In search of new biomarkers for nonalcoholic fatty liver disease (2016)

Watch a video presentation of this article Watch the interview with the author 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. 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 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. • Simple and inexpensive • 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 • Involves biomarkers that are not routinely done 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. • 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/m2 • 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/m2 • 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 Magnetic resonance imaging (MRI)–based techniques such as proton magnetic resonance spectroscopy (1H-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. 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-α, 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. 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 are therefore inexpensive. FibroTest and the enhanced liver fibrosis panel instead include special biomarkers that are not commonly measured at routine clinical practice. • Easily accessible clinical parameters • Simple and inexpensive • Needs independent adjustment of body mass index across ethnic groups • Easily accessible clinical parameters • Simple and inexpensive • Highest AUROC for F3-4 diseases among studies • Validated in Asian population • Commercially available but more expensive • Involves biomarkers that are not routinely done • 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 • Involves biomarkers that are not routinely done 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 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/m2, 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 • 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/m2 when using traditional M probe; XL probe can enhance accuracy • 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 • 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 • Good diagnostic accuracy and distinction of different fibrosis stages than ultrasound-based techniques • Not affected by obesity or ascites • Allow simultaneous structural examination and 1H-MRS for hepatic steatosis assessment • Limited by cost and availability • Less operator dependent • Quick • Good diagnostic accuracy in patients with mixed liver diseases • Can allow structural examination and hepatocellular carcinoma screening • Limited NAFLD data 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. 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. 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.