GASTROENTEROLOGY 2009;136:160 167 CLINICAL LIVER, BILIARY TRACT Performance of ELF Serum Markers in Predicting Fibrosis Stage in Pediatric Non-Alcoholic Fatty Liver Disease VALERIO NOBILI,* JULIE PARKES, GIANFRANCO BOTTAZZO, MATILDE MARCELLINI,* RICHARD CROSS, DANIEL NEWMAN,, FRANCESCO VIZZUTTI, MASSIMO PINZANI,,# and WILLIAM M. ROSENBERG** *Liver Unit and Direzione Scientifica Bambino Gesù Children s Hospital and Research Institute, Rome, Italy; Public Health Sciences and Medical Statistics, University of Southampton, United Kingdom; iqur Limited, United Kingdom; Department of Internal Medicine and # Center for Research, Higher Education and Transfer DENOThe, University of Florence, Italy; and **Institute of Hepatology, University College London, United Kingdom Background & Aims: Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in children and adolescents in industrialized countries. It is important to accurately determine the stage of fibrosis in these patients. The enhanced liver fibrosis (ELF) test has been validated for staging liver fibrosis in adult patients with chronic liver diseases, including NAFLD. We investigated the performance of this test in assessing liver fibrosis in children and adolescents with NAFLD, identified by biopsy. Methods: The ELF test was performed on a panel of serum samples collected from 112 consecutive subjects that were likely to have NAFLD (64 male, mean age of 13.8 3.3). A previously described and validated algorithm was used to analyze the data on hyaluronic acid (HA), amino-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels. Results: In pediatric patients with NAFLD, the ELF test predicted liver fibrosis stage with a high degree of sensitivity and specificity; results were superior to those reported for adults. The area under receiver operating characteristic curves/best possible ELF test cut-off values for the prediction of any (>stage 1), moderateperisinusoidal (>stage 1b), moderate-portal/periportal (>stage 1c), significant (>stage 2), or advanced (>stage 3) fibrosis were 0.92/9.28, 0.92/9.33, 0.90/9.54, 0.98/ 10.18 and 0.99/10.51, respectively. Conclusions: The ELF test can be used to accurately assess the level of liver fibrosis in pediatric patients with NAFLD. This information is important for identifying patients with progressive fibrosis that require further histopathological analysis or therapeutic follow-up. Pediatric nonalcoholic fatty liver disease (NAFLD) has become the most frequent chronic liver disease in children and adolescents in industrialized countries due to the growing prevalence of childhood obesity and overweight. 1,2 NAFLD affects 2.6% to 9.8% of children and adolescents, 3 5 especially in the presence of obesity. 6 The high prevalence of NAFLD and the likelihood of evolution to cirrhosis and its complications with the consequent need for liver transplantation warrant increased attention toward this disorder. 7 11 In addition, obesity and older age are independently associated with more advanced fibrosis, 12 and a rapid progression to cirrhosis has been reported in some children with NAFLD. 2,13 The evolution of NAFLD toward hepatic fibrosis and cirrhosis depends on the combination of necroinflammation and fibrogenesis that characterize nonalcoholic steatohepatitis (NASH). The distinction between simple NAFLD and NASH with the exclusion of competing causes of chronic liver disease is based on the histopathological analysis of liver tissue. 14 In this context, staging the degree of fibrosis is of particular importance because it defines a key feature of progressive chronic liver disease (CLD). In the past decade, major efforts have been directed at identifying noninvasive methods for the assessment of liver fibrosis as well as for the differentiation of NASH from simple NAFLD. 15 Although many of the noninvasive methods proposed have been criticized for Abbreviations used in this paper: ALP, alkaline-phosphate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under ROC; BMI, body mass index; CI, confidence interval; CLD, chronic liver disease; ELF, enhanced liver fibrosis; HA, hyaluronic acid; HABP, hyaluronic acid binding protein; HOMA, homeostatic model assessment; INR, international normalized ratio; ISI, insulin sensitivity index; IQR, interquartile range; -GT, gamma glutamil-transpeptidase; LR, positive likelihood ratio; LR, negative likelihood ratio; MAbs, monoclonal antibodies; OC, receiver operating characteristic OGTT, oral glucose tolerance test; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD Activity Score; NASH, nonalcoholic steatohepatitis; PIIINP, amino-terminal propeptide of type III collagen; PPV, positive predictive value; S, sensitivity; SD, standard deviation; Sp, specificity; NPV, negative predictive value; TIMP-1, tissue inhibitor of metalloproteinase 1. 2009 by the AGA Institute 0016-5085/09/$36.00 doi:10.1053/j.gastro.2008.09.013
January 2009 ELF TEST IN PEDIATRIC NAFLD 161 sub-optimal diagnostic accuracy, it is increasingly accepted that the application of combination of these methods may reduce the need for liver biopsy and provide a valuable adjunct to biopsy, especially in the longitudinal monitoring of disease progression. These considerations assume a particular relevance for children, in whom the use of liver biopsy is often perceived as bearing higher risk and less acceptable than in adults. Serum biomarkers and several algorithms based on combinations of serum markers related to cell damage, fibrogenesis, and hepatocellular failure are currently under evaluation, and/or further validation in different cohorts of patients with CLD including NASH. 15,16 However, the possibility of employing serum biomarkers for the prediction of hepatic fibrosis in children and adolescents, and particularly in those with suspected NASH, has not yet been reported. The enhanced liver fibrosis (ELF) test, first characterized and validated in a mixed cohort of patients with CLD, 17 has been validated further in cohorts of patients with NAFLD/NASH. 16 The performance of this panel of markers in fatty liver disease and in particular their ability to assess accurately mild, moderate, and severe fibrosis identified the ELF test as a suitable tool for the assessment of fibrosis in pediatric NAFLD. Accordingly, the aim of the present study was to investigate the performance of the ELF test in the assessment of liver fibrosis in children and adolescents with biopsy-proven NAFLD investigated in a specialized tertiary referral center. Materials and Methods Patients The study initially included 121 consecutive patients (68 male and 53 female, mean age of 14.1 3.7; age range, 3 17) diagnosed with NAFLD according to reported and widely accepted criteria 12 and referred to Bambino Gesù Children s Hospital and Research Institute between June 2004 and November 2006 for staging and grading of the disease. All patients showed serum aminotransferases either persistently or intermittently elevated (at least 2 abnormal determinations within 6 months prior to enrollment), associated with diffusely hyperechogenic liver tissue (so-called bright liver) at ultrasound examination, and hyperinsulinism. Insulin resistance was assessed by the homeostatic model assessment (HOMA) 18 and ISI-composite (insulin sensitivity index). 19 Children with at least 1 of the following conditions were excluded from the study: cardiopulmonary disease, chronic renal failure, recent active infections, chronic inflammatory diseases, autoimmune diseases, use of anti-inflammatory drugs, abnormal international normalized ratio (INR), and/or platelet count below 60 10 9 /L. 20 Secondary causes of steatosis 21 including previous bariatric surgery, alcohol abuse ( 140 g/wk), total parenteral nutrition or rapid weight loss, endocrinological diseases, inborn disorders, inflammatory bowel disease, and the use of drugs known to cause steatosis were excluded in all cases. The concomitant presence of other acute or CLD, and particularly viral hepatitis, vascular diseases of the liver, biliary tract disorders, autoimmune, genetic and other metabolic liver diseases were ruled out using standard clinical and laboratory evaluation as well as through histological examination of the liver biopsy. Laboratory tests, including bilirubin and albumin, platelet count, INR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltranspeptidase ( -GT), alkaline-phosphate (ALP), creatinine, fasting glucose and insulin, ferritin, cholesterol and triglycerides, and oral glucose tolerance test (OGTT), were performed in all subjects within 1 week before liver biopsy (mean interval, 3 1 days). After this extensive clinical evaluation, 4 patients were not enrolled in the study because of the presence of 1 or more exclusion criteria. Five additional patients were not considered in the statistical analysis because of the insufficient number of portal tracts in the liver biopsy specimen. Therefore, the study was conducted in a total of 112 patients (64 male and 48 female, mean age 13.8 3.3; age range, 3 17 y). Paired blood samples and liver specimens were obtained from each patient on the same study day. One milliliter of serum was aliquoted and stored at 80 C until testing for ELF biomarkers. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki (revision of Edinburgh, 2000) and was performed according to the recommendations of the Ethics Committee of the Bambino Gesù Children s Hospital and Research Institute. An informed consent was a priori obtained from a responsible guardian. Liver Histology The clinical indication for biopsy was either to assess the presence of NASH and degree of fibrosis or other likely independent or competing liver diseases. Liver biopsy was performed in all subjects after an overnight fast, using an automatic core biopsy 18-gauge needle (Biopince; Amedic, Sollentuna, Sweden) under general anesthesia 22 and ultrasound guidance. A Sonoline Omnia Ultrasound machine (Siemens, Munich, Germany) with a 5-MHz probe (5.0 C 50, Siemens) and a biopsy adaptor were employed. Two biopsy passes within different liver segments were performed for each subject. The length of liver specimens (in millimeters) was recorded. Only samples with a length 15 mm and including at least 10 to 11 complete portal tracts were considered adequate for the purpose of the study. Biopsies were evaluated by a single liver pathologist blinded for ELF test marker results. Biopsies were routinely processed (ie, formalinfixed and paraffin-embedded), and 5- m-thick sections of liver tissue were stained with hematoxylin-eosin, Van
162 NOBILI ET AL GASTROENTEROLOGY Vol. 136, No. 1 Gieson, PAS-D, and Prussian blue stain. Immunohistochemical staining with antibodies against alpha-1-antitrypsin was used to exclude alpha-1-antitrypsin deficiency associated liver disease. Fibrosis was scored by using the modified Brunt (Kleiner et al) (0, no fibrosis; 1, perisinusoidal/periportal fibrosis [1a mild zone 3 perisinusoidal, 1b moderate zone 3 perisinusoidal, 1c portal/ periportal]; 2, perisinusoidal and portal/periportal fibrosis; 3, bridging fibrosis; 4, cirrhosis) classification. 23 Subsequently, patients were stratified according to the NAFLD Activity Score (NAS) as proposed by Kleiner et al. 24 ELF Test The simplified ELF algorithm derived using the whole of the original ELF cohort (n 921) and externally validated in 7 independent populations of patients with chronic liver disease was used. 25 This panel comprises hyaluronic acid (HA), amino-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) combined in an algorithm. ELF Algorithm: Discriminant score 7.412 ln(ha) * 0.681 ln(p3np) * 0.775 ln(timp1) * 0.494 10 In contrast to the original ELF algorithm, the simplified algorithm omits age providing the advantage that age can be considered as a separate factor in prognostic analyses. In addition, omission of age facilitates automated analysis of samples in the absence of patient data, eliminating the necessity to record these demographic data and also reducing transcription errors and facilitating population screening. 16 TIMP-1, PIIINP, and HA (ELF test) were assayed using specifically manufactured highly sensitive enzyme-linked immunosorbent assays on an automated IMMUNO 1 immunoanalyzer (Siemens Medical Solutions Diagnostics, Tarrytown, NY). The assays are magnetic particle separation immunoassays and were identical to those used for the 2004 European Liver Fibrosis study. 17 The TIMP-1 and PIIINP assays each use 2 monoclonal antibodies (MAbs) that bind to independent binding sites on their respective antigens. The HA assay uses HA-binding protein (HABP), which is isolated from cow nasal septum, in the place of MAbs. The ELF markers were analyzed individually, and the results continually referred to a set of quality standards to ensure accurate analysis. The ELF assays require a total of 22.2 L of serum for a single determination, 3.5 L for HA, 15.0 L for PIIINP, and 3.7 L for TIMP-1. Statistical Analysis Data are presented as median values with interquartile ranges. The comparison between clinical and laboratory variables between fibrosis stages was conducted with analysis of variance. The diagnostic performance of the ELF test in the identification of fibrosis stage defined by the staging of liver histology was evaluated by calculating the area under receiver operating characteristic curves (AUROC). Different levels of severity of fibrosis were evaluated as follows: any fibrosis ( 1a), moderate fibrosis-perisinusoidal ( 1b), moderate fibrosis-portal/periportal ( 1c), significant fibrosis ( 2), and advanced fibrosis ( 3). Sensitivities, specificities, predictive values, and likelihood ratios (LRs) were derived. Data were compared using Jonckheere Terpstra and Mann-Whitney U tests where appropriate. To evaluate whether any clinical or laboratory variables could add to the diagnostic value of ELF in a pediatric population, ROC analyses were conducted to compare the AUROC of ELF and the published ELF simple marker algorithms in adults (see equations below). 16 In addition, multivariable logistic regression analysis using a backward stepwise selection approach was used to evaluate whether any clinical or laboratory variables could add to the diagnostic value of ELF. A 2-tailed P value of at least.05 was considered statistically significant. To evaluate the clinical utility of ELF at high sensitivities and specificities in an obese pediatric hospital population, a theoretical cohort of 1000 patients was used to derive the proportion that could avoid biopsy, with estimates of true/false results, using the disease prevalence from this study population. All analyses were performed using the SPSS software package version 14 (SPSS Inc, Chicago, IL, and STATA version 9.0 for Windows, College Station, TX). Combined ELF test and simple markers panel for distinguishing no fibrosis: Score 2.722 1.482 * ELF 0.062 * BMI 1.241 * diabetes IFG (1 yes, 0 no) 0.590 * AST ALT ratio 0.002 * platelets 0.043 * albumin. Combined ELF test and simple markers panel for distinguishing significant fibrosis: Score 5.257 2.408 * ELF 0.084 * BMI 1.848 * diabetes IFG (1 yes, 0 no) 1.839 * AST ALT ratio 0.012 * platelets 0.141 * albumin. Combined ELF test and simple markers panel for distinguishing advanced fibrosis: Score 20.870 5.506 * ELF 4.513 * diabetes IFG (1 yes, 0 no) 3.144 * AST ALT ratio 0.058 * BMI 0.026 * platelets 0.639 * albumin.
January 2009 ELF TEST IN PEDIATRIC NAFLD 163 Table 1. Clinical and Laboratory Findings in the Study Population Variable fibrosis 0 (n 37) 1a (n 8) 1b (n 6) 1c (n 44) 2 (n 9) 3 4 (n 8) P value (ANOVA) Age (y) 12.3 2.1 13.1 2.3 13.6 2.1 13.7 2.6 13.6 3.1 14.1 3.4 P.17 Male gender, n (%) 21 (56) 5 (62.5) 4 (66.6) 24 (54.5) 5 (55.5) 5 (62.5) P.15 a BMI (kg/m 2 ) 25.34 3.93 24.94 3.58 24.48 4.78 25.36 4.37 26.08 2.98 26.61 0.24 P.95 BMI Z-score 1.84 0.64 1.66 0.60 1.81 0.67 1.86 0.58 1.83 0.5 1.86 0.48 P.12 Obese, n (%) 13 (35.1) 3 (37.5) 3 (50) 20 (45.4) 2 (22.2) 5 (62.5) P.64 a Overweight, n (%) 24 (64.8) 5 (62.5) 3 (50) 24 (54.5) 7 (77.7) 3 (37.5) P.61 a AST (U/L) n.v. 5 40 50 16 52 10 56 19 51 25 40 10 45 16 P.57 ALT (U/L) n.v. 5 40 76 67 82 57 88 47 86 64 62 59 84 55 P.76 AST/ALT 1,n(%) 13 (35) 3 (37) 2 (33) 14 (32) 3 (33) 3 (37) P.53 a INR n.v. 0.9 1.1 1.07 0.18 1.12 0.14 0.97 0.11 1.09 0.19 1.02 0.12 1.10 0.09 P.71 Bilirubin (mg/dl) n.v. 0.25 1.00 0.67 0.34 0.69 0.23 0.77 0.15 0.65 0.39 0.78 0.19 0.77 0.32 P.83 Albumin (g/dl) n.v. 3.5 5.5 4.18 0.28 4.3 0.37 4.23 0.26 4.19 0.29 4.21 0.3 4.16 0.27 P.92 -GT (U/L) n.v. 5 45 26 19 33 24 26 17 28 22 15 34 28 23 P.40 ALP (U/L) b 571 216 588 127 700 139 611 166 681 153 593 148 P.46 Fasting glucose (mg/dl) n.v. 100 81 8 77 6 79 8 80 9 82 7 78 4 P.63 Fasting insulin ( IU/L) n.v. 6.00 27.00 c 11 5.3 12.2 6.1 10.8 5.9 11.7 5.4 12.4 4.9 11.2 6 P.71 HOMA-IR n.v. 3 d 2.3 1.20 2.6 0.81 2.1 1.3 2.7 0.9 2.6 0.98 2.4 1.11 P.52 ISI n.v. 6 4.2 2.3 4.6 1.7 3.9 2.5 4.8 2.2 4 2 3.8 1.9 P.15 Ferritin (ng/ml) n.v. 9 290 58.79 26.5 52.28 35.43 63.33 31.2 70.15 10.8 68.3 28 67 15 P.18 Cholesterol (mg/dl) n.v. 200 e 160 42 158 40 182 34 168 35 143 51 183 29 P.29 Triglycerides (mg/dl) n.v. 160 e 97 65 114 92 112 68 109 71 86 76 150 39 P.36 Platelet Count ( 10 9 /L) n.v. 150 450 308 58 302 73 342 119 336 83 281 47 297 18 P.18 ELF 7.92 0.44 8.34 0.60 8.77 0.42 8.78 0.39 9.71 0.42 11.08 0.07 P.0001 NOTE. Results are expressed as mean SD. Fibrosis was scored according to the modified Brunt score. Patients were considered overweight or obese if the BMI was greater than the 85th percentile and equal to the 97th or greater than 97th percentile, respectively. ANOVA, analysis of variance; BMI, body mass index; AST, alanine aminotransferase; ALT, aspartate aminotransferase; INR, international normalized ratio; -GT, gamma-glutamyl transpeptidase; ALP, alkaline phosphates; HOMA-IR, homeostatic model assessment-insulin resistance; ISI, insulin sensitivity index; ELF, enhanced liver fibrosis; n.v., normal values. a Chi-square. b n.v. below 1 year of age 50 100 U/L; 200 850 U/L after 1 year of age until puberty. c Analyzed by ADVIA Centaur (Siemens, Milan, Italy). d For HOMA-IR, normal pediatric values see reference 26. e For cholesterol and triglycerides normal pediatric values see references 27 and 27 28, respectively. Results Patient Characteristics The major clinical and biochemical parameters are reported in Table 1. All the enrolled patients showed insulin resistance, and type 2 diabetes was present in 2 cases. The presence of NAFLD was confirmed histopathologically in all subjects. The relatively high prevalence of significant fibrosis (ie, Brunt 2) in our study population (15%, see Table 1) was likely related to the pressure to select cases in which liver biopsy was indicated. In the group of patients with advanced fibrosis, none had a clinical history of decompensation (ie, ascites, portal hypertension-related bleeding, encephalopathy). The length of liver specimens was on average ( SD) 20.7 2.3 and included 14.9 3.2 complete portal tracts. All histological sections were considered adequate for evaluation with the employed scoring system by the pathologist. Histopathological features and stratification of patients according to the NAS score are shown in Table 2. The ability of the ELF panel in discriminating stages of fibrosis according to the modified Brunt scoring system is shown in Figure 1. Performance of the ELF test in the prediction of different fibrosis stages according to the modified Brunt scoring system is shown in Table 3. Accuracy of the ELF Test for the Prediction of Any Fibrosis (ie, >1a) Tables 3 and 4 show the accuracy of the ELF test for the diagnosis of any fibrosis. The data-driven best cut-off for the diagnosis any fibrosis was 9.28 (Table 4). Accuracy of the ELF Test for the Prediction of Moderate Fibrosis-Perisinusoidal (ie, >1b) The accuracy of the ELF test for the diagnosis of fibrosis 1b is illustrated in Tables 3 and 4. The datadriven best cut-off for the diagnosis of mild fibrosis was 9.33 (Table 4). Accuracy of the ELF Test for the Prediction of Moderate Fibrosis-Portal/Periportal (ie, >1c) The accuracy of the ELF test for the diagnosis of fibrosis 1c is illustrated in Tables 3 and 4. The datadriven best cut-off for the diagnosis of moderate fibrosis was 9.54 (Table 4). Accuracy of the ELF Test for the Prediction of Significant Fibrosis (ie, >2) The accuracy of the ELF test for the diagnosis of significant fibrosis is illustrated in Tables 3 and 4. The data-driven best cut-off for the diagnosis of significant fibrosis was 10.18 (Table 4). Accuracy of the ELF Test for the Prediction of Advanced Fibrosis (ie, >3) The accuracy of the ELF test for the diagnosis of advanced fibrosis is illustrated in Tables 3 and 4. The data-driven best cut-off for the diagnosis of advanced fibrosis was 10.51 (Table 4). Multivariable logistic regression analysis showed that no clinical-analytical variable could add significantly to
164 NOBILI ET AL GASTROENTEROLOGY Vol. 136, No. 1 the discriminative value of ELF for any stage of fibrosis investigated. The AUROC performance of the ELF plus simple markers was not significantly better than ELF alone for the identification of any, significant, and advanced fibrosis (data not shown). The performance of the ELF panel was evaluated in a theoretical cohort of 1000 obese pediatric subjects in the hospital setting. If thresholds are used to rule in fibrosis (upper threshold with high specificity and high positive predictive value) or rule out fibrosis (lower threshold with high sensitivity and high negative predictive value) with a high degree of accuracy, the clinical utility of ELF can be evaluated. Using ELF to identify advanced fibrosis at data-derived thresholds of 10.51 and 11.56 (sensitivity and specificity of 100%, respectively) in this cohort, 93% would have avoided a liver biopsy all of which were correctly classified, and 7% would have had an indeterminate classification (ie, had a value between these thresholds). For the identification of significant fibrosis (using thresholds with a sensitivity and specificity of Table 2. Histological Findings Fibrosis, n(%) Score Inflammation, n(%) Ballooning, n(%) Steatosis, n(%) 0 37 (33) 0 15 (13.4) 20 (17.9) 0 (0) 1 18 (16) 14 (12.5) 12 (10.7) 2 4 (3.6) 3 (2.7) 16 (14.3) 3 0 (0) 9 (8) 1a 8 (7.1) 0 3 (2.7) 5 (4.4) 0 (0) 1 4 (3.6) 1 (0.9) 4 (3.6) 2 1 (0.9) 2 (1.8) 3 (2.7) 3 0 (0) 1 (0.9) 1b 6 (5.4) 0 0 (0) 2 (1.8) 0 (0) 1 4 (3.6) 1 (0.9) 0 (0) 2 2 (1.8) 3 (2.7) 3 (2.7) 3 0 (0) 3 (2.7) 1c 44 (39.3) 0 9 (8) 15 (13.4) 0 (0) 1 26 (23.2) 20 (17.9) 12 (10.7) 2 9 (8) 9 (8) 21 (18.7) 3 0 (0) 11 (9.8) 2 9 (8) 0 2 (1.8) 5 (4.4) 0 (0) 1 6 (5.3) 3 (2.7) 5 (4.4) 2 1 (0.9) 1 (0.9) 2 (1.8) 3 0 (0) 2 (1.8) 3 6 (5.4) 0 2 (1.8) 5 (4.4) 0 (0) 1 4 (3.6) 0 (0) 3 (2.7) 2 0 (0) 1 (0.9) 3 (2.7) 3 0 (0) 0 (0) 4 2 (1.8) 0 0 (0) 1 (0.9) 0 (0) 1 2 (1.8) 0 (0) 1 (0.9) 2 0 (0) 1 (0.9) 0 (0) 3 0 (0) 1 (0.9) NAS 2 (NASH excluded) 37 (33) 3 4 (borderline) 45 (40) 5 (NASH) 30 (27) NOTE. Histological features including steatosis, inflammation (portal and lobular), hepatocytes, ballooning, and fibrosis were scored according to the scoring system for NAFLD developed by the NIH-sponsored NASH Clinical Research Network. 24 n, number of patients; NAS, NAFLD activity score. Figure 1. Box plots of ELF test values in relation to the degree of fibrosis. ELF test values are reported on the Y-axis, and the degree of fibrosis is on the X-axis (modified Brunt score). The top and bottom of the boxes represent the 25th and 75th quartiles, respectively. The length of the box represents the IQR within which 50% of the values are located. The line through the middle of each box represents the median. Whisker bars show minimum and maximum nonextreme values. * P.001 among fibrosis stage 0 versus 1b, 1a versus 1c, 1b versus 2, 1c versus 2, 2 versus 3, and 3 versus 4. Abbreviations: ELF, enhanced liver fibrosis; IQR, interquartile range. 100%, respectively), 88% would have avoided a liver biopsy all of which were correctly classified, and 12% would have had an indeterminate classification. If ELF was used to delineate moderate (portal/periportal)/severe fibrosis (using thresholds with a sensitivity and specificity of 90%, respectively), 75% would have avoided a liver biopsy, 6% would be incorrectly classified, and 25% would have had an indeterminate classification. If a sensitivity and specificity of 90% is chosen for the detection of any fibrosis, 87% of patients would avoid a biopsy with 77% correctly classified, and 13% would have an indeterminate classification. For the identification of moderate (perisinusoidal)/moderate (portal/periportal)/severe fibrosis (using thresholds with a sensitivity and specificity of 90%, re- Table 3. AUC for ELF Test in Identification of Different Fibrosis Stages According to the Modified Brunt Scoring System Modified brunt Subjects in each cut (n 112) AUC (95% CI) 0vs 1a 37/75 0.92 (0.86 0.97) 1a vs 1b 45/67 0.92 (0.87 0.97) 1b vs 1c 51/61 0.90 (0.84 0.96) 1c vs 2 95/17 0.98 (0.96 1.00) 2 vs 3 104/8 0.99 (0.97 1.00) NOTE. Different levels of severity of fibrosis were defined as follows: any fibrosis ( 1a), moderate fibrosis-perisinusoidal ( 1b), moderate fibrosis-portal/periportal ( 1c), significant fibrosis ( 2), and advanced fibrosis ( 3). AUC, area under ROC curve; CI, confidence interval.
January 2009 ELF TEST IN PEDIATRIC NAFLD 165 Table 4. Diagnostic Performance of the ELF Test for Identifying Different Degrees of Fibrosis Fibrosis ELF S (%) Sp (%) PPV (%) NPV (%) LR LR FP/FN (%) Any fibrosis >1a 9.19 91 65 84 77 2.6 0.14 12/6 9.28 88 81 90 77 4.6 0.15 6/8 9.44 83 89 94 72 7.6 0.19 3/12 Moderate fibrosis-perisinusoidal >1b 9.33 90 76 85 83 3.7 0.07 15/4 9.55 85 91 93 80 9.4 0.17 5/6 9.95 54 96 95 58 13.5 0.48 3/19 Moderate fibrosis-portal/periportal >1c 9.54 90 82 86 88 5.0 0.12 8/2 9.71 80 88 89 79 6.7 0.23 5/11 9.93 57 90 88 64 5.7 0.48 4/23 Significant fibrosis >2 10.09 100 88 61 100 8.3 0.01 9/0 10.18 94 93 70 99 13.4 0.07 6/1 10.30 82 100 100 97 82.0 0.18 0/3 Advanced fibrosis >3 10.51 100 98 80 100 50.0 0.01 2/0 10.78 50 99 80 96 50.0 0.51 5/6 11.56 25 100 100 95 25.0 0.75 0/5 NOTE. This table shows the diagnostic accuracy of the ELF test in predicting any, moderate-perisinusoidal, moderate-portal/periportal, significant and advanced fibrosis. Performance of the selected best ELF test values is indicated in boldface. ELF, enhanced liver fibrosis; S, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; LR, positive likelihood ratio; LR, negative likelihood ratio; FN, false negative; FP, false positive. spectively), 89% would have avoided a liver biopsy, 10% would be incorrectly classified, and 11% would have had an indeterminate classification. Therefore, use of 2 thresholds (high and low) rather than a single threshold can reduce the false-positive and negative results but at the cost of reducing the correct avoidance of biopsy and a higher number of patients who cannot be allocated at a high sensitivity and specificity. Comparisons of clinical utility of single ELF thresholds and using 2 thresholds as mentioned are presented in Figure 2. Figure 2. Performance of the ELF test for the prediction of different degrees of liver fibrosis (scored according to modified Brunt on the X-axis) in a theoretical cohort of 1000 pediatric NAFLD hospitalized subjects. Abbreviations: ELF, enhanced liver fibrosis; NAFLD, nonalcoholic fatty liver disease. Complications Related to the Procedures No major complications were associated with percutaneous liver biopsy. Thirty-eight patients (34%) experienced a self-limiting abdominal and/or right shoulder pain, and 10 patients (9%) required a single dose of intravenous analgesic drug (tramadol). There were no complications associated with blood sampling for the ELF test. Discussion Liver biopsy is currently the standard method to assess the presence of fibrosis and inflammation, (ie, to identify NAFLD patients at risk of developing progressive disease). In addition to the well- known patient hazard and technical limitations, however, liver biopsy is not feasible as a first-line diagnostic approach in increasingly large populations of adults and children with suspected NASH. The use of liver biopsy in children is generally perceived as bearing higher risk, and timing for liver biopsy for the diagnosis of NASH remains controversial with no clear standard protocols established. 2 Accordingly, in children even more than in adults, there is an urgent need to develop clinical/biochemical discriminators able to stratify patients requiring histopathological assessment, clinical follow-up, and treatment. Although several noninvasive approaches have been proposed to discriminate NAFLD from NASH and to detect the presence of fibrosis in adult patients, 15,29 no information is available concerning the clinical utility of these methods in pediatric cohorts affected by any form of CLD. Consequently, the present study was undertaken to evaluate the clinical utility of the ELF test, an algorithm including 3 direct markers of fibrosis, with excellent performance that had been preliminarily shown and recently validated in adult patients with NAFLD. 16,17
166 NOBILI ET AL GASTROENTEROLOGY Vol. 136, No. 1 The results herein reported indicate that, in our cohort of pediatric NAFLD, the ELF panel is able to predict liver fibrosis stages with a high degree of sensitivity and specificity and is superior to those recently shown in adults. 16 Due to the low representation of cases with advanced fibrosis (ie, 3), however, these results should be interpreted with caution. In an attempt to overcome this limitation in the interpretation of the results, a mathematical model analyzing a theoretical cohort of 1000 pediatric patients was introduced. According to this analysis, the use of the ELF panel showed an adequate diagnostic accuracy also for the prediction of significant and advanced fibrosis. It is obvious that these findings need to be validated in other populations of patients with pediatric NAFLD before proposing the use of the ELF panel in clinical practice. However, the limitation related to the paucity of cases with advanced fibrosis and cirrhosis, which is typical of pediatric cohorts with this type of liver disease, could be resolved only by performing large multicenter studies aimed at recruiting a sufficient number of cases with advanced fibrosis. Considering the lower stages of fibrosis (ie, below stage 2), it should be noted that the ability of the ELF panel to discriminate between different stages, and in particular, the identification of subjects with at least moderate fibrosis-portal/periportal (ie, 1c), may result in clinically relevant information for the selection of cases with possible progressive fibrogenic outcome suitable for further histopathological analysis and/or therapeutic follow-up. Some considerations can be made to explain the relatively higher level of performance of the ELF panel in pediatric NAFLD compared with adult NAFLD. First, the effects of borderline comorbidities and the aging process on extrahepatic extracellular matrix turnover and organ fibrogenesis typical of adult cohorts are less likely in children and adolescents, thus making pediatric NAFLD a relatively uncontaminated liver disease. Second, the pattern of fibrogenesis in pediatric NAFLD differs from that observed in adults, with a more prominent involvement of the periportal area and a mononuclear rather than polymorphonuclear inflammatory infiltrate. 24,30 Third, the hepatic wound healing process in children may be characterized by different dynamics and associated with a more efficient degradation and remodeling of scar tissue. 31 33 Along these lines, it is of interest that the expression of at least 1 of the 3 markers included in the ELF panel (ie, TIMP-1) during the chronic wound healing process varies with progressive aging, 33 and it may be characterized by a wider range of expression in typical adult cohorts (ie, within an age range of 20 to 70 years). Another key feature of the pediatric cohort investigated in the present study is the predominant presence of lower degrees of fibrosis and particularly stages 0 to 1c according to the modified Brunt classification, 24 which was employed for its superior applicability to the pattern of fibrosis progression typical of pediatric NAFLD. The presence of lower degrees of fibrosis is characteristic of pediatric NAFLD populations, 12,34,35 mostly because of the disease s short natural history. This generally limited fibrogenic progression of the disease makes the applicability of most of the marker panels proposed in adult NAFLD/NASH, that include clinical and biochemical parameters distinctive of advanced stages (eg, albumin, platelet count, prothrombin time), unreliable. Accordingly, the inclusion of simple biochemical tests shown to enhance the performance of the ELF panel in adult NAFLD 16,36 did not significantly improve the performance of the ELF panel in the pediatric cohort investigated in the present study. Therefore, the use of serum biomarkers directly reflecting hepatic extracellular matrix handling seems to be the most appropriate for detecting fibrosis in children and adolescents. An important point to be discussed is the applicability of the results of the present study to primary care settings, where the general assessment of obese children is directed at identifying the minority at risk of liver fibrosis from the vast majority with simple fatty liver, and the possibility of using a reliable noninvasive test would have a major impact in clinical practice. The patient population analyzed in the present study was highly selected on the basis of clinical and biochemical evidence of NAFLD, and chronic liver diseases presenting with apparently similar features or possibly contributing to disease progression as cofactors were carefully excluded. In addition, although the presence of significant fibrosis, and in particular cirrhosis, was relatively low, the prevalence of fibrosis in this cohort is likely to be much higher than in a population of unselected overweight children, in which the performance of this test is unknown. Accordingly, further studies in a primary care setting will be required before the ELF panel could be recommended as a largescale screening test. The present study provides for the first time an evaluation of the discriminant performance of an algorithm of serum markers directly related to extracellular matrix turnover for predicting fibrosis in a pediatric population with NAFLD. Taken together, the results of the study suggest that the ELF panel offers considerable promise in its ability to detect liver fibrosis in children and adolescents affected by NAFLD. It is worth stressing once again that the clinical relevance of this type of test is not to substitute liver biopsy but actually to identify subjects in whom liver biopsy is correctly indicated. Further characterization of this test s performance in larger and lessselected cohorts of patients and its possible use in combination with other noninvasive methodologies such as imaging and transient elastography 37 may reveal synergies that could offer more efficient characterization of patients and a more effective clinical management.
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Lifestyle intervention and antioxidants in children with nonalcoholic fatty liver disease: a randomized, controlled trial. Hepatology 2008;48:113 128. 36. Angulo P, Hui JM, Marchesini G, et al. The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45:846 854. 37. Nobili V, Vizzutti F, Arena U, et al. Accuracy and reproducibility of transient elastography for the diagnosis of fibrosis in pediatric non-alcoholic steatohepatitis. Hepatology 2008;48:442 448. Received April 23, 2008. Accepted September 11, 2008. Address requests for reprints to: Valerio Nobili, Liver Unit, Research Institute, Bambino Gesù Children s Hospital Piazza S. Onofrio, 4, 00165 Rome, Italy. e-mail: nobili66@yahoo.it; fax: 39 0668592192. The authors are indebted to all the patients and their legal guardians who participated in this study. The authors disclose the following: Dr Richard Cross is an employee of iqur Limited; Prof. William M. Rosenberg is founder and holds stock in iqur Limited. M.P. and W.M.R. contributed equally to this paper.