National Medical Policy

National Medical Policy Subject: Policy Number: FIBROSpect, FIBROSURE, ActiTest and Other Non-invasive Testing for Liver Fibrosis NMP198 Effective Date*: January 2005 Updated: August 2015 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate Medicaid Manuals for coverage guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link National Coverage Determination (NCD) National Coverage Manual Citation Local Coverage Determination (LCD)* Article (Local)* X Other Refer to state and other applicable manuals/criteria for specific guidance for Hepatitis C medication None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 1

If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement Please refer to specific hepatitis C medication prior authorization criteria for acceptable tests for hepatic fibrosis, as they may differ by plan. (For example: Medi-Cal considers magnetic resonance elastography (MRE) medically necessary as a measure of hepatic fibrosis related to the use of hepatitis C drugs.) Health Net, Inc. considers serological testing [e.g., FIbroSure/Fibrospect, ActiTest, Hepascore, aspartate aminotransferase (AST) to platelet ratio (APRI)] medically necessary for detecting or monitoring hepatic fibrosis in persons with hepatitis C or other chronic liver diseases. Health Net, Inc. considers radiologic testing [e.g., transient elastography (ultrasound based elastography or FibroScan) and acoustic radiation force impulse (ARFI)] imaging medically necessary to monitor or detect hepatic fibrosis in persons with hepatitis C or other chronic liver diseases. Tests not listed are considered investigational at this time due to a lack of large-scale clinical studies in peer-reviewed literature validating their utility. Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures will be replaced by ICD-10 code sets. Health Net National Medical Policies will now include the preliminary ICD-10 codes in preparation for this transition. Please note that these may not be the final versions of the codes and that will not be accepted for billing or payment purposes until the October 1, 2015 implementation date. ICD-9 Codes (may not be all inclusive) 070.41 Acute hepatitis C with hepatic coma 070.44 Chronic hepatitis C with hepatic coma 070.51 Acute hepatitis C without mention of hepatic coma 070.54 Chronic hepatitis C without of hepatic coma 571.0-571.9 Chronic liver disease and cirrhosis ICD-10 Codes B17.0-B17.9 Other acute viral hepatitis B18.0-B18.9 Chronic viral hepatitis K70.10-K709 Alcoholic liver disease CPT Codes There is no specific CPT for FIBROSpect, HCV-FIBROSURE, or ActiTest FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 2

82172 Apolipoprotein, each 82247 Bilirubin; total 82977 Glutamyltransferase, gamma (GGT) 83010 Haptoglobin; quantitative 83519 Immunoassay, analyte quantitative by radiopharmaceutical technique (ie RIA) 83883 Nephelometry, each analyte elsewhere specified 84450 Transferase; aspartate amino (AST) (SGOT) 84460 Transferase; alanine amino (ALT) (SGPT) 0001M HCV FibroSURE, LabCorp, Infectious disease, HCV, 6 biochemical assays (ALT, A2-macroglobulin, apolipoprotein A-1, total bilirubin, GGT, and haptoglobin) utilizing serum, prognostic algorithm reported as scores for fibrosis and necroinflammatory activity in liver 0002M ASH FibroSURE, LabCorp, Liver disease, 10 biochemical assays (ALT, A2-macroglobulin, apolipoprotein A-1, total bilirubin, GGT, haptoglobin, AST, glucose, total cholesterol and triglycerides) utilizing serum, prognostic algorithm reported as quantitative scores for fibrosis, steatosis, and alcoholic steatohepatitis (ASH) 0003M NASH FibroSURE, LabCorp, Liver disease, 10 biochemical assays (ALT, A2-macroglobulin, apolipoprotein A-1, total bilirubin, GGT, haptoglobin, AST, glucose, total cholesterol and triglycerides) utilizing serum, prognostic algorithm reported as quantitative scores for fibrosis, steatosis, and non-alcoholic steatohepatitis (NASH) HCPCS Codes N/A Scientific Rationale Update August 2015 Rudler et al (2014) sought to extend the validation of FibroTest (FT) and transient elastography (TE) as markers of occurrence of cirrhosis without complications (F4.1), esophageal varices (F4.2), and severe complications (F4.3) in patients with chronic hepatitis B (CHB). The second aim was to validate a previous definition of an inactive carrier based on normal FT and ActiTest (normal-ft-at). The third aim was to assess the long-term dynamics of fibrosis in patients with sustained virological response in a 10-year updated individual data of 1434 patients pooled from two prospective cohorts. Of the 1312 patients without a history of complications, varices had occurred after 10 years in 14 patients (F4.2, incidence of 1.7%, 95% CI [0.6-2.8]), and severe complications in 25 (F4.3 3.7% [1.8-5.7]), including hepatocellular carcinoma (HCC) in 21 (3.7% [1.5-5.8]). Using Cox-multivariate analysis adjusted for treatment, viral load, HBeAg status and ALT, FT, and TE were predictive of liver complications (n=37; AUROC=0.83 [0.71-0.90]; p<0.0001) and (n=8/844; AUROC=0.82 [0.72-0.89]; p<0.0001) respectively. Normal FT-AT better identified patients with lower fibrosis progression than the ALT-based standard: 3/163 (1.8%) vs. 16/181 (8.8%; p=0.004) in the Paris cohort, and 5/195 (2.6%) vs. 15/228 (6.6%; p=0.05) in the Bordeaux cohort. Of the 582 responders, 23 had complications (incidence 6.2% [3.2-9.1]) including 19 HCC (5.8% [2.6-9.0]) and 10 with varices (2.6% [0.8-4.4]). Of the 138 responders with advanced fibrosis, only 31% (15-47%) had fibrosis regression. The authors concluded the FibroTest and TE identified three categories of cirrhosis with increasing morbidity. Normal FibroTest and ActiTest were better able to identify inactive hepatitis B carriers than the standard definition. Despite virological response, the overall incidence of cirrhosis increased, with a remaining 5.8% risk of HCC. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 3

Zarenski et al (2014) aimed to evaluate the capacity of FibroSURE, one of the two noninvasive fibrosis indices commercially available in the United States, to identify HBV infected patients with moderate to severe fibrosis. Forty-five patients who underwent liver biopsy at a single tertiary care center were prospectively enrolled and had FibroSURE performed within an average interval of 11 days of the biopsy. Of the 45 patients, 40% were Asian, 40% were African American, and 13% were Caucasian; 27% were co-infected with HIV and 67% had no or mild fibrosis. The authors found FibroSURE to have moderate capacity to discriminate between patients with moderate to high fibrosis and those with no to mild fibrosis (area under receiver operating characteristic [AUROC] curve=0.77; 95% confidence interval [CI] [0.61, 0.92]). When the authors combined the fibrosis score determined by FibroSURE with aspartate aminotransferase (AST) measurements and HIV co-infection status, the discriminatory ability significantly improved reaching an AUROC of 0.90 (95% CI [0.80, 1.00]). FibroSURE also had a good ability to differentiate patients with no or mild from those with moderate to high inflammation (AUROC=0.83; 95% CI [0.71, 0.95]). The authors concluded FibroSURE in combination with AST levels has an excellent capacity to identify moderate to high fibrosis stages in chronic HBV-infected patients. These data suggest that FibroSURE may be a useful substitute for liver biopsy in chronic HBV infection. Salkic et al (2014) aimed to systematically review studies describing the diagnostic accuracy of FibroTest/Fibrosure (FT) for predicting chronic hepatitis B (CHB)-related fibrosis. The authors included 16 studies (N=2494) and 13 studies (N=1754) in the heterogenous meta-analysis for liver fibrosis and cirrhosis, respectively. The area under the hierarchical summary receiver operating curve for significant liver fibrosis and for all included studies was 0.84 (95% confidence interval (CI): 0.78-0.88). At the FT threshold of 0.48, the sensitivity, specificity, and diagnostic odds ratio (DOR) of FT for significant fibrosis were 61 (48-72%), 80 (72-86%), and 6.2% (3.3-11.9), respectively. The area under the hierarchical summary receiver operating curve for liver cirrhosis and for all included studies was 0.87 (95% CI: 0.85-0.90). At the FT threshold of 0.74, the sensitivity, specificity, and DOR of FT for cirrhosis were 62 (47-75%), 91 (88-93%), and 15.7% (8.6-28.8), respectively. The reviewers concluded FibroTest is of value in exclusion of patients with CHB-related cirrhosis, but has suboptimal accuracy in the detection of significant fibrosis and cirrhosis. It is necessary to further improve the test or combine it with other noninvasive modalities in order to improve accuracy. Batheja et al (2015) compared fibrosis seen on liver biopsy to MR elastography (MRE) stiffness measurements in normal controls and patients with abnormal transaminases and chronic liver disease. The control group consisted of 22 healthy liver transplant donors who by definition had normal transaminases. The patient group (32 patients) was recruited from the Mayo Clinic Arizona hepatobiliary clinic over a 3-year span. All subjects underwent a liver biopsy as part of their evaluation and agreed to MRE within 35 days of biopsy. Non-parametric tests were used to compare the MRE-predicted liver fibrosis to the fibrosis noted on liver biopsy. Analysis included 54 subjects (32 patients with chronic liver disease and 22 healthy liver donor candidates). MRE median liver stiffness measurements increased per histologic liver fibrosis stage (one-way ANOVA p = 0.001), with significant correlation between increasing fibrosis stage and stiffness values. Median MRE for control group (2.13 kpa; mean = 2.3 ± 0.6 kpa) was significantly lower than for patient group (3.7 kpa; mean = 4.1 ± 2.1 kpa) (p = 0.003). Significantly, lower median stiffness was seen in zero-to-moderate (F0-2, n = 22) vs. severe fibrosis stages (F3-4, n = 10) 2.80 vs. 5.9 kpa, respectively (p < 0.05). Using a 3.7-kPa cut-off value, the FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 4

predicted sensitivity and specificity for detecting F0-2 from F3-4 were 91% and 80%, respectively. The authors concluded the analysis supports previous findings that MRE is a non-invasive and effective method for detection and assessment of liver fibrosis, particularly for discrimination between F0-2 stages and F3-4 stages. MRE may represent a valuable tool to finely discern hepatic fibrosis non-invasively. Scientific Rationale - Update August 2014 Noninvasive tests of hepatic fibrosis are gaining acceptance for use in staging of fibrosis in patients with chronic liver disease. Though liver biopsy has been considered the gold standard, is not without drawbacks, including risk of complications (bleeding, pain, injury to the hepatic system), sampling error associated with small tissue sample size, and intra/inter-observer variation in histological examination. Lai et al (2011) states that liver biopsy is 80% accurate, with a risk of both understaging and overstaging and cirrhosis can be missed in ~20% of patients. Liver biopsy can establish the presence of concomitant diseases (such as hemochromatosis, alcoholic hepatitis, nonalcoholic steatohepatitis/nonalcoholic fatty liver disease, and hepatic sarcoidosis) and the degree to which these conditions contribute to the patient's liver disease. Noninvasive tests with the highest potential to be widely used are those that have been extensively validated in independent cohorts of patients, incorporate analytical validation, contain precise information on the diagnostic accuracy and potential causes of unreliable results, and are useful for establishing disease outcomes, which ultimately is the most relevant endpoint (Ratziu 2010). The most studied panels are the aspartate aminotransferase (AST) to platelet ratio (APRI), FibroTest/FibroSure, Hepascore, and FibroSpect. The APRI has the advantage of being easily calculated using data available from routine laboratory tests. Most of the testing has focused on specific populations (such as chronic hepatitis C), but they are also being used in patients with other other chronic liver diseases, such as nonalcoholic fatty liver disease, primary sclerosing cholangitis and for monitoring patients taking medications associated with chronic liver damage. Some methods, such as the Fibrotest/Fibrosure and transient elastography are widely used in Europe, and are gaining a growing role in routine clinical practice, especially in chronic hepatitis C. To further improve the ability to correctly assess the degree of hepatic fibrosis, combination of multiple serologic panels, serologic panels with radiographic imaging and the use of algorithms are being studied. For example, the Sequential Algorithm for Fibrosis Evaluation (SAFE) combines the APRI and Fibrotest-Fibrosure tests in a sequential fashion to test for fibrosis and cirrhosis. Sebastiani et al (2009) reports on a large multicenter study validating this algorithm to detect significant fibrosis (stage F2 or greater by the F0 F4 METAVIR scoring system), its accuracy was 90.1%, the area under the receiver operating characteristic curve was 0.89 (95% CI 0.87 0.90), and it reduced the number of liver biopsies needed by 46.5%. When the algorithm was used to detect cirrhosis, its accuracy was 92.5%, the area under the curve was 0.92 (95% CI 0.89 0.94), and it reduced the number of liver biopsies needed by 81.5%. FibroTest has also been evaluated in combination with ultrasound-based transient elastography. Castéra et al (2005, 2011) reported on a study of 183 patients with chronic HCV, the combination of these tests demonstrated an area under the ROC curve of 0.88 for F 2, 0.95 for F 3, and 0.95 for F = 4 [14]. When the elastography and FibroTest results agreed, liver biopsy examination confirmed the stage of fibrosis in 84 percent of cases for F 2 fibrosis, in 95 percent for F 3 FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 5

fibrosis, and in 94 percent for F = 4 fibrosis. The authors concluded that it is likely that a combination of serum biomarkers and elastography will improve the accuracy of fibrosis detection. Manning (2008) and Rizzo et al (2011) note that noninvasive tests are not focused on exact staging of disease, but rather on dividing patients into categories of mild fibrosis (metavir stage 0 to 1) vs. significant fibrosis (metavir stage 2 or greater) and cirrhosis. They are able to exclude significant disease and to diagnose cirrhosis with areas under the receiver operating characteristic curve of 0.85 0.95. According to UptoDate (June 2014), some practitioners still utilize liver biopsy for assessment of fibrosis stage, however other experts do not routinely biopsy patients with HCV. As treatment for HCV continues to become less toxic and more effective, there is less need to precisely stage the patient s liver disease through biopsy. Decisions for treatment may be based on history, physical exam, laboratory tests, and noninvasive assessment of liver fibrosis (such as FibroSure or ultrasound-based transient elastography). Liver biopsy is still needed if laboratory testing and imaging studies are inconclusive. In August 2014, the Infectious Disease Society of America s (IDSA) American Association for the Study of Liver Disease (AASLD) Practice Guidelines for Hepatitis C revised it recommendations for pretreatment assessment (accessed at: http://www.hcvguidelines.org/full-report/when-and-whom-initiate-hcv-therapy): An assessment of the degree of hepatic fibrosis, using noninvasive testing or liver biopsy, is recommended. Rating: Class I, Level A An accurate assessment of fibrosis is vital in assessing the urgency for treatment. The degree of hepatic fibrosis is one of the most robust prognostic factors used to predict disease progression and clinical outcomes. (Everhart, 2010) Those with substantial fibrosis (defined as Metavir F2 or higher) should be given priority for therapy in an effort to decrease the risk of clinical consequences such as cirrhosis, liver failure, and hepatocellular cancer. However, those with severe fibrosis (Metavir stage F3 and F4) are most in need of immediate therapy. In addition to urgency for antiviral therapy, individuals with severe fibrosis require surveillance monitoring for liver cancer, esophageal varices, and hepatic function. (Garcia-Tsao, 2007); (Bruix, 2011) Although liver biopsy is the diagnostic standard, sampling error and observer variability limit test performance, particularly when inadequate sampling occurs. Up to one-third of bilobar biopsies had a difference of at least 1 stage between the lobes (Bedossa, 2003.) In addition, the test is invasive and minor complications are common, limiting patient and practitioner acceptance. Serious complications such as bleeding, although rare, are well recognized. Noninvasive tests to stage the degree of fibrosis in patients with chronic HCV infection include models incorporating indirect serum biomarkers (routine tests), direct serum biomarkers (components of the extracellular matrix produced by activated hepatic stellate cells), and vibration-controlled transient liver elastography. No single method is recognized to have high accuracy alone and each test must be interpreted carefully. A recent publication of the Agency for Healthcare Research and Quality found evidence FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 6

in support of a number of blood tests; however, at best they are only moderately useful for identifying clinically significant fibrosis or cirrhosis. (Selph, 2014) Vibration-controlled transient liver elastography is a noninvasive way to measure liver stiffness and correlates well with measurement of substantial fibrosis or cirrhosis in patients with chronic HCV infection. A cutoff value of 8.7 kpa correlates with Metavir F2 or higher fibrosis stage; greater than 9.5 kpa with F3; and 14.5 or higher kpa with F4 or cirrhosis. The measurement range does overlap between stages. (Ziol, 2005) The most efficient approach to fibrosis assessment is to combine direct biomarkers and vibration-controlled transient liver elastography. (Boursier, 2012) A biopsy should be considered for any patient who has discordant results between the 2 modalities that would affect clinical decision making. For example, 1 shows cirrhosis and the other does not. The need for liver biopsy with this approach is markedly reduced. Alternatively, if direct biomarkers or vibration-controlled transient liver elastography are not available, the aspartate aminotransferase-to-platelet ratio index (APRI) or fibrosis-4 index (FIB-4) can help identify those most likely to have F3 or F4 fibrosis stage. (Sebastiani, 2009); (Castera, 2010); (Chou, 2013b) An APRI above 2.0 or FIB-4 above 3.25 has a high specificity for advanced fibrosis or cirrhosis, although neither test is sensitive enough to rule out substantial fibrosis if values are below these thresholds. (Chou, 2013b) Biopsy should be considered in those in whom more accurate fibrosis staging would impact treatment decisions. Individuals with clinically evident cirrhosis do not require additional staging (biopsy or noninvasive assessment). Scientific Rationale Update April 2014 Hepatic fibrosis occurs in response to chronic liver injury. This process may ultimately progress to cirrhosis with its accompanying consequences of portal hypertension and impaired hepatic function. Hepatic fibrosis was originally thought to be irreversible, but it is now recognized as a dynamic process with the potential for significant resolution. Liver biopsy can provide objective, semi-quantitative information regarding the amount and pattern of collagen or scar tissue in the liver, which can assist with treatment and monitoring plans. The Metavir fibrosis score (0-4) and Ishak fibrosis score (0-6) are commonly used to score the amount of hepatic collagen. A liver biopsy can also help assess the severity of liver inflammation, or of hepatic steatosis, and help exclude competing causes of liver injury. However, the procedure has a low but real risk of complications, and sampling artifact makes its serial use in some patients less desirable. Noninvasive tests of hepatic fibrosis attempt to predict the stage of hepatic fibrosis that would be seen histologically. There are several histologic scoring systems for chronic liver disease. Many use five-point scales, such at the Metavir score: F0: No fibrosis F1: Portal fibrosis without septa FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 7

F2: Few septa F3: Numerous septa without cirrhosis F4: Cirrhosis Patients are typically considered to have significant fibrosis if their Metavir fibrosis score is F2. The fibrosis score is also defined by Ishak and is recommended because of its prognostic value in overall survival. This scoring system uses a 0-6 scale: F0 Fibrosis score 0-4 (none to moderate fibrosis) F1 Fibrosis score 5-6 (severe fibrosis or cirrhosis) Noninvasive tests of hepatic fibrosis are primarily proposed for staging of fibrosis in patients with chronic liver disease, and are considered for patients with chronic viral hepatitis at the time of initial evaluation, to determine the likelihood of advanced liver fibrosis. In patients who are not successfully treated, subsequent testing may be useful to determine if there is progression of fibrosis. The tests are also being proposed in patients with other chronic liver diseases, such as nonalcoholic fatty liver disease and primary sclerosing cholangitis. There are two general categories of noninvasive tests for fibrosis, serologic panels of tests and radiologic tests. Serologic testing is more widely available. However, while progress has been made in improving the accuracy of serum markers of hepatic fibrosis, they cannot yet replace direct histologic analysis (i.e., liver biopsy). When available, radiologic measurement of elasticity can be used alone or in combination with serologic testing. A combination of serologic testing and ultrasound-based transient elastography is suggested. There are four commercial serum marker systems that have been validated: FibroTest/FibroSure (marketed in the United States by LabCorp), Hepascore (Quest Diagnostics), FibroSpect (Prometheus Corp), and the European Liver Fibrosis Study Group panel (not available in the United States). In addition, the aspartate aminotransferase to platelet ratio (APRI) has also been studied. The APRI has the advantage of being easily calculated using data available from routine laboratory tests. In the case of proprietary panels, the blood samples may need to be sent out for analysis. All the serum tests have limitations: They typically reflect the rate of matrix turnover, not deposition, and thus tend to be more elevated when there is high inflammatory activity. By contrast, extensive matrix deposition can go undetected if there is minimal inflammation. None of the markers are liver-specific, and concurrent sites of inflammation may contribute to serum levels. Serum levels are affected by clearance rates, which may be impaired either due to sinusoidal endothelial cell dysfunction or impaired biliary excretion. They are surrogates, not biomarkers. Radiologic methods for assessing hepatic fibrosis include ultrasound-based transient elastography (i.e., FibroScan), magnetic resonance elastography, acoustic radiation force impulse imaging, and cross sectional imaging. Ultrasound-based transient FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 8

elastography is the most widely used. Where available, these tests are often used in conjunction with serologic panels. A variety of serologic markers have been evaluated to predict the degree of fibrosis in the liver, and panels have been developed that combine assays of multiple markers to improve predictive ability. The most studied panels are the aspartate aminotransferase (AST) to platelet ratio (APRI), FibroTest/FibroSure, Hepascore, and FibroSpect. Overall, studies of the various panels suggest they have good ability to differentiate patients with significant fibrosis (F2-F4) from those without significant fibrosis (F0-F1). A disadvantage of these panels is that they are not able to reliably differentiate between the different stages of fibrosis, and indeterminate outcomes are common (up to 50 percent with the FibroTest). No panel has yet emerged as standard of care, and the choice of panel is often dictated by local availability. Serologic markers of hepatic fibrosis can broadly be categorized as indirect or direct: Indirect markers reflect alterations in hepatic function but do not directly reflect extracellular matrix metabolism. (eg., platelet count, coagulation studies, and liver aminotransferases). Direct markers of fibrosis reflect extracellular matrix turnover. (eg., procollagen types I and III, hyaluronic acid, and tissue inhibitor of metalloproteinase). Hepascore involves a combination of bilirubin, GGT, hyaluronic acid, alpha-2- macroglobulin, age, and sex. In one study, Hepascore was no more accurate than the FibroTest in patients with alcoholic liver disease. In other studies, Hepascore was useful for predicting fibrosis in hepatitis C. Tests continue to be developed to detect fibrosis serologically, though they have not been studied as extensively as the APRI or FibroTest/FibroSure. Some of these have been derived for use in specific groups of patients (eg, testing for fibrosis in patients with Non-alcoholic Fatty Liver Disease) [NAFLD). FIB-4 index The FIB-4 index combines biochemical values (platelet count, ALT, and AST) and age. It had good predictive accuracy for advanced fibrosis in at least two studies involving patients with chronic HCV. In another report, it performed better than other serologic markers for predicting advanced fibrosis in patients with NAFLD. It also appears to be helpful for predicting outcomes in patients with NAFLD. In a retrospective series with 320 patients with NAFLD, the area under the ROC curve for predicting adverse liver-related outcomes was 0.81 and for predicting death or liver transplantation was 0.67. NAFLD fibrosis score The NAFLD fibrosis score is another score used to assess the probability of fibrosis in patients with NAFLD. It takes into account the patient's age, body mass index, blood glucose levels, aminotransferase levels, platelet count, and albumin. In a validation study, a high NAFLD fibrosis score cutoff (>0.676) was associated with a positive predictive value for advanced fibrosis (F3-F4) of 82 percent (sensitivity 43 percent, specificity 96 percent), and a low cutoff value (<-1.455) was associated with a negative predictive value of 88 percent (sensitivity 77 percent, specificity 71 percent). It has also been used to predict outcomes in patients with NAFLD. In the same retrospective study described above, the area under the ROC curve for predicting adverse liverrelated outcomes was 0.86 and for predicting death or liver transplantation was 0.70. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 9

PGA index The PGA index combines the measurement of the prothrombin index, GGT level, and apolipoprotein A1 level (PGA). It was devised originally as a simple biological index for detection of alcoholic liver disease. It has been validated in patients with a variety of chronic liver diseases, but particularly alcoholic liver disease. Its accuracy for the detection of cirrhosis has ranged between 66 and 72 percent. FibroIndex The FibroIndex is derived from the platelet count, AST, and gammaglobulin measurements. It has been proposed as a marker of significant fibrosis in chronic HCV. Its accuracy is still being determined. Forns index The Forns index takes into account age, GGT, cholesterol and platelet count. It has primarily been studied in patients with HCV. It appears to have performance characteristics similar to those seen with the APRI. Fibrometer The Fibrometer test involves a combination of the platelet count, prothrombin index, AST, alpha-2-macroglobulin, hyaluronic acid, blood urea nitrogen, and age. It performed well in predicting severe fibrosis in patients with chronic viral hepatitis, but was no better than the FibroTest in predicting severe fibrosis in alcoholic liver disease. BARD score The BARD score was developed to predict fibrosis in patients with NAFLD. The BARD score takes into account BMI, the AST/ALT ratio, and the presence of diabetes mellitus [50]. In a series of 126 patients with NAFLD, the positive and negative predictive values of the BARD score for advanced fibrosis were 69 and 96 percent, respectively, with an area under the ROC curve of 0.87. In another study, the areas under the ROC curves for the BARD score predicting liver-related adverse outcomes and death or liver transplantation were 0.73 and 0.66, respectively. Proteomics and glycomics Patterns of proteins or glycoproteins can be assessed by mass spectroscopy of serum samples. These methods represent "surrogate" markers of fibrosis, and in fact, the identities of the peaks are generally not known. Nevertheless, impressive correlations have been reported. In a study from Belgium, combining serum glycomics with FibroTest resulted in a sensitivity for predicting cirrhosis of 100 percent and a specificity of 75 percent. Professional Societies The Hepatitis C Virus-Human Immunodeficiency Virus (HCV-HIV) International Panel: In 2007, an international panel of experts issued updated guidelines for the management of HIV/HCV-infected patients and concluded that, in most cases, liver biopsy is not a prerequisite for treatment of chronic HCV infection. The expert panel also concluded that a combination of noninvasive methods accurately predicts hepatic fibrosis in most cases. In a 2006 technical review on the management of HCV, the American Gastroenterological Association stated that neither clinical nor laboratory markers, individually or in combination, predict accurately the degree of necroinflammatory activity or the level of fibrosis in the liver. Therefore, despite sampling error, liver biopsy remains the gold standard for determining histologic grade and stage. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 10

In 2012, the American Gastroenterological Association (AGA), the American Association for the Study of Liver Diseases (AASLD) and American College of Gastroenterology (ACG) notes: A joint practice guideline on the diagnosis and management of nonalcoholic fatty liver disease (NAFLD) states that serum aminotransferase levels and imaging tests such as ultrasound (US), computed tomography (CT), and MR do not reliably assess steatohepatitis and fibrosis in patients with NAFLD (Chalasani et al., 2012). Specific reference was made to magnetic resonance elastography (MRE) only to point out that this test is rarely used in clinical practice. MRE was neither endorsed nor opposed. Screening for NAFLD in adults at high risk for NAFLD is not recommended due to uncertainties surrounding serum marker tests and standard US and treatment options, along with lack of knowledge related to the long-term benefits and costeffectiveness of screening. American Association for the Study of Liver Disease (AASLD) and Infectious Diseases Society of America (IDSA, 2014) in their Recommendations for Testing, Managing, and Treating Hepatitis C notes the following: Evaluation for other conditions that may accelerate liver fibrosis, including HBV and HIV infections, is recommended for all persons with HCV infection. Evaluation for advanced fibrosis, using liver biopsy showing fibrosis corresponding to a Metavir score of >2 or Ishak shore >3, imaging, or noninvasive markers, is recommended in all persons with HCV infection to facilitate an appropriate decision regarding HCV treatment strategy and determine the need for initiating additional screening measures (eg, hepatocellular carcinoma [HCC] screening). Non-invasive methods frequently used to estimate liver disease severity include a liver-directed physical exam (normal in most patients), routine blood tests (eg, serum alanine transaminase, albumin, bilirubin, international normalized ratio levels, and complete cell blood counts with platelets), serum fibrosis marker panels, liver imaging (eg, ultrasound, computed tomography scan), and liver elastography. Simple blood tests (eg, serum aspartate aminotransferase/platelet ratio index). Liver elastography can provide instant information regarding liver stiffness at the point-of-care but can only reliably distinguish cirrhosis from non-cirrhosis. (Castera, 2012). o (Liver stiffness can be measured noninvasively to assess liver fibrosis in patients with chronic hepatitis C. In patients with chronic liver diseases, level of fibrosis predicts liver-related complications and survival.) There is no specific mention of FIBROSpect, HCV FIBROSURE, ActiTest, ASH FibroSure or NASH FibroSure in these guidelines. Scottish Intercollegiate Guideline Network (SIGN): HCV management guidelines issued by the SIGN in 2006 concluded that, when used in panels, surrogate markers are able to determine whether an individual has high or low levels of fibrosis, but cannot differentiate the stage of fibrosis reliably. The SIGN guidelines also stated FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 11

that biochemical markers should not be used as an alternative to liver biopsy for staging of intermediate grades of fibrosis ; however, biochemical tests may be used as an alternative to liver biopsy to diagnose cirrhosis or to direct screening for complications of fibrosis. World Health Organization (WHO): In its 2006 clinical protocol for the management of HCV and HIV coinfection, the WHO stated that noninvasive tests including combined serum markers are quite reliable, better accepted by patients than biopsies and could potentially save approximately 50% of patients from being biopsied. CDC Guidelines Since the 2003 CDC guidance on Hepatitis C was published, there have been two developments with important implications for HCV testing: 1. Availability of a rapid test for HCV antibody: The OraQuick HCV Rapid Antibody Test (OraSure Technologies) is a rapid assay for the presumptive detection of HCV antibody in fingerstick capillary blood and venipuncture whole blood. Its sensitivity and specificity are similar to those of FDA approved, laboratoryconducted HCV antibody assays. In 2011, a Clinical Laboratory Improvements Amendments waiver was granted to the test by FDA. The waiver provides wider testing access to persons at risk for HCV infection, permitting use of the assay in nontraditional settings such as physician offices, hospital emergency departments, health department clinics, and other freestanding counseling and testing sites. 2. Discontinuation of RIBA HCV. The Chiron RIBA HCV 3.0 Strip Immunoblot Assay (Novartis Vaccines and Diagnostics) that was recommended for supplemental testing of blood samples after initial HCV antibody testing is no longer available. As a result, the only other FDA-approved supplemental tests for HCV infection are those that detect HCV viremia. Supplemental Testing for HCV Antibody If testing is desired to distinguish between true positivity and biologic false positivity for HCV antibody, then, testing may be done with a second HCV antibody assay approved by FDA for diagnosis of HCV infection that is different from the assay used for initial antibody testing. HCV antibody assays vary according to their antigens, test platforms, and performance characteristics, so biologic false positivity is unlikely to be exhibited by more than one test when multiple tests are used on a single specimen. Acute hepatitis C and hepatitis C, past or present, are nationally notifiable conditions, and are subject to mandated reporting to health departments by clinicians and laboratorians, as determined by local, state or territorial law and regulation. Surveillance case definitions are developed by the Council of State and Territorial Epidemiologists in collaboration with CDC. In all but a few jurisdictions, positive results from HCV antibody and HCV RNA testing that are indicative of acute, or past or present HCV infection, are reportable. Specific policies for laboratory reporting are found at health department websites. Research, development, and validation, studies are ongoing to inform the best practices for detecting HCV viremia and for distinguishing between resolved HCV infection and biologic false positivity for HCV antibody in persons in whom HCV RNA FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 12

is not detected. Outcomes of these studies will provide comprehensive guidance on testing, reporting, and clinical management, and will improve case definitions for disease notification and surveillance. In 2011, FDA approved boceprevir (Victrelis, Merck & Co.) and telaprevir (Incivek, Vertex Pharmaceuticals) for treatment of chronic hepatitis C genotype 1 infection, in combination with pegylated interferon and ribavirin, in adult patients with compensated liver disease. Boceprevir and telaprevir interfere directly with HCV replication. Persons who complete treatment using either of these drugs combined with pegylated interferon and ribavirin are more likely to clear virus (i.e., have virologic cure), compared to those given standard therapy based on pegylated interferon and ribavirin. Viral clearance, when sustained, stops further spread of HCV and is associated with reduced risk for hepatocellular carcinoma and all-cause mortality. Other compounds under study in clinical trials hold promise for even more effective therapies. Because antiviral treatment is intended for persons with current HCV infection, these persons need to be distinguished from persons whose infection has resolved. HCV RNA in blood, by nucleic acid testing (NAT), is a marker for HCV viremia and is detected only in persons who are currently infected. Persons with reactive results after HCV antibody testing should be evaluated for the presence of HCV RNA in their blood. Accurate testing to identify current infection is important to: 1. Help clinicians and other providers correctly identify persons infected with HCV, so that preventive services, care and treatment can be offered; 2. Notify tested persons of their infection status, enabling them to make informed decisions about medical care and options for HCV treatment, take measures to limit HCV-associated disease progression (e.g., avoidance or reduction of alcohol intake, and vaccination against hepatitis A and B), and minimize risk for transmitting HCV to others; and 3. Inform persons who are not currently infected of their status and the fact that they are not infectious. The testing sequence in this guidance is intended for use by primary care and public health providers seeking to implement CDC recommendations for HCV testing. In most cases, persons identified with HCV viremia have chronic HCV infection. This testing sequence is not intended for diagnosis of acute hepatitis C or clinical evaluation of persons receiving specialist medical care, for which specific guidance is available. A reactive result indicates one of the following: 1. Current HCV infection, 2. Past HCV infection that has resolved, or 3. False positivity. A reactive result should be followed by nucleic acid testing (NAT) for HCV RNA. If HCV RNA is detected, that indicates current HCV infection. If HCV RNA is not detected, that indicates either past, resolved HCV infection, or false HCV antibody positivity. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 13

Advantages and Limitations of Biopsy and Noninvasive Tests for Detecting Hepatic Fibrosis Advantages Disadvantages Liver Biopsy Direct observation of fibrosis Examines 1/50,000th of hepatic parenchyma Accurate for detecting cirrhosis Contraindicated in ascites, coagulopathy Sampling error and observer variation Unsuitable for longitudinal monitoring Serum TE MRE Markers Noninvasive Noninvasive Noninvasive Examines indirect or direct markers of fibrosis Can be accurate for detecting cirrhosis Delays in test result generation with send-out proprietary tests False-positive values with hemolysis, inflammation, Gilbert syndrome Indices may change with disease progression or response to therapy Examines 1 4 cm area over right liver edge Accurate for detecting cirrhosis Failure rate with obesity, narrow rib spaces False-positive values with inflammation, congestion Liver stiffness does change with disease progression or response to therapy Examines multiple areas within right and left liver Accurate for detecting cirrhosis Limited by claustrophobia and typical MRI contraindications False-positive values with inflammation, congestion Liver stiffness does change with disease progression or response to therapy Schmeltzer PA, Talwalkar JA. Noninvasive Tools to Assess Hepatic Fibrosis: Ready for Prime Time? Gastroenterology Clinics - Volume 40, Issue 3 (September 2011). Interpretation of results of tests for hepatitis C virus (HCV) infection and further actions suggested FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 14

Test Outcome Interpretation Further Action HCV antibody nonreactive No HCV antibody detected Sample can be reported as nonreactive for HCV antibody. No further action required. If recent HCV exposure in person tested is suspected, test for HCV RNA.* HCV antibody reactive Presumptive HCV infection A repeatedly reactive result is consistent with current HCV infection, or past HCV infection that has resolved, or biologic false positivity for HCV antibody. Test for HCV RNA to identify current infection. HCV antibody reactive, HCV RNA detected HCV antibody reactive, HCV RNA not detected Current HCV infection No current HCV infection Provide person tested with appropriate counseling and link person tested to medical care and treatment. No further action required in most cases. If distinction between true positivity and biologic false positivity for HCV antibody is desired, and if sample is repeatedly reactive in the initial test, test with another HCV antibody assay. In certain situations follow up with HCV RNA testing and appropriate counseling. *Note: If HCV RNA testing is not feasible and person tested is not immunocompromised, follow-up testing for HCV antibody should be done to demonstrate seroconversion. If the person tested is immunocompromised, consider testing for HCV RNA. Per the CDC, it is recommended before initiating antiviral therapy to retest for HCV RNA in a subsequent blood sample to confirm HCV RNA positivity. For persons who might have been exposed to HCV within the past 6 months, testing for HCV RNA or follow-up testing for HCV antibody is recommended. For persons who are immunocompromised, testing for HCV RNA can be considered. To differentiate past, resolved HCV infection from biologic false positivity for HCV antibody, testing with another HCV antibody assay can be considered. Repeat HCV RNA testing if the person tested is suspected to have had HCV exposure within the FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 15

past 6 months or has clinical evidence of HCV disease, or if there is concern regarding the handling or storage of the test specimen. Studies Bota et al. (2013) completed a meta-analysis with the goal to compare the diagnostic performance of acoustic radiation force impulse (ARFI) elastography and transient elastography (TE) in the assessment of liver fibrosis using liver biopsy (LB) as 'gold-standard'. Thirteen studies including 1,163 patients with chronic hepatopathies were included in the analysis. Inability to obtain reliable measurements was more than three times as high for TE as that of ARFI (6.6% vs. 2.1%, P < 0.001). For detection of significant fibrosis, (F >= 2) the summary sensitivity (Se) was 0.74 (95% CI: 0.66-0.80) and specificity (Sp) was 0.83 (95% CI: 0.75-0.89) for ARFI, while for TE the Se was 0.78 (95% CI: 0.72-0.83) and Sp was 0.84 (95% CI: 0.75-0.90). For the diagnosis of cirrhosis, the summary Se was 0.87 (95% CI: 0.79-0.92) and Sp was 0.87 (95% CI: 0.81-0.91) for ARFI elastography, and, respectively, 0.89 (95% CI: 0.80-0.94) and 0.87 (95% CI: 0.82-0.91) for TE. The diagnostic odds ratio of ARFI and TE did not differ significantly in the detection of significant fibrosis [mean difference in rdor = 0.27 (95% CI: 0.69-0.14)] and cirrhosis [mean difference in rdor = 0.12 (95% CI: 0.29-0.52)]. Acoustic radiation force impulse elastography seems to be a good method for assessing liver fibrosis, and shows higher rate of reliable measurements and similar predictive value to TE for significant fibrosis and cirrhosis. D'Ambrosio et al. (2013) Transient elastography (TE) is proposed as a validated noninvasive tool to evaluate hepatic fibrosis in patients with hepatitis C virus (HCV) infection. Whether TE may sense changes of liver fibrosis following therapeutic HCV eradication has never been evaluated. 37 HCV cirrhotics with paired pre- and postsustained virological response (SVR) liver biopsies (LB) underwent TE at the time of post-svr LB. Liver fibrosis was staged with the METAVIR scoring system and the area of fibrosis (%) was assessed morphometrically. Thirty-three patients had valid TE measurements after 61 (48-104) months from an SVR, and 20 (61%) of them had cirrhosis regression. On post-svr LB, the median area of fibrosis was 2.3%, being significantly reduced from baseline (p <0.0001). Median TE value was 9.8 kpa being lower in regressed vs. not regressed patients (9.1 kpa vs. 12.9 kpa, p = 0.01). TE was <12 kpa in 5 (38%) F4 patients and in 19 (95%) <=F3 patients (p = 0.0007). The diagnostic accuracy of TE for diagnosing F4 after treatment was 61% sensitivity, 95% specificity, 12.3 LR+, 0.4 LR-, and AUROC 0.77. A significant correlation was found between TE and both fibrosis stage (r = 0.56; p = 0.001) and morphometry (r = 0.56, p = 0.001) as well as between fibrosis stage and area of fibrosis (r = 0.72, p = 0001). Following therapeutic eradication of HCV, the predictive power of the viremic cut-off of 12 kpa was low as a consequence of liver remodelling and fibrosis reabsorption. LB still remains the only reliable approach to stage liver fibrosis following an sustained virological response (SVR). Ferraioli et al. (2013, World Journal of Gastroenterology) completed a single center cross-sectional study. Consecutive patients with chronic viral hepatitis scheduled for liver biopsy were enrolled. Transient elastography (TE) was carried out by using FibroScan. Liver biopsy was performed on the same day as TE. Fibrosis was staged according to the Metavir scoring system. The diagnostic performance of TE was assessed by using receiver operating characteristic (ROC) curves and the area under the ROC curve analysis. Two hundred and fifty-two patients met the inclusion criteria. Six (2%) patients were excluded due to unreliable TE measurements. 246 (171 men and 75 women) patients were analyzed. One hundred and ninety-five FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 16

(79.3%) patients had chronic hepatitis C, 41 (16.7%) had chronic hepatitis B, and 10 (4.0%) were coinfected with human immunodeficiency virus. ROC curve analysis identified optimal cut-off value of TE as high as 6.9 *kpa for F >= 2; 7.9 kpa for F >= 3; 9.6 kpa for F = 4 in all patients (n = 246), and as high as 6.9 kpa for F >= 2; 7.3 kpa for F >= 3; 9.3 kpa for F = 4 in patients with hepatitis C (n = 195). Cut-off values of TE obtained by maximizing only the specificity were as high as 6.9 kpa for F >= 2; 9.6 kpa for F >= 3; 12.2 kpa for F = 4 in all patients (n = 246), and as high as 7.0 kpa for F >= 2; 9.3 kpa for F >= 3; 12.3 kpa for F = 4 in patients with hepatitis C (n = 195). The cut-off values of TE obtained in this single center study are comparable to that obtained in a recently published meta-analysis that included up to 40 studies. (*The results are immediately available and expressed in kilopascals (kpa), corresponding to the median value of ten validated measurements and range from 2.5 to 75 kpa, with normal values around 5.5 kpa. kpa - Mean values of liver stiffness (in kpa) by inflammation strata. When ultrasound-based transient elastography is used in a clinical setting, commonly used cutoff for significant fibrosis (F) and cirrhosis are >7 kpa and >11 to 14 kpa, respectively.) Han et al. (2013) Liver stiffness measurement (LSM) using transient elastography (Fibroscan) are proposed to identify individuals with potential underlying liver disease. The authors evaluated the prevalence of abnormal LSM values as assessed using LSM and its predictors in HIV-infected asymptomatic patients receiving combined antiretroviral treatment (cart) without HBV/HCV coinfection. The authors prospectively recruited 93 patients who had consistently been undergoing cart for more than 12 months. LSM values >5.3 kpa were defined as abnormal. Thirty-nine (41.9%) had abnormal LSM values. On multivariate correlation analysis, the cumulative duration of boosted and unboosted protease inhibitors (PIs) were the independent factors which showed a negative and positive correlation to LSM values, respectively (beta = -0.234, P = 0.023 and beta = 0.430, P<0.001). In multivariate logistic regression analysis, the cumulative exposure duration of boosted-pis and - glutamyltranspeptidase levels were selected as the independent predictors which showed a negative and positive correlation with abnormal LSM values, respectively (odds ratio [OR], 0.941; 95% confidence interval [CI], 0.889-0.997; P = 0.039 and OR, 1.032; 95% CI, 1.004-1.060; P = 0.023). The high percentage of HIV-infected asymptomatic patients receiving cart without HBV/HCV coinfection had abnormal LSM values. The cumulative exposure duration of boosted-pis and -GT level were independent predictors which showed a negative and positive correlation with abnormal LSM values, respectively. Singh et al. (2013) Liver stiffness measurement (LSM), using elastography, can independently predict outcomes of patients with chronic liver diseases (CLDs). However, there is much variation in reporting and consistency of findings. The authors performed a systematic review and meta-analysis to evaluate the association between LSM and outcomes of patients with CLDs prospectively for at least 6 months and reported the association between baseline LSM and subsequent development of decompensated cirrhosis or hepatocellular carcinoma (HCC), as well as mortality. Summary relative risk (RR) estimates per unit of LSM and 95% confidence intervals (CIs) were estimated using the random effects model. Our final analysis included 17 studies, reporting on 7,058 patients with CLDs. Baseline LSM was associated significantly with risk of hepatic decompensation (6 studies; RR, 1.07; 95% CI, 1.03-1.11), HCC (9 studies; RR, 1.11; 95% CI, 1.05-1.18), death (5 studies; RR, 1.22; 95% CI, 1.05-1.43), or a composite of these outcomes (7 studies; RR, 1.32; 95% CI, 1.16-1.51). Considerable heterogeneity was observed among studies, which could not be explained by variations in study locations, etiologies and stages of CLD, FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 17

techniques to measure liver stiffness, adjustment for covariates, or method of imputing relationship in the meta-analysis. Based on a meta-analysis of cohort studies, the degree of liver stiffness is associated with risk of decompensated cirrhosis, HCC, and death in patients with CLDs. LSM therefore might be used in risk stratification. Platon et al. (2013, Journal of GI and Liver Diseases) completed a study aimed to establish the liver stiffness (LS) cut-off values and their performance in the prediction of the fibrosis stage in chronic hepatitis C (CHC) patients, to find the anthropometric and biochemical factors leading to overestimation of the fibrosis stage and to analyze the factors leading to the technique failure. 1,202 consecutive CHC patients were prospectively included in the study. All of them underwent percutaneous liver biopsy for grading and staging the disease (METAVIR) and were referred to LS measurement 1 day prior to biopsy. LS values varied between 2.8-75 kpa. Transient elastography success rate (SR) ranged between 0-100% (84.82+/- 24.46%). In 27 patients (2.2%), no valid measurement was obtained; high BMI influenced independently the measurement failure. In 11.2% of cases, the SR was <60%, but 10 valid measurements were nevertheless recorded; the female sex and high BMI were the only factors independently leading to a SR<60%. ROCs for the diagnosis of fibrosis F>=1, F>=2, F>=3, and F=4 were 0.879, 0.889, 0.941 and 0.970, for the cut-off values of 5.3 kpa, 7.4 kpa, 9.1 kpa and 13.2 kpa respectively, and they did not significantly differ from the adjusted ROC values. The patients with false positive results were younger and had significantly higher serum aminotransferase (ALT, AST) and gamma glutamyl-transpeptidase levels than the patients with concordant results. The multivariate analysis showed that only high ALT levels influenced independently the occurrence of false positive results. Transient elastography is a useful non-invasive method for the assessment of fibrosis in CHC patients. However, it must be interpreted in the clinical and biochemical context, in order to insure high-quality results. Adebajo et al. (2012) Ultrasound-based transient elastography (TE) is a promising noninvasive alternative to liver biopsy for the detection of hepatic fibrosis due to recurrent hepatitis C virus (HCV) after liver transplantation (LT). However, its overall test performance in various settings remains unknown. The aim of this study was to perform a systematic review and diagnostic accuracy meta-analysis of studies comparing ultrasound-based TE to liver biopsy for the detection of hepatic fibrosis due to a recurrent HCV infection after LT. Electronic and manual bibliographic searches (including scientific abstracts) were performed to identify potential studies. A meta-analysis was conducted to generate pooled estimates of the sensitivity values, specificity values, likelihood ratios, and diagnostic odds ratios of individual studies. The extent of the heterogeneity and the reasons for it were assessed. Six fully published studies were identified for analysis. Five studies that evaluated significant fibrosis were identified. Among these studies, the pooled estimates were 83% for sensitivity [95% confidence interval (CI) = 77%-88%], 83% for specificity (95% CI = 77%-88%), 4.95 for the positive likelihood ratio (95% CI = 3.4-7.2), 0.17 for the negative likelihood ratio (95% CI = 0.09-0.35), and 30.5 for the diagnostic odds ratio (95% CI = 12.8-72.4). For the 5 studies that assessed cirrhosis, the pooled estimates were 98% for sensitivity (95% CI = 90%-100%), 84% for specificity (95% CI = 80%-88%), 7 for the positive likelihood ratio (95% CI = 2.8-17.3), 0.06 for the negative likelihood ratio (95% CI = 0.02-0.19), and 130 for the diagnostic odds ratio (95% CI = 36.5-462.1). A diagnostic threshold (or cutoff value) bias was identified as an important cause of heterogeneity for the pooled results of both patient groups. In conclusion, ultrasound-based TE has FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 18

excellent diagnostic accuracy for identifying cirrhosis due to a recurrent HCV infection after LT. The detection of significant fibrosis is more accurate for these patients versus patients whose native liver is chronically infected with HCV. Zarski et al. (2012) Blood tests and transient elastography (Fibroscan) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC). This was a multicentre prospective independent study of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan. Two experienced pathologists independently reviewed the liver biopsies (mean length = 25 +/- 8.4 mm). Performance was assessed using ROC curves corrected by Obuchowski's method. Fibroscan was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan, Fibroscan performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowski's measure showed Fibrometer (0.86), Fibrotest (0.84), Hepascore (0.84), and interpretable Fibroscan (0.84) to be the most accurate tests. The combination of Fibrotest, Fibrometer, or Hepascore with Fibroscan or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROC's ranged from 0.82 (Fibrometer) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer and Hepascore) to 0.83 (FIB-4) for cirrhosis. Contrarily to blood tests, performance of Fibroscan was reduced due to uninterpretable results. Fibrotest, interpretable Fibroscan, Fibrometer, and Hepascore perform best and similarly for diagnosis of significant fibrosis and cirrhosis. Castera et al. (2011) Two index studies suggesting the interest of TE in the assessment of liver fibrosis were conducted in pts with chronic hepatitis C. Other groups have confirmed these results with a strong correlation of liver stiffness values with Metavir fibrosis stages and AUROCs ranging from 0.75 to 0.91 for the diagnosis of significant fibrosis and from 0.90 to 0.98 for cirrhosis. It should be stressed, however, that despite high AUROC values, a substantial overlap of liver stiffness values was observed between adjacent stages of hepatic fibrosis, particularly for lower fibrosis stages. The authors felt that the most rational way of using these tools is to make a compromise: use non-invasive methods to classify those pts in whom they perform with high accuracy, limiting liver biopsy to the subset of patients in whom precise non-invasive staging is not possible. Liver biopsy and non-invasive methods, particularly TE, should be employed as an integrated system to allow a more efficient and convenient management of patients with chronic hepatitis. Friedrich-Rust et al. (2008) Completed a meta-analysis including 50 studies. The mean ROC for the diagnosis of significant fibrosis, severe fibrosis, and cirrhosis were 0.84 (95% confidence interval [CI], 0.82-0.86), 0.89 (95% CI, 0.88-0.91), and 0.94 (95% CI, 0.93-0.95), respectively. For the diagnosis of significant fibrosis, a reduction of heterogeneity of ROC was found when differentiating between the underlying liver diseases (P <.001). Transient elastography can be performed with excellent diagnostic accuracy and independent of underlying liver disease for diagnosis of cirrhosis. Testing to detect hepatic fibrosis FibroTest, marketed as FibroSure in the United States, is the best evaluated of the multiparameter blood tests. The test incorporates haptoglobin, bilirubin, GGTP, FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 19

apolipoprotein A-I, and α2-macroglobulin and has been found to have high positive and negative predictive values for diagnosing advanced fibrosis in patients with chronic hepatitis C. One study showed that use of a more sensitive index cut-off had a sensitivity of 90%, specificity of 36%, positive predictive value of 88%, and negative predictive value of 40% for the diagnosis of bridging fibrosis in patients with chronic hepatitis C. The test has similar performance characteristics in patients with chronic hepatitis B and alcoholic liver disease and has been shown to predict advanced fibrosis in patients taking methotrexate for psoriasis. The newer FIBROSpect II assay incorporates hyaluronate, tissue inhibitor of metalloproteinase 1, and α2-macroglobulin. In a group of patients with chronic hepatitis C, FIBROSpect II had a sensitivity of 72% and a specificity of 74% for identifying advanced fibrosis. Transient elastography (TE) ) is a non-invasive technique that uses both ultrasound and low-frequency elastic waves to quantify liver fibrosis. A meta-analysis showed that TE performed best at differentiating cirrhosis from absence of cirrhosis but was less accurate for the estimation of lesser degrees of fibrosis. Transient elastography has also been shown to be accurate for identifying advanced fibrosis in patients with chronic hepatitis C, PBC, hemochromatosis, NAFLD, and recurrent chronic hepatitis after liver transplantation. The HCV FibroSure test has been developed and tested as a non-invasive measure of fibrosis, with the main body of literature published by the same group of investigators who developed the test. Data on the diagnostic accuracy and predictive value is variable. Although the negative predictive value for the FibroSure was reported as 100% by the authors who developed the test, another group of investigators reported an 89% negative predictive value, suggesting that 11% of patients would potentially forego initial antiviral therapy. There are less published data regarding the ASH FibroSure and NASH FibroSure tests and the FibroSpect test. In one study, the negative predictive value (NPV) of FibroSpect was 75.8%, which is substantially lower than that reported for FibroSure. Because of the limited evidence on these other tests, the diagnostic accuracy and predictive ability is uncertain. There were no studies identified that actually used the results of any of the tests to either clinically manage patients or to reduce the number of biopsies performed. Further, clinical practice guidelines do not address these tests. The scientific data is insufficient to permit conclusions on whether multianalyte assays with algorithmic analyses can improve health outcomes; therefore, these tests, including HCV FibroSure, ASH FibroSure, NASH FibroSure, and FibroSpect, are considered investigational. Per Curry et al. (UpToDate, 2013) Noninvasive tests of hepatic fibrosis have been used in clinical scenarios. The majority of studies of serologic markers and radiologic tests have looked at the use of these tests for staging of fibrosis in patients with chronic liver disease. Noninvasive testing is proposed for patients presenting for evaluation of chronic viral hepatitis. There are two general categories of noninvasive tests for fibrosis, serologic panels of tests and radiologic tests. When evaluating patients for hepatic fibrosis using noninvasive approaches, we typically use a combination serologic testing FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 20

(i.e., Hepascore) and ultrasound-based transient elastography. The specific tests chosen will depend on local availability. A variety of serologic markers have been evaluated to predict the degree of fibrosis in the liver, and panels have been developed that combine assays of multiple markers to improve predictive ability. The most studied panels are the aspartate aminotransferase (AST) to platelet ratio (APRI), FibroTest/FibroSure, Hepascore, and FibroSpect. The APRI has the advantage of being easily calculated using data available from routine laboratory tests. Studies suggest they have good ability to differentiate patients with significant fibrosis (F2-F4) from those without significant fibrosis (F0-F1). The panels may also be able to monitor changes in fibrosis over time. Radiologic methods for staging hepatic fibrosis are emerging as promising tools. The methods include ultrasound-based transient elastography, magnetic resonance elastography, acoustic radiation force impulse imaging, and crosssectional imaging. Ultrasound-based transient elastography is the most studied radiologic method for staging hepatic fibrosis. When ultrasound-based transient elastography is used in a clinical setting, commonly used cutoff for significant fibrosis and cirrhosis are >7 kpa and >11 to 14 kpa, respectively. Using multiple serologic panels or combining serologic panels with radiographic imaging may improve the ability to correctly assess the degree of a patient s fibrosis. In addition, it may be possible to improve the diagnostic performance of these panels if they are used in stepwise combination. Magnetic resonance elastography (MRE) involves the three-dimensional analysis of wave propagation and tissue deformation in patients suspected of or known to have chronic liver disease resulting from viral hepatitis or alcohol abuse. MRE is based on the principle that fibrosis changes the elasticity and viscosity of tissue. By assessing the propagation of acoustic waves through liver tissue, the extent of fibrosis is proposed to be indirectly derived. The paucity of selected peer-reviewed studies suggests that the mean liver stiffness as measured by magnetic resonance elastography (MRE) increases with the stage of liver fibrosis determined by histopathology, however, no consistent methods for differentiating the stages of fibrosis have been determined. As a result, the facilities that adopt this test will find it difficult to interpret the test results, and the facilities that currently perform MRE may reach divergent conclusions from similar test results. The National Cancer Comprehensive Network (NCCN), Clinical Practice Guidelines in Oncology, on Hepatobiliary Cancers, Version 2. 2014, do not mention anything about elastography, FibroScan, FIBROSpect, HCV-FIBROSURE, ActiTest, serum marker testing, or any other non-invasive tests. Scientific Rationale Update December 2013 The HCV FibroSure test (i.e., FibroTest) has been developed and extensively tested as a non-invasive measure of fibrosis, with the main body of literature published by the same group of investigators who developed the test. Data on the diagnostic accuracy and predictive value is variable. Although the negative predictive value for the FibroSure was reported as 100% by the authors who developed the test, another group of investigators reported an 89% negative predictive value, suggesting that 11% of patients would potentially forego initial antiviral therapy. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 21

FibroTest and FibroSure are identical proprietary tests marketed under different names in Europe and America, respectively. ActiTest is a modification of FibroTest. The tests have primarily been studied in patients with HCV. Transient elastography, also known as FibroScan, rapidly and noninvasively measures mean hepatic tissue stiffness. Ultrasound-based transient elastography fulfills many features desirable of noninvasive assessment of hepatic fibrosis; it is quick, inexpensive, reproducible, painless, and examines a large mass of liver tissue, thereby reducing sampling error. The use of elastography for diagnosing liver disease in HIV/HCV-coinfected patients is a promising modality. This technique is widely available in Europe, however it is restricted to only a few research centers in the United States. FibroScan was approved by the U.S. Food and Drug Administration in April 2013. FibroScan measures liver stiffness using a section of liver that is approximately 1 cm in diameter and 5 cm long, which is 100 times greater in size than a standard liver biopsy, and thus may be more representative of the entire hepatic parenchyma. In cirrhotic patients, liver stiffness measurements range from 12.5 to 75.5 kpa. However, the clinical relevance of these values is unknown. ASH FibroSURE (i.e., ASH Test) uses a combination of 10 serum biochemical markers of liver function together with age, gender, height, and weight in a proprietary algorithm and is proposed to provide surrogate markers for liver fibrosis, hepatic steatosis, and alcoholic steatohepatitis (ASH). The biochemical markers include alpha-2 macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, GGT, ALT, AST, total cholesterol, triglycerides, and fasting glucose. The test has been available in Europe under the name ASH Test and is exclusively offered by LabCorp in the U.S. as ASH FibroSure. NASH FibroSURE (i.e.,nash Test) uses a proprietary algorithm of the same 10 biochemical markers of liver function in combination with age, gender, height, and weight and is proposed to provide surrogate markers for liver fibrosis, hepatic steatosis, and NASH. The biochemical markers include alpha-2 macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, GGT, ALT, AST, total cholesterol, triglycerides, and fasting glucose. The test has been available in Europe under the name NASH Test and is exclusively offered by LabCorp in the U.S. as NASH FibroSure. There are less published data regarding the ASH FibroSure and NASH FibroSure tests and the FibroSpect test. In one study, the negative predictive value (NPV) of FibroSpect was 75.8%, which is substantially lower than that reported for FibroSure. Because of the limited evidence on these other tests, the diagnostic accuracy and predictive ability is uncertain. Polynard et al. (2012) assessed the performance of a newer test, the Elasto- FibroTest (EFT), combining FibroTest and liver stiffness measurement (LSM). The authors used a data base of 1289 patients with biopsy and 604 healthy volunteers to analyze the Elasto-FiberTest (EFT). Authors concluded for the diagnosis of cirrhosis the Elasto-FibroTest had higher performances compared to FibroTest or FibroScan alone. However, no improvement in performance has been observed for the diagnosis of advanced fibrosis vs. FibroTest alone. In the 2012 guidelines on the Diagnosis and Management of Non-Alcoholic Fatty Liver Disease (NAFLD), the practice guidelines by the American Association for the Study of Liver Diseases and the American College of Gastroenterology do not FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 22

address the FibroTest or other available analyses. There is strong evidence outlined in the guidelines that include imaging and liver biochemistries for the detection of NAFLD. In the guidelines for the Prevention and Management of Gastroesophageal Varices and Variceal Hemorrhage in Cirrhosis approved by the American Association for the Study of Liver Diseases and the American College of Gastroenterology, the FibroTest is described as having limited predictive accuracy. Guidelines recommend that large prospective studies of noninvasive markers need to be performed, and until the data is available, endoscopic screening is still the main means of assessing for the presence of esophageal varices. There is a paucity of studies identified that actually used the results of any of the tests to either clinically manage patients or to reduce the number of biopsies performed. Further, clinical practice guidelines do not address these tests. The scientific data is insufficient to permit conclusions on whether multianalyte assays with algorithmic analyses can improve health outcomes; therefore, these tests, including HCV FibroSure, ASH FibroSure, NASH FibroSure, and FibroSpect, are considered investigational. The National Cancer Comprehensive Network (NCCN, Version 2.2013) Guidelines on Hepatobiliary Cancer, have no mention of serum marker testing for liver fibrosis or of the elastography noted as investigational within the policy statement. Scientific Rationale Update December 2012 There is a double-blind, randomized, placebo-controlled multi-center, phase II parallel dose-ranging Clinical Trial on Antifibrotic Activity Of GI262570 In Chronic Hepatitis C Subjects. The ClinicalTrials.gov Identifier is NCT00244751, and was last verified in November 2009. The purpose of this study is to examine the safety and effectiveness of GI262570 compared to placebo, in improving specific tests that indicate the degree of liver fibrosis. Subjects who are enrolled in the study must have had prior treatment with interferon (either pegylated or standard interferon) plus ribavirin for at least 12 weeks to treat their hepatitis C, but either failed to clear the virus or didn't tolerate the treatment. One of the secondary outcome measures is Change from baseline in serum FibroSure (FibroTest/ActiTest) score. Although it states that this is completed, there are no study results noted. Although liver biopsy is still the gold standard to diagnose liver fibrosis, the serum assay approaches remain promising, in part because these tests may represent an integrated readout of liver activity, rather than a minute sampling of the type obtained by conventional liver biopsy. However, additional peer-reviewed studies are needed. Scientific Rationale Update December 2011 While tremendous progress has been made in improving the accuracy of serum markers of hepatic fibrosis, they cannot yet supplant direct analysis of the liver. The ideal fibrosis marker is one that is specific, biologically based, noninvasive, easily repeated in all patients, correlates well with disease severity and outcome, and is not confounded by co-morbidities or drugs. Although this ideal has nearly been reached, no serum test has emerged as the perfect marker of fibrosis; all the serum tests have the following limitations: FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 23

They typically reflect the rate of matrix turnover, not deposition, and thus tend to be more elevated when there is high inflammatory activity. In contrast, extensive matrix deposition can go undetected if there is minimal inflammation. None of the molecules are liver-specific, so that concurrent sites of inflammation may contribute to serum levels. Serum levels are affected by clearance rates, which may be impaired either due to sinusoidal endothelial cell dysfunction or impaired biliary excretion. They are surrogates, not biomarkers. There are currently four commercial serum marker systems that have been most extensively validated: the Fibrospect (Prometheus Corp), the Fibrotest (marketed in the United States by Labcorp), Hepascore (Quest Diagnostics), and the European Liver Fibrosis Study Group panel (not available in the United States), which uses a diagnostic algorithm and assays developed by Siemens Healthcare Diagnostics. In addition, a growing list of other noninvasive tests have been developed using a variety of standard serum hematologic or chemistry in varying combinations. There are few discernible differences among the three major serum assays, and their value can be summarized as the following: The tests are accurate (>95 percent) in determining the near absence (F0 or F1) of fibrosis in HCV or presence of cirrhosis, but not for intermediate stages. Thus in select clinical circumstances the findings from these tests may obviate the need for biopsy or help guide decisions about treatment with antiviral combinations. Less is known about their value in patients with liver diseases other than HCV, but data are beginning to emerge. They suffer from a variable but significant number of indeterminate outcomes (up to 50 percent with the Fibrotest). There is no evidence that any test can discern changes in fibrosis content over time in an individual patient, although these tests are proving valuable in cohort studies, where mean changes in serum values among a group of patients will correlate with changes in fibrosis. Costa et al. (2010) Three gene polymorphisms, interferon-lambda-3 (IL28B), inosinetriphosphatase (ITPA) and bilirubinuridine diphosphateglucuronosyltransferase (UGT1A1) are associated with treatment (interferon and ribavirin) efficacy and adherence in patients with chronic hepatitis C. The hypothesis was that fibrosis stage estimated with FibroTest instead of biopsy was still an independent predictive factor of sustained virologic response (SVR) when these new polymorphisms were assessed. Patients receiving standard of care treatment were retrospectively analyzed with determination of IL28B, ITPA, and UGT1A1 polymorphisms. Baseline prognostic factors were combined using logistic regression analysis in a training group (157 patients) and validated in a validation group (79 patients). The combination of the five most predictive factors (HCV genotype 2/3, IL28B genotype, FibroTest, ActiTest and viral load) in the training population had AUROC for SVR=0.743 (0.655-0.810; P<0.0001 vs. random), which was validated in the validation population, AUROC=0.753 (0.616-849; P=0.0007 vs. random, not different from training P=0.88). FibroTest remained significant [OR=4.20 (2.59-12.50); P=0.03] after assessment of the IL28B CC, HCV genotype and viral load. Fibrosis stage assessed by FibroTest is an independent predictor of SVR, after accounting for the IL28B genetic polymorphism. A combination of five baseline FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 24

biomarkers could simplify the baseline prediction of SVR. However, additional, clinical studies are needed with the various serum biomarkers that are being promoted to analyze hepatic fibrosis. Comparative peer-reviewed studies with these biomarkers and with direct analysis of the liver, are necessary to compare the accuracy of this testing. Cholongitas et al. (2010) systematically reviewed the literature regarding noninvasive tests (NIT) following liver transplantation. These investigators identified 14 studies evaluating NIT based on serum markers and/or liver imaging techniques: 10 studies assessed NIT in recipients with recurrent HCV infection for fibrosis and 4 studies evaluated predictors of progression of fibrosis in recurrent HCV. Transient elastography had good discrimination for significant fibrosis (median AUROC: 0.88). Among the serum NIT, APRI had good performance (median AUROC: 0.75). Trnasient elastography performed better than serum (direct and indirect) NIT for significant fibrosis with median AUROC 0.88 (versus 0.66, p < 0.001), median sensitivity 0.86 (versus 0.56, p = 0.002), median NPV 0.90 (versus 0.74, p = 0.05) and median PPV 0.80 (versus 0.63, p = 0.02). Transient elastography compared to indirect serum NIT, had better performance, but was not superior to APRI score. Finally, direct, compared to indirect NIT, were not significantly different except for specificity: median: 0.83 versus 0.69, respectively, p = 0.04. The authors concluded that NIT could become an important tool in clinical management of liver transplant recipients, but whether they can improve clinical practice needs further evidence. Their optimal combination with liver biopsy and assessment of collagen content requires investigation. The 2012 National Cancer Comprehensive Network (NCCN) does not mention FIBROSpect, HCV-FIBROSURE, or ActiTest in their 2012 guidelines on Hepatobiliary Cancer. Scientific Rationale Update March 2011 FibroTest and ActiTest are noninvasive tests used in determining the level of fibrosis and the degree of necroinflammatory activity in the liver. Uyar et al. (2010) investigated whether these tests could be alternatives to liver biopsy. Fifty patients were included in the study. Serum samples were obtained and liver needle biopsy was performed on the same day. Levels of fibrosis in FibroTest and levels of activity in ActiTest, both determined via serum biochemical markers, were compared with levels of fibrosis and activity in histopathological examination. For statistical analyses, Mc Nemar chi square test and Spearman's correlation tests were used. There was a significant positive correlation between fibrosis in biopsy and the level of fibrosis in FibroTest in patients with hepatitis B virus (HBV) (rho: 0.67, P < 0.0001). However, no significant correlation was determined between the activity in biopsy and the degree of activity in ActiTest (rho: 0.29, P < 0.05). No significant correlation was determined between both fibrosis and activity established in biopsy and the results of FibroTest and ActiTest in the group of patients with hepatitis C virus (HCV) (rho: 0.22, P < 0.05 and rho: 0.15, P < 0.05, respectively). The results suggest that novel and safer noninvasive biochemical tests are needed as an alternative to histopathology in patients infected with HBV and HCV. Consequently, we believe that liver biopsy maintains its place as a gold standard in determining the histopathological condition of the liver. Shaheen and colleagues (2007) compared FibroTest and another marker (FibroScan) to biopsy in patients with hepatitis C related fibrosis. For significant fibrosis, FibroTest had a sensitivity of 47% and a specificity of 90%. There was lesser FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 25

accuracy for earlier stages of fibrosis. The authors noted these tests are not ready to replace liver biopsy and additional studies should be conducted. Scientific Rationale March 2010 Liver biopsy is regarded as the gold standard for determining the extent of fibrosis and defining liver disease status, however, it is subject to sampling error and requires special expertise for interpreting the histopathology. Risks associated with liver biopsy include pain, bleeding and perforation of other organs. Alternative, less invasive means for the diagnosis or clinical management of liver disease, including hepatitis C, are being investigated. There are several serum marker panels currently available, including, Fibrospect, Fibrosure, Fibrotest, Fibromax, Actitest and Hepascore. These markers are useful for establishing the two ends of the fibrosis spectrum (minimal fibrosis and cirrhosis) but are less helpful in assessing the midranges of fibrosis or for tracking fibrosis progression. The FibroTest-ActiTest (BioPredictive) uses a panel of six markers (i.e., alpha-2 macroglobulin, haptoglobulin, apolipoprotein A1, total bilirubin, GGT and ALT). The FibroMax (BioPredictive) panel includes the same six markers along with AST, fasting glucose, triglycerides and total cholesterol. The Hepascore (Quest Diagnostics) is based on serum levels of α2-macroglobulin, hyaluronic acid, gamma glutamyl transferase (GGT), and total bilirubin, along with age and sex. According to American Association for the Study of Liver Disease (Apr. 09): A liver biopsy should be considered in patients with chronic hepatitis C infection if the patient and health care provider wish information regarding fibrosis stage for prognostic purposes or to make a decision regarding treatment [Class IIa (Weight of evidence/opinion is in favor of usefulness/efficacy); Level B (Data derived from a single randomized trial, or nonrandomized studies)] Currently available noninvasive tests may be useful in defining the presence or absence of advanced fibrosis in persons with chronic hepatitis C infection, but should not replace the liver biopsy in routine clinical practice [Class IIb (Usefulness/efficacy is less well established by evidence/ opinion; Level C (Only consensus opinion of experts, case studies, or standard-of-care.)] Guajardo-Salinas et al (2009) performed a retrospective chart review of all patients undergoing Roux-en-Y gastric bypass with routine liver biopsy performed at Valley Baptist medical center during a 24-month period (2005-2006). Of 129 liver biopsies, only 25.7% had some degree of steatosis, but about 55% had nonalcoholic steatohepatitis (NASH), and 30.9% had liver fibrosis. Of those patients with liver fibrosis, only 6.9% had moderate to severe fibrosis (stages 2-4), and only one patient had cirrhosis (0.7%). Of the 129 patients, only 92 had a FIBROspect score II in their chart, and they ranged from 9 to 95, with a mean of 28.3. Of these patients, 36 had a score less than 20, and none had significant fibrosis in their biopsy. FIBROspect II(R) score (cutoff <20) had a negative predictive value (NPV) of 100%), positive predictive value (PPV) of 15%, sensitivity of 100%, and specificity of 42% to predict stage 2 fibrosis or higher. Becker et al (2009) investigated the hepascore test in patients with chronic hepatitis C virus infection. Patients were assigned to training (n=203) or validation (n=188) sets. Liver fibrosis was staged according to the METAVIR scoring system. The Hepascore algorithm utilizes data on age, sex, as well as total bilirubin, gamma- FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 26

glutamyl transferase, alpha2-macroglobulin and hyaluronic acid levels. The investigator reported theat the ability of Hepascore to predict significant fibrosis (F2-4) as determined by the area under the ROC was similar in training (0.83) and validation sets (0.81) and was comparable to results seen in previous studies. A cutoff score of > 0.55 was best for predicting significant fibrosis with a sensitivity and specificity of 82% and 65%, and positive and negative predictive values of 70% and 78%. When compared with two simple indices FIB-4 (age, platelets, AST and ALT) and APRI (AST and platelets), Hepascore performed better at excluding advanced fibrosis using a low cutoff score but worse at predicting fibrosis using a high cutoff score. An algorithm using Hepascore followed by FIB-4 or APRI, spared 103 of 391 individuals a liver biopsy missing advanced fibrosis in only 1 patient. Smith and Sterling (2009) performed a systematic review of non-invasive measures of assessing fibrosis in individuals with chronic hepatitis C virus and their ability to replace biopsy. 151 studies were identified: 87 using biochemical, 57 imaging and seven breath tests either alone or in combination. The authors reported that although no single non-invasive test or model developed to date can match that information obtained from actual histology (i.e. inflammation, fibrosis, steatosis), combinations of two modalities of non-invasive methods can reliably differentiate between minimal and significant fibrosis, and thereby avoid liver biopsy in a significant percentage of patients. Fontanges et al (2008) compared liver biopsy and Actitest and Fibrotest for the evaluation of hepatic activity and fibrosis in patients with chronic hepatitis in routine practice. Routine blood tests, Actitest and Fibrotest and liver biopsy were performed in 96 patients with chronic hepatitis C. Receiver operating characteristics (ROC) curves were used to assess the diagnostic value of the biochemical tests in comparison with the METAVIR classification. The study population was predominantly male (63.5%) with a mean age of 48 years; 83.3% of the patients had genotype 1 hepatitis C virus infection. Treatment status was naive (62.5%), nonresponders (17.7%), relapsers (7.3%), or unknown (12.5%). The comparison of F0-F2 versus F3-F4 estimated the negative predictive value at 92% and the positive predictive value at 52% for a cut-off of 0.455. Discrepancies in activity score were more frequently due to a higher score of the biochemical test compared to biopsy (18 cases out of 19). Discrepancies for fibrosis were observed in 18 patients with a higher score for biochemical test in eight and a higher score for liver biopsy in 10 cases. A significant increase of gamma-glutamyl-transferase (GGT) and alanine aminotransferase (ALT) was observed in case of biochemical test overestimation of activity, and a significant increase of alpha2-macroglobulin and GGT in case of biochemical test overestimation of fibrosis. The authors concluded tat although the diagnostic value of biochemical tests for necrotico-inflammatory activity and fibrosis was good compared with the histological analysis of liver biopsy, clinicians must interpret the results of Actitest and Fibrotest with caution in patients with a significant elevation of ALT, and/or GGT and/or alpha2-macroglobulin which could overestimate hepatic injury. Halfon et al (2008) updated a previous meta-analysis of the FibroTest (FT) diagnostic value, its advantages and limitations and provide an overview of the prognostic value of FT in chronic hepatitis C (CHC), chronic hepatitis B (CHB) and alcoholic liver disease (ALD). For diagnostic value, the main endpoint was the FT area under the ROC curves (AUROCs) for the diagnosis of bridging fibrosis (F2/F3/F4 vs F0/F1), standardized for the spectrum of fibrosis. Sensitivity analysis integrated the nonstandardized observed AUROCs, the independency of authors, size (length) of FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 27

biopsy, prospective design, correctness of procedures, co-morbidities, and timelag between biopsy and serum sampling. For prognostic value, the main endpoint was the FT AUROC for the prognostic value of liver complications or death related to liver disease. A total of 38 diagnostic studies were included, which pooled 7985 subjects who had undergone both FT and biopsy (4600 HCV, 1580 HBV, 267 NAFLD, 524 ALD and 1014 mixed). The mean standardized AUROC was 0.84 (95% CI, 0.83-0.86), with no differences in terms of causes of liver disease: HCV 0.84 (0.82-0.87); HBV 0.81 (0.78-0.83); NAFLD 0.84 (0.76-0.92); ALD 0.87 (0.82-0.92); and mixed 0.85 (0.81-0.89). Three prognostic studies were also included. FT was found to have higher or similar prognostic value compared with biopsy in patients with CHC, CHB or ALD. Liver biopsy remains the standard method for the assessment and diagnosis of liver fibrosis and inflammation. Although numerous noninvasive biochemical markers have been studied in the published, peer-reviewed literature as possible markers of fibrosis and/or inflammation, none has been proven to accurately differentiate and predict liver histology and fibrosis severity. The role of these markers has yet to be determined in the diagnosis and management of liver disease. Scientific Rationale March 2009 Shaheen and Myers (2008) performed a systematic review and meta-analysis of the diagnostic accuracy of fibrosis marker panels in patients with HIV/hepatitis C coinfection. Random effects meta-analyses and areas under summary receiver operating characteristics curves (AUC) examined test accuracy for detecting significant fibrosis (F2 to F4) and cirrhosis. Heterogeneity was explored using metaregression. Five studies (n = 574) including 4 fibrosis measures (APRI [n = 4 studies], Forns' [n = 2], FibroTest [n = 1], SHASTA [n = 1]) met the inclusion criteria. The prevalence of significant fibrosis and cirrhosis were 51 % and 16 %, respectively. For the prediction of significant fibrosis, the summary AUC was 0.82 (95 % CI 0.78 to 86) and diagnostic odds ratio was 7.8 (5.1 to 11.9). For cirrhosis, these figures were 0.83 (0.69 to 0.97) and 11.0 (4.6 to 26.2), respectively. Metaregression including study factors (methodological quality and biopsy adequacy), patient characteristics (age, gender, CD4 count), and fibrosis measure failed to identify important predictors of accuracy. The authors concluded that available fibrosis marker panels have acceptable performance for identifying significant fibrosis and cirrhosis in HIV/HCV-coinfected patients but are not yet adequate to replace liver biopsy. They noted that additional studies are needed to identify the optimal measure. The FibroSure test has been developed and extensively tested by the same group of investigators. However, in the only study that attempted independent validation, the diagnostic performance of the FibroSure test was inferior to that reported by the original investigators, (Rossi et al. [2003]. There are less published data regarding the FibroSpect test. The clinical utility is related to how the results of either test can be used to improve patient management. Although the negative predictive value for the FibroSure was reported as 100% by the authors who developed the test, (Imbert-Bismut et al. [2007], another group of investigators reported a 89% negative predictive value (Rossi et al. [2003]), suggesting that 11% of patients would potentially forego initial antiviral therapy. The negative predictive value of FibroSpect was reported as 75.8%. (Patel et al. [2004]) There were no studies identified that actually used the results of either the FibroSure or FibroSpect in the management of patients to direct liver biopsy decisions. Therefore, there is FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 28

inadequate scientific data to permit conclusions regarding either FibroSure or FibroSpect. Scientific Rationale March 2008 Patel et al. (2008) prospectively evaluated the FIBROSpect II (FSII) biomarker panel versus pathology assessment and a quantitative measure of fibrosis. Liver biopsy specimens and serum were obtained from 252 CHC patients, including 50 posttransplant, from 3 tertiary centers. Biopsy specimens were scored centrally and independently at each site, along with central quantification of fibrosis by digitized morphometry. Serum tests were performed blinded to clinical or histologic evaluation. The mean biopsy specimen length was 1.95 +/- 0.87 cm; prevalence of stage F2 through F4 fibrosis was 77%. Agreement between central and site readings for individual stages was modest (k = 0.674), with concordant readings in 106 of 248 (43%) biopsy specimens. The area under the receiver operating characteristic curve for FSII and morphometry for stages F2 through F4 for concordant biopsy specimens were 0.823 and 0.728, respectively. Sensitivity and specificity for FSII were 83.5% and 66.7%, respectively, with an accuracy of 80.2%. The aspartate aminotransferase to platelet ratio index sensitivity and specificity for predicting F2 through F4 were 30.4% and 100%, respectively, the indeterminate rate was 40.4%, and the accuracy rate was 48.4%. The accuracy of FSII in concordant biopsy specimens in the posttransplant cohort was 73%. Serum biomarkers can differentiate mild from moderate-to-severe fibrosis. This prospective study validates the performance characteristics of FSII in CHC patients and a posttransplant cohort. Assessing the diagnostic utility of biomarkers is limited by variability in methods to quantify fibrosis and poor interobserver agreement for histologic staging. Zaman et al. (2007) performed a study to prospectively validate a panel of serum fibrosis markers (FIBROSpect SM II) that has been recently developed. Serum was obtained from 108 consecutive hepatitis C virus (HCV) (15% with HCV/ETOH) patients. The performance of FIBROSpect II (consisting of 3 fibrosis markers: hyaluronic acid, tissue inhibitor of metalloproteinases 1, and alpha-2-macroglobulin) in differentiating mild (F0-F1) from significant (F2-F4) fibrosis was assessed by comparing the panel results with performed liver biopsy. The prevalence of significant fibrosis in the study group was 36.1%. The diagnostic value of the serum marker panel to detect significant fibrosis as assessed by area under the receiver operating characteristic (ROC) curve was 0.826. Performance characteristics are as follows: sensitivity 71.8%, specificity 73.9%, positive predictive value 60.9%, negative predictive value 82.3%, and overall accuracy of 73.1%. This prospective study supports the clinical utility of serum markers in detecting fibrosis and validates the performance of FIBROSpect II in a prospective cohort of patients. The high negative predictive value of the test provides a reliable alternative to rule out severe fibrosis. FIBROSpect II may be useful in assessing fibrosis progression in patients who have had a baseline liver biopsy in the past, including those patients with non- HCV-related liver diseases. However, this must be studied prospectively before it can be recommended. It will also be important to prospectively validate these results using a community-based setting with different relative prevalence of different stages of HCV in order to further assess its clinical utility. Moreover, it will be of interest to determine how the FIBROSpect II might change in patients receiving antiviral therapy. Wilson et al. (2006) Baseline liver biopsies were obtained (1996-1998) on a random sample of 210 out of 1667 HCV-positive injection drug users (IDUs). Subjects were followed biannually, with a second biopsy offered to those eligible. Paired biopsies FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 29

were scored 0 to 6 (modified Ishak score). Predictive values of blood markers FibroSURE, aspartate aminotransferase-to-platelet-ratio index (APRI) and alanine aminotransferase (ALT) were assessed for detection of liver fibrosis. Among 119 prospectively followed IDUs, 96% were African American; 97% HCV genotype 1a/b; 27% HIV-infected, and median age was 42 years. 90.7% did not have significant liver fibrosis at first biopsy. Although predictive value for detecting insignificant fibrosis at first biopsy was greater than 95% for FibroSURE, APRI, and ALT, specificities were 88.9%, 72.7%, and 72.7%, respectively. After 4.2 years median follow-up, 21% had progression of fibrosis, which was significantly associated with serum level of HCV RNA and ALT. No serological test had predictive value greater than 40% for contemporaneous or future significant fibrosis. Even initial biopsy result had only a 30.4% value for predicting future significant fibrosis. Significant liver fibrosis and progression were detected in some, but not most, IDUs in this cohort. In this setting with low fibrosis prevalence, FibroSURE, ALT, and APRI tests predict insignificant fibrosis; however, further work is needed to find noninvasive markers of significant liver fibrosis. Rockey et al. (2006) As novel therapies for liver fibrosis evolve, non-invasive measurement of liver fibrosis will be required to help manage patients with chronic liver disease. Although liver biopsy is the current and time-honored gold standard for measurement of liver fibrosis, it is poorly suited to frequent monitoring because of its expense and morbidity, and its accuracy suffers from sampling variation. At the current writing, serum markers and imaging methods are available and increasingly in use as alternatives to biopsy. However, many questions remain about their indications and accuracy, and more investigation is required before they are put into widespread use. The development of safe, and reliable noninvasive fibrosis measurement tools remains a research priority in clinical hepatology. Scientific Rationale Update March 2007 The use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) continue to be investigated as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C. However, the studies fail to provide sufficient information to reach conclusions regarding the use of these markers, used in various combinations, as a replacement for liver biopsy. In addition, the studies fail to reach conclusions concerning how the results of the fibrosis biomarker tests impact treatment decisions and health outcomes. Christensen et al. (2006) investigated FIBROSpect II (FS) results with liver fibrosis scores to determine if this test is sufficiently accurate to be a viable alternative to liver biopsy. A total of 142 serum specimens were evaluated for fibrosis with Fibrospect II and were compared with Knodell and Ishak fibrosis scores. FS reports an index score ranging from 0.1 to 1.0, which corresponds to the probability of progressive liver fibrosis. Using a FS index cut-off of 0.42, 50 of 54 patients with Ishak 3-6 were classified as having advanced fibrosis and 58 of 88 patients with Ishak 0-2 as having no/mild fibrosis, resulting in a sensitivity of 93%, specificity of 66%, and an overall test accuracy of 76%. With a 38% prevalence of advanced fibrosis, the negative predictive value was 94% and positive predictive value was 63%. A biopsy length of > or = 2 cm was associated with higher concordance between FS results and liver fibrosis scores. The investigator concluded that Fibrospect II was clinically useful in ruling out advanced fibrosis in hepatitis C by identifying patients with mild disease in whom treatment could be deferred. The FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 30

investigator noted the limitation of this test is its decreased sensitivity and specificity in the middle of the test's reporting range between scores of 0.42 and 0.80. Bourliere et al. (2006) investigated the diagnostic accuracy of noninvasive indexes in comparison with liver histology in 235 hepatitis C virus (HCV)-infected patients. FibroTest (FT), aspartate aminotransferase to platelet ratio index (APRI) and Forns score were assessed in the cohort and compared with liver histology performed on the same day. The main end point was the area under characteristic curves (AUCs) for the diagnosis of significant fibrosis (F2-F4) and cirrhosis (F4) by the METAVIR classification. For the diagnosis of significant fibrosis, respective AUCs of FT, APRI and Forns score were 0.81 (95% confidence interval: 0.76-0.86), 0.71 (0.67-0.79) and 0.76 (0.70-0.82); for cirrhosis prognosis, AUCs of FT and APRI were 0.82 (0.77-0.87) and 0.81 (0.76-0.86) (AUCs not significantly different). Using each index independently, all patients were classified by FT, 214 (91%) patients were classified by APRI and 129 (55%) by Forns score. The investigator concluded that the combination of all tests with liver biopsy allowed 225/235 (96%) patients to be correctly classified. The combination of all tests without liver biopsy allowed 191/235 (81.3%) patients to be correctly classified; liver biopsy remained mandatory in some patients (18.7%). The American Association for the Study of Liver Diseases (AASLD) practice guideline on Diagnosis, Management and Treatment of Hepatitis C (2004) states: Although liver fibrosis markers are commercially available, they are currently insufficiently accurate to support their routine use. Until sensitive serum markers can be developed that will define all stages of fibrosis and mirror the information derived from liver biopsy, the procedure remains the only means of defining the severity of damage from HCV infection in many patients. A 2006 technical review on the management of hepatitis C by the American Gastroenterological Association states Neither clinical nor laboratory markers, individually or in combination, predict accurately the degree of necroinflammatory activity or the level of fibrosis in the liver. Therefore, despite sampling error, liver biopsy remains the gold standard for determining histologic grade and stage. Scientific Rationale Approximately four million Americans are infected and 8-10,000 die each year from hepatitis C. Most patients have the infection for many years without symptoms. Approximately 20% of untreated patients develop cirrhosis after 20 years and 50% after 50 years. Effective treatment with interferon and ribavirin can now eradicate the virus in about 40% of patients. Pegylated interferon plus ribavirin eradicates the virus in about 50-60%. Hepatitis C is the leading reason for liver transplantation in the United States. Each year 1-3% of cirrhotic patients develop cancer of the liver called hepatocellular carcinoma. Deaths are expected to triple in the next few decades. The natural history of cirrhosis is such that chronic liver injury leads first to fibrosis, and, if present over long periods of time, to cirrhosis. Various noninvasive tests of hepatic fibrosis have been examined for monitoring patients with chronic HCV infection. These include routinely available laboratory tests, such as liver-associated chemistries, platelet count, and prothrombin time. No single test or panel of serologic markers can provide an accurate assessment of intermediate stages of hepatic fibrosis. Similarly, quantitative tests of liver function and radiologic imaging of the liver are sensitive for diagnosing advanced cirrhosis but are not useful in FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 31

assessing hepatic fibrosis and early cirrhosis. In the absence of sensitive noninvasive markers of fibrosis, liver biopsy still remains essential for direct assessment of the degree of hepatic fibrosis. Liver biopsy provides a unique source of information on fibrosis and assessment of histology. Liver enzymes have shown little value in predicting fibrosis. Extracellular matrix tests can predict severe stages of fibrosis but cannot consistently classify intermediate stages of fibrosis. Moreover, only liver biopsy provides information on possible contributions of iron, steatosis, and concurrent alcoholic liver disease to the progression of chronic hepatitis C toward cirrhosis. Since a favorable response to current antiviral therapy occurs in 80 percent of patients infected with genotype 2 or 3, it may not always be necessary to perform liver biopsy in these patients to make a decision to treat. All patients with chronic hepatitis C are potential candidates for antiviral therapy. Treatment is recommended for patients with an increased risk of developing cirrhosis. These patients are characterized by detectable HCV RNA levels higher than 50 IU/mL, a liver biopsy with portal or bridging fibrosis, and at least moderate inflammation and necrosis. The majority also have persistently elevated ALT values. But liver biopsy has three major limitations, which are the risk of adverse events, sampling error, and inter- and intra- pathologist variability. The small risk of bleeding or other complications can be reduced but not eliminated by ultrasound or CT guidance of the biopsy. Thus, there is considerable interest in the development of noninvasive markers of liver fibrosis as alternatives to liver biopsy in patients with chronic hepatitis C and other chronic liver diseases, including chronic hepatitis B, alcoholic liver disease, or non-alcoholic steatosis. Recent studies suggest improvement of the diagnostic accuracy of biochemical markers, and that liver biopsy should no longer be considered mandatory in patients with chronic hepatitis. These biochemical and serologic tests appear to be best at predicting no or minimal fibrosis and at predicting advanced fibrosis/cirrhosis, and are poor at predicting intermediate levels of fibrosis. Considerable interest has been displayed in the area of tumor markers for fibrosis assessment. FIBROSpect (Prometheus Laboratories) is a panel that uses a combination of components in the fibrogenic cascade, such as hyaluronic acid, TIMP- 1 (tissue inhibitor of metalloproteinase), and alpha-2-macroglobulin. According to the manufacturer, the test is intended to differentiate mild liver fibrosis from more severe disease. HCV Fibrosure (LabCorp) uses a combination of six serum biochemical markers (alpha2-macroglobulin, haptoglobin, gamma-glutamyl transpeptidase [GGT], total bilirubin, apolipoprotein A1, plus alanine aminotransferase [ALT]) plus age and gender in a patented algorithm to determine the degree of liver fibrosis and the level of ongoing necroinflammatory activity. FibroTest (BioPredictive) is a biochemical marker of liver fibrosis and ActiTest (BioPredictive) is a biochemical marker of inflammation and necrosis of the liver. Rossi et al (2003) reported on the results of FibroTest scores of 125 patients with hepatitis C. Of these, 57 had FibroTest scores either less than 0.1 (indicating no fibrosis) or greater than 0.6 (indicating significant fibrosis). Although 33 of the 125 patients had FibroTest scores <0.1 and were therefore deemed unlikely to have fibrosis, 6 (18%) had significant fibrosis. Conversely, of the 24 patients with scores >0.6 who were likely to have significant fibrosis, 5 (21%) had mild fibrosis. The investigators concluded that 57 (46%) could have avoided liver biopsy, but discrepant results were recorded in 11 of those 57 (19%). In other words, discrepancies with the biopsy gold standard were found in one-fifth of patients. There FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 32

are no prospective clinical outcome studies of the HCV-FibroSure in the management of patients with hepatitis C or other chronic liver diseases. Poynard et al. (2004), by reviewing a total of 16 publications, sought to summarize the diagnostic values of FibroTest (FT) and ActiTest (AT) from the scientific literature, to respond to frequently asked questions by performing original new analyses (including the range of diagnostic values, a comparison with other markers, the impact of genotype and viral load, and the diagnostic value in intermediate levels of injury), and to develop a system of conversion between the biochemical and biopsy estimates of liver injury. They found that the areas under the receiver operating characteristics curves (AUROC) ranged from 0.73 to 0.87. For the diagnosis of significant histological activity, the AUROCs ranged from 0.75 to 0.86. At a cut off of 0.31, the FT negative predictive value for excluding significant fibrosis was 91%. At a cut off of 0.36, the ActiTest negative predictive value for excluding significant necrosis was 85%. The AUROCs of FT-AT for consecutive stages of fibrosis and grades of necrosis were the same for both moderate and extreme stages and grades. Based on these results, they recommended that the FibroTest and ActiTest can be used as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C, and that in clinical practice, liver biopsy should be recommended only as a second line test, i.e., in case of high risk of error of biochemical tests. Patel et al (2004) have reported the results of thier study assessing the utility of FibroSpect II in a cohort of 244 patients with hepatitis C-related liver disease. They found that this test had a 71% to 87% positive predictive value for differentiating mild fibrosis from more severe disease. Again, these findings are consistent with the general tenet that these tests are able to differentiate advanced liver disease from minimal disease, but highlight the concept that they lack the ability to discriminate among intermediate degrees of fibrosis, thus, indeterminate values become problematic in clinical practice. During the Digestive Disease Week meeting (May 15-20, 2004) Friedman emphasized the evolution of proteomics in this field, suggesting that fingerprints of the protein composition in the blood may be able to accurately reflect the fibrogenic activity of the liver. Recent work in this area has suggested the feasibility of this approach. This point was considerably strengthened by Hui et al (2004) who assessed the utility of a novel non-electrophoresis-based proteomic technology, SELDI-TOF MS (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry), in predicting different degrees of liver fibrosis in chronic hepatitis B. The results were striking in that they were able to identify 30 proteomic profiles that were significantly associated with fibrosis in patients with hepatitis B. When these investigators divided subjects into those with moderate/severe fibrosis and those with cirrhosis, they were able to demonstrate that the method had a sensitivity and specificity of greater than 90% for prediction of either moderate/severe fibrosis or cirrhosis. These data suggest that this methodology may be extremely useful in the future for more precise assessment of fibrosis risk. At present, methods to accurately assess fibrosis noninvasively are in evolution, and although controversy about their usefulness exists, it is anticipated that, as the methods become more refined, such noninvasive measurement of fibrosis will be readily feasible. For the moment, however, liver biopsy continues to be the gold standard, whereas, the use of the biochemical markers of liver fibrosis must remain only as second line tests in those patients deemed at high risk for liver biopsy. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 33

The National Institutes of Health Consensus Statement on Management of Hepatitis C (NIH, 2002) concluded that: liver biopsy is useful in defining baseline abnormalities of liver disease and in enabling patients and healthcare providers to reach a decision regarding antiviral therapy.noninvasive tests are not adequate substitutes for liver biopsy, specific serum markers of fibrosis and inflammation are not currently widely available or well validated. No single test or panel of serologic markers can provide an accurate assessment of intermediate stages of hepatic fibrosis. Similarly, quantitative tests of liver function and radiologic imaging of the liver are sensitive for diagnosing advanced cirrhosis but are not useful in assessing hepatic fibrosis and early cirrhosis. Review History Medical Advisory Council January 11, 2005 April 2006 Update - no changes March 2007 Update - no changes March 2008 Update no revisions March 2009 Update no revisions. Codes Reviewed. March 2010 Update no revisions March 2011 Update. Added Medicare Table. No revisions. December 2011 Update - no revisions. December 2012 Update no revisions December 2013 Update Added ASH FibroSure and NASH FibroSure as investigational as serum markers for hepatic fibrosis. Added transient elastography, (i.e. FibroScan) as investigational. Codes updated April 2014 Update. Added magnetic resonance elastography as investigational since there is a paucity of peer reviewed literature to support this. Codes updated. August 2014 Revised policy statement to consider various tests (FIbroSure/Fibrospect, ActiTest, Hepascore, APRI, FibroScan, ARFI) as medically necessary August 2015 Update no revisions This policy is based on the following evidence-based guidelines: 1. National Institutes of Health (NIH). Management of hepatitis C: 2002. Rockville, MD: National Institutes of Health (NIH); August 26, 2002. 2. Dienstag JL, McHutchison JG. American Gastroenterological Association technical review on the management of hepatitis C. 2006 Jan;130(1):231-64. 3. Strader DB, Wright T, Thomas DL, Seeff LB. American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004 Apr;39(4):1147 4. The National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. Chronic Hepatitis C: Current Disease Management. NIH Publication: 97-4230. 5. Ghany MG, Strader DB, Thomas DL, Seeff LB, American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009 Apr;49(4):1335-74. Available at: FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 34

http://www.aasld.org/practiceguidelines/documents/bookmarked%20practice% 20Guidelines/Diagnosis_of_HEP_C_Update.Aug%20_09pdf.pdf 6. Hayes. Health Technology Brief. FIBROSpect II Test (Prometheus Laboratories Inc.) for Diagnosis of Liver Fibrosis in Patients with Hepatitis C. 2010. Updated March 23, 2012. Archived April 26, 2013. 7. National Cancer Comprehensive Network (NCCN). Hepatobiliary Cancer. Version 2.2012. Updated Version 2.2013. Update 2.2015 8. Chalasani, N, Younossi, Z, Lavine, JE, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology. 2012 Jun;142(7):1592-609. 9. American Association for the Study of Liver Disease (AASLD), Infectious Disease Society of America (IDSA). Recommendations for Testing, Managing, and Treating Hepatitis C. March 12, 2014. Available at: http://www.hcvguidelines.org/full-report-view 10. CDC. Viral Hepatitis Resource Center: 2011 HCV Symposium. U.S. Department of Health and Human Services, CDC; 2011. Available at: http://www.cdc.gov/hepatitis/resources/mtgsconf/hcvsymposium2011.htm. 11. CDC. Viral hepatitis surveillance, United States, 2009 2011. U.S. Department of Health and Human Services, CDC; 2012. Available at: http://www.cdc.gov/hepatitis/statistics/2010surveillance/index.htm. 12. Centers for Disease Control & Prevention (CDC). Testing Recommendations for Chronic Hepatitis C Virus Infection. Guidelines and Recommendations. Testing for HCV infection: An update of guidance for clinicians and laboratorians. MMWR. May 7, 2013; Vol. 62. Available at: http://www.cdc.gov/mmwr/pdf/wk/mm62e0507a2.pdf 13. Centers for Disease Control & Prevention (CDC). Morbidity and Mortality Weekly Report (MMWR). Testing for HCV Infection: An Update of Guidance for Clinicians and Laboratorians. Weekly. May 10, 2013 / 62(18);362-365. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6218a5.htm 14. Ferraioli G, Tinelli C, dal Bello B, et al. Performance of liver stiffness measurements by transient elastography in chronic hepatitis. World Journal of Gastroenterology. 19 (1) (pp 49-56), 2013. 15. Hayes. Search & Summary. Ultrasound Transient Elastography (FibroScan; Echosens SA) for Detection of Hepatic Fibrosis in Patients with Hepatitis C. December 20, 2013. 16. Hayes. Medical Technology Directory. Magnetic Resonance Elastography for Detecting and Staging Liver Fibrosis. January 9, 2013. Updated January 24, 2014. Update Jan 2015 17. National Cancer Comprehensive Network (NCCN). NCCN Clinical Practice Guidelines in Oncology. Hepatobiliary Cancers. Version 2. 2014. 18. Soriano V, Puoti M, Sulkowski M, et al. Care of patients coinfected with HIV and hepatitis C virus: 2007 updated recommendations from the HCV-HIV International Panel. AIDS. 2007;21(9):1073-1089. 19. Hayes Medical Technology Directory. Ultrasound Transient Elastography for Detecting Hepatic Fibrosis in Patients with Hepatitis C. December 2014 References Update August 2015 1. Al-Hamoudi WK, Abdelrahman AA, Helmy A, et al. The role of Fibroscan in predicting the presence of varices in patients with cirrhosis. Eur J Gastroenterol Hepatol. 2015 Jul 16. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 35

2. Batheja M, Vargas H, Silva AM, et al. Magnetic resonance elastography (MRE) in assessing hepatic fibrosis: performance in a cohort of patients with histological data. Abdom Imaging. 2015 Apr;40(4):760-5. 3. Castera L. Noninvasive Assessment of Liver Fibrosis. Dig Dis. 2015;33(4):498-503. 4. Chrostek L, Panasiuk A. Liver fibrosis markers in alcoholic liver disease. World J Gastroenterol. 2014 Jul 7;20(25):8018-23. 5. Fernandez M, Trépo E, Degré D, et al. Transient elastography using Fibroscan is the most reliable noninvasive method for the diagnosis of advanced fibrosis and cirrhosis in alcoholic liver disease. Eur J Gastroenterol Hepatol. 2015 May 18. 6. Fitzpatrick E, Dhawan A. Noninvasive biomarkers in non-alcoholic fatty liver disease: current status and a glimpse of the future. World J Gastroenterol. 2014 Aug 21;20(31):10851-63. 7. Huet N, Denis I, Martino Aet al. Ultrasonographic assessment of liver fibrosis with computer-assisted analysis of liver surface irregularities. Diagn Interv Imaging. 2015 Apr 23. 8. Lignon G, Boursier J, Delumeau S, et al. Screening for significant chronic liver disease by using three simple ultrasound parameters. Eur J Radiol. 2015 Aug;84(8):1466-72. 9. Lynch M, Higgins E, McCormick PA, et al. The use of transient elastography and FibroTest for monitoring hepatotoxicity in patients receiving methotrexate for psoriasis. JAMA Dermatol. 2014 Aug;150(8):856-62 10. Ozyalvacli G, Kucukbayrak A, Kurt M, et al. Non-invasive fibrosis tests are correlated with necroinflammatory actvity of liver in patients with chronic hepatitis B. Clin Ter. 2014;165(3):e199-204. 11. Patel K, Remlinger KS, Walker TG, et al. Multiplex protein analysis to determine fibrosis stage and progression in patients with chronic hepatitis C. Clin Gastroenterol Hepatol. 2014 Dec;12(12):2113-20. 12. Poynard T, Vergniol J, Ngo Y, et al. Staging chronic hepatitis B into seven categories, defining inactive carriers and assessing treatment impact using a fibrosis biomarker (FibroTest) and elastography (FibroScan). J Hepatol. 2014 Nov;61(5):994-1003. 13. Salkic NN, Jovanovic P, Hauser G, Brcic M. FibroTest/Fibrosure for significant liver fibrosis and cirrhosis in chronic hepatitis B: a meta-analysis. Am J Gastroenterol. 2014 Jun;109(6):796-809. 14. Shiraishi A, Hiraoka A, Aibiki T, et al. Real-time tissue elastography: noninvasive evaluation of liver fibrosis in chronic liver disease due to HCV. Hepatogastroenterology. 2014 Oct;61(135):2084-90. 15. Zaleska-Dorobisz U, Pawluś A, Kucharska M, Inglot M. SWE elastography in assessment of liver fibrosis. Postepy Hig Med Dosw (Online). 2015 Feb 15;69:221-6. 16. Zeremski M, Dimova RB, Benjamin S, et al. FibroSURE as a noninvasive marker of liver fibrosis and inflammation in chronic hepatitis B. BMC Gastroenterol. 2014 Jul 3;14:118. References Update August 2014 1. Rizzo L et al. Comparison of transient elastography (TE) and acoustic radiation force impulse (ARFI) for non-invasive staging of liver fibrosis in patients with chronic hepatitis C. Am J Gastroenterol 2011;106:2112 20 2. Curry, M.P., Afdhal, N.H, (2014) Tests used for the noninvasive assessment of hepatic fibrosis. UptoDate 3. Manning DS, Afdhal NH. Diagnosis and quantitation of fibrosis. Gastroenterology 2008;134:1670 1681. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 36

4. Lai, M., MD, MPH, Afdhal, N.H., MD. Staging Liver Fibrosis in Hepatitis C: A Challenge for This Decade. Am J Gastroenterology Vol 106 Dec 2011 5. Sebastiani G, Halfon P, Castera L, et al. SAFE biopsy: a validated method for large-scale staging of liver fibrosis in chronic hepatitis C. Hepatology 2009; 49:1821 1827. 6. Chronic Hepatitis C Virus (HCV) Infection: Treatment Considerations from the Department of Veterans Affairs. National Hepatitis C Resource Center Program and the Office of Public Health. (March 27, 2014; data last reviewed on March 6, 2014; last revised on May 13, 2014) accessed August 3, 2014 at http://www.hepatitis.va.gov/pdf/2014hcv.pdf 7. American Association for the Study of Liver Disease/Infectious Disease Society of America (ASLD/IDSA/IAS USA). Recommendations for testing, managing, and treating hepatitis C. http://www.hcvguidelines.org. Accessed August 3, 2014. 8. Castéra L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128:343. 9. Ratziu V. Serum fibrosis markers: death by validation or a leap of faith? J Hepatol 2010;53:222-224. References from the AASLD (http://www.hcvguidelines.org/fullreport/when-and-whom-initiate-hcv-therapy) August 2014 1. Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology. 2003;38(6):1449-1457. 2. Boursier J, de L, V, Zarski JP, et al. Comparison of eight diagnostic algorithms for liver fibrosis in hepatitis C: new algorithms are more precise and entirely noninvasive. Hepatology. 2012;55(1):58-67. 3. Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology. 2011;53(3):1020-1022. 4. Castera L, Sebastiani G, Le BB, de L, V, Couzigou P, Alberti A. Prospective comparison of two algorithms combining non-invasive methods for staging liver fibrosis in chronic hepatitis C. J Hepatol. 2010;52(2):191-198. 5. Chou R, Wasson N. Blood tests to diagnose fibrosis or cirrhosis in patients with chronic hepatitis C virus infection. Ann Intern Med. 2013b;159(5):372. 6. Chou R, Wasson N. Blood tests to diagnose fibrosis or cirrhosis in patients with chronic hepatitis C virus infection: a systematic review. Ann Intern Med. 2013;158(11):807-820. 7. Everhart JE, Wright EC, Goodman ZD, et al. Prognostic value of Ishak fibrosis stage: findings from the hepatitis C antiviral long-term treatment against cirrhosis trial. Hepatology. 2010;51(2):585-594. 8. Garcia-Tsao G, Sanyal AJ, Grace ND, Carey W. Prevention and management of gastroesophageal varices and variceal hemorrhage in cirrhosis. Hepatology. 2007;46(3):922-938. 9. Sebastiani G, Halfon P, Castera L, et al. SAFE biopsy: a validated method for large-scale staging of liver fibrosis in chronic hepatitis C. Hepatology. 2009;49(6):1821-1827. 10. Selph S. and Chou R. Impact of contacting study authors on systematic review conclusions: diagnostic tests for hepatic fibrosis. http://www.ncbi.nlm.nih.gov/books/nbk198806/. Accessed on July 11, 2014 by the AALSD 11. Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C. Hepatology. 2005;41(1):48-54. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 37

References Update April 2014 1. Adebajo CO, Talwalkar JA, Poterucha JJ, et al. Ultrasound-based transient elastography for the detection of hepatic fibrosis in patients with recurrent hepatitis C virus after liver transplantation: a systematic review and metaanalysis. Liver Transpl. 18 (3) (pp 323-331), 2012. 2. Bachir T. Detection of Liver Fibrosis With Magnetic Resonance Imaging (MRI). National Library of Medicine (NLM) Identifier: NCT01600105. Updated July 17, 2012. ClinicalTrials.gov. Available at: http://clinicaltrials.gov/ct2/show/nct01600105 3. Backus LI, Boothroyd DB, Phillips BR, et al. A sustained virologic response reduces risk of all-cause mortality in patients with hepatitis C. Clin Gastroenterol Hepatol 2011;9:509 16. 4. Bensamoun SF, Leclerc GE, Debernard L, et al. Cutoff values for alcoholic liver fibrosis using magnetic resonance elastography technique. Alcohol Clin Exp Res. 2012. Epub ahead of print. December 6, 2012. 5. Bota S, Herkner H, Sporea I, et al. Meta-analysis: ARFI elastography versus transient elastography for the evaluation of liver fibrosis. Liver International. 33 (8) (pp 1138-1147), 2013. 6. Castera L, Winnock M, Pambrun E, et al. Comparison of transient elastography (FibroScan), FibroTest, APRI and two algorithms combining these non-invasive tests for liver fibrosis staging in HIV/HCV coinfected patients: ANRS CO13 HEPAVIH and FIBROSTIC collaboration. HIV Medicine. 15 (1) (pp 30-39), 2014. 7. Castera L. Non-invasive assessment of liver fibrosis in chronic hepatitis C. Hepatol Int. Jun 2011; 5(2): 625 634. 8. Colombo M. Prevention of hepatocellular carcinoma and recommendations for surveillance in adults with chronic liver disease. UpToDate. February 21. 2014. 9. Cooper C, Lester R, Thorlund K, et al. Direct-acting antiviral therapies for hepatitis C genotype 1 infection: a multiple treatment comparison metaanalysis. QJM 2013;106:153 63. 10. D'Ambrosio R, Aghemo A, Fraquelli M, et al. The diagnostic accuracy of Fibroscan for cirrhosis is influenced by liver morphometry in HCV patients with a sustained virological response. Journal of Hepatology. 59 (2) (pp 251-256), 2013. 11. Eramova I, Matic S, Munz M, et al. World Health Organization (WHO). Regional Office for Europe. Management of hepatitis C and HIV coinfection. Clinical protocol for the WHO European Region. Protocol 6. In: HIV/AIDS Treatment and Care. Clinical Protocols for the WHO European Region. Copenhagen: World Health Organization Europe; 2007;226-272. 12. Friedrich-Rust M, Ong MF, Martens S, et al. Performance of transient elastography for the staging of liver fibrosis: a meta-analysis. Gastroenterology. 2008;134:960 974. 13. Gale HB, Dufour DR, Qazi NN, Kan VL. Comparison of serial hepatitis C virus detection in samples submitted through serology for reflex confirmation versus samples directly submitted for quantitation. J Clin Microbiol 2011;49:3036 9. 14. Han SH, Kim SU, Kim CO, et al. Abnormal Liver Stiffness Assessed Using Transient Elastography (Fibroscan) in HIV-Infected Patients without HBV/HCV Coinfection Receiving Combined Antiretroviral Treatment. PLoS ONE. 8 (1), 2013. Article Number: e52720. 15. Lee CK, Perez-Atayde AR, Mitchell PD, et al. Serum biomarkers and transient elastography as predictors of advanced liver fibrosis in a United States cohort: The Boston Children's Hospital experience. J Pediatr. 2013 Jun 4. 16. Morgan RL, Baack B, Smith BD, et al. Eradication of hepatitis C virus infection and the development of hepatocellular carcinoma. A meta-analysis of observational studies. Ann Intern Med 2013;158:329 37. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 38

17. Paar D. Incorporation of the AST platelet ratio index (APRI) into the HCV evaluation and treatment pathway in the Texas Department of Criminal Justice (TDCJ). Infectious Diseases in Corrections Report (IDCR). 2007;9(19). 18. Platon ML, Stefanescu H, Feier D, et al. Performance of unidimensional transient elastography in staging chronic hepatitis C. results from a cohort of 1,202 biopsied patients from one single center. Journal of Gastrointestinal and Liver Diseases. 22 (2) (pp 157-166), 2013. 19. Poordad F, Dieterich D. Treating hepatitis C: current standard of care and emerging direct-acting antiviral agents. J Viral Hepat 2012;19:449 64. 20. Sheth SG, Chopra S. Epidemiology, clinical features, and diagnosis of nonalcoholic steatohepatitis. Last reviewed June 2012. UpToDate 21. Ramirez R, Valero S, Escrich C, et al. A cross-sectional epidemiological study of chronic HCV liver disease stage in HIV-coinfected patients using noninvasive techniques: Co Trans- GEENI Study. Curr HIV Res. 10 (8) (pp 688-693), 2012. 22. Shivkumar S, Peeling R, Jafari Y, et al. Accuracy of rapid and point-of-care screening tests for hepatitis C: a systematic review and meta-analysis. Ann Intern Med 2012;157:558 66. 23. Scottish Intercollegiate Guidelines Network (SIGN). Management of hepatitis C. A national clinical guideline. Edinburgh (Scotland): Scottish Intercollegiate Guidelines Network. December 2006. 24. Shaheen AA, Wan AF, Myers RP. FibroTest and FibroScan for the prediction of hepatitis C-related fibrosis: a systematic review of diagnostic test accuracy. Am J Gastroenterol. 2007;102:2589 2600. 25. Singh S, Fujii LL, Murad MH, et al. Liver stiffness is associated with risk of decompensation, liver cancer, and death in patients with chronic liver diseases: a systematic review and meta-analysis. Clin Gastroenterol Hepatol. 2013;11(12):1573. 26. Talwalkar JA, Kurtz DM, Schoenleber SJ, et al. Ultrasound-based transient elastography for the detection of hepatic fibrosis: systematic review and metaanalysis. Clin Gastroenterol Hepatol. 2007;5:1214 1220. References Update December 2013 1. Barbero-Villares A, Mendoza Jimenez-Ridruejo J, Taxonera C, et al. Madrid Group for the Study of Inflammatory Bowel Disease ENICMAD. Evaluation of liver fibrosis by transient elastography (Fibroscan) in patients with inflammatory bowel disease treated with methotrexate: A multicentric trial. Scand J Gastroenterol. 2012;47(5):575-579. 2. Cassinotto C, Lapuyade B, Aït-Ali A, et al. Liver fibrosis: noninvasive assessment with acoustic radiation force impulse elastography, comparison with FibroScan M and XL probes and FibroTest in patients with chronic liver disease. Radiology 2013; 269:283. 3. Nguyen, NH, Nguyen, V, Trinh, HN, et al. Treatment eligibility of patients with chronic hepatitis B initially ineligible for therapy. Clin Gastroenterol Hepatol. 2013 May;11(5):565-71. 4. Poynard T, de Ledinghen V, Zarski JP, et al. Performances of Elasto- FibroTest((R)), a combination between FibroTest((R)) and liver stiffness measurements for assessing the stage of liver fibrosis in patients with chronic hepatitis C. Clinics and research in hepatology and gastroenterology. 2012 Oct;36(5):455-63. 5. Sheth SG, Chopra S. Epidemiology, clinical features, and diagnosis of nonalcoholic steatohepatitis. UpToDate. June 2012. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 39

6. Pesce A, Scilletta R, Branca A, et al. Does transient elastography (FibroScan ) have a role in decision making in hepatocellular carcinoma? HPB (Oxford). 2012;14(6):403-408. 7. Zarski JP, Sturm N, Guechot J, et al. Comparison of nine blood tests and transient elastography for liver fibrosis in chronic hepatitis C: the ANRS HCEP-23 study. J Hepatol 2012; 56:55. References Update December 2012 1. Adebajo CO, Talwalkar JA, Poterucha JJ, et al. Ultrasound-based transient elastography for the detection of hepatic fibrosis in patients with recurrent hepatitis C virus after liver transplantation: a systematic review and metaanalysis. Liver Transpl 2012; 18:323. 2. Clinicaltrials.gov. Antifibrotic Activity Of GI262570 In Chronic Hepatitis C Subjects. ClinicalTrials.gov Identifier: NCT00244751. November 2009. Available at: http://www.clinicaltrials.gov/ct2/show/nct00244751?term=actitest&rank=2 3. Crespo G, Fernández-Varo G, Mariño Z, et al. ARFI, FibroScan, ELF, and their combinations in the assessment of liver fibrosis: a prospective study. J Hepatol 2012; 57:281. 4. Myers RP, Pomier-Layrargues G, Kirsch R, et al. Discordance in fibrosis staging between liver biopsy and transient elastography using the FibroScan XL probe. J Hepatol 2012; 56:564. 5. Prometheus Therapeutic and Diagnositcs. PROMETHEUS FIBROSpect II. 2012. 6. Usluer G, Erben N, Aykin N, et al. Comparison of non-invasive fibrosis markers and classical liver biopsy in chronic hepatitis C. Eur J Clin Microbiol Infect Dis. 2012 Aug;31(8):1873-8. References Update December 2011 1. Cholongitas E, Tsochatzis E, Goulis J, Burroughs AK. Noninvasive tests for evaluation of fibrosis in HCV recurrence after liver transplantation: A systematic review. Transpl Int. 2010;23(9):861-870. 2. Costa JM, Telehin D, Munteanu M, et al. HCV-GenoFibrotest: a combination of viral, liver and genomic (IL28b, ITPA, UGT1A1) biomarkers for predicting treatment response in patients with chronic hepatitis C. Clin Res Hepatol Gastroenterol. 2011 Mar;35(3):204-13. Epub 2011 Feb 26. 3. Curry MP, Afdhal NH. Noninvasive assessment of hepatic fibrosis. UpToDate. July 29, 2011. Updated October 2, 2012. Updated October 18, 2013. Updated December 13, 2013. 4. Degos F, Perez P, Roche B, et al. Diagnostic accuracy of FibroScan and comparison to liver fibrosis biomarkers in chronic viral hepatitis: A multicenter prospective study (the FIBROSTIC study). J Hepatol. 2010;53(6):1013-1021. 5. Feldman: Sleisenger and Fordtran's Gastrointestinal and Liver Disease, 9th ed. 2010 Saunders, An Imprint of Elsevier. Tests to Detect Hepatic Fibrosis. 6. Martinez SM, Fernández-Varo G, González P, et al. Assessment of liver fibrosis before and after antiviral therapy by different serum marker panels in patients with chronic hepatitis C. Aliment Pharmacol Ther. 2011 Jan;33(1):138-48 7. McPherson: Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed., 2011 Saunders, An Imprint of Elsevier. Diagnosis of Liver Diseases. 8. Myers RP, Elkashab M, Ma M, Transient elastography for the noninvasive assessment of liver fibrosis: A multicentre Canadian study. Can J Gastroenterol. 2010;24(11):661-670. 9. Terrault NA, Chopra S. Screening for and diagnostic approach to hepatitis C virus infection. UpToDate. January 18, 2011. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 40

10. Thabut D, Moreau R, Lebrec D. Noninvasive assessment of portal hypertension in patients with cirrhosis. Hepatology. 2011;53(2):683-694. References Update March 2011 1. Zhang YG, Wang BE, Wang TL, et al. Assessment of hepatic fibrosis by transient elastography in patients with chronic hepatitis B. Pathol Int. 2010;60(4):284-290. 2. Stebbing J, Farouk L, Panos G, et al. A meta-analysis of transient elastography for the detection of hepatic fibrosis. J Clin Gastroenterol. 2010;44(3):214-219. 3. ClinicalTrials.gov. Search for Clinical Trials Database: fibrospect. 2010. Available at: http://www.clinicaltrials.gov. 4. Patel K. An independent and prospective comparison of two commercial fibrosis marker panels (HCV FibroSURE and FIBROSpect II) during albinterferon alfa-2b combination therapy for chronic hepatitis C. J Viral Hepat. 01-MAR-2009; 16(3): 178-86. 5. Uyar C. Comparison of FibroTest-ActiTest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis B and C. Indian J Pathol Microbiol. 01-JUL-2010; 53(3): 470-5. References Update March 2010 1. Becker L, Salameh W, Sferruzza A, et al. Validation of hepascore, compared with simple indices of fibrosis, in patients with chronic hepatitis C virus infection in United States. Clin Gastroenterol Hepatol. 2009 Jun;7(6):696-701. 2. Fontanges T, Bailly F, Trepo E, et al. Discordance between biochemical markers of liver activity and fibrosis (Actitest-Fibrotest) and liver biopsy in patients with chronic hepatitis C. Gastroenterol Clin Biol. 2008 Sep 3 3. Gressner OA, Beer N, Jodlowski A, Gressner AM. Impact of quality control accepted inter-laboratory variations on calculated Fibrotest/Actitest scores for the non-invasive biochemical assessment of liver fibrosis. Clin Chim Acta. 2009 Nov;409(1-2):90-5. 4. Guéchot J, Lasnier E, Sturm N, et al. Automation of the Hepascore and validation as a biochemical index of liver fibrosis in patients with chronic hepatitis C from the ANRS HC EP 23 Fibrostar cohort. Clin Chim Acta. 2010 Jan;411(1-2):86-91 5. Halfon P, Munteanu M, Poynard T. FibroTest-ActiTest as a non-invasive marker of liver fibrosis. Gastroenterol Clin Biol. 2008 Sep;32(6 Suppl 1):22-39. 6. Guajardo-Salinas GE, Hilmy A. Prevalence of Nonalcoholic Fatty Liver Disease (NAFLD) and Utility of FIBROspect II to Detect Liver Fibrosis in Morbidly Obese Hispano-American Patients Undergoing Gastric Bypass. Obes Surg. 2009 Dec 3. 7. Menter A. An independent and prospective comparison of two commercial fibrosis marker panels (HCV FibroSURE and FIBROSpect II) during albinterferon alfa-2b combination therapy for chronic hepatitis C. - J Am Acad Dermatol - 01- SEP-2009; 61(3): 451-85 8. Smith JO, Sterling RK. Systematic review: non-invasive methods of fibrosis analysis in chronic hepatitis C. Aliment Pharmacol Ther. 2009 Sep 15;30(6):557-76 9. Wu SD, Wang JY, Li L. Staging of liver fibrosis in chronic hepatitis B patients with a composite predictive model: a comparative study. World J Gastroenterol. 2010 Jan 28;16(4):501-7. References Update March 2009 FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 41

1. Shaheen AA, Myers RP. Systematic review and meta-analysis of the diagnostic accuracy of fibrosis marker panels in patients with HIV/hepatitis C coinfection. HIV Clin Trials. 2008;9(1):43-51. 2. Sebastiani G, Vario A, Guido M et al. Performance of noninvasive markers for liver fibrosis is reduced in chronic hepatitis C with normal transaminases. J Viral Hepat 2008; 15(3):212-8. 3. Patel K, Nelson DR, Rockey DC et al. Correlation of FIBROSpect II with histologic and morphometric evaluation of liver fibrosis in chronic hepatitis C. Clin Gastroenterol Hepatol 2008; 6(2):242-7. 4. Mehta P, Ploutz-Snyder R, Nandi J et al. Diagnostic accuracy of serum hyaluronic acid, FIBROSpect-II, and YKL-40 for discriminating fibrosis stages in chronic hepatitis C. Am J Gastroenterol 2008; 103(4):928-36. 5. Kotlyar DS, Blonski W, Vinod K. et al. Noninvasive Monitoring of Hepatitis C Fibrosis Progression. Clinics in Liver Disease - Volume 12, Issue 3 (August 2008). 6. Re VL, Kostman JR, Amorosa VK, et al. Management Complexities of HIV/Hepatitis C Virus Coinfection in the Twenty-First Century. Clinics in Liver Disease - Volume 12, Issue 3 (August 2008). References Update March 2008 1. Patel K, Nelson DR, Rockey DC, et al. Correlation of FIBROSpect II with histologic and morphometric evaluation of liver fibrosis in chronic hepatitis C. 2008 Feb; 6(2): 242-7. Epub 2008 Jan 9. 2. Nourani S, Pockros PJ. How should hepatitis C be managed in patients aged 65 years and older? Nature Clin Practice Gastroenterol Hepatol. 2007;4:22-23. 3. Rossi E, Adams LA, Bulsara M, Jeffrey GP. Assessing liver fibrosis with serum marker models. Clin Biochem Rev. 2007; 28(1): 3 10. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1904421. 4. de Franchis R, Dell'Era A. Non-invasive diagnosis of cirrhosis and the natural history of its complications. Best Pract Res Clin Gastroenterol. 2007;21(1):3-18. 5. Zaman A, Rosen H, Ingram K, et al. Assessment of FIBROSpect II to Detect Hepatic Fibrosis in Chronic Hepatitis C Patients. The American Journal of Medicine - Volume 120, Issue 3 (March 2007). 6. Berrutti M, Ciancio A, Smedile A, et al. Assessment of liver fibrosis in the clinical setting: Something is changing? Minerva Gastroenterol Dietol. 2007;53(1):111-114. 7. Wilson LE, Torbenson M, Astemborski J, et al. Progression of liver fibrosis among injection drug users with chronic hepatitis C. Hepatology. 43(4): 788-95, 2006 Apr. 8. Rockey DC, Bissell DM. Hepatology. 2006 Jun;43(6):1401-2. Noninvasive measures of liver fibrosis. Hepatology. 2006 Feb;43(2 Suppl 1):S113-20. References Update March 2007 1. Christensen C, Bruden D, Livingston S, et al. Diagnostic accuracy of a fibrosis serum panel (FIBROSpect II) compared with Knodell and Ishak liver biopsy scores in chronic hepatitis C patients. J Viral Hepat. 2006 Oct;13(10):652-8. 2. Ngo Y, Munteanu M, Messous D, et al. A prospective analysis of the prognostic value of biomarkers (FibroTest) in patients with chronic hepatitis C. Clin Chem. 2006 Oct;52(10):1887-96. 3. Thuluvath PJ, Krok KL. Noninvasive markers of fibrosis for longitudinal assessment of fibrosis in chronic liver disease: are they ready for prime time? Am J Gastroenterol. 2006 Jul;101(7):1497-9. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 42

4. Halfon P, Bourliere M, Penaranda G, Cacoub P. Serum markers of non-invasive fibrosis in chronic hepatitis C virus infection. Rev Med Interne. 2006 Oct;27(10):751-61. 5. Sene D, Limal N, Messous D, et al. Biological markers of liver fibrosis and activity as non-invasive alternatives to liver biopsy in patients with chronic hepatitis C and associated mixed cryoglobulinemia vasculitis. Clin Biochem. 2006 Jul;39(7):715-21. 6. Leroy V, Hilleret MN, Sturm N, et al. Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C. J Hepatol. 2007 Jan 26. 7. Sebastiani G, Vario A, Guido M, Alberti A. Sequential algorithms combining noninvasive markers and biopsy for the assessment of liver fibrosis in chronic hepatitis B. World J Gastroenterol. 2007 Jan 28;13(4):525-31. 8. Bourliere M, Penaranda G, Renou C, et al. Validation and comparison of indexes for fibrosis and cirrhosis prediction in chronic hepatitis C patients: proposal for a pragmatic approach classification without liver biopsies. J Viral Hepat. 2006 Oct;13(10):659-70. 9. Thabut D, Le Calvez S, Thibault V, et al. Hepatitis C in 6,865 patients 65 yr or older: a severe and neglected curable disease? Am J Gastroenterol. 2006 Jun;101(6):1260-7. 10. Halfon P, Bourliere M, Deydier R, et al. Independent prospective multicenter validation of biochemical markers (fibrotest-actitest) for the prediction of liver fibrosis and activity in patients with chronic hepatitis C: the fibropaca study. Am J Gastroenterol. 2006 Mar;101(3):547-55. References - Initial 1. Munteanu M, Messous D, Thabut D, et al. Intra-individual fasting versus postprandial variation of biochemical markers of liver fibrosis (FibroTest) and activity (ActiTest). Comp Hepatol. 2004. 2. Poynard T, Munteanu M, Imbert-Bismut F, et al. Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem. 2004 Jun 10. 3. Imbert-Bismut F, Messous D, Thibaut V, et al. Intra-laboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors. Clin Chem Lab Med. 2004;42(3):323-333. 4. Le Calvez S, Thabut D, Messous D, et al. The predictive value of Fibrotest vs. APRI for the diagnosis of fibrosis in chronic hepatitis C. Hepatology. 2004;39(3):862-863; author reply 863. 5. Patel K, Nelson DR, Afdahl N, et al. Prospective evaluation of a serum (FibroSpect II) panel to predict fibrosis in chronic HCV Patients. Gastroenterology. 2004;126 (suppl 2):A-708. [S1645] 6. Friedman SL. Prognosticating in the future: Genomics and beyond. In: AGA Clinical Symposium -- Prognostic Markers of Liver Disease. Program and abstracts of Digestive Disease Week 2004; May 15-20, 2004; New Orleans, Louisiana. 7. Hui AY, Poon TCW, Chan HLY, et al. Serum proteomic profiling predicts liver fibrosis in chronic hepatitis B. Gastroenterology. 2004;126(suppl 2):A-669. [Abstract 145] 8. Le Calvez S, Thabut D, Messous D, et al. The predictive value of Fibrotest vs. APRI for the diagnosis of fibrosis in chronic hepatitis C. Hepatology 2004, 39:862-863. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 43

9. Munteanu M, Messous D, Thabut D, et al. Intra-individual fasting versus postprandial variation of biochemical markers of liver fibrosis (Fibrotest) and activity (Actitest). Comp Hepatol 2004, 3:3. 10. Callewaert N, Van Vlierberghe H, Van Hecke A, et al. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat Med 2004, 10:429-434. 11. Poynard T, Imbert-Bismut F, Munteanu M, et al. Overview of the diagnostic value of biochemical markers of liver fibrosis (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic hepatitis C. Comp Hepatol. 2004 Sep 23;3(1):8. 12. Poordad FF. FIBROSpect II: a potential noninvasive test to assess hepatic fibrosis. Expert Rev Mol Diagn. 2004 Sep;4(5):593-7. 13. Hui AY, Poon TCW, Chan HLY, et al. Serum proteomic profiling predicts liver fibrosis in chronic hepatitis B. Gastroenterology. 2004;126(suppl 2):A-669. 14. Myers RP, Tainturier MH, Ratziu V, et al. Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B. J Hepatol. 2003;39(2):222-230. 15. Rossi E, Adams L, Prins A, et al. Validation of the FibroTest biochemical markers score in assessing liver fibrosis in hepatitis C patients. Clin Chem. 2003;49(3):450-454. 16. Wai CT, Greenson JK, Fontana RJ, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology. 2003;38:518-526. 17. Afdhal NH: Diagnosing fibrosis in hepatitis C: is the pendulum swinging from biopsy to blood tests? Hepatology 2003, 37:972-974. 18. Colloredo G, Guido M, Sonzogni A, Leandro G: Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease. J Hepatol 2003, 39:239-244. 19. Bedossa P, Dargère D, Paradis V: Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology 2003, 38:1449-1457. 20. Poynard T, McHutchison J, Manns M, et al. Biochemical surrogate markers of liver fibrosis and activity in a randomized trial of peginterferon alfa-2b and ribavirin. Hepatology 2003, 38:481-492. 21. Myers RP, Benhamou Y, Imbert-Bismut F, et al. Serum biochemical markers accurately predict liver fibrosis in HIV and hepatitis C virus-coinfected patients. AIDS 2003, 17:1-5. 22. Myers RP, de Torres M, Imbert-Bismut F, et al. Biochemical markers of fibrosis in patients with chronic hepatitis C: a comparison with prothrombin time, platelet count and the age-platelet index. Dig Dis Sci 2003, 48:146-153. 23. Thabut D, Simon M, Myers RP, et al. Noninvasive prediction of fibrosis in patients with chronic hepatitis C. Hepatology 2003, 37:1220-1221. 24. Rossi E, Adams L, Prins A, et al. Validation of the FibroTest biochemical markers score in assessing liver fibrosis in hepatitis C patients. Clin Chem 2003, 49:450-454. 25. Poynard T, Imbert-Bismut F, Ratziu V, et al. Fibrotest even better than liver biopsy? Clin Chem 2003. [Electronic letter. Response]. 26. Poynard T, Imbert-Bismut F, Ratziu V, et al. An overview of biochemical markers' (Fibrotest-Actitest) diagnostic value in chronic liver diseases: a non-invasive alternative to liver biopsy. Hepatology 2003, 38:559A. 27. Thabut D, Imbert-Bismut F, Cazals-Athem D, et al. Diagnostic value of fibrosis biochemical markers (Fibrotest) for the prediction of portal hypertension in liver disease. Hepatology 2003, 38:282A. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 44

28. Halfon P, Bourliere M, Deydier R, et al. Independent prospective multicenter validation of biochemical markers (Fibrotest-Actitest) for the prediction of liver fibrosis and activity in patients with chronic hepatitis C. Hepatology 2003, 38:188A. 29. Myers RP, Tainturier MH, Ratziu V, et al. Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B. J Hepatol 2003, 39:222-230. 30. Murawaki Y, Ikuta Y, Okamoto K, et al. Diagnostic value of serum markers of connective tissue turnover for predicting histological staging and grading in patients with chronic hepatitis C. J Gastroenterol 2001, 36:399-406. 31. Halfon P, Imbert-Bismut F, Messous D, et al. A prospective assessment of the inter-laboratory variability of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) in patients with chronic liver disease. Comp Hepatol. 2002;1(1):3. 32. Dienstag J: The role of liver biopsy in chronic hepatitis C. Hepatology 2002, 36:S152-S160. 33. Regev A, Berho M, Jeffers LJ, et al. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol 2002, 97:2614-2618. 34. Gebo KA, Herlong HF, Torbenson MS, et al. Role of liver biopsy in management of chronic hepatitis C: A systematic review. Hepatology 2002, 36:S161-S172. 35. Poynard T, Imbert-Bismut F, Ratziu V, et al. Biochemical markers of liver fibrosis in patients infected by Hepatitis C Virus: Longitudinal validation in a randomized trial. J Viral Hepatitis 2002, 9:128-133. 36. Myers RP, Ratziu V, Imbert-Bismut F, et al. Biochemical markers of liver fibrosis: a comparison with historical features in patients with chronic hepatitis C. Am J Gastroenterol 2002, 97:2419-2425. 37. Imbert-Bismut F, Ratziu V, Pieroni L, et al. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet. 2001 Apr 7;357(9262):1069-1075. 38. Bravo AA, Sheth SG, Chopra S: Liver biopsy. N Engl J Med 2001, 344:495-500. 39. Imbert-Bismut F, Ratziu V, Laurence Pieroni L, et al. MULTIVIRC Group: Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 2001, 357:1069-1075. 40. Fortunato G, Castaldo G, Oriani G, et al. Multivariate discriminant function based on six biochemical markers in blood can predict the cirrhotic evolution of chronic hepatitis. Clin Chem 2001, 47:1696-1700. 41. Poynard T, Ratziu V, Bedossa P: Appropriateness of liver biopsy. Can J Gastroenterol 2000, 14:543-548. 42. Ono E, Shiratori Y, Okudaira T, et al. Platelet count reflects stage of chronic hepatitis C. Hepatol Res 1999, 15:192-200. 43. Poynard T, Bedossa P: Age and platelet count: a simple index for predicting the presence of histological lesions in patients with antibodies to hepatitis C virus. METAVIR and CLINIVIR Cooperative Study Groups. J Viral Hepat 1997, 4:199-208. 44. Bedossa P, Poynard T: An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology 1996, 24:289-293. 45. The French METAVIR Cooperative Study Group: Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 1994, 20:15-20. General Purpose. Important Notice FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 45

Health Net's National Medical Policies (the "Policies") are developed to assist Health Net in administering plan benefits and determining whether a particular procedure, drug, service or supply is medically necessary. The Policies are based upon a review of the available clinical information including clinical outcome studies in the peer-reviewed published medical literature, regulatory status of the drug or device, evidence-based guidelines of governmental bodies, and evidence-based guidelines and positions of select national health professional organizations. Coverage determinations are made on a case-by-case basis and are subject to all of the terms, conditions, limitations, and exclusions of the member's contract, including medical necessity requirements. Health Net may use the Policies to determine whether under the facts and circumstances of a particular case, the proposed procedure, drug, service or supply is medically necessary. The conclusion that a procedure, drug, service or supply is medically necessary does not constitute coverage. The member's contract defines which procedure, drug, service or supply is covered, excluded, limited, or subject to dollar caps. The policy provides for clearly written, reasonable and current criteria that have been approved by Health Net s National Medical Advisory Council (MAC). The clinical criteria and medical policies provide guidelines for determining the medical necessity criteria for specific procedures, equipment, and services. In order to be eligible, all services must be medically necessary and otherwise defined in the member's benefits contract as described this "Important Notice" disclaimer. In all cases, final benefit determinations are based on the applicable contract language. To the extent there are any conflicts between medical policy guidelines and applicable contract language, the contract language prevails. Medical policy is not intended to override the policy that defines the member s benefits, nor is it intended to dictate to providers how to practice medicine. Policy Effective Date and Defined Terms. The date of posting is not the effective date of the Policy. The Policy is effective as of the date determined by Health Net. All policies are subject to applicable legal and regulatory mandates and requirements for prior notification. If there is a discrepancy between the policy effective date and legal mandates and regulatory requirements, the requirements of law and regulation shall govern. * In some states, prior notice or posting on the website is required before a policy is deemed effective. For information regarding the effective dates of Policies, contact your provider representative. The Policies do not include definitions. All terms are defined by Health Net. For information regarding the definitions of terms used in the Policies, contact your provider representative. Policy Amendment without Notice. Health Net reserves the right to amend the Policies without notice to providers or Members. In some states, prior notice or website posting is required before an amendment is deemed effective. No Medical Advice. The Policies do not constitute medical advice. Health Net does not provide or recommend treatment to members. Members should consult with their treating physician in connection with diagnosis and treatment decisions. No Authorization or Guarantee of Coverage. The Policies do not constitute authorization or guarantee of coverage of particular procedure, drug, service or supply. Members and providers should refer to the Member contract to determine if exclusions, limitations, and dollar caps apply to a particular procedure, drug, service or supply. Policy Limitation: Member s Contract Controls Coverage Determinations. Statutory Notice to Members: The materials provided to you are guidelines used by this plan to authorize, modify, or deny care for persons with similar illnesses or conditions. Specific care and treatment may vary depending on individual need and the benefits covered under your contract. The determination of coverage for a particular procedure, drug, service or supply is not based upon the Policies, but rather is subject to the facts of the individual clinical case, terms and conditions of the member s contract, and requirements of applicable laws and regulations. The contract language contains specific terms and conditions, including pre-existing conditions, limitations, exclusions, benefit maximums, eligibility, and other relevant terms and conditions of coverage. In the event the Member s contract (also known as the benefit contract, coverage document, or evidence of coverage) conflicts with the Policies, the Member s contract shall govern. The Policies do not replace or amend the Member s contract. Policy Limitation: Legal and Regulatory Mandates and Requirements The determinations of coverage for a particular procedure, drug, service or supply is subject to applicable legal and regulatory mandates and requirements. If there is a discrepancy between the Policies and legal mandates and regulatory requirements, the requirements of law and regulation shall govern. Reconstructive Surgery CA Health and Safety Code 1367.63 requires health care service plans to cover reconstructive surgery. Reconstructive surgery means surgery performed to correct or repair abnormal structures of the body FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 46

caused by congenital defects, developmental abnormalities, trauma, infection, tumors, or disease to do either of the following: (1) To improve function or (2) To create a normal appearance, to the extent possible. Reconstructive surgery does not mean cosmetic surgery," which is surgery performed to alter or reshape normal structures of the body in order to improve appearance. Requests for reconstructive surgery may be denied, if the proposed procedure offers only a minimal improvement in the appearance of the enrollee, in accordance with the standard of care as practiced by physicians specializing in reconstructive surgery. Reconstructive Surgery after Mastectomy California Health and Safety Code 1367.6 requires treatment for breast cancer to cover prosthetic devices or reconstructive surgery to restore and achieve symmetry for the patient incident to a mastectomy. Coverage for prosthetic devices and reconstructive surgery shall be subject to the co-payment, or deductible and coinsurance conditions, that are applicable to the mastectomy and all other terms and conditions applicable to other benefits. "Mastectomy" means the removal of all or part of the breast for medically necessary reasons, as determined by a licensed physician and surgeon. Policy Limitations: Medicare and Medicaid Policies specifically developed to assist Health Net in administering Medicare or Medicaid plan benefits and determining coverage for a particular procedure, drug, service or supply for Medicare or Medicaid members shall not be construed to apply to any other Health Net plans and members. The Policies shall not be interpreted to limit the benefits afforded Medicare and Medicaid members by law and regulation. FIBROSpect, HCV-FIBROSURE, ActiTest Aug 15 47