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1
HEPATIC FIBROSIS ASSESSMENT USING
MULTIPARAMETRIC BIOMARKER TESTS
PROF. DR. RALF LICHTINGHAGEN
MEDICAL UNIVERSITY HANNOVER
Institute for Clinical Chemistry
Topics
• Etiologies, natural history of e.g. NAFLD
• Fibrosis development, extracellular matrix (ECM)
• Gold standard liver biopsy
• ECM markers of liver fibrosis
• Multiparametric scores from standard measurands
• Multiparametric scores from innovative measurands
• Guidelines (e.g. viral hepatitis, NAFLD)
Etiology Group Quantitative
Importance in
Middle Europe
Example
Toxic frequent Alcohol (ARLD)
Metabolic frequent and *) NASH
Malignant frequent HCC, Metastases
Viral medium Hepatitis B und C
Autoimmune rare AIH, PBC
Hereditary rare Hemochromatosis
Vasculary very rare Budd-Chiari-
Syndrome
East
Asia
Etiologies of Chronic Liver Disease
*) NAFLD: rapidly growing indication for liver transplantation
Need for screening strategy in high risk poplation (obesity, type 2 diabetes)
(Wrong RJ et al. Gastroenterology 2015)
Natural History of NAFLD
NAFLD with
20-30% in general
population
STEATOSIS(90-95%)
NON-ALCOHOLIC STEATOHEPATITIS(NASH) (5-10%)
FIBROSIS (38%)
Cirrhosis (30%)
Decompensation (5-10%) Liver Cancer (1-2%)(in 10 y from cirrhosis development)
Stable Non cirrhoticdisease liver cancer
(<1%)
Type 2 diabetesCardiovascular disease
HEPATIC FIBROSIS ASSESSMENT USING
MULTIPARAMETRIC BIOMARKER TESTS
PROF. DR. RALF LICHTINGHAGEN
Institute for Clinical Chemistry
Etiology group Quantitative
Importance in
Middle Europe
Example
toxic frequent Alkohol
metabolic frequent and NASH
malignant frequent HCC, Metastases
viral medium Hepatitis B und C
autoimmune rare AIH, PBC
hereditary rare Hämochromatosis
vasculary very rare Budd-Chiari-
Syndrome
Perisinusoidal space
Pathophysiology ofLiver Fibrosis
Fibrotic Liver
Normal Liver
Fibronectin
Collagen IV
Collagen VI
Perlecan
Fibrillar Collagen
Collagen I
Collagen III
Neobasal membrane
•Laminin
•Entactin
•Collagen IV
•Perlecan
The Hepatic Extracellular Matrix (ECM)
2
Liver Biopsy
Gained information
• Degree of fibrois (Staging)
• Degree of inflammation (Grading)
• Etiology
Complications
• Secondary hemorrhage
• Injury to internal organs
• Hurt
• Death
Journal Year Grading Staging
Knodell RG et al. Hepatology 1981 I-III, 0 – 18 0 – 4
Ishak K et al. J Hepatol 1995 ABCD, 0-18 0 – 6
Scheuer PJ J Hepatol 1991 0 – 4 0 – 4
Desmet VJ et al. Hepatology 1994 0 – 4 0 – 4
METAVIR Group Hepatology 1994 0 – 3 0 – 4
Histological Staging of Chronic Liver Diseases
F0
F0
F1 F2 F3
F2
F4
F3
F5 F6
septa
porto-portal porto-central
F1 F4
portal fibrosis
portal triad central vein fibrotic matrix
Sampling Error of Liver Biopsy
Biopsy size (cm)Biopsy size (cm)0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Bedossa P et al., Hepatology 2003
Se
nsi
tiv
ity
(%
)
100
80
60
40
Se
nsi
tiv
ity
(%
)
100
80
60
40
20
0
F0
F1
F2
F3
F4
METAVIR
15 25 mm
15 25 mm
65
75
Hepatopathologist vs. Community Pathologist
Robert M et al., Clin Gastroenterol Hepatol 2009
Chronic hepatitis C, n=391
Aims in Laboratory Diagnosis of Fibrosis
Detection of liver cirrhosis
Further diagnostics(e.g. varicose vein screening)
HCC monitoring Indication for liver transplantation
Detection of fibrosis progression
Therapy management
Detection of well-defined fibrosis stages
Indication for therapy
Methods of Non Invasive Fibrosis Detection
Clinical Chemistry
Liver Function Tests Fibrosis MetabolismECM components and enzymes
• Transaminases• GGT• Cholesterol• Platelets• α2 Macroglobulin• Haptoglobin• Bilirubin• Apo A1
mono- vs. multiparametric
• Hyaluronan• PIIINP (Collagen III)
• TIMP-1• Laminin• MMP-2• Tenascin• Undulin• 7S (Collagen IV)
Imaging
Elasticity ClassicalImaging
• FibroScan• MR Elastograph.• ARFI• 2D SWE…
• Ultrasound &Doppler
• MRI• CT…
Vs. gold standard liver biopsy(amount of fibrosis (staging),inflammation, etiology)
3
Expression Profiles of Components of the Hepatic ECM
100
1
10
MMP-3
MMP-9
MMP-11
MMP-13
TIMP-1
TIMP-2
TIMP-3
Type I Collagen
Type IV Collagen
Laminin
X-f
old
In
cre
ase
1
10
100MMP-1
MMP-2
MMP-7
MMP-14
N F0 F 1-4 F5/6 Ci N F0 F 1-4 F5/6 Ci
Disease Stage
Real time RT-PCR data
formatrix metalloproteinases
(MMPs),
specific inhibitors (TIMPs),
ECM proteins
(collagens, laminin)
from liver tissue
F0-F6: liver biopsies
(histological stagingaccording to Ishak et al.)
Ci: liver resections of
decompensated cirrhosis
N: healthy donor livers
Lichtinghagen et al. Clinical Science 2003
TIMP-1 (0.706)
MMP-2 (0.589)
Hyalur. (0.714)
0,0 0,2 0,4 0,6 0,8 1,00,0
0,2
0,4
0,6
0,8
1,0
1-Specificity
Sen
siti
vit
y
Fibrosis
TIMP-1 (0.901)
MMP-2 (0.972)
Hyalur. (0.910)
0,0 0,2 0,4 0,6 0,8 1,00,0
0,2
0,4
0,6
0,8
1,0
1-Specificity
Sen
siti
vit
y
Cirrhosis
Diagnostic Validity of HA, TIMP-1, and MMP-2
in Liver Fibrosis and Cirrhosis
Böker et al. Clin Chim Acta 2002
Single serum marker measurements for
fibrosis and cirrhosis detection
Diagnostic Validity of ECM-Markers
for the Detection of Liver Fibrosis
FIBROSISPrevalence: 46%
(n=59)
TIMP-1 Hyaluronan Pro-MMP-2
Sensitivity 52 / 67 %
(35 -81)
48 %
(31 -65)
7 / 7 %
(1 – 22)
Specificity 88 / 68 %
(53 - 96)
84 %
(70 – 94)
100 / 97 %
(85 - 100)
Vgl: GGT AST ALT Albumin
Sensitivity 65 78 96 27Specificity 53 40 16 90
CirrhosisPrevalence: 30%
(n=78)
TIMP-1 Hyaluronan Pro-MMP-2
Sensitivity 100 / 100 %
(88 - 100)
90 %
(71 – 98)
75 / 83 %
(55 – 94)
Specificity 75 / 56 %
(44 – 84)
73 %
(63 – 82)
96 / 100 %
(89 - 100)
Vgl: GGT AST ALT Albumin
Sensitivity 73 81 88 73Specificity 47 60 11 86
! !
Diagnostic Validity of ECM-Marker
for the Detection of Liver Cirrhosis
Multiparametric Scores from
Standard measurands
Innovative measurands
Transient Elastography
Non invasive Diagnosis of Hepatic FibrosisNew Developments?
Routine Diagnostics for Hepatitis C: e.g. ALT
Normal ALT Increased ALT
Shiffman ML et al., J Infect Dis 2000
NoFibrosis
Portal Fibrosis Bridge Fibrosis
Cirrhosis
Mild Fibrosis
40% 23%
26% 6%6%
No Fibrosis
PortalFibrosis
Bridge Fibrosis
Cirrhosis
Mild Fibrosis
19% 19%
24%
16%
22%
4
Routine Diagnostics for NAFLD: e.g. ALT
80 % of NAFLD patients have
normal range liver enzyme levels.
Men: ALT <30 U/lWomen: ALT <19 U/l
Liver enzyme levels do not correlatewith histological staging and grading.
NO SIMPLE BLOOD TESTS DIFFERENTIATE NASH ORFIBROSIS FROM SIMPLE STEATOSIS!!!
Despite of advanced fibrosis there are no obious symptoms.
(Browning JD et al Hepatology 2004)
Special diagnostics is well validated in chronicviral hepatitis:
HCV > HBV or HCV/HIVClarification of cirrhosis >> sign. fibrosis
Non-invasive Diagnostic of Liver Fibrosis
Established Routine Diagnostics – Special Diagnostics
Adequate staging is not possible with
available routine diagnostics.
• Liver biopsy and elastography require access
to specialist services
Limitations for screening and monitoring tools
in at-risk patients
• Serological tests for liver fibrosis that permit
more widespread use are neccessary
• Rely on vigilance of non-hepatologists to
identify at-risk individuals
Non-invasive Diagnostic of Liver Fibrosis
Established Routine Diagnostics – Special DiagnosticsNon invasive Diagnosis of Hepatic Fibrosis
New Developments?
Multiparametric Scores from
Standard Measurands
innovative Measurands
Transient Elastography
Aspartate-to-Alanine-Aminotransferase-Ratio (AAR)
Wai CT et al., Hepatology 2003 | Giannini E et al., Hepatology 2003
• 239 HCV patients
• Correlation AAR vs. fibrosis in histologyrs= 0.57
• Prediction of significant fibrosisc-Index= 0.82AAR 1 PPV= 0.89
• Prediction of cirrhosisc-Index= 0.91
Fibrosis Scores from Standard Measurands
AST-to-Platelet-Ratio-Index (APRI)
Wai CT et al., Hepatology 2003Forns-Index (FI)
Forns X et al., Hepatology 2002
Platelets [109/l]APRI =
AST [/UNL] × 100
7,811 - 3,131 ln(Platelets) + 0.781 ln(γGT)+ 3.467 ln(Age) – 0.014 Cholesterol
FI =
FIB-4-Index (FIB-4)
Sterling RK et al., Hepatology 2006
FIB-4 =Platelets [109/l] x ALT [U/L]
Age [J] x AST [U/l]
5
AST-to-Platelet-Ratio-Index (APRI)
Wai CT et al., Hepatology 2003
Patientsn = 192, chronic hepatitis C, treatment naïv
Validating collective (n=72)
Significant fibrosis Cut-offs <0.5 | >1.5
Sensitivity 91%, Specificity 95%
51% of patients classifiable
Cirrhosis Cut-offs <1.0 | >2.0
Sensitivity 89%, Specificity 93%
81% of patients classifiable
r=0,60, p<0,01
0
3
6
9
AP
RI
0 1 2 3 4 5 6
Ishak-Fibrose-Score
https://www.hepatitisc.uw.edu/page/clinical-calculators/apri
Forns Index
Cut-off <4.2: Sensitivity 94%, Specificity 51%
Cut-off >6.9: Sensitivity 30%, Specificity 95%Significant Fibrosis
51% (39+12) of patients classifiable
Forns X et al., Hepatology 2002
n = 351, chronic hepatitis C, treatment naïv
Validation collective (n=125)Patients
no significantfibrosis
Significantfibrosis
Notclassifiable
|6.9
|4.2Cut-off:
39% 12%49%
https://www.hepatitisc.uw.edu/page/clinical-calculators/fib-4
Cut-off <1.45:
NPV 90% (adv. fibrosis)
Cut-off >3.2:
Specificity 97%, PPV 65%
70% patients classifiableavoid biopsy
accuracy 86%
FIB-4 Index (Calculator)
Fibrosis-Scores I
Delta Fibrosis Score (DFS)1, if Albumin <1.19 (x LNL)
+1, if γGT >0.5 (x UNL)
+1, if CHE < 1.46 (x LNL)+1, if Age >42
0-4 points, n=72, 2 ± 1.1AUC = 0,87
Fibrosis Cirrhosis
Non-invasive Fibrosis-Score (DFS) for Delta-Hepatitis
Lutterkort et al. Liver International 2016
6
NAFLD Fibrosis Score
n=480 AUC 0.88
n=253 AUC 0.82
Advanced Fibrosis
AgeBMI
hyperglycemia
AST/ALTplatelets
albumin
Angulo et al. Hepatology 2007
NAFLD Fibrosis Score (Calculator)
http://nafldscore.com
Multiparametric Scores from
Standard Measurands
innovative Measurands
Transient Elastography
Non invasive Diagnosis of Hepatic FibrosisNew Developments?
Fibrosis-Scores II
ELF-Score (European-Liver-Fibrosis-Group)
Rosenberg WMC et al. Gastroenterology 2004
Scheuer-Fibrosis-Score
ELF
-Sc
ore
• European cross-sectional study, 1021 patients, all etiologies
• P-III-NP, Kollagene IV, VI, Laminin, HA, Tenascin, MMP-2, MMP-9, TIMP-1
• 3 independent pathologists
ELF-Score:Age, P-III-NP,
Hyaluronat, TIMP-1
• Detection: significant fibrosis
Sensitivity 90%
• Exclusion: significant fibrosis
NPV 92%
• ELF-Score better than match
between pathologists
Enhanced Liver Fibrosis (ELF)-Score: Interpretation of Results
Lichtinghagen et al.; J Hepatol 2013
ELF Values
Normal 6.7 - 9.8
Cut-off value 1: 7.7
Cut-off value 2: 9.8
Cut-off value 3*): 11.3
*)evaluated for chronic hepatitis C
7
Enhanced Liver Fibrosis (ELF)-Score: Diagnostic Validity Enhanced Liver Fibrosis (ELF)-Score: Influencing Factors
ELF-Score
Reference Intervals
Total (400) 6.7 - 9.8
Morning (129) 6.6 - 9.5
Afternoon(236) 6.7 - 9.9
Standard Normal Deviate Test:
no partition of reference intervals
Enhanced Liver Fibrosis (ELF)-Score: Influencing Factors
ELF Reference Intervals
Total: 6.7 - 9.8
Men: 7.0 - 9.8
Women: 6.6 – 9.3
Standard Normal Deviate Test:
Partition of reference intervals
Enhanced Liver Fibrosis (ELF)-Score: Influencing Factors
ELF Reference Intervals
Total (n=400): 6.7 - 9.8
<30 y*) (n=213):6.6 – 9.2
>30 y*) (n=175):6.9 – 10.1
Most significant influencing factor
Standard Normal Deviate Test:
Partition of reference intervals
Mean ELF increase about 0.3 per decade
*) Strongly depending on the composition
of the evaluation collective (n=400)
ELF and Outcome in Chronic Liver Disease
Parkes J et al., Gut 2010
n = 457, chronic liver disease, different entities
7 year follow up (39 deaths, 61x liver relevant outcome)Patients
ELF-Score
Low: 4.14 – 8.33
Intermediate I: 8.34 – 10.43Intermediate II: 10.43 -12.51
High: 12.52 – 16.67
AUC 5J / 6J / 7J – survival
Biopsy 0.83 / 0.81 / 0.82ELF 0.86 / 0.87 / 0.87
ELF and Outcome in ARLD
It is 7x more likely to have a liver related outcome in 7 years,
if ELF score is > 11.3 than those with score <7.7
From: Julie Parkes (Southhampton) at Siemens Healthineers Science & Innovation Days 2017
Day et al., (in press).
8
FibroTest®
FibroTest =4,467 log(α2-Macroglobulin) - 1,357 log(Haptoglobin)+ 1,017 log(-GT) + 0,0281 Age + 1,737 log(Bilirubin)- 1,184 Apolipoprotein A1 + 0,301 (if male) - 5,540
Poynard T, US Patent Application 2004
Correlation with Staging (METAVIR)
Imbert-Bismut F et al., Lancet 2001
Patients n = 205, chronic hepatitis CValidating collective (n=134)
Method Comparison APRI, FibroTest and Fibroscan
FibroscanAPRI FibroTest
Castéra L et al. Gastroenterology 2005
• 183 patients • Chronic hepatitis C
Comparison of ELF, FibroTest und FibroScan
Friedrich-Rust M et al., BMC Gastroenterology 2010
n = 74 chron. liver diseases (36 HCV, 10 HBV, 28 PBC)
Liver biopsie METAVIR (F0 (4), F1 (21), F2 (18), F3 (20), F4 (11)Patients
F>2 F=4
AUC (Staging) F >2 F =4
ELF 0.81 0.92
(0.69-0.93) (0.83-1.0)
Fibrotest 0.72 0.91
(0.59-0.85) (0.82-0.99)
Elastography 0.85 0.94(0.74-0.96) (0.86-1.0)
FibroGene for Hepatitis B, C, and NAFLD
AUROC Fibrogene > AUROC APRI, Forns, FIB-4, NFS
AUROC FibroGene HCV > HBV / NAFLD
FibroMeter: Different Marker Sets for Different Etiologies
Fibrometer (NAFLD)
Fibrometer (chronic viral hepatis
Multiparametric Scores from
Standard Measurands
innovative Measurands
Transient Elastography
Non invasive Diagnosis of Hepatic FibrosisNew Developments?
9
Biomarkers vs. Imaging
What is the matching score for my diagnostic problem?
When is the biopsy not necessary?
What do current guidelines say?
SAFE-Biopsy AlgorithmsPre-Screening - Screening
Sebastiani G et al., Hepatology 2009
Signifikant fibrosis
• 90% diagn. Efficacy
• AUC 0,89 (0,87 – 0,90)
Cirrhosis
• 93% diagn. Efficacy• AUC 0,92 (0,89 – 0,94)
Multi-center study, n=2035, chronic hepatitis C
Pre-Screening
Screening
Pre-screening options(?)
APRI, FIB-4, Forns
Screening options (?)
ELF, Fibrometer, Fibrotest
EASL-ALEH-Guidelinesfor Hepatitis C
J Hepatol 2015
EASL-EASD-EASOGuidelinesfor NAFLD
Important:
Non-invasive
differentiation
NAFLD
vs.
NASH
J Hepatol 2016
Fibrosis Assessment in NAFLD
Kaswala et al. Dig Dis Sci 2016
10
CONCLUSION on Diagnostics of Liver Fibrosis
• Use of fibrosis scores enables earlydetection of significant fibrosis
• Number of recommended biopsiescan be reduced
• Use evaluated scores for different entitiesof chronic liver diseases
• Note interference with histol. grading
• Combination with imaging methodsfor diagnostic safety according toof current guidelines.