In Search of New Biomarkers for Nonalcoholic Fatty Liver Disease

REVIEW In Search of New Biomarkers for Nonalcoholic Fatty Liver Disease Ting-Ting Chan, M.R.C.P., and Vincent Wai-Sun Wong, M.D. Nonalcoholic fatty liver disease (NAFLD) affects 15% to 40% of the general adult population and has become an important cause of cirrhosis and hepatocellular carcinoma. Liver biopsy was traditionally the primary method to assess the severity of NAFLD. Features of interest include hepatic steatosis, necroinflammation, and fibrosis. In particular, nonalcoholic steatohepatitis (NASH), the more progressive form of NAFLD, is defined as the presence of steatosis, lobular inflammation, and ballooning. 1 In natural history studies, fibrosis, and to a lesser extent NASH, correlates with long-term survival and liver-related complications. 2 However, liver biopsy is invasive and poorly accepted by patients. It is also unrealistic to perform repeated biopsies in routine practice. Biomarkers are therefore needed for prognostication and disease monitoring. Although not currently achieved, biomarkers that reflect how a patient feels, functions, or survives can be used as surrogate endpoints in clinical trials. The latter can greatly improve the current drug development process. In this short review, we describe currently available biomarkers and highlight new developments in this field. BIOMARKERS OF STEATOSIS The diagnosis of NAFLD depends on the detection of hepatic steatosis. In contrast, the degree of steatosis correlates poorly with liver injury and clinical outcomes. 2 In fact, steatosis often decreases as a patient s condition progresses to cirrhosis. For the same reason, although some early-phase studies used noninvasive tests of steatosis to evaluate new treatments, it is unclear whether Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristic curve; CAP, controlled attenuation parameter; FIB-4, Fibrosis-4; GGT, gamma-glutamyl transpeptidase; 1 H-MRS, proton magnetic resonance spectroscopy; MRI, magnetic resonance imaging; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. From the Department of Medicine and Therapeutics and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong. This study was supported by the General Research Fund of the Research Grant Council of Hong Kong (project reference 477813). Potential conflict of interest: V. Wong has received lecture fees from Echosens. Received 2 May 2016; accepted 15 May 2016 Contract grant sponsor: Research Grant Council of Hong Kong; contract grant number: 477813. View this article online at wileyonlinelibrary.com VC 2016 by the American Association for the Study of Liver Diseases 19 CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD

TABLE 1. SERUM BIOMARKERS AND PREDICTION MODELS OF STEATOSIS Fatty liver index A formula comprising body mass index, triglycerides, and GGT NAFLD liver fat score A formula comprising metabolic syndrome, type 2 diabetes, fasting serum insulin, AST, and AST/ALT SteatoTest A proprietary formula comprising a 2 -macroglobulin, haptoglobin, apolipoprotein A1, total bilirubin, GGT, fasting glucose, triglycerides, cholesterol, and ALT, adjusted for patient s age, sex, weight, and height Suitable for epidemiological studies Inadequate distinction of the severity of steatosis Suitable for epidemiological studies Insulin not routinely done Inadequate distinction of the severity of steatosis Suitable for epidemiological studies and individual diagnosis Relatively expensive Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transpeptidase. this approach can select good candidates for further development. Table 1 summarizes the serum biomarkers and prediction models of steatosis. These models use routine clinical parameters and/or biomarkers to predict the presence of NAFLD. Apart from clinical application, it is possible to retrospectively calculate these scores from existing cohorts for NAFLD research. Because of its availability and moderate accuracy, abdominal ultrasonography is the primary test to diagnose NAFLD in routine practice (Table 2). In recent years, controlled attenuation parameter (CAP) by FibroScan has also been developed for steatosis measurement. 3 The measurement is based on the fact that the amplitude of ultrasound waves is attenuated more rapidly in a fatty liver. CAP can also be measured with liver stiffness simultaneously, allowing evaluation of the severity of NAFLD at the same time. Magnetic resonance imaging (MRI) based techniques such as proton magnetic resonance spectroscopy ( 1 H- MRS) and MRI-proton density fat fraction are probably the most accurate noninvasive tests to quantify liver fat and are sensitive to changes in steatosis over time. The cost and availability of the techniques are the main challenges. BIOMARKERS OF STEATOHEPATITIS NASH is the active form of NAFLD and the target for treatment. The development of NASH biomarkers is therefore important for selecting patients for treatment and also for monitoring treatment response. Compared with steatosis and fibrosis, noninvasive tests of NASH are less well developed. Serum cytokeratin 18 fragment reflects hepatocyte apoptosis and is one of the most extensively validated NASH biomarkers. Its overall accuracy is modest. 4 Other evaluated biomarkers include other cell death markers (e.g., soluble Fas, intact cytokeratin 18), adipokines (e.g., adiponectin, tumor necrosis factor-a, interleukin-6, adipocyte fatty acid binding protein), metabolic markers (e.g., homeostasis model assessment of insulin resistance, fibroblast growth factor 21), and inflammatory markers (e.g., C-reactive protein). Because NASH is a multisystem disease and the relative contribution of different disease processes varies from patient to patient, these biomarkers tend not to perform as well when validated in independent cohorts. Combined biomarker panels are probably required. BIOMARKERS OF FIBROSIS Like other chronic liver diseases, NAFLD can also progress to advanced fibrosis and cirrhosis starting at perisinusoidal regions. Patients with NASH-related cirrhosis have similar mortality as those with cirrhosis from other causes. 5 It is therefore important to detect and assess severity of fibrosis in NFALD, to allow a window of complication screening and treatment evaluation. Table 3 summarizes the serum biomarkers and predicted models of fibrosis. The NAFLD fibrosis score and Fibrosis-4 (FIB-4) index use easily accessible clinical parameters and 20 CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD

TABLE 2. RADIOLOGICAL TESTS OF STEATOSIS Abdominal ultrasonography CAP MRI Can be done together with liver stiffness measurement by FibroScan 1 H-MRS or MRI-estimated proton density fat fraction Widely available and relatively inexpensive Can screen for hepatocellular carcinoma at the same time Moderate distinction of the severity of steatosis Operator dependent May miss steatosis <20% Performs less well in patients with body mass index >40 kg/m 2 Quick procedure, relatively inexpensive after the initial installment Moderate distinction of the severity of steatosis Objective and reproducible Detect steatosis >10% Machine may be unavailable at some centers Performs less well in patients with body mass index >35 kg/m 2 Good distinction of the severity of steatosis Objective and reproducible Detects steatosis as low as <1% and sensitive to change over time Not affected by obesity or ascites Can screen for hepatocellular carcinoma at the same session Expensive; service may not be available at some centers TABLE 3. SERUM BIOMARKERS AND PREDICTION MODELS OF FIBROSIS NAFLD fibrosis score FIB-4 index FibroTest Enhanced liver fibrosis panel A formula comprising age, hyperglycemia, body mass index, platelet count, albumin, and AST/ALT ratio A formula comprising age, platelet count, AST, and ALT A proprietary formula comprising a 2 -macroglobulin, apolipoprotein A1, haptoglobin, total bilirubin, and GGT A proprietary formula comprising hyaluronic acid, procollagen III amino-terminal peptide, tissue inhibitor of matrix metalloproteinase 1, and age Easily accessible clinical parameters Needs independent adjustment of body mass index across ethnic groups Easily accessible clinical parameters Highest AUROC for F3-4 diseases among studies Validated in Asian population Commercially available but more expensive Affected by other causes of hyperbilirubinemia and elevated GGT, e.g., hemolysis, alcohol intake Commercially available but more expensive Good distinction of the severity of fibrosis Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristic curve; GGT, gamma-glutamyl transpeptidase. are therefore inexpensive. FibroTest and the enhanced liver fibrosis panel instead include special biomarkers that are not commonly measured at routine clinical practice. A number of radiological tests have been validated for NAFLD in recent years (Table 4). Transient elastography by FibroScan is the most widely available tool for liver 21 CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD

TABLE 4. RADIOLOGICAL TESTS OF FIBROSIS Transient elastography Using FibroScan to measure the velocity of shear wave in liver parenchyma and thus liver stiffness measurement Highly reproducible Validated across patients with different liver diseases Inaccurate in patients with acute hepatitis, congestive heart failure, and biliary obstruction Performs less well in obese subjects with body mass index >35 kg/m 2 when using traditional M probe; XL probe can enhance accuracy Acoustic radiation force impulse Measure attenuation of ultrasound wave energy as a result of tissue absorption and scattering Can perform hepatocellular carcinoma screening at the same time Not affected by external factors, e.g., obesity, ascites Higher variability because of small region of interest Shear wave elastography Generates shear wave directly at liver parenchyma with ultrasound beam Can perform hepatocellular carcinoma screening at the same time Operators can choose suitable areas of examination interest, e.g., free of blood vessels or focal lesions Not affected by external factors, e.g., obesity, ascites Magnetic resonance elastography MRI of shear wave propagation Good diagnostic accuracy and distinction of different fibrosis stages than ultrasound-based techniques Not affected by obesity or ascites Allow simultaneous structural examination and 1 H-MRS for hepatic steatosis assessment Limited by cost and availability Multiparametric MRI Using T 1 mapping for fibrosis/inflammation Less operator dependent imaging; T 2 for liver iron quantification Quick and 1 H-MRS for liver fat quantification Good diagnostic accuracy in patients with mixed liver diseases Can allow structural examination and hepatocellular carcinoma screening Limited NAFLD data stiffness measurement. It is reproducible and highly accurate for cirrhosis and less so for significant fibrosis. While liver stiffness measurement may fail in obese subjects with a body mass index greater than 30 kg/m 2, the success rate can still approach 80-90% when the XL probe is used. 6 Acoustic radiation force impulse accesses the internal energy vibration, whereas shear wave elastography differs from transient elastography in terms of direct generation of waveforms at liver parenchyma. Magnetic resonance elastography is superior in distinction of different fibrosis staging but limited by availability and cost. 7 Recently, the multiparametric MRI has been developed to measure hepatic steatosis, iron content, inflammation, and fibrosis at the same time, but its applicability in NAFLD patients requires further studies. 8 GENETIC BIOMARKERS Studies using next-generation sequencing have identified a few genes associated with NAFLD, among which patatin-like phospholipase domain-containing protein 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) have been most extensively validated across different ethnicities. 9,10 These gene polymorphisms are associated with both hepatic steatosis and liver injury, but not with increased cardiovascular risk. Currently, these genetic biomarkers remain as research tools; their clinical application is yet to be defined. FUTURE PERSPECTIVES Most biomarkers were tested against liver histology in cross-sectional studies. Their performance as monitoring tools is largely unknown. Because there is now an increasing number of clinical trials on NASH, this would be a good opportunity to develop biomarkers that can be used serially to monitor for disease progression and assess treatment response. In the future, changes in biomarkers must be validated against patients symptoms, functions, and survival. 22 CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD

CONCLUSION Abdominal ultrasonography remains the primary test for the diagnosis of NAFLD, after excluding other liver diseases as appropriate. Serum biomarkers of fibrosis and physical measurement of liver stiffness can be used to exclude advanced fibrosis. The development of biomarkers of NASH should now be a research priority. This would ultimately reduce the need for liver biopsy and improve patient care. CORRESPONDENCE Vincent Wong, Department of Medicine and Therapeutics, 9/F, Clinical Sciences Building, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, Hong Kong. E-mail: wongv@cuhk.edu.hk REFERENCES 1) Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 2012;55: 2005-2023. 2) Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 2015;149:389-397.e310. 3) Kwok R, Tse YK, Wong GL, Ha Y, Lee AU, Ngu MC, et al. Systematic review with meta-analysis: non-invasive assessment of non-alcoholic fatty liver disease---the role of transient elastography and plasma cytokeratin-18 fragments. Aliment Pharmacol Ther 2014;39: 254-269. 4) Shen J, Chan HL, Wong GL, Choi PC, Chan AW, Chan HY, et al. Non-invasive diagnosis of non-alcoholic steatohepatitis by combined serum biomarkers. J Hepatol 2012;56:1363-1370. 5) Bhala N, Angulo P, van der Poorten D, Lee E, Hui JM, Saracco G, et al. The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology 2011;54:1208-1216. 6) Wong VW, Vergniol J, Wong GL, Foucher J, Chan AW, Chermak F, et al. Liver stiffness measurement using XL probe in patients with nonalcoholic fatty liver disease. Am J Gastroenterol 2012;107: 1862-1871. 7) Cui J, Heba E, Hernandez C, Haufe W, Hooker J, Andre MP, et al. Magnetic resonance elastography is superior to acoustic radiation force impulse for the diagnosis of fibrosis in patients with biopsyproven nonalcoholic fatty liver disease: a prospective study. Hepatology 2016;63:453-461. 8) Pavlides M, Banerjee R, Sellwood J, Kelly CJ, Robson MD, Booth JC, et al. Multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease. J Hepatol 2016;64: 308-315. 9) Sookoian S, Pirola CJ. Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 2011;53:1883-1894. 10) Pirola CJ, Sookoian S. The dual and opposite role of the TM6SF2- rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis. Hepatology 2015;62:1742-1756. 23 CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD