topics...etiology group quantitative importance in middle europe example toxic frequent alkohol...

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)

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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%

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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?


Standard Measurands

innovative Measurands


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]

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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

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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


Standard Measurands




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

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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


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


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).

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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


Standard Measurands




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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

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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.

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