Alcoholic Fatty Liver Disease

721 Current Molecular Medicine 2016, 16, 721-737

REVIEW ARTICLE ISSN: 1566-5240 eISSN: 1875-5666

Volume 16, Number 8

Current Molecular Medicine The international journal for in-depth reviews on Molecular Medicine

Non-Invasive Assessment of Liver Injury in NonAlcoholic Fatty Liver Disease: A Review of Literature

Impact Factor: 2.912

BENTHAM SCIENCE

M. Maida1, F.S. Macaluso1, F. Salomone2 and S. Petta*,1 1

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Section of Gastroenterology, DIBIMIS, University of Palermo, Palermo, Italy; Division of Gastroenterology, Azienda Sanitaria Provinciale di Catania, Catania, Italy

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DOI: 10.2174/1566524016666161 004143613

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Received: February 9, 2016 Revised: September 6, 2016 Accepted: September 26, 2016

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ARTICLE HISTORY

Abstract: NAFLD (Non-Alcoholic Fatty Liver Disease) is an increasingly significant public health issue, regarded as the most relevant liver disease of the twenty-first century. Approximately 20%-30% of NAFLD subjects develop a NASH (Non-Alcoholic SteatoHepatitis), a condition which can potentially evolve to liver cirrhosis and hepatocellular carcinoma. For these reasons a proper evaluation of liver damage is a key point for diagnosis and prognosis and liver biopsy still remains the “gold standard” procedure both for discrimination between steatosis and steatohepatitis and assessment of the degree of liver fibrosis. Nonetheless, given it is an invasive, painful and costly procedure, a great research efforts have been made in order to develop non-invasive methods for the assessment of NAFLD presence and/or severity by serum markers and imaging techniques. In this review we aimed to perform a comprehensive review of the literature about strengths and weaknesses of the main tools available for the non-invasive assessment of NAFLD patients.

NASH, and the precise assessment of the severity of fibrosis [5]. Nonetheless, it has clear disadvantages, namely it is an invasive, painful and costly procedure with potential life-threatening complications for the patient, and it has well-known inherent limitations [6, 7]. Given all the above, it is not surprising that great research efforts have been made in order to codify non-invasive methods for the assessment of NAFLD presence and/or severity [8]. In this context, we can discriminate between two main groups of non-invasive methodologies [9]. The first one includes the so-called “serum markers” - i.e. parameters measurable in serum - which can be further divided into indirect and direct markers. Indirect markers are based on single or algorithmic elaboration of common alterations in liver function tests or other simple biochemical parameters, independently of the extracellular matrix turnover. Conversely, direct markers reflect fatty acid uptake/accumulation and the actual extracellular matrix metabolism within the liver. The second group includes parameters derived from instrumental devices, including not only the current liver imaging techniques (ultrasound, computed tomography, magnetic resonance), but also novel tools which use principles of physics to explore liver parenchyma, such as Fibroscan and acoustic radiation force impulse (ARFI). In addition, the non-invasive assessment of NAFLD has been recently enlarged by novel markers/indexes directly associated with the severity of liver disease, and by the identification of single nucleotide polymorphisms (SNPs) associated with NAFLD susceptibility and/or severity which may be helpful, alone or variably combined, for such purpose.

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NAFLD is regarded as the most relevant liver disease of the twenty-first century. Indeed, it has been estimated that NAFLD covers about one billion individuals worldwide [1], being the number one cause of elevation of aminotransferases in the Western world, where at least one third of the population is affected [2]. Although simple fatty liver can be considered a benign condition, approximately 20%-30% of NAFLD subjects develop a state of non-alcoholic steatohepatitis (NASH), a condition which can potentially lead to severe sequelae including end-stage liver disease and hepatocellular carcinoma [3]. In addition, several lines of evidence clearly demonstrated that all NAFLD/NASH patients are at high risk of non-hepatic disorders such as cardiovascular disease, type 2 diabetes, kidney failure and colorectal cancer [4]. As a consequence, a comprehensive evaluation of liver damage – in particular the discrimination between simple steatosis and NASH and the evaluation of the severity of fibrosis - is a crucial point in the management of patients with NAFLD. In this line, the ideal clinical aim should be the prediction not only of liver, but also of not liver-related prognosis of these patients.

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INTRODUCTION

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Keywords: NAFLD, NASH, steatosis, non-invasive assessment, liver biopsy.

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Current Molecular Medicine

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Liver biopsy still remains the “gold standard” procedure for the quantification of the amount of steatosis, the distinction between simple steatosis and *Address correspondence to this author at the Section of Gastroenterology, DIBIMIS, University of Palermo, P.zza delle Cliniche 2, 90127 Palermo, Italy; Tel: +39 0916552145; Fax: +39 0916552156; E-mail: [email protected] 1875-5666/16 $58.00+.00

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Current Molecular Medicine, 2016, Vol. 16, No. 8

Maida et al.

However, both old and new non-invasive scores/tools are far from being “ideal”, i.e. none of them has perfect accuracy in detecting the presence of the disease, nor high specificity and cost-effectiveness, and none of them is a highly reliable baseline predictor of both liver and systemic disease progression. Therefore, great efforts should be made in the following years to fill this gap.

Interestingly, diagnostic accuracy of US for the detection of steatosis seems lower in a setting of chronic hepatitis C compared with NAFLD, not only in terms of predictive values, but also of sensitivity and specificity of the technique [19]. However, US can be considered the first diagnostic imaging tool for the diagnosis of fatty liver due to its low cost, and to acceptable sensitivity and specificity values, even if some limitations should be emphasized. In particular, US is an operator-dependant technique with relevant inter and intra-observer disagreement rates [20], and it does not provide reproducible quantitative information, despite some studies proposed the use of scoring systems for fatty liver gradation (“mild”, “moderate” and “severe”) [14] which, however, have never been formally validated.

In this review we aimed to perform a comprehensive review of the literature about the main strategies available for the non-invasive assessment of NAFLD patients, highlighting strengths and limitations of every tool. Furthermore, we also tried to depict the future directions of this complex research scenario.

DISEASE:

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As previously stated, the gold standard for the detection and quantification of fatty liver remains liver biopsy [5]. However, its accuracy and reliability can be influenced not only by its intrinsic limitations (sampling errors, insufficient width and length of biopsy sample, variability in the definition of pathological features, inter-observer and/or intra-observer disagreement), but also by the fact that quantification of steatosis by biopsy is derived from a small fraction of tissue which may not be representative of the real liver fat content [6, 7]. These issues, together with the obvious impossibility to perform liver biopsy in the general population, have raised the clinical necessity to detect the presence of fatty liver also with non-invasive modalities.

Furthermore, magnetic resonance (MR) is useful for fatty liver evaluation and, differently from CT, it is not associated with radiation exposure. In this line, proton magnetic resonance spectroscopy (1H MRS) is the technique with the better accuracy for steatosis detection, showing overall a very accurate diagnostic performance [23, 24]. These results were further confirmed by a recent meta-analysis which demonstrated the superiority of MR and, particularly, of 1H-MRS on the other imaging techniques for an accurate evaluation of hepatic steatosis [25]. Therefore, although its high costs, MR and 1H MRS can be used when a more accurate evaluation of steatosis is needed, obviously after a first-line evaluation with US.

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NAFLD should be suspected not only in all patients with an overt metabolic syndrome (MS), but also in those with just some of the features which define the MS [10, 11]. Of note, NAFLD is an asymptomatic condition, and the diagnostic workup often is induced by abnormal liver tests or by the incidental evidence of fatty liver detected by an US examination performed for various reasons. Anyway, it is important to underline that about 80% of patients with NAFLD have normal liver tests [12], and that screening for NALFD is currently not codified, even in high-risk group, mostly due to uncertainties surrounding the cost-effectiveness of diagnostic tests and the optimal available treatments [13].

Among the several properties, computed tomography (CT) has also the capability to detect the presence of fatty liver [21]. Even if some studies reported an acceptable diagnostic performance of CT for the diagnosis of steatosis, this instrument is not useful in clinical practice because other hepatic disorders – iron overload for example - could affect the accuracy of CT for steatosis detection [22] and, above all, because CT is associated with unacceptable radiation exposure.

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NON-ALCOHOLIC FATTY LIVER NON-INVASIVE ASSESSMENT

Imaging Techniques Ultrasound (US) is the most accessible instrument to diagnose the presence of hepatic steatosis. Liver fat accumulation is commonly reflected by the detection of a typical ultrasonographic picture - i.e. the Bright Liver Echo Pattern - which results from the increased reflectivity of the parenchyma induced by triglycerides inclusions [14]. Even if variable results have been reported in the literature about its accuracy [15-17], US can be regarded as a technique with acceptable diagnostic performance when compared to liver histology as reference, particularly in presence of moderate-severe degrees of steatosis [18].

Finally, controlled attenuation parameter (CAP) is a novel technique for steatosis detection and quantification which measures the degree of US attenuation induced by hepatic fat using a process based on vibration control transient elastography. In a preliminary study of 115 patients with various chronic liver diseases, CAP was able to accurately detect >10%, >33% and >67% steatosis with area under receiver operating characteristics curves (AUCs) of 0.91, 0.95 and 0.89, respectively [26]. Subsequently, several studies have confirmed these encouraging results [27-30], even if some limitations of the technique have been reported. Indeed, the results obtained with CAP showed poor accuracy for the differentiation of adjacent degrees of steatosis, and conflicting results were observed when CAP performances were compared with those obtained with common serum indices such as Fatty Liver Index or SteatoTest [28, 29].

Non-Invasive Assessment of Liver Injury in Non-Alcoholic Fatty Liver Disease

Serum and Clinical Markers

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of 0.87 in the estimation and 0.86 in the validation group [33].

NON-ALCOHOLIC STEATOHEPATITIS: NONINVASIVE ASSESSMENT

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Although the “gold standard” for the diagnosis of NASH remains liver biopsy, several non-invasive tools have been developed for the distinction between simple steatosis and the features of liver necroinflammation and injury which define NASH. They include several serum markers and markers of apoptosis, with various diagnostic performances. Overall, NASH test and the evaluation of CK-18 fragments are the most studied and validated tools in this setting. More recently, some imaging techniques have been applied to attempt a non-invasive diagnosis of NASH - with intriguing results - even if current evidences are still not enough to support their use in clinical practice. Serum Markers Among all scores based on the evaluation of serum markers and aimed at NASH diagnosis, NASH test is the most investigated panel. It results from the combination of thirteen parameters - age, sex, height, weight, triglycerides, total cholesterol, α2macroglobulin, apolipoprotein A1, haptoglobin, gammaglutamyltranspeptidase (GGT), ALT, AST, and total

Fatty Liver Index (FLI) NAFLD Liver Fat

Parameters

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Diagnostic End-point

Overall AUC (single value or range)

Ultrasonography

Fatty Liver

0.78-0-84

1H MRS

Fatty Liver

0.87

WC, TG

Ultrasonography

Fatty Liver

0.72-0-80

Bedogni, 2006 [31]

GGT, BMI, WC,

Kim, 2011 [32]

TG,

Kotronen, 2009 [33]

Score

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Author, Year [Ref.]

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Name of the Test

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Main serum and clinical markers for the non-invasive assessment of non-alcoholic fatty liver disease.

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Table 1.

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The main algorithms/scores codified for the prediction of fatty liver are resumed in Table 1. They include the Fatty Liver Index (FLI) [31, 32], the Kotronen score [33], the Lipid Accumulation Product (LAP) [32, 34], the Korean study score [35], the Hepatic Steatosis Index (HSI) [36], and the SteatoTest [37]. Even if overall these scores showed a good diagnostic performance, it is necessary to underline that most of them still need external validation, and that, even if potentially applicable in large scale epidemiological studies, their utility in the individual patient – and thus in clinical routine practice - is poor. Among them, the FLI and the Kotronen score – algorithms easy to be computed - are probably the most representative ones. FLI is calculated using four commonly available variables - BMI, waist circumference, GGT and serum triglyceride levels – and its diagnostic accuracy is expressed by an AUC of 0.84 [31]. Such good performance has been reported also by other groups, which applied FLI in population studies [32, 38]. The Kotronen score is considered one of the most robust algorithm in this setting because it underwent internal validation and used 1H MRS as accurate reference standard for steatosis detection, differently from the other scores, whose reference was US. It is based on a combination of clinical and serum markers – presence of MS, type 2 diabetes, fasting serum insulin levels, AST, and the AST/ALT ratio – which reached an AUC


Diabetes, insulin levels, MS, AST,

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AST/ALT

Bedogni, 2010 [34]

Korean Study

Park, 2011 [35]

GGT, BMI, AST/ALT, TG

Ultrasonography

Fatty Liver

0.79

Hepatic Steatosis Index (HSI)

Lee, 2010 [36]

BMI, AST/ALT, gender, diabetes

Ultrasonography

Fatty Liver

0.81

SteatoTest

Poynard, 2005 [37]

BMI, GGT, haptoglobin,

Liver Biopsy

Steatosis ≥ 5%

0.72-0.86

N

Lipid Accumulation Product (LAP)

Kim, 2011 [32]

Score

α2macroglobulin, apolipoprotein A1, bilirubin, cholesterol, TG, glucose, corrected for age and gender Abbreviations: 1H MRS: Proton Magnetic Resonance Spectroscopy; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body Mass Index; GGT: Gamma-glutamyl transpeptidase; MS: Metabolic Syndrome; TG: Triglycerides; WC: Waist circumference.


Maida et al.

bilirubin - and it was elaborated originally in a group of 160 patients, and then validated in 97 NAFLD patients, resulting in an AUC of 0.79 for the diagnosis of NASH in both groups [39]. Subsequently, NASH test was further validated by other investigators [40], maintaining a good diagnostic performance as highlighted by a recent meta-analysis [41], which documented an AUC for NASH diagnosis of 0.84 in the pooling based on individual patients data. Nonetheless, even if NASH test has the advantage of its extensive validation, the elevated number of parameters requested to calculate the score limits its applicability in clinical practice. Interestingly, Hyssalo and colleagues [42] recently elaborated a new “NASH Score”, a model including PNPLA3 genotype, AST and fasting insulin. The score – which firstly included a genetic variable - was elaborated in a cohort of 296 consecutive Finnish bariatric surgery patients who underwent a liver biopsy, and then validated in an Italian cohort comprising of 380, mainly non-bariatric, patients. The investigators reported an overall AUC for NASH prediction of 0.77 in Finns and 0.76 in Italians.

Markers of Apoptosis

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Imaging Techniques

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Recent studies highlighted a potential utility of some imaging techniques for the non-invasive discrimination between simple steatosis and NASH. In this line, a great diagnostic accuracy (AUC of 100%) has been reported for contrast-enhanced US using Levovist [60]; the rationale of this promising - but preliminary - result lies in the lower accumulation of Levovist microbubbles in the liver parenchyma of NASH patients compared with NAFLD, probably due to the difference in the amount of fibrosis between these conditions. Furthermore, proton-decoupled phosphorus 31 3.0-T MR spectroscopy – a technique able to detect NADPH liver content – has been shown to be able to differentiate simple steatosis from NASH and cirrhosis, because NADPH liver content is increased in patients with NASH and in those with cirrhosis compared with patients with simple fatty liver [61]. Other authors have proposed a sonographic score that combines attenuation of the echo amplitude, enlarged splenic diameter and presence of focal fat sparing [62]. Finally, a recent study evaluated the diagnostic accuracy of MR elastography for the early detection of NASH among NAFLD patients: results were very interesting (AUC: 0.93), and the accuracy of the technique was high also in absence of liver fibrosis [63].

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Other than NASH test and NASH score, a great number of non-invasive panels for the diagnosis of NASH have been reported [43-47], as summarized in Table 2. Some have been derived by general clinical settings of NAFLD patients, while others have been elaborated by cohorts including NAFLD patients with severe obesity and/or bariatric populations. Even if the overall diagnostic accuracy of these tests is acceptable - with reported AUCs ranging from 0.63 to 0.93 – they are lacking of robust validation data, and this issue obviously limits their recommendation in clinical practice.

investigators elaborated the Nice Model, a simple panel based on CK-18 fragments plus ALT and presence of the MS [57], reporting an AUC of 0.88 and 0.83 in the training and in the validation group, respectively. Another study observed a good diagnostic performance (AUC of 0.93 in the training group, and of 0.79 in the validation cohort) of a panel composed by CK-18 fragments and soluble Fas levels [58]. Interestingly, an excellent accuracy was also obtained with the evaluation of total (cleaved and uncleaved) CK-18 levels - expression of both apoptosis and necrosis processes - by using a specific ELISA assay [59]. Overall, these data demonstrate the great potentiality for an accurate diagnosis of NASH of total or cleaved CK-18 levels, alone or in combination with other variables. However, it should be underlined that CK-18 testing is not currently available in clinical practice, and that the cut-offs used for the diagnosis of NASH are not fully standardized yet. As a consequence, although serum/plasma CK-18 evaluation is probably the most promising tool for the non-invasive detection of NASH, nowadays it cannot be recommended in clinical practice.

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Apoptosis of hepatocytes has recently emerged as one of the most relevant feature in the pathogenesis of NASH [48, 49]. Starting from these basic knowledges, caspase generated cytokeratin-18 (CK-18) fragments a product of apoptosis in liver cells - have been investigated as potential non-invasive markers of NASH in clinical studies. The first reports showed that CK-18 fragments levels were significantly higher in patients with NASH compared to individuals with simple fatty liver or to healthy controls, being the AUC for NASH prediction very high (0.93) [50]. These results have been validated in several studies [51-53], and further confirmed by a recent meta-analysis that showed a summary AUC for the prediction of NASH of 0.82 [54]. In line with these encouraging results, in order to enhance the diagnostic accuracy of the test, other authors attempted to combine CK-18 levels with other variables. Younossi and colleagues [55] found that a combination of cleaved CK-18 with serum levels of adiponectin and resistin accurately predicted NASH diagnosis, with an AUC of 0.85. Shen and colleagues [56] reported a good diagnostic performance for the non-invasive diagnosis of NAFLD and NASH of a panel composed by CK-18, adipocyte fatty acid binding protein and fibroblast growth factor 21. Other

FIBROSIS IN NON-ALCOHOLIC FATTY LIVER DISEASE: NON-INVASIVE ASESSMENT Once again, liver biopsy is the “gold standard” procedure for the assessment of fibrosis in patients with NAFLD. Anyway, several non-invasive tools have been developed to attempt to detect and quantify the severity of liver fibrosis among NAFLD patients, including demographic/serum markers and specific fibrosis biomarkers. These scores resulted in variable but overall acceptable - diagnostic accuracy for the


Table 2.


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Main clinical scores for the non-invasive prediction of non-alcoholic steatohepatitis.

Name of the Test NASH test

Author, Year [Ref.]

Parameters

Criteria for NASH Diagnosis


Kleiner classification

0.69-0-84


0.77-0.76

Poynard, 2006 [39]

Age, sex, height,

Lassailly, 2011 [40]

weight, TG, ALT, AST,

Poynard, 2012 [41]

cholesterol, GGT, α2macroglobulin, apolipoprotein A1, haptoglobin, total bilirubin

NASH score

Hyysalo, 2014 [42]

PNPLA3 genotype, AST, fasting insulin

Miele, 2009 [43]

Age, TIMP-1, hyaluronic acid


0.93

/

Palekar, 2006 [44]

Age, AST, BMI, AST/ALT, gender,

Brunt classification

0.71-0-78

Sumida, 2011 [45]

hyaluronic acid Sumida, 2011 [45]

Ferritin, insulin, type

Hypertension, ALT, HOMA score

Campos, 2008 [47]

Race, hypertension,

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NASH Clinical Scoring System

Dixon, 2001 [46] Sumida, 2011 [45]

0.63-0.90


0.80

Steatosis and at least one among ballooning with lobular inflammation, perisinusoidal fibrosis, and Mallory bodies

0.73-0.90

diabetes, sleep

CK-18, adiponectin,

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Younossi, 2008 [55]

Nice Model /

Anty, 2010 [57]

Tamimi, 2010 [58]

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Shen, 2012 [56]

Steatosis, ballooning and intralobular hepatocyte necrosis or moderate fibrosis

0.71-0.73

CK-18, ALT, MS


0.83-0.88

CK-18, soluble Fas

Brunt classification

0.79-0.93

CK-18, FGF-21

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apnea, AST, ALT

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0.78-0.85

Steatosis and at least two among ballooning, lobular inflammation, and pericellular fibrosis

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HAIR

Steatosis with lobular inflammation and ballooning

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IV collagen 7S

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NAFIC

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Abbreviations: ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body Mass Index; CK-18: Cytokeratin-18; FGF-21: Fibroblast Growth Factor21; GGT: Gamma-glutamyl transpeptidase; HOMA: Homeostasis Model assessment; MS: Metabolic Syndrome; TG: Triglycerides; TIMP-1: TIMP metallopeptidase inhibitor 1.

diagnosis of severe fibrosis, while their performance is significantly lower for the diagnosis of mild or significant fibrosis. Among these algorithms, FIB-4, NAFLD fibrosis score (NFS) and Fibro-Test have been validated most widely. Furthermore, a strong contribute to the non-invasive diagnosis of fibrosis in NAFLD has been recently given by well-characterized and reliable instrumental tools – namely Fibroscan and ARFI. Demographic/Serum Markers Among demographic/serum markers tested as noninvasive tools for the estimation of liver fibrosis in NAFLD, the most validated scores are FIB-4 and NFS. The FIB-4 uses simple parameters - namely age, platelets, ALT and AST – which are sufficient to guarantee a good diagnostic performance for the diagnosis of severe fibrosis, as confirmed by several reports [64-67]. Furthermore, FIB-4 has been recently validated as a surrogate marker of fibrosis in a

Japanese NAFLD population: investigators reported the best accuracy (AUC: 0.87) and the best negative predictive value (98%) of FIB-4 for the non-invasive diagnosis of fibrosis as compared with other scores [68]. Similarly, the NFS is a panel based on six readily available variables - age, BMI, AST/ALT ratio, platelet count, hyperglycemia, albumin - and calculated using a pre-defined formula available online (http://nafldscore.com). This scoring system was constructed using a large cohort of 733 biopsy-proven NAFLD patients [69], and it showed a good accuracy (AUC: 0.84) for the diagnosis of severe fibrosis. The good performance of this panel has been further validated by several independent studies [64-66, 68, 70-72] and by a recent meta-analysis including 13 studies, which confirmed the good accuracy of the test (AUC: 0.85) for the prediction of advanced fibrosis [54]. In addition to FIB-4 and NFS, several non-invasive scores for fibrosis assessment in NAFLD patients have

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Maida et al.

been developed (Table 3). Overall, they had a less extensive validation, and results were sometimes contrasting across different studies. Among them, we should mention the BARD score [45, 64, 65, 68, 71, 7375], the AST-to-platelet ratio index (APRI) [63, 64, 67, 69, 75-77], the AST⁄ALT ratio (AAR) [64, 65, 68, 70, 78, 79], and the FibroMeter [76, 80].

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Imaging Techniques In the last years different imaging techniques have been developed for the non-invasive assessment of liver fibrosis in patients with chronic liver diseases including those with NAFLD. New emerging diagnostic tools are transient elastography (TE) and acoustic radiation force impulse (ARFI).

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Fibrosis Biomarkers

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Several panels including fibrosis biomarkers have been evaluated in patients with NAFLD, with variable results. Anyway, they are quite expensive, and not easy to apply in the individual patient. As a consequence, currently they are poorly used in clinical practice. Among them, FibroTest is the best validated panel. It results from the combination of five biochemical markers – GGT, haptoglobin, apolipoprotein A1, α2-macroglobulin, total bilirubin corrected for age and gender [89]. All in all, the diagnostic accuracy of the test seems acceptable, with an estimated AUC of 0.84 for the diagnosis of advanced fibrosis in NAFLD patients [90], and of 0.720.85 in bariatric ones [41]. In addition to FibroTest, other non-invasive scores using fibrosis biomarkers have been tested in NAFLD settings, even if they did not receive large validation (Table 3). Here we should mention the European Liver Fibrosis (ELF) test - combining age with serum levels

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TE (Fibroscan, Echosens, Paris, France) is a noninvasive method for assessment of liver fibrosis, which employs ultrasound-based technology, and can be considered as a rapid, low cost, accurate and noninvasive technique [95-103]. A recent meta-analysis [103] confirmed the excellent diagnostic performance of LSM in discriminating cirrhosis from all other stages of fibrosis, with a progressive reduction in its diagnostic accuracy when severe fibrosis (F3–F4) or significant fibrosis (F2–F4) were to be distinguished from the milder stages. TE has been mostly validated in patients with chronic hepatitis C, however in the last years a growing number of studies have been also performed in both pediatric [104] and adult NAFLD patients [105110]. The larger study assessing the performance of TE in NAFLD were performed by Wong and colleagues in a cohort of 246 biopsy-proven NAFLD patients from two ethnic groups, observing an AUC of 0.84 for the diagnosis of moderate fibrosis, of 0.93 for severe fibrosis, and of 0.84 for cirrhosis [106]. In this study, the authors also identified in 7.0 kPa, 8.7 kPa and 10.3 kPa the best LSM cut-off discriminating significant fibrosis, severe fibrosis and cirrhosis respectively [106]. Our group also evaluated the performance of TE in NAFLD in a mono-centric cohort of 169 patients, identifying in a LSM value >7.25 kPa the best cut-off for predicting significant fibrosis (AUC 0.79), and in a LSM value >8.75 kPa the best cut-off for severe fibrosis (F3-F4) (AUC 0.870) [110]. According to the good performance of TE in fibrosis diagnosis also in NAFLD, a 2010 metaanalysis demonstrated an AUC of TE of 0.84 for significant fibrosis and of 0.94 for severe fibrosis [54].

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Furthermore, some authors evaluated the capability of prediction of single parameters previously associated with the severity of liver fibrosis in NAFLD, such as GGT and ferritin. For example, a small study on NAFLD patients observed that GGT alone had an AUC of 0.74 for the prediction of advanced fibrosis [81], while ferritin levels have been defined as independent predictor of advanced fibrosis in some series [82-84]. However, the diagnostic role of serum ferritin has been recently questioned by a brilliant study by Angulo and colleagues [85]: using three cut-off levels for ferritin values (1.0-, 1.5-, and 2.0-fold the upper limit of normal), authors showed that the AUCs of the test were low (less than 0.60) for the detection of fibrosis of any stage, and that the diagnostic performances reported for other non-invasive scoring systems did not change after the inclusion of serum ferritin values. Musso and colleagues [66] showed a good diagnostic accuracy of visceral adiposity index (VAI) index elaborated using BMI, waist circumference, triglycerides and HDL-cholesterol [86] - for the diagnosis of severe fibrosis (AUC: 0.84), even if the association between VAI index and histological damage was not confirmed by another study [87]. Finally, our group recently demonstrated how the combination of VAI index with HOMA score is useful for the prediction of significant fibrosis in patients with NAFLD [88]. Specifically, we found that the prevalence of significant fibrosis progressively increased from patients with a VAI ≤ 2.1 and HOMA ≤ 3.4 (26%) to those with a VAI > 2.1 and HOMA > 3.4 (83%).

of three markers of matrix turnover, namely hyaluronic acid, tissue inhibitor of metalloproteinase 1, and aminoterminal peptide of pro-collagen III [91, 92] – the HepaScore [67], the NAFIC [45], and the ALT to HbA1c ratio [93]. A recent meta-analysis has reported that the ELF – together with FibroTest and NFS - had significantly better diagnostic accuracy than the BARD score, and their AUCs did not significantly differ from each other [54]. Furthermore, interesting results have been recently highlighted from the evaluation of CK-18 fragments levels - extensively validated markers of NASH among NAFLD patients, as above discussed – as non-invasive predictors of fibrosis. In this line, higher CK-18 fragments serum levels were associated with the severity of liver fibrosis, and, interestingly, the evaluation of total CK-18 levels revealed a good diagnostic performance also for the discrimination between low degrees of fibrosis [94]. More research should be performed in this field.


Table 3.


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Main serum and fibrosis biomarkers for the non-invasive assessment of liver fibrosis among patients with non-alcoholic fatty liver disease.

Name of the Test

Author, Year [Ref.]

Parameters

Diagnostic End-points


FIB-4

McPherson, 2010 [64]

AST, ALT, age, platelets

Significant Fibrosis

0.74-0-75

Severe Fibrosis

0.80-0.86


0.67-0.90

Shah, 2009 [65] Musso, 2012 [66] Adams, 2011 [67] Sumida, 2012 [68] NAFLD Fibrosis Score (NFS)

Angulo, 2007 [69]

AST/ALT, Age, hyperglicemia, BMI, platelets, albumin

McPherson, 2010 [64] Sumida, 2012 [68]

Severe Fibrosis

0.64-0.93

Cirrhosis

0.80-0-94


0.60-0-68

Severe Fibrosis

0.67-0.90

Cirrhosis

0.62-0.74

Shah, 2009 [65] Wong, 2008 [70] Fujii, 2009 [71] Qureshi, 2008 [72]

Harrison, 2009 [73]

AST/ALT ratio, BMI, Diabetes

O

Fujii, 2009 [71] Ruffillo, 2011 [74] McPherson, 2010 [64]

U

Shah, 2009 [65]

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Raszeja-Wyszomirska, 2010 [75] Sumida, 2012 [68]

Loaeza-del-

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Shah, 2008 [65]

Wong, 2010 [78]

Sumida, 2012 [68]

0.56-0.89

Severe Fibrosis

0.56-0.90

Cirrhosis

0.74-0.85


0.63

Shah, 2009 [65]

Severe Fibrosis

0.58-0.83

Sumida, 2012 [68]

Cirrhosis

0.66-0.81

Age, AST, ALT, platelets, weight, fasting glucose, ferritin


0.93-0.95

Severe Fibrosis

0.92-0.95

Cirrhosis

0.88-0.94

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McPherson,2010 [64]

AST/ALT


N

AST⁄ALT Ratio (AAR)

Fo rD

McPherson, 2010 [64] Wong, 2008 [70]

AST/platelets

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Castillo, 2008 [77]

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Cales, 2009 [76]

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AST-to-Platelet Ratio Index (APRI)

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Sumida, 2011 [45]

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BARD

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Musso, 2012 [66]

Wong, 2008 [70] Wong, 2010 [78] Bass, 2010 [79] FibroMeter

Cales, 2009 [76] Cales, 2010 [80]

Visceral Adiposity Index (VAI)

Musso, 2012 [66]

BMI, WC, HDL, TG

Severe Fibrosis

0.84

/

Petta, 2012 [88]

VAI, HOMA


Good accuracy as ruler in/out

FibroTest

Ratziu, 2006 [89]

Haptoglobin, α2-


0.65-0.86

Lassailly, 2011 [40]

macroglobulin,

Severe Fibrosis

0.72-0.92

Poynard, 2007 [90]

apolipoprotein

Poynard, 2012 [41]

A1, total bilirubin, GGT, plus correction for age and gender

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(Table 3) Contd. Name of the Test

Author, Year [Ref.]

European Liver Fibrosis (ELF) test HepaScore

NAFIC

/

Parameters

Diagnostic End-points

Rosenberg, 2004 [91]

Age, hyaluronic


0.90

Guha, 2008 [92]

acid, TIMP-1, P3NP

Severe Fibrosis

0.87-0.93

Adams, 2011 [67]

Age, GGT. gender,


0.72

bilirubin, hyaluronic

Severe Fibrosis

0.81

acid, α-2 macroglobulin

Cirrhosis

0.90

Sumida, 2011 [45]


Ferritin, insulin,


0.83

type IV collagen 7S

Severe Fibrosis

0.86

ALT/HbA1C

Any fibrosis

0.90

Gholam, 2007 [93]

Abbreviations: ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body Mass Index; GGT: Gamma-glutamyl transpeptidase; HbA1c: Glycated hemoglobin; HOMA: Homeostasis Model Assessment; P3NP: amino terminal peptide of procollagen III; TG: Triglycerides; TIMP-1: TIMP metallopeptidase inhibitor 1; WC: Waist circumference.

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as a non-invasive method for liver fibrosis assessment among patients with chronic hepatitis C, ARFI showed a similar or even higher performance compared to fibroscan [116-121]. In this line the performance of ARFI has been also investigated in NAFLD patients [122-124]. Yoneda and colleagues showed that in 50 NAFLD patients the ARFI AUC for severe fibrosis and for cirrhosis was 0.973 and 0.976, respectively [122]. Accordingly, a larger study evaluating ARFI in 172 NAFLD patients showed that this technique had an AUC of 0.90 for diagnosis of severe fibrosis, also highlighting as liver stiffness measured by ARFI is reliable regardless of BMI [123]. Finally, Friedrich-Rust et al. compared TE and ARFI in a cohort of 57 patients with NAFLD showing no significant difference of results between the two techniques [124].

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Nevertheless, as shown by literature data, a relevant limit of TE is that several factors are able to affect LSM and therefore its interpretation in clinical practice. Specifically, two of the above cited studies identified severe steatosis [107, 108] as a factor reducing the reliability of TE. In this line we identified BMI as the major determinant of diagnostic errors in predicting significant and severe fibrosis both by over- or underestimating the stage of fibrosis in NAFLD [110]. One more limitation of TE is that, using the standard probe, LSM is not reliable in about 5%-15% of the NAFLD population, mostly due to obesity that represents a very frequent finding among NAFLD patients. The recent introduction of the XL probe for obese patients seems to have reduced this problem [111], even if data from literature clearly show as even if this probe increases the reliability of LSM in the setting of patients with a high BMI, in the same manner it does not improve the accuracy in predicting the stage of ﬁbrosis, as assessed by using liver biopsy as the ‘‘gold standard’’, having both the M and XL probes comparable AUC for different stages of fibrosis [112114].

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Finally another limitation of TE in assessing fibrosis in NAFLD, is that the presence of severe steatosis can lead to overestimations of LSM values. A recent study performed by our group on a cohort of 253 patients affected by NAFLD showed that in patients with low amounts of fibrosis (F0-F1 and F0-F2), median LSM values were significantly higher in subjects with severe steatosis (≥66% at liver biopsy) compared to those without (F0-F1 6.9 versus 5.8, P=0.04; F0-F2 7.4 versus 6.0, P=0.001). The trend was also confirmed when US was used for detection of liver steatosis instead of histology. This data confirm as the presence of severe steatosis, detected by histology or by US, can lead to overestimations of liver fibrosis assessed by transient elastography, suggesting a cautious use of this technique in this setting of patients [115]. Another non-invasive imaging technique potentially useful for fibrosis prediction in NAFLD is ARFI. It is a new technological implementation based on the use of shear acoustic waves remotely induced by the radiation force of a focused ultrasonic beam [116]. When tested

In conclusion, these data show as ARFI is reliable and accurate for diagnosis of fibrosis in NAFLD patients and with a similar performance compared to TE. The features of ARFI, i.e. its reliability in all patients independently of BMI combined with its ability to sample many different areas of liver parenchyma being more representative of the whole liver - could suggest a better performance respect to TE, even if large scale validation studies are needed to further confirm the diagnostic performance of ARFI in NAFLD patients. All results of studies investigating fibroscan and/or ARFI for the non-invasive diagnosis of liver fibrosis in NAFLD patients are summarized in Table 4.

Finally, other imaging techniques have been investigated in the prediction of fibrosis in NAFLD, even if they are more expensive, not widely available, and most of them have not been internally or externally validated yet. Among these, in vivo proton-decoupled 31 P 3.0-T MR spectroscopy has been proved able to discriminate NAFLD patients with cirrhosis according to a recent study [125]. Beside this, retrospective studies have shown that two-dimensional magnetic resonance elastography (2D-MRE), a novel MR method for assessment of liver stiffness, correlates with advanced fibrosis in patients with NAFLD. A cross-sectional study performed on a


Table 4.


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Non-invasive evaluation of liver fibrosis by fibroscan or ARFI in patients with non-alcoholic fatty liver disease. Author, Year [Ref.]

N° of Patients

Diagnostic End-point/s

Diagnostic Cutoff/s

Overall Population AUC

Factors Affecting LSM

FIBROSCAN M PROBE

Nobili, 2008 [103]

52

Any/significant/severe fibrosis

5.1/7.4/10.2

0.97/0.99/1

-

Yoneda, 2008 [104]

97

Any/significant/severe fibrosis/cirrhosis

5.9/6.6/9.8/17.5

0.92/0.86/0.90 /0.99

-

Ziol, 2009 [106]

13

-

-

-

steatosis

Wong, 2010 [105]

246

Significant/severe fibrosis/cirrhosis

7/8.7/10.3

0.84/0.93/0.95

Liver biopsy length, F0- F2 fibrosis

Gaia, 2011 [107]

72

Any/significant/severe fibrosis/cirrhosis

5.5/7/8/10.5

0.77/0.80/0.75 /0.94

steatosis

Lupsor, 2010 [108]

72

Any/significant/severe fibrosis

5.3/6.8/10.4

0.87/0.78/0.97

ALT

Petta, 2011 [109]

169

Significant/severe fibrosis

7.2/8.7

0.79/0.87

BMI

Yoneda, 2010 [121]

54

Severe fibrosis/Cirrhosis

Myers, 2012 [110]

127


-

7.8/-/22.3

0.86/0.87/0.85

-

-

0.80/0.86/0.93 (for NAFLD and other CLD together)


-

0.80/0.73/0.93

-


6.9/8.4

-

steatosis


6.4/-/16

0.85/0.90/0.95

-

120


-

0.78/0.83/0.92 (for NAFLD and other CLD together)

-

Friedrich- Rust, 2012 [123]

61


-

0.82/0.84/0.93

-

Yoneda, 2010 [121]

54

Severe fibrosis/Cirrhosis

1.77/1.90

0.97/0.97

-

Palmeri, 2011 [122]

172

Severe fibrosis

4.24

0.90

-


61


-

0.66/0.71/0.74 Right lobe

-


61


-

0.62/0.60/0.90 Left lobe

-

Myers, 2012 [121]

127

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253


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61

Petta, 2015 [114]

De Leding- hen, 2012 [112]

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ARFI

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0.99/0.99

U

9.9/16

De Leding- hen, 2012 [112]

FIBROSCAN XL PROBE

O

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cohort of 102 patients with biopsy-proven NAFLD recently assessed the performance of two-dimensional magnetic resonance elastography (2D-MRE) respect to 8 non-invasive tools (AST to ALT ratio, APRI, BARD, FIB-4, NAFLD fibrosis score, Bonacini CDS, Lok index, and the NASH CRN model) for prediction of advanced fibrosis [126]. The area under ROC curve (AUROC) was 0.957 for 2D-MRE and between 0.796 and 0.861 for the other clinical prediction rules, and always better for 2D-MRE when compared in head-to-head comparisons to other non-invasive tools (P < 0.05) [126].

Indeed, in NASH-related publications of the last 2 decades, several definitions of NASH have been used, and a recent article by Younossi and colleagues reported data on the agreement between four definitions of NASH [129] as follows: 1) The original definition proposed in 1999, which is based on steatosis plus hepatocyte ballooning, Mallory-Denk bodies, or fibrosis [130]; 2) The definition proposed by Younossi et al. in this publication, which is based on steatosis plus centrolobular ballooning, Mallory-Denk bodies, or fibrosis [129];

Another cross-sectional analysis of a prospective study including 117 consecutive patients with biopsyproven NAFLD found similar results testing 2D-MRE for non-invasive diagnosis of advanced fibrosis [127].

4) Brunt’s definition, which is based on steatosis and lobular inflammation [132].

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Among all four definitions, the reported κ statistic ranged from an almost perfect agreement between Younossi’s and the original definition (κ = 0.896), to a moderate and fair respectively for NAS ≥ 5 (κ = 0.511) and Brunt’s (κ = 0.365) definition [129].

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These data are promising and suggest that 2D-MRE has an excellent accuracy in determining advanced fibrosis in patients with NAFLD. In addition, in the next future, further studies will assess the role of novel and more advanced MRE methods (such as 3D-MRE) for the non-invasive diagnosis and staging of fibrosis of NASH.

3) The definition based on a non-alcoholic steatohepatitis activity (NAS) score ≥ 5 [131];

Limitations of Non-Invasive Diagnosis in Patients with Non-Alcoholic Fatty Liver Disease

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Another relevant issue potentially affecting and limiting the performance of non-invasive tools for the evaluation of NASH and the severity of liver fibrosis, is the schematic approach by which we use to consider separately NASH and fibrosis diagnosis. This method, however, does not take into account that the majority of patients with fibrosis has NASH, and that mechanisms leading to both NASH and fibrosis are in part similar.

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Confirming this, there is a large overlap of biochemical parameters used in the presented panels for fibrosis and NASH, and related to both NASH presence and fibrosis severity. Therefore, in NAFLD patients, it would be more useful to identify a proper definition of “significant liver damage”, beyond the isolate diagnosis of NASH or the isolate evaluation of liver fibrosis.

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In conclusion, considering all available instrumental tools, only Fibroscan, and ARFI after proper validation, could be considered as useful tools in non-invasive assessment of fibrosis in NAFLD. Anyway, for a correct use of these tools, the “results” arisen from the tests need always a proper interpretation taking into account the demographic, clinical and biochemical status of the individual patient from which it has been derived.

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As showed so far, different tools have been developed for the non-invasive diagnosis of NASH and staging of fibrosis among NAFLD patients with a good diagnostic performance expressed in terms of AUC. However, in order to obtain a good knowledge regarding their application, it is important to underline that a number of limitations could affect their diagnostic performance and their use in clinical practice. In particular, most of these studies included NAFLD patients enrolled at tertiary referral centers, or have been performed in specific subsets of patients very different from the entire NAFLD population, for example obese subject candidates for bariatric surgery. In addition, almost all these panels have currently been internal validated, while only a few numbers of scores were been externally validated. Another relevant limitation related to the interpretation and the general applicability of the results from studies evaluating performance of non-invasive scores used in NAFLD patients, lies in the difficulty to perform the histological diagnosis of NASH, due to the heterogeneity in histological scores and the low agreement among pathologists [128].

Proposed Diagnostic Algorithm Despite the presence of several proposed noninvasive scores/tools for the diagnosis of and the characterization of liver damage in NAFLD, currently there is no consensus regarding the diagnostic approach to stage the severity of liver disease in NAFLD patients. Therefore, combining the available and above quoted literature data, we would propose a feasible, easy to perform and relatively low cost diagnostic approach to be applied in these patients. In this purpose, we identified two non-invasive tools that showed individually a high diagnostic performance in staging liver fibrosis, and that are respectively easy to calculate and easy to perform: NAFLD fibrosis score and fibroscan. Considering the uncertainty rates of these tools in the NAFLD fibrosis staging, we suggest to use both tests in order to further improve the diagnostic accuracy of each, when used individually.


Therefore we suggest to perform both NAFLD fibrosis score and Fibroscan in every NAFLD patient. Specifically, in patients in which both tests excluded a severe liver fibrosis, we are confident on the lack of disease severity and therefore we recommend followup and lifestyle therapy. Similarly, in patients with both tools suggesting a severe liver fibrosis we are confident on the presence of severe liver damage and we recommend follow-up, lifestyle therapy and an eventual experimental therapy in a context of randomized clinical trials. In contrast, in case of discordance between the two non-invasive tools, we suggest to perform liver biopsy to the characterization of liver fibrosis (Fig. 1).

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used tools have a good accuracy for the diagnosis of severe fibrosis, while are less performing for the diagnosis of significant fibrosis. However, since this last stage of disease has a high likelihood to progression toward severe fibrosis/cirrhosis, it needs an accurate characterization. Finally, one more limit of our proposed algorithm lies in the fact that it is not reliable in about 10%-20% of NAFLD patients, due to the lower reliability of Fibroscan in obese patients. A further effort to optimize the proposed algorithms, could be obtained by a proper selection of “at risk patients” to be filtered for diagnostic evaluation. Unfortunately, at the moment we are not able to clearly discriminate these patients, even though recently there is growing knowledge on the impact of genetic background in determining liver damage in NAFLD patients. In this regard, a great number of studies, and a recent meta-analysis [134], showed that the rs738409 C>G polymorphism of patatin-like phospholipase-3 (PNPLA3)/adiponutrin, is more prevalent among NAFLD patients compared with healthy controls, and it is also associated, with a diagnosis of NASH, and with severity of both steatosis and fibrosis, in patients with biopsy-proven NAFLD.

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Besides this, we recently observed that the IL28B rs12979860 GG genotype, is associated with the severity of liver damage in NAFLD [135], and that patients carrying both IL28B rs12979860 CC and PNPLA3 rs738409 GG genotypes are at very high risk of severe histological liver damage compared with those without both unfavorable genotypes [135].

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This proposed algorithm could be very useful in reducing the number of unnecessary liver biopsies that should be only performed in those patients with contrasting results. The diagnostic performance of this combined strategy for ruling-in or ruling-out F3-F4 fibrosis has been recently tested in 321 Italian patients with a histological diagnosis of NAFLD (179 Sicilian-training cohort, and 142 northern Italy-validation cohort). The AUCs of LSM, NFS, LSM plus NFS, were 0.857, 0.803, 0.878 respectively in the Sicilian cohort, and 0.848, 0.730 and 0.844 respectively in the northern Italy cohort. In the training cohort, the combination of LSM plus NFS was the best performing strategy, providing false positive, false negative and uncertainty area rates of 0%, 1.1% and 48% respectively. Similar results were obtained in the validation cohort, confirming as the combination of LSM with NFS, two complementary, easy-to-perform, and widely available tools, is able to accurately diagnose or exclude the presence of severe liver fibrosis, also reducing of about 50–60% the number of needed diagnostic liver biopsies [133].


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Nevertheless, this proposed algorithm needs to be externally validated and has different caveats. First, it is not able to discriminate patients with simple fatty liver from those with NASH. Second, it has been designed in order to diagnose severe liver fibrosis, because the

In the next future, and after a proper validation, all these data could help us to identify NAFLD patients at higher risk, in which non-invasive tests could perform better. In conclusion, waiting for a validated genetic tool, our proposed algorithm (Fig. 1) is in line with that

NAFLD patients

Non-invasive combined evaluation by NAFLD fibrosis score and Fibroscan (or ARFI)

NAFLD fibrosis score or Fibroscan (or ARFI) suggestive for F3-F4

Liver biopsy

NAFLD fibrosis b score and Fibroscan (or ARFI) both not suggestive for F3-F4

NAFLD fibrosis score and Fibroscan (or ARFI) both suggestive for F3-F4

No Liver biopsy

Fig. (1). Proposed algorithm for the assessment of severe liver fibrosis.

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recommended by AASLD 2012 guidelines on NAFLD [13], and suggest to consider liver biopsy in patients with NAFLD who are at increased risk to have steatohepatitis and advanced fibrosis.

patients, are available in this setting. In this line, the ELF panel has been reported to be associated with a doubling of the risk of liver-related mortality or morbidity at 6 year follow-up in NAFLD patients [140]. Similarly, in chronic hepatitis C patients, TE and FibroTest showed a high accuracy in prediction of death, liver-related death, and liver transplantation, even if this results need to be replicated and validated also in NAFLD patients [141].

Non-Invasive Prediction of Liver and not LiverRelated Morbidity and Mortality Over time major efforts of research were focused on non-invasive predictors of liver damage in NAFLD, contrariwise only few studies were focused on baseline and prospective non-invasive prediction of liver and no liver-related events and deaths.

We have formerly shown as VAI index is able to predict fibrosis in NAFLD patients [87]. In this regard, a recent study showed that VAI was accurate for predicting endothelial dysfunction, in a little cohort of NAFLD patients [66].

This issue is very relevant because NAFLD is strongly associated with cardiovascular and systemic dysfunctions including diabetes and colorectal cancer [54, 136].

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Gastaldelli and colleagues [38] showed that in a large European cohort of nondiabetic subjects, high values of FLI are associated with increased intima media thickness, coronary heart diseases risk, and reduced insulin. Furthermore, Kozakova and colleagues showed that carotid atherosclerosis was independently associated with older age, FLI≥60, and current smoking [142]. One more French study point out as FLI is able to independently predict diabetes occurrence in a cohort of about 4000 non-diabetic subjects [143].

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Finally, another non-invasive marker potentially useful for non-invasive baseline and prospective prediction of not liver-related events and complications in NAFLD patients is sCD36 [144]. According to recent evidences, it is reasonable to speculate that sCD36 represents a new biomarker of a phenotype of insulin resistance, carotid atherosclerosis and fatty liver, even if prospective studies are needed to better understand the role of sCD36 as early biomarker in this setting.

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Relevant data were also reported in a recent metaanalysis including 221 patients from ten studies [138]. Interestingly the authors showed a rate of fibrosis progression in 37.6% of cases, demonstrating that older age and inflammation on initial biopsy were independent predictors of progression towards advanced fibrosis [138]. Anyway all the above quoted studies did not evaluate liver mortality.

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Considering the progression of liver disease severity in NAFLD patients, some studies showed that disease progression was related to both baseline severity of liver disease and metabolic factors; for example a recent study showed that reduction or increase in BMI and waist circumference were independently associated respectively with nonprogressive or progressive disease activity and fibrosis [137].

Another non-invasive tool that seems able to predict no liver-related dysfunctions and death is FLI, a noninvasive marker of liver steatosis and its severity.

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Conversely, Bhala and colleagues recently investigated the long-term morbidity and mortality of 247 patients with NAFLD with advanced fibrosis or cirrhosis in a prospective, multicentric study [139]. The authors showed that platelet count, stage 4 fibrosis, lower platelet count, lower serum cholesterol and alanine aminotransferase (ALT) levels were associated with liver-related complications, while aspartate aminotransferase/ALT ratio >1 and older age were associated with overall mortality, and higher serum bilirubin levels and stage 4 fibrosis were associated with liver-related mortality [139]. These data could suggest as the severity of baseline fibrosis holds a relevant role in predicting events and mortality in NAFLD patients. Confirming this, another recent study performed by Younossi and colleagues showed a 30.6% overall mortality and a 8.6% liver-related mortality in a cohort of 209 NAFLD patients with a median follow up of approximately 12 years, pointing out the role of severe fibrosis as the only histological feature independently associated with liver-related deaths [129]. All these data should encourage to assess the performance of non-invasive fibrosis tools in prediction of events and mortality in NAFLD patients. However, only few data, and mostly not obtained in NAFLD

CONCLUSION It is likely that the prevalence of NAFLD will increase in the next future due to the epidemic of obesity. This will represent a relevant problem for national health institutes worldwide, due to the higher liver and not liver-related morbidity and mortality observed in these patients, especially in those with NASH and/or advanced fibrosis. Consequently, many efforts have been performed in order to assess the performance of non-invasive tools in the evaluation of liver disease severity, with the aim to avoid unnecessary liver biopsies. Up to date several non-invasive scores for NAFLD diagnosis have been developed. Nevertheless, US remains the more common instrument used for the diagnosis of NAFLD in clinical practice, even if this technique is affected by low accuracy for lower grades of steatosis with a low inter and intra-observer agreement.


[1] [2]

[3]

Some of these scores were also evaluated both in the prediction of cardiovascular morbidity, liver and not liver-related events and death, with promising results.

[4] [5]

On the basis of this background, many efforts should be targeted in the future, in order to improve the diagnostic accuracy of non-invasive tools for the baseline and prospective evaluation of liver and systemic damage in NAFLD patients.

[6]

[7]

Probably in the future it will be necessary a further assessment of these score by gender analysis, considering separately males and females.

[10]

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[11]

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In conclusion, it will be very useful in the next future, generating as accurate as simple algorithms, to be validated on a large scale for baseline and prospective prediction of liver and systemic damage, with the aim to simplify the diagnosis of NAFLD.

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[12]

[13]

[14]

[15]

[16]

[17]

AUTHOR CONTRIBUTIONS All authors contributed to writing the paper and had full control over preparation of manuscript; all authors approved the final draft manuscript.

CONFLICT OF INTEREST

[18]

[19]

The authors confirm that this article content has no conflicts of interest. [20]

ACKNOWLEDGEMENTS Declared none.

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[9]

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Specifically we think it would be very useful to assess and to take in consideration the follows: the genetic background, the ARFI when available, the dietary intake with particular attention to fructose consumption [149], the presence of ectopic fat deposition and dorsocervical fat [150], and the serum levels of uric acid and vitamin D [151].

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In the next future, as well as a gender-specific diagnostic approach in NAFLD patients, combined strategies that consider both old and new non-invasive tools will also be necessary.

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REFERENCES

Concerning the staging of liver fibrosis, liver stiffness measurement by fibroscan and NFS represent two easy and simple tests, mostly validated in NAFLD, and able to accurately identify patients with severe liver fibrosis. However, these tools less accurate for the diagnosis of lower stages of fibrosis, with a certain degree of uncertainty in different populations.

This is supported by evidence of an increasing prevalence of NAFLD according to age in females but not in males [145], and by evidence of the protective role of estrogens on liver damage [146]. In addition data on a cohort of hepatitis C infected females shows as the change in hormonal balance due to fertile and menopausal status are followed by increase in proinflammatory cytokines and by a more rapid liver damage progression [147, 148].


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