Technology Insight: noninvasive assessment of liver fibrosis by biochemical scores and elastography

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1 Technology Insight: noninvasive assessment of liver fibrosis by biochemical scores and elastography Massimo Pinzani*, Francesco Vizzutti, Umberto Arena and Fabio Marra review SUMMARY Although histopathological analysis of liver tissue is still the reference standard for the evaluation of disease progression in patients with chronic liver disease, a distinct change in clinical practice is occurring. The tendency to substitute histopathological analysis of liver biopsies with complex, surrogate noninvasive measures of disease progression has grown to such a level that clarification and guidance on their use is needed. This Review provides an overview of the proposed noninvasive diagnostic methodologies and their possible integration with the standard invasive procedures used for the evaluation of disease progression (i.e. liver biopsy and the measurement of portal pressure). A concise analysis of what has been proposed for the differentiation of simple fatty liver from nonalcoholic steatohepatitis and its possible fibrogenic evolution is also included. In particular, the Review focuses on the methods easily available as part of daily clinical practice in hepatology biochemical markers and transient elastography (i.e. liver stiffness measurement). Keywords fatty liver, liver fibrosis, noninvasive assessment, serum markers, transient elastography Review criteria PubMed was searched in April 2007 and subsequently in August 2007 for full papers and abstracts published in English-language journals in the past 15 years, using the following keywords alone or in combination: liver fibrosis, cirrhosis, chronic hepatitis, chronic liver disease, liver biopsy, serum markers, transient elastography, NAFLD/NASH, non-invasive, HVPG and fatty liver. The authors personal collections of articles on the above topics were also extensively consulted. The reference list was updated on 15 September M Pinzani is Professor of Medicine and responsible for Unit 6 (Hepatology), and F Marra is Associate Professor of Medicine, Center for Research, High Education and Transfer DENOThe, Università degli Studi di Firenze, Florence, Italy. F Vizzutti is a Gastroenterology Specialist and in charge of the Hepatic Hemodynamic Laboratory, and U Arena is a Gastroenterology Specialist and in charge of the Ultrasound Laboratory, Dipartimento di Medicina Interna at the Università degli Studi di Firenze. Correspondence *Dipartimento di Medicina Interna, Università di Firenze, Viale GB Morgagni 85, Firenze, Italy m.pinzani@dmi.unifi.it Received 2 July 2007 Accepted 24 October doi: /ncpgasthep1025 INTRODUCTION Progressive fibrosis and cirrhotic transformation of liver tissue are common pathological outcomes of most chronic liver diseases (CLDs). The reference standard for the evaluation of disease progression in patients with CLD is histopathological analysis of liver tissue, but there is now a growing tendency in clinical practice to use noninvasive methods instead. Several noninvasive approaches serum markers, transient elastography (i.e. liver stiffness measurement; LSM) and a revisitation of classical imaging techniques have been proposed as a replacement for, or to be used in combination with, histopathological analysis of liver biopsies. The large number of publications on these noninvasive methodologies confirms the interest in, and need for, this type of innovation; however, the complexity of these surrogate noninvasive measures of disease progression has resulted in the need for clarification and guidance on their use and interpretation. This article provides an overview of the noninvasive diagnostic methodologies and how they might be combined with standard invasive procedures liver biopsy and portal pressure measurement for the evaluation of disease progression. The article also includes a concise analysis of proposals for how to differentiate simple fatty liver from nonalcoholic steatohepatitis (NASH) and its possible fibrogenic evolution. Although there is extensive research activity in the area of liver imaging, this article considers only what is available to clinical hepatologists. In the future, it is likely that magnetic resonance spectroscopy and PET scans will allow comprehensive and dynamic measurement of disease progression in patients with CLD, but their current stage of development does not yet offer significant advantages in clinical practice. At present, similar considerations apply to attempts to develop diagnostic and/or prognostic genomic, proteomic and metabolomic markers. The reader should be aware that it was beyond the scope of this article to perform a systematic february 2008 vol 5 no 2 nature clinical practice GASTROENTEROLOGY & HEPATOLOGY 95

2 review or a meta-analysis of the data available in the literature. It is indeed possible that several key questions and points of controversy might only be answered satisfactorily by performing such analyses. NONINVASIVE METHODOLOGIES: GOOD FOR ANY STAGE OF FIBROTIC EVOLUTION? Most attention has been focused on whether noninvasive methodologies can detect no or minimal (i.e. F0F1), significant (i.e. F2) or advanced (i.e. F3F4) disease according to the METAVIR scoring system for chronic hepatitis C. 1 This approach is reasonable because these stages of development, particularly those resulting from chronic HCV infection, are suitable for treatment with increasingly available and effective antiviral agents. At their present stage of development, all noninvasive methodologies have been shown to be effective in predicting the presence of either no or minimal disease or advanced disease, rather than being able to assess precisely the progression of fibrosis in a step-wise fashion. Since many studies discussed in this Review have relied on a retrospective analysis, it is likely that more precise indications of whether or not noninvasive methods can assess fibrosis progression will derive from ongoing longitudinal studies and from a systematic review and/or meta-analysis of the studies published so far. The usefulness of performing a systematic review and/or meta-analysis has been illustrated by the systematic review published by Abdel Aziz et al. 2 Regardless of whether or not they can assess fibrosis progression, it is quite clear that serum markers, LSM and standard imaging techniques perform quite well at detecting advanced fibrosis and cirrhosis. It could, therefore, be proposed that these systems form part of the clinical workup for patients with advanced CLD. Indeed, there is a need to differentiate advanced fibrosis (i.e. F3F4) from the later stages of CLD (i.e. when the patient is in a clinical stage defined as compensated cirrhosis and, subsequently, when clinically significant portal hypertension and relative complications develop). The current histological assessment of the fibrotic development of CLD tends to define METAVIR F4 as a sort of end-stage of the fibrogenic process (i.e. the cirrhotic transformation of liver tissue). The transition from this initial stage of cirrhosis to clinical decompensation, however, can take several years or even decades. In this initial stage of cirrhosis, no effective diagnostic tools are available that can be used instead of, or in conjunction with, the measurement of the hepatic venous pressure gradient (HVPG). STANDARD INVASIVE PROCEDURES: ARE THEY REAL GOLD STANDARDS? The introduction and evaluation of different noninvasive measures for the assessment of disease progression in patients with CLD is based on using histopathological analysis of liver biopsies as a reference standard. In theory this is correct, since the quantification of the amount of fibrosis can be considered a valid clinical and/or pathological outcome measure. There are, however, several limitations to analyzing liver biopsies (summarized in Supplementary Table 1 online), particularly the fact that a single biopsy sample is representative of no more than 1/50,000 of the whole liver. These limitations make a fair comparison with serum markers or LSM rather difficult. Standard histopathological analysis can also have the same difficulty as noninvasive measures in discriminating adjacent stages of fibrosis (i.e. F2 vs F1 or F3 vs F2). 3,4 To minimize the limitations associated with analyzing biopsies and using histopathological analysis of biopsies as a reference standard, it is essential that all the proposed recommendations concerning the size of the biopsy and the number of analyzed portal tracts are followed when assessing histopathological staging. 3,5 In addition, noninvasive methods (e.g. blood test and LSM) should be performed within a reasonable length of time from the liver biopsy, ideally within 24 hours. There is also a need to improve and develop further the current histopathological scoring systems. For example, efforts should be directed at staging fibrosis beyond stage F4, as the current system makes no distinction between initial cirrhosis (i.e. thin bridging fibrous septa, with limited or no neoangiogenesis, surrounding large parenchymal nodules) and advanced and end-stage cirrhosis, for which the tissue angioarchitecture is greatly altered. 6 Along these lines, it has now been shown that the amount of scar tissue associated with advanced hepatic fibrosis could be more reliably quantified by using morphometric image analysis and that this technique could be used in future therapeutic trials of agents to inhibit fibrosis progression. 7 Another important issue is whether the METAVIR scoring system, as well as other standard 96 nature clinical practice GASTROENTEROLOGY & HEPATOLOGy PINZANI ET AL. february 2008 vol 5 no 2

3 scoring systems, should be applied to the rapid fibrogenic process that occurs in patients with recurrent HCV infection after liver transplantation. It has become more and more evident that, in this setting, a panlobular capillarization of sinusoids develops in parallel with portal tract expansion and septum development. 8 As the METAVIR score largely reflects only the morphology of portal tracts, there are clinical cases in which a METAVIR score of F2 corresponds to a HVPG of 15 mmhg (i.e. a severe degree of portal hypertension). 9 The use of morphometric image analysis could, therefore, also be suggested in this clinical setting. Measurement of the HVPG, which is currently used for the evaluation of portal hypertension, has been suggested to be a reliable end-point for the assessment of the therapeutic benefit of antiviral therapy in patients with advanced hepatic fibrosis caused by chronic HCV infection. 8, 1013 In the absence of significant fibrotic evolution, the HVPG does not exceed 5 mmhg: a gradient of more than 5 mmhg is always associated with significant fibrosis. When considering whether to treat patients with advanced fibrosis who have a HVPG of 510 mmhg, measurement of HVPG could provide relevant indications of whether there is improvement, stabilization or worsening within the stage of compensated cirrhosis. Along these lines, Nagula and co-workers highlighted a significant correlation between HVPG and both the thickness of fibrotic septa and the size of regenerative nodules in human cirrhotic liver. 14 The major advantages and disadvantages of HVPG measurement are detailed in Supplementary Table 1 online. In view of the fact that, within the range of 510 mmhg, portal hypertension is a direct consequence of the fibrotic and/or cirrhotic transformation of liver tissue, measurement of HVPG could be superior to liver biopsy as a reference standard in advanced stages of CLD, although this idea is controversial and needs to be substantiated in prospective studies including a large number of patients. Overall, it seems that the major limitation of HVPG measurement is logistic: it is expensive, requires a dedicated setting and very experienced operators, and hence is available only in specialized centers. SERUM MARKERS: THE WAY TO GO? A large scientific and commercial investment has been made in the past 10 years to develop serum markers that are able to predict the fibrotic stage of CLD. Among the proposed markers, some reflect alterations in hepatic function, but do not directly reflect extracellular matrix (ECM) metabolism so-called indirect markers. Other markers are directly linked to changes in ECM turnover occurring during fibrogenesis so-called direct markers. 15 It should be noted that both indirect and direct markers have intrinsic limitations. Indirect markers often include parameters such as bilirubin concentration or platelet count that are altered predominantly in the very advanced stages of the disease. Direct markers, the application of which is based on the mechanisms of hepatic fibrogenesis, might reflect changes in ECM turnover occurring concomitantly in other organs and systems (e.g. arthritis or atherosclerosis). The most established and/ or recently proposed systems that use serum biochemical markers are listed and analyzed in Tables 1, 2 and 3. In attempts to establish the diagnostic potential and accuracy of these tests, it is important to consider the main end-point of the pilot and relative validation studies (i.e. is the test designed to stage fibrosis accurately without a liver biopsy? Is the test to confirm or to exclude cirrhosis? Is the test to confirm normality or near normality?). In most of the studies listed in Tables 1, 2 and 3, the area under the curve (AUC) for the receiver operating characteristic (ROC) curve is used as a measure of test performance, with optimal values being as close to 1.0 as possible. Nevertheless, the reported median AUC in differentiating mild and/or no fibrosis from significant fibrosis in validation populations is around 0.77, which is far from high diagnostic accuracy. 16 All tests, however, show an improved performance when the end-point is to differentiate cirrhosis from noncirrhosis, with a median AUC in validation sets of approximately In a modeling analysis that included most of the proposed serum marker panels, Parkes and co-workers showed that when positive predictive value (PPV) and negative predictive value (NPV) thresholds of 90% and 95% were considered, liver biopsy could have been correctly avoided in an average of only 35% of the study population, with the stage of fibrosis misclassified in 20% of patients and impossible to classify in the rest because of intermediate values. 16 Overall, it is very important to note that, although direct, indirect and combined serum february 2008 vol 5 no 2 PINZANI ET AL. nature clinical practice GASTROENTEROLOGY & HEPATOLOGY 97

4 Table 1 Indirect serum markers of hepatic fibrosis and their possible interpretation. Index Parameters CLD and number of patients Forns 64 Age, plt, γgt, cholesterol HCV; t = 351 v = 125 APRI 65 AST, plt HCV; t = 192 v = 78 FT, FS 66 Haptoglobin, α2-mc, apo-a1, γgt, bilirubin, γ-globulin HCV, HBV; t = 205 v = 134 Fibroindex 67 Plt, AST, γgt HCV; t = 240 v = 120 Testa 68 FPI 69 FIB-4 70 Bonacini 71 Plt, spleen diameter AST, cholesterol, past alcohol intake, HOMA, age Plt, AST, ALT, age ALT, AST, INR, plt Calculation Interpretation a PPV/NPV (%) b AUC ln(plt) ln(γgt) ln(age) (cholesterol) >6.9 Scheuer 24 <4.2 Scheuer 01 ([AST/ULN]/plt [ 10 9 /l]) 100 >1.5 Ishak Ishak 02 Logistic regression index (proprietary) (plt [ 10 4 /mm 3 ]) (AST [IU/L]) (γgt[g/dl) F F3F F F F1F F F0F F F0F F2F3 HCV; 75 Plt count/spleen diameter >1750 Ishak Ishak >2 HCV; t = 176 v = 126 HCV or HIV; t = 555 v = 277 HCV; 79 E*/1 + e*, where * = (1.827 ln[ast]) + (0.081 age) + (0.768 [past alcohol use graded as 02]) + (0.385 HOMA) <0.2 F0F1 0.8 F2F4 (Age AST)/(plt count ALT 1/2 ) <1.45 Ishak <46 >3.25 Ishak 46 Sum (range 011) of (plt score) + (ALT/AST score) + (INR score) plt (x10 9 /l): >340 = 0; = 1; = 2; = 3; = 4; 4099 = 5; <40 = 6 ALT/AST ratio: >1.7 = 0; = 1; = 2; <0.6 = 3 INR: <1.1 = 0; = 1; >1.4 = 2 Pohl 72 AST, ALT, plt HCV; 211 Positive if: AST/ALT 1 and platelet count < /l Sheth 73 Park 74 AST, ALT AST, ALT HCV; 139 HCV; 153 AP 75 Plt, age HCV; t = 500 v = 120 AST/ALT AST/ALT Age score + plt score (010 possible score) age: <30 = 0; 3039 = 1; 4049 = 2; 5059 = 3; 6069 = 4; 70 = 5. Plt (x10 9 /l): 225 = 0; = 1; = 2; = 3; = 4; <125 = 5 PPV = 66 NPV = 96 PPV = 91 NPV = 90 PPV = 78 PPV = 76 PPV = 76 PPV = 67 PPV/NPV = 61/85 NPV = 91 NPV = 92 NPV = 94 NPV = 61.7 PPV = 90 t = 0.86 v = 0.81 t = 0.80 v = 0.88 F2F4 t = 0.83 v = 0.87 t = 0.83 v = 0.82 NPV = 79 c PPV = 78.9 c 0.74 NPV = 77.4 PPV = 87 NPV = 90 PPV = 65 t = 0.84 v = >8 Knodell 34 PPV = 92.9 NR positive F3-F4 PPV = 93 NR 1 Scheuer 4 1 Scheuer 4 PPV = 100 PPV = 73.7 NR F2F4 PPV = 96 t = 0.76 v = 0.69 a Fibrosis stages refer to the METAVIR system (F0F4) unless otherwise indicated. b Importantly, PPV and NPV vary based on the prevalence of the disease. c The predictive values were calculated by using the value reported in reference 65. Abbreviations: ALT, alanine aminotransferase; AP, age-platelet; apo-a1, apolipoprotein A1; APRI, ASTplatelet ratio index; AST, aspartate aminotransferase; AUC, area under the receiver operator curve; CLD, chronic liver disease; FPI, fibrosis probability index; FS, Fibrosure (Laboratory Corporation of America, Burlington, NC); FT, Fibrotest; γgt, γ-glutamyltransferase; HOMA, homeostatic model assessment; INR, international normalized ratio; α2-mc, α2-macroglobulin; NPV, negative predictive value; NR, not reported; plt, platelet count; PPV, positive predictive value; t, training group; ULN, upper limit of normal; v, validation group. marker systems measure rather different biomarkers, they are all characterized by an AUC for the ROC clustering around As already pointed out, it is likely that the explanation for this diagnostic equivalency lies in the inaccuracy of using liver biopsy as a reference standard, either in absolute terms or relative to the lack of adequate standards in the validation studies performed so far. 17 Accordingly, any discussion of sensitivity, specificity, AUCs and cut-off values for biomarkers versus biopsy should include a parallel discussion of the discordant cases. When there is discordance between a biomarker and a biopsy, the cause of discordance can be biomarker failure or the failure of a biopsy to detect the real stage of fibrotic evolution. 18 In our opinion, another relevant interpretation problem concerns the different spectrum of fibrosis stages (i.e. spectrum bias ) that accounts for most of the 98 nature clinical practice GASTROENTEROLOGY & HEPATOLOGy PINZANI ET AL. february 2008 vol 5 no 2

5 Table 2 Direct serum markers of hepatic fibrosis and their possible interpretation. Index Parameters CLD and number of patients MP3 76 PIIINP, MMP-1 HCV; (logpiiinp [ng/ml]) (logmmp- 1 [ng/ml]) ELF 20 PIIINP, HA, TIMP-1 Calculation Interpretation a PPV/NPV (%) b AUC <0.30 F0F2 >0.40 F3F4 <0.30 F0F1 >0.40 F2F4 Mixed; 1,021 Discriminant analysis (proprietary) >0.102 Scheuer 34 <0.102 Scheuer 02 NPV = 95 PPV = 66 NPV = 75 PPV = 91 PPV = 35 NPV = 92 a Fibrosis stages refer to the METAVIR system (F0F4) unless otherwise indicated. b Importantly, PPV and NPV vary based on the prevalence of the disease. Abbreviations: AUC, area under the receiver operator curve; CLD, chronic liver disease; ELF, European liver fibrosis; HA, hyaluronan; MMP, matrix metalloproteinases; NPV, negative predictive value; PIIINP, N-terminal propeptide of type III procollagen; PPV, positive predictive value; TIMP, tissue inhibitors of metalloproteinases Table 3 Combined indirect and direct serum markers of hepatic fibrosis and their possible interpretation. Index Parameters CLD and number of patients SHASTA 77 FM 19 Hepascore 78 FSII 79 HA, AST, albumin plt, PI, AST, HA, α2-mc, gender, age HA, α2-mc, γgt, age, gender HA, α2-mc, TIMP-1 Calculation Interpretation a PPV/NPV (%) b AUC HCV/HIV; (1 if HA 4185 ng/ml, 0 otherwise) (1 if HA >85 ng/ml, 0 otherwise) (1 if albumin <3.5 g/dl, 0 otherwise) (1 if AST>60 IU/l, 0 otherwise) Mixed; t = 383 v = 120 HCV; t = 117 v = 104 HCV; t = 294 v = 402 Viral CLD: plt (G/L) PI (%) AST (IU/I) α2-mc (mg/dl) HA (µg/l) urea (mmol/l) age (years) alcoholic CLD: PI (%) α2-mc(mg/dl) HA (µg/l) age (years) y/1 + y, where y = exp [ ( age) sex) + ( α2mc) + ( HA) + ( bilirubin) ( γgt)] Logistic regression index (proprietary) >0.8 Ishak 3 <0.3 Ishak 2 PPV = 100 NPV = Prediction of PPV = 86.3/ t = 0.88 CSF, i.e. F2 c 96.6 d v = F2F4 <0.5 F0F1 42 F2F4 <40 F0F1 PPV = 88 NPV = 98 PPV = 77.4 NPV = 78 t = 0.85 v = 0.82 a Fibrosis stages refer to the METAVIR system (F0F4) unless otherwise indicated. b Importantly, PPV and NPV vary based on the prevalence of the disease. c Calès P and collaborators prefer to compare tests using the test performance profile that details the performance as a function of histological fibrosis stages. d Respectively for virus (HCV and HBV) and alcohol induced CLD. Abbreviations: AST, aspartate aminotransferase; AUC, area under the receiver operator curve; CLD, chronic liver disease; CSF, clinically significant fibrosis; FM, Fibrometer; FSII, FIBROSpectII; γgt, γ-glutamyltransferase; HA, hyaluronan; α2-mc, α2-macroglobulin; NPV, negative predictive value; PI, prothrombin index; plt, platelet count; PPV, positive predictive value; SHASTA, serum HA-AST-albumin; t, training group; TIMP, tissue inhibitors of metalloproteinases; v, validation group heterogeneity between studies. For example, if extreme stages of fibrosis (i.e. F0 and F4) are over-represented in a study, then the specificity and sensitivity achieved will automatically be higher than in a study that includes only patients with adjacent stages of fibrosis. Sensitivity and/ or standardization analysis should, therefore, be performed according to these differences in stage prevalence, defining advanced (i.e. F3) and nonadvanced (i.e. F2) fibrosis. Among the studies listed in Tables 1, 2 and 3, only two notably the Fibrometer study 19 and the European Liver Fibrosis (ELF) study 20 included a wide range of liver diseases besides HCV-related CLD. The algorithm for the prediction of fibrosis stage proposed in the Fibrometer study is characterized by a high diagnostic efficacy for alcoholic liver disease (92.0), whereas the algorithm of the ELF study performs excellently in nonalcoholic fatty liver disease (NAFLD), hemochromatosis and recurrence of HCV-related CLD after liver transplantation. All of the etiological groups (i.e. alcoholic fatty liver disease, NAFLD, hemochromatosis and recurrence of HCV-related CLD) were underrepresented in these two studies, however, and the performance of the Fibrometer and ELF algorithms unquestionably needs to be confirmed and validated in large populations. In both studies, the actual extent of fibrosis was for the first time quantified by computerized morphometry. The amount of fibrosis quantified february 2008 vol 5 no 2 PINZANI ET AL. nature clinical practice GASTROENTEROLOGY & HEPATOLOGY 99

6 by the imaging assessment was, however, correlated with the noninvasive test in the Fibrometer study only. An advancement in understanding the clinical usefulness of serum tests could derive from their evaluation in prospective studies that use a combination of different noninvasive tests. Along these lines, Sebastiani and co-workers reported that using a stepwise combination of different algorithms (APRI [aspartate aminotransferaseplatelet ratio index], Fibrotest, Forns index) in cohorts of patients with chronic hepatitis B or C could reduce the need for liver biopsy in 5080% and 5070% of cases, respectively. 21,22 Moreover, Leroy and co-workers prospectively compared six noninvasive scores for the diagnosis of liver fibrosis in patients with chronic hepatitis C. 23 They found that the best combination of scores (including MP3, Fibrotest and APRI) could select one-third of patients with either the absence of significant fibrosis or the presence of advanced fibrosis with more than 90% certainty. In summary, at the present stage of development, the diagnostic accuracy of systems using serum biomarkers has been proved useful for the detection (or exclusion) of significant fibrosis or cirrhosis mainly in patients with chronic HCV infection. It is clear, however, that these tests might reduce but not eliminate the need for liver biopsy, and that platelet count per se allows the exclusion of cirrhosis with a fairly similar degree of accuracy. 24,25 LIVER STIFFNESS MEASUREMENT: MORE DIAGNOSTIC FLEXIBILITY? In the past 5 years, a noninvasive medical device based on transient elastography (Fibroscan, Echosens SA, Paris, France) has received increasing interest. This system has been proposed for the measurement of liver stiffness, which is considered a direct consequence of the fibrotic evolution of CLD. 26 As a result, several studies aimed at evaluating the clinical usefulness and diagnostic accuracy of LSM have been published in the past 2 years (Table 4) The results of the two pioneer studies performed in cohorts of patients with chronic hepatitis C suggested that this system could be useful in assessing the presence of significant fibrosis (i.e. F2) and in suggesting the presence of cirrhosis. 27,35 In a more recent report, Fraquelli and coworkers provided important information for the establishment of practical guidelines in the use of LSM as a diagnostic tool in patients with CLD. 36 Firstly, LSM is characterized by high intraobserver and interobserver agreement (0.98), which probably makes it the most reproducible among those systems currently available. Importantly, the interobserver, and most importantly, intraobserver agreement are influenced by variables such as body mass index (BMI, particularly when 28) and hepatic steatosis. LSM should, therefore, be used with caution as a surrogate for liver biopsy when assessing liver fibrosis in the presence of these clinical features. The study by Fraquelli and co-workers also strongly suggests that factors other than fibrosis might impact on the results of LSM. 36 Indeed, the extent of necroinflammatory activity influences the LSM, with a steady increase of LSM values in parallel with the degree of histological activity. The influence of the extent of necroinflammation is also strongly suggested by a report showing that significant variations in liver stiffness occur during a flare in alanine aminotransferase levels in patients with chronic viral hepatitis 38 and confirmed by results obtained in patients with acute viral hepatitis. 39 There is, in addition, an important methodological matter concerning the use of LSM. Indeed, no study published so far has addressed the issue of what a normal LSM value is (i.e. no measurements have been obtained in large control populations, e.g. blood donors), as has been done for Fibrotest. 40 Accordingly, the concept of what a normal LSM is relies on values obtained in patients classified as F0. Two considerations are inspired by analysis of the data reported in Table 4. For degrees of fibrosis preceding the cirrhotic stage, and particularly in HCV-related CLD, reported LSM values are not very different in the published series. Values show a broad interval for the prediction of F4, however, probably caused by the difficulty of assessing the degree of fibrotic evolution beyond this stage. This last point raises the issue of the potential use of LSM for the evaluation of disease progression in patients with histologically proven cirrhosis (i.e. a clinical stage lacking adequate diagnostic implementation). Carrion and co-workers 28 reported that the LSM reflects both the extent of fibrosis and the elevation of portal pressure in terms of HVPG in patients with recurrent hepatitis C after liver transplantation. Since then, it has been confirmed that there is an excellent correlation between LSM and HPVG values <10 mmhg, 100 nature clinical practice GASTROENTEROLOGY & HEPATOLOGy PINZANI ET AL. february 2008 vol 5 no 2

7 Table 4 Proposed liver stiffness measurement cut-off values for the prediction of different stages of fibrosis in chronic liver disease. Authors Etiology LSM cut-off values (kpa) PPV/NPV a AUC <F2 F2 F3 F4 (%) Castera L et al. 27 HCV Carrion JA et al. 28 HCV post OLT Foucher J et al. 29 CLD Gomez-Dominguez E CLD et al /48 87/81 77/95 79/92 50/100 90/52 90/80 91/92 88/50 78/76 80/ F F = F F F = F F F = F F F = F4 Ganne-Carrie N et al. 31 CLD / = F4 de Ledinghen V et al. 32 HCV-HIV 4.5 Kim KM et al. 33 CLD Yoneda M et al. 34 NAFLD 5.6 Ziol M et al. 35 HCV Fraquelli M et al. 36 CLD Corpechot C et al. 37 PBC/PSC NR 88/96 96/50 78/95 33/ / / / /100 88/56 71/93 78/97 NR NR NR 91/79 84/95 78/ F F = F F F = F F F F = F F F = F F F = F F F = F4 a Importantly, PPV and NPV vary based on the prevalence of the disease. Abbreviations: AUC, area under the receiver operator curve; CLD, chronic liver disease (i.e. hepatitis B and C, ASH and NASH); LSM, liver stiffness measurement; NAFLD, nonalcoholic fatty liver disease; NPV, negative predictive value; NR, not reported; OLT, orthotopic liver transplantation; PBC, primary biliary cirrhosis; PPV, positive predictive value; PSC, primary sclerosing cholangitis. but that the correlation becomes poor for HPVG values 12 mmhg. 41 Although the correlation between LSM and HPVG values needs to be further substantiated in a large cohort of patients, it could have implications for the clinical assessment of patients with CLD preceding the development of clinically significant portal hypertension. In this context, transient elastography could be useful for differentiating stages of the progressive cirrhotic evolution of liver tissue once the histological stage of cirrhosis has been reached. In some studies this possible use of transient elastography has been extended to the prediction of the most common complication of cirrhosis. Foucher et al. have proposed that transient elastography be used for the detection of cirrhosis and prediction of related complications, including the presence of esophageal varices and variceal bleeding. 42 Other authors have suggested that the LSM can predict the presence of large esophageal varices and could be useful for selecting patients for endoscopic evaluation. 43 The poor correlation between LSM and HVPG values 12 mmhg, however, means that is it unlikely that the LSM will be able to predict the presence and the size of esophageal varices with a sufficient degree of accuracy. 41 Important indications for the use of the LSM are likely to emerge from studies combining it with other noninvasive tests. In the study by Castéra and co-workers, the performance of LSM in combination with two biochemical markers Fibrotest and APRI was prospectively evaluated in a cohort of patients with chronic hepatitis C. 27 february 2008 vol 5 no 2 PINZANI ET AL. nature clinical practice GASTROENTEROLOGY & HEPATOLOGY 101

8 Box 1 Clinical and biochemical parameters associated with advanced fibrosis in patients with nonalcoholic fatty liver disease Clinically determinable factors Age Sex Elevated BMI Visceral obesity Presence of diabetes Presence of hypertension Presence of metabolic syndrome Laboratory tests AST/ALT ratio Low platelet count Indexes of insulin resistance (HOMA, QUICKI, OGIS) Elevated ferritin levels Hyaluronic acid Anti-MDA antibodies Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HOMA, homeostatic model assessment; MDA, malondialdehyde; OGIS, oral glucose insulin sensitivity index; QUICKI, quantitative insulin-sensitivity check index. The best performance was obtained by combining LSM with Fibrotest: liver biopsy confirmed the results in 84% ( F2), 95% ( F3) and 94% ( F4) of cases. Overall, it is becoming more and more evident that the use of the LSM will allow greater diagnostic flexibility and the possibility of integration with other diagnostic methodologies for the clinical assessment of patients with advanced fibrosis and cirrhosis. NONINVASIVE EVALUATION OF FIBROSIS IN NAFLD: STILL A DREAM? NAFLD encompasses a spectrum of diseases ranging from simple steatosis with or without nonspecific inflammation, to a more severe entity, NASH, which is associated with fibrosis and carries a significant risk of progression to cirrhosis and its complications. 44 The diagnosis of NASH is essentially based on histopatology and it is more dependent on liver biopsy than other CLDs. In addition, the problem of sample variability in liver biopsies is more prominent in patients with NAFLD than in those with hepatitis C. 45 This represents a key clinical problem because patients with NASH and fibrosis have to be closely followed up and, given the extremely high prevalence of this condition in the general population, the possibility of noninvasive assessment would have an important impact on both public health and health economics. Although there are currently no sophisticated and extensively validated diagnostic algorithms available for patients with NAFLD, several crosssectional studies evaluating patients with NASH and fibrosis have allowed the identification of clinical and biochemical parameters associated with advanced stages of fibrosis in patients with NAFLD (Box 1) Although NAFLD has some indicators in common with other CLDs, it is evident that parameters related to the clinical features of the metabolic syndrome also have a major impact on the fibrotic evolution of NAFLD. Interestingly, age and insulin resistance almost invariably emerged as risk factors for NAFLD in the cross-sectional studies, 46,47 but correlated less strictly with fibrosis progression in longitudinal studies, 50,56 reflecting the complexity of understanding fibrosis dynamics in this CLD and the importance of longitudinal studies in general. The role of autoantibodies in recognizing adducts with oxidative-stress-related products recalls data previously described for alcoholic liver disease, 52 while an increase in ferritin levels has been interpreted as a proxy of inflammatory activity rather than a marker of iron overload. 53 A small number of studies have provided performance data for tests that identify fibrosis in patients with NAFLD (Table 5). 20,47,55,5759 Importantly, in only a few studies 20,55,59 were the results of the training set confirmed in an independent validation set. Moreover, interpretation of the available data is not always easy, particularly because these series often report on small numbers of patients and the assessment of fibrosis stage varies across different reports. It is also important to note that performance of the tests varies based on the prevalence of severe fibrosis in the population tested and this variation further hampers the possibility of extrapolating the results. This difficulty is particularly important when considering that patients with NAFLD might be seen in settings (e.g. diabetes or obesity clinics) in which the prevalence of advanced fibrosis is generally lower than in a hepatology referral centre. Of note, the NAFLD Fibrosis Score (NFS) 55 includes the presence or absence of diabetes, among other parameters. 102 nature clinical practice GASTROENTEROLOGY & HEPATOLOGy PINZANI ET AL. february 2008 vol 5 no 2

9 Table 5 Serum markers of fibrosis in nonalcoholic fatty liver disease. Index Number of patients Parameters BAAT b Age, BMI, serum ALT, triglycerides ELF c Age, HA, TIMP-1, PIIINP Staging method Fibrosis stage Interpretation cut off Sensitivity (%) Specificity (%) PPV NPV AUC (%) a (%) a METAVIR F 2 vs 1 2 NS NS Modified Scheuer F 3 vs HA b Hyaluronic acid Brunt F 3 vs NFS b Hyperglycemia, 253 d BMI, platelet count, albumin, AST:ALT, age NS b Type IV collagen 7S, hyaluronic acid FT b 97 d Total bilirubin, GGT, gender, age, α2-macroglobulin, apolipoproteina1, haptoglobin Brunt F 3 vs 2 <1.455 >0.676 Brunt F 3 vs 2 Coll 5 or HA 50 Coll 5 and HA 50 Brunt/ Kleiner F 2 vs 1 F 3 vs a Importantly, PPV and NPV vary based on the prevalence of the disease. b Training group. c Combination of training group and validation group. d Validation group. Abbreviations: ALT, alanine aminotransferase; AST aspartate aminotransferase; AUC, area under receiver operator curve; BAAT, BMI, ALT, Age, Triglycerides; BMI, body mass index; ELF, European liver fibrosis, FT, FibroTest; HA, hyaluronan; PIIINP, N-terminal propeptide of type III procollagen; NFS, NAFLD fibrosis score; NPV, negative predictive value; NS, not stated; PIIINP, N-terminal propeptide of type III procollagen; PPV, positive predictive value; TIMP, tissue inhibitors of metalloproteinases NS NS NS NS c 0.82 b NS Overall, it is quite clear that the noninvasive serum markers proposed for NAFLD will also allow the identification or exclusion of patients with severe fibrosis, although a large proportion of the population is likely to fall in an indeterminate area. Regardless, the key diagnostic tool in NAFLD is a noninvasive test that can differentiate the presence of NASH from simple steatosis. Along these lines, several tests have been proposed and await adequate validation. Some tests, such as the NashTest, have been derived from those already proposed for predicting the stage of fibrosis with the inclusion of clinical parameters typical of NAFLD. 60 An alternative and original approach was reported in a study of 44 patients that evaluated levels of plasma caspase-3-generated cytokeratin-18 fragments, a biomarker of hepatocyte apoptosis. 61 Levels of cytokeratin-18 fragments identified patients with NASH versus those with simple steatosis with remarkably high specificity and acceptable sensitivity. Conversely, the algorithm proposed by Bedogni et al. relies on typical NAFLD parameters such as BMI, waist circumference, and triglycerides, 62 but was designed simply to detect steatosis in the general population and not to identify NASH. In addition, it has been suggested that the ASH/ NASH index (ANI), which is a score based on the aspartate aminotransferase:alanine aminotransferase (AST/ALT) ratio, BMI and gender, is helpful for differentiating ASH from NASH. 63 Finally, although the use of the LSM is severely hampered by elevated BMI and liver fat content, a study by Yoneda et al. 34 (Table 4) in 67 patients with NAFLD has shown that there are progressive increases in liver stiffness along the stages of fibrosis, and that LSM has excellent sensitivity and specificity for the identification of patients with cirrhosis. CONCLUSIONS The present stage of development of different noninvasive tools for the assessment of liver fibrosis is a testament to the large effort being made to find a better clinical definition of the fibrogenic progression of chronic hepatitis C as well as other liver fibrogenic disorders. Some major considerations have arisen from the experience accumulated so far. First, all noninvasive methodologies have sufficient to excellent diagnostic accuracy for the detection (or exclusion) of advanced fibrosis and cirrhosis, but none allow a step-wise follow-up of the fibrogenic evolution of CLD according to the existing histopathological staging systems. In other words, due to the absence of a true gold standard, achieving 90% diagnostic accuracy remains a goal for the future. february 2008 vol 5 no 2 PINZANI ET AL. nature clinical practice GASTROENTEROLOGY & HEPATOLOGY 103

10 Discordant noninvasive tests Biopsy if results influence management Suspected chronic liver disease Apply two unrelated noninvasive tests No significant fibrosis (i.e. F0F1) No biopsy Follow or treat Concordant noninvasive tests Gray area (i.e. F2F3) Biopsy if results influence management Cirrhosis (i.e. F4) No biopsy HCC and varices screening Figure 1 Proposed algorithm for the noninvasive assessment of fibrosis evolution in patients with chronic hepatitis C. The algorithm is proposed to stimulate constructive debate. Abbreviation: HCC, hepatocellular carcinoma. In addition, none of the currently available tests has a well-defined prognostic value, such as for the prediction of decompensation or death. Second, owing to spectrum bias and the possible causes of discordance with histopathological assessment, applying the different proposed cut-off values in clinical practice is currently hazardous. Third, the combination of two unrelated noninvasive tests (i.e. a biochemical test combined with the LSM) might provide a useful system for the initial assessment of fibrosis evolution in patients with CLD. Figure 1 illustrates one possible algorithm for the noninvasive assessment of fibrosis evolution in patients with chronic hepatitis C: the algorithm is proposed to stimulate constructive debate. The definition of intermediate stages of fibrosis (i.e. F2 and F3) as gray area highlights the current difficulty in the clinical use of the proposed noninvasive tests. Regardless, it is becoming more and more evident that rational and prudent use of the proposed noninvasive methodologies will reduce the need for liver biopsy in a significant percentage of patients and represents diagnostic progress. Supplementary information in the form of a table and reference list is available on the Nature Clinical Practice Gastroenterology & Hepatology website. KEY POINTS Several noninvasive approaches serum markers, transient elastography and imaging have been proposed as a replacement for, or accompaniment to, histopathological analysis of liver biopsies for the assessment of liver fibrosis All proposed noninvasive methodologies have sufficient-to-excellent diagnostic accuracy for the detection (or exclusion) of advanced fibrosis and cirrhosis, but none allow a stepwise follow-up of the fibrogenic evolution of chronic liver disease according to the existing histopathological staging systems; in addition, none of the currently available tests has a well-defined prognostic value, such as the prediction of decompensation or death Due to spectrum bias and the possible causes of discordance with the histopathological assessment, applying the different proposed cut-off values in clinical practice is currently hazardous Combining two unrelated noninvasive tests (i.e. a biochemical test and liver stiffness measurement) might be useful for the initial assessment of fibrosis evolution in patients with chronic liver disease Most noninvasive methods could be instrumental in staging advanced fibrosis from F3/F4 and beyond A noninvasive approach to NAFLD would need to differentiate NASH from bland steatosis in addition to detecting clinically relevant fibrosis and monitoring disease progression References 1 The French METAVIR Cooperative Study Group (1994) Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 20: Abdel Aziz M et al. (2007) FibroTest and FibroScan for the prediction of hepatitis C-related fibrosis: a systematic review of diagnostic test accuracy. Am J Gastroenterol 102: Regev A et al. (2002) Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol 97: Bedossa P et al. (2003) Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology 38: Cadranel JF et al. (2000) Practices of liver biopsy in France: results of a prospective nationwide survey. Hepatology 32: Standish RA et al. (2006) An appraisal of the histopathological assessment of liver fibrosis. Gut 55: Goodman ZD et al. (2007) Progression of fibrosis in advanced chronic hepatitis C: evaluation by morphometric image analysis. Hepatology 45: Samonakis DN et al. (2007) Hepatic venous pressure gradient to assess fibrosis and its progression after liver transplantation for HCV cirrhosis. Liver Transpl 13: nature clinical practice GASTROENTEROLOGY & HEPATOLOGy PINZANI ET AL. february 2008 vol 5 no 2

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