Hepatic steatosis, the accumulation of lipids in. Long-Term Follow-up of Patients With NAFLD and Elevated Liver Enzymes

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1 Long-Term Follow-up of Patients With NAFLD and Elevated Liver Enzymes Mattias Ekstedt, 1 Lennart E. Franzén, 2 Ulrik L. Mathiesen, 3 Lars Thorelius, 4 Marika Holmqvist, 5 Göran Bodemar, 1 and Stergios Kechagias 6 Nonalcoholic fatty liver disease (NAFLD) is the most common cause of elevated liver enzymes in patients of developed countries. We determined the long-term clinical and histological courses of such patients. In a cohort study, 129 consecutively enrolled patients diagnosed with biopsyproven NAFLD were reevaluated. Survival and causes of death were compared with a matched reference population. Living NAFLD patients were offered repeat liver biopsy and clinical and biochemical investigation. Mean follow-up (SD) was 13.7 (1.3) years. Mortality was not increased in patients with steatosis. Survival of patients with nonalcoholic steatohepatitis (NASH) was reduced (P.01). These subjects more often died from cardiovascular (P.04) and liver-related (P.04) causes. Seven patients (5.4%) developed end-stage liver disease, including 3 patients with hepatocellular carcinoma. The absence of periportal fibrosis at baseline had a negative predictive value of 100% in predicting liver-related complications. At follow-up, 69 of 88 patients had diabetes or impaired glucose tolerance. Progression of liver fibrosis occurred in 41%. These subjects more often had a weight gain exceeding 5 kg (P.02), they were more insulin resistant (P.04), and they exhibited more pronounced hepatic fatty infiltration (P.03) at follow-up. In conclusion, NAFLD with elevated liver enzymes is associated with a clinically significant risk of developing end-stage liver disease. Survival is lower in patients with NASH. Most NAFLD patients will develop diabetes or impaired glucose tolerance in the long term. Progression of liver fibrosis is associated with more pronounced insulin resistance and significant weight gain. (HEPATOLOGY 2006;44: ) See Editorial on Page 802 Hepatic steatosis, the accumulation of lipids in hepatocytes, can occur in association with a wide range of toxins, drugs, and diseases. 1 In routine clinical practice most cases have previously been Abbreviations: NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. From the 1 Division of Gastroenterology and Hepatology, Department of Molecular and Clinical Medicine, University Hospital, Linköping, Sweden; 2 Department of Pathology, Clinical Research Centre, University Hospital, Örebro, Sweden; 3 Department of Internal Medicine, County Hospital, Oskarshamn, Sweden; 4 Division of Radiology, Department of Medicine and Care, University Hospital, Linköping, Sweden; 5 Division of Social Medicine and Public Health Science, Department of Health and Society, University Hospital, Linköping, Sweden; and 6 Division of Internal Medicine, Department of Medicine and Care, University Hospital, Linköping, Sweden. Received May 1, 2006; accepted June 28, Supported by the Research Council of Southeast Sweden, grant F Address reprint requests to: Stergios Kechagias, M.D., Ph.D., Division of Internal Medicine, Department of Medicine and Care, University Hospital, SE Linköping, Sweden. stergios.kechagias@lio.se; fax: (46) Copyright 2006 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. associated with excessive alcohol consumption, but during recent years hepatic steatosis has most commonly been associated with obesity, insulin resistance, and hyperlipidemia, all components of metabolic syndrome. 2 Nonalcoholic fatty liver disease (NAFLD) is now recognized as one of the most common causes of chronic liver disease worldwide. Patients with NAFLD are often identified by asymptomatic elevation of liver enzymes, most frequently of serum alanine aminotransferase (ALT), and nonalcoholic hypertransaminasemia, in which viral or other causes of liver disease are excluded, has been used as a surrogate marker for NAFLD. 3,4 Analysis of data from the Third National Health and Nutrition Examination Survey (NHANES III) shows the prevalence of elevated serum aminotransferases to be 7.9% in the United States, with most instances unexplained and thus attributed to NAFLD. 5 Obesity has been established as a major risk factor, albeit not a prerequisite, for NAFLD. Because obesity is reaching epidemic proportions worldwide, the population at risk for developing chronic liver disease and its complications from NAFLD will most likely increase over time. Thus, there is a critical need to better understand the natural history of 865

2 866 EKSTEDT ET AL. HEPATOLOGY, October 2006 NAFLD. Published follow-up studies where repeat liver biopsies were performed have either included relatively small numbers of patients, and/or repeat biopsies were obtained after a rather modest length of follow-up Our aim was to describe the long-term clinical and histological development of NAFLD in patients originally referred because of chronically elevated liver enzymes. Patients and Methods Subjects. We studied in detail all patients (n 212) referred consecutively between 1988 and 1993 to the Department of Gastroenterology and Hepatology, University Hospital in Linköping, or the Department of Internal Medicine, Oskarshamn County Hospital for evaluation of persistently ( 6 months) elevated serum ALT and/or aspartate aminotransferase (AST), defined as 41 U/L (0.70 kat/l), and/or elevated serum alkaline phosphatase, 106 U/L (1.80 kat/l). A diagnostic workup was performed including physical examination, laboratory investigations, and liver biopsy. 12 One hundred and forty-four patients were diagnosed with hepatic steatosis without any other concomitant liver disease or medication associated with fatty infiltration of the liver. Seven of these subjects reported at baseline current or previous average weekly alcohol consumption 140 g and were excluded from follow-up. The remaining 137 patients originally diagnosed with NAFLD constituted the cohort of this follow-up study. Data Collection. Each subject in the study cohort was currently identified by linking his or her unique personal identification number to the National Registry of Population. All medical records from primary care health centers and hospitals were reviewed. Special attention was given to development of chronic diseases and signs of alcohol abuse. Subjects who had died during follow-up were identified and their causes of death were obtained by reviewing their medical records and the information obtained from the Registry of Causes of Death. All participants in the study cohort who were still living were asked to participate in this follow-up study. Those who accepted were offered clinical and biochemical investigation and ultrasonography and a biopsy of the liver. Subjects had blood drawn for a complete blood count and analysis of prothrombin, thyroid-stimulating hormone, transferrin, iron, transferrin saturation, ferritin, AST, ALT, alkaline phosphatase, gamma glutamyl transferase, bilirubin, fasting total cholesterol, low-density lipoprotein, high-density lipoprotein, triglycerides, fasting plasma glucose, fasting serum insulin, and plasma protein electrophoresis including albumin, 1 -antitrypsin, ceruloplasmin, and immunoglobulins. In addition, blood was obtained for detection of hepatitis B surface antigen, anti hepatitis C virus (HCV) antibodies, hepatitis B virus DNA, HCV RNA, transglutaminase antibodies, antinuclear antibodies, smooth muscle antibodies, and mitochondrial antibodies. Moreover, genomic DNA isolated from anticoagulated venous blood was used to identify the C282Y, H63D, and S65C mutations in the HFE gene as well as the Z and S mutations in the Pi gene. Subjects were considered to have diabetes mellitus if they were receiving dietary or drug treatment for this disease. The remaining subjects had a 75-g oral glucose tolerance test after an overnight fast. The prevalence of liver-related complications of those in the general population in the same age range (36-80 years) as the NAFLD cohort was estimated by obtaining data from the Swedish Hospital Discharge Register. Included were all individuals living in the same geographical area as the NAFLD cohort who had been hospitalized in 2004 with primary or secondary diagnoses (according to the International Classification of Diseases, Tenth Revision) of cirrhosis, chronic hepatic failure, portal hypertension, hepatorenal syndrome, ascites, esophageal varices, or hepatocellular carcinoma. Liver Biopsy and Histopathological Evaluation. Liver biopsies of all subjects were performed percutaneously with ultrasound guidance and 1.6-mm Biopince needles on an outpatient basis. All biopsies at baseline and at follow-up were read by the same liver pathologist (L.E.F.), who was blinded to patient details. Liver histology was scored according to the system developed by Brunt et al., 13 except that acidophil bodies and glycogenated nuclei were not assessed and that PAS-D Kupffer cells were scored as present or absent. Moreover, quantitative steatosis was measured as the percentage of the liver biopsy containing fat. 14 Nonalcoholic steatohepatitis (NASH) was defined as steatosis plus any stage of fibrosis or as steatosis plus lobular inflammation plus ballooning degeneration. 15 Statistical Analysis. Statistical analyses were performed using SPSS software (version 11.5 for Windows). For continuous variables, differences between 2 groups were evaluated with the Student t test when data were normally distributed and with the Mann-Whitney U test when the assumption of normality was not met. For dichotomous variables, differences were tested using the 2 test corrected for continuity or Fisher s exact test. Observed survival and causes of death of NAFLD patients until September 30, 2005, were compared with those of a reference population, which was obtained from Statistics Sweden. The reference population comprised all those

3 HEPATOLOGY, Vol. 44, No. 4, 2006 EKSTEDT ET AL. 867 (n 44,745) of the same age and sex living in the same county as each patient with NAFLD at baseline. Survival curves were constructed according to the Kaplan-Meier method. One-sample log-rank tests were used for comparison with the reference population. Causes of death were compared using the z test with Bonferroni correction. A P value.05 was considered statistically significant. Ethical Considerations. Written informed consent was obtained from all participating subjects. The study design was approved by the ethics committee at the University Hospital in Linköping. Results Study Population. Eight subjects who denied previous or current alcohol consumption 140 g/wk at baseline were reclassified as having alcoholic liver disease at follow-up based on information in their medical records or self-reported alcohol consumption 140 g/wk. Thus, of the 137 patients originally diagnosed with NAFLD, 129 patients were considered to have had NAFLD when included in the study. Baseline clinical and biochemical characteristics of this cohort are shown in Table 1. Of these 129 patients, 104 were alive at follow-up, and 88 agreed to participate in this study (Fig. 1). Follow-up started March 10, 2003, and was completed September 30, Mean follow-up time SD was years from time of diagnosis of NAFLD, with a total of 1,202 person-years. Clinical and biochemical characteristics of the 88 patients who went to a consultation at follow-up are shown in Table 1. Sixteen patients were alive but did not return for follow-up. The baseline clinical, biochemical, and histological parameters of this group were not significantly different than those of the participatants in the follow-up study. Histology at Baseline. At baseline 71 patients (55%) fulfilled criteria for NASH. Four of these patients had cirrhosis. Twelve patients (9%) had steatosis with unspecific inflammation, and 46 (36%) had simple steatosis. Patients with NASH were significantly older than patients with steatosis with or without unspecific inflammation ( vs years, respectively, P.001). There were no other significant differences in baseline clinical and biochemical parameters between the 2 histological groups. Survival. A total of 26 subjects with NAFLD died during the follow-up period (including 1 patient who died shortly after diagnostic workup at follow up). Of these, 19 had NASH at baseline, including 1 patient with cirrhosis, and 7 had steatosis with or without unspecific inflammation. At the end of the follow-up period, survival of NAFLD patients was significantly lower than that of the reference population (78% vs. 84%, respectively; P.006; Fig. 2A). Subgroup analysis showed that survival among NASH patients was significantly lower than the corresponding reference population (70% vs. 80%, respectively, P.01; Fig. 2B), whereas survival did not differ significantly between patients with steatosis with or without unspecific inflammation and the corresponding reference population (Fig. 2C). Causes of Death and Liver-Related Morbidity and Mortality Among Subjects With NAFLD. Eleven (15.5%) patients with NASH died from cardiovascular disease, 4 (5.6%) from extrahepatic malignancy, 2 (2.8%) from liver-related causes (metastatic hepatocellular carcinoma and variceal hemorrhage), 1 (1.4%) from neurological disease, and 1 (1.4%) from respiratory disease. A comparison of the causes of death of patients with NASH with those of the corresponding reference population showed it was significantly more common for patients with NASH to die from liver-related causes (2.8% vs. 0.2%, respectively, P.04) and from cardiovascular disease (15.5% vs. 7.5%, respectively, P.04). Five non- NASH patients (8.6%) died from cardiovascular disease, 1 (1.7%) from extrahepatic malignancy, and 1 (1.7%) from renal disease. No significant differences in causes of death were found between non-nash patients and the corresponding reference population. Although 25 patients died during follow-up and 16 patients with NAFLD chose not to participate in the follow-up study, their medical records from hospitals and primary care health centers were available. A review of those records showed that no NAFLD patient who was alive but did not participate in the follow-up study had developed liver-related complications. Of the NAFLD patients who died during follow-up, 2 had developed cirrhosis-related complications prior to death. One female patient subsequently died from variceal hemorrhage, and one male patient who died from acute myocardial infarction had previously developed ascites, and cirrhosis was diagnosed with postmortem biopsy. Among the 88 NAFLD patients who participated in the follow-up study, 5 had developed cirrhosis-related complications (ascites in 2 patients, ascites and hepatocellular carcinoma in 2 patients, of whom 1 died shortly after diagnostic workup at follow-up, and hepatocellular carcinoma in 1 patient who had undergone successful orthoptic liver transplantation during follow-up). Thus, of 129 NAFLD patients, 7 (5.4%) developed cirrhosis-related complications during follow-up. There were no significant differences in baseline clinical, biochemical, and histological parameters between the 7 patients who developed end-stage liver disease during follow-up and the 15 patients who died from

4 868 EKSTEDT ET AL. HEPATOLOGY, October 2006 Table 1. Clinical and Biochemical Features of Cohort at Baseline and at Follow-Up [Mean SD or n (%)] At Baseline (n 129) At Follow-Up (n 88) Age (years) Sex (male) 87 (67%) 62 (70%) BMI (kg/m 2 ) Overweight 72 (56%) 49 (56%) Obese 37 (29%) 29 (33%) Previously diagnosed diabetes 11 (8.5%) 37 (42%) Diabetes diagnosed at consultation visit NA 14 (16%) IGT diagnosed at consultation visit NA 18 (20%) Hypertensive 93 (72%) 83 (94%) Manifest cardiovascular disease 14 (11%) 16 (18%) Hypertriglyceridemia 74 (57%) 35 (40%) Metabolic syndrome NA 52 (59%) ALT (U/L) AST (U/L) AST/ALT ratio ALP (U/L) Bilirubin (mg/dl) Albumin (g/dl) Platelet count ( 10 9 /L) Prothrombin (INR) Ferritin ( g/l) Glucose (mg/dl) NA IR HOMA NA Triglycerides (mg/dl) Cholesterol (mg/dl) HDL (mg/dl) NA LDL (mg/dl) NA Negative for HBsAg/anti-HCV 129/129 88/88 Negative for HBV DNA/HCV RNA NA/NA 88/88 Positive for ANA/SMA/AMA 37/23/0 12/8/0 Positive for transglutaminase antibodies NA 0 Mutation in the HFE gene * (C282Y homozygosity/c282y compound heterozygosity) NA 2/0 Mutation in the Pi gene (ZZ/SZ/MZ/MS) NA 0/0/10/3 Ceruloplasmin 0.20 g/l 0 0 NOTE. Conversions: ALT, AST, and ALP (U/L) kat/l; bilirubin (mg/dl) 17.1 mol/l; albumin (g/dl) 10 g/l; glucose (mg/dl) mmol/l; triglycerides (mg/dl) mmol/l; cholesterol, HDL, and LDL (mg/dl).026 mmol/l. Abbreviations: NS, not significant; NA, not available; BMI, body mass index; IGT, impaired glucose tolerance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; INR, international normalized ratio; IR HOMA, insulin resistance according to homeostasis model assessment 16 ; HDL, high-density lipoprotein; LDL, low-density lipoprotein; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV; hepatitis C virus; ANA, antinuclear antibodies, SMA, smooth muscle antibodies; AMA, mitochondrial antibodies. Overweight was defined as BMI 25 kg/m 2 but 30 kg/m 2, obesity as BMI 30 kg/m 2, diabetes as fasting plasma glucose 126 mg/dl requiring treatment or plasma glucose 199 mg/dl 2 h after oral administration of 75 g of glucose, impaired glucose tolerance as plasma glucose 140 mg/dl but 199 mg/dl 2 h after oral administration of 75 g of glucose, hypertension as blood pressure 130/85 or requiring treatment, and hypertriglyceridemia as fasting triglycerides 150 mg/dl. Metabolic syndrome was defined as having at least 3 of the following 17 : (1) waist circumference 102 cm in men or 88 cm in women; (2) fasting triglycerides 150 mg/dl; (3) fasting HDL 40 mg/dl in men or 50 mg/dl in women; (4) blood pressure 130/85 mm Hg or a diagnosis of hypertension; (5) fasting glucose 110 mg/dl or a diagnosis of diabetes mellitus. * C282Y, H63D, and S65C mutations in the HFE gene were identified from genomic DNA. Z and S mutations in the Pi gene were identified from genomic DNA. cardiovascular disease without previous development of end-stage liver disease. During 2004, 62 of every 100,000 inhabitants of the same geographical area were hospitalized with diagnoses of liver-related complications. Of these, 57% lacked etiologic diagnosis. The 2 most common etiologic diagnoses were alcoholic liver disease and hepatitis C, accounting for 37% and 3%, respectively, of the diagnoses. Metabolic and Cardiovascular Characteristics of Study Cohort. At baseline most patients were overweight or obese. Fasting plasma glucose was not measured at baseline, and thus the prevalence of diabetes at onset of the study cannot be reported (Table 1). The corresponding clinical parameters at follow-up are shown in Table 1. Histology at Baseline Versus Clinical Outcome. Of the 71 patients with NASH at baseline, 7 (10%) developed end-stage liver disease during follow-up, whereas none of the 58 patients with steatosis with or without unspecific inflammation developed complications related to chronic liver disease during follow-up. Subgroup analysis showed no patient who had NAFLD without fibrosis (n 60) or stage 1 fibrosis (n 31) at baseline had

5 HEPATOLOGY, Vol. 44, No. 4, 2006 EKSTEDT ET AL. 869 Fig. 1. Details about patients studied, showing reasons for exclusions. a Both patients were diagnosed with hepatocellular carcinoma at follow-up. One patient died shortly after diagnostic work-up at follow-up. b One patient developed hepatocellular carcinoma and underwent orthoptic liver transplantation during follow-up. PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; AAT, 1 -antitrypsin. developed complications related to chronic liver disease during follow-up. One of the 4 patients (25%) with cirrhosis at baseline, 3 of the 22 patients (14%) with stage 2 fibrosis at baseline, and 3 of the 12 patients (25%) with stage 3 fibrosis at baseline were found to have developed end-stage liver disease during follow-up. Forty-two patients with NASH at baseline returned for follow-up. Of these, 30 patients (71%) had diabetes. Of the patients with steatosis with or without unspecific inflammation, 21 (46%) had diabetes. This difference was statistically significant (P.01). Moreover, manifest cardiovascular disease at follow-up was significantly more common among patients with NASH at baseline than among those without NASH (29% vs. 9%, respectively, P.02). Histological Development. Follow-up biopsies were obtained from 68 patients after years (range years). Three patients, one of whom had undergone orthoptic liver transplantation, did not undergo a repeat liver biopsy because they had cirrhosis at baseline. Moreover, 2 patients without cirrhosis at baseline did not undergo a liver biopsy at follow-up because they were diagnosed with ascites and hepatocellular carcinoma at follow-up. These 2 patients were considered as having fibrosis that had progressed in stage. Fifteen patients who returned for follow-up refused a repeat liver biopsy (Fig. 1). The clinical and biochemical parameters of these patients were not significantly different than those of the patients who agreed to repeat liver biopsies, nor were the baseline histological features. Two patients had normal liver histology at follow-up. Overall, quantitative steatosis was significantly lower at follow-up (12.5% 9.7% vs. 8.8% 7.4%, P.004). Between the first and second biopsies, fibrosis stage progressed in 29 patients (41%), was unchanged in 30 pa-

6 870 EKSTEDT ET AL. HEPATOLOGY, October 2006 tients (43%), and regressed in 11 patients (16%); see Table 2. The presence of necroinflammatory changes at baseline was not associated with progression in fibrosis stage at follow-up (Table 2). A separate analysis of the 36 patients without fibrosis at baseline who underwent liver biopsy at follow-up showed only a small number exhibited histological features associated with hepatic necroinflammation at baseline. Despite this, 17 patients (47%) had developed fibrosis at follow-up (Table 2). Those patients whose fibrosis had progressed in stage at follow-up had significantly higher ALT levels (P.005), significantly higher AST levels (P.003), and significantly lower platelet counts (P.003). Moreover, at follow-up subjects with progressive fibrosis had a weight gain exceeding 5 kg significantly more often (P.02), were significantly more insulin resistant according to homeostasis model assessment 16 (P.04), and had significantly more pronounced hepatic fatty infiltration (P.03; Table 3). We were not able to find any association between baseline clinical and biochemical parameters with future progression of fibrosis. Fig. 2. (A) Overall survival of NAFLD patients originally referred because of elevated liver enzymes. (B) Survival of NASH patients. (C) Survival of patients with nonalcoholic liver steatosis with and without unspecific inflammation. Survival of patients was compared with that of a reference population comprising all subjects of the same age and sex living in the same county as each patient at baseline. Discussion In person-years, this is the largest reported study of a follow-up series of biopsy-proven NAFLD patients originally referred because of elevated liver enzymes. Although we cannot rule out unknown biases in referral for hepatological exploration, we believe that our study has several methodological strengths. First, all patients referred because of elevated liver enzymes were consecutively enrolled. Second, all patients underwent liver biopsy at baseline, and thus the diagnoses of NAFLD were based on histological criteria. Third, clinical and histological follow-up exceeded 10 years for all patients, and time to follow-up did not vary considerably between patients. The natural history of patients with biopsy-proven NAFLD and elevated liver enzymes previously has not been well defined. One of the main findings of the present study was that survival among these patients was lower than that of the matched reference population. A previous study 18 reported an association between NAFLD without fibrosis or inflammation with a benign clinical course without excess mortality. We confirmed this finding; but in addition, we have shown that NASH, which was more common in the older patients in our NAFLD cohort, is associated with increased overall mortality. Recently, Adams et al. 19 reported that survival of NAFLD patients was lower than the expected survival in

7 HEPATOLOGY, Vol. 44, No. 4, 2006 EKSTEDT ET AL. 871 Table 2. Changes in Fibrosis Stage Between First and Second Biopsies; Comparison of Baseline Necroinflammatory Parameters Between Patients With Progressive Fibrosis and Patients With Nonprogressive Fibrosis (n 70); and Baseline Necroinflammatory Parameters of Patients Without Fibrosis at Baseline (n 36) Fibrosis Stage at Follow-Up [n (%)] Fibrosis Stage at Baseline F0 F1 F2 F3 F4 F0 (n 36) 19 (53%) 8 (22%) 6 (17%) 3 (8%) 0 F1 (n 19) 5 (26%) 9 (47%) 3 (16%) 1 (5%) 1 (5%) F2 (n 11) 0 5 (45%) 1 (9%) 2 (18%) 3 * (27%) F3 (n 4) (25%) 1 (25%) 2 * (50%) F4 Necroinflammation Progressive Fibrosis (n 29) Nonprogressive Fibrosis (n 41) P Lobular inflammation 4 (14%) 1 (3%) NS Portal inflammation 9 (31%) 7 (17%) NS Periportal inflammation 4 (14%) 3 (7%) NS Hepatocellular ballooning 3 (10%) 2 (5%) NS Mallory s hyaline 1 (3%) 1 (2%) NS Steatosis Without Fibrosis (n 36) Fibrosis Present at Follow-Up (n 17) Fibrosis Absent at Follow-Up (n 19) Lobular inflammation 1 (6%) 0 NS Portal inflammation 2 (12%) 1 (5%) NS Periportal inflammation 1 (6%) 1 (5%) NS Hepatocellular ballooning 1 (6%) 1 (5%) NS Mallory s hyaline 0 0 NS Abbreviations: F0, no fibrosis; F1, expanded portal tracts and/or focally or extensively present zone 3 perisinusoidal/pericellular fibrosis; F2, focal or extensive periportal fibrosis and zone 3 perisinusoidal/pericellular fibrosis; F3, portal fibrosis with focal or extensive bridging fibrosis and zone 3 perisinusoidal/pericellular fibrosis; F4, cirrhosis; NS; not significant. * Two of these 5 patients were diagnosed with ascites and hepatocellular carcinoma and did not undergo liver biopsy at follow up. Three of 4 patients with cirrhosis at baseline were alive, all of whom returned for follow-up. Repeat liver biopsy was not performed in these patients. Two of the patients had not developed complications related to chronic liver disease during follow-up, and 1 had undergone orthoptic liver transplantation because of hepatocellular carcinoma. Dichotomous variables were tested using Fisher s exact test. the general population. In their study the NAFLD diagnosis of most patients was confirmed by imaging studies rather than histologically, and follow-up time was highly variable. In the present study we have extended their findings, showing increased mortality of patients with NASH but not of patients with steatosis. Moreover, we were able to show that the higher mortality of NASH patients was primarily a result of cardiovascular disease and, to a lesser extent, liver-related causes. Another main finding of our study, consistent with the increased cardiovascular mortality among NAFLD patients, is that most NAFLD patients (78%) were diagnosed with diabetes or impaired glucose tolerance (IGT) at follow-up. Although we do not know with certainty how many NAFLD patients had diabetes or IGT at baseline, these findings indicate liver enzyme elevation due to NAFLD is strongly associated with future onset of type 2 diabetes or IGT. Given the strong association between insulin resistance and NAFLD, it is reasonable to recommend lifestyle modifications to all patients with NAFLD. Not only do such modifications reduce the risk of developing type 2 diabetes, 20,21 but an intense dietary intervention, in particular, may also improve liver histology in those with NAFLD. 22 Interestingly, 3 of the 7 patients who developed end-stage liver disease during follow-up were diagnosed with hepatocellular carcinoma. All 3 had previously been diagnosed with diabetes. These findings are in accordance with those of a previous report that identified diabetes as an independent risk factor for hepatocellular carcinoma. 23 Our data primarily indicate that the association between diabetes and hepatocellular carcinoma is a result of the high prevalence of NAFLD in patients with diabetes. With the increasing prevalence of NAFLD, particularly at younger ages, the modestly increased relative risk of mortality and the low absolute risk of end-stage liver disease that these patients have may be of considerable public health significance in the near future. In the present study, 29 NAFLD patients (41%) showed progression in the stage of their fibrosis. It is difficult to compare these results with those of other studies because previous investigations of fibrosis change in NAFLD over time were limited to small numbers of patients and the patients generally had undergone sequential biopsies because of clinical indications, potentially biasing

8 872 EKSTEDT ET AL. HEPATOLOGY, October 2006 Table 3. Clinical, Biochemical, and Histological Features at Follow-Up of Patients With Progressive Fibrosis and Patients With Nonprogressive Fibrosis [Mean SD or n (%)] Progressive Fibrosis (n 29) Nonprogressive Fibrosis (n 41) P Age (years) NS Follow-up time (years) NS Sex (male) 21 (72%) 29 (71%) NS BMI (kg/m 2 ) NS BMI (97%) 34 (83%) NS Weight gain 5 kg 16 (55%) 10 (24%).02 IGT 7 (24%) 7 (17%) NS Diabetes 15 (52%) 24 (58%) NS Hypertension 28 (97%) 38 (93%) NS Metabolic syndrome 18 (62%) 21 (51%) NS Alcohol consumption (g/week) NS ALT (U/L) AST (U/L) AST/ALT ratio NS ALP (U/L) NS Bilirubin (mg/dl) NS Albumin (g/dl) NS Platelet count ( 10 9 /L) Prothrombin (INR) NS Ferritin ( g/l) NS Glucose (mg/dl) NS IR HOMA Triglycerides (mg/dl) NS Cholesterol (mg/dl) NS HDL (mg/dl) NS LDL (mg/dl) NS Mutation in the HFE gene * 6 (21%) 16 (39%) NS Mutation in the Pi gene 5 (17%) 4 (10%) NS Quantitative steatosis (%) NOTE. Conversions: ALT, AST, and ALP (U/L) kat/l; bilirubin (mg/dl) 17.1 mol/l; albumin (g/dl) 10 g/l; glucose (mg/dl) mmol/l; triglycerides (mg/dl) mmol/l; cholesterol, HDL, and LDL (mg/dl) mmol/l. Abbreviations: NS, not significant; BMI, body mass index; IGT, impaired glucose tolerance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; INR, international normalized ratio; IR HOMA, insulin resistance according to homeostasis model assessment 16 ; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Metabolic syndrome was defined as having at least 3 of the following 17 : (1) waist circumference 102 cm in men or 88 cm in women; (2) fasting triglycerides 150 mg/dl; (3) fasting HDL 40 mg/dl in men or 50 mg/dl in women; (4) blood pressure 130/85 mm Hg or a diagnosis of hypertension; (5) fasting glucose 110 mg/dl or a diagnosis of diabetes mellitus. * The C282Y, H63D, and S65C mutations in the HFE gene were identified from genomic DNA. Z and S mutations in the Pi gene were identified from genomic DNA. Continuous variables were normally distributed (except IR HOMA ) and thus were analyzed with the unpaired Student t test (IR HOMA was analyzed with the Mann-Whitney U test). Dichotomous variables were analyzed using the 2 test corrected for continuity. results toward patients with more severe or atypical disease In a recently published study, 11 progression of fibrosis was found in 37% of 103 NAFLD patients. However, follow-up was short (mean 3.2 years). Moreover, this study included patients who were markedly obese and few had simple steatosis, making it likely that the participants were not reflective of the general NAFLD population with elevated liver enzymes. Although most patients in the present study were nonobese, more pronounced insulin resistance at follow-up was associated with progression of liver fibrosis. This is consistent with the findings of previous studies. 8,11,24 We have shown that NAFLD patients with progressive fibrosis were, in addition to being more insulin resistant, significantly more likely to have a weight gain exceeding 5 kg and more severe hepatic fatty infiltration at follow-up. A previous study 7 reported a relatively benign histological course among NAFLD patients without fibrosis. According to the results of the present study, this view should be modified. Although none of the 60 NAFLD patients without fibrosis at baseline developed clinically manifest liver disease during follow-up, 17 (47%) of the 36 patients who underwent repeat liver biopsy at follow-up had developed fibrosis, including 3 who had developed bridging fibrosis. A limitation of all NAFLD studies investigating hepatic histology is the significant sampling variability of routine liver biopsy. 25 We cannot with certainty quantify the effect of sampling error on our results. A total of 31 subjects without cirrhosis at baseline who did not have end-stage liver disease at follow-up did not undergo repeat liver biopsy. Their clinical, biochemical, and baseline histological parameters were not different, and thus it is unlikely this group had a different histological course than the group of patients who underwent liver biopsy at follow-up. Although a clinicobiological score for the selection of candidates for liver biopsy in NAFLD has been proposed in a cross-sectional study, 8 in the present long-term follow-up study we were not able to find parameters useful in the clinical setting that could predict the progression of fibrosis in a patient with NAFLD. The results of liver biopsy at baseline was the best predictor of developing cirrhosis-related complications at follow-up, with a positive predictive value of 18% in patients with periportal fibrosis and a negative predictive value of 100% in patients without established periportal fibrosis. References 1. 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