In this a historical cohort study, patients residing in Olmsted County, Rochester, Minnesota, United States who had been diagnosed with NAFLD-fatty liver (HICDA Code 05710420), fatty liver hypertrophy (HICDA Code 05710421), fatty liver cirrhosis (HICDA Code 05710422), fatty liver steatohepatitis (HICDA Code 05710423) or NASH (HICDA Code 05710431), or fatty liver steatohepatitis (HICDA Code 05710-42-43) or steatosis (HICDA Code 02790-44-1) between January 1, 1980 and January 1, 2000 were drawn from the Rochester Epidemiology Project (REP) master diagnostic index. The REP index is a unique database system of medical diagnoses of the population living in Olmsted County, Minnesota[13]. Although fatty liver was recognized prior to 1980, this liver condition was better characterized in 1980[14]; therefore, we chose to identify patients after this date.

From the 479 patients with NAFLD assessed, 302 patients (63%) greater than 18 years old were included. All of these patients were followed, and their medical charts were reviewed, until August 31, 2009 or the date when the first primary endpoint occurred. By using a standardized case record form, we recorded a detailed history and physical examination and use of statins and metformin during the follow-up period.

We excluded NAFLD patients who lacked the data needed for the NFS calculation, patients with pre-existing poor outcomes including overt CHD or overt liver complications at the time of NAFLD diagnosis and patients with duration of follow-up of less than 5 years. One hundred seventy-seven NAFLD patients were excluded due to missing the data needed for the NFS calculation (n = 95), overt CHD confirmed at baseline (n = 63), liver cirrhosis with complications confirmed at baseline (n = 11) and duration of follow-up of less than 5 years (n = 8).

The diagnosis of NAFLD was based on a liver biopsy showing steatosis in at least 5% of hepatocytes or fatty infiltration of the liver confirmed by imaging study (ultrasound, computed tomography, or magnetic resonance imaging) and the exclusion of liver disease of other etiologies, including alcohol-induced liver disease (history of excessive alcohol consumption greater than 20 gm/d), drug-induced liver disease, autoimmune or viral hepatitis and cholestatic or metabolic/genetic liver disease[1]. Cirrhosis was defined based on the pathological term for the chronic liver diseases[15]. In our study, 85% of patients (n = 256) were diagnosed by liver imaging. Liver biopsy was performed in 46 patients (15% of 302 patients). The staging of fibrosis was divided into fibrosis stage 0 to stage 4 using the Brunt criteria[16]. The NAFLD patients with a histological liver fibrosis stage of 1-2 were classified as “mild liver fibrosis,” and those with a histological fibrosis stage of 3-4 were classified as “advanced liver fibrosis”[16].

The primary endpoints were all-cause mortality, cardiac complications, and/or liver complications. Cardiac complications included new-onset CHD events as recorded in the medical records, defined as congestive heart failure, unstable angina, myocardial infarction, flow-limiting stenosis from angiography or angina requiring revascularization during the follow-up period and need for hospitalization[17]. Liver complications were diagnosed by clinical signs and symptoms[18], including the presence of ascites, variceal bleeding, a severe grade of hepatic encephalopathy, liver failure or hepatocellular carcinoma, that occurred during the follow-up and required hospitalization with or without death[19]. All causes of death listed on the death certificates or pathological findings (underlying, intermediate, immediate and other major conditions) were recorded using the 10^th revision of the International Classification of Diseases (ICD-10). The presence of metabolic syndrome (MetS) was defined by using the 2001 National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII) criteria and the new definition, which requires the presence of at least three of the five features[20,21]. The NFS is composed of 6 variables, including age, hyperglycemia, BMI, platelet count, albumin, and AST/ALT ratio as independent indicators of advanced liver fibrosis[9]. NAFLD fibrosis score = -1.675 + 0.037 × age (year) + 0.094 × BMI (kg/m²) + 1.13 × IFG/diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio - 0.013 × platelet count (×10⁹/L) - 0.66 × albumin (g/dL)[9].

In this study, the NFS was used to classify advanced liver fibrosis into 2 categories[9]. NAFLD patients with a score less than -1.5 were classified as “low probability of advanced liver fibrosis,” and those patients with a score of at least -1.5 were classified as “intermediate or high probability of advanced liver fibrosis”[9]. Because the information required for determination of the NFS was not always available on the same day, the scores were calculated at the time of NAFLD diagnosis using data from the medical records from visits within 3 mo of the “true” NAFLD diagnosis date and the last follow-up date. If more than one assessment for a given variable was available in the medical records during this time period, the value closest to the “true” follow-up date was used for the NFS calculation[9]. According to the 3 subgroups of low, intermediate and high probability of fibrosis, we used parameters of NFS < -1.5 for low, NFS ≥ -1.5 to NFS < 0.67 for intermediate and NFS ≥ 0.67 for high probability of fibrosis at baseline.

At the end of the follow-up period, we classified the patients into 3 subgroups according to the progression pattern of the liver fibrosis by comparing the NFS at baseline to the end of follow-up. The first group had stable fibrosis, defined as stability of the NFS during the follow-up. The second group had regression of fibrosis, defined as a reduction of the NFS to a milder stage of fibrosis during the follow-up. Lastly, the third group had fibrosis progression, defined as an increase of the NFS to a more advanced stage during the follow-up.

We assumed that the overall mortality rate in NAFLD would be 12%, with liver-related complications being observed in at least 3% and cardiac complications in 11% of patients based on results from a previous study[4]. We anticipated that at least 150 patients, or 50% of the total cohort, would be classified as “low probability of advanced liver fibrosis” using the NFS, and at least 150 would be classified as “intermediate or high probability of advanced liver fibrosis”[12]. However, a portion of patients were expected to develop new CHD events, liver complications and death. We anticipated that 15% of patients would be identified as having experienced a primary endpoint. We assumed that, at most, 10% of patients with a low probability of advanced liver fibrosis would experience events within 5 years and at least 20% of patients with an intermediate or high probability of advanced liver fibrosis would experience events within 5 years to obtain a statistical power of at least 82%.

Patients were categorized by the NFS into 2 groups of probability of advanced liver fibrosis. Differences between the primary endpoints of the two groups of low and high NFS were compared by using the χ² test. Differences between NAFLD patients with and without primary endpoints were tested by independent t tests for continuous variables and were tested by the χ² test for proportions. Continuous outcomes were presented as the mean ± SD, and categorical data were presented as numbers (percentage). Logistic regression analysis was used to identify the factors significantly associated with death among NAFLD patients. Only those variables with a P value < 0.1 by univariate analysis were included in multivariate analysis. To avoid overestimation of the model, we excluded those variables used as a part of the NFS calculation. We estimated receiver operating characteristics of related variables for the predicting of death to maximize the area under the curve (AUC). Two-sided P values < 0.05 were considered statistically significance. The rate of NFS change in each patient was calculated by the difference of NFS at the end of follow-up and at baseline divided by the duration of the follow-up time (Δ NFS/Δ time). Both phases of the study used the SPSS statistical software package (SPSS Version 15.0.1.1, Windows VISTA, July 3, 2007) for analysis. The study was approved by the Institutional Review Board (IRB) of the Mayo Clinic, Rochester, MN, United States, and all participants provided permission for their medical information to be used for research.

This study included three hundred two NAFLD patients aged 47 ± 13 year (range 21-86 year), of which 95% were white, and 44% were male. Obesity was present in 73%, and a history of diabetes mellitus type II and hypertension was observed in 16% and 41%, respectively. Eighty-five percent of patients (n = 256) were diagnosed by liver imaging. Liver biopsy was performed in 46 patients (15%). Mild liver fibrosis (stage F 0-2) was found in 34 patients (74%), while advanced fibrosis (stage F 3-4) was found in 12 patients (26%). NAFLD patients who underwent a liver biopsy had a significantly lower diastolic blood pressure, a lower BMI and a higher AST level than those without a liver biopsy (P < 0.05). The characteristics of the 302 patients based on the degree of advanced liver fibrosis estimated by the NFS at baseline are shown in Table 1. A low probability of advanced liver fibrosis (NFS < -1.5) was found in 60% of patients, while an intermediate or high probability of advanced liver fibrosis (NFS ≥ -1.5) was found in 40%. The mean ± SD value of NFS in patients with a low probability of advanced liver fibrosis was -2.6 ± 0.8 and was lower than in patients with an intermediate or high probability of advanced liver fibrosis (-0.4 ± 0.9, P < 0.0001). Patients with a low probability of advanced liver fibrosis had a lower CHD risk at baseline, as estimated by the Framingham risk score (FRS) calculation, compared to patients with an intermediate or high probability of advanced liver fibrosis (14% vs 19%, P = 0.003 ).

The mean follow-up of the total cohort was 11.9 ± 3.9 years for a total of 3594 person-years. Approximately 47% of patients with a low probability of advanced liver fibrosis at baseline progressed to an intermediate or high probability of advanced liver fibrosis at the end of follow-up, while 94% of patients with an intermediate or high probability of advanced liver fibrosis remained in the same group. At the end of the follow-up, patients with an intermediate or high probability of advanced liver fibrosis had a significantly higher BMI, more frequent diabetes, and more frequent histories of hypothyroidism, cholecystectomy and obstructive sleep apnea. Furthermore, the patients with an intermediate or high probability of advanced liver fibrosis were more likely to use metformin, glitazones and aspirin compared to those with a low probability of advanced liver fibrosis (P < 0.05).

A history of diabetes at baseline was found in 16%, while the proportion of patients with NAFLD who developed impaired fasting glycemia or diabetes during the follow-up was 89 patients (29%) with an average interval of 6.3 ± 4.2 year (range 0.2-17.2) from the diagnosis of NAFLD. Patients who developed impaired fasting glycemia or diabetes during the follow-up were more frequent male, had less hypertension, and had a lower NAFLD Fibrosis Score at baseline compared to those with diabetes at baseline (P < 0.05). The primary endpoints of both groups were not significantly different.

Patients with an intermediate or high probability of advanced liver fibrosis had a significantly higher glucose and a higher AST/ALT ratio at the end of follow-up compared to those with a low probability of advanced liver fibrosis. Hematocrit, platelet count, AST, ALT, albumin, cholesterol and low-density lipoprotein (LDL)-cholesterol level were significantly lower in patients with an intermediate or high probability of advanced liver fibrosis compared to those with a low probability of advanced liver fibrosis (Table 2). At the end of follow-up, 55 patients developed primary endpoints, including death or the presence of CHD or liver complications. Thirty of these patients had new-onset CHD, whereas 8 of 30 patients (27%) died from CHD-related complications during the follow-up period (Table 2). Patients with new CHD events (n = 30) were significantly older and had a higher systolic blood pressure (SBP), FRS at baseline, calculated % CHD risk at baseline, and NFS at the end of follow-up and exhibited a lower ALT than those patients without new CHD events (n = 272) (P < 0.05). In addition, the NFS at baseline was similar in patients with and without new CHD events (-1.2 ± 1.6 vs -1.8 ± 1.4, P = 0.07). Liver complications occurred in 6 patients, 5 of whom (83%) died during the follow-up. The liver complications included massive ascites requiring abdominal paracentesis (n = 3), hepatopulmonary syndrome and hepatocellular carcinoma (n = 1 each).

A total of 39/302 (13%) patients died during the follow-up period. The leading causes of death were non-hepatic malignancy (n = 13/39; 33.3%), CHD (n = 8/39; 20.5%), and liver related mortality (n = 5/39; 12.8%). The other 13 patients (33.3%) died from various causes (Table 3). The primary types of cancers were gastric cancer (n = 2), colon cancer (n = 2), pancreatic cancer (n = 2), breast cancer (n = 2), leiomyosarcoma of uterus (n = 1), diffuse B cell lymphoma (n = 1), endometrial cancer (n = 1), lung cancer (n = 1) and unknown primary cancer with liver metastasis (n = 1). Additionally, our study showed a significant, graded relationship between the NAFLD fibrosis score, classified into 3 subgroups (low, intermediate and high probability of liver fibrosis), and the occurrence of primary endpoints, as shown in Table 4.

Patients who died (n = 39) were significantly older with more frequent diabetes and had a higher SBP, NFS at baseline, FRS, glucose, and a lower diastolic blood pressure, ALT and albumin (Table 5). Moreover, they had greater NFS changes per year than those who survived (n = 263, P <0.05). Three models of multivariate analysis were used to identify the best fit model for predictors of death and are illustrated in Table 6. In model 1, we added 9 variables, including gender, systolic blood pressure, diastolic blood pressure, NFS at baseline, use of metformin, use of simvastatin, use of aspirin, presence of new-onset CHD and new-onset liver complications, without interaction among these variables. Model 2 included 10 variables, gender, systolic blood pressure, diastolic blood pressure, NFS at baseline, NFS changes per year, use of metformin, use of simvastatin, use of aspirin, presence of new-onset of CHD and new-onset of liver complications without interaction among these variables. Finally, model 3 added the interaction between NFS at baseline and NAFLD NFS changes per year with the use of aspirin, metformin, and simvastatin into model 2. We did not add the FRS into these models due to the repetition of several variables in the NFS and the FRS. Model 3 was the best fit model, which found that a higher NFS at baseline and more frequent new-onset CHD were significantly predictive of death (OR = 2.6 and 9.2, respectively; P < 0.0001). The use of metformin or simvastatin for at least 3 mo during the follow-up were associated with fewer deaths in patients with NAFLD (OR = 0.2 and 0.03, respectively; P < 0.05).

Table 7 showed the results of the comparison of NAFLD patients with and without death after excluding those with established type 2 diabetes at baseline (n = 254). After excluding patients with established type 2 diabetes at baseline, we found that a higher NFS at baseline and higher alkaline phosphatase remained significantly predictive of death (OR = 1.9 and 1.006; P <0.0001 and 0.012, respectively) as shown in Table 8. Additionally, Table 9 showed that non-diabetic patients with intermediate or high probability of liver fibrosis had a significantly higher rate of primary end point and all-cause death than those patients with low probability of liver fibrosis.

Using the ROC curves to predict death, we found that a baseline NFS of -0.9 was the best cutoff value with a sensitivity of 62%, specificity of 76%, positive predictive value of 28%, negative predictive value of 93% and AUC of 0.7 (Figure 1).

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Figure 1 Presence of death estimated by the nonalcoholic fatty liver disease fibrosis score at baseline. Using the receiver operating characteristics curve, the nonalcoholic fatty liver disease fibrosis score at baseline of -0.9 was the best cutoff value for predicting death based on a sensitivity of 62%, specificity of 76%, positive predictive value of 28%, negative predictive value of 93% and area under the curve of 0.7.

The median rate of annual NFS change for all patients was 0.1 (IQR of 0.02, 0.13). The rate of annual NFS change in patients with an intermediate or high probability of advanced liver fibrosis was significantly lower than in patients with a low probability of advanced liver fibrosis (0.06 vs 0.09, P = 0.004). The annual NFS change in patients who died was significantly higher than those in patients who survived (0.14 vs 0.07, P = 0.03). At the end of the follow-up, we classified the patients into 3 subgroups according to the progression pattern of the liver fibrosis by comparing the NFS at baseline to that at the end of follow-up. Most patients were in the stable-fibrosis (60%) and progressive-fibrosis (37%) groups, whereas only 3% were in the regressive-fibrosis group. The annual NFS change in the progressive-fibrosis group and the stable-disease group was 0.20 ± 0.02 and 0.05 ± 0.08, respectively (P = not significant).

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in Western countries. During an average 7.6 years of follow-up, 13% of the patients died, mainly from malignancy, coronary heart disease (CHD) and liver-related mortality.

Another study revealed that the survival of patients with nonalcoholic steatohepatitis was reduced and that these patients died significantly more often from CHD and liver-related causes. Patients with more advanced liver fibrosis tend to have more liver complications than those without liver fibrosis.

A study from Japan validated the NAFLD Fibrosis Score and found it to have an acceptable sensitivity, specificity, and positive and negative predictive values for advanced liver fibrosis of 100%, 83%, 63%, and 100%, respectively.

In this manuscript, the authors intend to evaluate whether the NAFLD fibrosis score could act as a prognostic predictor for mortality and liver complications among NAFLD patients. It is suggested that the NAFLD fibrosis score is a simple, non-invasive method for predicting prognosis in patients with NAFLD. In general, this paper is significant and of clinical importance.