Diagnosis of Nonalcoholic Fatty Liver Disease - Semantic Scholar

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a liver biopsy remains the only reliable way to precisely diagnose NASH and ... concise overview of the role of liver biopsy versus noninvasive diagnostic tools ...
Diagnosis of Nonalcoholic Fatty Liver Disease: Invasive versus Noninvasive Anna Wieckowska, M.D.,1 and Ariel E. Feldstein, M.D.2,3

ABSTRACT

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States and many other parts of the world. Its prevalence continues to rise—currently affecting 20 to 30% of adults and 10% of children in the United States. NAFLD represents a wide spectrum of conditions ranging from fatty liver, which in general follows a benign nonprogressive clinical course, to steatohepatitis or NASH, a more serious form of NAFLD that may progress to cirrhosis and end-stage liver disease. Although currently a combination of noninvasive clinically available laboratory and imaging tests may help in the diagnostic evaluation of a patient with suspected NAFLD, a liver biopsy remains the only reliable way to precisely diagnose NASH and establish the severity of liver injury and presence of fibrosis. It also provides important information regarding prognosis as well as response to therapeutic interventions. However, a liver biopsy is an invasive and costly procedure, which is poorly suited as a diagnostic test for a condition that may affect about one-third of the U.S. population. This review provides a concise overview of the role of liver biopsy versus noninvasive diagnostic tools for the differentiation of fatty liver from nonalcoholic steatohepatitis as well as for the determination of presence and extent of fibrosis. In particular, this review focuses on the methods currently available in daily clinical practice in hepatology and touches briefly on potential future markers under investigation. KEYWORDS: Fatty liver, nonalcoholic steatohepatitis, liver biopsy, noninvasive diagnostic test, biomarkers

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onalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in both children and adults and threatens to become a serious public health problem.1,2 NAFLD encompasses a wide spectrum of conditions associated with overaccumulation of fat in the liver ranging from fatty liver or NAFL (nonalcoholic fatty liver) to steatohepatitis or NASH (nonalcoholic steatohepatitis) to advanced fibrosis and cirrhosis.1 Although NAFL, which is the most common form of NAFLD, appears to follow a benign nonprog-

ressive clinical course, NASH is a potentially serious condition because as many as 25% of these patients may progress to cirrhosis and experience complications of portal hypertension, liver failure, and hepatocellular carcinoma.3–6 At present, the available noninvasive markers for NAFLD include a set of clinical signs and symptoms, easily available laboratory and radiological imaging tests, and combinations of clinical and blood test results. Although several of these markers are in general useful

1

Ohio 44195 (e-mail: [email protected]). Fatty Liver Disease; Guest Editor, Arun J. Sanyal, M.B.B.S., M.D. Semin Liver Dis 2008;28:386–395. Copyright # 2008 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. DOI 10.1055/s-0028-1091983. ISSN 0272-8087.

Department of Pediatric Gastroenterology, CHUQ, Quebec City, Quebec, Canada; 2Department of Pediatric Gastroenterology and 3 Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio. Address for correspondence and reprint requests: Ariel E. Feldstein, M.D., Departments of Pediatric Gastroenterology and Cell Biology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland,

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for the diagnostic evaluation of a patient with suspected NAFLD, they lack specificity and sensitivity to distinguish NAFL from NASH and determine the presence and stage of fibrosis.7 This represents a key clinical problem because patients with NASH and fibrosis are probably those who need close monitoring and followup as well as the potential targets for therapeutic intervention when specific treatments for this condition become available. Thus, a liver biopsy remains the gold standard for NAFLD diagnosis.8 However, as we will discuss in detail in the next section an invasive liver biopsy is poorly suited as a diagnostic test in such a prevalent condition because of its expense and risks of complications. In the future, the increasing understanding of the pathogenesis of disease progression in NAFLD may result in development of mechanism-based biomarkers that could provide a reliable noninvasive alternative to liver biopsy.

LIVER BIOPSY The histological criteria for NASH diagnosis have been evolving over the last 2 decades since it was initially described by Ludwig et al.9 The principal histological features of NASH include the presence of macrovesicular fatty changes of hepatocytes with displacement of the nucleus to the edge of the cell, ballooning degeneration of hepatocytes, and a mixed lobular inflammation.10 Other features such as perisinusoidal–pericellular fibrosis, Mallory hyaline, megamitochondria, acidophil bodies, glycogenated nuclei, can be present but are not always required to establish the diagnosis of NASH.11 Moreover, in some individuals, atypical features such as predominantly portal-based inflammation and fibrosis are present. This pattern has been observed more frequently in children and it is currently unknown whether it represents an early stage of steatohepatitis or a distinct clinicopathological condition.12 In a recent attempt to standardize the histological diagnostic criteria the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored NASH Clinical Research Network (CRN) developed the NAFLD activity score (NAS).13 This score was based on the classification proposed earlier by Brunt et al14 and consists of the unweighted sum of scores for each of the following lesions: steatosis, lobular inflammation, and hepatocellular ballooning. A NAS of  5 was almost universally associated with the diagnosis of NASH, and cases with a NAS of < 3 were largely considered ‘‘not NASH.’’ However, the NAS as numeric values was not intended to replace a pathologist’s diagnostic determination of steatohepatitis. The prognostic value of the NAS as well as the utility of this score to assess response to therapeutic intervention during treatment trials for which this score was designed, remains to be determined.

Liver fibrosis appears to be one of the most important prognostic factors in patients with NAFLD, as the presence of fibrosis suggests a more advanced and severe liver injury.3,15 Several scoring systems for fibrosis have been used in patients with NAFLD over time. The METAVIR score (F0 ¼ no fibrosis, F1 ¼ stellate enlargement of portal tracts without septa, F2 ¼ stellate enlargement of portal tracts with few septa, F3 ¼ septal fibrosis without cirrhosis, F4 ¼ cirrhosis), originally designed for patients with chronic hepatitis C16 has been used in several NAFLD studies. However, this system as well as others such as the Knodell and Scheuer does not take into account the unique pattern of fibrosis seen in this condition. In 1999, Brunt et al14 developed a staging system based on this concept that begins with the characteristic zone 3 perisinusoidal/pericellular ‘‘chicken wire’’ fibrosis (stage 1) and progresses through portal fibrosis (stage 2) and bridging fibrosis (stage 3) to cirrhosis (stage 4). Finally, the NIDDK CRN13 further refined the categories by subdividing the fibrosis stages and adapting the scoring system to an increasing array of histological presentations seen in NAFLD. Fibrosis scores for stage 1 were extended to include a distinction between delicate (1A) and dense (1B) perisinusoidal fibrosis, and to detect portal-only fibrosis, without perisinusoidal fibrosis (stage 1C), the latter pattern is more commonly encountered in the pediatric population. To date, clinically available noninvasive tests in general lack sensitivity and specificity for diagnosing and staging NASH (see below). Thus, histology remains the gold standard. A liver biopsy provides important information regarding the degree of liver damage, changes in the overall liver architecture, as well as severity of inflammatory activity and fibrosis. It also provides useful information regarding prognosis and may impact clinical management.17,18 Recent longitudinal natural history studies from both the United States and Europe have demonstrated that patients with histological fatty liver tend to have a benign nonprogressive clinical course, whereas only those patients with NASH and fibrosis at baseline may develop complications related to chronic liver disease during follow-up and experience a significant increase in overall and liver-related mortality.3–6 Thus, a diagnosis of NASH may result in a more aggressive therapeutic approach toward metabolic risk factors and recruitment into clinical trials. The presence of advanced fibrosis or cirrhosis would result in regular screening for complications of portal hypertension and hepatocellular carcinoma. Histological changes are also currently used as primary endpoints for monitoring response to therapy during therapeutic intervention trials. However, there is currently limited evidence that serial liver biopsies and the changes in the different grading and staging systems used in NAFLD are an accurate and reproducible method to determine the efficacy of a pharmacological agent or lifestyle intervention. A recent

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study from Ratziu and colleagues suggested that sampling variability (see te next section) may be an important confounder that needs to be considered at the time of designing a particular therapeutic trial.19

Liver Biopsy Limitations There are several important limitations of liver biopsy. First, it is obvious that being an invasive procedure, stressful for patients and their physician, and associated with potential significant complications occurring in 0.5% of cases is not suitable as a screening test in a condition that affects close to a third of the American population.20 There is also a mounting body of evidence that in NAFLD, as is the case with other chronic liver conditions such as hepatitis C, a needle liver biopsy that results in a tissue sample that is a tiny portion of the liver (1/50,000 of the total mass of the liver) may be subject to significant sampling variability.21–23 A series of studies looking at sampling error in NAFLD has suggested that this is more of an issue for the histological findings of necroinflammatory activity and hepatocyte ballooning, but less so for steatosis and fibrosis.21–23 Interestingly, one of these studies also showed that the kappa coefficient of agreement for the diagnosis of NASH was better when using the Brunt scoring system (k ¼ 0.82) than the NAS score (k ¼ 0.69).22 Finally, another important limitation of liver biopsy relates to the fact that histological analysis remains subjective, is influenced by skill and experience of the reading pathologist, and thus prone to intra- and interobserver variability.13,24 Presently, as the diagnosis and evaluation of NASH is mostly dependent on histological assessment, the important limitations of liver biopsy should be taken into account when designing clinical trials and studies on NAFLD natural history. More important, the development of alternative noninvasive markers assessing the global involvement of the liver is greatly needed.

CLINICAL FEATURES AND LABORATORY TESTS Most patients with NAFLD, including adults and children with either NAFL or NASH are asymptomatic at presentation.2,17,25 When present, clinical symptoms and physical findings are nonspecific and unreliable in assessing disease severity in patients with compensated liver disease. The most common signs and symptoms are fatigue, right upper quadrant pain, and hepatomegaly, as well as acanthosis nigricans, which tends to be seen more frequently in the pediatric population. Several clinical features as well as historical data have been shown to be independent risk factors for NASH and presence of fibrosis. The features more consistently found to be associated with disease severity include

obesity, older age, diabetes, and hypertension.1 However, the accuracy and utility of these variables to diagnose NASH and determine the presence of fibrosis have only been assessed in a relatively small number of studies in which specific clinical models or algorithms were created combining them with different biochemical markers. These models are reviewed below in the Panel Markers section. Laboratory tests that are routinely included in the evaluation of patients with suspected NAFLD include a serum panel of liver tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], gamma-glutamyl-transpeptidase [GGT], albumin), prothrombin time, and complete blood counts. Elevated serum ALT and AST levels are the primary abnormality seen in patients with NAFLD and tend to be higher in patients with NASH as compared with NAFL. However, liver aminotransferase levels are seldom higher than 5 times the upper limit of normal, and typically fluctuate with normal levels seen in more than two-thirds of NASH patients at any give time.26–28 Moreover, a study by Mofrad and colleagues clearly demonstrated that the entire histological spectrum of NAFLD can be seen in patients with normal ALT values.29 A subsequent study by Kunde et al30 formally evaluated the diagnostic accuracy of serum ALT for NASH diagnosis in 233 women undergoing gastric bypass surgery. In this study, they used their reference laboratory cutoff value of  30 U/L as well as the new proposed lower cutoff value of  19 U/L. A cutoff value  30 U/L showed a relatively good specificity but poor sensitivity of 40%. Decreasing the upper limit of normal as has been recently suggested improved the sensitivity, but resulted in a very high false-positive rate. More recently, Suzuki and colleagues assessed the diagnostic accuracy of serum aminotransferases (ALT and AST) changes in predicting histological changes over time.31 They used the dataset from the ursodeoxycholic acid (UDCA) NASH randomized trial for which baseline and end of treatment liver biopsies, as well as serial aminotransferases measurements were available.32 After adjusting for baseline histological features and baseline aminotransferases, the rate of ALT (or AST) changes was only correlated with changes in inflammation over the 24-month period of the trial, but not with steatosis or fibrosis. Aminotransferases information provided only a fair prediction of both histological improvement and worsening with AUC (area under the curve) of 0.72 and 0.77, respectively. These values were even worse when percent change of ALT and AST (24-month levels minus baseline levels, divided by baseline levels) rather than rate of changes using the serial measurement data was used with AUC of 0.66 and 0.7 in predicting improvement of inflammation, respectively.

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Serum ALP, GGT, or both are usually mildly elevated in many patients with NAFLD. However, their utility for the diagnosis of NASH is poor. Among other routine laboratory tests, a reversal of the AST/ALT ratio to more than one had been consistently reported to predict the presence of more advanced fibrosis.33,34 This is also true in a variety of other chronic liver diseases like chronic hepatitis C. However, in a similar fashion as the isolated aminotransferase levels, it has a relatively poor sensitivity and negative predictive value (NPV). Hypoalbuminemia, prolonged prothrombin time, and hyperbilirubinemia may be seen with cirrhotic NASH, but are not present until decompensated disease arises.

PANEL MARKERS In an effort to improve the accuracy to diagnose NASH noninvasively and to determine the stage of fibrosis, several groups have created panels using different combinations of a series of clinical and biochemical markers to generate various scoring systems. A proprietary algorithm that provides an estimate for either NASH diagnosis (Table 1) or the presence and extent of fibrosis (Table 2) has also been developed. The HAIR (Hypertension, ALT, Insulin Resistance) score was described in early work by Dixon and colleagues in a group of 105 severely obese patients undergoing gastric bypass surgery.35 The score was designed for prediction of NASH diagnosis and uses a combination of presence of hypertension, elevated ALT (> 40 U/L), and insulin resistance (defined as an insulin resistance index above 5). The presence of at least 2 parameters predicted NASH with high sensitivity and specificity. Palekar et al generated a clinical model to distinguish NASH from simple steatosis by combining 6 different variables including age (> 50 years), female gender, AST (> 45 U/L), body mass index (BMI > 30 kg/m2), and AST/ALT ratio (> 1), and serum hyaluronic acid (HA; > 55 mg/L).36 The AUC for this model was 0.76. The presence of 3 or more of these factors had a sensitivity and specificity for NASH

diagnosis of 74% and 66%, respectively. More recently, Gholam et al proposed a simplified model created using logistic regression analysis using only AST and diagnosis of diabetes, which was able to separate NASH from fatty liver with or without nonspecific inflammation in bariatric surgery patients with similar accuracy as the panels described in previous studies.37 In general, these as well as other studies with similar results (Table 1),38,39 show that a combination of liver function tests and metabolic syndrome indices are useful markers for NASH. However, none of these panels yet has been independently validated in different populations in a prospective fashion. Others have focused on models to predict the extent of fibrosis in NAFLD patients (Table 2). In general, most of these noninvasive panels performed well and show similar accuracy to detect advanced, severe fibrosis, but they have low NPVs for the presence of intermediate and early stages of fibrosis. This represents a central limitation for their clinical use as screening tests for patients with NAFLD, as patients in these categories are probably the ones that would benefit the most from therapeutic interventions. Ratziu and colleagues combined 4 clinical variables to generate the BAAT score,40 including age (> 50 years), BMI (> 28 kg/m2), ALT (> 2XN), and serum triglycerides (> 1.7 mmol/L). Each variable received a score of 0 or 1. Although a total score of 0 had sensitivity close to 100% and a specificity of 47%, a high total score of 4 gave a sensitivity of 14% and a specificity of 100% for detection of septal fibrosis. More recently, the same group has tested the utility of FibroTest-FibroSURE for prediction of liver fibrosis in patients with NAFLD.41 This proprietary panel has been extensively studied in chronic hepatitis C and combines 5 biochemical markers including a2macroglobulin, apolipoprotein A1, haptoglobulin, total bilirubin, and GGT. The score is computed (using an undisclosed formula) by entering patients age and sex along with the 5 components into a proprietary program. The test yielded an AUC for the diagnosis of advanced fibrosis of 0.86. A cutoff value of 0.3 had a 90% NPV for

Table 1 Panel Markers for Nonalcoholic Steatohepatitis (NASH) Diagnosis Test

N

Dixon et al35 (HAIR)

105

AUC for NASH Diagnosis

Components

0.90 (CI: N/S)

HTN, ALT, IR

80

0.76 (CI: 0.65, 0.87)

Age, female gender, AST, BMI, AST/ALT ratio, HA

Gholam et al37

97

0.82 (CI: N/S)

AST, diabetes

Poynard et al38

257

0.79 (CI: 0.69, 0.86)*

Same as FibroTest plus six other components

Palekar et al36

0.79 (CI: 0.67, 0.87)y

(NashTest) Zein et al39 (NPI)

177

0.87 (0.81, 0.93)*

(total of 13 measurements)z Age, HOMA, Log (AST x ALT), female sex, BMI

0.85 (0.75, 0.95)y *Training set. y Validation set. z Other components include height, weight, serum levels of triglycerides, cholesterol, ALT, and AST. AST, aspartate aminotransferase; ALT, alanine aminotransferase; AUC, area under the curve; BMI, body mass index; CI, confidence interval; HA, hyaluronic acid; HOMA, homeostatic model assessment; HTN, hypertension; IR, insulin resistance; N/S, not stated; NPI, NASH predictive index.

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Table 2 Panel Markers for Fibrosis Test

Staging System

N

AUC for Advanced Fibrosis

Components

BAAT score

Metavir

93

0.84 (CI: N/S)

Age, BMI, ALT, serum triglycerides

FibroTest

Modified Brunt

267

0.86 (CI: 0.77, 0.91)*

Age, sex, a2-macroglobulin, apolipoprotein A1,

NAFLD fibrosis

Modified Brunt

733

0.88 (CI: 0.85, 0.92)*

OELF

Scheuer

61

0.87 (CI: 0.66, 1.0)

Age, HA, PIIINP, TIMP-1

ELF

Modified Brunt

192

0.90 (0.84, 0.96)

HA, PIIINP, TIMP-1

0.75 (CI: 0.61, 0.83)y 0.82 (CI: 0.76, 0.88)y

score

haptoglobulin, total bilirubin, GGT Age, BMI, platelet count, albumin, AST/ALT ratio, IFG/diabetes

*Training set. y Validation set. ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the curve; BMI, body mass index; CI, confidence interval; HA, hyaluronic acid; IFG, increased fasting glucose; NAFLD, nonalcoholic fatty liver disease; N/S, not stated; PIIINP, aminoterminal peptide of procollagen III; TIMP-1, tissue 1 inhibitor of metalloproteinase.

advanced fibrosis, whereas a cutoff value of 0.7 had a 73% positive predictive value (PPV) for advanced fibrosis. The most frequent causes of FibroTest (FT) failure include Gilbert’s syndrome, cholestasis, and acute inflammation, which result in increases in bilirubin and haptoglobulin, respectively. Another cause of FT failure in patients with NAFLD that was not recognized previously in other populations is an abnormal apolipoprotein A1 concentration, which might be related to the frequent lipid abnormalities seen in these patients. Angulo and colleagues42 recently created a NAFLD fibrosis score to separate patients with or without advanced fibrosis in a large cohort of biopsy-proven NAFLD patients. An algorithm was constructed using 6 readily available laboratory and clinical variables including age, hyperglycemia, BMI, platelet count, albumin, and AST/ALT ratio. The AUC for this model was 0.88 and 0.82 in the estimation and validation group, respectively. Using this curve they generated two (high and low) cutoff values that allow the diagnosed of advanced fibrosis with high accuracy (NPV between 88 and 93%, PPV between 82 and 90%). However, similar to the FT study in which 33% of cases the presence or absence of advanced fibrosis could not be predicted, there were 25% of indeterminate cases in this series. Fibrosis is a dynamic process that may result in increased circulating levels of extracellular matrix (ECM) components. Several groups have used this reasoning to develop different blood tests using individual or a composite of ECM components. Suzuki et al43 determined the reliability of serum hyaluronic acid (HA) to predict the severity of hepatic fibrosis in 79 patients with histologically confirmed NAFLD. Serum HA was obtained at the time of liver biopsy. The logarithm of serum HA showed a significant positive correlation with the stage of fibrosis, and this association persisted after adjusting for age and serum albumin. The test was found to be useful for predicting severe fibrosis (stages 3 to 4) with AUC of 0.9 (95% CI, 0.83–0.97). The study could not evaluate the efficacy for moderate fibrosis (stage 2) due to the limited number of patients with this stage of

fibrosis and showed low accuracy for mild fibrosis. Lydatakis et al44 subsequently studied the utility of serum HA as well as laminin (LN) in 50 patients with NASH. Twenty-three of these patients had some degree of fibrosis and 27 had no evidence of fibrosis on liver biopsy. Both serum HA and LN were significantly higher in patients with NASH-fibrosis versus those without fibrosis. However, only the mean concentration of serum HA was found to be significantly different among patients with various stages of fibrosis. AUC were calculated to distinguish fibrotic-NASH versus NASH without fibrosis showing high accuracy. In contrast to the study by Suzuki, no analysis was performed to assess the accuracy of the test to separate different stages of fibrosis. Moreover, the proposed cutoff value to predict fibrosis significantly differed between the 2 above-mentioned studies. This lack of reproducibility may be due to many factors including differences in the population tested and in assay systems used. Further larger studies with sufficient number of patients with different stages of fibrosis are still required to better address both the accuracy and reproducibility of serum HA measurement as a marker of fibrosis in NAFLD. In a recent study, the European Liver Fibrosis study group examined a combination of multiple ECM related components in 1021 subjects including 61 patients with NAFLD.45 An algorithm combining age, HA, amino terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) was developed to detect severe fibrosis with high sensitivity as well as excellent NPV for absence of fibrosis. However, similar to the other panel markers for diagnosis of liver fibrosis the test showed a poor performance to predict early and intermediate stages of fibrosis. The same group recently validated the original panel and a simplified algorithm not containing age in a large independent cohort of patients with NAFLD.46 They reported that removing age did not alter the diagnostic performance of the panel. The AUC for the simplified algorithm was 0.90 for distinguishing severe fibrosis, although the addition of simple markers to the panel

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(age, BMI, presence of diabetes or impaired fasting glucose, AST/ALT ratio, platelets, and albumin) improved diagnostic performance with AUC of 0.98.46 These studies suggest that either a combination of clinical and biochemical markers or specific markers of fibrosis may be used for noninvasive staging of NAFLD patients. However, to date, all of these studies have been tested in a cross-sectional fashion and the role of these biomarkers for monitoring disease progression, response to therapy, and prognosis remains completely unknown.

IMAGING STUDIES Several imaging techniques have been advocated as noninvasive diagnostic tests for NAFLD as they can detect liver steatosis. Ultrasonography (US) is currently the preferred method for screening asymptomatic patients with elevated liver enzymes and suspected NAFLD. Ultrasonographic findings of fatty liver include hepatomegaly, diffuse increase in echogenicity of the liver parenchyma, and vascular blunting. Although several studies have demonstrated that the sensitivity, specificity, and PPV of this technique to detect steatosis is as high as 80 to 100%47, this technique has important limitations. First, it is operator-dependent and subject to significant intra- and interobserver variability.48 Second, US does not provide quantitative information of the degree of lipid accumulation. In this regard, recent studies like the one by Hamaguchi and colleagues49 have attempted to generate scoring systems to provide better semiquantitative information on not only the degree of steatosis, but also visceral obesity and the metabolic syndrome. Third, the sensitivity of US to detect steatosis decreases sharply if the degree of fat infiltration is 30% or less50 and in patients with morbid obesity in whom sensitivity lower than 40% has been reported.51 In the later, this is likely due to technical difficulties in performing US in such patients. Finally, the most important limitation of US is the inability to diagnose NASH and hepatic fibrosis. Both computerized tomographic (CT) scanning and magnetic resonance imaging (MRI) are sensitive techniques for quantification of steatosis. More recently, localized proton magnetic resonance spectroscopy (MRS), has been shown to be a noninvasive method that is highly accurate in measuring hepatic triglyceride content (HTGC).52–54 HTGC obtained by MRS closely coincides with biopsyderived triglyceride concentrations. Unfortunately, currently these technologies are limited to the assessment of liver fat and none of these imaging techniques has sufficient sensitivity and specificity for staging the disease and cannot distinguish between fatty liver and NASH with or without fibrosis.55 Recently, transient elastography, a technique for measuring tissue elasticity based on ultrasound technology has been applied for the noninvasive assessment of

hepatic fibrosis. Promising results have been shown on initial studies in chronic hepatitis C patients with AUC, specificities, and sensitivities similar to that of the Fibrotest and other panel markers of fibrosis.56 However, a recent study from France in more than 1000 male subjects undergoing transient elastography showed that the only independent risk factor for failure of the procedure was a BMI > 28 with an odds ratio close to 10,57 which the vast majority of patients with NAFLD will fit in. Moreover, it remains to be determined whether this technique is able to differentiate fibrosis from significant steatosis and its precision may not be sufficient for tracking changes in fibrosis over time and as a result of treatment. Thus, potentially combining transient elastography with noninvasive blood tests may be an attractive approach to provide a more accurate measure of fibrosis.

BIOMARKERS UNDER INVESTIGATION Due to the important limitations of the currently available noninvasive tests, several investigators have tried to identify potential novel biomarkers based on the current knowledge of the pathophysiologic mechanisms involved in disease progression in NAFLD (Fig. 1). An ideal biomarker should be simple, reproducible, inexpensive, readily available, and accurate for a particular disease process. In the case of NAFLD, this biomarker should serve for distinguishing NASH from NAFL and/ or determine the extent of liver fibrosis present. It should also be useful for monitoring disease progression over time, response to different therapeutic interventions, and prognosis. Finally, identification of such a biomarker may also help as a new tool in the development of effective new therapeutics as well as to identify the specific population that may obtain the best benefit from these treatments. We will briefly review data on some of these markers. For a more extended discussion, please refer to a recently published thorough review.7 Oxidative stress (OS) has long been recognized as a key mechanism responsible for liver damage and disease progression in NAFLD.58,59 Enhanced OS occurs in the liver of patients with NASH as well as animal models of NASH.60,61 Several oxidation pathways may play a role in the overproduction of reactive oxygen species (ROS) in NASH including mitochondrial, peroxisomal, cytochrome P-450, myeloperoxidase, and nitric oxide synthase. Each of these pathways may generate different oxidation products that could be potentially quantified. Several groups have attempted to elucidate whether measurement of systemic markers of OS may reflect the levels of OS present in the liver. Most groups have used either method to measure systemic levels of stable lipid byproducts of ROS activity such as lipid peroxides and thiobarbituric acid-reacting substance or ‘‘total antioxidant status’’ with mixed

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Figure 1 Potential rational targets for biomarkers development in nonalcoholic fatty liver disease based on pathophysiology of disease. Lipid overloading of hepatocyte is a prerequisite for the subsequent events that lead to liver injury and fibrogenesis. Mitochondrial dysfunction is thought to play a central role in the progression from steatosis to nonalcoholic steatohepatitis to cirrhosis. Different mechanisms have been proposed including an increased production of reactive oxygen species (1), and mitochondrial outer membrane permeabilization, resulting in a cascade of events leading to inflammation (2), hepatocellular apoptosis (3), fibrogenesis and fibrosis (4). ROS, reactive oxygene species; HSC, activated stellate cells.

results.62–65 Many questions remain unanswered such as what is the relative importance of each of these oxidation pathways in the liver damage seen in NASH. As ROS react rapidly and in situ in the environment they are produced, does measuring these markers in blood or breath reflect what is happening in the liver? A chronic low-grade inflammatory state characteristic of patients with the metabolic syndrome has been extensively associated with the development of steatosis as well as liver damage in NAFLD.66,67 A cytokine imbalance, and in particular an increase in the tumor necrosis factor- a (TNF-a)/adiponectin ratio may play an important role in the development of NASH.68–72 Several groups have investigated the circulating levels of these cytokines in blood from patients with NAFLD and their correlation with disease severity. However, there is currently limited data available on the accuracy and clinical usefulness of these markers for noninvasive NASH diagnosis. Two other inflammatory markers, interleukin-6 (IL-6) and C-reactive protein, which have been extensively studied as markers for insulin resistance, type 2 diabetes, and cardiovascular disease, have been tested in NAFLD patients with mixed results73,74 and larger future studies are still needed to better characterize the role of these as well as other cytokines/chemokines as potential biomarkers for NASH diagnosis in isolation or in combination as diagnostic panels. Hepatocyte apoptosis has been found to be a prominent feature in patients with NASH making it an interesting focus for biomarker development and for

therapeutic intervention.75–79 Recently, a specific byproduct of apoptosis in liver cells, caspase generated cytokeratin-18 fragments have been shown to be significantly elevated in patients with NASH as compared with fatty liver or healthy controls, the AUC for predicting NASH being 0.93.80 These results were validated in a study involving close to 300 patients from 8 different centers in the United States81 that are part of the NIDDK CRN. Thus, the CK-18 test holds significant promise and future studies to better determine the utility of this marker to predict disease progression, and monitor response to therapy are warranted. As important progress is made in the elucidation of the pathogenesis of NAFLD, new rational noninvasive serum biomarkers that reflect the pathogenesis of the disease will continue to be tested. Promising novel approaches, including proteomics, metabolomics, and genomics may help identifying these biomarkers that potentially may drive clinical decision making, supplementing or replacing currently available tests. To date, only a few small, single-center pilot studies have utilized these technologies in NAFLD.82 However, these types of studies are critically dependent on the collection of a large number of well-characterized cases and controls, which will most likely require multicenter collaborations. A good example of this is the recently reported study on genetic markers in over 900 patients with chronic hepatitis C, showing 2 single nucleotide polymorphisms significantly associated with advanced fibrosis.83 Finally, other exciting new

NAFLD DIAGNOSIS/WIECKOWSKA, FELDSTEIN

technologies are those related to molecular imaging with the ability to noninvasively demonstrate both the level of a specific molecular target and the functional state of the target in vivo. These techniques may potentially allow for direct determination of the extent of apoptosis, inflammation, and fibrosis in the liver of patients with NAFLD.

ACKNOWLEDGMENTS

This work was supported by NIH grant (DK076852) and the AGA Research Scholar Award (RSA) to A.E.F.

ABBREVIATIONS ALT alanine aminotransferase AST aspartate aminotransferase AUC area under the curve BMI body mass index ECM extracellular matrix GGT gamma-glutamyl-transpeptidase HA hyaluronic acid HTGC hepatic triglyceride content MRS magnetic resonance spectroscopy NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis NPV negative predictive value OS oxidative stress PPV positive predictive value ROS reactive oxygen species UDCA ursodeoxycholic acid US ultrasonography REFERENCES 1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002;346:1221–1231 2. Wieckowska A, Feldstein AE. Nonalcoholic fatty liver disease in the pediatric population: a review. Curr Opin Pediatr 2005;17:636–641 3. Adams LA, Lymp JF, St Sauver J, et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 2005;129:113–121 4. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413–1419 5. Ekstedt M, Franzen LE, Mathiesen UL, et al. Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology 2006;44:865–873 6. Dam-Larsen S, Franzmann M, Andersen IB, et al. Long term prognosis of fatty liver: risk of chronic liver disease and death. Gut 2004;53:750–755 7. Wieckowska A, McCullough AJ, Feldstein AE. Noninvasive diagnosis and monitoring of nonalcoholic steatohepatitis: present and future. Hepatology 2007;46(2):582–589 8. Ramesh S, Sanyal AJ. Hepatitis C and nonalcoholic fatty liver disease. Semin Liver Dis 2004;24:399–413

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