Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease affecting approximately 25% of the global population and is commonly recognized as the hepatic manifestation of the metabolic syndrome. The histological spectrum of NAFLD ranges from isolated steatosis to non-alcoholic steatohepatitis (NASH), with risk of developing fibrosis and subsequent cirrhosis and hepatocellular carcinoma. The gold standard for diagnosing NAFLD is liver biopsy. However, because of its invasive nature, several non-invasive methods have been developed and validated in evaluating fat and fibrosis in patients with NAFLD.

Liver fat content can be assessed using various methods. The conventional histopathological method consists of a visual semiquantitative approach in which the pathologist uses a four-point scale: grade 0 corresponds to fat deposition in <5% of hepatocytes and grade 1−3 (which is needed for the diagnosis of NAFLD) corresponds to ≥5%. An alternate approach is to quantitatively assess steatosis using stereological point counting (SPC) – which rely on liver biopsy. However, in vivo proton magnetic resonance spectroscopy (1H-MRS) is a reliable noninvasive method that can be used to quantitatively assess total hepatic lipid content, or proton density fat fraction (PDFF).

In Paper I we compared the conventional semiquantitative histological method (grade 0-3) with SPC and 1H-MRS. We found a strong positive correlation between 1H-MRS and SPC, whereas the correlations between 1H-MRS or SPC and histopathological grading were substantially weaker. Using the widely used cut-off value of PDFF ≥5%, all participants were found to have steatosis (specificity 100%, sensitivity 53%). Reducing the cut-off value to 3% maintained 100% specificity while increasing sensitivity to 79%.

In Paper IV we evaluated quantitative steatosis, by SPC, in 106 biopsy-proven NAFLD patients during a 20-year follow-up. SPC was independently associated with an increased risk of all-cause mortality and development of T2DM. Moreover, in the 59 patients with sequential biopsies (approximately 10 years apart), a reduction of quantitative hepatic steatosis decreased the all-time risk of developing T2DM.

NASH is commonly seen as a histological feature portending a worse prognosis in NAFLD. Interestingly, no dual biopsy study has ever shown that NASH predicts fibrosis progression. Yet, NASH is seen as a surrogate marker in pharmaceutical trials – were resolution in NASH is equivalent to future resolution of fibrosis.

In Paper II we conducted a long-term follow-up study (20 years) in a large cohort of biopsy-proven NAFLD patients (n=646), in a collaboration with Karolinska Institute. We could not ascertain that NASH had any effect on all-cause, or disease-specific mortality. However, higher stages of fibrosis predicted all-cause and disease specific mortality. In Paper III, we present 129 biopsy-proven NAFLD patients, in which we had prospective, longitudinal data. They were included between 1988 and 1993. All patients alive, were re-invited 2003-2005 and 2013-2015. Dual biopsies were present in 68 patients, and three consecutive biopsies were available in 33 patients. Results showed that NAFLD is a highly heterogeneous disease, with 9.3% developing end-stage liver disease and 16% progressing to advanced stages of fibrosis without any clinically significant baseline data predicting disease progression. In summary, when using 1H-MRS as a diagnostic method for NAFLD, the diagnostic cut-off should be reduced from 5% to 3%. Furthermore, quantitative amount of hepatic steatosis could be used to stratify patients with NAFLD related to future risk of developing T2DM. Moreover, we have shown that NASH does not predict future all-cause or disease-specific mortality nor end-stage liver disease, therefore a different surrogate marker should be used in clinical trials when assessing NAFLD improvement, so to not imbue false reliance in new therapies. Lastly, we have shown that NAFLD has a more dismal prognosis than previously reported, and that it is unexpectedly difficult to predict fibrosis progression in individual NAFLD patients, emphasizing the need for robust non-invasive biomarkers suitable to monitor large number of patients.