12/14/2016 The Bugaboos of Fatty Liver Disease: Ballooning and Fibrosis Hans Popper Hepatopathology Society Companion Meeting San Antonio, Tx March, 2017 David Kleiner, M.D., Ph.D. NCI/Laboratory of Pathology Challenges in the Diagnosis of Steatohepatitis • Ballooning – Importance and Characterization • Fibrosis – The Complexity of Fibrosis Progression in NASH Why is Ballooning So Important? 1 12/14/2016 Steatohepatitis is a Specific Pattern of Liver Injury • Steatosis and Inflammation – Necessary but not sufficient • Zone 3 centered hepatocellular injury – Required for specific diagnosis – Ballooning degeneration, Mallory-Denk bodies • Fibrosis – Characteristic pattern, but outcome, not cause – Pericentral/sinusoidal “chicken-wire” fibrosis then central- central and central-portal bridging Chronic Hepatitis C 2 12/14/2016 Ballooning is Associated with Long Term Survival, Whereas Steatosis is Not Angulo et al., Gastroenterology 149: 389; 2015 Steatosis Ballooning P = 0.607 P < 0.001 3 12/14/2016 Definition of Ballooning • Cell is enlarged beyond backround, non- steatotic hepatocytes • Cytoplasm is altered by clearing and clumping, not dominated by fat vacuoles • Mallory-Denk bodies may be present Agreement Feature Inter-rater Intra-rater Kappa Kappa Steatosis Grade 0.79 0.85 Lobular 0.45 0.91 Inflammation Portal Inflammation 0.45 0.7 Ballooning 0.56 0.7 Fibrosis 0.84 0.91 Diagnostic Classification 0.61 0.59 to 0.88 (no defined criteria) Hepatology 2011; 53: 810-820 4 12/14/2016 5 12/14/2016 So, what do you do with cells that don’t seem to fit all the criteria? Classical vs Non-Classical • Classical ballooning • Non-Classical ballooning – Enlarged (>1.5x nml) – Typically in zone 3, – Cytoplasmic clearing perivenular – Cytoplasmic clumping – Smaller and often in groups – May have Mallory- – Same cytoplasmic Denk bodies alterations – Lack Mallory-Denk bodies 6 12/14/2016 There are Significant Clinical and Histological Differences Between Categories of Ballooning Non-Classical Vs Classical Vs Severe Vs None Non-Classical Not-Severe (Classical) Steatosis ++++ Lobular Inflammation ++ ++++ ++++ Portal Inflammation +++ ++ ++ Fibrosis ++++ ++++ ++++ Age + Sex ++++ +++ BMI + + Diabetes ++++ Metabolic Syndrome ++++ ALT ++++ Alk Phos ++ ++ Glucose ++++ + Insulin ++ + HOMA-IR + ++ + +: p 0.05; ++: p 0.01; +++: p 0.001; ++++: p 0.0001 The Degree of Ballooning is Strongly Associated with Fibrosis 100% 90% 80% Stage 70% 4 60% 3 50% 2 40% 1C 1B 30% Percent of Percent of Cases 1A 20% None 10% 0% None Non-Classic Few, Classic Many, Severe Classic p < 0.0001 Clear Relationship of Ballooning Degree to Diagnosis 100% 90% 80% 70% 60% 50% Definite SH 40% Bdln SH Not SH Percent of Percent of Cases 30% 20% 10% 0% None Non-Classic Few, Many, Severe Classic Classic p < 0.0001 7 12/14/2016 Ballooning Summary • Ballooning is important to recognize because – It makes steatohepatitis a specific pattern of injury even in the absence of fibrosis – It is prognostically important • Ballooning has distinctive features that allow us to pick out specific balloon cells • There may be relevance to cells that meet some, but not all, of the criteria The Problem of Fibrosis Staging in NASH Staging Fibrosis NASH CRN classification 0 No fibrosis 1 Perisinusoidal or periportal (but not both) 1A Zone 3 perisinusoidal fibrosis (mild, Trichrome only) 1B Zone 3 perisinusoidal fibrosis (moderate, seen on H&E) 1C portal fibrotic expansion/periportal fibrosis 2 Both perisinusoidal and periportal 3 Bridging fibrosis (any type) 4 Cirrhosis 8 12/14/2016 Questions Questions Questions • Why is it so complicated? • Why don’t the different stages have progressively more fibrosis? • Why is stage 1 split between perisinusoidal and pericentral fibrosis? Pediatric Biopsies are More Likely to Show Periportal rather than Central Perisinusoidal Fibrosis Adult (429) Child (102) p<0.0001 Chi-square 35 30 25 20 15 Population 10 % 5 0 01a1b1c2 3 4 none Mild psf Mod psf periportal psf+pp bridging cirrhosis Fibrosis Fibrosis Progression In Viral Hepatitis None Periportal Bridging Cirrhosis Fibrosis Fibrosis 9 12/14/2016 Fibrosis Progression Pathways in NASH Perisinus., mod. 1B Perisinus., mild 1A Periportal/ Pericentral Bridging Cirrhosis None 2 3 4 0 Periportal Only 1C Diagnosis of Periportal Fibrosis in NAFLD/NASH • Extension of pericellular septations • Trapping of periportal hepatocytes Stage 2 Fibrosis 10 12/14/2016 Stage 2 Fibrosis The distinction between Stage 2 and Stages 1 and 3 is important 11 12/14/2016 Progression and Regression of Fibrosis “Red” = Progressed, “Green” = Regressed Stage at Last Follow-up Biopsy 01A1B1C2 3 4 0476 7 61730 1A 17 10 7 1 12 4 1 1B 13 3 12 0 7120 1C 7 003211 2 15 5 13 2 15 17 4 3 50711243 21 Stage at First Biopsy First at Stage 4 00000610 375 patients Long-term Prognostic Relevance of Liver Histology in Nonalcoholic Fatty Liver Disease Gastroenterology 2015;149:389-397 • International collaborative longitudinal study of patients with NAFLD • 619 patients followed for a median of 12.6 years (range 0.3-35.1) • Inclusion based on liver biopsy with at least 5% steatosis (minimum diagnostic criterion for NAFLD) read by single pathologist Cumulative probability (unadjusted) of outcomes by histological feature Mortality/liver transplantation Liver-related events (n = 619) (n = 615) Liver Biopsy Features Unadjusted cumulative P value Unadjusted cumulative P value (%)a (%)a Portal inflammation, grade <0.001 <0.001 0 25.5 1.9 1 29.3 3.5 2 50.0 11.8 Ballooning, grade <0.001 <0.001 0 28.3 2.6 1 32.7 5.3 2 42.5 10.3 NASH category <0.001 <0.001 Non-NASH 25.4 1.2 Borderline/suspicious 40 6.7 Definitive NASH 36.9 8.5 Fibrosis stage <0.001 <0.001 0 23.0 1.6 1 29.8 2.8 2 42.3 7.1 3 50.9 13.7 4 77.8 23.5 Steatosis and Lobular inflammation were not-significant 12 12/14/2016 Gastroenterology 2015;149:389-397 Inflammation is Redistributed between Stage 1B and Stage 2 in NASH Stage 1B Stage 2 90 80 70 60 % 50 40 30 20 10 0 012 123 Portal Inflammation Lobular Inflammation Hepatology 2014;59: 1393-1405 13 12/14/2016 Findings • Portal inflammation was strongly correlated with fibrosis stage and ductular reaction (DR) • Portal inflammation was enriched for macrophages and CD8+ T-cells • Infiltration of portal areas by macrophages was the earliest change and preceded the increased expression of proinflammatory cytokines • Conclusion: Detailed characterization of portal inflammation, especially in the DR/progenitor cell niche, may be important to understand progression of disease in NASH Hepatology 2014;59: 139 Portal Inflammation Becomes More Severe as Periportal Fibrosis Develops and Progresses Brunt et al., Hepatology 2009;49:809-820 None Mild More than Mild p<0.0001 45 40 35 30 25 20 Population 15 10 % 5 0 01a1b1c234 none Mild psf Mod psf periportal psf+pp bridging cirrhosis Fibrosis Portal Inflammation is Also Related to Long-Term Outcome Gastroenterology 2015;149:389-397 14 12/14/2016 Fibrosis Summary • Fibrosis progression in NAFLD/NASH is much more complex than progression viral hepatitis • The development of periportal fibrosis appears to be part of the progression pathway in most cases • The degree of periportal vs central perisinusoidal fibrosis appears to vary significantly • Nonetheless, in cross-sectional analysis, patients with stage 2 fibrosis have a worse prognosis than those with stage 1 • The appearance of periportal fibrosis is linked with the development of increased portal inflammation www.cancer.gov www.cancer.gov/espanol References • 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;149:389-397 e310. • 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;59:1393-1405. • 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;49:809-820. • Patton HM, Lavine JE, Van Natta ML, Schwimmer JB, Kleiner D, Molleston J, Nonalcoholic Steatohepatitis Clinical Research N. Clinical correlates of histopathology in pediatric nonalcoholic steatohepatitis. Gastroenterology 2008;135:1961-1971 e1962. • Neuschwander-Tetri BA, Clark JM, Bass NM, Van Natta ML, Unalp-Arida A, Tonascia J, Zein CO, et al. Clinical, laboratory and histological associations in adults with nonalcoholic fatty liver disease. Hepatology 2010;52:913-924. • Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research. Semin Liver Dis 2012;32:3-13. • Kleiner DE, Makhlouf HR. Histology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in Adults and Children. Clin Liver Dis 2016;20:293-312. 15 12/14/2016 Staging and Grading Systems in Use as Clinical Research Tools • 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;94:2467-2474. • 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;41:1313-1321. • Bedossa P, Poitou C, Veyrie N, Bouillot JL, Basdevant A, Paradis V, Tordjman J, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology 2012;56:1751-1759. 16.