Fibrosis Progression in Nonalcoholic Fatty Liver vs Nonalcoholic Steatohepatitis: A Systematic Review and Meta-analysis of Paired-Biopsy Studies

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Fibrosis Progression in Nonalcoholic Fatty Liver Versus Nonalcoholic Steatohepatitis: A Systematic Review and Meta-Analysis of Paired-Biopsy Studies Q27

Siddharth Singh,* Alina M. Allen,* Zhen Wang,‡ Larry J. Prokop,‡ Mohammad H. Murad,‡ and Rohit Loomba§ *Division of Gastroenterology and Hepatology, Department of Internal Medicine, ‡Knowledge and Evaluation Research Unit, Mayo Clinic, Rochester, Minnesota; §Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California, San Diego, California BACKGROUND & AIMS:

Little is known about differences in rates of fibrosis progression between patients with nonalcoholic fatty liver (NAFL) vs nonalcoholic steatohepatitis (NASH). We conducted a systematic review and meta-analysis of all studies that assessed paired liver biopsy specimens to estimate the rates of fibrosis progression in patients with nonalcoholic fatty liver disease (NAFLD) including NAFL and NASH.

METHODS:

Through a systematic search of multiple databases and author contact, up to June 2013, we identified studies of adults with NAFLD that collected paired liver biopsy specimens at least 1 year apart. From these, we calculated a pooled-weighted annual fibrosis progression rate (number of stages changed between the 2 biopsy samples) with 95% confidence intervals (CIs), and identified clinical risk factors associated with progression.

RESULTS:

We identified 11 cohort studies including 411 patients with biopsy-proven NAFLD (150 with NAFL and 261 with NASH). At baseline, the distribution of fibrosis for stages 0, 1, 2, 3, and 4 was 35.8%, 32.5%, 16.7%, 9.3%, and 5.7%, respectively. Over 2145.5 person-years of follow-up evaluation, 33.6% had fibrosis progression, 43.1% had stable fibrosis, and 22.3% had an improvement in fibrosis stage. The annual fibrosis progression rate in patients with NAFL who had stage 0 fibrosis at baseline was 0.07 stages (95% CI, 0.02–0.11 stages), compared with 0.14 stages in patients with NASH (95% CI, 0.07–0.21 stages). These findings correspond to 1 stage of progression over 14.3 years for patients with NAFL (95% CI, 9.1–50.0 y) and 7.1 years for patients with NASH (95% CI, 4.8–14.3 y).

CONCLUSIONS:

Based on a meta-analysis of studies of paired liver biopsy studies, liver fibrosis progresses in patients with NAFL and NASH.

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Keywords: Fibrosis; Cirrhosis; Nonalcoholic Steatohepatitis; Fatty Liver; Natural History.

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onalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States.1–6 It is defined as the presence of hepatic steatosis in at least 5% of hepatocytes on liver biopsy examination in individuals who consume little or no alcohol, after exclusion of other causes of liver disease.2 NAFLD can be classified broadly into 2 subtypes: nonalcoholic fatty liver (NAFL), which generally is considered to be benign with negligible risk of progression to advanced fibrosis and liver-related mortality, and nonalcoholic steatohepatitis (NASH), which generally is considered to be progressive with a substantial risk of progression to advanced fibrosis and liver-related mortality. Several longitudinal cohort studies have provided novel insight into the natural history of liver disease in

N

patients with NAFLD.5,7–10 Despite these advances, the fibrosis progression rate in NAFLD remains to be quantified and is poorly understood.9 Observational studies with paired liver biopsy specimens in patients with NAFLD, although prone to ascertainment and selection bias, offer some of the best available natural history data on rate and risk factors associated with progressive Abbreviations used in this paper: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; FPR, fibrosis progression rate; I2, inconsistency index; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; RCT, randomized controlled trial. © 2014 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2014.04.014

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fibrosis. Based on a pooled analysis of 10 studies including 221 patients with NASH alone, Argo et al11 estimated that 37.6% of patients have progressive fibrosis over 5.3 years; the mean rate of progression for the entire cohort was 0.03 stages per year. However, in this study, patients with NAFL were excluded. Although previous studies have suggested that NAFL may be benign, and does not lead to progressive fibrosis,12 emerging data suggest that fibrosis progression may be seen not only in NASH, but also in NAFL.13 There are limited data on the differences in the fibrosis progression rate in patients with NAFL vs NASH. Therefore, we aimed to perform a systematic review and meta-analysis of studies in patients with biopsy-proven NAFL and NASH who underwent paired liver biopsies at least 1 year apart and to quantify differences in fibrosis progression in patients with NAFL vs NASH.

Methods This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, and the process followed an a priori established protocol.14

Selection Criteria

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Studies included in the meta-analysis met the following inclusion criteria: (1) cohort studies and placebo-controlled randomized controlled trials (RCTs), (2) included adult patients (>18 y) with a histologic diagnosis of NAFLD at any stage of baseline fibrosis, (3) repeat liver biopsy was performed at least 1 year apart, and (4) contained sufficient information to allow estimation of the fibrosis progression rate (FPR) by each baseline fibrosis stage. We excluded the following studies: (1) the diagnosis of NAFLD and/or degree of fibrosis (either baseline or during follow-up evaluation) was established using noninvasive means; (2) participants in the active arm of a clinical trial (ie, patients randomized to potentially disease-modifying active intervention for NAFLD); (3) cross-sectional studies; (4) studies in which the time difference between paired biopsies was fewer than 12 months; and (5) studies in which there were insufficient data to allow estimation of FPR (ie, insufficient data on person-years of follow-up evaluation, or mean/median duration of follow-up period/time between 2 biopsies, or only contained information on mean change in fibrosis stage for the entire cohort). In case of multiple studies from the same cohort, we included data from the most recent comprehensive report.

Search Strategy We conducted a comprehensive search of multiple electronic databases from 1985 to June 2013 containing

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data on adults, with no language restrictions. The databases included Ovid Medline In-Process and Other NonIndexed Citations, Ovid MEDLINE, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, Web of Science, and Scopus. The search strategy was designed and conducted by an experienced medical librarian (L.J.P.) with input from the study’s investigators (S.S., R.L.), using controlled vocabulary supplemented with keywords, for cohort studies and placebo-controlled RCTs of NAFLD. The details of the search strategy are included in the Supplementary Table 1. The title and abstract of studies identified in the search were reviewed by 2 authors independently (S.S., A.M.A.) to exclude studies that did not address the research question of interest, based on prespecified inclusion and exclusion criteria (see earlier). The full text of the remaining articles was examined to determine whether it contained relevant information. Next, the bibliographies of the selected articles and review articles on the topic were searched manually for additional studies. Third, a manual search of conference proceedings of major gastroenterology and hepatology conferences (The Liver Meeting, organized by the American Association for the Study of the Liver; The International Liver Congress, organized by the European Association for the Study of the Liver; and Digestive Diseases Week, organized in conjunction with the American Gastroenterological Association) between 2008 and 2012 was conducted to identify additional studies published only in abstract form. Supplementary Figure 1 shows the schematic diagram of study selection.

Data Abstraction Data on the following study- and patient-related characteristics, as well as histologic classification and risk factors associated with progressive fibrosis, were abstracted onto a standardized form: (1) study characteristics: primary author, time period of study/year of publication, and country of the population studied; (2) patient characteristics: total number of patients with NAFLD who underwent paired biopsies, demographic and clinical characteristics at time of first biopsy (age, sex, body mass index or obesity, presence of diabetes, hypertension, metabolic syndrome), baseline laboratory characteristics (aspartate aminotransferase [AST], alanine aminotransferase [ALT], AST/ALT ratio, platelet count, ferritin, and measure of insulin resistance, using the homeostasis model of assessment–insulin resistance), and treatment undertaken by participants, including lifestyle changes and potentially disease-modifying therapy (vitamin E, thiazolidinediones, metformin); (3) histologic characteristics: proportion of patients with NASH and NAFL separately, baseline and follow-up fibrosis stage for individual patients in the study, time interval between paired biopsies to allow calculation of FPR (person-years, mean or median follow-up period of cohort, time

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difference between paired biopsies), attrition rate, criteria used for diagnosis and staging of fibrosis, proportion of patients with progression of fibrosis, stable fibrosis stage, and regression of fibrosis on repeat biopsy; and (4) risk factors at baseline associated with progressive and nonprogressive fibrosis (in univariate analysis of individual studies).

Quality Assessment The quality of included studies was assessed using a modified scale derived from the Newcastle–Ottawa scale.15 This quality score consisted of 8 questions, as follows: representative of the average adult in the community (1 point for unselected participants in population-based or multicenter studies; 0.5 points for unselected participants in single-center, hospital-based studies; and 0 points for participants in an RCT); large cohort size (1 point if cohort size was >50 patients with baseline and follow-up biopsies; 0.5 points if cohort size was between 25 and 50 patients; and 0 points if cohort size was <25 patients); histologic confirmation of NAFLD (1 point if standardized assessment of NAFLD had information on both NASH and NAFL separately; 0.5 points if standardized assessment of NAFLD was without distinction between NASH or NAFL; and 0 points if it reviewed only nonstandardized classification of NAFLD); histologic assessment of fibrosis progression (1 point if assessed using the Brunt classification or the NASH Clinical Research Network; 0.5 points if assessed using Ishak classification; and 0 points if assessed using nonstandardized classification); adequate follow-up evaluation of cohort for outcome to occur (1 point if mean follow-up period of entire cohort was >5 y; 0.5 points if cohort follow-up period was between 3 and 5 y; and 0 points if mean follow-up period of cohort was <3 y); attrition rate (1 point if >80% of cohort was followed up; 0.5 points if 50%–80% of cohort was followed up; and 0 points if >50% was lost to follow-up evaluation); clear information on potential disease-modifying therapy adopted by participants (1 point if there was adequate information on lifestyle modification as well as medication use; 0.5 points if incomplete information; and 0 points if no information on adoption of diseasemodifying therapy); reported risk factors associated with progression of fibrosis (1 point if factors were studied in multivariate models; 0.5 points if factors were reported in only univariate models; and 0 points if factors were associated with fibrosis progression not reported). A score of 6 or higher, 3 to 5, and 2 or less were considered suggestive of a high-, medium-, and lowquality study.

Outcomes Assessed Primary Outcome. The primary outcome measure was

estimation of the FPR in the entire cohort of patients

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with NAFLD, and separately in patients with biopsyproven NAFL and NASH, with baseline stage 0 fibrosis. Estimation of fibrosis progression rate. As previously reported by Ghany et al,16 the FPR was calculated as the number of stages migrated in paired biopsy specimens, over the time difference between the 2 biopsy samples. In this systematic review, we estimated the average FPR for each baseline fibrosis stage (calculated as the difference in fibrosis stage between the first and last biopsy divided by the time between biopsies in years) in patients with NAFL and NASH, with a special focus on patients with baseline stage 0 fibrosis, as well as clinical risk factors associated with the progression of fibrosis. Sensitivity Analyses. A priori hypotheses to assess the stability of FPR and sources of heterogeneity included the location of study (Western population vs Asian population) and study quality. Because of inherent differences in participants in an observational study and an RCT, these 2 groups of studies were analyzed separately. Covariate assessment. To identify risk factors associated with progressive fibrosis in patients with NAFLD, we performed a meta-analysis of differences in baseline clinical (age, sex, presence of diabetes/hypertension/ obesity/metabolic syndrome), laboratory (mean AST level, ALT level, AST:ALT ratio, ferritin level, platelet count, and the homeostasis model of assessment–insulin resistance), and biopsy features (grade of inflammation and steatosis) by comparing patients who showed fibrosis progression and those who did not.

Statistical Analysis We used the random-effects model described by DerSimonian and Laird17 to calculate summary FPR and 95% confidence intervals (CIs) for each stage of baseline fibrosis, using binomial distribution. We assessed heterogeneity between study-specific estimates using 2 methods.18 First, the Cochran’s Q statistical test for heterogeneity, which tests the null hypothesis that all studies in a meta-analysis have the same underlying magnitude of effect, was measured. Because this test is underpowered to detect moderate degrees of heterogeneity, a P value of less than .10 was considered suggestive of significant heterogeneity. Second, when heterogeneity was present, to estimate what proportion of total variances across studies was owing to heterogeneity rather than chance, the inconsistency index (I2) statistic was calculated. In this, a value of greater than 50% was suggestive of substantial heterogeneity.19 Once heterogeneity was noted, between-study sources of heterogeneity were investigated using subgroup analyses by stratifying original estimates according to study characteristics as described earlier. Publication bias was ascertained, qualitatively, by visual inspection of the funnel plot,20 and quantitatively by the Egger et al21 regression test. For all tests (except for heterogeneity and publication bias), a probability level of less than 0.05

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was considered statistically significant. All analyses were performed using STATA version 12 (StataCorp).

Results From a total of 1994 unique studies identified using the prespecified systematic search strategy, 11 observational studies were included in this analysis.7,8,12,13,22–28 Six studies were excluded owing to a lack of sufficient information to calculate the FPR despite the availability of paired liver biopsies. We were able to obtain additional information (which was not available in the published article) that was required to calculate the FPR by the presence of baseline NASH and NAFL for 3 studies through contact with the study authors.8,26,28 Only 2 RCTs provided sufficient data to estimate the FPR by baseline fibrosis stage, obtained by contacting the study’s lead author.29,30 Two RCTs provided data on the overall FPR but not by baseline stage.31,32 Two RCTs reported the proportion of placebo-treated patients who progressed or remained stable, but no additional information to estimate the FPR.33,34 Overall, these RCTs reported on 186 patients treated with placebo (158 with paired biopsies), of whom 38 (24.0%) had worsening fibrosis, 84 (53.2%) remained stable, and 36 (22.8%) had improvement in fibrosis stage on follow-up biopsy (Supplementary Tables 2 and 3); owing to a limited number of studies with a short duration of follow-up evaluation, an accurate estimate of the FPR could not be obtained. Eight placebo-controlled RCTs with paired biopsies performed 12 months apart provided data only on the mean change in fibrosis stage. Hence, RCTs were excluded from further analysis.

Characteristics and Quality of Included Studies Baseline characteristics. Table 1 describes the characteristics of the included studies. We identified 11 observational studies including 411 patients with biopsy-proven NAFLD (150 patients with NAFL and 261 patients with NASH). Overall, 366 patients had sufficient information for FPR estimation by baseline stage. Among the 11 studies included in this meta-analyses, 8 were derived from Western populations7,8,12,13,22–24,27 and 3 were derived from Asian populations.25,26,28 Nine of the 11 studies used Brunt’s classification for fibrosis staging suggesting good generalizability across studies.35 Supplementary Table 4 lists the definitions of NAFLD, NAFL, and NASH in the studies. Table 2 describes the baseline characteristics of the patients in the included studies. The mean age of participants at the time of initial biopsy ranged from 44 to 55 years; 53.0% of patients were men. The mean body mass index ranged from 27.4 to 37.7 kg/m2, and almost half of the patients (49.9%) were diabetic. Entire cohort changes in fibrosis and duration of follow-up period. At baseline, the distribution of fibrosis

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for stages 0, 1, 2, 3, and 4 was 35.8%, 32.5%, 16.7%, 9.3%, and 5.7%, respectively. Over 2145.5 person-years of follow-up evaluation, 33.6% (n ¼ 138) had progression (increased by at least 1 fibrosis stage as compared with baseline), 43.1% (n ¼ 177) remained stable, and 22.3% (n ¼ 96) had improvement (decreased by at least one fibrosis stage as compared with baseline) in fibrosis stage. Quality assessment. A detailed quality assessment of the studies included in the meta-analyses is provided in Supplementary Table 5. Most of the included studies were at moderate risk of bias, with 2 studies being at low risk of bias.8,28 None of the included studies were population-based, and only 1 observational study was a multicenter study.8 Three studies were restricted only to patients with NASH,22–24 and 2 studies included only patients with NAFL.12,13 Of the remaining 6 studies, 4 provided sufficient data (either in the primary publication or through contact with the authors) to allow estimation of the FPR for patients with NASH and NAFL separately8,25,26,28; we were not able to obtain this information in 2 studies.7,27 Five studies provided information on use of potential disease-modifying pharmacologic therapy and recommendations offered for lifestyle changes,22,24,25,28,29 however, the extent to which these recommendations were adopted could not be ascertained reliably.

Rate of Fibrosis Progression in Patients With Nonalcoholic Fatty Liver Versus Nonalcoholic Steatohepatitis Fibrosis progression rate in the entire cohort of nonalcoholic fatty liver disease patients. Eleven studies

provided sufficient information to estimate the FPR in 366 patients with NAFLD. Of these, 132 (36.1%) patients developed progressive fibrosis, 158 (46.2%) patients remained stable, and 76 (20.8%) patients had improvement in fibrosis. On meta-analysis, the overall annual FPR in patients with NAFLD with baseline stage 0 fibrosis (131 patients) was 0.13 stages (95% CI, 0.07–0.18), which corresponded to an average progression of 1 stage over 7.7 years (95% CI, 5.5–14.8 y) (Figure 1A). Fibrosis progression rate in nonalcoholic fatty liver. Six studies provided sufficient information to estimate the FPR in 133 patients with NAFL.8,12,13,25,26,28 Of these, 52 (39.1%) patients developed progressive fibrosis, 70 (52.6%) patients remained stable, and 11 (8.3%) patients had improvement in fibrosis. On meta-analysis, the overall annual FPR in patients with NAFL with baseline stage 0 fibrosis (81 patients) was 0.07 stages (95% CI, 0.02–0.11), which corresponded to an average progression of 1 stage over 14.3 years (95% CI, 9.1–50.0 y) (Figure 1B). Fibrosis progression rate in nonalcoholic steatohepatitis. Seven studies provided sufficient information to

estimate the FPR in 116 patients with NASH.8,22–26,28

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Table 1. Details of Included Observational Studies Study, year of publication

Rochester, MN; hospital-based Charlottesville, VA

Total patients with paired biopsies, n

Criterion for histologic staging

Change in fibrosis scores Worse, n (%)

Stable, n (%)

1980–2003

103; NAFL, 7; NASH, 96

Brunt

NR; 3.2 (3.0); 0.7–21

38 (37%) 35 (34%)

1997–1999

5; NAFL, 0; NASH, 5

Brunt; NASH CRN

22 p-y; 4.4; 3.0–6.4

70; NAFL, 36; NASH, 34

Brunt

1202 p-y; 13.8 (1.2); 10.3–16.3 29 (41%) 30 (43%)

7; NAFL, 0; NASH, 7

Brunt

57.2 p-y; 8.2; 5.5–11.9

4 (57%)

3 (60%)

0

Improved, Quality score n (%) (maximum, 8)a 30 (29%)

5

2 (40%)

5

Linköping, Sweden 1988–1993; follow-up evaluation, 2003 Glasgow, UK 1985–1994; follow-up evaluation, 1999 Buenos Aires, 1986–2002 Argentina Ishikawa, Japan 1997–2008

11 (16%)

6.5

0

3.5

22; NAFL, 0; NASH, 22

Brunt, Ishak

NR; 5.3 (2.7); 3.0–14.3

7 (32%) 11 (50%)

4 (18%)

5.5

39; NAFL, 22; NASH, 17

Brunt

NR; 2.4; 1.0–8.5

11 (28%) 16 (41%)

12 (31%)

4.5

San Antonio, TX

1985–2001

22; NAFL, 0; NASH, 22

Brunt

NR; 5.7; 1.4–15.7

7 (32%) 11 (50%)

4 (18%)

4.5

Hong Kong

1996–2004

17; NAFL, 3; NASH, 14

Brunt

97.8 p-y; 6.1; 3.8–8.0

9 (53%)

0

4.5

Paris, France

1998–2009

70; NAFL, 25b; NASH, 45 Kleiner and Brunt

102.7 p-y; 3.7 (2.1); NR

Paris, France

1988–1999

14; NAFL, 10; NASH, 4

Metavir

73 p-y; NR; 1.5–15.0

2 (14%)

8 (57%)

4 (29%)

1.5

Newcastle, UK

1978–1985

12; NAFL, 12; NASH, 0

NR

NR; 11.6; 7.6–16

1 (8%)

11 (92%)

0

1.5

Hong Kong

2006–2009

52; NAFL, 35; NASH, 17

Kleiner and Brunt

NR; 3.0; NR

3 (43%)

8 (47%)

20 (29%) 30 (42%)

14 (27%) 25 (48%)

20 (29%)

13 (25%)

5

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NOTE. The study by Harrison et al50 was not included in the overall analysis because it did not provide information on the FPR based on baseline stage. CRN, clinical research network; NR, not reported; p-y, person-years. a The details of quality assessment of individual studies is reported in Supplementary Table 3. b Fibrosis progression was reported only for the subset with NAFL.

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Adams et al,7 2005 Argo et al,22 2009 Ekstedt et al,8 2006 Evans et al,23 2002 Fassio et al,24 2004 Hamaguchi et al,25 2010 Harrison et al,50 2003 Hui et al,26 2005 Pais et al,13 2013 Ratziu et al,27 2000 Teli et al,12 1995 Wong et al,28 2010

Period of recruitment

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Location; setting

Duration of follow-up period, total p-y; mean (SD); range

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523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580

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Table 2. Baseline Characteristics of Patients Included in the Respective Studies

Study

Age, Sex mean (SD) (as % males), n 37 (38)

Ekstedt et al,8 2006 Evans et al,23 2002 Fassio et al,24 2004 Hamaguchi et al,25 2010 Harrison et al,50 2003 Hui et al,26 2005 Pais et al,13 2013 Ratziu et al,27 2000 Teli et al,12 1995 Wong et al,28 2010

Obesity Diabetes (as % total) (as % total), n

AST, mean (SD)

Metformin, n (%)

TZDs, Vitamin E, n (%) n (%) Nonpharmacologic interventions

-

67

42 (43)

75 (50)

1 (0.9%)

0

0

NR

NR

37.7 (6.5)

NR

60 (3)

94 (39), ALT

2 (40%)

1 (20%)

0

51 (13)

67 (47)

28.3 (3.8)

29

85 (60)

45 (23)

NR

Physical activity, high intensity, 40%; moderate intensity, 40%; low intensity, 20% NR

49 (10)

43 (3)

32.4 (5.0)

57

43 (3)

38 (18)

NR

NR

45 (13)

41 (9)

29.8 (24.0–38.2)a

45

36 (8)

73 (41–129),a ALT

Nutritional counseling

47 (20–79)a 56 (22)

27.8 (22.5–44.4)a

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77 (30)

40 (11–106)a

No specific disease-modifying pharmacologic therapy 4 (10%) 1 (2.6%) 0

Diet/insulin for diabetic patients

51 (33–64)a 59

33.8 (26.5–48.6)a

77

41

74.5 (13–158)a

NR

NR

42 (3) 52 (10)

65 (11) NR

29.3 (23–35.5)a 29 (3.6)

35 NR

24 (4) 35 (25)

104 (32–193),a ALT 3 (18%) 85 (43), ALT NR

49 (20–79)

34 (8)

29.1 (25.1–45.7)a

NR

16 (2)

NR

NR

NR

55 (26–79)a 55 (6)

NR

30

10 (1)

35 (26)

NR

NR

44 (9)

27.4 (3.7)

NR

50 (26)

61 (37–100),a ALT

16 (31%)

65 (34)

0

0

0 0

0

Q22

NR NR

Baseline dietician visit; advised to exercise 3 /wk

NOTE. The study by Harrison et al50 was not included in the overall analysis because it did not provide information on the FPR based on baseline stage. BMI, body mass index; NR, not reported; TZD, thiazolidinedione. a Median (range).

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Adams 45 (11) et al,7 2005 Argo NR et al,22 2009

BMI, mean (SD)

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Figure 1. Pooled fibrosis progression rate in patients with (A) nonalcoholic fatty liver disease, (B) nonalcoholic fatty liver, and (C) nonalcoholic steatohepatitis, and baseline stage 0 fibrosis. Effect size represents the annual rate of progression of fibrosis stage.

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Overall, 40 (34.5%) patients developed progressive fibrosis, 45 (38.8%) patients remained stable, and 31 (26.7%) patients had improvement in fibrosis. In a metaanalysis of patients with NASH with baseline F0 fibrosis (21 patients), the annual FPR was estimated at 0.14 stages (95% CI, 0.07–0.21), corresponding to an average 7.1 years (95% CI, 4.8–14.3 y) as time taken to progress by 1 stage (Figure 1C). Table 3 shows the overall FPR by baseline stage for patients with NAFLD, NAFL, and NASH, and shows generalizability across studies. Supplementary Table 6 shows the baseline and follow-up fibrosis stage for each individual study.

Subgroup and Sensitivity Analyses As expected, heterogeneity was observed in the FPR in patients with NAFLD and NAFL; however, NASH patients had less heterogeneity in FPR. (I2 for NAFLD, NAFL, and NASH was 88%, 81%, and 21%, respectively.) On subgroup analysis, there was no significant difference in the FPR in Western and Asian patients with NAFLD (Supplementary Table 7). Likewise, there were no significant differences in the FPR based on study quality. To assess whether any one study had a dominant effect on

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the meta-analytic FPR, each study was excluded and its effect on the main summary estimate and I2 test for heterogeneity was evaluated; no study markedly affected the overall FPR or degree of heterogeneity. Because substantial heterogeneity was observed in the overall analysis, evaluation for publication bias using a funnel plot was unremarkable.36 Explanation for heterogeneity in nonalcoholic fatty liver disease (rapid progressors and slow progressors). In

this study, we observed 2 subsets of patients with NAFLD stratified by the rate of fibrosis progression: rapid progressors and slow progressors. Rapid progressors included a small subset of patients with NAFLD (with baseline stage 0 fibrosis) who developed rapid progression to advanced fibrosis (stage 3 or 4 fibrosis). Of the 52 patients with NAFLD with stage 0 fibrosis at baseline who showed progression in their fibrosis stage at the follow-up liver biopsy, 11 (21.2%) patients progressed to stage 3 or 4 fibrosis over a mean (SD) follow-up period of 5.9 (3.7) years (Figure 2). The vast majority of patients with NAFLD showed slow progression in their fibrosis stage at the follow-up liver biopsy usually only by 1 or 2 stages. Of the 29 patients with NAFL with baseline stage 0 fibrosis who developed progressive fibrosis, 5 (17.2%)

Table 3. Overall Fibrosis Progression Rate by Baseline Fibrosis Stage in Patients With NAFLD, NAFL Alone, and NASH Alone Final fibrosis stage

Total stages of fibrosis progressed

Person-years of follow-up evaluation

þ91 þ43 6 16 8 þ104 þ134

968 628.4 331.8 153.4 63.8 2145.4 1596.4

þ48 þ20 3 0 0 þ75 þ68 þ18 þ13 4 6 1 þ20 þ31

Fibrosis progression rate (95% CI)

Time taken to progress by 1 stage (95% CI)

NAFLD (11 studies) Baseline fibrosis stage

0 1 0 (131) 79 28 1 (119) 26 44 2 (61) 9 17 3 (34) 2 5 4 (21) 0 0 Overall (366) Stage 0 plus stage 1

2 13 32 14 10 1

3 7 15 13 7 6

4 4 2 8 10 14

fibrosis (250)

0.13 (0.07–0.18) 0.10 (0.04–0.16) NA NA NA NA 0.12 (0.07–0.16)

7.7 (5.5–14.8) 10.0 (6.2–25.0) 8.3 (6.2–14.3)

751.3 112.6 40.7 0 0 904.6 863.9

0.07 (0.02–0.11) 0.15 (-0.09 to 40) NA NA NA NA 0.09 (0.04–0.14)

14.3 (9.1–50.0) NA 11.1 (7.1–25.0)

115.5 396.6 222.3 95.8 12.6 842.8 512.1

0.14 (0.07–0.21) 0.08 (-0.01 to 0.17) NA NA NA NA 0.10 (0.03–0.17)

NAFL (6 studies) Baseline fibrosis stage

0 1 0 (81) 52 16 1 (39) 6 13 2 (13) 2 3 3 (0) 0 0 4 (0) 0 0 Overall (133) Stage 0 plus stage 1

2 8 14 5 0 0

3 4 6 2 0 0

4 1 0 1 0 0

fibrosis (120)

NASH (7 studies) Baseline fibrosis stage

0 1 0 (21) 10 7 1 (49) 9 25 2 (25) 3 10 3 (16) 0 4 4 (5) 0 0 Overall (116) Stage 0 plus stage 1

2 2 9 4 4 0

3 1 5 4 2 1

fibrosis (70)

4 1 1 4 6 4

7.1 (4.8–14.3) NA

10.0 (5.9–33.3)

NOTE. If the lower limit of the 95% CI for fibrosis progression rate was negative (ie, the lower limit suggested there could be net regression of fibrosis stage), then the time taken to progress to fibrosis by 1 stage was not calculated.

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Risk Factors Associated With Progressive Fibrosis Ten studies reported baseline clinical, laboratory, and/or histologic features among patients with and without progressive fibrosis on paired biopsy specimens. Meta-analysis of these clinical and laboratory risk factors for progressive fibrosis could be performed for 7 studies (Supplementary Table 8). The presence of hypertension (odds ratio, 1.94; 95% CI, 1.00–3.74) and a low AST:ALT ratio at the time of baseline biopsy was associated with the development of progressive fibrosis (Table 4). Data on histologic variables associated with risk of fibrosis progression was not amenable to quantitative synthesis. Two7,8 (of 4 studies)7,8,24,26 observed that patients with a higher steatosis grade were more likely to develop progressive fibrosis; no association was found between baseline severity of necroinflammation and risk of progressive fibrosis.7,8,24,26

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Discussion Figure 2. Differential rate of fibrosis progression in patients with NAFLD, NAFL, and NASH, among patients with progressive fibrosis. Rapid progressors refers to a subset of patients with baseline stage 0 fibrosis who developed rapid progression to advanced fibrosis (stage 3 or 4 fibrosis); slow progressors refers to patients with baseline stage 0 fibrosis who had slow progression by 1 to 2 stages on follow-up biopsy.

patients were rapid progressors. Similarly, of the 11 patients with NASH with baseline stage 0 fibrosis who developed progressive fibrosis, 2 (18.2%) patients were rapid progressors.

Based on evidence derived from this systematic review and meta-analysis of paired liver biopsy studies, we show that both patients with NAFL and NASH may develop progressive liver fibrosis. The annual FPR in patients with NAFL vs NASH, with baseline stage 0 fibrosis, was 0.07 stages vs 0.14 stages, respectively, corresponding to an average progression of 1 stage over 14.3 vs 7.1 years, respectively. Among patients who developed progressive liver fibrosis on follow-up liver biopsy, we identified 2 potentially distinct subsets and classified them into rapid progressors (progression from stage 0 to bridging fibrosis or cirrhosis) and slow progressors (progression from stage 0 to stage 1 or 2 fibrosis). One in 5 patients who developed interval

Table 4. Meta-Analysis of Clinical Variables Comparing Patients With Progressive Fibrosis Versus Nonprogressive Fibrosis (Reference) Risk factor

Q25

No. of studies

Categoric variables Sex (males vs females) Diabetes (yes vs no) Hypertension (yes vs no) Metabolic syndrome (yes vs no) Continuous variables Age, y BMI, kg/m2 AST level, U/L ALT level, U/L AST:ALT ratio Platelet count (109/L) Ferritin level, mg/L HOMA-IR

Odds ratio (or weighted difference in means)

7 7 5 3

0.75 1.05 1.94 0.63

7 5 4 7 4 3 3 4

0.71 1.41 8.09 7.60 -0.08 -2.72 -77.15 0.11

P value

I2 (%)a

0.44–1.28 0.64–1.72 1.00–3.74 0.32–1.25

.29 .86 .05 .19

2.2 0.0 0.0 0.0

-2.37 to 3.80 -0.28 to 3.10 -1.84 to 18.02 -6.97 to 22.17 -0.16 to 0.00 -56.82 to 51.38 -368.6 to 214.3 -1.05 to 1.27

.65 .10 .11 .31 .06 .92 .60 .86

68.5 45.3 46.0 73.3 0.0 83.1 76.8 96.8

95% CI

BMI, body mass index; HOMA-IR, homeostasis model of assessment–insulin resistance. a Low value indicates low heterogeneity.

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fibrosis progression could be classified as a rapid progressor, whereas the rest of the patients were slow progressors; however, in the absence of information on individual patient data and small sample size, more detailed assessment on this differential rate of fibrosis progression was not feasible. Through a systematic review of the published literature, performed with additional data obtained through contact with the authors, we were able to estimate the rate of progression of fibrosis in patients with NAFL and NASH with baseline stage 0 fibrosis, separately. Although previously underappreciated, we observed that patients with NAFL can develop progressive fibrosis, albeit at a slower rate than patients with NASH. We were not able to identify specific risk factors for the development of progressive fibrosis in these patients with NAFL. It is possible that these may represent a subset of patients with NAFL who have mild lobular inflammation, without hepatocyte ballooning or fibrosis, not fulfilling criteria for definite steatohepatitis, but having persistent inflammation, which in turn may lead to progression to definite NASH in due course of time. Pais et al13 observed that among 16 patients with NAFL with mild lobular inflammation at baseline, 13 patients progressed to typical NASH or bridging fibrosis; on the other hand, of the 5 patients with simple steatosis alone, only 1 patient progressed to NASH. Our observations on FPR were seemingly different than that reported by Argo et al,11 in their meta-analysis of 10 studies in patients with NASH. They estimated an overall FPR of 0.03 stages per year (SD, 0.53 stages/y). In their analysis, they included a heterogenous cohort of patients with NASH at any stage of fibrosis at baseline, and estimated an overall fibrosis progression, regardless of baseline fibrosis stage. By combining patients at all stages of fibrosis to estimate a single FPR, their analysis was more likely to be affected by selection bias. Individual patient data on how patients moved across stages of fibrosis at baseline and follow-up evaluation was not available in their analysis. The progression of fibrosis in patients with NAFLD represents a complex interplay of genetic factors, extrinsic environmental and intrinsic microbial factors.37–39 The natural history of NAFLD is potentially modifiable through diet and lifestyle changes, and, hence, is not a universally progressive condition as was confirmed in our study.2,40 Previous studies have shown that age and diabetes are 2 of the major drivers of fibrosis progression.41–45 In this meta-analysis, age and diabetes did not reach statistical significance, but hypertension and the AST:ALT ratio were associated more closely with fibrosis progression. It is plausible that the AST:ALT ratio is perhaps a very sensitive discriminative marker of fibrosis progression early on in the natural history of NAFLD. Interestingly, we observed that among a subset of patients who progress, a small proportion of patients may progress rapidly to advanced fibrosis. This may have accounted for the considerable heterogeneity observed in our analysis. Although it is possible that this may be owing to a longer

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time interval between serial biopsies in these patients (we were not able to ascertain this in the absence of individual patient data) along with the inherent limitations of sampling variability of a liver biopsy, it is unlikely to be the only reason. It is probable that these groups of patients have inherent genetic or modifiable environmental factors putting them at risk for rapid progression, as has been observed in other chronic liver diseases by independent groups.46–49 This finding underscores the need for further research in identifying the pathogenetic factors that determine the differential rate of fibrosis progression between rapid and slow progressors, and efforts aimed at identifying this subset of patients would allow appropriate clinical follow-up evaluation and enrollment in clinical trials of disease-modifying pharmacologic agents. Because of limited data on potential rapid and slow progressors in the included studies, a systematic evaluation of such factors was not possible in the current analysis.

Strengths and Limitations The strengths of this review included the following: (1) comprehensive and systematic literature search with well-defined inclusion criteria; (2) estimation of the FPR in patients with NASH and NAFL separately; (3) estimation of the FPR by baseline stage of fibrosis to overcome the limitation of selection bias; (4) separate analysis of observational studies and placebo-controlled RCTs; (5) rigorous evaluation of study quality; and (6) qualitative and quantitative assessment of clinical risk factors associated with the development of progressive fibrosis. Several limitations of the included studies and, consequently, the meta-analysis merit further discussion. First, the studies were at high risk for selection bias. The high attrition rate, owing to a lack of patient consent or physician recommendation for a follow-up liver biopsy, may have influenced the study results. However, in the absence of accurate noninvasive biomarkers of NASH and fibrosis, paired liver biopsy studies offer the best insight into the natural history of this condition. Second, we were unable to estimate the FPR for patients with intermediate and advanced fibrosis at baseline. Clinical indications for repeat biopsy in patients with NAFLD are not well defined. It is conceivable that more healthconscious and informed patients, especially those with baseline intermediate or advanced fibrosis, may have opted for a repeat biopsy after they followed recommendations for a healthy lifestyle, resulting in a selection of patients who were slower to progress (or more likely to regress). It also is likely that patients who progressed from advanced fibrosis to decompensated cirrhosis were not advised to repeat a histologic examination, potentially underestimating the rate of fibrosis progression. To overcome this limitation, we specifically estimated the FPR in patients with stage 0 fibrosis. Third, the effect of lifestyle, including weight loss and physical activity, and

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potential disease-modifying therapy, which would alter the rate of fibrosis progression, was not captured adequately in individual studies. Likewise, risk factors associated with progressive fibrosis were not reported consistently and uniformly in individual studies. Our assessment of risk factors was based on a comparison of patients with progressive fibrosis and those without progressive fibrosis; ideally, a stratified analysis of the FPR based on putative risk factors would have been ideal (eg, comparing the FPR in diabetic vs nondiabetic, obese vs nonobese, and so forth), however, such data were not available. Fourth, none of the studies were populationbased, and the majority of the studies were single-site studies. There was no centralized reading of biopsy specimens, resulting in variability in the diagnosis of NAFLD and increasing the possibility of misclassification bias. However, despite these limitations, the majority of the studies used a common histologic scoring system that allowed for generalizability of results, in single academic centers with expert gastrointestinal pathologists. It conservatively can be estimated that more than 40% of patients with NAFLD will have progressive fibrosis. Although previously under-recognized, patients with NAFL without fibrosis also may progress to advanced fibrosis, at a rate of 1 stage over 14.3 years. In the absence of disease-modifying pharmacologic therapy and limited uptake of healthy lifestyle changes, and assuming (a conservative) linear progression of fibrosis, a vast proportion of young obese adults with NAFLD today may develop advanced fibrosis over the next 3 decades. This has serious implications for liver-related morbidity and mortality, and highlights a major unmet need for disease-modifying therapy in patients with NASH.

Implications for Clinical Practice and Future Research In this systematic review and meta-analysis of paired liver biopsy studies in patients with NAFLD, contrary to conventional paradigm, we found that both patients with NAFL and NASH develop progressive hepatic fibrosis, progressing by 1 fibrosis stage (from baseline stage 0 fibrosis) over 14.3 and 7.1 years, respectively. A small subset of these patients may develop rapidly progressive hepatic fibrosis. Based on this systematic review, assuming that 80 to 100 million Americans are afflicted with NAFLD in the United States, a significantly higher number of patients with NAFLD are at risk for progressive liver disease than previously appreciated. Effective therapies are needed to halt the progression of fibrosis in NAFLD.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Clinical

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Gastroenterology and Hepatology at www.cghjournal.org, and at http://dx.doi.org/10.1016/j.cgh.2014.04.014.

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Reprint requests Address requests for reprints to: Rohit Loomba, MD, MHSc, Associate Professor of Medicine, Division of Gastroenterology and Epidemiology, University of California at San Diego School of Medicine, UC 303, MC-0063, 9500 Gilman Drive, La Jolla, California 92093. e-mail: [email protected]; fax: (858) 534-3338. Acknowledgments The authors sincerely thank the following investigators for kindly sharing additional data from their cohort, which enabled this meta-analysis to be performed: Manel Abdelmalek, MD, MPH (Division of Gastroenterology/Hepatology, Duke University Medical Center, Durham, North Carolina); Henry Chan, MD (Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong SAR, China); Stergio Kechagias, MD, PhD (Division of Gastroenterology and Hepatology, Linköping University, Linköping, Sweden); Carmela Loguercio, MD (Department F. Magrassi e A. Lanzara, Second University of Naples, Naples, Italy); Wade Shields, DO (Division of Gastroenterology and Hepatology, Naval Medical Center, San Diego, California); and Vincent Wong, MBChB, MD (Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong SAR, China). Conflicts of interest The authors disclose no conflicts. Funding Supported in part by the American Gastroenterological Association Foundation–Sucampo–ASP Designated Research Award in Geriatric Gastroenterology, a T. Franklin Williams Scholarship Award, Atlantic Philanthropies, Inc, the John A. Hartford Foundation, the Association of Specialty Professors, and grants P30CA23100-28 and K23DK090303 (R.L.).

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1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450

2014

Fibrosis Progression in NAFLD

12.e1

1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508

Supplementary Figure 1. Flowchart summarizing study identification and selection.

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12.e2

1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566

Singh et al

Clinical Gastroenterology and Hepatology Vol.

-,

No.

-

Supplementary Table 1. Details of Search Strategy

Q28Q29

Ovid: database(s): Embase 1988 to 2013 week 28; Ovid MEDLINE in-process and other nonindexed citations and Ovid MEDLINE 1946 to present; EBM reviews, Cochrane Central Register of Controlled Trials June 2013; EBM reviews, Cochrane Database of Systematic Reviews 2005 to May 2013 Search strategy: No.

Searches

1 2 3

Fatty liver/ Exp nonalcoholic fatty liver/ (“Fatty liver” or steatohepatitis or ((NAFLD or NASH) and (liver* or hepat*)) or steatohepatitides or (liver adj2 steatos*) or “visceral steatos*”).mp. [mp ¼ ti, ab, sh, hw, tn, ot, dm, mf, dv, kw, nm, kf, ps, rs, ui, tx, ct] 1 or 2 or 3 Prognosis/ Disease course/ Disease progression/ Exp treatment outcome/ (Outcome* or “natural history” or prognos* or progression or (disease adj3 course) or (disease adj3 evolution)).mp. [mp ¼ ti, ab, sh, hw, tn, ot, dm, mf, dv, kw, nm, kf, ps, rs, ui, tx, ct] Or/5–9 4 and 10 Exp controlled study/ Exp randomized controlled trial/ ((Control$ or randomized) adj2 (study or studies or trial or trials)).mp. [mp ¼ ti, ab, sh, hw, tn, ot, dm, mf, dv, kw, nm, kf, ps, rs, ui, tx, ct] Meta analysis/ Meta-analys$.mp. Exp “systematic review”/ (Systematic* adj review$).mp. Exp cohort studies/ Exp prospective study/ (Prospective adj (study or studies or survey or surveys or analysis or analyses or trial or trials)).mp. Cohort*.mp. Or/12–22 11 and 23 From 11 keep 7438–11600 Limit 25 to (controlled clinical trial or meta-analysis or randomized controlled trial or systematic reviews) [limit not valid in Embase, CCTR, CDSR; records were retained] 24 or 26 Limit 27 to (book or book series or editorial or erratum or letter or note or addresses or autobiography or bibliography or biography or comment or dictionary or directory or interactive tutorial or interview or lectures or legal cases or legislation or news or newspaper article or overall or patient education handout or periodical index or portraits or published erratum or video-audio media or webcasts) [limit not valid in Embase, Ovid MEDLINE, Ovid MEDLINE in-process, CCTR, CDSR; records were retained] 27 not 28 From 11 keep 11601–11780 29 or 30 Limit 31 to (“all adult (19 plus years)” or “young adult (19 to 24 years)” or “adult (19 to 44 years)” or “young adult and adult (19–24 and 19–44)” or “middle age (45 to 64 years)” or “middle aged (45 plus years)” or “all aged (65 and over)” or “aged (80 and over)”) [limit not valid in Embase, CCTR, CDSR; records were retained] Limit 32 to (adult <18 to 64 years> or aged <65þ years>) [limit not valid in Ovid MEDLINE, Ovid MEDLINE in-process, CCTR, CDSR; records were retained] Limit 33 to yr ¼ “1985 -Current” Remove duplicates from 34

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

29 30 31 32

33 34 35

Results 34,015 10,820 53,319

Q30

53,319 732,245 275,761 383,328 1,648,861 4,710,658 4,767,844 11,780 4,168,131 709,672 5,388,418 117,927 191,750 62,017 149,408 1,579,728 656,470 793,561 721,146 7,253,882 3821 4163 240 3844 135

3709 180 3811 3492

2062 2052 1469

Scopus

1 TITLE-ABS-KEY(“fatty liver” or steatohepatitis or ((NAFLD or NASH) and (liver* or hepat*)) or steatohepatitides or (liver W/2 steatos*) or “visceral steatos*”) 2 TITLE-ABS-KEY(outcome* or “natural history” or prognos* or progression or (disease W/3 course) or (disease W/3 evolution)) 3 TITLE-ABS-KEY((meta W/1 analys*) OR (systematic* W/2 review*) OR (control* W/2 stud*) OR (control* W/2 trial*) OR (randomized W/2 stud*) OR (randomized W/2 trial*) or cohort* OR “prospective stud*” OR “prospective survey*” OR “prospective analys*”) 4 PUBYEAR Aft 1984 5 1 and 2 and 3 and 4 6 TITLE(child* or adolescent* or infant*) 7 TITLE(adult or adults) 8 6 and not 7 9 5 and not 8

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Q31

1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624

-

1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 Q32 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682

2014 10 11 12 13

Fibrosis Progression in NAFLD

12.e3

PMID(0*) OR PMID(1*) OR PMID(2*) OR PMID(3*) OR PMID(4*) OR PMID(5*) OR PMID(6*) OR PMID(7*) OR PMID(8*) OR PMID(9*) 9 and not 10 DOCTYPE(le) OR DOCTYPE(ed) OR DOCTYPE(bk) OR DOCTYPE(er) OR DOCTYPE(no) OR DOCTYPE(sh) 11 and not 12

Web of Science

1 Topic¼(“fatty liver” or steatohepatitis or ((NAFLD or NASH) and (liver* or hepat*)) or steatohepatitides or (liver NEAR/2 steatos*) or “visceral steatos*”) AND Topic¼(outcome* or “natural history” or prognos* or progression or (disease NEAR/3 course) or (disease NEAR/3 evolution)) AND Topic¼((meta NEAR/1 analys*) OR (systematic* NEAR/2 review*) OR (control* NEAR/2 stud*) OR (control* NEAR/2 trial*) OR (randomized NEAR/2 stud*) OR (randomized NEAR/2 trial*) or cohort* OR “prospective stud*” OR “prospective survey*” OR “prospective analys*”) Timespan¼1985-2013. Databases¼SCI-EXPANDED. 2 TI¼(child* or adolescent* or infant*) NOT TI¼(adult or adults) Databases¼SCI-EXPANDED Timespan¼1985-2013

3 (#1 not #2) AND Document Types¼(Article OR Abstract of Published Item OR Meeting Abstract OR Proceedings Paper) Databases¼SCIEXPANDED Timespan¼1985-2013

Supplementary Table 2. Details of Patients Included in Control Arm of Placebo-Controlled Randomized Controlled Trials of NAFLD Therapy

Study, year of publication

Duration, mo

Location

Abdelmalek, 2009

Minnesota and Florida

12

Aithal, 2008

Nottingham, UK

12

Ratziu, 2008

France

12

Sanyal, 2010

United States

24

Van Wagner, 2011

Illinois

12

Active treatment arm; no. of patients

Histologic staging system Brunt

Change in fibrosis scores in Placebo arm placebo-treated patients intervention; no. of patients (total/no. with paired biopsies) Worsened Stable Improved

Betaine; 17

Placebo and diet þ exercise counseling; 28/18 Brunt Pioglitazone; 31 Placebo and diet þ exercise counseling; 37/30 Brunt Rosiglitazone; 32 Placebo and diet þ exercise counseling; 31/31 NASH-CRN Vitamin E or Placebo and pioglitazone; diet þ exercise 164 counseling; 83/72 Brunt Pentoxifylline; 19 Placebo and diet þ exercise counseling; 7/7

NOTE. All participants in these trials had NASH. CRN, clinical research network.

Supplementary Table 3. Overall Fibrosis Progression Rate by Baseline Fibrosis Stage in Patients With Nonalcoholic Steatohepatitis, Enrolled in the Control Arm of Placebo-Controlled Randomized Controlled Trials Final fibrosis stage

Total stages Person-years of fibrosis of follow-up progressed evaluation

NAFLD (2 studies): all patients had NASH 0 1 2 3 4 Baseline 0 (8) 6 0 1 1 0 fibrosis stage 1 (9) 0 5 1 2 1 2 (4) 0 1 1 1 1 3 (7) 1 1 0 5 0 4 (0) 0 0 0 0 0 Overall (28) Stage 0 plus stage 1 fibrosis (17)

þ5 þ8 þ1 5 0 þ9 þ13

8 9 4 8 0 28 17

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7 (39%)

8 (44%)

3 (17%)

6 (20%)

18 (60%)

6 (20%)

6 (19%)

20 (65%)

5 (16%)

16 (22%)

34 (47%)

22 (31%)

3 (43%)

4 (57%)

0

1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740

1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770 1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796 1797 1798 12.e4

Supplementary Table 4. Definitions of NAFLD, NAFL, and NASH in Included Studies Study

NAFL

NASH

(1) Steatosis involving at least 10% of hepatocytes on biopsy, (2) ethanol consumption of <140 g/wk, (3) exclusion of patients with evidence of other liver disease using standard clinical, laboratory, and histologic criteria Only patients with NASH

Combination of steatosis with nonspecific inflammation (steatosis plus either lobular inflammation or ballooning, but not both) or bland steatosis (steatosis without lobular inflammation, ballooning or fibrosis) -

Either the presence of steatosis plus mixed lobular inflammation plus hepatocellular ballooning, or the presence of steatosis plus any stage of fibrosis

Simple steatosis or steatosis with nonspecific inflammation and absence of fibrosis

Evans et al,23 2002

Hepatic steatosis without any other concomitant liver disease, weekly alcohol consumption <140 g, or medication associated with fatty infiltration of the liver Only patients with NASH

Fassio et al,24 2004

Only patients with NASH

Hamaguchi et al,25 2010

Hepatic steatosis in the absence of known causes of fatty liver Histologic evidence of steatosis with or without the presence of necroinflammation and fibrosis, without known causes of fatty liver Hepatic steatosis >10% in the absence of known causes of fatty liver

Adams et al, 2005

Argo et al,22 2009

Hui et al,26 2005

Pais et al,13 2013

Ratziu et al,27 2000

Teli et al,12 1995 Wong et al,28 2010

Abnormal liver enzyme levels in overweight or obese patients, after exclusion of other causes of liver diseases Hepatic steatosis in the absence of excessive alcohol use Histologic evidence of steatosis with or without the presence of necroinflammation and fibrosis, without known causes of fatty liver

-

Hepatic steatosis without presence of ballooned hepatocytes Simple steatosis without necroinflammation or fibrosis Hepatic steatosis alone (bland steatosis) or steatosis without evidence of ballooning, with spotty lobular inflammation of grade 1 maximum (<2 foci/sox power field) and no fibrosis or fibrosis limited to mild periportal or perisinusoidal fibrosis Not adequately reported

Hepatic steatosis, with no features of steatohepatitis and fibrosis Fatty liver alone without necroinflammation

NASH characterized by fatty infiltration, inflammation, and variable degrees of fibrosis, Mallory hyaline, and apoptotic bodies Steatosis plus any stage of fibrosis or steatosis plus lobular inflammation plus ballooning degeneration Nonalcoholic steatosis with necroinflammation and/ or fibrosis Nonalcoholic macrovesicular steatosis (>10% of hepatocytes) and lobular inflammation plus ballooning degeneration, Mallory hyaline fibrosis, sinusoidal fibrosis, or a combination thereof Hepatic steatosis along with ballooned hepatocytes with lobular hepatitis Hepatic steatosis with some necroinflammatory activity and/or fibrosis Hepatic steatosis (>5%) co-existing with hepatocellular ballooning and lobular necroinflammation, with or without fibrosis Q34

Not adequately reported

Hepatocyte ballooning or intralobular hepatocyte necrosis

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Ekstedt et al,8 2006

Q33

Singh et al

NAFLD

6

-,

No. -

1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856

1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 -

Supplementary Table 5. Quality of Included Studies

Representative of the average adult in the community

Histologic confirmation of NAFLD

Histologic assessment of fibrosis progression

Adequate follow-up evaluation of cohort for outcome to occur

Attrition rate

Evans et al23

Fassio et al24

Hamaguchi Hui Pais Ratziu Teli Wong Abdelmalek et al25 et al29 Harrison41 et al26 et al13 et al27 et al12 et al28

0.5

0.5

1

0.5

0.5

0.5

1

0.5

0.5

0

0.5

0.5

0

1

0

1

0

0.5

0.5

0

0

0.5

0

0

1

0

0.5

1

1

1

1

1

1

1

1

0.5

0

1

1

1

1

1

1

0.5

0.5

1

1

1

0.5

0

1

1

0.5

0.5

1

1

1

0

1

1

0.5

0.5

1

0.5

0

0

0.5

0.5

0

0

0

0

0

0

0

0

1

0.5

Q35

12.e5

1 point for unselected participants in populationbased or multicenter studies, 0.5 points for unselected participants in single-center hospital-based study; 0 points for participants in an RCT or selective cohort 1 point if cohort size was >50 patients with baseline and follow-up biopsies, 0.5 points if cohort size was between 25 and 50 patients, 0 points if cohort size was <25 patients 1 point if standardized assessment of NAFLD with information on NASH and/or NAFL separately, 0.5 points if standardized assessment of NAFLD without distinction between NASH or NAFL, 0 points if reviewed only nonstandardized classification of NAFLD 1 point if assessed using Brunt classification or NASH-CRN, 0.5 points if assessed using Ishak or Metavir classification, 0 points if assessed using nonstandardized classification 1 point if mean follow-up evaluation of entire cohort >5 years, 0.5 points if cohort follow-up evaluation was between 3 and 5 years, 0 points if mean follow-up period of cohort was <3 years 1 point if >80% of cohort was followed up, 0.5 points if 50%–80% of cohort was

Adams Argo Ekstedt et al7 et al22 et al8

Fibrosis Progression in NAFLD

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Large cohort size

Scoring scheme

2014

Question

1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944 1945 1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972

1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 Scoring scheme

Evans et al23

Fassio et al24

Hamaguchi Hui Pais Ratziu Teli Wong Abdelmalek et al25 et al29 Harrison41 et al26 et al13 et al27 et al12 et al28

0.5

1

0

0

1

1

0

0.5

0.5

0

0

1

1

1

0.5

1

0

1

1

0.5

0.5

1

0

0

1

0

5

5

6.5

3.5

5.5

4.5

4.5

4.5

5

1.5

1.5

7

3.5

CRN, clinical research network.

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followed up, 0 points if >50% was lost to follow-up evaluation or if how many patients with NAFLD seen in clinic was not reported Information on 1 point if adequate information potential on lifestyle modification as well as medication use, 0.5 diseasemodifying points if incomplete information, 0 points if no therapy adopted by participants information on adoption of disease-modifying therapy Reported risk 1 point if factors were studied in factors multivariate models, 0.5 associated with points if factors were progression of reported in only in univariate fibrosis models, 0 points if factors associated with fibrosis progression were not reported Total score (maximum, 8; high quality, 6; medium quality, 3–5; and low quality, 2)

Adams Argo Ekstedt et al7 et al22 et al8

Singh et al

Question

12.e6

Supplementary Table 5. Continued

-,

No. -

2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088

-

2089 2090 2091 2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128 2129 2130 2131 2132 2133 2134 2135 2136 2137 2138 2139 2140 2141 2142 2143 2144 2145 2146

2014

Fibrosis Progression in NAFLD

12.e7

Supplementary Table 6. Fibrosis Progression in Individual Studies Final fibrosis stage

Study Adams et al7 Initial fibrosis stage

F0 F1 F2 F3 F4

(25) (21) (23) (18) (16)

F0 13 7 4 2 0

F1 5 2 4 1 0

(0) (1) (2) (2) (0)

F0 0 0 1 0 0

F1 0 0 1 0 0

F2 3 8 5 6 1

F3 2 4 7 5 5

F4 2 0 3 4 10

Final fibrosis F2 F3 0 0 1 0 0 0 0 0 0 0

F4 0 0 0 2 0

(36) (19) (11) (4) (0)

F0 19 5 0 0 0

Final fibrosis stage F1 F2 F3 8 6 3 9 3 1 5 1 2 0 1 1 0 0 0

F4 0 1 3 2 0

(4) (1) (2) (0) (0)

F0 2 0 0 0 0

Final fibrosis stage F1 F2 F3 1 1 0 0 1 0 0 1 1 0 0 0 0 0 0

F4 0 0 0 0 0

(3) (11) (4) (4) (0)

F0 2 0 0 0 0

Final fibrosis stage F1 F2 F3 0 0 0 8 0 3 2 1 1 2 0 0 0 0 0

F4 1 0 0 2 0

(0) (24) (6) (5) (4)

F0 0 3 0 0 0

Final fibrosis stage F1 F2 F3 0 0 0 10 10 1 4 2 0 2 2 1 0 0 1

F4 0 0 0 0 3

(11) (5) (1) (0) (0)

F0 5 0 0 0 0

Final fibrosis stage F1 F2 F3 5 1 0 3 2 0 0 0 0 0 0 0 0 0 0

F4 0 0 1 0 0

(10) (10) (5) (0) (0)

F0 6 0 0 0 0

Final fibrosis stage F1 F2 F3 3 1 1 5 4 0 4 1 0 0 0 0 0 0

F4 0 0 0 0 0

Overall Argo et al22 Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Ekstedt et al8 Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Evans et al23 Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Fassio et al24 (Ishak staging)

Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Hamaguchi et al25 (some reported as 1.5 stage)

Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Hui et al26 Initial fibrosis stage

F0 F1 F2 F3 F4

Overall Pais et al13 Initial fibrosis stage

F0 F1 F2 F3 F4

Fibrosis progression

P-y follow-up evaluation

þ25 þ9 þ1 -10 -7 þ18

80 67.2 73.6 57.6 51.2 329.6

0 þ1 -3 þ2 0 0

0 4.4 8.8 4.4 0 22

þ29 þ3 þ3 þ1 0 þ36

496.8 262.2 151.8 55.2 0 1202

þ3 þ1 þ1 0 0 þ5

32.8 8.2 16.4 0 0 57.2

þ4 þ6 -1 -2 0 þ7

15.9 58.3 21.2 21.2 0 116.6

0 þ10 -4 -6 -1 -1

0 57.6 14.4 12 9.6 93.6

þ7 þ2 þ2 0 0 þ11

67.1 30.5 6.1 0 0 97.8

þ7 (approximately) þ13 þ1 0 0

37 37 18.5 0 0

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2147 2148 2149 2150 2151 2152 2153 2154 2155 2156 2157 2158 2159 2160 2161 2162 2163 2164 2165 2166 2167 2168 2169 2170 2171 2172 2173 2174 2175 2176 2177 2178 2179 2180 2181 2182 2183 2184 2185 2186 2187 2188 2189 2190 2191 2192 2193 2194 2195 2196 2197 2198 2199 2200 2201 2202 2203 2204

12.e8

2205 2206 2207 2208 2209 2210 2211 2212 2213 2214 2215 2216 2217 2218 2219 2220 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 2242 2243 2244 2245 2246 2247 2248 2249 2250 2251 2252 2253 2254 2255 2256 2257 2258 2259 2260 2261 2262

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Supplementary Table 6. Continued Final fibrosis stage

Study

Fibrosis progression

P-y follow-up evaluation

þ21

102.7

0

0 0 0 0 0

20.8 (mean follow-up period, 5.2 inferred from total p-y) 52 0 0 0 73

þ2 0 0 0 0 þ2

139.2 0 0 0 0 139.2

þ14 -2 -6 -1 0 þ5

78 51 21 3 3 156

Overall Ratziu et al27 Initial fibrosis stage

F0 (4)

F0 4

F1 F2 F3 F4

4 0 0 0

(10) (0) (0) (0)

Final fibrosis stage F1 F2 F3 0 0 0

F4 0

4 0 0 0

1 0 0 0

0 0 0 0

1 0 0 0

Final fibrosis stage F0 F0 (12) 11 F1 (0) 0 F2 (0) 0 F3 (0) 0 F4 (0) 0

F1 0 0 0 0 0

F2 1 0 0 0 0

F3 0 0 0 0 0

F4 0 0 0 0 0

Final fibrosis stage F0 F0 (26) 17 F1 (17) 7 F2 (7) 4 F3 (1) 0 F4 (1) 0

F1 7 7 1 0 0

F2 0 1 0 1 0

F3 1 2 1 0 0

F4 1 0 1 0 1

Overall Teli et al12 Initial fibrosis stage

Overall Wong et al28 Initial fibrosis stage

Overall

P-y, person-years.

Supplementary Table 7. Subgroup Analysis Comparing Rates of Fibrosis Progression in Western and Asian Populations Location Western (8 studies)

Asian (3 studies)

Baseline fibrosis stage Stage 0 Stage 1 Stages 0 or 1 Stage 0 Stage 1 Stages 0 or 1

NAFLD 0.12 0.13 0.12 0.14 0.06 0.10

(0.06–0.18) (0.04–0.23) (0.06–0.17) (0.07–0.21) (-0.06 to 0.18) (0.07–0.14)

NAFL 0.05 0.35 0.09 0.11 0.06 0.09

(0.00–0.10) (0.20–0.50) (0.02–0.15) (0.01–0.22) (-0.19 to 0.32) (0.04–0.14)

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NASH 0.14 0.06 0.08 0.17 0.33 0.14

(0.00–0.29) (-0.02 to 0.14) (-0.01 to 0.17) (0.03–0.31) (-0.04 to 0.71) (0.07–0.21)

2263 2264 2265 2266 2267 2268 2269 2270 2271 2272 2273 2274 2275 2276 2277 2278 2279 2280 2281 2282 2283 2284 2285 2286 2287 2288 2289 2290 2291 2292 2293 2294 2295 2296 2297 2298 2299 2300 2301 2302 2303 2304 2305 2306 2307 2308 2309 2310 2311 2312 2313 2314 2315 2316 2317 2318 2319 2320

2321 2322 2323 2324 2325 2326 2327 2328 2329 2330 2331 2332 2333 2334 2335 2336 2337 2338 2339 2340 2341 2342 2343 2344 2345 2346 2347 2348 2349 2350 2351 2352 2353 2354 2355 2356 2357 2358 2359 2360 2361 2362 2363 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374 2375 2376 2377 2378 2379 2380 2381 2382 2383 -

Supplementary Table 8. Comparison Between Patients With Progressive and Nonprogressive Fibrosis Ekstedt et al8

PR (38)

Non-PR (35)

PR (7)

Non-PR (15)

Age, mean  SD Male sex, n Diabetes, n Hypertension, n Obesity, n, or mean  SD Metabolic syndrome, n AST level, mean  SD ALT level, mean  SD AST/ALT ratio, mean  SD Ferritin level, mean  SD HOMA-IR, mean  SD Platelet count, mean  SD

44  2 13 20 12 28

47  2 16 15 10 22

49  12 2 4

44  11 7 4

6

4c

19

20

18

21

-

-

3

71  34

74  44

42  17

31  13c

-

-

104  60

94  51

75  44

51  25

70  10

73  22

0.8  0.3

0.9  0.4

0.6  0.2

0.7  0.4

354  381

258  194

207  193 174  125

-

2.78  1.71 4.15  3.78

5.2  5.3

2.9  1.5c

228  85

205 þ 59

252  62c

197  72

PR (29)

Non-PR (41)

61  11 60  11 21 29 15 24 28 38 29.6  3.3 28.3  5.3

Harrison et al36b

Hamaguchi et al25a

Wong et al28

PR (7)

Non-PR (15)

PR (9)

Non-PR (8)

PR (14)

Non-PR (38)

49 6 3 6 31.8

51 7 6 5 34.7

46  8 5 2 0 29.8  2.9

36  7 6 2 0 27.2  3.7

45  10 9 6 9 27.7  4.4

44  9 25 20 17 27.3  3.4

7c

-

-

-

-

8

27

32  17

29  18

76

40c

-

-

-

-

40  38

48  14

95

64

128  41

90  35

51  15

63  16

-

-

0.73

0.76

-

-

-

-

-

46  84

397  103

-

-

-

-

-

-

-

-

3.9  2.4

3.4  1.4

-

-

-

-

1.7  0.5

2.1  0.5

-

-

243  52

230  80

-

-

-

-

-

-

0.53  0.12 0.59  0.14

PR (11)

Non-PR (16)

Hui et al26

51  9 48  15 5 12 7 11 4 3 30.9  3.6 27.5  2.5

Q36

Q37

Q38

BMI, body mass index; HOMA-IR, homeostasis model of assessment–insulin resistance; non-PR, patients with no progression of fibrosis; PR, patients with progressive fibrosis. a Compares progressors with stable repeat biopsy. b Study offers P value for differences between groups; for some studies, instead of the SD the range was reported. This was converted to a SD using the following formula: SD ¼ (maximum - minimum)/4 (derived from Hozo SP, et al. BMC Med Res Methodol 2005;5:13). c P < .05 between progressors vs nonprogressors.

Fibrosis Progression in NAFLD

REV 5.2.0 DTD
YJCGH53791_proof
19 May 2014
5:35 pm
ce

Study

Fassio et al24

2014

Adams et al7a

12.e9 2384 2385 2386 2387 2388 2389 2390 2391 2392 2393 2394 2395 2396 2397 2398 2399 2400 2401 2402 2403 2404 2405 2406 2407 2408 2409 2410 2411 2412 2413 2414 2415 2416 2417 2418 2419 2420 2421 2422 2423 2424 2425 2426 2427 2428 2429 2430 2431 2432 2433 2434 2435 2436 2437 2438 2439 2440 2441 2442 2443 2444 2445 2446