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?
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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 centralcentral and central-portal bridging
Chronic Hepatitis C
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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
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Definition of Ballooning
• Cell is enlarged beyond backround, nonsteatotic hepatocytes
• Cytoplasm is altered by clearing and
clumping, not dominated by fat vacuoles
• Mallory-Denk bodies may be present
Agreement
Feature
Steatosis Grade
Lobular
Inflammation
Inter-rater
Kappa
Intra-rater
Kappa
0.79
0.85
0.45
0.91
Portal Inflammation
0.45
0.7
Ballooning
0.56
0.7
Fibrosis
Diagnostic
Classification
0.84
0.91
0.61
0.59 to 0.88
(no defined criteria)
Hepatology 2011; 53: 810-820
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So, what do you do with cells that
don’t seem to fit all the criteria?
Classical vs Non-Classical
• Classical ballooning
–
–
–
–
Enlarged (>1.5x nml)
Cytoplasmic clearing
Cytoplasmic clumping
May have MalloryDenk bodies
• Non-Classical ballooning
– Typically in zone 3,
perivenular
– Smaller and often in groups
– Same cytoplasmic
alterations
– Lack Mallory-Denk bodies
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There are Significant Clinical and Histological
Differences Between Categories of Ballooning
Non-Classical Vs
None
Steatosis
Classical Vs
Non-Classical
Severe Vs
Not-Severe (Classical)
++++
++++
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%
Percent of Cases
90%
80%
Stage
70%
4
3
2
1C
1B
1A
None
60%
50%
40%
30%
20%
10%
0%
None
Non-Classic Few, Classic
Many,
Classic
Severe
p < 0.0001
Clear Relationship of Ballooning
Degree to Diagnosis
100%
Percent of Cases
90%
80%
70%
60%
Definite SH
Bdln SH
Not SH
50%
40%
30%
20%
10%
0%
None
Non-Classic
Few,
Classic
Many,
Classic
Severe
p < 0.0001
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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
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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)
35
p<0.0001
Chi-square
% Population
30
25
20
15
10
5
0
0
1a
none
Mild psf
1b
1c
Mod psf periportal
2
3
4
psf+pp
bridging
cirrhosis
Fibrosis
Fibrosis Progression In Viral
Hepatitis
None
Periportal
Fibrosis
Bridging
Fibrosis
Cirrhosis
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Fibrosis Progression Pathways
in NASH
Perisinus., mod.
1B
Perisinus., mild
1A
None
0
Periportal/
Pericentral
2
Bridging
3
Cirrhosis
4
Periportal Only
1C
Diagnosis of Periportal Fibrosis
in NAFLD/NASH
• Extension of
pericellular septations
• Trapping of periportal
hepatocytes
Stage 2 Fibrosis
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Stage 2 Fibrosis
The distinction between Stage 2
and Stages 1 and 3 is important
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Progression and Regression of Fibrosis
“Red” = Progressed, “Green” = Regressed
Stage at Last Follow-up Biopsy
Stage at First Biopsy
0
1A
1B
1C
2
3
4
0
47
6
7
6
17
3
0
1A
17
10
7
1
12
4
1
1B
13
3
12
0
7
12
0
1C
7
0
0
3
2
1
1
2
15
5
13
2
15
17
4
3
5
0
7
1
12
43
21
4
0
0
0
0
0
6
10
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
(n = 619)
Unadjusted cumulative P value
(%)a
Portal inflammation, grade
<0.001
0
25.5
1
29.3
2
50.0
Ballooning, grade
<0.001
0
28.3
1
32.7
2
42.5
NASH category
<0.001
Non-NASH
25.4
Borderline/suspicious
40
Definitive NASH
36.9
Fibrosis stage
<0.001
0
23.0
1
29.8
2
42.3
3
50.9
4
77.8
Steatosis and Lobular inflammation were not-significant
Liver Biopsy Features
Liver-related events
(n = 615)
Unadjusted cumulative P value
(%)a
<0.001
1.9
3.5
11.8
<0.001
2.6
5.3
10.3
<0.001
1.2
6.7
8.5
<0.001
1.6
2.8
7.1
13.7
23.5
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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
0
1
2
Portal Inflammation
1
2
3
Lobular Inflammation
Hepatology 2014;59: 1393-1405
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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
% Population
None
Mild
p<0.0001
More than Mild
45
40
35
30
25
20
15
10
5
0
0
1a
none
Mild psf
1b
1c
Mod psf periportal
2
3
4
psf+pp
bridging
cirrhosis
Fibrosis
Portal Inflammation is Also Related to
Long-Term Outcome
Gastroenterology 2015;149:389-397
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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
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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.
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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.
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