Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. It is a generic term characterized by the accumulation of fat in the liver and describes a range of clinical phenotypes. Non-alcoholic steatohepatitis (NASH) is the progressive manifestation of NAFLD (indicated by the NAFLD activity score), a diagnosis of NASH, confirmed by the histologic presence of steatosis, hepatocyte ballooning, and lobular inflammation. NASH patients have a worse prognosis and progress to cirrhosis more rapidly than patients with simple steatosis.

In addition, the life expectancy of NASH liver disease patients is associated with an increased risk of mortality and the development of clinical sequelae such as hepatocellular carcinoma and cardiovascular disease. The most important prognostic feature of NASH is the severity of associated liver fibrosis, which is currently assessed by liver biopsy. Progression of fibrosis due to NASH is not a linear process. Patients progress through the various stages of the disease, showing alternating phases of inflammation and fibrogenesis, as well as decreased activity and regression of fibrosis.

How many people have nonalcoholic steatohepatitis? 
Up-to-date, 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 nonalcoholic steatohepatitis treated?
Currently, 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 many mechanisms are currently under investigation as potential treatments for NASH.

How is nonalcoholic steatohepatitis diagnosed?
NAFLD and NASH are mainly diagnosed and monitored by liver biopsy. Hepatic steatosis can be detected by ultrasound. However, the diagnosis NASH can only be confirmed by liver biopsy to determine the pathology of steatohepatitis.

NASH is diagnosed by liver biopsy, which is inherently flawed because of its inherent sampling and observer errors. In addition, performing and evaluating a liver biopsy is a costly and labor-intensive procedure that carries the risk of bleeding. These disadvantages indicate that liver biopsy is not a suitable procedure for mass screening and for staging and risk stratification. Furthermore, as a small snapshot, liver biopsy provides little information about the dynamics of fibrogenesis in the liver or whether the disease is likely to progress rapidly or regress spontaneously in a patient.

Noninvasive biomarkers of liver status, such as NASH, which specifically target the dynamics of liver fibrosis, provide a novel tool to determine a patient's disease progression. Such liver disease biomarkers could be used for risk stratification of patients as well as prognostic enrichment of clinical trials targeting the highest risk patients, thereby increasing the likelihood of success. In addition, such serum biomarkers are direct measures of liver fibrosis dynamics that can be used as determinants of treatment efficacy. Nordic Bioscience offers an accurate liver fibrosis biomarker panel that our non-alcoholic steatohepatitis experts can analyze in our laboratory, such as the biomarker PRO-C3 NASH, which provides information about disease progression.

Serum biomarkers of liver function that specifically target the formation and degradation of collagens, the key components of fibrotic tissue, have a distinct advantage over traditional indirect biomarkers such as alanine transaminase and aspartate transaminase because they directly reflect the dynamics of hepatic fibrogenesis. For example, in our laboratories we can measure the progression of non-alcoholic steatohepatitis (NASH). The combination of a specific protease and protein associated with the pathology yields specific neo-epitopes that can be evaluated using neo-epitope Protein Fingerprint technology.

An example of a neo-epitope biomarker for NASH is PRO-C3 (ELISA or high-precision measurement on the Roche Cobas platform), which differs from PIIINP in that it specifically targets the neo-epitope released upon cleavage of the N-terminal pro-peptide by ADAMTS2. Therefore, PRO-C3 in NASH is considered a marker for true type III collagen formation as well as fibrogenic activity. C3M is a biomarker for type III collagen degradation and can be used to determine the extent of fibrolysis.

Visit our liver biomarker portfolio and choose a panel that fits your clinical research or drug development targets!

Increase in networking forming collagens of the basement membrane produced by endothelial cellsIncrease in networking forming collagens of the basement membrane produced by endothelial cells

Twenty collagen types have been identified in the stroma of the liver, and their localization reflects their function. Fibrillar collagens (such as type I, III, and V collagens) are located around the portal tract and the walls of the central veins and provide structural support. Basement membrane collagens, such as type IV collagen, are found in a network within the walls of the sinusoids.

Liver fibrosis is the most important histologic 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 for the development of cirrhosis and clinical sequelae. By using serum biomarkers of liver fibrosis that target different types of collagens in the liver, it is possible to determine the type of fibrosis that is forming, i.e., pro-reparative (more basement membrane matrix) or progressive (more interstitial matrix), as well as the dynamics driving disease progression.

PRO-C3 biomarker in fibrosis stages and the NAS activity scorePRO-C3 biomarker in fibrosis stages and the NAS activity score

NASH Disease activity and severity of fibrosis are determined by histologic examination. Pathologists usually use the NASH - Clinical Research Network (CRN) criteria. The NAFLD activity score (NAS) is an activity index calculated by summing the scores for macrovesicular steatosis, ballooning, and lobular inflammation.

PRO-C3 was shown by Daniels et al. (2019) to increase gradually from no fibrosis to stage 3 and 4 steatohepatitis, depending on fibrosis and NAS severity in NAFLD patients, indicating that PRO-C3 is a marker of fibrosis severity and disease activity.

Change in the PRO-C3 biomarker after 18 months of therapyChange in the PRO-C3 biomarker after 18 months of therapy

Disease progression and liver damage in NASH patients is characterized by worsening liver fibrosis and liver disease.

Bril et al (2019) have shown that changes in PRO-C3 directly reflect both worsening and improvement of liver fibrosis in NAFLD patients.

PRO-C3 biomarker changes after treatment of aldaferminPRO-C3 biomarker changes after treatment of aldafermin

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

PRO-C3, a marker of collagen type formation III, has been studied as a secondary endpoint in several clinical trials as a determinant of treatment efficacy. Harrison et al. 2020 demonstrated that treatment of NASH patients with aldafermin, an FGF-19 analog, significantly decreased levels 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.

PRO-C3 biomarker changes after treatment of resmetiromPRO-C3 biomarker changes after treatment of resmetirom

High levels of biomarkers of fibrogenesis, such as PRO-C3 in NASH, indicate 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 PRO-C3 levels with treatment with resmetirom (a selective thyroid hormone receptor-β agonist).

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

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