- Top of page
- Patients and Methods
- Supporting Information
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide.[1-3] NAFLD encompasses a wide disease spectrum from simple steatosis to non-alcoholic steatohepatitis (NASH). Although simple steatosis is largely benign, patients with NASH have active liver injury and may progress to cirrhosis, liver failure and hepatocellular carcinoma.[4-6] Increased overall and liver-specific mortality is also observed mainly in NASH patients.[7, 8] Thus, it is important to distinguish between simple steatosis and NASH.
Liver biopsy is the gold standard for diagnosing NAFLD and NASH. However, it is an invasive procedure which might result in major complications like haemorrhage, and may not be acceptable to some patients. It is also not a suitable method for repeated assessments. Reliable non-invasive tests of NASH are urgently needed.
Several biomarkers have been developed to differentiate NASH from simple steatosis.[9-16] Among them, blood cytokeratin-18 (CK-18) fragment, CK18Asp396 neo-epitope (M30) has been validated in different cohorts.[9, 17-20] M30 is a fragment which is cleaved by caspases during cell apoptosis from CK-18, a major intermediate filament protein in hepatocytes. Detecting serum M30 level reflects the degree of hepatocellular apoptosis, a characteristic feature of NASH.[21, 22] Our previous study revealed that M30 has high overall accuracy in differentiating NAFLD from control subjects and moderate accuracy in differentiating NASH from simple steatosis.
Recent studies suggest that other cell death markers may also be useful in the prediction of NASH. M65 and M65ED, which detect both caspase-cleaved and uncleaved CK-18, reflect total cell death including apoptosis and necrosis. Joka and colleagues suggested that M65/M65ED may have superior performance to M30 in detecting mild fibrosis and steatosis. However, that study was limited by the inclusion of different liver diseases and the small number of NAFLD patients. As such, the performance of M65 and M65ED in detecting NASH compared with M30 is currently unknown. In addition, limited by cross-sectional design, previous studies could not address whether these biomarkers can be used for serial monitoring.
In this study, we aimed to evaluate the performance of serum CK-18 M30, M65 and M65ED in assessing NAFLD in a large histological cohort. We also aimed to test the performance of these biomarkers in predicting histological changes in 36 months.
- Top of page
- Patients and Methods
- Supporting Information
In this study, we demonstrated that apoptosis biomarker CK-18 M30 and total cell death biomarkers M65/M65ED had similar accuracy in predicting NAFLD and NASH. M65 and M65ED were superior in differentiating minimal and moderate steatosis grade. Changes of M30 and M65ED were associated with disease progression. Changes of M65ED may also predict fibrosis progression.
Apoptosis and necrosis are both important modes of cell death in liver disease. While necrosis is the typical consequence of acute injuries such as ischemia/reperfusion or acute drug-induced hepatotoxicity, apoptosis represents programmed cell death. Apoptosis was thought to be a characteristic feature of NASH.[21, 22] However, Joka and colleagues suggested recently that necrosis might also contribute to the liver damage in NAFLD and NASH by showing biomarkers detecting both apoptosis and necrosis might be superior to detecting apoptosis alone. However, this study included patients with different liver diseases, and NAFLD patients were underrepresented. The role of different cell death markers was also discussed in other liver diseases, such as HBV infection and acute liver failure.[34, 35]. A study in US suggested that the overall diagnostic accuracy of M65 for NASH was higher than that of M30, but that was not confirmed by another study in Turkey. Nevertheless, the correlation between the biomarkers and individual histological features of NAFLD, in particular the evaluation of mild disease, was not reported. In the current study, we confirmed that M30, M65 and M65ED had similar overall accuracy in predicting NASH. M65 and M65ED had higher discriminating power in detecting mild steatosis and fibrosis. As studies on biopsy-proven NAFLD usually include patients with risk factors of advanced disease such as metabolic syndrome, the proportion of patients with NASH is relatively high. When the biomarkers are applied to primary care setting, the NPV in excluding NASH will be even higher.
Our study was unique in including a subgroup of patients with paired liver biopsies 36 months apart. We were therefore able to evaluate the performance of the biomarkers as a monitoring tool for NAFLD patients. The changes in all three biomarkers correlated well with changes in NAS and could be used to predict progression to NASH. Changes in M65 and M65ED were also associated with progression of liver fibrosis. Notably, the AUROCs for predicting disease progression were both higher than 0.8 for M30 and M65ED. These results could be promising as non-invasive tests are more acceptable for long-term and repeated monitoring of disease progression. Importantly, while the changes in CK-18 correlated with histological changes, baseline CK-18 level alone failed to predict disease progression. This indicates that NASH is a dynamic process. Change in disease activity is possible with lifestyle modifications.[4, 37, 38] Therefore, serially performing these biomarkers may be helpful to monitor disease progression.
While the cell death markers are elevated in NAFLD and particularly in NASH patients, it should be noted that they are nonspecific markers that may be elevated in other situations such as chronic hepatitis B and acute liver failure.[34, 35] However, the diagnosis of NAFLD is usually clinically apparent by the time these cell death markers are applied in real-life practice. In addition, the diagnostic accuracy of these biomarkers for NASH remains modest. Future work is needed to develop new biomarkers and evaluate the possibility of using multiple biomarkers as in the case of fibrosis testing.[39, 40]
Our study had several limitations. First, we used liver biopsy as the reference standard, which might suffer from sampling bias. Future studies are required to test these biomarkers against other reference standards such as clinical outcomes. Second, no liver biopsy was performed in control subjects. However, biopsy on healthy people is unethical. Instead, liver disease was excluded in controls stringently by history, laboratory tests and 1H-MRS. Third, some biopsy samples were shorter than 2 cm. However, after excluding samples with short biopsy length, the AUROCs of M30, M65 and M65ED in predicting NASH were 0.67 (0.54–0.81), 0.75 (0.62–0.87) and 0.73 (0.60–0.86) respectively. The performance was similar to the whole study population. Fourth, the paired liver biopsies cohort was relatively small, this resulted the wide statistical variations; a larger prospective longitudinal study should be conducted to further clarify the utility of cell death biomarkers in disease progression prediction. Finally, CK-18 ELISA assays from different companies have not been systematically compared. Our findings cannot be directly extrapolated to other assays. However, we chose the assays that were used in recent similar studies to facilitate comparison across studies.[9, 24]
In conclusion, apoptosis marker CK-18 M30 and total cell death marker M65/M65ED have similar high diagnostic performance in NAFLD and similar moderate accuracy in predicting NASH. Changes in the biomarkers also correlate with histological progression. However, development of new biomarkers is still required to improve the diagnostic accuracy.