1. Is Liver Biopsy the Gold Standard?
I want to thank the organizers of this conference for inviting me to speak. Today, I am going to discuss if liver biopsy is the gold standard.
Is Liver Biopsy the Gold Standard?
Mamta K. Jain, MD, MPH

2. What are the indications for a liver biopsy?
Diagnosis of liver disease
Assess severity of liver disease
Assess response to treatment
In order to understand why the liver biopsy may be considered we need to examine why a liver biopsy may be done. One reason is the diagnoses liver disease. Second, a biopsy may be done to assess severity of liver disease, and finally, a bx is done to assess response to treatment.
Add slide that liver bx is not needed in everyone
What are the indications for doing a liver bx in HBV and HCV?

3. Hepatitis B
Initial trials used pre-treatment and post-treatment biopsy to assess response to nucleoside therapy
Knodell index grades histological activity from 0-22
Periportal bridging necrosis (0-10)
Focal necrosis (0-4)
Portal inflammation (0-4)
Fibrosis (0-4)
Treatment response based on 2 point decrease in necroinflammatory activity
composed of the first three parameters
measured (0-18)
Historically, liver biopsies have been used in clinical trials. In HBV, a pre- and post bx was done to assess response to nucleoside therapy. Now we use virologic markers to assess response to therapy.
So when lamivudine was in clinical trials for HBV, the knodell index was used to assess response. It was a grade from The components of the histological activity is
Periportal briding necorosis, forcal necrosis, portal inflammation, and fibroiss.
Treatment response was based on 2 point decrease in necroinflammation which was composed of the first three parameters and is measured on scale of 0-18.
Lai et al. New Engl J Med 1998;339:61-8

4. Hepatitis C
Clinical trials have used viral response as primary endpoint
Liver biopsies pre- and post- treatment were done for
Confirmation of chronic hepatitis, r/o other causes, identify cirrhosis (staging)
Change in histological activity
Knodell HAI
METAVIR
In HCV, the clinical trials have always used viral response as primary endpoint. However pre-and post liver biopsies were done for
1. Confirmation of chronic hepatitis and to rule out other causes like alcohol, fatty liver, and the identify cirrhosis (staging). A secondary endpoint of efficacy was change in HAI score m
McHutchison et al. New Engl J Med 1998;339:
Poynard et al. Lancet 1998;352:

5. Relationship Between Liver disease Stage and Fibrosis
Fibrosis– excess collagen
Collagen proportion of liver fibrosis correlates with hepatic venous pressure gradient (HVPG)
Increasing fibrosis has prognostic value
Histological assessment of fibrosis is by trichrome or reticulin stains but does not correlate with quantitative amount of hepatic collagen
Fibrosis is part of histologic staging of disease severity
Germani et al. Histopathology 2010; 57:

6. Relationship Between Liver Disease Stage and Fibrosis
Staging describes features that depend on architectural changes, not just degree of fibrosis
Histopathologic assignment of liver disease stage is a different process from measurement of liver fibrosis
Both measurements are complementary but are imperfectly correlated
I want to take a moment to talk about the difference of fibrosis vs. staging
Staging describes the features that depend on architectural changes. It is not just the degree of fibrosis that is present on the biopsy.
Histopathologic assignment of liver disease stage is a different process from measurement of liver fibrosis
Both processes are complementary but are imperfectly correlated.
Thus measurement of fibrosis is not the same as measurement of cirrhosis
Germani et al. Histopathology 2010; 57:

7. This is a biopsy specimen of a normal liver. Normal architrecture.

8. Now in contrast, you can see on this biopsy that there are nodules characteristic of cirhosis.

9. Size of Liver Biopsy
Smaller biopsy is associated with greater sampling error
Error reduced by increasing sample size and number of biopsies performed
Study found 25 mm biopsy had error rate of 25%
Optimal size 40 mm
But only 16% of samples are >20 mm
Now let talk a little about the limitations of the liver bx
Size of liver bx affects identification of staging. Error can be reduced…
In one study 25 mm bx had an error rate of 25%
Optimal size if 4 mm but only 16% fo the samples are >20 mm
Bedossa et al. Hepatology 2003;38:

10. Sampling Error
Studies have shown 10-30% of cirrhosis was understaged by percutaneous liver biopsy
Study of biopsy of the left and right lobe of the liver found discrepancy in 50% of the samples
Inaccurate by 1 stage
Underestimation of inflammation
In addition to size of the bx, there is some inherent problems with sampling error especially in disease which may have hetrogenous pattern. Studies have shown that 10-30% of cirrhosis is missed by liver bx
One study in which both the left and right lobe of the liver was biopsied, there was as much to 50% discrepancy in the read in 50% of the samples
Maharaj et al. Lancet 1986; 1:523-25
Poniachik et al. Gastrointest endosc 1996; 43:
Regev et al. Am J Gastroenterol 2002; 97:

11. Intra-observer Variability
Intra-observer variability seen in up to 10% of samples and was 1 stage or 1 grade.
Use of scoring system has made staging more consistent
Another problem is variability of characterizing the stage and grade on liver bx which was seen in 10%
Use of a scoring system has made staging more consistent
Regev et al. Am J Gastroenterol 2002; 97:

12. Complications
HALT-C reported complications of liver biopsy in HCV patients with advanced liver disease
1.1 % serious adverse events
0.6% due to bleeding (most common)
More common if platelet <60,000
INR>1.3
Finally, liver bx are invasive procedures and have complications.
In the HALT-C study reported complications of liver bx in HCV patients with advanced liver disease
They had complication rate of 1.1% SAE
And .6% was due to bleeding, which was also the most common
Usually the bleeding was more likely to occur if plt <60K or INR>1.3
Seeff et al. Clin Gastroentrol Hepatol 2010; 8:

13. Pro and Cons for Liver Biopsy
Pros
Cons
Steatosis assessment and quantification
Fibrosis assessment and architectural distortion
Iron level measurement
Diagnose other pathology
Using special stains
other liver disease (viral hepatitis + NAFLD, EtOH, etc)
Invasive
Risk of complications 1-5%
Mortality .01% to 0.1%
Limitations
Sampling error
Intra-observer variability
To summarize
Pros including assessment and quantification of steatosis, assessment of fibrosis and architectural distoration,
Measure iron levels, stains can also be used to dignoase other pathology
And also bx can allow you to dx other disease which may not have serolgical markers
The cons include invasiveness which has been reported between 1-5%
If bx is obtained there are issues with inadequate staging and grading due to sample size, inherent differences sampling, and variability among pathologist with regard to reading.

14. What are the alternatives?
Radiology CT
MRI US
Hepatic elastography
Serum markers of fibrosis
Indirect
Direct
Now what are the alternatives
Radiographic techniques include , such as CT, MRI, Ultrasound. Hepatic elastography has been used to assess fibrosis. And then there are serum markers of fibrosis which use indirect and direct markers

15. Pros and Cons for Radiology
Non-invasive
US with Doppler can be used to support diagnosis of cirrhosis
Accuracy 82-88%
Insufficient resolution to detect earlier stages of fibrosis
Aube et al. J Hepatol 1999; 30:
Gaiani et al. J Hepatol 1997;27:

16. Hepatic Elastography Emerging technology to stage hepatic fibrosis
Elastography technique measures liver stiffness of hepatic tissue (FibroScan; Echosens, Paris, France)
Ultrasound wave that produces elastic shear
velocity of the shear wave is related to tissue stiffness
more rigid, the faster the wave travels
Increased rigidity is marker of progressive fibrosis
Sandrin et al. Ultrasound Med Biol 2003;29:

17. Liver Fibrosis Measurement of Stiffness in Patients with HCV
Pt distribution for METAVIR fibrosis stage, activity grade, and steatosis
AUROC curve
Liver fibrosis measurmeent of stiffness
Ziol et al. Hepatology 2005; 41:48-54.

19. Liver Stiffness and HVPG
HVPG– predictor of survival and decompensation in cirrhotic patients
Liver stiffness measurement (LSM) predicted severe portal hypertension in HCV patients
AUROC curves for prediction of HVPG
>10mmHg was 0.99
>12mmHg was 0.92
LSM Cut-off values 13.6kPa sensitivity 97%
LSM Cut-off values 17.6kPa sensitivity 94%
Vizzutti et al. Hepatology 2007; 45:

20. Pro and Cons for FibroScan
Pros
Cons
Non-invasive
Able to assess a much larger proportion of the liver
Serial measurements to evaluate fibrosis progression
Poor performance in mild to moderate disease
Cannot be used in
Patients with ascites
Morbid obesity (BMI>40)
Cost
Cannot distinguish between stage 0-II or III-IV

21. Serum Markers of Fibrosis
Ideal biomarker
Liver specific
Independent of metabolic alterations
Detect fibrosis regardless of cause
Sensitive enough to distinguish between fibrosis stages
Reflective of dynamic changes
Friedman. J Hepatol. 2003; 38 (Suppl 1); S38-S53.

22. Indirect Markers of Fibrosis
FibroTest/FibroSure
Alpha-2 globulin
Alpha-2 macroglobulin
Gamma globulin
Apoliprotein A1
Gamma-glutamyl transferase (GGT)
Total bilirubin
ActiTest
Fibrotest +ALT
Forns index
APRI
Fib-4
AST/ALT ratio
AST/ALT with plts
Afdhal and Shiffman hepatitis.

23. FibroTest Classifies fibrosis into 1 of 3 categories
Mild (METAVIR F0-F1)
Significant (METAVIR F2-F4)
Indeterminate
HCV
Detect F2 or higher stage
75% sensitivity
85% specificity
Accuracy 46%
Imbert-Bismut Lancet. 2001;357:

24. ActiTest FibroTest + ALT Reflects necro-inflammation and fibrosis
Better at identifying more advanced fibrosis associated with histological inflammation
Meta-analysis of HCV patients found both FibroTest and ActiTest reliable alternatives for liver biopsy.
Poynard et al. Comp Hepatol 2004;3:8.
Halfon et al. Am J Gastroenterol. 2006;101:

25. Forns Test Uses 4 common clinical measurements Patient age
Cholesterol level
Platelet count
Gamma-glutamyl transpeptidase
Studies HCV patients to
96% NPV in mild fibrosis
66% PPV in F2-F4
Able to accurately exclude mild fibrosis
Inferior to FibroTest
Forns et al. Hepatology. 2003; 34 (4 pt 1):
Thabut et al. Hepatology. 2003:37:

26. APRI Uses clinical variables ALT and platelet count NPV PPV
Significant fibrosis 86%
Cirrhosis 98%
PPV
Significant fibrosis 88%
Cirrhosis 57%
Able to exclude cirrhosis
Wai et al. Hepatology. 2003; 38:

27. Fib-4 Index Uses clinical variables
Platelets
ALT
AST
Age
<1.45 NPV 94.7% to exclude severe fibrosis (F3-F4) with sensitivity 74.3%
>3.25 PPV significant fibrosis 82% with specificity of 98%
Fib-4 <1.45 or >3.25 (62% of all cases) was highly correlated to FibroTest in 92% and 76%
Vallet-Pichard et al. Hepatology. 2007; 46:

28. Indirect Methods Pros Cons Non-invasive Less expensive
Useful in HCV for determining significant fibrosis (METAVIR stage F2-F4) when HCV treatment is recommended
May be most useful in fibrosis that is unevenly distributed
Not sensitive enough to distinguish between stages
Degree of fibrosis does not linearly correlate with biopsy stage
May be better to evaluate for inflammation (i.e., FibroTest)

29. Direct Markers
Measure qualitative and quantitative changes in extracellular matrix markers
Markers matrix deposition
Procollagen type I carboxy-terminal peptide
Procollagen type III amino-terminal peptide
Tissue inhibitor of metalloproteinase
Transforming growth factor-beta
Collagen type IV
Markers of matrix removal
Procollagen Type IV C peptide
Procollagen Type IV N peptide
Matrix metalloproteinase
Other markers
Hyaluronic acid
YkL-40
Combination biomarker assay
FibroSpect
ELF
SHASTA
None of these markers are liver specific and are influenced by metabolism
Afdhal and Shiffman hepatitis.

30. Is Liver Biopsy a Gold Standard?
Perhaps it depends on why biopsy is being done
Yes, if it is :
To diagnose a disease (excluding viral hepatitis)
To exclude other concomitant disease
To measure iron stores
To quantify steatosis
To get special stains for diagnosis
To stage liver disease if fibrosis is evenly distributed

31. Is Liver Biopsy a Gold Standard?
No, if it is:
To determine cirrhosis
To evaluate for significant fibrosis
To determine stage if fibrosis is unevenly distributed
To follow longitudinally

32. Future
Hepatitis C
With new DAA with high SVR rates, perhaps a liver biopsy is not needed?
Biopsy is not typically performed in genotype 2 and 3 if patient willing to be treated
If liver biopsy and elastography are not equivalent, then what impact does this have on patient characterization in clinical trials?
Clinical trials are using FibroScan in Europe instead of liver biopsy
Non-invasive tests need to be validated as predictors of poor outcomes and not just on correlation to liver biopsy stage
Hepatitis B
Will we still need biopsy to determine disease activity to assess for treatment?
May not need biopsy in HIV/HBV who were started on ART with Truvada (treatment based on viral load and not on biopsy)

33. Conclusions Liver biopsy still plays an important role in
Diagnosis of some types of liver disease
Quantification of steatosis
Measurement of iron stores
To distinguish between earlier stages of disease
Fibrosis can be determined by other means such as elastography or other non-invasive tests