The development of non invasive biomarkers of disease has become a major focus of interest in nonalcoholic fatty liver disease (NAFLD). The large prevalence of the disease and the invasive nature of the investigation means that screening with liver biopsy is impractical. In addition to screening, the differentiation of those with simple steatosis vs steatohepatitis and fibrosis is clinically important as the prognosis of each differs. Serum biomarkers may be a combination of simple markers derived from large data sets or direct markers of disease activity. Serum markers of inflammation, apoptosis and oxidative stress in addition to fibrosis have been extensively studied in patients with NAFLD. Other techniques such as transient elastography, magnetic resonance elastography and acoustic radiation force imaging are becoming more established as noninvasive methods of detecting fibrosis in a variety of chronic liver conditions in addition to NAFLD. Newer high throughput methods such as proteomics and glycomics allow the nonhypothesis-driven identification of novel markers and may also potentially contribute to our understanding of the pathogenesis of the condition. This review addresses some of the methodological issues which need to be considered in the search for the ideal biomarker. It is likely that a combination of serum biomarkers and techniques such as transient elastography may provide the optimal diagnostic discrimination however this remains to be proven in large studies.

Ultimately 10% to 28% of nonalcoholic steatohepatitis (NASH) patients develop cirrhosis and hepatocellular carcinoma[1-3]. The criterion standard for diagnosis and assessing progression of disease is liver histology, though this has inherent limitations. Still, the decision “if or when” to perform and repeat a liver biopsy in patients with nonalcoholic fatty liver disease (NAFLD) remains controversial. The prevalence of the condition is such that the resources needed to perform liver biopsy on every patient with NAFLD would be enormous. Liver biopsy often requires admission to hospital and sedation. Risks include bleeding and very rarely death[4]. For the same reason, repeated biopsy is not a suitable tool for regularly monitoring progression of disease or response to treatment. In addition, biopsy samples only 1/50000 of the liver, raising the possibility of sampling error[5].

There has been much focus on the development and validation of noninvasive biomarkers of NAFLD in recent years. There is an urgent need for a less invasive method than biopsy of screening the population, stratifying disease severity and following disease progression. This is particularly relevant in the paediatric population. Many markers of inflammation, hepatocyte apoptosis, fibrosis and oxidative stress are under investigation. The European Association for the Study of the Liver special topic conference on NAFLD called for a renewed focus on noninvasive biomarkers of disease[6]. In common with all biomarkers which are “biological markers of disease presence and progression”[7], important characteristics include; sufficient sensitivity to identify those with disease, specificity to exclude those without disease, cost-effectiveness, ease of use and reproducibility. There are several different approaches to the identification of biomarkers: the first is the use of clinical or biochemical markers that have been derived from large association studies. The second is the use of algorithms including markers of extracellular matrix turnover in the case of fibrosis and inflammation/cell death in the case of inflammatory change. The third is the non-hypothesis driven new-technology based approach such as microarray techniques, proteomics and glycomics[8,9] (Figure 1).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Serum biomarkers of disease activity may measure inflammation, apoptosis, oxidative stress or fibrosis. This is a schematic representation of the pathophysiological processes in nonalcoholic fatty liver disease (NAFLD), markers of which may be demonstrable in serum. LDL: Low density lipoprotein; IL: Interleukin; TBARS: Thiobarbituric acid reactive substances; TIMP1: Tissue inhibitor of MMP 1.

The pathophysiology and evolution of the particular pathological condition is an important consideration in the development and evaluation of biomarkers. In the case of NAFLD; there are two potential targets. The first is the differentiation of simple steatosis from steatohepatitis. This is important as the prognosis of those with simple steatosis is different from those with NASH[10]. The second issue is the identification of fibrosis stage. This is the main determinant of prognosis and knowing the extent of fibrosis is useful in making treatment decisions, in patient selection for treatment studies and in monitoring progression/regression. Most longitudinal cohort studies in NAFLD have shown that prognosis is determined by stage and rate of progression of fibrosis rather than the presence of necro-inflammation[1,2,11]. Clinical importance lies with being able to differentiate between no/minimal fibrosis (F0/F1), significant fibrosis (F2), severe fibrosis (F3) and cirrhosis (F4).

Important issues to be considered in the design and validation of any noninvasive markers include the inherent limitations of liver biopsy as the criterion standard and the differences in prevalence of different disease stages (spectrum bias).

Variations in size of biopsy tissue, number of portal tracts and fragmentation will all influence accuracy of liver biopsy in determining the true stage of fibrosis as described previously[12,13]. In the case of NAFLD the degree of steatosis and inflammation is assessed separately to fibrosis and scoring systems such as the NASH activity score is used to distinguish simple steatosis from steatohepatitis. Both intra and interobserver variability may also significantly affect the score[14]. Thus, the ability of noninvasive biomarkers to differentiate between fibrosis stages is limited by the criterion standard.

Some of these issues in terms of scoring variability may be overcome using techniques such as collagen proportionate area quantification, however the limitations of a short or nonrepresentative biopsy remain.

The ideal outcome measure for any noninvasive biomarker is disease outcome over time, such as has been reported by Parkes et al[15]. Long-term outcomes (morbidity/mortality/need for transplantation) are the optimal measures, though are not feasible in shorter term studies.

Large adult series have suggested scoring systems using age, BMI, insulin resistance, aspartate aminotransferase/alanine aminotransferase (AST/ALT), platelet count and albumin to differentiate mild from severe disease[16-19] (Table 1). These simple markers are neither sensitive nor specific enough in isolation[20,21]. A growing understanding of the pathophysiology of the disease has allowed the investigation of more specific, mechanism-based biomarkers. These biomarkers focus on the specific pathways involved in the progression of the disease process: hepatocyte apoptosis, oxidative stress, inflammation and fibrosis[8,22,23] (Figure 2).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 An approach to using noninvasive markers of disease to assess severity of disease in nonalcoholic fatty liver disease.

It is the severity and rate of progression of fibrosis rather than inflammation per se that determines outcome in the majority of cases[55,56]. The importance of staging disease in the context of fibrosis across liver disease in general is thus manifold. Firstly in the development of treatment decision algorithms; this is particularly relevant in adult viral hepatitis. Secondly functional tests may be even better than biopsy or measurement of hepatic vein pressure gradient in predicting outcome and thus planning appropriate follow up and services[57,58]. Finally the diagnosis of cirrhosis is important so that surveillance for varices and hepatocellular carcinoma may be instigated. These issues are clearly applicable across the spectrum of chronic liver disease, not alone NAFLD[59,60].

Other biomarkers measure the degree of extracellular matrix (ECM) turnover. Using such ECM markers is a more direct method of assessing fibrogenic activity, and will tend to measure a dynamic process rather than a static one. Hyaluronic acid is one of the most validated markers of fibrosis in liver disease, synthesised by stellate cells and metabolised by sinusoidal endothelial cells[72,73]. Hyaluronic acid was found to be an accurate marker of fibrosis in NAFLD[74,75].

Combinations of both clinical markers and ECM turnover include the FibroTest^®[54,76,77], an algorithm of 13 markers derived from regression analysis including haptoglobin, α2-macroglobulin, apolipoprotein A1, bilirubin, γ-glutamyl transpeptidase, age and gender. It has an AUROC of 0.84 for advanced fibrosis in NAFLD[78].

The European Liver Fibrosis test (ELF)™ combining hyaluronic acid, procollagen III N-terminal peptide and TIMP1 was first derived by Rosenberg et al[79] in a cohort of over 1000 patients with chronic liver disease including NAFLD and has since been validated in other NAFLD cohorts with the addition of several simple markers to improve accuracy[80]. Importantly this test has been shown to correlate well with outcome[15].

Table 2 summarises previous studies investigating serum biomarkers of fibrosis in NAFLD[81-84].

Biomarkers of NAS and fibrosis have also been reported by a few paediatric studies as referenced below. These studies are relatively limited by the size of the cohorts involved and are mostly validation of adult biomarkers.

Transient elastography (Fibroscan^®) has been shown to be a useful method for detection of liver fibrosis. This technique uses both ultrasound (5 MHz) and low frequency (50 Hz) elastic waves with a propagation velocity directly related to the stiffness of the liver; i.e., the stiffer the medium, the faster the wave. The low frequency vibrations are transmitted to the skin by placement of the probe at the intercostal space where a liver biopsy would be performed. A shear wave is induced which propagates into the liver. The wave passes through tissue 2.5-6.5 cm below skin surface, (in those 0 to 7 years a modified probe which can measure 2.5-5.5 cm is used). A pulse-echo acquisition is then used to measure the propagating wave’s velocity which is proportional to tissue stiffness represented by the equation for Young’s elastic modulus E (3pv²) (p = density, v = shear velocity). Machine based software determines whether each measurement is successful or not. Requirements for accurate evaluation of liver stiffness include an interquartile range of +/- 30% of the median value and ratio of successful measurements to the total no of acquisitions > 60%.

Transient elastography (TE) has been well validated and was the subject of a recent systematic review of 50 studies which concluded that Fibroscan^® has excellent diagnostic capability across different liver diseases for cirrhosis[104]. There was some variability for diagnosis of lesser degrees of fibrosis.

In NAFLD, a number of studies have demonstrated the efficacy of TE in distinguishing severity of fibrosis. In a study of 246 adults with NAFLD, TE had an AUROC of 0.84, 0.93 and 0.95 in distinguishing significant fibrosis, severe fibrosis and cirrhosis respectively[82]. A Japanese study demonstrated similar results[105]. A recent report of 52 children with NAFLD has shown an AUROC of 0.977, 0.992 and 1 for distinguishing any, significant and severe fibrosis[106]. Feasibility and reproducibility of transient elastography is an issue when patients have a BMI > 30[107,108]. An XL probe is now available for better accuracy in this scenario[108,109] demonstrating reliable measurements in 73% using the XL probe vs 50% with the S probe[108].

MR may be useful in detection of steatosis as above however the differentiation of patients with advanced disease from those with simple steatosis requires assessment of fibrosis. Similarly to transient elastography, MR elastography (MRE) may be a useful tool in this regard. Kim et al[112] report a comparison of MRE to 6 laboratory based models of fibrosis in 142 patients with liver biopsy-confirmed NAFLD. The cut off for advanced fibrosis in this cohort was 4.15 kPa with an AUROC of 0.954, a sensitivity of 0.85 and specificity of 0.929. They found that MRE could potentially be a useful tool but did not meet the sensitivity or specificity of the NAFLD fibrosis score or the FIB-4 score.

Chen et al[113] studied 58 patients with NAFLD and found that liver stiffness using a threshold of ≥ 2.74 kPA could differentiate patients with NASH from simple steatosis with a sensitivity of 94% and a sensitivity of 73% (AUROC 0.94).

The use of relatively new, high throughput techniques such as proteomics, glycomics and microarray studies in the derivation of panels of biomarkers associated with a disease may also give an insight into pathophysiology of the condition.

Younossi et al[114] found 34 different expression of genes in those with NASH vs controls. Four were confirmed using real time reverse transcription PCR. Sreekumar et al[115] found 16 genes expressed differently in NASH-associated cirrhosis vs other aetiologies; mainly genes which were involved in the anti-oxidant response as well as fat and carbohydrate metabolism. Yoneda et al[116] performed a microarray analysis of NASH vs simple steatosis and found expression of 27 genes at higher levels in NASH. The upregulated gene sets included those responsible for the platelet derived growth factor, hepatic nuclear factor 3 and the smad4 pathways.

Proteomic studies use pattern recognition with subtraction. Several previous studies have reported different protein peaks in the serum of those with NASH vs simple steatosis[117,118]. Two important proteomic studies using liver tissue and serum respectively of adult patients with and without NAFLD revealed an increased expression of lumican, (a keratan sulphate proteoglycan involved in collagen cross-linking and epithelial-mesenchymal transition) in patients with NASH vs normal and simple steatosis[119,120]. Yu et al[121] used proteomics to demonstrate that higher baseline haemoglobin values were associated with the development of NAFLD in a prospective study of 6944 subjects.

Glycosylation is the post-translational modification of secreted proteins with carbohydrate moieties conveying structural diversity and with a possible role in protein folding and in cell to cell interaction including migration, solubility and receptor attachment[122,123]. Changes in glycosylation serve as a particularly good marker of liver dysfunction for a number of reasons. Most glycoproteins in serum (aside from IgG) are made in the liver. Thus, the N-glycome profile will reflect any changes in either the liver or B cell function. In addition, both the asialoglycoprotein receptor and the mannose/O-linked beta-N-acetylglucosamine receptor in liver are important in clearing aberrantly glycosylated proteins from the serum. In the presence of architectural disarray, these receptors are decreased in number and thus there is a build-up of glycoproteins in serum[124]. With a systems biology approach to the analysis using high-throughput technology, serum N-glycomics may prove to be valuable biomarkers of disease.

Previously reported glycomic analysis of liver disease include the development of the GlycoCirrhotest[125], the GlycoFibrotest[126], and the GlycoHCC test[127] which can predict the presence of cirrhosis, fibrosis and hepatocellular carcinoma respectively due to difference in N-glycome patterns. Two recent studies have investigated the potential of Glycomics in non-invasive evaluation of NAFLD[128-130].

Glycomics was also demonstrated to have a tole n biomarker discovery in paediatric NAFLD[131].

In view of the high prevalence of NAFLD in the population, in both adults and children, and the fact that up to a one third will develop end stage liver disease and/or hepatocellular carcinoma, it is important that we develop noninvasive methods to diagnose and monitor this liver condition. A differentiation needs to be made between those with advance disease/or are at risk of developing advanced disease from those who have simple steatosis and are unlikely to progress. Liver biopsy is not a practical tool for this mass screening though the disease is still defined histologically. Noninvasive biomarkers either in blood or imaging techniques show promise in this context and in many centres are used routinely. It is possible that a combination of blood biomarkers with methods such as transient elastography or acoustic radiation force impulse may yield the highest diagnostic discrimination. New techniques such as proteomics and glycomics may not only allow development of novel markers but also allow us a better insight into the pathophysiology of the condition.