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

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?

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 0-22. 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

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;339:1485-92 Poynard et al. Lancet 1998;352:1426-32

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; 57:773-784

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; 57:773-784

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

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

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;38:1449-1457

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; 43:568-571 Regev et al. Am J Gastroenterol 2002; 97:2614-2618.

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; 97:2614-2618.

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; 8:877--83.

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.

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

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; 30:472-478 Gaiani et al. J Hepatol 1997;27:979-985

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;29:1705-1713.

Liver Fibrosis Measurement of Stiffness in Patients with HCV Pt distribution for METAVIR fibrosis stage, activity grade, and steatosis AUROC curve 0.97-0.99 0.91-0.95 0.79-0.81 Liver fibrosis measurmeent of stiffness Ziol et al. Hepatology 2005; 41:48-54.

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; 45:1290-97.

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

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.

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 2006 www.CCO hepatitis.

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;357:1069-1075.

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;101:547-555

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): 986-992. Thabut et al. Hepatology. 2003:37:1220-1221

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: 518-526.

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: 32-36.

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)

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 2006 www.CCO hepatitis.

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

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

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)

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