Curr Gastroenterol Rep (2015)7:3 DOI 10.1007/s11894-015-0447-z
PEDIATRIC GASTROENTEROLOGY (S ORENSTEIN, SECTION EDITOR)
A Comprehensive Review of Noninvasive Liver Fibrosis Tests in Pediatric Nonalcoholic Fatty Liver Disease Sana Mansoor 1 & Elizabeth Collyer 1 & Naim Alkhouri 1
# Springer Science+Business Media New York 2015
Abstract Nonalcoholic fatty liver disease (NAFLD) and its spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and fibrosis have been increasing in the pediatric population. The presence and severity of fibrosis in patients with NAFLD are important prognostic factors for the risk of disease progression to cirrhosis. The gold standard for staging liver fibrosis is a liver biopsy. However, given the risks of this procedure, especially in the pediatric population, the development of noninvasive markers to diagnose and monitor progression of NAFLD is desirable. This paper will review recently developed noninvasive methods for diagnosing liver fibrosis in children with NAFLD. These include simple fibrosis scores, advanced biochemical markers, and radiologic imaging studies. Simple fibrosis scores use readily available laboratory tests; available one include AST/ALT ratio, AST to platelet ratio index (APRI), fibrosis (FIB)-4 index, NAFLD fibrosis score (NFS), pediatric NAFLD fibrosis index (PNFI), and pediatric NALFD fibrosis score (PNFS). Advanced biochemical markers include biomarkers of hepatocyte cell death such as cytokeratin 18 fragment levels, and markers of extracellular matrix turnover such as the Enhanced
This article is part of the Topical Collection on Pediatric Gastroenterology * Naim Alkhouri [email protected] Sana Mansoor [email protected] Elizabeth Collyer [email protected] 1
Cleveland Clinic Children’s Hospital, 9500 Euclid Avenue, Cleveland, OH 44195, USA
Liver Fibrosis (ELF) test and hyaluronic acid. Radiologic imaging studies estimate liver stiffness as a surrogate for liver fibrosis; these include transient elastography (TE), magnetic resonance elastography (MRE), and acoustic radiation force impulse imaging (ARFI). Keywords Nonalcoholic fatty liver disease (NAFLD) . Liver fibrosis . Noninvasive markers
Introduction: the Epidemic of Nonalcoholic Fatty Liver Disease in Children Nonalcoholic fatty liver disease (NAFLD) has emerged as a medical challenge for pediatricians during the past two decades and is currently considered the most common form of chronic liver disease in children in the USA [1]. Epidemic proportions of children affected with NAFLD have prompted extensive research that has led to significant advances in areas of disease pathogenesis and natural history as well as diagnostic and therapeutic interventions. The strong association of NAFLD with obesity, insulin resistance, and metabolic syndrome is well established. A landmark study by Schwimmer et al. utilizing pediatric autopsies on 742 subjects between the ages of 2 and 19 years revealed a prevalence of NAFLD of 9.6 % in North American children [2•]. A similar prevalence of NAFLD was found in another study using the National Health and Nutrition Examination Survey (NHANES) database in which up to 8 % of adolescents 12–19 years of age were found to be affected [3]. Moreover, the prevalence is markedly higher in studies involving obese pediatric patients, with reported ranges of 14–83 % [2•, 3–8].
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Stages of Liver Fibrosis in NAFLD NAFLD includes a disease spectrum ranging from steatosis, or simple lipid accumulation in hepatocytes, to a more advanced injury state called nonalcoholic steatohepatitis (NASH), which is a consolidation of steatosis in the presence of cell injury, inflammation, and fibrosis. The eventual stage of hepatic injury on this spectrum is cirrhosis with its secondary complications, including portal hypertension, liver failure, and hepatocellular carcinoma (HCC). Several studies have now characterized the progression of untreated hepatic fibrosis in pediatric NAFLD to eventual cirrhosis during childhood. The prevalence rate of cirrhosis reported among such a cohort ranges widely between 0 and 10 % [9•, 10, 11•]. Thus, the presence of fibrosis in NAFLD is possibly the most important prognostic factor determining disease progression and complications [12, 13]. The most commonly used histologic scoring method for staging of hepatic fibrosis is the Metavir score, which includes five categories based on what is characteristically seen on histologic examination of liver biopsy: F0—no fibrosis; F1—portal fibrosis without septae; F2—portal fibrosis with septae; F3—numerous septae without cirrhosis; and F4—cirrhosis [14, 15•, 16]. Fibrosis stages F2–F4 are considered clinically significant and fibrosis stages F3–F4 are considered advanced fibrosis. It is important when interpreting studies on predictive models and markers for fibrosis to determine their primary objective identification of any fibrosis, clinically significant fibrosis, or advanced fibrosis.
Diagnosis of Liver Fibrosis in Children with NAFLD: Finding the Holy Grail At present, liver biopsy remains the only definitive diagnostic tool to determine the stage of liver fibrosis in children with NAFLD. There is an urgent need to develop diagnostic tools to stage fibrosis that are less invasive and more widely acceptable, cost-effective, and more readily available. Over the past decade, significant strides have been made in developing noninvasive diagnostic markers for liver fibrosis, ranging from scores that use simple laboratory tests to more complex serum biochemical markers, and imaging modalities based on the principle of measuring liver stiffness as well as a combination of these different modalities. In the remainder of this review, we aim to describe these markers and their utility in the pediatric population as studied by various groups. Table 1 summarizes the various diagnostic modalities including degree of accuracy and cost effectiveness among other features. These are discussed in more detail below. A common means by which to compare the diagnostic accuracy of different tests is by using receiver operating characteristic (ROC) analysis which is a graphical representation of the compromise
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between false-negative and false-positive rates for every possible cutoff, described by calculating the area under the ROC curve (AUROC). A diagnostic test is considered ideal when the AUROC is 1. Simple Fibrosis Scores The presence of significant fibrosis may be the most important factor determining the prognosis of NAFLD and its risk of progression to end-stage liver disease. Several noninvasive fibrosis scoring systems comprised of routinely measured clinical and laboratory variables have been developed in adult patients with NAFLD to identify those with advanced fibrosis. These include the AST/ALT ratio, the NAFLD fibrosis score (NFS), the AST to platelet ratio index (APRI), and the fibrosis (FIB)-4-score [14]. However, recent data from our group and others have suggested that these adult scores may not be accurate for predicting advanced fibrosis in children [15•, 16]. The first noninvasive score of liver fibrosis designed specifically for the pediatric population was developed in an Italian cohort by Nobili et al. in 2009 and is called the pediatric NAFLD fibrosis index (PNFI) [17•]. This score uses simple clinical parameters including age, waist circumference, and triglycerides. The authors reported an AUROC of 0.80 for prediction of liver fibrosis. However, when the PNFI was tested in South Korean children in an attempt to externally validate the score, it performed poorly, producing an AUROC value of 0.41 for predicting clinically significant fibrosis (F2 or higher). Unfortunately, the PNFI was not different between those with and without significant fibrosis (7.61±2.08 and 7.71±2.79, respectively; p=0.314). A novel step forward in this field is the development of another model for predicting hepatic fibrosis designed specifically for children by our group in 2014 [18]. This singlecenter study included a large cohort of children (n=242, mean age 12.4±3.1 years) with biopsy-proven NAFLD. Several simple fibrosis scores were initially calculated for each child (APRI, NFS, FIB-4 index, AST/ALT ratio). After comparison of these scores, the data extracted were used to build a new model using multivariable logistic regression analysis. This new model titled pediatric NAFLD fibrosis score (PNFS) was found to have AUROC value of 0.74 for advanced fibrosis, superior to other markers tested. The PNFS incorporates simple determinants such as alkaline phosphatase, GGT, and platelets which are easily available in a primary care setting. Some of the limitations of this study of the PNFS include its subjects being limited to a largely Caucasian population from a single tertiary care center and variations in alkaline phosphatase values due to several factors unrelated to fibrosis, such as vitamin D deficiency. Therefore, external validation is needed for the PNFS before it is widely implemented.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Discriminant score=−7.412+[(ln(HA)*0.681)+ (ln(P3NP)*0.775)+(ln(TIMP-1)*0.494)]+10
Ultrasound-based technique to measure shear wave velocity/liver stiffness (Fibroscan)
Magnetic resonance based imaging to measure liver stiffness
Ultrasound-based technique to measure shear wave velocity/liver stiffness
Cytokeratin 18 (CK-18)
Hyaluronic acid (HA)
Enhanced Liver Fibrosis Test (ELF)
Transient elastography (TE)
MR elastography (MRE)
Acoustic radiation force impulse (ARFI)
−ð0:02*GGTÞ0 plateletsÞphosphataseÞ=dlÞ:539* log
−ð1:1*logðPlateletsÞÞ
þð0:002*Alkaline PhosphataseÞ
p¼
pffiffiffiffiffiffiffiffiffiffi ez 100z ¼ 1:1 þ 0:34* ALT 1 þ ez
10
Pediatric NAFLD fibrosis score (PNFS)
1 1þe−lp
Linear predictor (lp)=−6.539*loge [age (years)] +0.207*waist h i(cm)+1.957*loge [triglycerides (mg/dl)]−10.074
Pediatric NAFLD fibrosis index (PNFI)
PNFI ¼
Calculation
ARFI cutoff value of >2.0 m/s predicts advanced fibrosis ( ≥F3)
Liver stiffness cutoff value of 2.71 kPa predicts significant fibrosis (≥F2)
A p=26 % or greater gives specificity of 92 %, sensitivity of 31 %, PPV 41 %, and NPV 88 % for predicting advanced fibrosis A p=8 % confers specificity of 33 %, sensitivity of 97 %, PPV 20 %, and NPV 99 % for ruling out advanced fibrosis Originally tested as a biomarker for NASH. Recommended cutoff [19] of 233 gives maximal sensitivity and specificity of 85 % and 86.9 %, respectively, with PPV of 93 % and NPV of 71.6 % for NASH diagnosis HA ≥1200 ng/ml: absence of fibrosis was unlikely 7 % (95 % CI 1–14 %) HA ≥2100 ng/ml made F2–F4 likely 89 % (95 % CI 75–100 %) ELF score ≥9.28=presence of any fibrosis ELF score ≥10.18=presence of significant fibrosis ELF score ≥10.51=presence of advanced fibrosis TE value 5–7 kPa: stage 1 fibrosis TE value 7–9 kPa: stage 1 or 2 fibrosis TE value >9 kPa: stage 3 or 4 fibrosis
Range 0–10 ≤3 no fibrosis ≥9 presence of any fibrosis
Interpretation
+
Varied AUROC: 0.85 in initial study; externally validated: AUROC 0.41 for determining clinically significant fibrosis PPV 98.5 (95 % CI 91.8 to 100) to rule in liver fibrosis without liver biopsy [17•] AUROC value of 0.74 for advanced fibrosis [18]
++
++
+++
AUROC 0.95 [21]
AUROC 0.92 for detecting any fibrosis AUROC 0.98 for detecting significant fibrosis AUROC 0.99 for detecting advanced fibrosis [24•]
TE cutoff of 5.1 confers 97 % sensitivity and 91 % specificity for ≥stage 1 fibrosis TE cutoff >7.4 give 100 % sensitivity and 92 % specificity for ≥stage 2 fibrosis TE cutoff 10.2 gives 100 % sensitivity and specificity for ≥stage 3 fibrosis [27] AUROC 0.92 for detecting significant fibrosis (≥stage 2) with sensitivity of 88 % and specificity of 85 % [28] 100 % sensitivity, 39 % specificity
+++
+++
++ AUROC 0.75 for the presence of any fibrosis (unpublished data) AUROC 0.77 for advanced fibrosis
+
Cost
Accuracy
Summary table of markers for evaluating liver fibrosis in pediatric patients. Costs rated on a scale of + for minimal cost, ++ for moderate, and +++ for most costly tests
Marker
Table 1
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Advanced Biochemical Markers Another category of fibrosis scores includes those that use more sophisticated biochemical markers of hepatocyte cell death and extracellular matrix turnover. These scores include cytokeratin 18 (CK-18) fragment levels, hyaluronic acid (HA), and the Enhanced Liver Fibrosis (ELF). Unfortunately, these may not be readily available, as compared to the previously discussed simple fibrosis scores. Caspase-cleaved CK-18, a marker of hepatocyte apoptosis, has been extensively validated in adult studies to be a reliable marker of NASH. This finding was also reproduced in children by Feldstein et al. who found significantly higher levels of CK-18 in subjects with NASH compared to those with no NASH, with an AUROC of 0.93 [19]. However, the only pediatric study looking at utility of this marker in liver fibrosis in NAFLD is from Poland by Lebensztein et al. [20]. They found that among a cohort of 52 children, 19 children who had biopsy-proven liver fibrosis also had significantly higher CK-18 levels than in children without fibrosis, with AUROC value for differentiating no fibrosis from presence of fibrosis of 0.666. HA is a glycosaminoglycan synthesized by extracellular matrix-producing cells such as activated hepatic stellate cells, hence making it a direct marker of liver injury. The most convincing data for the usefulness of HA levels come from an Italian study by Nobili et al. who found that while evaluating a group of 100 children with NAFLD and biopsy-proven liver fibrosis, that HA values ≥1200 ng/ml made presence of fibrosis (F1) likely, while values of HA ≥2100 ng/ml made significant fibrosis (F2, F3, F4) very likely. Once again, more studies are needed to confirm these findings [21]. Thirdly, the ELF test is used for the diagnosis of liver fibrosis. ELF is an algorithm involving three direct markers of fibrogenesis and extracellular matrix turnover, HA, aminoterminal pro peptide of type III collagen (PIIINP) and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels. ELF has been well-studied and established in adults to be an excellent marker of liver fibrosis in chronic liver disease (CLD) from various etiologies including NAFLD [22, 23]. In children too, ELF showed excellent results for prediction of fibrosis in a group of 112 consecutive children deemed likely to have NAFLD that was later confirmed via biopsy [24•]. In fact, the high degree of sensitivity and specificity found for ELF values in children exceeded those for adults for all stages of fibrosis including presence of any fibrosis (AUROC 0.92), significant fibrosis (0.98), and advanced fibrosis (0.99). Imaging Studies to Diagnose Fibrosis The rise in popularity of minimally invasive diagnostic and surgical procedures has led to advances in radiographic
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techniques over the past decade. These advances include the advent of imaging modalities for diagnosis of hepatic fibrosis. Among the most well-known and well-studied modalities are transient elastography (TE), magnetic resonance elastography (MRE), and acoustic radiation force impulse imaging (ARFI) (Fig. 1). TE, using the FibroScan® apparatus, has been used widely to evaluate liver fibrosis in Europe for several years, but was only approved by the FDA recently in 2013. It works by measuring the shear wave velocity of a sound wave of approximately 50 MHz that is directed from a small transducer at the end of an ultrasound probe into the liver. Another transducer on the same probe measures the shear wave (in m/s) as the wave passes through the liver. The value is then converted into kilopascals to indicate liver stiffness [25]. Two major pediatric studies by de-Lĕdinghen et al. and Nobili et al. have shown encouraging results of using TE for prediction of fibrosis in pediatric liver disease [26, 27]. The latter Italian study used a group of 52 children (mean age 13.6±2.4 years) with biopsyproven NAFLD and demonstrated excellent accuracy for AUROC values for TE to predict the presence of any fibrosis (F1-4, AUROC 0.977), significant fibrosis (F24, AUROC 0.992), and advanced fibrosis (F3-4, AUROC 1.000). Another advantage of TE is the fact that the results can be obtained in real time. The study, however, did suggest that, at this time, liver biopsy may still be necessary for some uncertain TE values [27]. It has been noted that the mean BMI of this Italian cohort is significantly less obese than that typically reported for the US population [28]. MRE is another promising noninvasive tool for measuring liver stiffness and assessing fibrosis that has demonstrated excellent results in adults with liver fibrosis [29, 30]. Recently, a group from Cincinnati Children’s Hospital published a pilot study of 35 children and adolescents (median age 13 years), undergoing MRE and liver biopsy for evaluation of CLD [28]. Their results show excellent accuracy of MRE for detecting significant fibrosis AUROC of 0.92. A major advantage of MRE over TE is that MRE is independent of abdominal wall fat deposition, which can be a limiting factor for transient elastography. This pilot study is yet to be followed by larger studies to validate the outcomes as well as a dedicated study to assess fibrosis specifically in children with NAFLD is also needed. Cost effectiveness of MRE is another aspect which needs to be addressed. Lastly, ARFI is an ultrasound-based approach using short bursts of high-intensity acoustic pulses that produce shear waves through the liver tissue. The velocity of these waves then correlates with liver stiffness. Two prospective pediatric cohort studies have shown ARFI to be a good noninvasive modality. Hanquinet et al. recruited 39 children with biopsyproven chronic liver disease and 103 healthy controls and
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Fig. 1 Comparison of different imaging studies to diagnose liver fibrosis in children with NAFLD
found the mean value for ARFI to be 1.12 m/s in controls and 1.99 m/s in those with chronic liver disease. Furthermore, they found that an ARFI cutoff value of less than 2 m/s yielded a sensitivity of 100 % to distinguish between children with mild and severe (F>2) fibrosis [31]. A similar study by Noruegas found ARFI to be a useful test to determine the stage of fibrosis in children with CLD ≥F1 (AUROC 0.834), ≥F2 (AUROC 0.818), and F4 (AUROC 0.983) [32]. Both studies revealed that the reliability of this modality was higher in patients with advanced fibrosis than in those with less severe fibrosis. Combinations of Different Fibrosis Tests There has been a recent trend in studying the utility of a combination of two or more noninvasive fibrosis tests in children with NAFLD. The premise is to start with a simple fibrosis score that can be calculated by any clinician using readily available clinical values as a screening test and if the results are inconclusive to move on to more complex tests including advanced biochemical markers and imaging studies. Such examples include combining PNFI with TE in children with biopsy-proven NAFLD. In this population, the detection rate for clinically significant fibrosis utilizing the combination of PNFI and TE was found to be as high as 98 % [33]. PNFI paired with ELF has also proven to be another successful combination for detecting significant liver fibrosis: AUROC 0.944 for the prediction of fibrosis in children (compared to individual AUROC values for PNFI and ELF of 0.761 and
0.924, respectively) [34•]. Recently, a Portuguese group of researchers have looked at the combination of ARFI with several markers and found that ARFI in combination with AST/ALT ratio index may potentially serve as an accurate method for detecting significant fibrosis in children (AUROC value 0.83 in their study) [35].
Future Directions A quick review of adult data highlights numerous markers which are yet to be applied and studied in the pediatric population. These include indirect markers such as Fibrotest, ActiTest, Hepascore, BARD score, proteomics, and glycomics. Examples of more direct markers of liver fibrosis include Fibrospect II, Procollagen type I carboxy-terminal peptide (PICP), laminin, YKL-40, tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2, transforming growth factor (TGF) alpha and beta, platelet-derived growth factor (PDGF), and others. Likely, future developments in this field are multiple and exciting. Shear wave elastography (SWE), a novel radiological tool, has been recently tested in a large pediatric cohort with promising results [36]. In a Turkish study that compared 50 healthy controls and 76 children with CLD, SWE (in kPa) had sensitivity of 91.5 %, specificity of 94 %, PPV 93.2 %, and NPVof 94 % for diagnosing liver fibrosis.
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Conclusions Once thought to be a hallmark of advanced liver disease only seen in adults, liver fibrosis is now being seen increasingly in children as a complication of NAFLD. As this population increases, importance is placed on the best diagnostic tool for accurately detecting clinically significant liver fibrosis in children. In the future, it would be ideal to have noninvasive markers that allow avoidance of a liver biopsy and its complications. In this review, we examined the accuracy of several noninvasive markers of variable complexities and origins in children, with the conclusion that none of the markers is yet able to match the precision of tissue sampling. However, some performed very well and may serve as promising noninvasive markers for detecting this important clinical entity. There were limitations of several studies we examined, with the most common limitation being small sample size and lack of external validation or reproducibility. Larger studies need to be undertaken to validate the efficacy of those markers that demonstrated promising results.
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Compliance with Ethics Guidelines 12. Conflict of Interest Sana Mansoor, Elizabeth Collyer, and Naim Alkhouri declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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PEDIATRIC GASTROENTEROLOGY (S ORENSTEIN, SECTION EDITOR)
A Comprehensive Review of Noninvasive Liver Fibrosis Tests in Pediatric Nonalcoholic Fatty Liver Disease Sana Mansoor 1 & Elizabeth Collyer 1 & Naim Alkhouri 1
# Springer Science+Business Media New York 2015
Abstract Nonalcoholic fatty liver disease (NAFLD) and its spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and fibrosis have been increasing in the pediatric population. The presence and severity of fibrosis in patients with NAFLD are important prognostic factors for the risk of disease progression to cirrhosis. The gold standard for staging liver fibrosis is a liver biopsy. However, given the risks of this procedure, especially in the pediatric population, the development of noninvasive markers to diagnose and monitor progression of NAFLD is desirable. This paper will review recently developed noninvasive methods for diagnosing liver fibrosis in children with NAFLD. These include simple fibrosis scores, advanced biochemical markers, and radiologic imaging studies. Simple fibrosis scores use readily available laboratory tests; available one include AST/ALT ratio, AST to platelet ratio index (APRI), fibrosis (FIB)-4 index, NAFLD fibrosis score (NFS), pediatric NAFLD fibrosis index (PNFI), and pediatric NALFD fibrosis score (PNFS). Advanced biochemical markers include biomarkers of hepatocyte cell death such as cytokeratin 18 fragment levels, and markers of extracellular matrix turnover such as the Enhanced
This article is part of the Topical Collection on Pediatric Gastroenterology * Naim Alkhouri [email protected] Sana Mansoor [email protected] Elizabeth Collyer [email protected] 1
Cleveland Clinic Children’s Hospital, 9500 Euclid Avenue, Cleveland, OH 44195, USA
Liver Fibrosis (ELF) test and hyaluronic acid. Radiologic imaging studies estimate liver stiffness as a surrogate for liver fibrosis; these include transient elastography (TE), magnetic resonance elastography (MRE), and acoustic radiation force impulse imaging (ARFI). Keywords Nonalcoholic fatty liver disease (NAFLD) . Liver fibrosis . Noninvasive markers
Introduction: the Epidemic of Nonalcoholic Fatty Liver Disease in Children Nonalcoholic fatty liver disease (NAFLD) has emerged as a medical challenge for pediatricians during the past two decades and is currently considered the most common form of chronic liver disease in children in the USA [1]. Epidemic proportions of children affected with NAFLD have prompted extensive research that has led to significant advances in areas of disease pathogenesis and natural history as well as diagnostic and therapeutic interventions. The strong association of NAFLD with obesity, insulin resistance, and metabolic syndrome is well established. A landmark study by Schwimmer et al. utilizing pediatric autopsies on 742 subjects between the ages of 2 and 19 years revealed a prevalence of NAFLD of 9.6 % in North American children [2•]. A similar prevalence of NAFLD was found in another study using the National Health and Nutrition Examination Survey (NHANES) database in which up to 8 % of adolescents 12–19 years of age were found to be affected [3]. Moreover, the prevalence is markedly higher in studies involving obese pediatric patients, with reported ranges of 14–83 % [2•, 3–8].
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Stages of Liver Fibrosis in NAFLD NAFLD includes a disease spectrum ranging from steatosis, or simple lipid accumulation in hepatocytes, to a more advanced injury state called nonalcoholic steatohepatitis (NASH), which is a consolidation of steatosis in the presence of cell injury, inflammation, and fibrosis. The eventual stage of hepatic injury on this spectrum is cirrhosis with its secondary complications, including portal hypertension, liver failure, and hepatocellular carcinoma (HCC). Several studies have now characterized the progression of untreated hepatic fibrosis in pediatric NAFLD to eventual cirrhosis during childhood. The prevalence rate of cirrhosis reported among such a cohort ranges widely between 0 and 10 % [9•, 10, 11•]. Thus, the presence of fibrosis in NAFLD is possibly the most important prognostic factor determining disease progression and complications [12, 13]. The most commonly used histologic scoring method for staging of hepatic fibrosis is the Metavir score, which includes five categories based on what is characteristically seen on histologic examination of liver biopsy: F0—no fibrosis; F1—portal fibrosis without septae; F2—portal fibrosis with septae; F3—numerous septae without cirrhosis; and F4—cirrhosis [14, 15•, 16]. Fibrosis stages F2–F4 are considered clinically significant and fibrosis stages F3–F4 are considered advanced fibrosis. It is important when interpreting studies on predictive models and markers for fibrosis to determine their primary objective identification of any fibrosis, clinically significant fibrosis, or advanced fibrosis.
Diagnosis of Liver Fibrosis in Children with NAFLD: Finding the Holy Grail At present, liver biopsy remains the only definitive diagnostic tool to determine the stage of liver fibrosis in children with NAFLD. There is an urgent need to develop diagnostic tools to stage fibrosis that are less invasive and more widely acceptable, cost-effective, and more readily available. Over the past decade, significant strides have been made in developing noninvasive diagnostic markers for liver fibrosis, ranging from scores that use simple laboratory tests to more complex serum biochemical markers, and imaging modalities based on the principle of measuring liver stiffness as well as a combination of these different modalities. In the remainder of this review, we aim to describe these markers and their utility in the pediatric population as studied by various groups. Table 1 summarizes the various diagnostic modalities including degree of accuracy and cost effectiveness among other features. These are discussed in more detail below. A common means by which to compare the diagnostic accuracy of different tests is by using receiver operating characteristic (ROC) analysis which is a graphical representation of the compromise
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between false-negative and false-positive rates for every possible cutoff, described by calculating the area under the ROC curve (AUROC). A diagnostic test is considered ideal when the AUROC is 1. Simple Fibrosis Scores The presence of significant fibrosis may be the most important factor determining the prognosis of NAFLD and its risk of progression to end-stage liver disease. Several noninvasive fibrosis scoring systems comprised of routinely measured clinical and laboratory variables have been developed in adult patients with NAFLD to identify those with advanced fibrosis. These include the AST/ALT ratio, the NAFLD fibrosis score (NFS), the AST to platelet ratio index (APRI), and the fibrosis (FIB)-4-score [14]. However, recent data from our group and others have suggested that these adult scores may not be accurate for predicting advanced fibrosis in children [15•, 16]. The first noninvasive score of liver fibrosis designed specifically for the pediatric population was developed in an Italian cohort by Nobili et al. in 2009 and is called the pediatric NAFLD fibrosis index (PNFI) [17•]. This score uses simple clinical parameters including age, waist circumference, and triglycerides. The authors reported an AUROC of 0.80 for prediction of liver fibrosis. However, when the PNFI was tested in South Korean children in an attempt to externally validate the score, it performed poorly, producing an AUROC value of 0.41 for predicting clinically significant fibrosis (F2 or higher). Unfortunately, the PNFI was not different between those with and without significant fibrosis (7.61±2.08 and 7.71±2.79, respectively; p=0.314). A novel step forward in this field is the development of another model for predicting hepatic fibrosis designed specifically for children by our group in 2014 [18]. This singlecenter study included a large cohort of children (n=242, mean age 12.4±3.1 years) with biopsy-proven NAFLD. Several simple fibrosis scores were initially calculated for each child (APRI, NFS, FIB-4 index, AST/ALT ratio). After comparison of these scores, the data extracted were used to build a new model using multivariable logistic regression analysis. This new model titled pediatric NAFLD fibrosis score (PNFS) was found to have AUROC value of 0.74 for advanced fibrosis, superior to other markers tested. The PNFS incorporates simple determinants such as alkaline phosphatase, GGT, and platelets which are easily available in a primary care setting. Some of the limitations of this study of the PNFS include its subjects being limited to a largely Caucasian population from a single tertiary care center and variations in alkaline phosphatase values due to several factors unrelated to fibrosis, such as vitamin D deficiency. Therefore, external validation is needed for the PNFS before it is widely implemented.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Discriminant score=−7.412+[(ln(HA)*0.681)+ (ln(P3NP)*0.775)+(ln(TIMP-1)*0.494)]+10
Ultrasound-based technique to measure shear wave velocity/liver stiffness (Fibroscan)
Magnetic resonance based imaging to measure liver stiffness
Ultrasound-based technique to measure shear wave velocity/liver stiffness
Cytokeratin 18 (CK-18)
Hyaluronic acid (HA)
Enhanced Liver Fibrosis Test (ELF)
Transient elastography (TE)
MR elastography (MRE)
Acoustic radiation force impulse (ARFI)
−ð0:02*GGTÞ0 plateletsÞphosphataseÞ=dlÞ:539* log
−ð1:1*logðPlateletsÞÞ
þð0:002*Alkaline PhosphataseÞ
p¼
pffiffiffiffiffiffiffiffiffiffi ez 100z ¼ 1:1 þ 0:34* ALT 1 þ ez
10
Pediatric NAFLD fibrosis score (PNFS)
1 1þe−lp
Linear predictor (lp)=−6.539*loge [age (years)] +0.207*waist h i(cm)+1.957*loge [triglycerides (mg/dl)]−10.074
Pediatric NAFLD fibrosis index (PNFI)
PNFI ¼
Calculation
ARFI cutoff value of >2.0 m/s predicts advanced fibrosis ( ≥F3)
Liver stiffness cutoff value of 2.71 kPa predicts significant fibrosis (≥F2)
A p=26 % or greater gives specificity of 92 %, sensitivity of 31 %, PPV 41 %, and NPV 88 % for predicting advanced fibrosis A p=8 % confers specificity of 33 %, sensitivity of 97 %, PPV 20 %, and NPV 99 % for ruling out advanced fibrosis Originally tested as a biomarker for NASH. Recommended cutoff [19] of 233 gives maximal sensitivity and specificity of 85 % and 86.9 %, respectively, with PPV of 93 % and NPV of 71.6 % for NASH diagnosis HA ≥1200 ng/ml: absence of fibrosis was unlikely 7 % (95 % CI 1–14 %) HA ≥2100 ng/ml made F2–F4 likely 89 % (95 % CI 75–100 %) ELF score ≥9.28=presence of any fibrosis ELF score ≥10.18=presence of significant fibrosis ELF score ≥10.51=presence of advanced fibrosis TE value 5–7 kPa: stage 1 fibrosis TE value 7–9 kPa: stage 1 or 2 fibrosis TE value >9 kPa: stage 3 or 4 fibrosis
Range 0–10 ≤3 no fibrosis ≥9 presence of any fibrosis
Interpretation
+
Varied AUROC: 0.85 in initial study; externally validated: AUROC 0.41 for determining clinically significant fibrosis PPV 98.5 (95 % CI 91.8 to 100) to rule in liver fibrosis without liver biopsy [17•] AUROC value of 0.74 for advanced fibrosis [18]
++
++
+++
AUROC 0.95 [21]
AUROC 0.92 for detecting any fibrosis AUROC 0.98 for detecting significant fibrosis AUROC 0.99 for detecting advanced fibrosis [24•]
TE cutoff of 5.1 confers 97 % sensitivity and 91 % specificity for ≥stage 1 fibrosis TE cutoff >7.4 give 100 % sensitivity and 92 % specificity for ≥stage 2 fibrosis TE cutoff 10.2 gives 100 % sensitivity and specificity for ≥stage 3 fibrosis [27] AUROC 0.92 for detecting significant fibrosis (≥stage 2) with sensitivity of 88 % and specificity of 85 % [28] 100 % sensitivity, 39 % specificity
+++
+++
++ AUROC 0.75 for the presence of any fibrosis (unpublished data) AUROC 0.77 for advanced fibrosis
+
Cost
Accuracy
Summary table of markers for evaluating liver fibrosis in pediatric patients. Costs rated on a scale of + for minimal cost, ++ for moderate, and +++ for most costly tests
Marker
Table 1
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Advanced Biochemical Markers Another category of fibrosis scores includes those that use more sophisticated biochemical markers of hepatocyte cell death and extracellular matrix turnover. These scores include cytokeratin 18 (CK-18) fragment levels, hyaluronic acid (HA), and the Enhanced Liver Fibrosis (ELF). Unfortunately, these may not be readily available, as compared to the previously discussed simple fibrosis scores. Caspase-cleaved CK-18, a marker of hepatocyte apoptosis, has been extensively validated in adult studies to be a reliable marker of NASH. This finding was also reproduced in children by Feldstein et al. who found significantly higher levels of CK-18 in subjects with NASH compared to those with no NASH, with an AUROC of 0.93 [19]. However, the only pediatric study looking at utility of this marker in liver fibrosis in NAFLD is from Poland by Lebensztein et al. [20]. They found that among a cohort of 52 children, 19 children who had biopsy-proven liver fibrosis also had significantly higher CK-18 levels than in children without fibrosis, with AUROC value for differentiating no fibrosis from presence of fibrosis of 0.666. HA is a glycosaminoglycan synthesized by extracellular matrix-producing cells such as activated hepatic stellate cells, hence making it a direct marker of liver injury. The most convincing data for the usefulness of HA levels come from an Italian study by Nobili et al. who found that while evaluating a group of 100 children with NAFLD and biopsy-proven liver fibrosis, that HA values ≥1200 ng/ml made presence of fibrosis (F1) likely, while values of HA ≥2100 ng/ml made significant fibrosis (F2, F3, F4) very likely. Once again, more studies are needed to confirm these findings [21]. Thirdly, the ELF test is used for the diagnosis of liver fibrosis. ELF is an algorithm involving three direct markers of fibrogenesis and extracellular matrix turnover, HA, aminoterminal pro peptide of type III collagen (PIIINP) and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels. ELF has been well-studied and established in adults to be an excellent marker of liver fibrosis in chronic liver disease (CLD) from various etiologies including NAFLD [22, 23]. In children too, ELF showed excellent results for prediction of fibrosis in a group of 112 consecutive children deemed likely to have NAFLD that was later confirmed via biopsy [24•]. In fact, the high degree of sensitivity and specificity found for ELF values in children exceeded those for adults for all stages of fibrosis including presence of any fibrosis (AUROC 0.92), significant fibrosis (0.98), and advanced fibrosis (0.99). Imaging Studies to Diagnose Fibrosis The rise in popularity of minimally invasive diagnostic and surgical procedures has led to advances in radiographic
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techniques over the past decade. These advances include the advent of imaging modalities for diagnosis of hepatic fibrosis. Among the most well-known and well-studied modalities are transient elastography (TE), magnetic resonance elastography (MRE), and acoustic radiation force impulse imaging (ARFI) (Fig. 1). TE, using the FibroScan® apparatus, has been used widely to evaluate liver fibrosis in Europe for several years, but was only approved by the FDA recently in 2013. It works by measuring the shear wave velocity of a sound wave of approximately 50 MHz that is directed from a small transducer at the end of an ultrasound probe into the liver. Another transducer on the same probe measures the shear wave (in m/s) as the wave passes through the liver. The value is then converted into kilopascals to indicate liver stiffness [25]. Two major pediatric studies by de-Lĕdinghen et al. and Nobili et al. have shown encouraging results of using TE for prediction of fibrosis in pediatric liver disease [26, 27]. The latter Italian study used a group of 52 children (mean age 13.6±2.4 years) with biopsyproven NAFLD and demonstrated excellent accuracy for AUROC values for TE to predict the presence of any fibrosis (F1-4, AUROC 0.977), significant fibrosis (F24, AUROC 0.992), and advanced fibrosis (F3-4, AUROC 1.000). Another advantage of TE is the fact that the results can be obtained in real time. The study, however, did suggest that, at this time, liver biopsy may still be necessary for some uncertain TE values [27]. It has been noted that the mean BMI of this Italian cohort is significantly less obese than that typically reported for the US population [28]. MRE is another promising noninvasive tool for measuring liver stiffness and assessing fibrosis that has demonstrated excellent results in adults with liver fibrosis [29, 30]. Recently, a group from Cincinnati Children’s Hospital published a pilot study of 35 children and adolescents (median age 13 years), undergoing MRE and liver biopsy for evaluation of CLD [28]. Their results show excellent accuracy of MRE for detecting significant fibrosis AUROC of 0.92. A major advantage of MRE over TE is that MRE is independent of abdominal wall fat deposition, which can be a limiting factor for transient elastography. This pilot study is yet to be followed by larger studies to validate the outcomes as well as a dedicated study to assess fibrosis specifically in children with NAFLD is also needed. Cost effectiveness of MRE is another aspect which needs to be addressed. Lastly, ARFI is an ultrasound-based approach using short bursts of high-intensity acoustic pulses that produce shear waves through the liver tissue. The velocity of these waves then correlates with liver stiffness. Two prospective pediatric cohort studies have shown ARFI to be a good noninvasive modality. Hanquinet et al. recruited 39 children with biopsyproven chronic liver disease and 103 healthy controls and
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Fig. 1 Comparison of different imaging studies to diagnose liver fibrosis in children with NAFLD
found the mean value for ARFI to be 1.12 m/s in controls and 1.99 m/s in those with chronic liver disease. Furthermore, they found that an ARFI cutoff value of less than 2 m/s yielded a sensitivity of 100 % to distinguish between children with mild and severe (F>2) fibrosis [31]. A similar study by Noruegas found ARFI to be a useful test to determine the stage of fibrosis in children with CLD ≥F1 (AUROC 0.834), ≥F2 (AUROC 0.818), and F4 (AUROC 0.983) [32]. Both studies revealed that the reliability of this modality was higher in patients with advanced fibrosis than in those with less severe fibrosis. Combinations of Different Fibrosis Tests There has been a recent trend in studying the utility of a combination of two or more noninvasive fibrosis tests in children with NAFLD. The premise is to start with a simple fibrosis score that can be calculated by any clinician using readily available clinical values as a screening test and if the results are inconclusive to move on to more complex tests including advanced biochemical markers and imaging studies. Such examples include combining PNFI with TE in children with biopsy-proven NAFLD. In this population, the detection rate for clinically significant fibrosis utilizing the combination of PNFI and TE was found to be as high as 98 % [33]. PNFI paired with ELF has also proven to be another successful combination for detecting significant liver fibrosis: AUROC 0.944 for the prediction of fibrosis in children (compared to individual AUROC values for PNFI and ELF of 0.761 and
0.924, respectively) [34•]. Recently, a Portuguese group of researchers have looked at the combination of ARFI with several markers and found that ARFI in combination with AST/ALT ratio index may potentially serve as an accurate method for detecting significant fibrosis in children (AUROC value 0.83 in their study) [35].
Future Directions A quick review of adult data highlights numerous markers which are yet to be applied and studied in the pediatric population. These include indirect markers such as Fibrotest, ActiTest, Hepascore, BARD score, proteomics, and glycomics. Examples of more direct markers of liver fibrosis include Fibrospect II, Procollagen type I carboxy-terminal peptide (PICP), laminin, YKL-40, tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2, transforming growth factor (TGF) alpha and beta, platelet-derived growth factor (PDGF), and others. Likely, future developments in this field are multiple and exciting. Shear wave elastography (SWE), a novel radiological tool, has been recently tested in a large pediatric cohort with promising results [36]. In a Turkish study that compared 50 healthy controls and 76 children with CLD, SWE (in kPa) had sensitivity of 91.5 %, specificity of 94 %, PPV 93.2 %, and NPVof 94 % for diagnosing liver fibrosis.
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Conclusions Once thought to be a hallmark of advanced liver disease only seen in adults, liver fibrosis is now being seen increasingly in children as a complication of NAFLD. As this population increases, importance is placed on the best diagnostic tool for accurately detecting clinically significant liver fibrosis in children. In the future, it would be ideal to have noninvasive markers that allow avoidance of a liver biopsy and its complications. In this review, we examined the accuracy of several noninvasive markers of variable complexities and origins in children, with the conclusion that none of the markers is yet able to match the precision of tissue sampling. However, some performed very well and may serve as promising noninvasive markers for detecting this important clinical entity. There were limitations of several studies we examined, with the most common limitation being small sample size and lack of external validation or reproducibility. Larger studies need to be undertaken to validate the efficacy of those markers that demonstrated promising results.
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Compliance with Ethics Guidelines 12. Conflict of Interest Sana Mansoor, Elizabeth Collyer, and Naim Alkhouri declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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