REVIEWS. Introduction. FibroMax complete diagnosis of liver injury. Abbreviations

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1 REVIEWS Noninvasive Biomarkers for the Screening of Fibrosis, Steatosis and Steatohepatitis in Patients with Metabolic Risk Factors: FibroTest-FibroMa Eperience Mona Munteanu, Vlad Ratziu 2, Rachel Morra, Djamila Messous 3, Francoise Imbert-Bismut 3, Thierry Poynard 2 ) Biopredictive, Paris, France; 2) Hepatology Department, Pitié Salpêtrière Hospital, Paris, France; 3) Biochemistry Department, Pitié Salpêtrière Hospital, Paris, France Introduction Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis occurring in those who do not consume high amounts of alcohol. The prevalence of NAFLD is high in the general population with metabolic risk factors and insulin resistance being mainly associated with overweight, diabetes, hyperlipidemia and hypertension [, 2]. Two histological patterns of NAFLD have been described: fatty liver alone and, in a minority of patients, necroinflammatory form of NAFLD known as non-alcoholic steatohepatitis (NASH) that may progress to cirrhosis and hepatocellular carcinoma []. Although confirmation of the diagnosis of NAFLD can be achieved by imaging methods, they do not provide data on staging the disease (steatohepatitis and fibrosis), which until recently required a liver biopsy for confirmation. Due to the high prevalence of NAFLD in at risk populations [], the limitations of biopsy [3, 4] and the developing of reliable noninvasive blood tests [5-8], liver biopsy should no longer be considered mandatory as first-intention screening of liver lesions [9, 0]. FibroMa complete diagnosis of liver injury Three simple blood tests were developed to provide Abbreviations ALD: alcoholic liver disease; ALT: alanine-aminotrasnferase; AST: aspartate aminotransferase; AUROC: area under the ROC curve; BMI: body mass inde; GGT: gamma-glutamyl transpeptidase; HBV hepatitis B virus; HCV: hepatitis C virus; HIV: human immune-deficiency virus; HOMA-IR: homeostatic model assessment-estimated insulin resistance; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis. J Gastrointestin Liver Dis June 2008 Vol.7 No 2, 87-9 Address for correspondence: Mona Munteanu Groupe Hospitalier Pitié Salpêtrière Service d Hépato-Gastroentérologie Bd de l Hôpital 7503 Paris, France mona.munteanu@biopredictive.com an estimate of liver fibrosis and of its aggravating factors, steatosis and nonalcoholic steatohepatitis (NASH): FibroTest, SteatoTest and NashTest, respectively. FibroMa (Biopredictive, Paris, France) is the association of these three tests on the same result sheet and provides physicians with simultaneous and complete estimation of the liver injury associated with NAFLD []. All FibroMa s tests are patented algorithms owned by the French public health organization (Assistance Publique - Hôpitau de Paris, APHP) and all are provided with instructions for use, securing their interpretation. SteatoTest SteatoTest (score from 0 to, see Table I gives a quantitative estimation of steatosis of different origins: NAFLD (overweight, insulin resistance, hyperlipidemia or diabetes mellitus), chronic viral hepatitis and alcoholic liver disease (ALD) [7]. SteatoTest combines 0 blood components readily available in every laboratory (see Table II) with age, se and BMI and has several advantages over other proposed diagnostic tools, such as being cheaper than biopsy or magnetic resonance imaging. SteatoTest had a better value for the diagnosis of steatosis than classical markers of steatosis: AUROC (se) = 0.80 (0.02) for SteatoTest versus 0.66 (0.02) for GGT (p<0.000), 0.6 (0.02) for ALT (p<0.000), and 0.65 (0.03) for ultrasonography (p<0.00). Moreover, concordance with biopsy for steatosis detection was better for SteatoTest than for ultrasonography (kappa coefficient 0.44 versus 0.32, respectively, p=0.02) [7]. The better performance of SteatoTest than regular ultrasonography could be eplained by the quite low sensitivity, the observer variability and the low accuracy of ultrasonography, especially in patients with advanced fibrosis [2]. Moreover, one study with repeated SteatoTest and biopsy showed that SteatoTest is a sensitive marker for the followup of treated patients [7]. This first result was confirmed thereafter in a prospective trial in NAFLD patients with Advanced fibrosis was defined as portal fibrosis with few septa (F2), many septa (F3) or cirrhosis (F4).

2 88 Munteanu et al rosiglitazone versus placebo showing significant steatosis regression at the SteatoTest in the treated group versus placebo [3]. Recently, an original independent study used the SteatoTest as a reference noninvasive method instead of the biopsy, in order to validate a new imagistic method for liver steatosis quantification (the hepatorenal ultrasound inde, HRI) [4]. NashTest The NashTest is intended to detect probable NASH that associates necrosis and inflammation. NASH and fibrosis lesions may coeist with steatosis in a minority of NAFLD patients [8]. The utility of the NashTest may be to separate those patients with more severe injury from those with little injury for identifying patients at the greatest risk for disease progression. NashTest was constructed for the diagnosis of NASH in the three categories according to the histological NAS score of Kleiner [5]: NASH, Borderline NASH and No NASH (Table I and Table II). In the validation study on NAFLD patients (n=257), the value of NashTest for the diagnosis of NASH was fair (AUROC = 0.80) with no differences between the training and the validation group for the diagnosis of no NASH, Borderline NASH and NASH. An independent validation on a multicenter population found a similar value (AUROC = 0.83) for the diagnosis of NASH (de Ledinghen Victor, CYTOL study group, personal database). In a study with two concomitant percutaneous liver biopsies in NAFLD patients, authors demonstrated a high risk of misdiagnosis of NASH and staging inaccuracies of liver biopsy mainly because the histological lesions could be unevenly distributed throughout the liver parenchyma [4]. Because of biopsy limitations, NashTest together with SteatoTest and FibroTest were used in large scale studies to screen liver injury without biopsy, in at risk populations like hyperlipidemics, diabetics and morbidly obese [6-8]. FibroTest FibroTest (score from 0 to, see Table I and Table II) is a universal marker, fully validated to provide a quantitative estimation of fibrosis that occurs in the most frequent liver diseases: NAFLD, ALD and chronic viral hepatitis (HBV, HCV and HIV-coinfected) [6]. The last meta-analysis showed in NAFLD patients a mean standardized AUROC for advanced fibrosis of 0.84 (95% CI, ) without differences between the causes of liver disease. Moreover, the AUROC for the diagnosis of the intermediate adjacent stages F2 vs F did not differ from that of the etreme stages F3 vs F4 or F vs F0 [6]. The FibroTest was directly compared [9] to another The histological NASH score (NAS) is defined as the sum of the scores for steatosis (0 3), lobular inflammation (0 3), and ballooning (0 2); cases with scores of 0 to 2 were diagnosed as no NASH, cases with scores of 5 or greater were diagnosed as NASH and intermediate cases with scores of 3 or 4 were probable NASH (borderline) [5]. Table I. Conversion between FibroTest score and fibrosis stages (Panel A), SteatoTest score and steatosis grades (Panel B) and between NashTest score and nonalcoholic steatohepatitis categories (Panel C) in patients with non-alcoholic liver disease Panel A Fibro Test Fibrosis stage estimate* F F3-F F F F-F F F0-F F0 *According to METAVIR scoring system Panel B SteatoTest Estimate of steatosis percentage S2S3>32% S2 6-32% S -5% S0 0% Panel C NashTest Estimate of nonalcoholic steatohepatitis class 0.75 Nonalcoholic steatohepatitis (NASH) 0.50 Borderline NASH 0.25 No NASH Table II. Biomarkers combined in FibroTest and FibroMa Biomarkers FibroTest FibroMa (for SteatoTest and NashTest) Age Gender Alpha 2 macroglobulin, g/l Haptoglobin, g/l Apolipoprotein A, g/l Total Bilirubin, µmol/l GGT, IU/l ALT, IU/l, AST, IU/l Total cholesterol, mmol/l Triglycerides, mmol/l Fasting glucose, mmol/l Weight, kg Height, m surrogate score of fibrosis in NAFLD: NAFLD fibrosis score (NFS) an independently validated non patented panel [20]. The FibroTest demonstrated a better value for the diagnosis of advanced fibrosis and cirrhosis in two different

3 FibroTest-FibroMa Eperience 89 retrospective analyses: AUROCs (95%CI): 0.89 ( ) for FibroTest vs ( ) for NFS (p=0.035) in the diagnostic cohort and 0.80 ( ) for FibroTest vs. NS 0.70 ( ) for NFS (p=0.0) in the therapeutic trial cohort [9]. Moreover, recently the FibroTest demonstrated its value for the long-term and short-term prediction of complications and death in chronic viral hepatitis [2-23]. For all these reasons, the FibroTest was widely used as a non invasive alternative to liver biopsy, with 300,000 tests ordered between September 2002 and May 2008 (Biopredictive data on file, Jean Marie Castille, personal communication). Large scale screening studies using FibroMa Several studies used FibroMa (FibroTest, SteatoTest and NashTest) to screen liver injury in identified at risk populations with one or several metabolically risk factors: hyperlipidemics, diabetics and morbidly obese patients. Hyperlipidemic patients screening for fibrosis, steatosis and NASH Hyperlipidemic patients have multiple risk factors of insulin resistance and abnormal serum levels of hepatic enzymes are frequent and often associated with elevated blood pressure, body mass inde, fasting glucose and uric acid [24]. A study using FibroMa was the first to assess prevalence and factors associated with liver injury in a large number of hyperlipidemic patients (n=909) followed prospectively in a lipid centre (all sera were analyzed retrospectively) [6]. Advanced fibrosis was identified by FibroTest in 2.8%, advanced steatosis by SteatoTest in 30.% and NASH by NashTest in 7% hyperlipidemic patients. Age, male gender, waist circumference, triglycerides increase and HDL-cholesterol decrease were the most significant clinical characteristics associated with presumed fibrosis. The highest prevalence of advanced steatosis was observed among diabetic hyperlipidemics and those with a high HOMA-IR and central obesity. The NASH prevalence in hyperlipidemics with metabolic syndrome was 0-fold higher in patients with the metabolic syndrome compared with those without (P < 0.000) [6]. The study found a highly significant association between the number of metabolic syndrome 2 factors and liver disease prevalence the highest being for hyperlipidemics with type 2 diabetes. This study stressed that the biomarkers could be useful when two metabolic syndrome factors or more are present because a clear increase of risk of liver injury. Hyperlipidemic patients were referred when they presented with either plasma low-density lipoprotein (LDL)-cholesterol levels. above 60 mg/dl or triglyceride levels above 50 mg/dl. 2 The definition of the metabolic syndrome was based on the ATPIII classification [25]. Diabetic patients screening for fibrosis, steatosis and NASH Patients with type 2 diabetes present a known risk for NAFLD leading to advanced fibrosis, cirrhosis and liver cancer [26-27]. A recent study estimated the efficacy to detect fibrosis with a noninvasive fibrosis biomarker (FibroTest) of a screening of diabetic patients without a history or clinical signs of liver disease, seen prospectively in a tertiary center (n=3 cases) [7]. All patients with presumed advanced fibrosis were reinvestigated by a hepatologist using elastography (FibroScan, Echosens, France) and, if necessary, ultrasonography, endoscopy, or liver biopsy. The prevalence of confirmed advanced fibrosis was 2.8% with 5 cases of cirrhosis, 4 cases of hepatocellular carcinoma and one cholangiocarcinoma. In the population with type 2 diabetes 45 years or older, the prevalence of confirmed advanced fibrosis was higher (4.3%) and of hepatocellular carcinoma was 5.7 of 000 [7]. The prevalence of steatosis at the SteatoTest and of NASH at the NashTest was higher in diabetics with confirmed advanced fibrosis (86% and 38%, respectively) compared to diabetics with no or minimal fibrosis (4% and 6%, respectively, all p <0.000). Among the confirmed advanced fibrosis diabetic patients, one quarter had normal GGT (false-negatives), and twothirds had normal ALT (false-negatives) confirming the low sensitivity to detect fibrosis of transaminases and GGT compared to combinations of biomarkers like FibroTest. The study strongly suggested the need for screening of advanced fibrosis, using validated biomarkers such as FibroTest, in type 2 diabetic patients aged 45 years or older in order to prevent liver-related mortality. Morbidly obese patients screening for fibrosis, steatosis and NASH Obesity is a major health issue worldwide since studies suggest it is associated with overall increased morbidity and mortality [28]. BMI seems correlated with NAFLD prevalence [29] with obesity being the most frequent risk factor associated with histological NAFLD at autopsy [30]. A review of available studies in severely obese patients submitted to bariatric surgery found that the prevalence of liver steatosis and non-alcoholic steatohepatitis (NASH) ranged from 85% to 98% and from 24 to 98%, respectively, with unepected cirrhosis in % to 7% [3]. A recent study proposed to estimate the diagnostic value of biomarkers of liver injury in morbidly obese (MO) patients addressed to bariatric surgery for BMI 40kg/m² or higher, or for BMI 30kg/m² or higher and at least one risk factor [8]. MO patients (n=94) had peroperatory biopsy and concomitant biomarkers (FibroMa) and were compared to a nonmorbidly obese group (nonmo) with NAFLD (n=234) that had had percutaneous biopsy and biomarkers. The FibroTest value for the diagnosis of advanced fibrosis was not different in MO patients to that in nonmo [AUROCs 0.76 (0.07) vs (0.03), respectively, p=ns]; similar results were obtained for NASH diagnosis at the NashTest

4 90 Munteanu et al [AUROCs 0.72 (0.09) vs (0.06), respectively, p=ns] and for severe steatosis at the SteatoTest [AUROCs 0.8 (0.06) vs (0.05), respectively, p=ns] after adjustment of etreme BMI. The estimated prevalence of advanced fibrosis, steatosis and NASH with biomarkers in MO patients was 3%, 54% and %, respectively. These preliminary results suggest that biomarkers could be useful for the screening of liver injury in MO patients, a population difficult to investigate and for which the peroperatory biopsy demonstrates major limitations [32]. Limitations of biomarkers and precautions of use FibroMa biomarkers have several limitations related to the non-interpretability of results in about 5% of cases. One study identified the causes of failure of the FibroTest [33]. The most frequent cause leading to false negative result was high haptoglobin in acute inflammation or sepsis. The most frequent cause of false positive results was etremely low haptoglobin associated with intravascular hemolysis and high bilirubin in hemolysis and Gilbert disease. The biomarkers had to be deferred for: acute hemolysis, acute hepatitis, acute inflammation or etra hepatic cholestasis. Results are provided with security algorithms identifying situations of failure and isolated etreme values of one of the components; presence of warnings on the result sheet should lead to caution in interpreting the results. The reliability of results depends also on compliance of laboratories with the preanalytical and analytical conditions recommended by the quality chart of BioPredictive [34] Several studies have validated these analytical and preanalytical recommendations [35, 36]. According to a prospective study, fasting is not mandatory for the FibroTest dosage but is mandatory for FibroMa (SteatoTest and NashTest) [37]. Advantages of non invasive markers in trials Recently a trial with rosiglitazone versus placebo in patients with NASH, tested the impact on liver injury (steatosis and fibrosis) assessed concomitantly with biomarkers and biopsy at baseline and after one year of treatment [3]. Results showed more improved steatosis between baseline and end-of- treatment in the treated group compared to placebo at biopsy (47% vs. 6%, p=0.04) and at biomarkers (mean SteatoTest 0.7 vs. 0.64, p=0.04 in the treated group). There was no observed fibrosis histological improvement at biopsy in the treated group; meanwhile FibroTest score improved significantly in the treated group (0.44 vs. 0.34, p=0.04) compared to placebo (p=ns). These preliminary results suggest that biomarkers may be a more sensitive method than biopsy for detecting early histological changes. Recently, an original study assessed the hepatorenal ultrasound inde (HRI) for quantification of liver steatosis taking the SteatoTest as a standard instead of the biopsy [4]. This noninvasive approach was justified knowing that biopsy in NAFLD showed sampling variability for steatosis, with discordance in 22% of patients [4]. Several advantages of biomarkers in trials could be that they are at least as accurate as a 5 mm biopsy fragment, have no observer variability and no severe adverse events, being cheaper and repeatable and leading to less missing data, refusal and bias. Conclusion In France, according to a nationwide survey, the majority of hepatologists have changed their practice using noninvasive biomarker (FibroTest) and elastography instead of liver biopsy in chronic hepatitis C management. The French health authorities officially have agreed with this noninvasive practice [38, 39]. The screening for significant liver injury in patients with NAFLD will be an important medical challenge in the years to come, due to the epidemics of obesity and diabetes. A large-scale prospective screening with FibroTest- FibroMa on the general population is ongoing in France and preliminary data showed impaired fasting glucose as being one of the major risk factors associated with advanced fibrosis (Thierry Poynard personal database). From a practical point of view, the screening of liver injury could be recommended in patients with at least two metabolic syndrome factors and in type 2 diabetics aged 45 years or more. Transaminases or GGT should not be used as screening tests for the diagnosis of liver injury. The use of biomarkers should help the clinician to reassure patients with only steatosis and to identify among patients with steatosis, those with severe injuries such as advanced fibrosis and NASH in order to prevent liver-related mortality. The FibroTest and FibroMa have proven their efficacy as noninvasive biomarkers in large scale studies, being readily available in more than 50 countries and easy to perform in any routine analysis laboratory [34]. Conflicts of interest TP is the inventor of FibroTest and is a consultant and has a capital interest in Biopredictive, the company marketing FibroTest. RM and MM are Biopredictive employees. Acknowledgement We thank all the medical staff of the Diabetology Unit, Anti-fibrosis Hepatology Unit and Biomarkers Unit (Biochemistry and Biopredictive). References. Sanyl AJ. AGA technical review on nonalcoholic fatty liver disease. Gastroenterology 2002;23: Angulo P. Nonalcoholic fatty liver disease. Rev Gastroenterol Me 2005;70: Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology 2003;38:

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