THE NATURAL HISTORY OF NONALCOHOLIC FATTY LIVER DISEASE WITH ADVANCED FIBROSIS OR CIRRHOSIS: AN INTERNATIONAL COLLABORATIVE STUDY

Participants & setting

Two-hundred and forty seven Child-Pugh class A patients with biopsy-confirmed NAFLD and advanced fibrosis or cirrhosis comprised the NAFLD cohort. This cohort was recruited from 1984 to 2006. Patients were previously untreated and consecutively biopsied at 4 centers: Mayo Clinic Rochester, USA (n = 105); Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK (n = 57); Westmead Hospital, Sydney, Australia (n = 51) and University of Turin, Italy (n = 34). The comparator cohort consisted of 264 patients diagnosed with HCV infection and advanced fibrosis or cirrhosis who were also Child-Pugh class A enrolled from 1987 to 2005. HCV infection was confirmed by a positive polymerase chain reaction at baseline in all patients. HCV subjects were seen and consecutively biopsied at Westmead Hospital, Sydney (n = 209) and University of Turin, Italy (n = 55). They were either non-responders to antiviral treatment (n= 224; 85%) or had never had any treatment for HCV infection (n=40; 15%). These criteria were chosen because sustained virological response to anti-HCV therapy improves the outcomes of these patients. The study was approved by appropriate regulatory bodies at all centers and written informed consent was obtained from all patients for participation in medical research.

NAFLD was diagnosed based on: (i) elevated aminotransferases for at least six months; (ii) liver biopsy showing changes consistent with advanced fibrotic NAFLD (detailed below), and (iii) exclusion of other etiologies including viral, autoimmune, cholestatic, genetic, metabolic, alcoholic or drug-induced liver diseases. These other etiologies were excluded using specific biochemical, clinical, radiological, and/or histological criteria. All patients had current and past consumption of ethanol less than 20 grams per day on direct questioning of both the patients and a close relative.

A complete medical history and physical examination was undertaken. Body mass index (BMI) was calculated using the formula: weight (in kilograms)/height² (in metres). Waist circumference (to the nearest half centimetre) was measured at the midpoint between the lower border of the ribcage and the iliac crest. Serum measurements included routine liver biochemistry (ALT and AST levels, total bilirubin, albumin, alkaline phosphatase, and gamma glutamyl transpeptidase); complete blood count; fasting glucose; fasting insulin; total cholesterol, HDL cholesterol, and total trigycerides; serology for hepatitis B and C viruses; iron studies; autoantibodies; alpha 1 antitrypsin levels and phenotype; and ceruloplasmin levels. Components of the metabolic syndrome including central obesity, hyperglycemia, hypertrigylceridemia, hypertension and low HDL cholesterol were recorded.

Liver histology

Liver biopsies were stained with hematoxylin and eosin, Masson’s trichrome, and special stains for iron and copper. Liver biopsies were read by a single liver pathologist in each participating center. Histological features of NAFLD such as steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored as previously described.(14, 15) Only those patients that had steatosis of at least 5% plus stage 3 (septal/bridging) or stage 4 (cirrhosis) fibrosis were included in this analysis. Other histological changes of steatohepatitis such as inflammation and ballooning were not required as inclusion criteria.

For HCV, the degree of fibrosis was scored according to the METAVIR scale (16): briefly, stage 0: no fibrosis; stage 1: enlarged portal tract without septa; stage 2: enlarged portal tract with rare septa; stage 3: numerous septa without cirrhosis; stage 4: cirrhosis.

Only those patients with fibrosis stage 3 or 4 disease were included. There was no clinical, serological or radiological evidence of HCC at recruitment in any patient with NAFLD or HCV infection.

Data Collection

The start date for analyses was the date of liver biopsy, with any events occurring in the first 6 months excluded. Patients were monitored every 3-6 months and followed up until death or liver transplantation, whichever occurred first, or until data analysis. For those lost to follow-up, up-to date clinical information was sought by: (i) contact with the primary care physician, (ii) telephone interview, and (iii) checking the respective death and transplant registries. Patients were censored at the time of death or transplantation or last clinic visit, and those lost to follow-up were censored at the time last seen. All clinical outcomes were confirmed by a physician at each center utilising the patient records and physician diagnoses.

The following outcomes were assessed: (i) liver complications including liver failure, gastro-esophageal varices (+/− hemorrhage), ascites, encephalopathy, hepatopulmonary syndrome and HCC; (ii) liver-related death or liver transplantation (for calculation of survival probability, transplantation was considered as an equivalent end-point); (iii) all-cause mortality; and (iv) total vascular events (including myocardial infarction, stroke, and vascular deaths).(13)

HCC was diagnosed if the following were present: (1) pathological changes consistent with HCC identified by histological examination of liver tissue obtained by fine-needle aspiration, liver biopsy, or liver explant at transplantation or autopsy, or (2) one or more hepatic space-occupying lesions that had vascular patterns typical of HCC by angiography, triple-phase computed tomography or magnetic resonance imaging.

All patients were followed according to standards of care and guidelines without experimental or therapeutic interventions for NAFLD or HCV. Weight management was performed with lifestyle intervention such as dietary modification and exercise recommended at the outpatient clinic in patients with overweight/obesity. Other treatments, such as oral hypoglycemics, cholesterol-lowering medications, and anti-hypertensive medications were only given in the context of management of concomitant diabetes mellitus, hypercholesterolemia or hypertension, respectively. Neither drug use nor lifestyle interventions were recorded systematically after baseline.

Statistical Analysis

Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago IL). Results are reported as means ± standard deviation or frequency (percentage) as appropriate. Continuous variables were compared using two-tailed students’ t-test. Categorical data were compared using the Chi square test. Variables with a p-value of ≤ 0.1 on univariate analysis were further analyzed by multiple logistic regression in order to determine the independent determinants of outcome variables. The cumulative probabilities of morbidity and mortality (including 95% confidence intervals [CI]) were analysed using the Kaplan-Meier method and using the date of the liver biopsy as time zero (baseline). The effect of individual variables upon morbidity and mortality were compared using log-rank tests. When assessing the effects of multiple variables on survival, Cox-regression was used.

Baseline characteristics

Table 1 summarizes the baseline characteristics of the patient population. Among cases with NAFLD, the mean age was 54.7 years, 60.5% were female, 91.5% were of white ethnicity, and the mean BMI was 32.8 kg/m². More than half had concomitant metabolic morbidity including 50% with diabetes, 44.1% with hypertension and 12.1% with previous vascular disease. At baseline, metformin was used in a third (34.4%) of patients, and statins in one fifth (21.5%) of patients. Among cases with HCV, the mean age was 48.3 years, 35.2% were female, 72.3% were white, and the mean BMI was 27.3 kg/m². The average BMI was significantly lower in patients with HCV infection and they had lower levels of glucose, cholesterol, and triglycerides, and higher levels of ALT and AST than patients with NAFLD. Although the NAFLD cohort had lower levels of albumin and higher platelet count, other parameters indicative of liver function such as bilirubin, INR and MELD score were similar between the two groups as was the proportion of patients with fibrosis stage 3 and 4 (Table 1).

Liver-related complications and cardiovascular events

The 247 NAFLD participants had a mean (±SD) follow-up of 85.6 (±54.5) months (range 6-297 months). Complete follow-up was achieved in 235 (95.1%) patients and 12 (4.9%) patients were lost to follow-up. During follow-up, 48 (19.4%) patients developed liver-related complications, with some developing more than one complication. Twenty six (10.5%) cases developed gastroesophageal varices, 19 (7.7%) developed ascites, liver failure, hepatopulmonary syndrome and/or encephalopathy, and 6 (2.4%) developed HCC (4 of which were initially in stage 4 fibrosis). Thirteen (5.3%) had subsequent myocardial infarctions (one being fatal), and 4 (1.6%) developed strokes (all ischemic in etiology).

The 264 HCV participants had a mean (±SD) follow-up of 74.9 (±47.1) months (range 6-238 months). Complete follow-up was achieved in 254 (96.2%) patients, and 10 (3.8%) patients were lost to follow-up. During follow-up, 47 (17.8%) patients developed liver-related complications. Nine (3.4%) developed gastroesophageal varices, 20 (7.6%) developed ascites, liver failure and/or encephalopathy, and 18 (6.8%) patients developed HCC (10 of which were initially in stage 4 fibrosis). Nine (3.4%) had subsequent myocardial infarctions and one (0.3%) had a stroke (diagnosed as ischemic in etiology).

In the NAFLD cohort, the probability of liver-related complication-free survival was 98.0%, 93.4% and 81.5% at 12, 36 and 120 months respectively, whereas in the HCV cohort was 98.4%, 93.5% and 76.5% at 12, 36 and 120 months respectively (Figure 1A). In unadjusted comparison, there was a trend for more liver-related complications in the cohort with HCV infection than in the cohort with NAFLD (p=0.09; Figure 1A); there appeared to be stronger evidence of a difference between groups when adjusted in age, sex, body mass index and diabetes (p=0.03; Figures 1B & C). HCC occurred more commonly in HCV than in NAFLD (18 [6.8%] vs. 6 [2.4%] respectively, p=0.03), with time-to-event illustrated in Supplementary Figure 1. There was no significant difference in total vascular events between NAFLD and HCV groups (17 (6.9%), vs. 10 (3.8%); p=0.17).

Mortality (liver-related and all-cause) and liver transplantation

In the NAFLD cohort, there were a total of 33 deaths or liver transplants (13.4%). Of the 14 liver-related deaths and transplantations, three were related to HCC; there were four deaths related to other cancers, and one definite vascular death. The probability of overall survival was 99.6%, 96.7% and 81.6% at 12, 36 and 120 months respectively (Figure 2A). In the HCV cohort, there were a total of 25 deaths or liver transplants (9.5%). Of the 21 liver-related deaths and transplantations, 12 were related to HCC; there was one definite vascular death, and three deaths due to unknown causes. The probability of overall free survival was 99.2%, 98.3% and 82.0% at 12, 36 and 120 months respectively (Figure 2A). The overall mortality was similar in both cohorts (p=0.38; Figure 2A), with no evidence of differences after adjustment by differences between groups in age, sex, body mass index, diabetes and dislipidemia (p=0.6; Figures 2B & C).

Liver-related complications and overall mortality by fibrosis stage

In the NAFLD group there was strong evidence of differences between fibrosis stage 3 and 4 for total liver-related complications (p<0.001) and some evidence for overall mortality (p=0.05) as illustrated in Supplementary Figures 2A and 2B. In the HCV group there was little evidence of differences between fibrosis stage 3 and 4 for total liver-related complications (p=0.18) and some evidence for overall mortality (p=0.04) as illustrated in Supplementary Figures 3A and 3B.

Independent predictors for liver-related complications in the NAFLD cohort

Univariate models to characterise differences in the NAFLD group are shown in Supplementary Table 1 and a summary of the multivariate predictive factors for all categories of outcome are shown in Table 2. Stage 4 fibrosis, past history of coronary heart disease, lower serum levels of cholesterol, lower levels of ALT, and lower platelet count were all independently associated with total liver-related complications. Independent predictors were also identified for the development of ascites (lower platelet count), encephalopathy (older age), gastroesophageal varices (stage 4 fibrosis; lower levels of ALT; lower platelet count; lower levels of cholesterol), and myocardial infarction (past medical history of hypercholesterolemia, lower HDL-cholesterol). No factors were identified as predictors for HCC or stroke. All these differences remained unaffected when the center variable was included in the models.

Independent predictors for mortality in the NAFLD cohort

Univariate models to characterise differences in the NAFLD cohort are shown in Supplementary Table 2 and a summary of the multivariate predictive factors are shown in Table 2. By multivariate analysis and adjusting for center, older age and higher AST/ALT ratio were independently associated with overall mortality. Stage 4 fibrosis and higher serum bilirubin levels were independently associated with liver-related mortality. History of diabetes mellitus and hypercholesterolemia were associated with vascular events (ie, non-fatal myocardial infarction, non-fatal stroke, and vascular death) and vascular-related death.