*2.2. Steatohepatitis*

The transition from simple hepatic steatosis to NASH is the most crucial step during the development of severe liver disease with poor prognosis and the higher risk to get fibrosis and progress to end-stage liver disease. Thus, the assessment of NASH and the possibility to distinguish between the dynamic changes from NAFLD to NASH are ongoing challenges. Precise diagnosis still depends on liver biopsy with huge variability between pathologists. For that reason, Bedossa et al. developed the Fatty Liver Inhibition of Progression (FLIP) algorithm, which requires pathologists to follow generalized criteria for scoring. The FLIP algorithm considers histologically steatosis, disease activity and fibrosis scores [50]. Very recently, Canbay and colleagues established a novel machine learning approach to assess the severity of NAFLD and distinguish between NAFLD and NASH. In this study NAFLD was defined as the NAFLD activity score (NAS) ≤ 4 and NASH as NAS ≥ 4. With the help of an ensemble feature selection approach (EFS) they identified age, HbA1c, γ-GT, adiponectin and the apoptosis marker M30 to be the biomarkers highly associated with the prediction of NAFLD. The developed CHeK score, which is available at http://CHek.heiderlab.de is not only able to detect NASH, but also to monitor the development from NAFLD to NASH and can be used to screen patients in a long-term follow up during disease progression or therapy [51].

Besides histological scoring, the development from NAFLD to NASH involves a variety of different molecular, cellular and hormonal changes. Numerous blood biomarker and panels were investigated and developed trying to detect and reflect disease severity and underlying pathways. The apoptosis marker cytokeratin 18 (CK18) is a very well-studied individual blood biomarker so far. NASH patients show a significant increase of plasma CK18 indicating hepatocyte death through apoptosis and necroptosis compared to NAFLD patients [63]. CK18 is the main intermediate filament protein in hepatocytes and is released upon the initiation of cell death [64]. While the whole length CK18 is predominantly released upon hepatic necrosis, caspase cleaved CK18 (M30) is mainly produced by apoptotic cells [65]. Although CK18 is considered to be one of the most promising biomarkers, several studies showed that the sensitivity to predict NASH is 66%, while the specificity is 82% [66,67]. In addition, the ability of M30 to predict NASH and distinguish between NAFLD and NASH was calculated as 0.82 [68]. To increase the reliability of CK18 as a noninvasive biomarker for NASH a study shows that the combination with serum levels of the apoptosis-mediating surface antigen FAS

(sFAS) further increases the accuracy [69]. However, the optimal cut-off serum concentrations still vary between different studies and require further investigation.

NASH is predominantly characterized by pathological alterations in glucose and lipid metabolism. These alterations include modifications in adipokines (such as leptin, adiponectin and resistin) and liver-derived lipid hormones like the fibroblast growth factor 21 (FGF21), which is secreted upon peroxisome proliferator-activated receptor-α (PPARα) activation [70,71]. FGF21 was found to be significantly elevated in patients with mild to moderate hepatic steatosis. Serum levels were directly linked to increased intrahepatic triglyceride accumulation and liver damage [72,73]. However, FGF21 is known to also increase in sepsis and systemic inflammation [74]. Further, adipokines were shown also to reflect visceral adiposity leading to a moderate specificity value of 62% with a specificity of 78% [68]. Further studies even show a drop of FGF21 levels with increasing liver inflammation [75].

The most evident difference between simple steatosis and advanced steatohepatitis is the absence of an inflammatory infiltrate. As a hallmark of NASH, a variety of inflammatory markers are elevated in patients with NASH, while disease is progressing. Increasing serum levels of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and several interleukins such as, IL-6 and IL-8 were proposed as clinical markers. Although, they all correlate with the observed inflammatory status in NASH, none of them reached statistically significant values adjusted by the FDR on univariable analysis to be approved as a diagnostic marker yet because of their insensitivity to NASH specific inflammatory changes.

Recently, the transcription factor forkhead box protein A (FOXA1), also known as hepatocyte nuclear factor 3-α, was described as a potential new biomarker as it is involved in mediating homeostasis and metabolism by targeting genes in liver, adipose tissue and pancreas [76]. Moya et al. could show that FOXA1 acts anti-steatotic by lowering fatty acid uptake and is suppressed in patients with NAFLD and insulin resistance [77]. Therefore, the authors proposed this protein as sensitive noninvasive biomarker of liver fat accumulation, mitochondrial membrane potential and the production of reactive oxygen species (ROS). The limitation coming along with using a transcription factor as biomarker is, that FOXA1 is not secreted into the serum.

Oxidative stress, which is indicated by excessive ROS production, is one of the most important mechanisms underlying the disease pathogenesis of NASH finally leading to lipid oxidation and inflammation [78]. Based on changes in lipid catabolism and de novo lipogenesis the oxNASH score was calculated including the linoleic acid:13-hydroxyoctadecadienoic acid (13-HODA) ratio together with the patient characteristics age, BMI and AST level. This score reached diagnostic accuracy with an AUROC 0.74–0.83 [79]. Because mass spectroscopy is needed for the measurement of the described parameters, the oxNASH score is not commonly used today. In line with biomarkers targeting products, which are secreted due to an altered lipid metabolism, insulin-like growth factor binding protein 1 (IGFBP-1) was recently suggested as a potential serum marker for NAFLD and NAFLD-related fibrosis. It is exclusively upregulated in the liver in response to hepatic inflammation and oxidative stress and regulated by insulin [80]. On this basis, Regué et al. could show that the global deletion of the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2 or IGF2 mRNA-binding protein 2, IMP-2) lead to a resistance to obesity and fatty liver in mice treated with a high fat diet (HFD) due to reduced adiposity [81]. A limitation of those markers is that elevations might be not exclusively related to NAFLD-induced conditions, but also the metabolic syndrome and insulin resistance in general. Anyhow this is an interesting starting point for future investigations also in regard to therapeutic interventions and the understanding of the mechanisms that lead to steatosis.

The expression of ferritin is generally known to be increased in patients with NAFLD and metabolic syndrome. It was further shown to be independently associated with increased steatosis grades, NASH and NASH fibrosis with an AUROC of 0.62 [82,83]. This accuracy can be increased to an AUROC of 0.81 when including AST, BMI, type 2 diabetes, presence or absence of hypertension and platelet count to ferritin levels [84]. The broad and long-lasting search for novel biomarkers to diagnose NASH, which are modestly accurate, show the multiple factors involved in NAFLD and the complexity of disease mechanisms. To date the combination of several biomarkers drastically increases diagnostic preciseness. Especially for NASH, panels like the Nash Test (NT) include baseline patient characteristics such as age, gender, height, weight and serum levels of triglycerides, cholesterol, transaminases, total bilirubin, α2-macroglobulin, haptoglobin, apolipoprotein A1, γ-GT [85].

Overall, most of the actual biomarkers and panels need further validation on cohorts with patients, including several different ethnicities and various starting points and outcomes. Up to now most validation studies work with patients undergoing bariatric surgery. Also choosing the best cut-off value for the specific serum markers is still not optimal. This points to the urgent need of basic research studies to help better understanding the underlying mechanisms and key molecules involved in the development of NAFLD and progression to NASH and end-stage liver disease.
