Serum biomarkers for nonalcoholic fatty liver disease: Are we there yet?
Authors: Ken Liu, Weiqi Xu, Vincent Wai‐Sun Wong
Journal: Hepatology (2016)

Nonalcoholic fatty liver disease (NAFLD) is already the most common liver disease in the Western world, where overweight or obese adults make up the growing majority.1 Its comparable impact in other regions, such as Asia, is increasingly being recognized.2 As we enter into an era of improving global hepatitis B vaccination coverage and effective therapies to either control or eradiate chronic viral hepatitis, the proportional burden of NAFLD is set to rise dramatically and demand our attention. Accordingly, NAFLD has already become the second-leading indication for liver transplantation in the United States.3 Distinguishing between nonalcoholic fatty liver (NAFL) and the more-progressive nonalcoholic steatohepatitis (NASH) is clinically important given that NASH is currently the target for pharmacological treatment. Routine imaging techniques, such as ultrasound scan, computed tomography, and magnetic resonance imaging, can accurately detect NAFLD and even quantify hepatic steatosis in the case of magnetic resonance spectroscopy. However, these investigations cannot diagnose NASH or liver fibrosis. Currently, the gold standard for diagnosing and differentiating the NAFLD spectrum is a liver biopsy. Its invasiveness, cost, and low patient acceptance make it unfeasible to be used for routine screening, much less for repeated assessments to monitor disease progression or response to treatment. Even when a liver biopsy is successfully performed, its shortcomings attributed to sampling error or inter- and intraobserver variability are well documented.4 Hence, noninvasive tests, such as serum biomarkers, are clearly needed to prioritize "at-risk" patients who require liver biopsy or ideally replace liver biopsy all together. Opportunities exist for biomarkers to make an impact across the whole NAFLD spectrum, such as diagnosing NAFL, diagnosing NASH, and assessing fibrosis (Fig. 1). In this issue of Hepatology, Ajmera et al.5 evaluate 32 potential plasma biomarkers of disease activity and severity in a large, well-characterized cohort of adult patients with biopsy-proven NAFLD. The 648 patients were recruited from two multicenter studies conducted by the NASH Clinical Research Network: the NAFLD database study and the pioglitazone or vitamin E for nonalcoholic steatohepatitis (PIVENS) trial. All participants underwent robust confirmation and phenotyping of NAFLD by baseline liver histology, which was reviewed centrally by a group of nine blinded hepatopathologists. Plasma samples drawn within 6 months of the liver biopsy were then analyzed using a Luminex-based biomarker multiplex assay. Given that all biomarkers evaluated were chosen based on a priori knowledge, the positive findings in this study have largely been demonstrated previously. However, the investigators were seeking to find the most impactful biomarkers in NAFLD and their intercorrelation—a feat only achieved with a large sample size and biomarker panel as in the current study. Diagnosis of definite NASH, which was present in 58% of patients, was the primary outcome. Its individual histological components (steatosis, lobular inflammation, and hepatocyte ballooning) were also studied. On multivariate analysis, only increased activated plasminogen activator inhibitor 1 (PAI-1) had a strong association with definite NASH. This finding confirms previous observations by Verrijken et al. in a study of 273 overweight or obese subjects with biopsy-proven NAFLD.6 PAI-1 is a serine protease inhibitor of tissue plasminogen activator and urokinase, the initiators of fibrinolysis. In relation to the liver, PAI-1 plays a role in fibrogenesis by reducing plasmin-mediated activation of matrix metalloproteinases, which are required for degradation of extracellular matrix. Interestingly, because of its procoagulant properties, PAI-1 has been hypothesized as a potential link between NAFLD and its associated cardiovascular risk.6 For diagnosis of significant fibrosis (F2-4), increased interleukin-8 (IL-8) and soluble IL-2 receptor alpha demonstrated the strongest associations, among others. Whereas the link between these markers of inflammation and advanced fibrosis or cirrhosis has been observed in other chronic liver diseases, their potential value in NASH is confirmed in this study.7, 8 IL-8 is a potent chemokine involved in neutrophil recruitment, degranulation, and liver inflammation. Its association with hepatocyte ballooning (which occurs in the context of steatohepatitis) is also a novel finding, although not entirely unexpected. It should be noted that these associations between biomarkers and their endpoints, although significant, have modest odds ratios (1.2-1.3 per 0.5-SD increment). This highlights that no single biomarker can be used as a stand-alone test in diagnosing NASH. Instead, it is likely that these biomarkers will be used in a panel along with other biomarkers, clinical parameters, and/or imaging techniques. Formal c-statistics analysis to document the reliability and accuracy of the strongest biomarkers, and comparison with more-traditional prediction models such as the NAFLD fibrosis score, would have been informative in this study. Given that this is a study of predominantly white subjects (three quarters of participants), independent validation is needed before one can generalize the findings to other ethnicities. Differences in genetic polymorphisms, which influence lipid metabolism, redox pathways, inflammatory recruitment, fibrogenesis, and therefore downstream biomarkers, have been described between ethnicities.9 Although the investigators performed a cross-sectional study, 90% of subjects in the PIVENS cohort actually underwent an end-of-treatment liver biopsy at 96 weeks. This would have been an opportunity to evaluate the temporal relationship between biomarkers and disease activity and hence their usefulness as a monitoring tool. After all, when applied in the real world, clinicians need biomarkers for not only selecting patients for treatment, but also monitoring treatment response. Along the same line, before new biomarkers can be applied at the clinic, we need to understand their variability over time and clinical confounders. In the current analysis, some of the biomarkers were correlated with different metabolic factors. This by itself may only reflect the mechanism through which the biomarkers were linked to NASH and fibrosis. Further analysis is needed to evaluate whether some clinical factors and concomitant drugs may affect the diagnostic performance of these markers. Aside from limitations in their performance, the utility of biomarkers is somewhat held back by the lack of evidence-based treatment options for NASH. Indeed, lifestyle intervention is the only established therapy and should be encouraged in all individuals regardless of their NAFLD severity. However, even though risk stratification of patients currently would not alter management significantly, this information may help engage and motivate patients to enact lifestyle modifications. Additionally, reliable biomarkers may serve as new tools for patient selection or evaluation of response in randomized, clinical trials. Given that NASH is a complex heterogeneous disease, it is likely that biomarkers of treatment response will be closely related to the mechanism of action of a specific treatment rather than being broadly applicable. With promising new therapies currently being tested, the discovery of noninvasive biomarkers to direct future treatment is becoming increasingly urgent.10 Finally, the importance of biomarkers associated with liver disease severity should be tempered by perspective. Given that fibrosis progression in NASH is slow (mean increase of 0.09 fibrosis stages per year) and not universal (41% of patients), non-liver-related death remains far more common than liver-specific death or hepatocellular carcinoma combined.1 Is there any value in diagnosing NASH or even advanced fibrosis with a biomarker in a patient who will die from cardiovascular disease? The focus and validation of serum biomarkers currently falls in the realm of diagnosis, whereas a shift in focus to the prediction of clinical outcomes is needed. At the end of the day, the most important issue is the ability to predict whether a patient will develop hepatic complications (or even cardiovascular events) in the future. As such, we look forward to further longitudinal studies before serum biomarkers become prime time. Ken Liu, M.B.B.S., F.R.A.C.P.1,2 Weiqi Xu, M.Med.1 Vincent Wai-Sun Wong, M.D.1 1Institute of Digestive Disease The Chinese University of Hong Kong Hong Kong Department of Medicine and Therapeutics The Chinese University of Hong Kong Hong Kong State Key Laboratory of Digestive Disease The Chinese University of Hong Kong Hong Kong 2Faculty of Medicine The University of Sydney Sydney, NSW, Australia