Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome, it is an umbrella term that is characterized by the accumulation of fat within the liver and describes a range of clinical phenotypes. Non-alcoholic steatohepatitis (NASH) is the progressive manifestation of NAFLD, a diagnosis of NASH which is confirmed by the histological presence of steatosis, hepatocyte ballooning and lobular inflammation. NASH patients have a worse prognosis and progress towards cirrhosis at a faster rate compared to those with simple steatosis. Furthermore, NASH is associated with an increased risk of mortality and the development of clinical outcomes such as hepatocellular carcinoma (HCC) and cardiovascular disease (CVD). The key prognostic feature of NASH is the severity of associated liver fibrosis, which is currently evaluated by liver biopsy. Fibrosis progression due to NASH is not a linear process, patients fluctuate through the many stages of the disease, exhibiting alternating phases of inflammation and fibrogenesis and reduced activity and fibrosis regression.

How many have non-alcoholic steatohepatitis? 
The global prevalence of NAFLD is estimated to be around 24% and is set to increase in parallel with obesity and type II diabetes. Of those with NAFLD, 20% have the progressive subtype NASH.

How is non-alcoholic steatohepatitis treated?
At present, there is no approved therapy for the treatment of NASH. Pioglitazone and vitamin E have shown limited efficacy and are used off-label to treat patients. Over 50 compounds targeting a plethora of mechanisms are currently under investigation as a potential treatment for NASH.

How is non-alcoholic steatohepatitis diagnosed?
NAFLD and NASH are primarily diagnosed and monitored through the use of a liver biopsy. Hepatic steatosis can be determined through ultrasound; however, a diagnosis of NASH can only be confirmed through the use of a liver biopsy.

NASH is diagnosed through the use of a liver biopsy, which is an inherently flawed modality due to its intrinsic sampling and observer error. Furthermore, performing and evaluating a liver biopsy is a costly and labor-intensive procedure that has a risk of bleeding. Collectively these drawbacks indicate that the liver biopsy is not a suitable modality for mass screening and for staging and risk stratification. Additionally, the small-sized snapshot nature of the liver biopsy provides little information about the dynamics of liver fibrogenesis, or whether a patient is likely to rapidly progress in disease severity or spontaneously regress.

Non-invasive biomarkers specifically targeting the dynamics of liver fibrosis provide a novel tool through which patient trajectory can be determined. Such biomarkers could be used to risk-stratify patients, as well as for prognostic enrichment of clinical trials targeting the most at need and at-risk patients thereby increasing the likelihood of success. Furthermore, as such markers are direct measurements of the dynamics of liver fibrosis that may be used as determinants of treatment efficacy.

Serum biomarkers that specifically targets the formation and degradation of collagens, the key component of fibrotic tissue, have a conspicuous advantage over traditional indirect biomarkers, such as alanine transaminase (ALT) and aspartate transaminase (AST), as they directly reflect the dynamics of hepatic fibrogenesis. The combination of a specific pathology associated protease and a protein yields specific neo-epitopes that can be exploited by neo-epitope Protein Fingerprint™ technology. An example of a neo-epitope biomarker is PRO-C3, which is distinct from PIIINP in that it specifically targets the neo-epitope exposed when ADAMTS2 cleaves off the N-terminal pro-peptide. Therefore, PRO-C3 is considered a marker of true type III collagen formation as well as fibrogenic activity. C3M is a biomarkers of type III collagen degradation and can be used to determine the level of fibrolysis.

20 types of collagen have been identified in the liver, their localization reflects their function, fibrillar collagens (such as type I, III, and V collagen) are located around the portal tract and the walls of the central veins providing structural support. BM collagens, such as type IV collagen, can be found in a network within the walls of the sinusoids. Liver fibrosis is the key histological feature of NASH, the early stages of NASH-fibrosis are characterized by the development of reparative basement membrane rich collagen fibrils around hepatocytes in a “chicken-wire” pattern. The later stages of NASH-fibrosis are characterized by the development of interstitial matrix rich bridging septa and an increased risk of developing cirrhosis and clinical outcomes. By using biomarkers targeting different types of collagens within the liver, one can determine the nature of the fibrosis being formed, i.e. pro-reparative (more basement membrane matrix) or progressive (more interstitial matrix) as well as the dynamics driving disease progression.

NASH disease activity and fibrosis severity are established via histological evaluation, pathologists typically use the criteria outlined by the NASH-Clinical Research Network (CRN). An activity index is calculated through the summation of the scores given for macrovesicular steatosis, ballooning and lobular inflammation, referred to as NAFLD activity score (NAS) activity score. PRO-C3 was shown by Daniels et al (2019) to increase in a stepwise manner according to fibrosis and NAS severity in NAFLD patients demonstrating that PRO-C3 is a marker of fibrosis severity and disease activity.

Disease progression in NASH patients is characterised by a worsening of liver fibrosis, Bril et al (2019) demonstrated that changes in PRO-C3 directly reflected both worsening and improvement of liver fibrosis in NAFLD patients.

Protein Fingerprint biomarkers allow pharmacodynamic profiling of novel treatments by measuring protein formation and degradation fragments directly in a serum sample.

PRO-C3, a marker of type III collagen formation, has been evaluated as a determinant of treatment efficacy in multiple clinical trials as a secondary endpoint. Harrison et al 2020 demonstrated that treatment of NASH patients with aldafermin, a FGF-19 analogue, significantly reduced the level of PRO-C3.

Harrison et al showed at AASLD 2019 that aldafermin reduced the ratio of fibrogenesis (PRO-C3) to fibrolysis (C3M, a marker of type III collagen degradation) in NASH patients.

High levels of fibrogenesis biomarkers, such as PRO-C3, are indicative of high disease activity and can be used to enrich clinical trials with patients likely to respond to therapy. Harrison et al demonstrated at EASL 2018 that patients with PRO-C3 greater than or equal to 17.5 ng/mL had a significant reduction in their PRO-C3 level due to resmetirom (a selective thyroid hormone receptor-β agonist) treatment.

Similar predictive results for Resmetirom were observed by Madrigal Pharmaceuticals at Global NASH Congress 2020 in the extension study when using a baseline level of ≥14 ng/mL PRO-C3. Consequently, PRO-C3 levels >14 ng/mL have been listed as inclusion criteria for the phase III NAFLD clinical trial MAESTRO-NAFLD1 (NCT04197479).

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