Discussion
To the best of our knowledge, this is the first study that compared the performance of clinical scoring systems and serum biomarkers for predicting NASH in patients with biopsy-confirmed NAFLD who have persistent nALT levels. In contrast to the few available retrospective studies that have included patients with NAFLD with nALT levels,6 7 our study involves a relatively large cohort of well-characterized patients with biopsy-proven NAFLD, including also a substantial number of patients with persistent nALT levels.
We found that measurement of either serum CK18-M30 or GP73 levels alone was not able to accurately identify NASH in the subgroup of patients with NAFLD with persistent nALT levels. However, we found that serum CK18-M30 levels can be useful during an initial screening test, and then sequentially combined with a newly developed G-NASH model that includes measurement of serum GP73 and other clinical/biochemical parameters in its equation. Indeed, our results show that the G-NASH model has good diagnostic accuracy in identifying NASH, and by using this model it should be possible to accurately identify those subjects who require a liver biopsy.
Serum ALT levels are the most widely used laboratory test for assessing and monitoring liver injury. Both in China and in many other countries, serum ALT levels are often used by primary care clinicians for referring patients to gastroenterologists/hepatologists for further investigation. However, the fact that increased serum ALT levels can be present only in a proportion of patients with NAFLD (be it simple steatosis or NASH) necessitates finding alternative non-invasive tests to refine specialist referral pathways.7 Previous efforts have been made to discover novel biomarkers or test panels to better distinguish NASH from simple steatosis. Some studies have suggested that measurement of serum CK18-M30 levels may be useful,26 27 although one study has concluded that CK18-M30 measurement alone lacked sufficient sensitivity for diagnosing NASH.28 Similarly, in the present study, we found that a substantial number of patients with persistent nALT (53 out of 105) had NASH on histology, and more importantly serum CK18-M30 levels alone did not have sufficiently high diagnostic accuracy for identifying NASH in this group of patients. Thus, based on these findings, we could make the following inferences: (1) screening for NASH is required in patients with NAFLD who have persistent nALT due to the progressive potential of their liver disease; and (2) serum CK18-M30 measurement alone does not have sufficient sensitivity for detecting the early stages of NASH in this group of patients.
We have also tested the performance of serum GP73 levels in identifying NASH in patients with NAFLD with persistent nALT. A recent study pertaining to hepatocellular carcinoma development showed increased GP73 secretion by hepatocyte endoplasmic reticulum (ER) stress.29 In view of the recognized relevance of ER stress in NAFLD pathogenesis, it is also possible to speculate that upregulation of gene expression and increased serum levels of GP73 may also occur in NASH.30 Additionally, it has been reported that interferon gamma (IFN-γ) was responsible for increased expression of GP73 in hepatocytes,31 and elevated IFN-γ-secreting T helper cell 17 infiltration was also found in the hepatic tissue relative to peripheral blood mononuclear cells.32 Collectively, these findings might, at least in part, explain the significant association we observed between serum GP73 levels and the presence of NASH.
Increased platelet volume may occur with inflammatory states,33 and alterations in serum zinc34 and thyroxine levels35 have been noted to be associated with NAFLD. Zinc is mostly found in skeletal muscle rather than the liver, and fluctuation of serum zinc levels may occur with sarcopenia,36 which has been noted with NAFLD.37 Therefore, since NASH is a proinflammatory state, there is emerging evidence supporting the inclusion of SDPV, serum zinc and thyroxine levels in the prediction model.
In clinical practice, the use of liver biopsy is limited mostly due to its poor acceptability to patients, high costs, and high risk to benefit ratio. Among patients with NAFLD with nALT levels, this issue is even more pertinent, as clinicians are more inclined to investigate only patients with abnALT levels. Since it is known that most of the available non-invasive tests/methods perform poorly in diagnosing NASH, better non-invasive tests are increasingly needed, especially for screening the subgroup of patients with NAFLD with nALT levels, who may be otherwise misdiagnosed as having less harmful disease by non-specialists. In this regard, serum CK18-M30 levels and other non-invasive clinical scoring systems have been previously investigated for their ability to accurately identify NASH in patients with NAFLD and abnALT.27 38 However, these non-invasive clinical scoring systems are complex combinations that include ‘omics’ methodology, which is usually not available in clinical practice.
To resolve this conundrum, we have proposed a sequential non-invasive approach by using serum CK18-M30 measurement first and then applying the G-NASH model. In our cohort of patients with biopsy-confirmed NAFLD, serum GP73 had better sensitivity than serum CK18-M30 alone for the diagnosis of NASH among those patients with persistent nALT, thus suggesting that serum GP73 might be superior to CK18-M30 alone in diagnosing the early stages of NASH. However, it is noteworthy that increased serum CK18-M30 might reflect prominent hepatic necroinflammation, which typically occurs in NASH, but not NAFL.39 Therefore, we propose that serum CK18-M30 measurement alone could be used first to screen out apparent cases of NASH, with subsequent use of the G-NASH model (which includes serum GP73 concentration along with BMI and other laboratory parameters) for accurately differentiating NASH from simple steatosis in the remaining patients. Notably, this proposed combined and sequential approach for non-invasively identifying NASH was effective in reducing unnecessary liver biopsies with an acceptable misdiagnosis rate.
Among the major limitations of our single-center, observational study, we need to mention the lack of an external validation cohort to test our proposed strategy of using a sequential combination of serum CK18-M30 and the G-NASH model for non-invasively identifying patients with NASH among NAFLD with persistent nALT. In addition, serum CK18-M30 and GP73 levels are not measured in routine clinical practice. Our study also included a relatively small number of patients (n=53) with NASH who had persistent nALT. With this small number of subjects we were unable to stratify the analyses by sex in order to test the prediction model in men and women, separately. Patients included in our study were of Chinese Han ethnicity, precluding extrapolation of these results to other ethnic groups. Thus, our results need to be verified in larger studies that also include other ethnic groups.
In conclusion, our results show that ~50% of patients with NAFLD with persistent nALT levels have NASH, and the sequential combination of serum CK18-M30 measurement and the G-NASH model can be used to identify NASH. Not only does this approach accurately identify patients with NASH, but importantly it also reduces the number of patients who may be subjected to an unnecessary liver biopsy.