, will that alter the natural history of the disease? It is important to keep these questions in mind as we develop effective therapies for NAFLD. References 1. Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, Mills PR, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Longterm Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 2015;149: e Brunt EM, Kleiner DE, Wilson LA, Unalp A, Behling CE, Lavine JE, Neuschwander-Tetri BA, et al. Portal chronic inflammation in nonalcoholic fatty liver disease (NAFLD): a histologic marker of advanced NAFLD-Clinicopathologic correlations from the nonalcoholic steatohepatitis clinical research network. Hepatology 2009;49: Gadd VL, Skoien R, Powell EE, Fagan KJ, Winterford C, Horsfall L, Irvine K, et al. The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology 2014;59: Gramlich T, Kleiner DE, McCullough AJ, Matteoni CA, Boparai N, Younossi ZM. Pathologic features associated with fibrosis in nonalcoholic fatty liver disease. Hum Pathol 2004;35: Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41: Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research. Semin Liver Dis 2012;32: Neuschwander-Tetri BA, Loomba R, Sanyal AJ, Lavine JE, Van Natta ML, Abdelmalek MF, Chalasani N, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2015;385:

66 Elisabetta Bugianesi, MD, PhD University of Turin Torino Italy Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Pre-cirrhotic NASH Introduction NAFLD is a complex condition related to systemic metabolic derangements common to other organs and systems, including type 2 diabetes, cardiovascular and chronic kidney disease. However, in a subset of patients the liver becomes the target of multiple hits leading to nonalcoholic steatohepatitis (NASH). The presence of steatohepatitis and significant fibrosis are considered harbingers of adverse hepatic outcomes in individuals with NAFLD and are associated with an increased risk for morbidity and mortality through progression to endstage liver disease and HCC [1]. The non-hepatic companion diseases represent the major comorbidities and cardiovascular disease is the main cause of mortality in NAFLD and in the early stages of fibrotic NASH [2]. Liver-related morbidity and mortality only increase significantly beyond stage 1 fibrosis, especially with the emergence of cirrhosis [1, 2]. Thus, prevention and therapy of NASH must address two largely independent targets: the metabolic complications to prevent non-liver related mortality, and moderate to advanced fibrosis to prevent liver-related mortality. Metabolic parameters A clinically meaningful outcome in pre-cirrhotic NASH should have beneficial effects on many of the metabolic risk factors that contribute to the development of liver-related complications [2, 3]. Whether a decrease in insulin resistance also results in an improvement in hepatic fibrosis is debated, but improving the conditions that created NASH may subsequently shut-off the fibrogenic process. Weight reduction is the most straightforward clinical indicator of a meaningful benefit both for the metabolic complications as well as for NASH. A seminal study demonstrated that diet and lifestyle interventions led to resolution of NASH in 90% and improvement of fibrosis in 45% of those patients who lost 10% of body weight [4]. As far as the metabolic syndrome, there are multiple standard fasting or dynamic parameters of glucose homeostasis and lipid alterations that can be measured. Significant reduction of hemoglobin A1C and LDL levels below the risk threshold are well established parameters to monitor the risk of metabolic and CVD related complications. The presence and/or development of T2DM is considered a major determinant of fibrosis progression, hence its prevention or its tight control should also result in a better control of the liver disease [1]. In non-diabetic individuals, improvement in insulin resistance can be demonstrated by surrogate measures, such as homeostatic model assessment of insulin resistance. The adipose tissue insulin resistance index (the product of free fatty acids and fasting insulin) can be a dynamic marker of lipids overflow to the liver, which is not necessary reflected by fasting lipid levels [5]. Clinical data suggest that an improvement in insulin sensitivity coexists with an improvement in hepatic necroinflammation, probably due to a reduction in lipotoxicity or to increases in adipokines with anti-inflammatory properties [5]. Metabolic improvement is usually associated with a reduction in steatosis. Imaging modalities for a precise quantification of steatosis are now available using magnetic resonance spectroscopy [6] and MRI-based proton density fat fraction (PDFF) [7]. The latter has the advantage of being easier to implement, even on different MRI machines. Paired 62

67 histology-mri-pdff data from a subset of the FLINT trial has shown that a 5 6% absolute change in PDFF correlates with steatosis improvement or aggravation as measured by liver biopsy [8]. Another study has shown the same correlation with histology for a 30% relative change [9]. Parameters of Liver damage Most liver related outcomes occur once cirrhosis has developed and progression to liver cirrhosis has been accepted by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as part of the composite primary endpoint (suggesting an impact on clinical liver outcomes) in clinical trials [3]. Validated surrogates are biomarkers for which there are data to show that the biomarker does predict clinical outcomes. For example, hemoglobin A1C is a validated surrogate endpoint in diabetes. Resolution of NASH (i.e., absence of ballooning with no or minimal inflammation by histology) and improvement in fibrosis have been accepted as surrogate endpoints, but they do require liver biopsy [3]. The use of noninvasive biomarkers in this context is still considered experimental, and there are no noninvasive biomarkers that meet the requirements for a surrogate likely to predict clinical benefit. Liver disease activity In NASH, progression to cirrhosis is not linear but it is rather the extent and length of the reparative process relative to the inflammatory periods that may determine the speed of fibrosis progression. This may explains why surrogate serum markers of inflammation, such as ALT, show an overall correlation with the risk of fibrosis progression in larger cohorts, but have a limited ability to predict progression/regression on an individual basis. Nevertheless, in those patients with NASH and elevated serum aminotransferases levels, a sustained reduction (at least by 40%), or better a normalization of ALT, can be taken into consideration to define a clinically meaningful end-point [10]. Conversely, a lack of ALT change is generally considered a negative signal indicative of ongoing disease activity. CK-18 fragments are not fully reliable marker of steatohepatitis and changes in CK-18 are difficult to interpret due to the large range of variability [11]. Overall, there are no good serum or imaging markers of steatohepatitis, improvement in liver cell injury or cell death. Liver fibrosis The histologic feature that appears to best predict mortality in NASH is the presence of significant fibrosis [12]. Antifibrotic effects are even harder to assess without histology, because they are slowly evolving processes and it may take years before a meaningful reduction in fibrosis becomes detectable. In general, simple panels such as the NAFLD fibrosis score (NFS) and FIB-4 can reliably exclude advanced fibrosis but are unreliable at diagnosing advanced fibrosis [13]. Direct fibrogenic markers, such as the Enhanced Liver Fibrosis (ELF ) test and Fibrotest, two commercial panels, could be more useful for this purpose, but they have not been sufficiently validated [13]. In fact NFS, FIB-4, FibroTest, and ELF can also rapidly decline due to the decrease in inflammation in the absence of a reduction in fibrosis, as shown in patients treated for viral hepatitis [10]. A number of imaging techniques including elastometry (Fibroscan) or magnetic resonance elastography (MRE) hold promise in the evaluation of fibrosis, although larger studies are required [13]. These techniques relies on changes in liver stiffness (or elasticity ) due to the parenchymal rigidity caused by collagen deposition. However, inflammation and ALT levels influence liver stiffness beyond the amount of fibrotic scarring and may confound early changes in elastometry values [10]. A better performance is expected from 2D magnetic resonance elastography technology. It has been suggested that a 15% relative reduction in stiffness, measured by magnetic resonance elastography, represents a one stage reduction in fibrosis measured by histology [14]. More data will be available in the near future from 63

68 clinical trials. Recently a multiparametric magnetic resonance imaging proved to be a promising method to grade the severity of steatohepatitis and to predict clinical events [15]. However, these preliminary studies await independent confirmation from larger trials. Other experimental Markers Lately, much effort is invested in the search and validation of serum markers that predict the dynamics of fibrogenesis and fibrolysis. One of the novel markers developed and validated in the EPOS (European Project on Steatohepatitis) cohort, Pro-C3, is a direct marker of collagen synthesis and is increasingly used in proof-of-concept clinical studies to predict the antifibrotic potential of novel agents for the treatment of NASH [16]. Other potential approaches to detect changes in liver damage/fibrosis include: epigenetic factors such as differential DNA methylation and circulating cell-free DNA methylation signatures in plasma [17]; microrna (mirna), another genetic marker that appears to be relatively stable and can be detected in plasma following release from injured tissue [18]; metabolomics and lipidomic approaches based on the rationale that dysregulation of liver lipid metabolism is central to the development of NAFLD/NASH [19]. However, the direct markers discussed above require further validation before they are likely to be widely used in routine clinical practice. Conclusion Advanced fibrosis and cirrhosis have become the major liver-related clinical endpoints in NASH. The major challenge of ongoing clinical trials will be to demonstrate that achieving reasonably likely surrogates, such as resolution of NASH and an improvement in fibrosis stage, does indeed translate into a reduction in the rate of progression to cirrhosis. Academy and industry are currently engaged in the common effort to discover and validate surrogates biomarkers able to predict clinical outcomes. References 1. A. Marengo, R. Younes, E. Bugianesi Progression and Natural History of Nonalcoholic Fatty Liver Disease in Adults. Clin Liver Dis. 2016;20(2): L.A. Adams, Q.M. Anstee, H. Tilg, G. TargherNon-alcoholic fatty liver disease and its relationship with cardiovascular disease and other extrahepatic diseases Gut, 66 (2017), pp A.J. Sanyal, S.L. Friedman, A.J. McCullough, L. Dimick-Santos, American Association for the Study of Liver Diseases, United States Food and Drug Administration Challenges and opportunities in drug and biomarker development for non-alcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop Hepatology, 61 (2015), pp E. Vilar-Gomez, Y. Martinez-Perez, L. Calzadilla-Bertot, A. Torres-Gonzalez, B. Gra- Oramas, L.Gonzalez-Fabian, et al.weight loss through lifestyle modification significantly reduces features of nonalcoholic steatohepatitis Gastroenterology, 149 (2015), pp A. Gastaldelli, S.A. Harrison, R. Belfort-Aguilar, L.J. Hardies, B. Balas, S. Schenker, et al. Importance of changes in adipose tissue insulin resistance to histological response during thiazolidinedione treatment of patients with nonalcoholic steatohepatitis Hepatology, 50 (2009), pp L.S. Szczepaniak, P. Nurenberg, D. Leonard, J.D. Browning, J.S. Reingold, S. Grundy, et al. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population Am J Physiol Endocrinol Metab, 288 (2005), pp. E462-E S. Meisamy, C.D. Hines, G. Hamilton, C.B. Sirlin, C.A. McKenzie, H. Yu, et al. Quantification of hepatic steatosis with T1-independent, T2-corrected MR imaging with 64

69 spectral modeling of fat: blinded comparison with MR spectroscopy Radiology, 258 (2011), pp M.S. Middleton, E.R. Heba, C.A. Hooker, M.R. Bashir, K.J. Fowler, K. Sandrasegaran, et al. Agreement between magnetic resonance imaging proton density fat fraction measurements and pathologist-assigned steatosis grades of liver biopsies from adults with nonalcoholic steatohepatitis Gastroenterology, 153 (2017), pp J. Patel, R. Bettencourt, J. Cui, J. Salotti, J. Hooker, A. Bhatt, et al. Association of noninvasive quantitative decline in liver fat content on MRI with histologic response in nonalcoholic steatohepatitis Ther Adv Gastroenterol, 9 (2016), pp V. Ratziu A critical review of endpoints for non-cirrhotic NASH therapeutic trials. J Hepatol Feb;68(2): K. Cusi, Z. Chang, S. Harrison, R. Lomonaco, F. Bril, B. Orsak, et al. Limited value of plasma cytokeratin-18 as a biomarker for NASH and fibrosis in patients with nonalcoholic fatty liver disease J Hepatol, 60 (2014), pp P. Angulo, D.E. Kleiner, S. Dam Larsen, L.A. Adams, E.S. Bjornsson, P. Charatcharoenwitthaya, et al. Liver fibrosis, but no other histologic features, is associated with long term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 2015;149: EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol Jun;64(6): C.C. Park, P. Nguyen, C. Hernandez, R. Bettencourt, K. Ramirez, L. Fortney, et al. Magnetic resonance elastography vs transient elastography in detection of fibrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease Gastroenterology, 152 (2017), p. e M. Pavlides, R. Banerjee, J. Sellwood, C.J. Kelly, M.D. Robson, J.C. Booth, et al. Multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease J Hepatol, 64 (2016), pp M.J. Nielsen, S.S. Veidal, M.A. Karsdal, D.J. Ørsnes-Leeming, B. Vainer, S.D. Gardner, et al.plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression in patients with chronic hepatitis C Liver Int, 35 (2015), pp M. Zeybel, T. Hardy, Y.K. Wong, et al. Multigenerational epigenetic adaptation of the hepatic wound-healing response. Nature Medicine 2012;18: C.J. Pirola, T. Fernandez Gianotti, G.O. Castano, et al. Circulating microrna signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis. Gut 2015;64: J. Barr, J. Caballeria, I. Martinez-Arranz, et al. Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression. J Proteome Res 2012;11:

70 Mohammad Shadab Siddiqui, MD Virginia Commonwealth University Glen Allen, VA Defining a Clinically Meaningful Change in Circulating Biomarker, Imaging and Elastography Based Assessment of Cirrhosis Due to NASH Untreated nonalcoholic steatohepatitis (NASH) may lead to end stage liver disease or cirrhosis. The natural history of cirrhosis includes an asymptomatic phase (compensated cirrhosis) followed by decompensated phase characterized by development of complications of portal hypertension and liver dysfunction and is associated with higher risk of liver related mortality when compared to compensated cirrhosis 1. Clinically meaningful treatment outcomes in patients with NASH cirrhosis should be to (1) improve liver related mortality, (2) reduce risk of hepatic decompensation, (3) transition patients from decompensated state to compensated state and (4) fibrosis regression. However, since the rate of transition from compensated state to decompensated state is 5-7%/year and median survival is 12 years, it is impractical to use these as endpoints for clinical trials in all patients with NASH related cirrhosis 1. Since severity of hepatic fibrosis and portal hypertension have been linked to these risk of hepatic decompensation and mortality, these are often used as surrogates in clinical trials in patients with compensated cirrhosis as changes in these are easier to demonstrate over shorter duration of time 2 4. In contrast, the morbidity and mortality in patients with decompensated cirrhosis is high, thus, endpoints focusing on reducing mortality, transplant free survival and decompensating events is appropriate 1,5. Liver biopsy remains the gold standard for detecting and quantifying hepatic fibrosis, however, several non-invasive imaging and serum based biomarkers have emerged for fibrosis assessment 6. The diagnostic accuracy of magnetic resonance elastography (MRE) and vibration based transient elastography (VCTE) is high. Using VCTE, the liver stiffness measurement cutoff value of 14kPa had area under the receiver operating characteristic (AUROC) curve of 0.92 and PPV value of 0.73 and NPV of Similarly, the MRE has a high diagnostic accuracy for identifying patients with NASH cirrhosis (AUROC 0.97) 7. However, the sample size of patients with cirrhosis in these studies is low and additional data is required to generate optimal cutoffs for identifying cirrhosis. Non-invasive serum-based fibrosis models, such has FIB-4 and NAFLD Fibrosis Score (NFS), have high diagnostic performance for identification of cirrhosis and their performance is better at ruling out than ruling in a disease 7,8. The diagnostic performance of these non-invasive models for detecting presence of cirrhosis is lower than MRE and VCTE 7,8. Several other serum-based biomarkers such as PIIINP, pro-c3, and enhanced liver fibrosis (ELF TM ) test have been evaluated for detecting presence of cirrhosis in patients with NASH, however, more data is required before they can be recommended 6. The hallmark of portal hypertension is an increase in pressure gradient between the portal vein and the inferior vena cava, which is measured by hepatic venous pressure gradient (HVPG) 9. HVPG is directly linked to severity of portal hypertension, risk of hepatic decompensation and liver related mortality 10. A HVPG measurement of > 6mmHg indicates portal hypertension and HVPG > 10 mmhg represents clinically significant portal hypertension. In patients with cirrhosis, Model for End Stage Liver Disease (MELD) score is a predictor hepatic decompensation and mortality 11, however, methacetin breath test (MBT) was recently shown to be a better predictor of clinical decompensation that MELD score alone

71 Reduction in HVPG has been linked to reduced risk of cirrhosis related complications including risk of variceal hemorrhage and ascites, thus is an attractive target to study in clinical trials 13,14. Additionally, in patients with compensated cirrhosis impact of therapy on fibrosis regression via liver biopsy can be evaluated. However, aside from HVPG and histological assessment, there is limited data evaluating the longitudinal change in radiological, non-invasive fibrosis models, serum-based biomarkers and functional assessment of the liver on clinical outcomes. In a posthoc analysis of two therapeutic clinical trials in patients with NAFLD, reduction in body weight was associated with reduction in MRE and serum ALT levels 15. Similarly, reduction in body weight and fibrosis improvement was associated with improvement in serum ALT, NFS and platelet count 16. These studies were conducted in patients without cirrhosis, and although these results are not easily applicable to patients with NASH cirrhosis, they provide framework for linking clinically significant changes in patients with non-invasive biomarkers. References 1. D Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies. J Hepatol 2006;44: Hagstrom H, Nasr P, Ekstedt M, et al. Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD. J Hepatol August Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 2015;149: e Vilar-Gomez E, Calzadilla-Bertot L, Wai-Sun Wong V, et al. Fibrosis Severity as a Determinant of Cause-specific Mortality in Patients With Advanced Nonalcoholic Fatty Liver Disease. Gastroenterology May Jepsen P, Vilstrup H, Andersen PK, et al. Comorbidity and survival of Danish cirrhosis patients: A nationwide population-based cohort study. Hepatology 2008;48: Shadab Siddiqui M, Harrison SA, Abdelmalek MF, et al. Case definitions for inclusion and analysis of endpoints in clinical trials for NASH through the lens of regulatory science. Hepatology October Imajo K, Kessoku T, Honda Y, et al. Magnetic Resonance Imaging More Accurately Classifies Steatosis and Fibrosis in Patients With Nonalcoholic Fatty Liver Disease Than Transient Elastography. Gastroenterology 2016;150: e7. 8. Xiao G, Zhu S, Xiao X, et al. Comparison of laboratory tests, ultrasound, or magnetic resonance elastography to detect fibrosis in patients with nonalcoholic fatty liver disease: A meta-analysis. Hepatology 2017;66: Bosch J, Abraldes JG, Berzigotti A, et al. The clinical use of HVPG measurements in chronic liver disease. Nat Rev Gastroenterol Hepatol 2009;6: Ripoll C, Groszmann R, Garcia-Tsao G, et al. Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis. Gastroenterology 2007;133: Wiesner RH, McDiarmid S V, Kamath PS, et al. MELD and PELD: application of survival models to liver allocation. Liver Transpl 2001;7: Stravitz RT, Reuben A, Mizrahi M, et al. Use of the methacetin breath test to classify the risk of cirrhotic complications and mortality in patients evaluated/listed for liver transplantation. J Hepatol 2015;63: D Amico G, Garcia-Pagan JC, Luca A, et al. Hepatic Vein Pressure Gradient Reduction and Prevention of Variceal Bleeding in Cirrhosis: A Systematic Review. Gastroenterology 2006;131: Garcia-Tsao G. Transjugular Intrahepatic Portosystemic Shunt in the Management of Refractory Ascites. Semin Intervent Radiol 2005;22:

72 15. Patel NS, Hooker J, Gonzalez M, et al. Weight Loss Decreases Magnetic Resonance Elastography Estimated Liver Stiffness in Nonalcoholic Fatty Liver Disease. Clin Gastroenterol Hepatol 2017;15: Vilar-Gomez E, Calzadilla-Bertot L, Friedman SL, et al. Serum biomarkers can predict a change in liver fibrosis 1 year after lifestyle intervention for biopsy-proven NASH. Liver Int 2017;37:

73 Vlad Ratziu, MD Sorbonne Université, Hospital Pitié Salpêtrière Institute for Cardiometabolism and Nutrition Paris France Current Clinical Trial Landscape in Early Development and Prognosis For 2025 Nonalcoholic steatohepatitis is a disease without a single, specific, diagnostic marker which means that multiple indicators need to be used to measure therapeutic efficacy. Moreover, drug candidates for nonalcoholic steatohepatitis target many distinct mechanisms that are believed to promote hepatic injury. Therefore, there is a wide range of endpoints that have to be reached, sequentially, as required by the drug development process. Some of these endpoints validate the mechanism of action, others are used to anticipate histological efficacy. Histological endpoints are still considered the best predictors of clinical outcomes but they can only be reliably tested in larger, late phase trials. The rationale and clinical data in favor of the endpoints used at different stages of therapeutic trials will be reviewed. The validity and limitations of current phase 2b histological endpoints, in particular that of a one stage reduction in fibrosis, for their ability to predict progression to cirrhosis which is the ultimate objective of therapeutic trials will be discussed. Therapeutic trials need to provide answers to precise questions and these questions are different at the various stages of drug development. Early phase trials (phase 1 and 2a) are designed to inform about pharmacokinetic parameters, early and short-term human safety data and pharmacodynamic aspects. These are common to most therapeutic fields or classes of drugs with little if any specificity for particular diseases such as NASH. In addition, phase 2a trials also demonstrate proof of principle, meaning measurable on target effects and their biological consequences. An outcome of phase 2a trials that is particularly important is the selection of ideally one or two doses that provide maximum efficacy with acceptable safety and tolerability. This will also allow to gain precious insight into the dose dependency of the therapeutic effect and the safety margin of a particular compound. These doses will be carried over in later stage trials. Typically phase 2b trials will explore whether the biological effects observed in earlier trials translate into hepatic histological improvement. Finally, large scale phase 3 and subsequent outcome trials will provide a comprehensive and statistically robust demonstration of the benefit in terms of histological improvement and clinical long-term outcomes. Since each trial will have to deliver information critical for designing the next step, a careful consideration of how the endpoints should be chosen and if they can be achieved within a given time frame is necessary. Many endpoints are currently being measured in NASH trials in an attempt to best predict the biological activity of a drug candidate and its efficacy in improving liver injury in NASH. One of the major breakthroughs in the field was the construction of a regulatory approval framework whereby endpoints that can be achieved within a reasonably short timeframe, compatible with clinical trials, were granted reasonably likely surrogate status. The second major breakthrough was the recognition of progression to cirrhosis as a major therapeutic objective on par with the documentation of liver-related events such as cirrhosis complications, liver transplantation and death. The major challenge of ongoing registrational trials will be to demonstrate that achieving reasonably likely surrogates such as resolution of NASH and an improvement in fibrosis stage does indeed translate into a reduction in the rate of progression to cirrhosis. While waiting for 69

74 regulatory acceptable biomarkers of fibrosis, a better histological definition of anti-fibrotic activity taking into account both stage improvement and stage worsening is critical in that regard. 70

75 Stephen A. Harrison, MD, FAASLD University of Oxford Oxford United Kingdom Current Clinical Trial Landscape in Late Development (phase 3) and Prognosis for 2025 Despite the high prevalence of NAFLD and NASH, there has historically been a paucity of interest in developing pharmacologic treatment for this common liver disease. With the advent of successful hepatitis C therapies and a clear tract to regulatory approval for NASH therapies, the treatment landscape for this disease has changed dramatically. Currently, four compounds are in phase 3 clinical trials with more expected in the near future. The objective of this talk is to identify the compounds in late stage development and then look to the future to see how this will affect the way we address NASH clinically. The compound that advanced to phase 3 first was obeticholic acid from Intercept Pharmaceuticals. This is an FXR agonist that has shown benefit in improving the NAFLD activity score and fibrosis in phase 2b 1. The phase 3 trial is an 18-month study assessing 2 doses of obeticholic acid versus placebo. It has fully enrolled the subpart H cohort. The primary endpoint is either resolution of NASH without worsening of fibrosis or improvement of fibrosis by 1 stage or more without worsening of NASH. Elafibranor, a PPAR α/δ agonist manufactured by Genfit, is also in phase 3. The phase 2b trial showed a significant benefit of Elafibranor 120mg on NASH resolution when compared to placebo 2. The phase 3 trial is an 18-month study assessing one dose of Elafibranor (120mg) versus placebo. The subpart H portion of the study has been fully enrolled. The primary endpoint of this study is NASH resolution without worsening of fibrosis. Allergan is currently studying the efficacy of Cenicriviroc (CVC), a CCR2-5 antagonist, for the treatment of NASH fibrosis in a large phase 3 trial. This study is currently enrolling its subpart H cohort. Data to support this trial comes from the phase 2b CENTAUR trial. Year one results were recently published 3 and the year 2 results were recently presented at this year s EASL and DDW. The year 1 data showed that despite no improvement in NASH, a statistically significant improvement of CVC on fibrosis compared with placebo was found and the year two exploratory analyses corroborated CVCs antifibrotic activity. The primary endpoint of the phase 3 trial is improvement of fibrosis by 1 stage or more without worsening of NASH. Selonsertib, an ASK-1 inhibitor made by Gilead, is the final compound currently in phase 3 development for NASH. This drug is currently being assessed in 2 large trials, both of which have completed enrollment. One trial is targeting stage 3 patients while the other is aimed at well compensated cirrhotic patients. Data to support these late stage trials comes from a 72- patient, 6 month paired liver biopsy trial in NASH patients with stage 2-3 fibrosis 4. A dose response relationship was seen in fibrosis improvement despite the fact that NASH did not improve. The primary endpoints for both of the phase 3 trials is improvement in fibrosis by one stage or more without worsening of NASH. It is anticipated that if these studies achieve their respective primary endpoints, the first approved therapies could be on the market sometime in mid to late This would represent the first regulatory approved therapy for NASH. While this is a significant accomplishment, 71

76 based on the phase 2b data, there will still be room for improvement, both in NASH resolution and fibrosis reduction. Several of the compounds currently in phase 2 development have different or variant mechanisms of action than the compounds listed above and are expected to enter phase 3 sometime in 2019/2020. This increases the possibility for combination therapy. By the time 2025 rolls around, it is my expectation that many compounds, comprising complimentary mechanisms of action will be in development. Some will combine to target the drivers of NASH, while others will combine to target both NASH and fibrosis. Ultimately, I doubt we will have approved combination therapy by 2025, but I do suspect we will be discussing phase 2 combination trials that show significant promise over monotherapy. References 1. Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2015; 385: Ratziu V, Harrison SA, Francque S, et al. Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening. Gastroenterology 2016; 150: Friedman SL, Ratziu V, Harrison SA, et al. A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology 2018; 67: Loomba R, Lawitz E, Mantry PS, et al. The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: A randomized, phase 2 trial. Hepatology 2017; In press. 72

77 Miriam B. Vos, MD, MSPH Emory University, Children's Healthcare of Atlanta Atlanta, GA Current Clinical Trial Landscape in Pediatrics and Prognosis for 2025 The prevalence of NAFLD in children continues to grow and a recent review has demonstrated that the need for treatment extends worldwide 1. As covered in earlier presentations in this conference, disease in pediatrics can be severe and appears to continue into adulthood, supporting the need for therapeutics that could cure it. The disease pathophysiology of NAFLD in children is similar to adults, making the range of treatments similar although with some important differences including less emphasis on fibrosis and more focus on eliminating pathological steatosis and inflammation, consistent with the earlier phase of the disease. As of June 2018, there are 14 pediatric NAFLD clinical trials listed on ClinicalTrials.gov as recruiting or not yet recruiting from across the world. Eleven of these are recruiting and 3 are not yet recruiting. Across these trials, the majority of the studies have hepatic fat measurement by MRI as the primary outcome and 3 studies have change in alanine aminotransferase (ALT) as the primary outcome. Both ALT and hepatic fat by MRI have pediatric data to support their use as surrogate markers of histologic response, which is itself a surrogate of future clinical benefit. ALT was recently examined in a study by Arsik et al and demonstrated that the average ALT over the course of therapy was strongly associated with improvement in NASH and fibrosis after two years in the TONIC clinical trial (In press, Children 2018). The use of MRI as a measurement of steatosis has been validated in several pediatric studies and appears to be more accurate than liver biopsy because of the issues with small sample size in liver biopsies 2. The amount of steatosis reduction that is clinically relevant is not yet determined. All of the currently active studies are early phase (1 and 2) and the design of studies ranges from crossover trials, single arm to small randomized, controlled clinical trials (RCT). The sample sizes are consistent with the early phase and range from 15 to 160 participants. Two trials that are currently recruiting are testing dietary change and one is testing exercise. This fits the need expressed in the recent pediatric NAFLD guidelines which states that lifestyle changes remains the primary treatment in children and at the same time stated there is insufficient data for which lifestyle changes are most effective 3. One of the trials is a RCT out of Los Angeles, CA testing a sugar reduction education (PI Goran). The other dietary RCT is comparing two different diets and is based in Stockholm, Sweden (PI Hagstrom). Of note, there is another dietary study recently completed without results yet posted on a low sugar diet as a treatment (PI Schwimmer and Vos). There is one exercise study in a RCT in Araba, Spain the investigators are comparing an exercise program to control (PI Labayen). There are 3 pediatric trials testing medications currently on Clinictrials.gov. Two of these are repurposed mediations approved for other conditions. Metformin is an insulin sensitizer previously tested in the TONIC trial that is now being tested in a RCT in New York, NY (PI Sopher) in 46 participants using liver fat by MRS as the primary outcome. Losartan is an angiotensin receptor blocker approved for hypertension in children and it will be tested in a RCT of 110 children in a trial run by the NASH CRN (NIH NIDDK). There is one trial of a novel therapeutic a single arm, multi-site trial of gemcabene, a novel drug targeting lipid synthesis and regulation in NAFLD based out of Atlanta, GA (PI Vos). There are also 4 trials testing either vitamins, supplements or a medical food. These types of treatments are particularly compelling in pediatrics because of the potential need for very long 73

78 term therapy and the low side effect profile of this category. Three are currently recruiting and this includes a trial of tomato juice enhanced with lycopene (Naples, Italy, PI Iorio), a trial of Hydroxytyrosol and vitamin E (Rome, Italy, PI Nobili) and a hyperimmune milk enhanced with anti-lps antibodies (Atlanta, GA, PI Vos). Lastly, there are 2 trials of weight loss surgery including a parallel assignment control study of vertical sleeve gastrectomy (Cincinnati, OH, PI Xanthakos) and a single arm trial of weight loss surgery (Boston MA, PI Corey). The number of pediatric NAFLD studies demonstrates the broad interest in and need for addressing this growing epidemic. The lack of phase 3 trials confirms the early stage of the pediatric NAFLD field. The future is promising, but the road to an FDA approved therapeutic is still quite long. With optimism, if one of the current phase 2 trials supports proceeding to a 2- year phase 3 trial in children, a therapeutic could be available before Another path would be if a drug is approved for adult NAFLD that can be translated to peds that could be a faster path but relies on the success of drugs currently under investigation. I expect by 2025 prevention of type II diabetes and decreasing future cardiovascular disease risk will increase in importance as the natural history studies provide better data supporting this. Lastly, by 2025 as our diagnostics and genetic testing improves and longer natural history data is available, phenotypes of pediatric NAFLD will be identified and treatment will be tailored to the specific phenotype, improving the effectiveness. References 1. Anderson EL, Howe LD, Jones HE, et al. The Prevalence of Non-Alcoholic Fatty Liver Disease in Children and Adolescents: A Systematic Review and Meta-Analysis. PLoS One 2015;10:e Middleton MS, Van Natta ML, Heba ER, et al. Diagnostic accuracy of magnetic resonance imaging hepatic proton density fat fraction in pediatric nonalcoholic fatty liver disease. Hepatology 2018;67: Vos MB, Abrams SH, Barlow SE, et al. NASPGHAN Clinical Practice Guideline for the Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease in Children: Recommendations from the Expert Committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN). J Pediatr Gastroenterol Nutr 2017;64:

79 POSTER ABSTRACT SUMMARIES 75

80 # 1 Somaya Albhaisi, MBBch Virginia Commonwealth University Health System Richmond, VA somaya.albhaisi@vcuhealth.org EXTRAHEPATIC NEOPLASMS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE Authors: A. Somaya 1 ; A. Sanyal 1 Institution(s): Virginia Commonwealth University Health System 1 Background: Patients with Non-alcoholic fatty liver disease (NAFLD) have increased overall mortality compared to matched control populations. The most common cause of death in patients with NAFLD is cardiovascular disease, followed by non-liver malignancy and liver-related death. It has been well established that hepatocellular carcinoma is a commonly observed complication of long-term NAFLD. Less data are available to address a potential link between NAFLD and extrahepatic cancers.thus, we conducted the current study to identify the prevalence and types of non-hepatic malignancies in NAFLD po pulation. Methods: We retrospectively reviewed 160 patients with NAFLD who were diagnosed with any type of non-hepatic malignancy between 1/1/2010 and 12/31/2016 at Virginal Commonwealth University. NAFLD diagnosis was based on imaging findings or liver biopsy results. We excluded patients with any type of liver malignancy. Extra-hepatic cancers were identified through coded diagnoses and outpatient oncology visits. Results: Among NAFLD patients, hematologic malignancies were the most prevalent (n=52, 32.5%), followed by gastrointestinal cancers (n=34, 21.3%), then breast cancer (n=23, 14.4%), then genitourinary cancers (n=18, 11.3%), then skin (10%), then Endocrine and other cancers (included parotid tumors, sarcoma and meningioma) (3% each), then renal cancers (2.5%), lung cancers (1.25%), and finally bone cancers (0.63%). Leukemia and Lymphoma were the most common hematologic malignancies (11% each). As anticipated, the most common digestive tract malignancy was colon cancer (9.4%), followed by ano-rectal cancers (3.8%), cholangiocarcinomas (2.5%), esophageal 1.9%), gastric, small bowel and pancreatic (1.3% each). Prostate and cervical cancers were the most common genitourinary malignancies (3.1%). 76

81 Conclusion: Patients with NAFLD are at high risk for extrahepatic malignancies, especially hematological and colorectal malignancies. Our study is limited because it was retrospective with no control group. We need prospective studies to address their association. References 1. Risk of liver and other types of cancer in patients with cirrhosis: a nationwide cohort study in Denmark.Sorensen HT, Friis S, Olsen JH, Thulstrup AM, Mellemkjaer L, Linet M, Trichopoulos D, Vilstrup H, Olsen J Hepatology Oct; 28(4): NAFLD and extrahepatic cancers: have a look at the colon. Herbert Tilg and Anna Mae Diehl. Gut Jun; 60(6): Nonalcoholic Cirrhosis Increased Risk of Digestive Tract Malignancies. A Population- Based Cohort Study. Li-Min Sun, Ming-Chia Lin, Cheng-Li Lin, Ji-An Liang, Long-Bin Jeng, 4. Chia-Hung Kao, and Chiao-Yi Lu. Medicine (Baltimore) Dec; 94(49): e Increased risk for malignant neoplasms among patients with cirrhosis. Kalaitzakis E, Gunnarsdottir SA, Josefsson A, Björnsson E. Clin Gastroenterol Hepatol Feb; 9(2): High prevalence of colorectal neoplasm in patients with non-alcoholic steatohepatitis. Wong 77

82 7. VW, Wong GL, Tsang SW, Fan T, Chu WC, Woo J, Chan AW, Choi PC, Chim AM, Lau JY, Chan FK, Sung JJ, Chan HL. Gut Jun;60(6): Relationship of non-alcoholic fatty liver disease to colorectal adenomatous polyps. Hwang ST, Cho YK, Park JH, Kim HJ, Park DI, Sohn CI, Jeon WK, Kim BI, Won KH, Jin W. J Gastroenterol Hepatol Mar;25(3): Non-Alcoholic Fatty Liver Disease and Extra-Hepatic Cancers. Claudia Sanna, Chiara Rosso, Milena Marietti, and Elisabetta Bugianesi. Int J Mol Sci May; 17(5): The Relationship between Nonalcoholic Fatty Liver Disease and Colorectal Cancer: The Future Challenges and Outcomes of the Metabolic Syndrome. Said O. Muhidin, Ahmed A. Magan, 2 Khalid A. Osman, Shareef Syed, and Mohamed H. Ahmed. J Obes. 2012; 2012: History of cirrhosis and risk of digestive tract neoplasms. Randi G, Altieri A, Gallus S, Franceschi S, Negri E, Talamini R, La Vecchia C. Ann Oncol Sep; 16(9): Gastric cancer prevalence in patients with liver cirrhosis. Zullo A, Romiti A, Tomao S, Hassan C, Rinaldi V, Giustini M, Morini S, Taggi F. Eur J Cancer Prev Jun; 12(3): Malignant lymphoproliferative disorders in liver cirrhosis. Lombardo L, Rota Scalabrini D, Vineis P, De La Pierre M Ann Oncol Mar; 4(3): Disclosure: Nothing to disclose. 78

83 # 2 Somaya Albhaisi, MBBCh Virginia Commonwealth University Richmond, VA somaya.albhaisi@vcuhealth.org STARVATION-INDUCED HEPATOPATHY IN A PATIENT WITH SEVERE ANOREXIA NERVOSA: A CASE REPORT Author: S. Albhaisi 1 Institution: Virginia Commonwealth University 1 Background: Anorexia nervosa is an eating disorder characterized by voluntary weight loss, distortion of body image and intense fear of gaining weight. Several studies have described hepatic complications in severely malnourished patients with this disease. These are usually related to an isolated elevation of transaminase levels, but nevertheless, very few cases of acute liver failure have been described. We report a case of severely anorexic female who developed acute liver damage with profound hypoglycemia. Methods: A 43-year-old woman with history of severe anorexia nervosa, systolic heart failure and anxiety disorder, presented as a transfer from another hospital for evaluation of progressively rising liver enzymes. Patient complained of nausea, vomiting, poor oral intake and loose watery stools for 10 days. She reportedly had normal liver function in the past. On admission, she presented with mild hypotension, hypothermia, hypoglycemia and hyponatremia. She denied taking any hepatotoxic or illicit drugs, or potentially harmful products. On exam, she appeared severely malnourished (Body Mass Index 9.8 kg/m2). Abdominal exam was negative. Results: Laboratory data demonstrated the following: Glucose 57 mg/dl, Total bilirubin 2.2 mg/dl, conjugated 1.5 mg/dl, Albumin 3.3 g/dl, AST 3400 units/l, ALT 2200 units/l, Alkaline Phosphatase 796 units/l, normal coagulation profile. All nutritional laboratory tests were normal. A complete infectious, autoimmune and genetic workup was negative. Abdominal ultrasound showed hepatic parenchymal disease without focal lesion. Magnetic resonance cholangiopancreatography showed iron deposition in liver and spleen. Thus, a diagnosis of acute liver injury associated with anorexia nervosa was considered the most plausible diagnosis. Patient declined a liver biopsy and psychiatry referral. She also refused enteral tube feeding and total parenteral nutrition. Instead she elected to work closely with a nutrition specialist to improve her oral intake. Endocrinology followed up closely for hypoglycemia which they thought was caused by substrate deficiency from reduced gluconeogenesis secondary to liver disease and reduced oral intake due to anorexia nervosa. A careful and progressive re-feeding was performed under the assistance of nutritionist. This resulted in slow improvement of hepatic panel. Conclusions: This case of anorexia nervosa illustrates the consequences of severe malnutrition on the liver, with marked elevation of liver enzymes and profound glycogen depletion. Although mild liver injury is not rare and is reported in up to 60% of patients with anorexia nervosa, high levels of serum transaminases are rather uncommon. Severe starvation can be regarded as a self-cannibalism action to cope with this stressful 79

84 condition. It can be associated with acute liver injury that is slowly reversible with careful enteral nutrition. References 1. Bridet, L., Beitia Martin, J. J., & Cabriada Nuno, J. L. (2014). Acute liver damage and anorexia nervosa: A case report. The Turkish Journal of Gastroenterology, 25(2), Kheloufi, M., Boulanger, C. M., Durand, F., & Rautou, P. E. (2014). Liver Autophagy in Anorexia Nervosa and Acute Liver Injury. BioMed Research International, Restellini, S., Spahr, L., & Rubbia Brandt, L. (2013). Severe starvation-induced hepatocyte autophagy as a cause of acute liver injury in anorexia nervosa: a case report. Case Reports in Hepatology, 2013(October 2015),

85 # 3 Andrea Dennis, PhD Perspectrum Diagnostics Oxford United Kingdom Andrea.Dennis@perspectum-diagnostics.com UTILITY AND VARIABILITY OF THREE NON-INVASIVE LIVER FIBROSIS IMAGING MODALITIES TO EVALUATE EFFICACY OF GR-MD-02 IN SUBJECTS WITH NASH AND BRIDGING FIBROSIS OR CIRRHOSIS DURING A PHASE-2 CONTROLLED STUDY Authors: S. Harrison 2 ; A. Dennis 3 ; M. Fiore 3 ; M. Kelly 3 ; D. Kelly 3 ; A. Paredes 4 ; J. Whitehead 4 ; P. Neubauer 4 ; P. Traber 1 ; D. Banerjee 4 Institution(s): Galectin Therapeutics Inc 1.; University of Oxford 2 ; Perspectum Diagnostics 3 ; San Antonio Military Medical Center 4 Background: Given the worldwide prevalence of NAFLD and the propensity to progress to NASH, there is a clear need to develop treatments to slow or reverse disease progression as an alternative to liver transplantation. GR-MD-02 (galactoarabino-rhamnogalaturonate), a new type of therapy for NASH with advanced fibrosis, has been shown to reduce hepatic fibrosis in animal studies, and lower serum biomarkers of NASH fibrogenesis in a phase 1 clinical trial [1]. The primary aim of this study was to determine the difference between four-month treatment with placebo and GR-MD-02 change in liver inflammation and fibrosis as measured by iron-corrected T1 (ct1) mapping. ct1 is a non-invasive, multi-parametric magnetic resonance imaging (MRI) protocol that has been shown to correlate with the extent of hepatic fibro-inflammatory disease [2,3]. The secondary aims were to determine the change in liver stiffness as measured by both magnetic resonance elastography (MRE) and shear-wave ultrasonic elastography, and to explore test-retest repeatability of the three non-invasive liver imaging modalities. Methods: Thirty subjects (13 females, years) with NASH and advanced fibrosis (Brunt fibrosis score 3) on prior liver biopsy performed within 12 months of study start were recruited. Subjects were randomized 1:1 to study drug (8 mg.kg-1 GR-MD-02 via IV infusion) or placebo groups. Study drug was administered biweekly over a 16-week period for a total of 9 infusions. Therapeutic efficacy was assessed ct1. Participants also underwent MR elastography (MRE) and ultrasound shear-wave elastography (Fibroscan; FS). Statistical analyses on primary and secondary end-points were performed using robust ANCOVA models adjusting for baseline measurement and additional covariates. Results: There was no statistically-significant change in ct1 (p=0.16) between study-drug and placebo groups, suggesting no efficacy of GR-MD-02 in reducing liver fibro-inflammatory disease in NASH patients with advanced fibrosis over a 16-week period. There was also no significant difference in change in liver stiffness, measured either by MRE (p=0.98) or FS- LSM (p=0.26), between study drug and placebo. Examination of the repeatability of the primary (ct1) and secondary (FS-LSM and MRE) endpoints revealed coefficient of variations of 3.5%, 44.0% and 12.7% respectively. Conclusion: 8 mg.kg-1 of GR-MD-02 had no statistically-significant effect on non-invasive 81

86 biomarkers of liver inflammation or fibrosis over a 4-month intervention period. The excellent reproducibility of the primary endpoint, ct1 (CoV = 3.5%), suggest that this imaging modality could be utilized for monitoring longitudinal change in patients with NASH. References 1. Harrison S, Marri S, Chalasani N, Kohli R, Aronstein W, Thompson G, et al. Randomised clinical study: GR-MD-02, a galectin-3 inhibitor, vs. placebo in patients having non-alcoholic steatohepatitis with advanced fibrosis. Aliment Pharmacol Ther. 2016;44(11 12): Banerjee R, Pavlides M, Tunnicliffe EM, Piechnik SK, Sarania N, Philips R, et al. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol. 2014;61(1): Pavlides M, Banerjee R, Tunnicliffe E, Kelly C, Collier J, Wang L, et al. Multi-parametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease severity. Liver Int. 2017;37(7): Disclosure Employee: Perspectum Diagnostics Ltd 82

87 # 4 Andrea Dennis, PhD Perspectrum Diagnostics Oxford United Kingdom Andrea.Dennis@perspectum-diagnostics.com REPEATABILITY AND REPRODUCIBILITY OF MULTIPARAMETRIC MAGNETIC RESONANCE IMAGING OF THE LIVER Authors: H. Wilman 1 ; A. Dennis 1 ; V. Bachtiar 1 ; J. Jacobs 1 ; R. Newbould 1 ; M. Gyngell 1 ; D. Kelly 1 ; M. Kelly 1 ; P. Neubauer 1 ; D. Banerjee 1 Institution(s): Perspectum Diagnostics 1 Background: MRI-based technology for quantitative analysis of hepatic fat, T2*, iron corrected T1 (ct1) and MR elastography has considerable utility in NASH clinical trials. To be clinically effective, a measurement should be consistent across sites with good reproducibility. Cross-vendor performance of MRE has been evaluated, and demonstrated a CoV of 10.7% [1]. ct1 has been shown to correlate with fibro-inflammatory disease and predict clinical outcomes [2,3,4]. We test the robustness of LiverMultiScan-derived metrics across different MRI scanner manufacturers, models, software versions and field strengths. Methods: ct1, T2* and Proton Density Fat Fraction (PDFF) maps were acquired from 61 participants on combinations of three Siemens (1.5T Avanto-Fit, VE11C, MyoMaps; 3T Prisma, VE11C, MyoMaps; 3T Skyra, VE11C, MyoMaps) and two Philips (1.5T Ingenia, 5.3.0, CardiacQuant; 3T Ingenia, 5.3.0, CardiacQuant) scanners. Participants were adult volunteers with mixed liver disease aetiology and those without any history of liver disease to represent a range of physiological values of these metrics. Two acquisition repeats were conducted for each scan, with subjects leaving the scanner in between. Participants were scanned on at least two different scanner models and field strengths in pseudorandomised order with up to 1- week between scans. Standardisation of T1 maps across scanner models and software versions was based on phantom-derived mappings from 90 acquisitions on the above MRI systems. Phantoms were purpose built to span the clinically-relevant range of liver T1s. Results: Standardised ct1 in participants demonstrated high reproducibility across different scanner models, software versions and field strengths (CoV 3.3%; bias 6.5 ms, 95% LoA of ms to 89.2 ms). Both T2* (CoV 6.6%; bias -1.7ms, 95% LoA of -6.6ms to 3.2 ms) and PDFF measurements (CoV 17%; bias 0.06%, 95% LoA of -0.69% to 0.82%) also showed excellent reproducibility at both field strengths and across different scanners models. Bland- Altman analysis of the T1 phantom measurements showed a clear reduction in bias (from -20ms to -4.7ms), tightening of the 95% Limits of Agreement (LoA: from -59.2ms 19ms, to -25.3ms 15.9ms) and reduction in mean coefficient of variation (CoV: 2.5% to 1.0%) after standardisation. Conclusion: We demonstrate standardised ct1 is a robust metric independent of vendor (Philips or Siemens) and field strength (1.5T or 3T). Combined with the excellent reproducibility of T2* and PDFF, LMS represents a robust and reliable non-invasive tool for characterising liver tissue. 83

88 References 1. Trout A, Serai S, Mahley A, Wang H, Zhang Y, Zhang B, et al. Liver Stiffness Measurements with MR Elastography: Agreement and Repeatability across Imaging Systems, Field Strengths, and Pulse Sequences. 2016;281(3): Banerjee R, Pavlides M, Tunnicliffe EM, Piechnik SK, Sarania N, Philips R, et al. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol. 2014;61(1): Pavlides M, Banerjee R, Sellwood J, Kelly C, Robson M, Booth J, et al. Multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease. J. Hepatol. 2016;64(2): Pavlides M, Banerjee R, Tunnicliffe E, Kelly C, Collier J, Wang L, et al. Multi-parametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease severity. Liver Int. 2017;37(7): Disclosure Employee: Perspectum Diagnostics Ltd 84

89 # 5 Anne-Marie Carreau, MD, MSc University of Colorado- Anschutz Medical Campus Denver, CO anne-marie.carreau@ucdenver.edu CLINICAL PREDICTION SCORE TO IDENTIFY HEPATIC STEATOSIS IN ADOLESCENTS WITH POLYCYSTIC OVARIAN SYNDROME Authors: A. Carreau 1 ; L. Pyle 1 ; Y. Garcia-Reyes 1 ; H. Rahat 1 ; T. Jensen 1 ; K. J. Nadeau 1 ; M. Cree-Green Institution(s): University of Colorado Anschutz Medical Campus 1 Background: Hepatic steatosis (HS) is common in obese girls with polycystic ovarian syndrome (PCOS) and may best predict future development of type 2 diabetes. To date, there are no non-invasive and inexpensive ways to identify HS in youth with PCOS. The onjective was to develop a new simple clinical prediction score for HS in young women with PCOS. Methods: The objective was to develop a new simple clinical prediction score for HS in young women with PCOS. 87 overweight/obese girls with PCOS (age years) had a fasting blood sample drawn, anthropometrics collected and abdominal magnetic resonance imaging performed for hepatic fat fraction. Girls were divided by the presence of HS (fat fraction 5.5% by MRI). Logistic regression with HS as the outcome and candidate predictors variables (BMI%ile, waist circumference, Homeostasis model assessment (HOMA), HDL, triglycerides, ALT, free testosterone, and SHBG) was performed. Backwards stepdown elimination identified variables that best predicted HS. A simplified model of the best variables was then created. Results: 52% of the girls had HS. Initial logistic regression with all candidate variables was discriminant for presence of HS (r2= 0.21), as was the simplified model that included BMI%ile, waist circumference, ALT and SHBG (r2= 0.37). A nomogram with each variable was created to calculate a prediction score with the simplified model. A score of 110 points was the optimal cut-off for sensitivity/specificity (sensitivity 0.82, specificity 0.69, positive predictive value 0.78, negative predictive value 0.74, AUC ROC 0.81). Conclusion: This new index can help clinicians to identify obese girls with PCOS at high risk of HS to better assess non-alcoholic fatty liver disease status and future cardiometabolic disease risk, follow-up and treatment. Disclosure: Nothing to disclose. 85

90 # 6 Mayra Pastore, PhD University of California, San Francisco San Francisco, CA mayra_pastore@yahoo.com CHARACTERIZING SEX DIFFERENCES OF ESTROGEN RECEPTOR-Α ACTIVITY IN GLUCOSE AND FATTY ACID HOMEOSTASIS IN NON-ALCOHOLIC FATTY LIVER DISEASE. Authors: M. Pastore 1 ; H. Escusa 1 ; H. Ingraham 1 Institution(s): University of California, San Francisco 1 Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) has paralleled the increased rates of obesity in the US. Estrogen signaling as mediated by estrogen receptor-α (ERα) is known to regulate female metabolism in both humans and animal models. Furthermore, when ovarian estrogen is eliminated, as in post-menopausal women or after ovariectomy in female mice, hepatic fat increases concomitant with insulin resistance. Although ERα is expressed in the female liver its role in hepatocytes remains controversial. Methods: Here, we manipulated ERα expression in both male and female hepatocytes to assess its potential role in NALFD. Adult Esr1f/f male and female mice were infected via retro-orbital injection with AAV8-control or Cre (ERKOLiver) to acutely knockout ERα specifically in the hepatocytes. Mice were placed on high fat diet (HFD) for 10 wks and housed in either standard conditions or placed in thermoneutral chambers followed by physiological and metabolic analyses. Results: Under standard housing conditions, both male and female mice showed unaltered glucose or insulin tolerance and hepatic/muscle glycogen content. Similarly, hepatic cholesterol and triglycerides were unchanged between the controls and ERKOLiver male mice. However, male ERKOLiver mice showed decreased glucose production as measured by the pyruvate tolerance test (PTT) with a concurrent reduction in phosphoenoylpyruvate carboxykinase (Pck1) transcript levels suggesting that the removal of estrogen signaling in male mice compromises gluconeogenesis. By contrast when housed in standard conditions female ERKOLiver mice displayed reduced circulating triglyceride levels but increased hepatic triglyceride content suggesting that loss of hepatocyte estrogen signaling promotes hepatic lipid accumulation. Females ERKOLiver mice kept in thermoneutral chambers were lighter than controls but showed significant ERKOLiver mice hepatic lipid accumulation which exceeded levels observed for ERKOLiver mice kept in standard housing conditions. Hepatic glycogen content was drastically reduced in ERKOLiver female mice while muscle glycogen levels remain the same. When fasted for 16 hrs ERKOLiver female mice displayed normal glucose tolerance, but showed initial signs of insulin resistance. Following PTT, ERKOLiver mice had robust glucose production and higher glucose levels, suggesting that there are major differences in male and female hepatic estrogen signaling as it impacts gluconeogenesis. 86

91 Conclusion: Collectively our findings reveal important estrogen-dependent sex differences in nutrient homeostasis in the liver and provide evidence that hormonal dysregulation may play a key role in the development and progression of NAFLD. References 1. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol. 2013;10: Zhang H, Liu Y, Wang L, et al. Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in the male rat. J Lipid Res. 2013;54: Shen M, Shi H. Sex Hormones and Their Receptors Regulate Liver Energy Homeostasis. Int J Endocrinol. 2015;2015: Disclosure: Nothing to disclose. 87

92 # 7 Anna Roskilly University of Leeds Leeds United Kingdom aroskilly@doctors.org.uk SLOW FIBROSIS PROGRESSION RATES IN PLACEBO-TREATED PARTICIPANTS WITH NASH IN RANDOMISED CONTROLLED TRIALS PREDICT LOW RATES OF CIRRHOSIS DEVELOPMENT Authors: A. Roskilly 1 ; A. Hicks 1 ; E. Taylor 2 ; R. Jones 1 ; R. Parker 1 ; I. Rowe 2 Institution(s): Leeds Teaching Hospitals NHS Trust 1 ; University of Leeds 2 Background: The natural history of non-alcoholic steatohepatitis (NASH) is incompletely understood. Defining the rate of progression of fibrosis is critical to understanding the likely benefits of treatments. Repeat biopsy studies have suggested that the fibrosis progression rate (FPR) in NASH is approximately 0.14 fibrosis stages per year but these studies are prone to selection bias. Placebo treated participants in randomised trials of new therapeutics for offer the potential to evaluate the FPR in well-defined populations with NASH. Accurately defining FPRs will allow the development of natural history models that predict the proportion of individuals with NASH who will ultimately develop cirrhosis. Aim: To estimate the proportion of individuals with NASH that will develop cirrhosis in their lifetime. Methods: Annual FPRs were calculated from a systematic review of placebo treated participants in randomised controlled trials. Probabilistic models were generated where fibrosis progressed from no fibrosis to cirrhosis in cohorts aged 30-year old at baseline. The rate of progression was randomly assigned to each simulated individual according to distributions defined by the FPRs described. Non-liver mortality was simulated using UK population data. The model outcomes for the placebo treated participants in randomised controlled trials were compared to model outcomes for the FPR described in a systematic review of non-randomised repeat biopsy studies. Results: 19 trials including 898 participants were identified in the systematic review. Annual FPRs were calculable for 18 trials with a pooled annual FPR of stages per year. Considering those trials at the lowest risk of bias (5 trials, 466 participants), the pooled annual FPR was 0.04 stages per year. Taking this value into the probabilistic model, 3.8% of individuals develop cirrhosis in their lifetime and the median age at development of cirrhosis was 75 years. In comparison, using the pooled annual FPR from non-randomised studies of 0.14 stages per year, 28% of individuals develop cirrhosis and the median age at cirrhosis development was 50 years (Figure). Increasing rates of non-liver mortality had only a minimal effect on the proportion of individuals developing cirrhosis. 88

93 Conclusion: Fibrosis progression rates in placebo treated participants with NASH enrolled in randomised controlled trials are significantly lower than in non-randomised repeat biopsy studies. In simulation models this predicts fewer than 1 in 25 individuals with NASH will develop cirrhosis in their lifetime, a 7-fold reduction in the proportion of individuals estimated to develop cirrhosis when compared with non-randomised studies. Current natural history models may overestimate the impact of NASH on the development of cirrhosis. There is therefore an urgent need to better define the natural history of NASH to inform the design of clinical trials and to define the likely benefits of pharmacological treatment. Disclosure Commercial Speaker's Bureau: Abbvie 89

94 # 8 Anna Roskilly University of Leeds Leeds United Kingdom aroskilly@doctors.org.uk FIBROSIS STAGE IMPROVEMENT IN NASH: A SYSTEMATIC REVIEW OF PLACEBO TREATED PARTICIPANTS IN RANDOMIZED CONTROLLED CLINICAL TRIALS Authors: A. Roskilly 2 ; E. Taylor 1 ; R. Jones 2 ; I. Rowe 1 Institution(s): University of Leeds1; Leeds Teaching Hospitals NHS Trust 2 Background: The severity of fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) is the critical determinant of liver related mortality. Consequently, improvements in liver fibrosis are being incorporated into clinical trial endpoints for new therapies for patients with non-alcoholic steatohepatitis (NASH). The aim of this study was to estimate the proportion of patients with NASH with fibrosis improvement in timeframes relevant to ongoing trials. Methods: We undertook a systematic review of placebo controlled randomized clinical trials (RCT) with a histological endpoint including patients with NASH. Data reported were extracted and the proportion of patients with fibrosis improvement, defined as at least 1 stage improvement, were calculated per protocol. A prespecified subgroup analysis explored fibrosis improvement in patients with fibrosis stage (F) 1 since those individuals with F0 at baseline could not improve fibrosis. The proportion and 95% confidence interval (95%CI) was calculated for each study and summarized as a pooled estimate. Results: We identified 31 RCT of therapies for patients with NASH with a histological endpoint. Of these, 19 trials (including 898 placebo treated participants) reported the proportion of those included where fibrosis improved. In these studies, a total of 717 placebo treated participants underwent repeat liver biopsy per protocol. There was substantial variation in the proportion of participants improving fibrosis (range 0 to 45%, Figure). The pooled proportion improving fibrosis in these 717 participants was 21.8% (95%CI %). 15 RCT reported fibrosis stage at baseline, and in these trials the proportion of participants with F0 was 18.8%. In a subgroup analysis excluding these participants, the pooled fibrosis improvement rate was 26.2% (95%CI %). 90

95 Conclusion: This study has important implications for current and future clinical trial design and interpretation. There is marked heterogeneity in the reported proportions of placebo treated participants with NASH improving fibrosis that requires further study. The pooled estimate for participants with F 1 at baseline improving fibrosis is 26.2%. This is consistent with previous reports of sampling variability in NASH, highlighting the deficiencies of liver biopsy to determine fibrosis endpoints in clinical trials in NASH. Validation of fibrosis stage improvement as a surrogate endpoint is an important outcome of the ongoing Phase 3 clinical trials. Disclosure Commercial Speaker's Bureau: Abbvie 91

96 # 9 Shikha Sundaram, MD MSCI Children's Hospital Aurora, CO shikha.sundaram@childrenscolorado.org NOCTURNAL HYPOXIA ACTIVATION OF THE HEDGEHOG SIGNALING PATHWAY AFFECTS PEDIATRIC NAFLD DISEASE SEVERITY Authors: M. Swiderska-Syn 1 ; R. Sokol 2 ; A. Diehl 3 ; A. Halbower 2 ; K. Capocelli 2 ; K. Robbins 2 ; Z. Pan 2 Institution(s): Medical College of South Carolina 1 ; Children's Hospital Colorado 2 ; Duke University 3 Background: Chronic intermittent hypoxia and dysregulation of the Hedgehog (Hh) pathway are associated with NAFLD progression. Objective: To understand the relationship between OSA/nocturnal hypoxia and dysregulation of the Hh signaling pathway in pediatric NAFLD. Methods: 31 adolescents with biopsy-proven NAFLD underwent polysomnogram, liver histology scoring, and laboratory testing. Sonic hedgehog (SHh), Indian Hh (IHh), Glioblastoma associated oncogene 2 (Gli2), (keratin 7- K7) and myofibroblasts (α-sma) were evaluated by immunohistochemistry. Results: Subjects with (68%) and without (32%) OSA/hypoxia had similar aminotransferases, lipids, inflammatory and insulin resistance markers. AST correlated with ShH (r=0.64), Gli2 (r=0.47), α-sma (r=0.55) and K7 (r=0.45), p<0.01, as did ALT (ShH, r=0.51; Gli2, r=0.43; α-sma, r=0.51, p<0.02). ShH correlated with NAS summary score (r=0.39), while IHh inversely correlated with inflammation (r=-0.478) and histologic grade (r=-0.43), p<0.03. Moreover, α- SMA correlated with the NAS summary score (r=0.43) and fibrosis stage (r=0.41), p<0.02. Subjects with OSA/hypoxia had higher mean SHh (4.0 ± 2.9 vs. 2.0 ± 1.5), Gli2 (74.2 ± 28.0 vs ± 11.8) and α-sma (6.2 ± 3.3 vs. 4.3 ± 1.2) levels, p <0.03. The severity of OSA (AHI) correlated with SHh (r=0.31, p=0.09) and Gli2 positively (r=0.37, p=0.04). The severity of hypoxia was also associated with increasing SHh (r=-0.53), GLI2 (r=-0.52), α SMA (r=-0.61), and K7(r=-0.42), p<0.02. Prolonged hypoxia (time with SaO2 < 90%) also strongly correlated with SHh (r=0.55), GLI2 (r=0.61), α SMA (r=0.7), and K7 (r=0.36), p<0.05. Conclusions: The Hh pathway is activated in pediatric NAFLD patients with nocturnal hypoxia and relates to disease severity. Tissue hypoxia may allow for functional activation of Hypoxia Inducible Factor 1α, with subsequent induction of genes important in epithelial-mesenchymal transition, including SHh, and NAFLD progression. Methods: 31 adolescents with biopsy-proven NAFLD underwent polysomnogram, liver histology scoring, and laboratory testing. Sonic hedgehog (SHh), Indian Hh (IHh), Glioblastoma associated oncogene 2 (Gli2), (keratin 7- K7) and myofibroblasts (α-sma) were evaluated by immunohistochemistry. Results: Subjects with (68%) and without (32%) OSA/hypoxia had similar aminotransferases, lipids, inflammatory and insulin resistance markers. AST correlated with ShH (r=0.64), Gli2 (r=0.47), α-sma (r=0.55) and K7 (r=0.45), p<0.01, as did ALT (ShH, r=0.51; Gli2, r=0.43; α-sma, r=0.51, p<0.02). ShH correlated with NAS summary score (r=0.39), while IHh inversely correlated with inflammation (r=-0.478) and histologic grade (r=- 92

97 0.43), p<0.03. Moreover, α- SMA correlated with the NAS summary score (r=0.43) and fibrosis stage (r=0.41), p<0.02. Subjects with OSA/hypoxia had higher mean SHh (4.0 ± 2.9 vs. 2.0 ± 1.5), Gli2 (74.2 ± 28.0 vs ± 11.8) and α-sma (6.2 ± 3.3 vs. 4.3 ± 1.2) levels, p <0.03. The severity of OSA (AHI) correlated with SHh (r=0.31, p=0.09) and Gli2 positively (r=0.37, p=0.04). The severity of hypoxia was also associated with increasing SHh (r=-0.53), GLI2 (r=-0.52), α SMA (r=-0.61), and K7(r=-0.42), p<0.02. Prolonged hypoxia (time with SaO2 < 90%) also strongly correlated with SHh (r=0.55), GLI2 (r=0.61), α SMA (r=0.7), and K7 (r=0.36), p<0.05. Conclusion: The Hh pathway is activated in pediatric NAFLD patients with nocturnal hypoxia and relates to disease severity. Tissue hypoxia may allow for functional activation of Hypoxia Inducible Factor 1α, with subsequent induction of genes important in epithelial-mesenchymal transition, including SHh, and NAFLD progression. Disclosure: Nothing to disclose. 93

98 The Liver Meeting has it all. High quality presentations. Top-tier research. Networking. Highlights of The Liver Meeting 2018 include: in 2018: Associates Day on Sunday, November 11 Trainee Day on Friday, November 9 Postgraduate Course on Saturday, November 10: A Hectic Day for a Hepatology Consultant Basic Science Symposium on Saturday, November 10: Matrix Biology and the Liver: Beyond Collagen and Fibrosis SIG Programming: Programs sponsored by our SIGs to drill down on highly specialized topics in hepatology. July 18 Advance Registration for AASLD Fellow Members July 25 Advance Registration for AASLD Members Register Online at: aasld.org/livermeeting The Liver Meeting will offer many opportunities to claim CME, CE and MOC this year. Stay tuned for more information on continuing education. August 1 General Advance Registration October 12 Advance Registration Deadline

99 HEPATIC FIBROSIS: NEW CONCEPTS AND CONTROVERSIES SEPTEMBER 14 15, 2018 DFW AIRPORT, TEXAS Program Chairs Don C. Rockey, MD, FAASLD Meena B. Bansal, MD Natalie J. Torok, MD, MSc, FAASLD This single topic conference will investigate the basic concepts underlying hepatic tissue fibrosis, with a focus on recent research and the development of new therapeutics. Offering an overview of the latest basic research, plus clinical updates and opportunities for networking and collaboration, this is a mustattend event for anyone with an interest in fibrosis. Come learn about hepatic fibrosis in depth and see what s in store for future treatments. Continuing Education 11.0 AMA PRA Category 1 Credits TM Visit aasld.org/stc to register and submit an abstract.

100 CALL FOR APPLICATIONS 2019 Research & Career Development Awards AASLD Foundation is investing even more in innovative hepatology research and in the people who study and treat liver disease. AASLD Foundation funds awards for researchers at all career levels, including: Early to mid-career investigators pursuing liver related research. Front-line providers seeking hepatology-focused training to adequately treat and care for liver disease patients. Travel to educational meetings, including the Abstract Travel Awards and the Emerging Liver Scholar Resident Travel Awards.* * These awards have different deadlines. To find out more and to apply, refer to the Travel Awards section at LEARN MORE AND APPLY AT aasldfnd.org/awards Application Deadlines Research Awards, Advanced/Transplant Hepatology Award, NP/PA Clinical Hepatology Fellowship: DECEMBER 4, 2018 (for funding beginning July 1, 2019) Bridge Award OCTOBER 5, 2018 (for funding beginning February 1, 2019) JANUARY 15, 2019 (for funding beginning May 1, 2019)