1. Lean Nonalcoholic Fatty Liver disease
목요세미나
Lean Nonalcoholic Fatty Liver disease
R3 김광우 / Pf. 김원

2. Background
Ref :

3. Background Lean Nonalcoholic Fatty Liver Disease (NAFLD) Prevalence
: Patients with a body mass index (BMI) <25 kg/m2
Prevalence
- NAFLD : 25%, world wide
- Lean NAFLD : varies from 7% in the United States to 19% in Asia
Ref. : Lisa B. VanWagner and Matthew J. Armstrong, Lean NAFLD: A not so benign condition? Hepatol Commun Jan; 2(1): 5–8.

4. Histological Severity and Clinical Outcomes of Nonalcoholic Fatty Liver Disease in Nonobese Patients
Jonathan Chung-Fai Leung, et al.
HEPATOLOGY 2017;65:54-64
Liver and Cardiovascular Damage in Patients With Lean Nonalcoholic Fatty Liver Disease, and Association With Visceral Obesity
Anna Ludovica Fracanzani, et al.
Clin Gastroenterol Hepatol Oct;15(10): e1.

5. Histological Severity and Clinical Outcomes of Nonalcoholic Fatty Liver Disease in Nonobese Patients
Jonathan Chung-Fai Leung, et al.
HEPATOLOGY 2017;65:54-64
Liver and Cardiovascular Damage in Patients With Lean Nonalcoholic Fatty Liver Disease, and Association With Visceral Obesity
Anna Ludovica Fracanzani, et al.
Clin Gastroenterol Hepatol Oct;15(10): e1.

6. Introduction NAFLD(Non-alcoholic fatty liver disease) Risk factors
One of the most common chronic liver disease
Progress to Non-alcoholic steatohepatitis (NASH), Cirrhosis, HCC
Increased risk of cardiovascular event, malignancies, mortality
Risk factors
Obesity
Metabolic syndromes (MetS)
Cardiovascular risk factors

7. Introduction Severity, fibrosis, related factors, prognosis of
More common in obese, but there is significant proportion of patients having relatively normal BMI
 “Lean” or “Non-obese NAFLD”
Not well understood
Mixed results
Severity, fibrosis, related factors, prognosis of
Obese VS Nonobese NAFLD

8. Patients and Methods Patients Prospective cohort study
Liver biopsy, non invasive test
Indication for liver biopsy
Unexplained elevation of liver enzymes
Evaluation of NAFLD severity
Participation in clinical trials
Aged 18 or above, histology-proven NAFLD
Excluded
HBsAg (+), HCV Ab (+) with HCV RNA (+)
Excessive alcohol consumption
Secondary fatty liver
Malignancy before baseline

9. Patients and Methods Clinical assessment
History taking, blood test, transient elastography (TE) done 1 day before liver biopsy
History taking : standardized questionnaire
BMI: nonobese – less than 25 kg/m2
Waist circumference (WC)
Liver biochemistry, glucose, insulin, lipids
MetS: 3 or more of the followings
Central obesity: men ≥ WC 90cm, women ≥ WC 80cm
Triglyceride > 1.7 mmol/L
HDL : men ≤ 1.03 mmol/L, women ≤ 1.29 mmol/L
BP ≥ 130/85 mmHg
Fasting plasma glucose ≥ 5.6 mmol/L
Receiving treatment

10. Patients and Methods Assessment of disease severity
Liver biopsy: 2 specimens, ≥ 15mm in length
Score: NASH Clinical Research Network System
NASH definition : Presence of steatosis, Lobular inflammation, Hepatocyte ballooning
Fibrosis staging : F0–4, (F3–4 : advanced)
Serum cytokeratin-18(CK-18)
: Marker of hepatocyte apoptosis and NASH
TE with Fibroscan
: Liver stiffness measurement (LSM)

11. Patients and Methods Long-term Outcomes Primary analysis
Prospectively followed yearly after baseline liver biopsy
Crosscheck : Local EMR system
Primary analysis
Cardiovascular events
Liver-related events
Malignancies
Mortality

12. Patients and Methods Statistical Analysis SPSS statistics software
Continuous variables & Ordinal variables :
Expression: mean ± SD, median (IQR)
Comparison: unpaired t-test, Mann-Whitney U test
Categorical variables: χ 2 test
Independent variables
NAFLD Activity Score (NAS): Ordinal logistic regression model
Advanced liver fibrosis : Binary logistic regression model
Clinical outcomes: Kaplan-Meier curves
Compare with obese vs. non-obese : Log-rank test
Total sample size 284  80% power to detect the difference at 5% significance level

13. Results

14. Results : Clinical characteristics of nonobese and obese patients

15. Results : NAFLD Activity in nonobese and obese patients

16. Results : Factors associated with disease activity in nonobese patients

17. Results : Fibrosis in nonobese and obese patients
Less-severe fibrosis
Lower liver stiffness by TE
Similar in advanced fibrosis

18. Results : Factors associated with advanced fibrosis in nonobese patients

19. Results : Disease progression in nonobese and obese patients

20. Results : prognosis of nonobese and obese patients

21. Results : prognosis of nonobese and obese patients
Clinical events occurred in 11.9% of obese patients
and 8.3% of nonobese patients (P = 0.190)

22. Discussion Nonobese NAFLD patients Similar prevalence of NASH
Lower NAS : lower degree of steatosis, hepatocyte ballooning
Less likely to have liver fibrosis
Similar proportions of patients with advanced fibrosis
High TG  high NAS
Creatinine (Cr.) : The only independent predictor of advanced fibrosis

23. Discussion Non-obese NAFLD histology : conflicting
More lobular inflammation & higher mortality?
Factors other than adiposity
High TG associated with high NAS
Only with steatosis, hepatocyte ballooning
No effect to lobular inflammation
High Cr. Level associated with advanced fibrosis
Associated with chronic kidney disease
In DM, early in nephropathy associated NAFLD, advanced fibrosis
Genetic factors : PNPLA3, TM6SF2
PNPLA3: hepatic steatosis  disease severity, progression
TM6SF2: disease progression
In this study: No relationship (small number of nonobese patients with advanced disease)

24. Discussion Obese: more clinical events occur
Cardiovascular, death  worse prognosis, high mortality
Obesity itself related with cardiovascular event
 Treatment : Weight loss
Nonobese: better prognosis, lower mortality
Longer study show higher mortality in nonobese
 Long term prognosis study needed

25. Discussion Strength Limitation Prospective design Systematic follow-up
Inclusion of histological, outcome data
Biopsy largely verified noninvasive assessments
Limitation
Patients with NASH or advanced fibrosis relatively small
Liver biopsy: only represents 1/50,000th of the entire liver
 sampling bias
Follow-up duration relatively short

26. Conclusions
Nonobese patients with NAFLD tend to have less-severe disease and may have a better prognosis than obese patients.
Hypertriglyceridemia, higher creatinine associated with advanced liver disease in nonobese patients.
Further research on the relationships between these risk factors and advanced liver disease is needed.

27. Histological Severity and Clinical Outcomes of Nonalcoholic Fatty Liver Disease in Nonobese Patients
Jonathan Chung-Fai Leung, et al.
HEPATOLOGY 2017;65:54-64
Liver and Cardiovascular Damage in Patients With Lean Nonalcoholic Fatty Liver Disease, and Association With Visceral Obesity
Anna Ludovica Fracanzani, et al.
Clin Gastroenterol Hepatol Oct;15(10): e1.

28. Introduction NAFLD(Non-alcoholic fatty liver disease)
The most common liver disease in western countries, affecting 20% to 34% of the general population
“Lean” NAFLD : NAFLD with a normal BMI (Body mass index)
Major challenge in Diagnosis of Lean NAFLD
Causes unclear
Scanty, conflicting data on the severity of liver, cardiovascular damage : different methods used

29. Introduction Genetic factors
Partly explain severe liver damage (NASH, advanced fibrosis), atherosclerotic disease
I148M variant of the patatin-like phospholipase domain-containing-3 (PNPLA3)
rs C>T genetic variant of the transmembrane 6 superfamily member 2 (TM6SF2)
Asian population : higher prevalence of PNPLA3 mutations in lean NAFLD

30. Introduction Primary objective Secondary outcome
Differences between lean and overweight/obese NAFLD
Factors : severe liver disease, cardiovascular disease
Secondary outcome
Evaluated the role of visceral obesity : WC
 On hepatic, vascular, metabolic alteration

31. Biopsy-proven NAFLD, Caucasian
Methods
Study Design and Participants
669 retrospective cohort
Biopsy-proven NAFLD, Caucasian
324 Milan, 323 Palermo, 22 Catania
Exclusion criteria:
Viral B and C hepatitis,
Wilson disease,
a1-antitrypsin deficiency,
Autoimmune hepatitis,
Genetic hemochromatosis,
Using drugs potentially causing steatosis, average Daily alcohol consumption (in grams of pure ethanol) higher than 20 g in women and 30 g in men (confirmed by at least 1 family member).
143
Lean
523
Overweight/Obese
Smoking : current, former, never-smoker

32. Methods Data acquisition Diabetes mellitus (DM) Insulin resistance
MetS
Lean/Overweight/Obese : <25 kg/m2 / kg/m2 / ≥ 30 kg/m2
WC (cm, men/women)
: group A (<94 / <80), group B ( / 80-88), group C (>102 / >88)
Carotid atherosclerosis assessment
: Mean carotid artery intima-media thickness (cIMT)
Liver histology
: grade steatosis, lobular inflammation, hepatocellular ballooning / stage fibrosis
0-4 / NAS 0-8
Insulin, adiponectin, and adipose tissue insulin resistance
Genotyping
: rs C>G (I148M PNPLA3), rs C>T (E167K TM6SF2),
singlenucleotide polymorphisms

33. Methods Data acquisition : Statistical analysis
χ 2 test : categoric variables, compared in men and women
Mann-Whitney or Kruskal-Wallis test : quantitative variables, compared in men and women)
Multiple linear or logistic regression analyses : significant at univariate analyses (p <0.05) but including only one of the variables with a similar role
Hosmer-Lemeshow test: Goodness-of-fit model
Receiver operating characteristic (ROC) curve analysis : evaluated the diagnostic performance of PNPLA3 in lean NAFLD

34. Results : Characteristics of Lean and Overweight/Obese Nonalcoholic Fatty Liver Disease

35. Results : Factors Related to More Severe Liver and Cardiovascular Disease Multivariable Analysis
Table 2. Variables Associated With NASH (Present in 240 Patients, 25 Among Lean and 215 Among Overweight/Obese) at Multivariate Analysis in Lean or Overweight/Obese NAFLD Patients

36. Results : Factors Related to More Severe Liver and Cardiovascular Disease Multivariable Analysis
Table 3. Variables Associated With Fibrosis ≥ 2 (Present in 246 Patients, 25 Among Lean and 221 Among Overweight/Obese) at Multivariate Analysis in Lean or Overweight/Obese NAFLD

37. Results : Factors Related to More Severe Liver and Cardiovascular Disease Multivariable Analysis
Table 4. Variables Associated With cplaques (Present in 201 Patients, 26 Among Lean and 175 Among Overweight/Obese) at Multivariate Analysis in Lean or Overweight/Obese NAFLD

38. Results : Role of Visceral Obesity

39. Results : Role of Visceral Obesity
Lean NAFLD with higher WC
Compared with overweight/obese group
DM, cplaques, fibrosis score of 2 or greater
Not reach significance
Compared with non-visceral obesity
NASH, fibrosis score of 2 or greater, MetS, cplaques
Reach significance

40. Results : Adiponectin, Adipose Tissue Insulin Resistance
Lean patient : higher adiponectin
Patients (overall, overweight/obese) with NASH : lower adiponectin, not significant
Adipose tissue insulin resistance
Overweight/obese
Visceral obesity (higher WC)
Patients with NASH : even each lean and overweight/obese
Adjusting for age, sex : Correlated with NASH / not fibrosis score of 2 or greater, carotid plaques

41. Discussion Conflicting data in lean NAFLD Lean NAFLD Asian NAFLD 1)
17% Severe liver damage : NASH, fibrosis score of 2 or higher
27% Carotid plaques
Conflicting data in lean NAFLD
Asian NAFLD 1)
20%, by magnetic resonance spectroscopy
43% NASH, by NAS score 3 or greater
US NAFLD 2)
7%, by ultrasonography
0.1% NASH, by increased ALT level + DM / Insulin resistance
1) Wei JL, Leung JC, Loong TC, et al. Prevalence and severity of nonalcoholic fatty liver disease in non-obese patients: a population study using proton-magnetic resonance spectroscopy. Am J Gastroenterol 2015;110:1306–1314; quiz 1315.
2) Younossi ZM, Stepanova M, Negro F, et al. Nonalcoholic fatty liver disease in lean individuals in the United States. Medicine (Baltimore) 2012;91:319–327.

42. Discussion Lean NAFLD with Higher WC
DM, cplaques, fibrosis score of 2 or greater
Coronary artery disease : worse survival at normal weight with central obesity than normal/overweight/obese without central obesity 3)
Ectopic fat accumulation : associated with
Insulin resistance
Proinflammatory cytokines
Endothelial dysfunction
Inflammatory gene : increased with the progression of histologic liver damage
3) Coutinho T, Goel K, Correa de Sa D, et al. Combining body mass index with measures of central obesity in the assessment of mortality in subjects with coronary disease: role of “normal weight central obesity”. J Am Coll Cardiol 2013;61:553–560.

43. Discussion Lean NAFLD with PNPLA3 polymorphism
PNPLA3 GG polymorphism : only independent variable associated with NASH, fibrosis score of 2 or greater
 More severe liver damage
No differences prevalence of gene : lean vs overweight/obese
PNPLA3 GG polymorphism
More susceptible to environmental factors : manifest liver disease
Associated significantly with NASH in lowest WC
Maybe major role in lean NAFLD with lower WC
Future study with larger number of patients needed

44. Discussion Limitation Strength Relatively small number
Not quantify the cytokines related between fatty liver and cardiovascular disease
Strength
Availability of a well-characterized cohort of patients, with contemporary assessment of liver histology, cardiovascular damage
Subset of Caucasian, other factors than metabolic alteration could play a role

45. Conclusion
20% of patients with lean NAFLD to have NASH, significant fibrosis (score 2 or greater), carotid atherosclerosis
Genetic polymorphism play a key role in the severity of disease, but data preliminary
Prompt the genetic characterization of lean patients with NAFLD to confirm this hypothesis

46. Thank you for your attention