1. Michel BEAUGRAND

2. EVALUATION OF LIVER FIBROSIS BLOOD TESTS, LIVER BIOPSY AND FIBROSCAN
M. Beaugrand
Service d’Hépatologie
Hopital J. Verdier
BONDY 93143
et Université Paris XIII
PHC JANUARY 07

3. EVALUATION OF FIBROSIS : WHY ?
Causal agent
Chronic liver
Disease
Cirrhosis
Decompensation Hepatocellular
2 % to 5 % per year carcinoma
2 % to 5 % per year
Fibrosis
Distorted architecture
Portal hypertension
Liver failure
Carcinogenesis
FIBROSIS
Death
4 % per year

4. ASSESSMENT OF FIBROSIS : WHY ?
Management of individual patients
Significant fibrosis Treatment
Cirrhosis Screening for varices and HCC
Evaluation of treatments
Antiviral and antifibrotic drugs
Screening for cirrhosis or extensive fibrosis
In high risk patients

5. EVALUATION OF FIBROSIS : HOW ?
- Liver biopsy
- Blood tests
Genuine serum markers of fibrosis :
by products of extracellular matrix
metabolism.
Probabilistic indexes = surrogate markers.
- Fibroscan ( transcient elastography)

6. LIVER BIOPSY : LIMITATIONS
Acceptability
- patients are often reluctant
- even some doctors are reluctant
Cost
Morbidity
Reliability
- liver sample size ideally ≥ 25 mm
- pathologist’s experience
- suboptimal reproductibility of scoring systems
- quantitative assessment unpractical

7. LENGH OF LIVER BIOPSY Biopsy length Bedossa et al, Hepatology 2003
Liver biopsy is the gold standard for evaluating liver fibrosis but it has some limitations. A shown by bedossa et al using virtual biopsy from liver explants, a 25 mm long biopsy correctly staged in 75% of the cases and 40 mm were necessary to obtain a 100%reliability.
Biopsy length
Bedossa et al, Hepatology 2003

8. LIVER BIOPSY : PROS AND CONS
. Not influenced by extrahepatic conditions
. Allows analysis of elementary lesions and comobidities
. May allow assessment of fibrogenesis (molecular biology)
CON
. Sampling error
. Dependant of pathologist’s experience
. Unavalaible in large parts of the world

9. BLOOD TESTS Matrix related PIIINP, collagen type IV, laminin
Hyaluronic acid, MMP, TIMP
Non maxtrix related
AST, ALT, gamma GT
Bilirubin, prothrombine, platelets
Gammaglobulins, ferritin
Alpha 2 macroglobulin, haptoglobin
Apo A1, cholesterol, HOMA

10. BLOOD TESTS Poynard, 1991 Prothrombine, GGT, Apo A1 PGA
Bonacini, 1997 AST/ALAT, platelets, prothrombine
Imbert-Bismut, 2001 bili, GGT, hapto., a2MG, apoA1 Fibrotest
Luo, 2002 glob/alb, platelets, AST/ALT
Forns, 2002 age, GGT, cholesterol, platelets
Kaul, 2002 sex, ang.spider angiomas, AST, platelets
Wai, 2003 AST/platelets APRI
Sud, 2004 age, AST, cholesterol, HOMA, alcohol
Lainé, 2004 hyaluronate, transferin
Rosenberg, 2004 age, hyalur., col IV, col VI, laminin, PIIINP, TIMP-1 ELF
Patel, 2004 TIMP-1, a2MG, hyaluronate Fibrospect
Leroy, 2004 PIIIMP, MMP1
Hui, 2005 BMI, platelets, albumin, bilirubin
Lok, 2005 AST/ALT, platelets, INR
Adams, 2005 bili, GGT, hyaluronate, a2MG, age, sex Hepascore
Cales, 2005 AST, platelets, prothrombine, hyaluronate, Fibrometre
urea, age

11. Imbert-Bismut et al. Lancet 2001;357:2069-75
BLOOD TESTS
Fibrotest ®
Metavir
index
VHC (n= 339)
Imbert-Bismut et al. Lancet 2001;357:

12. Imbert-Bismut et al. Lancet 2001;357:2069-75
BLOOD TESTS
Fibrotest ®
HCV (n=339)
Métavir ≥ F2
AUC = 0,827
Index < 0,10 NPV 100%
Index > 0,60 PPV > 90%
Liver Biopsy: - 46%
Imbert-Bismut et al. Lancet 2001;357:

13. BLOOD TESTS Ref. Test AUC Construction Validation Imbert-Bismut, 2001
HCV : F0-1 vs F2-3-4
Ref.
Test
AUC
Construction
Validation
Imbert-Bismut, 2001
Fibrotest
0.83
0.85
Forns, 2002
0.86
0.81
Wai, 2003
APRI
0.80
0.88
Patel, 2004
Fibrospect
Rosenberg, 2004
ELF
0.78
Leroy, 2004
0.82
Sud, 2004
0.84
Adams, 2005
Hepascore

14. HCV : F0-1-2-3 vs F4 (cirrhosis)
BLOOD TESTS
HCV : F vs F4 (cirrhosis)
Ref.
Test
AUC
Construction
Validation
Kaul, 2002
0.94
Wai, 2003
APRI
0.89
Rosenberg, 2004
ELF
Le Calvez, 2004
Fibrotest
0.92
Adams, 2005
Hepascore
Lok, 2005
0.78
0.81

15. BLOOD TESTS : PROS AND CONS
. Easy, non invasive,not too costy
. Allow to split patients in 3 groups
- those without significant fibrosis
- those with extensive fibrosis or cirrhosis
- intermediate
CON
. Studied mainly in naive patients with HCV chronic hepatitis
. External validation lacking (except fibrotest)
. Require standardisation of biochemical methods
. Poorer performances in HBV patients or coinfected HCV-HIV
. Possible influence of extrahepatic conditions
. No international consensus

16. ELASTOMETRY (FIBROSCAN)
2.5 cm
4 cm
1 cm 
Volume
LB x 100
Probe
Sandrin et al. Ultrasound Med Biol 2003;29:1-8

17. HOW TO MEASURE ELASTICITY ?
To generate an elastic
Shear vawe
To measure its spead Vs
Elasticity
E  VS2

18. Volume of exploration > 3 cm3
Probe position
2.5 cm
4 cm
1 cm 
Volume of exploration
Probe

19. PATIENTS WITH HCV CHRONIC HEPATITIS+
patients
with no
ascites
251 patients
included
53 patients excluded
biopsy not suitable for fibrosis stage assessment: less than 10 portal tracts in the absence of cirrhosis
23 patients excluded:
unreliable stiffness measurement: success rate less than 60% upon 10 measurements
Small biopsy
126 patients
Large biopsy
125 patients
327 patients consecutive patients were included from 4 french liver units. There was one exclusion criteria which was the presence of ascitis because elastic waves do not propagate through liquids. 23 stiffness meazsurement were unsuccessfull (too narrow intercostal space or too wide adipose tissue). 53 patients were further exlcluded because the biopsy was too small to accurately grade fibrosis

20. Fibrosis stage (METAVIR)
BOX PLOTS. N=251
100
Legend
maximum
Stiffness (kPa)
(logarithmic scale) 10
median
IQR
On this figure, median value of liver stiffness are compared with consensus METAVIR stage.
Liver stiffness was significantly correlated to fibrosis stage You can see that patients with F4 metavir score which means cirrhosis are well separated from other stages but overlaps are observed between F1 and 2 and F2 and F3.
minimum 1
Fibrosis stage (METAVIR)

21. ROC CURVES - F≥2 : 0.79 (0.73-0.84) AUROC ( CONFIDENCE INTERVALS 95%)

22. UNI AND MULTIVARIATE ANALYSIS
Univariate analysis (Kendall’s coefficient)
Fibrosis Activity Steatosis
Stiffness r
p <
Fibrosis r
p - <
On the other hand we checked that activity (which represents inflammatory infiltrate) and steatosis did not significantly affect liver stiffness measurements. I
Multivariate analysis (multiple regression)
Only fibrosis was significantly correlated to liver stiffness measurement.

23. VALIDATION OF DIAGNOSIS ACCURACY IN AN INDEPENDENT HCV POPULATION
Total number of included patients: 639
Number of unreliable liver samples: 86 (13%)
Number of unreliable LSM: 59 (9%)
Patients kept for statistical analysis : 494
Area under ROC curves
(95% confidence interval)
F01 versus F234 = 0.84 ( )
F012 versus F34 = 0.93 ( )
F0123 versus F4 = 0.96 ( )
METAVIR F 1 2 3 4 % 6 39 31 10 14 A 56 35
Steatosis S
1-10
11-30
31-100 47 27 15
Univariate Spearman correlation
METAVIR F: 0.70 (p << 0.001)
METAVIR A: 0.45 (p << 0.001)
Steatosis: 0.35 (p << 0.001)

24. LIVER BIOPSIES > 30 mm - 103 Patients - Results Causes : 71 VHC
14 VHB
15 VHC+HIV
2 VHB+HIV
1 VHC+VHB
Fibrosis Score :
F0 F1 F2 F3 F4 N
- Results
 F2  F3 = F4
AUROC

25. CUT-OFF VALUES*
The optimum thresholds were chosen to maximize the sum of sensitivity and specificity.
Threshold Sensitivity Specificity LR
(kPa)
F 
F 
F =
Choosing tresholds to maximize sensitivity and specificity
8,7 kpascal defined F superior or equal to 2 with a 55 percent sensitivity and 84% specificity etc….
* Obtained by the jack-knife method.

26. FIBROSIS AREA (morphometry)
- Patients
69 patients with chronic hepatitis C
without ascites, and previous anti-viral treatment
METAVIR
F1 F2 F3 F4
- Results
Spearman correlation test
p < 0.001
Parameters r
Fibrosis area METAVIR score 0.66
Elasticity METAVIR score 0.65
Elasticity fibrosis area 0.74
 Liver elasticity is closely correlated to fibrosis area.

27. FIBROSCAN VERSUS BLOOD TESTS
FibroTest
APRI
Elasticité (kPa)
Score METAVIR de fibrose
Score METAVIR de fibrose
Score METAVIR de fibrose
Castera Al. Gastroenterology 2005

28. CONCORDANCE WITH LIVER BIOPSY
AUROC
F01/F234 F012/F34 F0123/F4
APRI
FibroTest
FibroScan
Combinaison FibroTest+FibroScan
Percentage of concording results
F01/F234 F0123/F4
FibroTest 80 81
FibroScan 73 83
Combinaison FibroTest+FibroScan

29. PROPOSED COMBINATION OF NON INVASIVE METHODS
FibroScan + FibroTest
Concordance
Discordance
Fibrose minime (FS < 7.1 kPa + FT < F2)
Fibrose modérée (FS  7.1 kPa + FT  F2)
Fibrose sévère (FS  9.5 kPa + FT  F3)
Biopsy
Follow-up
Treatment
Treatment HCC screening
treatment
Castera et Al. Gastroenterlogy 2005

30. FIBROSCAN IN HBV PATIENTS
15 non interpretable biopsies
14 LSM considered as non reliable
Statistical analysis on 173 patients
AUROC
F01 versus F234: 0.81 ( )
F012 versus F34: 0.93 ( )
F0.123 versus F4: 0.93 ( )

31. FIBROSCAN PROS AND CONS
-easy, quick, not too costy
- very specific for extensive fibrosis or cirrhosis
- Allows to split patients between 3 groups
. Without significant fibrosis
. With extensive fibrosis or cirrhosis
. Intermediate
- closely related to the area of fibrosis in patients with chronic hepatitis
CON
- high rate of failure in obese patients
- doesn’t take in account liver architecture
- disturbed in acute conditions

32. SCREENING IN HIGH RISK PATIENTS
LSM
Blood tests
Confirmation of cirrhosis LB
LSM>13 kPa
oui
Absence of cirrhosis
Suspected
cirrhosis
non
227 patients in alcoholic abstinence program 41 34 33

33. FOLLOW-UP OF LSM IN TREATED PATIENTS WITH CHRONIC HEPATITIS C
BEFORE TREATMENT
END OF TREATMENT
6 MONTHS AFTER
All (n=85)
14.1 ± 7.2
10.9 ± 6.5
11.2 ± 8.6
SVR
12.0 ± 6.7
9.1 ± 3.7
7.5 ± 2.4 RR
14.6 ± 5.6
11.5 ± 5.0
12.8 ± 7.2 NR
16.9 ± 8.1
13.3 ± 9.1
16.1 ± 12.2

34. CONCLUSION
1) Liver biopsy has been partly challenged by non invasive methods for assessment of liver fibrosis
2) Non invasive methods have their own limitations : 2 might be better than one
3) Fibroscan could become a useful tool for assessing fibrosis variations