Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis

1006 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology Original research Diagnostic accuracy of noninvasive tests for advanced fibrosis in patients with NAFLD: an individual patient datametaanalysis Ferenc Emil Mózes ,1 Jenny A Lee,2 Emmanuel Anandraj Selvaraj,1,3,4 Arjun Narayan Ajmer Jayaswal,1 Michael Trauner ,5 Jerome Boursier ,6,7 Céline Fournier,8 Katharina Staufer,5,9,10 Rudolf E Stauber,11 Elisabetta Bugianesi ,12 Ramy Younes ,13 Silvia Gaia,12 Monica Lupșor- Platon,14 Salvatore Petta ,15 Toshihide Shima,16 Takeshi Okanoue,16 Sanjiv Mahadeva ,17 Wah- Kheong Chan,17 Peter J Eddowes,18,19 Gideon M Hirschfield,20 Philip Noel Newsome ,18,21,22 Vincent Wai- Sun Wong ,23 Victor de Ledinghen,24,25 Jiangao Fan,26 Feng Shen,26 Jeremy F Cobbold,3,4 Yoshio Sumida,27 Akira Okajima,28 Jörn M Schattenberg,29 Christian Labenz ,29 Won Kim,30 Myoung Seok Lee,31 Johannes Wiegand ,32 Thomas Karlas ,32 Yusuf Yılmaz ,33,34 Guruprasad Padur Aithal ,19,35 Naaventhan Palaniyappan,19,35 Christophe Cassinotto ,36 Sandeep Aggarwal,37 Harshit Garg,37 Geraldine J Ooi ,38 Atsushi Nakajima ,39 Masato Yoneda,39 Marianne Ziol,40 Nathalie Barget,41 Andreas Geier ,42 Theresa Tuthill,43 M. Julia Brosnan,43 Quentin Mark Anstee,44 Stefan Neubauer,1 Stephen A. Harrison,1 Patrick M Bossuyt,2 Michael Pavlides ,1,3,4 the LITMUS Investigators To cite: MózesFE, LeeJA, SelvarajEA, etal. Gut 2022;71:1006–1019. ►Additional supplemental material is published online only. To view, please visit the journal online (http:// dx. doi. org/ 10. 1136/ gutjnl- 2021- 324243). For numbered affiliations see end of article. Correspondence to Dr Michael Pavlides, Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, Oxfordshire, UK; michael. pavlides@ cardiov. ox. ac. uk Received 28 January 2021 Revised 23 April 2021 Accepted 29 April 2021 Published Online First 17May2021 © Author(s) (or their employer(s)) 2022. Reuse permitted under CC BYNC. No commercial reuse. See rights and permissions. Published by BMJ. ABSTRACT Objective Liver biopsy is still needed for fibrosis staging in many patients with nonalcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSMVCTE), Fibrosis- 4 Index (FIB- 4) and NAFLD (nonalcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. Design Individual patient data metaanalysis of studies evaluating LSMVCTE against liver histology was conducted. FIB- 4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. Results Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSMVCTE, FIB- 4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB- 4 cutoffs (<1.3; ≥2.67) followed by LSMVCTE cutoffs (<8.0; ≥10.0 kPa) to rulein or ruleout advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63–68) and 86% (84–87) with 33% needing a biopsy to establish a final diagnosis. FIB- 4 cutoffs (<1.3; ≥3.48) followed by LSM cutoffs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37–39) and specificity of 90% (89–91) with 19% needing biopsy. Conclusion Sequential combinations of markers with a lower cutoff to ruleout advanced fibrosis and a higher cutoff to rulein cirrhosis can reduce the need for liver biopsies. INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome with high prevalence worldwide.1 Most patients remain asymptomatic for long periods of time (years/decades) with slowly progressive disease, but Significance of this study What is already known on this subject? ►Patients with nonalcoholic fatty liver disease (NAFLD) and advanced fibrosis (F3–4) are at risk of disease progression and adverse clinical outcomes. ►Noninvasive tests with predefined cutoffs are used as screening biomarkers to identify those at low risk of advanced fibrosis who can be safely managed in primary care. ►Liver biopsy is still needed in secondary care to further identify those with cirrhosis who would benefit from surveillance for hepatocellular cancer and screening for oesophageal varices. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1007 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology a minority2 progress to cirrhosis, liver failure and hepatocellular carcinoma (HCC). NAFLD comprises several histological features ranging from simple steatosis to steatosis with lobular inflammation and ballooned hepatocytes (steatohepatitis), both of which can be accompanied by varying degrees of fibrosis. The currently accepted reference standard for diagnosing NAFLD is liver biopsy as its diagnostic features are based on histology.3 Liver biopsy, however, is invasive and carries a risk of complications,4 is limited by sampling variability5 and high observer dependent variability in pathological reporting.6 7 NAFLD is often diagnosed after incidental findings of elevated liver transaminases on blood tests, or liver steatosis or cirrhosis on imaging. One challenge clinicians face is to identify which of these patients are at high risk of progression or clinical outcomes, as they would benefit from specialist followup. There is now substantial evidence showing that those with at least advanced fibrosis (F3–4) are at higher risk of liverrelated events in later life.8–10 A large body of evidence also exists on how noninvasive tests (NITs) could be used to riskstratify patients for the presence of advanced fibrosis. These approaches usually involve sequential application of two NITs, with the first tier of a simple, inexpensive, serumbased test performed in the community (eg, Fibrosis- 4 Index (FIB- 4) or NAFLD Fibrosis Score (NFS)), followed by a second tier of liver stiffness measurement (LSM) (eg, vibration controlled transient elastography: VCTE), or a proprietary serumbased test (eg, enhanced liver fibrosis test; ELF). A lower and an upper threshold are usually used in each tier of testing to rule out (those with a NIT result less than the lower threshold) or rule in (those with a NIT result more than the upper threshold) patients at high risk of advanced fibrosis. Patients with indeterminate results in both tiers of testing would need a liver biopsy for risk stratification. The main value of these approaches lies in their high negative predictive value to rule out patients with low risk of advanced fibrosis who can be safely managed in primary care. Despite the increasing evidence to support these approaches, some aspects of their application require further clarifications. First, there is no consensus on which NIT thresholds to use for this purpose. For example, FIB- 4 upper cutoffs of 3.2511 and 2.6712 have been described, while other investigators omit the FIB- 4 upper cutoff altogether.13 There is also some uncertainty about the performance of NITs in specific patient subgroups, such as those with diabetes or obesity. Furthermore, for patients who are ruled in as being at high risk of advanced fibrosis (F3–4), liver biopsy is often needed to identify those with cirrhosis who would need surveillance for HCC.14 Developing approaches that can minimise the need for liver biopsy in secondary care is therefore an area of unmet need. To address these problems, we conducted an individual patient data metaanalysis (IPDMA) with three main aims: (1) to evaluate the performance of LSMVCTE and compare it to the performance of FIB- 4 and NFS as screening tests to rule out advanced fibrosis; (2) to evaluate NIT combination strategies to minimise the number of cases that would need a liver biopsy in secondary care; (3) to explore factors that influence diagnostic accuracy. METHODS This IPDMA was reported in accordance with the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta- Analyses- IPD Statement15 and was registered as PROSPERO CRD42019157661. Criteria for considering studies for the IPD meta-analysis Patients Studies reporting data on adults (≥18 years) with NAFLD and paired liver histology and LSMVCTE were eligible. When studies reported study groups of participants with unselected aetiologies, only IPD of those with NAFLD were sought. Index tests The index test of main interest was LSMVCTE performed with FibroScan (Echosens, France). Results for serumbased biomarkers NSF,16 FIB- 4,17 aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio18 and AST- to- platelet ratio index (APRI)19) were also computed where data were available. Online supplemental table 1 summarises the definition of NITs considered in this IPDMA. Universally accepted cutoffs for diagnosing different groups of fibrosis stages do not exist (several suggested cutoffs are presented in online supplemental table 2). For LSMVCTE, <7.9 kPa and ≥9.6 kPa are the most used for respectively ruling out and in, advanced fibrosis.20 Reference standard Only studies reporting histological classification of liver fibrosis based on the nonalcoholic steatohepatitis Clinical Research Network (NASH CRN) staging system were considered.21 Target conditions Advanced fibrosis (F3–4) and cirrhosis (F4) were the target conditions of interest. To fulfil the aims of the study, cutoffs Significance of this study What are the new findings? ►Existing noninvasive tests cutoffs are validated for their use as screening biomarkers to rule out advanced fibrosis in a study group of 5735 patients. ►The sequential combination of Fibrosis- 4 Index (FIB- 4) (<1.3; ≥2.67) and liver stiffness measurement by vibration controlled transient elastography (LSMVCTE) (<8.0 kPa; ≥10.0 kPa) which is increasingly used in routine practice has a false negative rate of 9% for advanced fibrosis. ►The diagnostic performance of LSMVCTE for advanced fibrosis is influenced by biopsy quality, body mass index and presence of type 2 diabetes. ►An algorithm combining FIB- 4 and LSMVCTE sequentially with lower cutoffs to rule out advanced fibrosis (FIB- 4 <1.3; LSMVCTE <8.0 kPa) and with upper cutoffs to rulein and positively diagnose cirrhosis without the need for liver biopsy with specificity of 95% (FIB- 4 ≥3.48; LSMVCTE ≥20.0 kPa) or 98% (FIB- 4 ≥4.63; LSMVCTE ≥28.0 kPa) can reduce the need for liver biopsies from 33% to 19% or 24%, respectively. How might it impact on clinical practice in the foreseeable future? ►The noninvasive test cutoffs for the diagnosis of cirrhosis can be incorporated into clinical practice as they have been validated in a large group of patients. ►Application of these cutoffs can lead to a decrease in the need for liver biopsies in secondary care. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1008 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology were selected to rule out or rule in advanced fibrosis, and to rule out advanced fibrosis or rule in cirrhosis. Study design All study designs were considered if they were reporting on patients with NAFLD undergoing both liver biopsy and LSMVCTE within 6 months. No language restrictions were applied. Establishing collaborations Authors of eligible studies were contacted by email and reminders were sent if a response was not received within 2 weeks. Only data from studies that received ethical approval were used. Additional ethical approval was not sought for the metaanalysis as only anonymised data were provided. Data verification Range checks of measurement values provided for individual patients were carried out and authors were asked to provide clarifications where necessary. Missing data were queried until received or confirmed as unavailable. Missing data were handled in the analysis by pairwise deletion. LSMVCTE with median stiffness ≥7.1 kPa and IQR- to- median LSM ratio >30% were considered unreliable.22 These were included in the main analysis and were later compared in a subgroup analysis to reliable measurements, to assess whether they can be reliably used to diagnose advanced fibrosis. Authors were provided with a template table of required data (online supplemental table 3) and were asked to deduplicate data were possible. We also checked for duplicate entries and where identified these were removed. Data analysis Quality and bias assessment The quality of studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS- 2).23 IPD meta-analysis The original data sets were merged, a study identification variable was added, and descriptive statistical analysis of the data sets was conducted. Dichotomous variables are displayed as percentages. Continuous variables are reported as means with SD, or medians with IQRs according to the distribution of the data. Analyses were done per protocol, as we did not have information on failed LSMVCTE. To express the diagnostic performance of NITs, nonparametric, empirical receiver operating characteristic (ROC) curves were constructed for the target conditions of interest. Diagnostic performance was expressed as the area under the ROC curve (AUROC) with 95% CI, based on De Long’s method. AUROCs were compared using De Long’s test statistic. Thresholds to maximise the Youden index (ie, sensitivity+specificity−1), for 90% sensitivity, and for 90% specificity were reported. The diagnostic performance of previously published cutoffs was also evaluated. Sequential combinations of serum biomarkers and LSMVCTE were evaluated, by computing sensitivity, specificity and proportions of misclassified and indeterminate patients. Positive and negative predictive values (PPV and NPV) were estimated for prevalences within the range of those reported in the original studies. The number of false positive and false negative results for 100 theoretical cases was also reported. The main analysis was conducted to maximise data for each NIT. For a valid comparison of the performance of NITs, a separate analysis was conducted in the subgroup of patients where all three of VCTE, FIB- 4 and NFS were available in each participant. To fulfil the aim of developing testing strategies that reduce the number of patients in need of a liver biopsy, lower cutoffs for ruling out advanced fibrosis and upper cutoffs for ruling in cirrhosis were used. The rationale for this approach is illustrated in online supplemental figure 1. The upper cutoffs for identifying cirrhosis were chosen at 95% and 98% specificity in a derivation set and tested in a validation set. Derivation and validation sets were obtained by random sampling from the IPD study group in a 3:2 ratio. These upper cutoffs were combined with lower cutoffs from the literature for ruling out advanced fibrosis and the algorithm was tested in the whole IPD study group. For ease of reference, we also examined the cutoffs of 8 kPa and 10 kPa (corresponding to the most common VCTE cutoffs in the literature of 7.9 kPa and 9.6 kPa rounded to the nearest integer) and also rounded our cirrhosis cutoffs to the nearest integer to facilitate application in clinical practice. Only testpositive and testnegative patients were included in the calculation of diagnostic performance indices, and patients in the indeterminate group were excluded from calculations. Subgroup analysis was performed according to biopsy length (<20 mm, ≥20 mm), number of portal tracts in biopsy samples (<11, ≥11), biopsy quality (intermediate: 10 mm ≤length <20 mm; high: length ≥20 mm and ≥11 tracts), age (four quartiles), sex, body mass index (BMI; BMI <25 kg/m2, 25 kg/m2≤ BMI <30 kg/m2, BMI ≥30 kg/m2), presence of type 2 diabetes mellitus (T2DM), continent of provenance (Europe, Asia), probes used (M, XL), reliability criteria for LSMVCTE (reliable (median LSM <7.1 kPa or median LSM ≥7.1 kPa and IQR/median LSM <0.30) vs unreliable (median LSM ≥7.1 kPa and IQR/median LSM ≥0.30)22 ; reliable (IQR/median LSM <0.30) vs unreliable (IQR/median LSM ≥0.30)), and aminotransferase levels (ALT or AST<40, 40≤ALT or AST<100, ALT or AST≥100; ALT<40 and AST<40, ALT≥40 or AST≥40. All statistical analyses were performed using R (V.1.2.1335, R Foundation for Statistical Computing, Vienna, Austria) with the pROC package24 25; 95% CIs were calculated using 500 stratified bootstrap replicates using the boot package.26 27 VCTE probe types The analysis to account for probe type is described in the online supplemental materials. Patient and public involvement Patients and the public were not involved in the conduct of this study as there was no direct patient participation in the study. RESULTS Search process and data collection Ten thousand three hundred ninetytwo articles were identified in a search performed for a larger systematic review evaluating the diagnostic performance of LSMVCTE and other index tests for the staging of fibrosis and diagnosis of NASH in adult patients with NAFLD. After removing duplicates, and screening titles, abstracts, and full texts, 59 studies examining VCTE were identified. The authors of 37 studies shared useable data (figure 1). Authors of more than one study supplied data in a single dataset and, overall, we received 30 data sets including data from 6571 patients. After removing duplicates (n=628) and patients with copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1009 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology missing biopsy (n=14) or LSMVCTE (n=194) data, the final dataset consisted of 5735 unique patients. Study and population characteristics The characteristics of the 30 data sets are summarised in table 1. Studies were conducted in Europe (67%), Asia (40%) and Australia (3%). Data availability is shown in online supplemental table 3. FIB- 4 and NFS were determined in 5393 (94%) and 3248 (57%) cases, respectively. Median age was 54 years, 2570 (45%) patients were women, 33% had diabetes and 43% had BMI ≥30 kg/m2. Overall, 30% had advanced fibrosis and 11% had cirrhosis. Details of the IPD study group are included in table 2, and online supplemental tables 4 and 5. Study quality The methodological quality of the studies assessed with the QUADAS- 2 tool is summarised in online supplemental figures 2 and 3. Only one study had low risk of bias or low applicability concerns in all QUADAS- 2 domains.28 The flow and timing domain were judged to have high risk or unclear risk of bias in 65% of studies, as these either excluded technical failures from their final diagnostic performance analysis or did not report them. Validating the diagnostic performance of LSM by VCTE and serum-based tests for detecting advanced fibrosis LSMVCTE, FIB- 4, NFS, APRI and AST/ALT had corresponding AUROCs of 0.85, 0.76, 0.73, 0.70, 0.64 for identifying advanced fibrosis (table 3), and 0.90, 0.80, 0.78, 0.72, 0.69 for the identification of cirrhosis (online supplemental table 6). LSMVCTE performed significantly better (p<10−15) in detecting both advanced fibrosis and cirrhosis than all serumbased tests. This relationship was preserved when performing a head- to- head comparison of LSMVCTE, FIB- 4 and NFS in the same group of patients (online supplemental tables 7 and 8). When considering cutoffs from the literature, we evaluated lower and higher cutoffs separately. For any given test, as would be expected, low thresholds yielded higher sensitivity and high thresholds were associated with higher specificity (online supplemental table 9). Indicative PPV and NPV are also provided for the range of prevalences (5%–50%) reported in the primary studies (online supplemental tables 10–14). APRI and AST/ALT ratio had only modest diagnostic performance for advanced fibrosis (AUROC ≤0.70, table 3), and were therefore not considered further. None of the thresholds regarded in isolation resulted in both a high sensitivity (≥80%) and high specificity (≥80%) (figure 2, table 3, online supplemental tables 9 and 15, and online supplemental figure 4). Therefore, we explored the use of a lower and an upper cutoff. LSMVCTE literature cutoffs performed well in only two cases (<7.1 kPa and ≥14.1 kPa: 83% sensitivity, 90% specificity; and <7.9 kPa and ≥9.6 kPa: 84% sensitivity, 78% specificity), while for other LSMVCTE, NFS and FIB- 4 thresholds a high specificity was observed (FIB- 4: 91% for <1.3 and ≥2.67, 95% for <1.3, ≥3.25) but sensitivity was <60% (table 4). In addition, the proportion of indeterminate cases was >30% for serumbased NITs. Threshold pairs derived from the IPD study group did not reduce the proportion of misclassified and indeterminate patients seen with literaturebased threshold pairs (table 4). We further evaluated the performance of LSMVCTE, FIB- 4 and NFS to diagnose advanced fibrosis in sequential combinations of serumbased NITs and LSMVCTE. When selecting threshold combinations for FIB- 4 and NFS available in the literature (<1.3 & ≥2.67,<1.3 & ≥3.25 for FIB- 4;<−1.455 & ≥0.676 for NFS) and pairing them with the best threshold pair for LSMVCTE (<7.9 kPa & ≥9.6 kPa, identified as the one with highest sensitivity and lowest indeterminate proportion), the proportion of patients in the indeterminate group was 5%. While both the FIB- 4+LSM VCTE and NFS+LSM VCTE sequential combinations had specificity >80%, their sensitivity was ≤80% (table 5). A better sensitivity was reached by using thresholds derived from the IPD study group (<0.88 & ≥2.31 for FIB- 4;<−2.55 & ≥0.28 for NFS), but the proportion of indeterminate cases was near 20% in those cases and the proportions of patients needing LSMVCTE was also larger than when using literature cutoffs (table 5). Algorithms to minimise the need for liver biopsy In the derivation set, the cutoffs for 95% and 98% specificity for the diagnosis of cirrhosis were respectively 20.4 kPa and 27.6 kPa for LSMVCTE, 3.48 and 4.63 for FIB- 4 and 1.01 and 1.57 for NFS. These cutoffs performed similarly in the validation set (online supplemental tables 16 and 17). Figure 1 Preferred Reporting Items for Systematic Reviews and Meta- Analyses flow chart illustrating the identification and selection process for studies finally included in this individual patient data metaanalysis. IPD, individual patient data; LSM, liver stiffness measurement; VCTE, vibration controlled transient elastography. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1010 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology Algorithms combining FIB- 4 (lower cutoff of 1.3 as described in the literature and upper cutoffs of 3.48 and 4.63 as described above) and LSM by VCTE (lower cutoff rounded to 8.0 kPa and upper cutoffs rounded to 20.0 kPa and 28.0 kPa, as described above) were then compared with the traditional way of applying these tests, also with rounded cutoffs for LSM by VCTE (8 kPa and 10 kPa) (figure 3). This approach increased the number of patients requiring a LSM Table 1 Details of individual patient data included in this metaanalysis Data set ID Country Study design Number of participants (n) Age (year) BMI (kg/m2) WC (cm) M/F Recruitment interval Hardware used Probe used Agrawal et al49 UK MC, P, CS 25 47.8 (19–70) 27.7 (15.8–35.7) 95.4 (39–120) 18/7 2009–2012 – M Aykut et al50 Turkey SC, P, CS 88 46.0 (24–62) 30.3 (18.3–41.8) 101.5 (70–143) 50/38 – FibroScan 502 Touch M Boursier et al51–53 France MC, P, CS 1063 56.1 (18–83) 31.6 (16.7–55.5) 108.3 (58–174) 613/450 – – M or XL Cassinotto et al 54 France SC, P, CS 61 55.9 (22–81) 30.1 (16.7–46.6) 103.6 (72–125) 40/21 2010–2012 – M and XL Cassinotto et al 55 France MC, P, CS 286 56.6 (18–80) 32.2 (20.3–57.4) 109.8 (68–168) 171/115 2011–2015 – M and XL Chan et al56 Malaysia SC, P, CS 146 50.4 (18–73) 29.4 (6.9–41.2) 98.3 (79–127) 80/66 2012–2013 FibroScan 502 Touch M Chan et al57 Malaysia, Hong Kong MC, P, CC 153 54.0 (24–76) 29.9 (20.1–44.8) 98.4 (69–141) 68/85 – FibroScan 502 Touch M and XL Eddowes et al31 58 UK MC, P, CS 358 53.3 (19–77) 34.2 (19.5–53.2) 117.2 (65–158) 206/152 – – M or XL Eddowes et al59 UK MC, P, CS 50 50.2 (18–73) 33.6 (23.6–47.8) 109.4 (89–132) 28/22 2014–2015 – M or XL Gaia et al60 Italy SC, P, CS 68 46.8 (28–65) 28.0 (21.2–40.2) – 48/20 2007–2009 – M Garg et al61 India SC, P, CS 76 38.2 (20–65) 45.2 (32.3–73.8) – 16/60 2014–2016 FibroScan 502 Touch XL Karlas et al62 Germany SC, P, CS 41 45.7 (28–64) 47.7 (33.7–60.1) – 13/28 – FibroScan 502 XL Labenz et al63 Germany SC, P, CS 126 47.4 (20–73) 31.6 (23.2–50.4) – 72/54 – FibroScan 402 M or XL Lee et al64 Korea SC, P, CS 94 55.5 (19–82) 27.2 (19.1–36.3) – 41/53 2014–2015 – M or XL Lupsor et al65 Romania SC, P, CS 72 42.4 (20–69) 29.7 (21.0–41.5) 102.4 (60–124) 51/21 2007–2009 – M Mahadeva et al66 Malaysia SC, P, CS 131 49.9 (23–73) 28.7 (18.6–43.1) 93.5 (43–128) 66/65 2009–2010 – M Okajima et al67 Japan SC, P, CS 173 56.3 (18–81) 27.2 (16.5–40.3) – 84/89 2013–2015 – M Ooi et al68 Australia MC, P, CS 82 44.5 (18–67) 46.2 (29.1–74.0) 136.5 (101–192) 23/59 2015–2016 – M or XL Pavlides et al69 UK SC, P, CS 70 53.5 (25–77) 34.5 (23.0–57.3) 112.5 (80–149) 42/28 2011–2015 – M or XL Petta et al70 71 Italy MC, P&R, CS 234 45.5 (15–78) 28.2 (15.7–40.7) 99.4 (69–126) 169/65 2008–2013 – M Petta et al28 France, Hong Kong, Italy MC, P, CS 260 54.6 (15–87) 29.4 (16.5–46.6) 100.9 (74–148) 122/138 – – M Petta et al72 Italy MC, P, CS 474 45.5 (19–77) 29.2 (15.2–49.5) 99.6 (47–164) 275/199 – – M Seki et al73 Japan SC, P, CS 181 57.7 (16–82) 27.1 (16.9–38.1) 95.1 (71–117) 91/90 2013–2015 – M Shen et al74 China MC, P, CS 101 59.0 (16–67) 27.0 (20.1–37.3) 92.9 (75–120) 74/27 2012–2014 FibroScan 502 M Staufer75 Austria MC, P, CS 186 49.6 (19–83) 32.5 (19.0–56.9) – 106/80 2011–2016 FibroScan 502 Touch M or XL Wong et al76–79 Hong Kong, France MC, P, CS 464 53.8 (20–83) 30.5 (17.3–48.0) 102.0 (71–148) 201/263 2009–2017 – M and XL Wong et al20 Hong Kong, France MC, P, CS 273 51.6 (21–77) 28.8 (16.5–54.0) 96.2 (65–144) 147/126 2003–2009 – M Yoneda et al80 Japan MC, P, CS 97 52.1 (19–76) 26.5 (17.9–38.5) – 41/56 <2008 – M Younes et al81 Italy MC, P, CS 289 44.8 (15–78) 28.8 (17.5–41.7) 98.9 (47–128) 199/90 – – M Ziol et al82 France SC, P, CS 13 49.3 (39–60) 29.4 (23.8–34.6) – 10/3 2003–2005 – – –, Data not available; BMI, body mass index; CC, casecontrol; CS, crosssectional; F, females; M, males; MC, multicentre; P, prospective; R, retrospective; SC, singlecentre; WC, waist circumference. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1011 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology (from 34% to 40% and 44%) but decreased the number of patients needing liver biopsy (from 33% to 19% and 24% when using the 95% and 98% specificity cutoffs, respectively) (online supplemental table 18 and figure 3). Subgroup and sensitivity analyses In subgroup analysis for the diagnosis of advanced fibrosis (online supplemental table 19), NITs performed better in patients with lower BMI (AUROCs LSMVCTE: 0.91, p<0.005; FIB- 4: 0.81, p<0.001; NFS: 0.76, p<0.025), without T2DM (LSMVCTE: 0.87, p<10−6; FIB- 4: 0.77, p<0.01), and with biopsies shorter than 20 mm (LSMVCTE: 0.87, p<0.005; FIB- 4: 0.80, p<0.001; NFS: 0.79, p<0.05), or with fewer than 11 portal tracts (LSMVCTE: 0.86, p=0.01; FIB- 4: 0.79, p=0.04; NFS: 0.78, p<0.005). Diagnostic performance was also lower in patients in the youngest age quartile (<43 years, AUROC: 0.58, p<0.001) and in women (AUROC: 0.71, p=0.03) for NFS, while continent of provenance did not have a significant effect for any NITs. In patients with normal levels of ALT (ALT<40) FIB- 4 performed worse (AUROC: 0.73) than in patients with ALT≥40 and ALT<100 (AUROC: 0.77, p<0.01). NFS Table 2 Demographic details of the entire cohort, and patients without (F0–2) and with (F3–4) advanced fibrosis Entire cohort (N=5735) F0–2 (N=4013) F3–4 (N=1722) Females (%) 45 43 48 BMI ≥30 kg/m2 (%) 43 45 53 Waist circumference (cm) 103 (15) 102 (15) 106 (14) Diabetes (%) 33 30 58 Age (years)* 54 (19) 50 (19) 59 (14) BMI (kg/m2)* 30 (7) 29 (8) 30 (7) Biopsy data Steatosis S0/S1/S2/S3 (%) 3/35/36/26 3/36/36/25 2/32/38/28 Ballooning B0/B1/B2 (%) 24/47/29 30/49/21 10/45/45 Inflammation I0/I1/I2/I3 (%) 13/60/24/3 17/62/20/1 5/55/34/6 NAS score† 4 (2) 4 (2) 5 (1) NASH (%) 50 43 67 Liver function tests ALT (IU/L)* 55 (48) 53 (48) 60 (48) AST (IU/L)* 40 (30) 36 (25) 50 (34) Platelets (×109/L)† 230 (72) 241 (67) 205 (75) Albumin (g/L)† 43 (9) 43 (7) 43 (13) GGT (IU/L)* 69 (87) 62 (78) 87 (102) NITs LSM (kPa)* 10.7 (6.1) 6.7 (3.5) 13.3 (12.0) FIB- 4* 1.7 (1.2) 1.1 (0.9) 1.9 (1.7) NFS† −1.5 (1.7) −1.9 (1.6) −0.6 (1.8) APRI* 0.6 (0.4) 0.4 (0.3) 0.6 (0.6) AST/ALT* 0.8 (0.4) 0.7 (0.4) 0.8 (0.5) *Data are reported as median (IQR). †Data are reported as mean (SD). ALT, alanine aminotransferase; APRI, AST- to- platelet ratio index; AST, aspartate aminotransferase; BMI, body mass index ; FIB- 4, Fibrosis- 4 Index; GGT, gammaglutamyltransferase; LSM, liver stiffness measurement; NAS, NAFLD activity score; NASH, nonalcoholic steatohepatitis; NFS, NAFLD (nonalcoholic fatty liver disease) Fibrosis Score; NIT, noninvasive test. Table 3 Diagnostic performance of noninvasive tests for advanced fibrosis (F3–F4) LSM by VCTE (n=5489) FIB- 4 (n=5393) NFS (n=3248) APRI (n=5477) AST/ALT (n=5434) Advanced fibrosis, % 30 30 29 30 30 AUROC 0.85 (0.84–0.86) 0.76 (0.74–0.77) 0.73 (0.71–0.75) 0.70 (0.69–0.72) 0.64 (0.62–0.65) YI 90% Se 90% Sp YI 90% Se 90% Sp YI 90% Se 90% Sp YI 90% Se 90% Sp YI 90% Se 90% Sp Threshold 9.1 7.4 12.1 1.44 0.88 2.31 −1.39 −2.55 0.28 0.49 0.29 0.91 0.64 0.51 1.34 Sensitivity, % 77 (75–79) 90 (89–91) 55 (52–57) 69 (67–72) 90 (88–91) 38 (36–41) 75 (72–78) 90 (88–92) 29 (26–32) 67 (64–69) 90 (89–92) 32 (30–34) 75 (73–77) 90 (87–91) 16 (14–18) Specificity, % 78 (76–79) 60 (59–61) 90 (89–91) 70 (69–72) 39 (37–40) 90 (89–91) 63 (61–65) 36 (33–37) 90 (89–91) 63 (62–65) 29 (28–30) 90 (89–91) 47 (45–48) 25 (23–26) 90 (89–91) Misclassified, % 22 (22–23) 31 (31–32) 21 (20–21) 30 (30–31) 46 (46–47) 26 (25–26) 34 (34–36) 48 (49–50) 28 (28–29) 36 (36–37) 53 (53–54) 27 (27–28) 45 (45–46) 56 (56–57) 32 (32–33) For each noninvasive test thresholds were selected according to Youden’s index (YI), and fixed at 90% sensitivity (90% Se) and 90% specificity (90% Sp). 95% CIs were estimated with 500 bootstrap replicates. ALT, alanine aminotransferase; APRI, AST- to- platelet ratio index; AST, aspartate aminotransferase; FIB- 4, Fibrosis- 4 Index; LSM, liver stiffness measurement; NFS, NAFLD (nonalcoholic fatty liver disease) Fibrosis Score; VCTE, vibration controlled transient elastography. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1012 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology performed better in patients with AST<40 (AUROC: 0.76), than in patients with AST≥100 (AUROC: 0.65, p<0.01). FIB- 4 performed better in patients with at least one abnormal aminotransferase measurement (AUROC: 0.72, p=0.014). For cirrhosis, the trends were similar, except that for the diagnosis of cirrhosis, LSM by VCTE performed better in the youngest age group (AUROC: 0.97, p<10−4) and NIT diagnostic performance was independent of aminotransferase levels (online supplemental table 20). The diagnostic performance of LSMVCTE was significantly lower in patients with unreliable LSMs (p<10−8; both for advanced fibrosis and cirrhosis) when applying the Boursiercriteria,22 but not when only considering IQR/median LSM <0.30. The proportion of unreliable results was 12% both in the advanced fibrosis and cirrhosis groups (online supplemental table 21). There was no difference in the diagnostic performance of LSMVCTE between the M and XL probes in the subgroup of patients who had undergone LSM by both probes (online supplemental table 22). In a sensitivity analysis of patients with LSM matched to BMI (only M probe measurements if BMI <30 kg/m2 and only XL probe measurements if BMI ≥30 kg/m2), there was no significant difference between the diagnostic performance of LSMVCTE when comparing to the entire IPD study group (online supplemental table 23). Figure 2 Distribution of sensitivities and specificities over the possible threshold ranges for liver stiffness measurement (LSM) by vibration controlled transient elastography (VCTE) (A), Fibrosis- 4 Index (FIB- 4) (B) and NAFLD (nonalcoholic fatty liver disease) Fibrosis Score (NFS) (C) when considering the diagnosis of advanced fibrosis. Insets show the distribution of cutoffs identified from the literature. Horizontal dashed lines are representing the minimum acceptable criteria for considering a test as having high sensitivity (≥80%) and high specificity (≥80%). copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1013 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology DISCUSSION Through an extensive collaboration network with authors of primary studies we were able to collect the largest dataset of its kind ever to be reported on. This includes a diverse set of study groups from Europe, Asia, and Australia, 30% of whom had advanced fibrosis. We believe that our findings are therefore relevant for patients typical of secondary care in these territories and may be applied in the development of new strategies or in the consolidation of existing practices in evaluating patients for referral to secondary care. A few studies evaluated the diagnostic performance of LSMVCTE and other NITs, but most report on fewer than 500 patients. One similarly large study reported on patients screened for inclusion in clinical trials, where the prevalence of advanced fibrosis was 71%,29 making it difficult to make generalisations about its applicability in routine practice or compare its results to ours. A smaller study with 1073 patients with NAFLD of whom 29% had advanced fibrosis30 examined the diagnostic performance of LSM by VCTE. The authors of that study reported AUC and specificity values similar to our findings, however they reported increased sensitivity. Other smaller studies reported similar prevalence of advanced fibrosis and similar AUROCs for LSMVCTE.31–34 Overall, the diagnostic performance of LSMVCTE for advanced fibrosis was good (AUROC=0.85), while that of FIB- 4 and NFS in the same group was moderate (AUROC=0.76 for FIB- 4, AUROC=0.73 for NFS). None of the studied NITs had both sufficiently high sensitivity and specificity (≥80%) when used with single cutoffs. Diagnostic performance was higher for detecting cirrhosis, as reported in previous studies.31 35 36 LSMVCTE had the highest sensitivity and specificity, both in the case of a single cutoff (9.1 kPa obtained by maximising the Youden index; 77% and 78%) and for two cutoffs (<7.4 kPa & ≥12.1 kPa; 84% and 87%). Of the LSMVCTE cutoff pairs tested,<7.1 kPa and ≥14.1 kPa, first published by Eddowes et al.,31 performed well for advanced fibrosis, with sensitivity of 83% and specificity of 90%, but with a proportion of 39% of patients ending up with an indeterminate result, similar to 41% indeterminate patients reported in the original paper.31 LSMVCTE thresholds identified in our study group (<9.1 kPa; <7.4 kPa & ≥12.1 kPa) were similar to thresholds reported in the literature (<9.9 kPa; <7.1 kPa & ≥14.1 kPa, <7.9 kPa & ≥9.6 kPa). However, thresholds for FIB- 4 (<1.44; <0.88 & ≥2.31) and NFS (<−1.39; <−2.55 & ≥0.28) defined in our IPD study group spanned a wider range than those reported in the literature (<1.3 & ≥2.67 or <1.3 & ≥3.25 for FIB- 4;<−1.455 & ≥0.676 for NFS). Our findings are in line with the existing literature suggesting that sequential combinations of NITs increase sensitivity and specificity.29 Additionally, we have found NFS+LSM VCTE and FIB- 4+LSM VCTE combinations to have similar sensitivity and specificity as recently reported by Boursier et al.37 Such combined testing strategies can reduce the number of indeterminate cases and reduce the costs associated with liver biopsies. Furthermore, we propose an approach that could minimise the need for liver biopsies further, by using upper cutoffs with 95% and 98% specificity for the identification of cirrhosis. The rationale for this approach is explained in the online supplemental discussion. When using the 95% specificity cutoff, the proportion of patients needing liver biopsy decreases from 33% to 19% (figure 3). However, in this approach, 345 of 656 patients ‘ruledin’ as having cirrhosis do not have histologically diagnosed cirrhosis. While this may seem like a high proportion Table 4 Diagnostic accuracy of pairs of cutoffs from the literature for liver stiffness measurement (LSM) by vibration controlled transient elastography (VCTE), Fibrosis- 4 Index (FIB- 4) and NAFLD (nonalcoholic fatty liver disease) Fibrosis Score (NFS) for diagnosing advanced fibrosis LSM by VCTE (n=5489) FIB- 4 (n=5393) NFS (n=3248) Advanced fibrosis, % 30 30 29 AUROC 0.85 (0.84–0.86) 0.76 (0.74–0.77) 0.73 (0.71–0.75) Source Anstee et al29 Eddowes et al31 Wong et al 76 Wong et al 20 This study Shah et al 83 McPherson et al84 This study Angulo et al 16 This study Thresholds <9.9, ≥11.4 <7.1, ≥14.1 <10, ≥15 <7.9, ≥9.6 <7.4, ≥12.1* <1.3, ≥2.67 <1.3, ≥3.25 <0.88, ≥2.31* <−1.455, ≥0.676 <−2.55, ≥0.28* Sensitivity, % 69 (67–71) 83 (80–86) 59 (57–61) 84 (82–87) 84 (81–87) 54 (52–56) 44 (42–46) 80 (76–83) 47 (44–50) 74 (70–79) Specificity, % 86 (85–88) 90 (88–92) 94 (93–96) 78 (76–80) 87 (85–88) 91 (89–92) 95 (93–96) 79 (77–81) 91 (89–93) 78 (76–81) Misclassified, % 17 (16–19) 7 (6–8) 12 (11–13) 17 (16–19) 10 (9–11) 12 (11–13) 10 (9–11) 10 (9–11) 11 (10–13) 10 (8–11) Indeterminate, % 7 (6–8) 39 (37–40) 18 (17–19) 13 (12–14) 31 30–33) 34 (33–35) 39 (37–40) 52 (50–53) 39 (37–41) 56 (54–59) *Cut- offs determined from the individual patient data study group. Lower cutoffs correspond to a lower limit of 90% sensitivity, upper cutoffs correspond to a lower limit of 90% specificity. 95% CIs were determined with 500 bootstrap replicates. AUROC, area under the receiver operating characteristic. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from 1014 MózesFE, etal. Gut 2022;71:1006–1019. doi:10.1136/gutjnl-2021-324243 Hepatology of patients with false positive results, this must be interpreted in the light of two factors. First, the limitations of liver biopsy could mean that these patients are falsely classified as not having cirrhosis histologically. Furthermore, patients without cirrhosis on histology and with high NIT values could have equivalent risks as patients with cirrhosis on histology. For example, it is known from the hepatitis C literature38 that patients without cirrhosis on liver biopsy but with a high FIB- 4 (>3.25) still had a significant risk of developing HCC after hepatitis C treatment, demonstrating that NITs can have added benefit beyond the histological diagnosis of cirrhosis alone. The rate of false positive results for cirrhosis can be decreased by choosing cutoffs with higher specificity, but this will come at the expense of doing more biopsies. Despite this encouraging result, this is an area where more information is needed, particularly longitudinal data comparing the prognostic value of LSMVCTE and other NITs against histology, and ultimately, the cost effectiveness of the various cutoffs would need to be evaluated. Surprisingly, subgroup analyses showed that the diagnostic accuracy of NITs was better in cases with poor biopsy quality. This finding is difficult to explain but a similar observation was reported previously in a large group of patients screened for clinical trials.29 The use of local biopsy reports as reference standard and the wellknown observerdependent variability of biopsy interpretation, even among expert pathologists,7 are factors that may have contributed to our finding. Spectrum bias was excluded as a source of this finding due to a nearidentical proportion of patients in both the advanced fibrosis and cirrhosis group having short biopsies (online supplemental table 5). Subgroup analysis showed better diagnostic performance of NITs in patients with lower BMI,39 40 and patients without diabetes, in keeping with other studies.41 42 This effect is likely to be primarily driven by BMI as there is thought to be a causal association between BMI and T2DM. NIT performance was impacted by age, with all NITs performing worse in the younger quartile of our study group for advanced fibrosis, but the trend was reversed for cirrhosis where NITs performed better in those younger than 43 years of age. The age dependence of FIB- 4 and NFS is expected, as age is one of the parameters included in the algorithms, and has indeed been previously described.13 43 It is, however, difficult to explain why performance of NITs is better in the younger age group for the diagnosis of cirrhosis. Our study has several strengths, including the large size of the IPD study group and composition with prevalence of advanced fibrosis of 30%, which makes it relevant to routine practice. Furthermore, the proportion of unreliable VCTE measurements in our study was 12%, in keeping with the literature.22 However, we acknowledge some limitations. We did not have any data from the USA and very few studies from Australia, so the results could not be globally applicable, due to differences in BMI across study populations. In addition, due to the nature of our study, we had to use the locally provided histology results possibly introducing bias. Furthermore, we covered a large chronological period, during which LSMVCTE application underwent significant changes, initially with the introduction of the XL probe, followed by the advice to measure skin- to- capsule distance (SCD) and the introduction of the Automatic Probe Selection tool. There was therefore some heterogeneity in the performance of LSMVCTE, with early studies using only the M probe to assess all patients, while only a subset of studies assessed SCD to guide probe selection. Furthermore, one third of the included studies was carried out in France, as the technology used for LSM by VCTE originates from there. Lastly, our data confirm that LSMVCTE had superior accuracy to serumbased tests, and this is independent of probe type, sex, ALT, AST, and participants’ continent of origin. There was, however, some dependence on the presence of T2DM, BMI and for the detection of cirrhosis, and we did not check for subgroupspecific cutoffs, but these should be explored in future studies. Our study examined some of the most widely available NITs. While it cannot be considered exhaustive, it can be regarded as the benchmark against which newer NITs can be tested. This is particularly important as new tests are continuously being developed (FibroTest- FibroSURE, ActiTest,44 ELF45). Furthermore, newer tests are also needed for patients with ‘at risk’ NASH (NASH+F2–3) who would be candidates for clinical trials or treatments, once approved therapies become available (FAST score,46 NIS4,47 cTAG48). In conclusion, our study provides further validation of the use of sequential combination of FIB- 4 and LSMVCTE to rule out patients with NAFLD and advanced fibrosis who can be managed in primary care. We have shown how the use of upper cutoffs to rule in cirrhosis in combination with lower cutoffs to Table 5 Diagnostic performance of combinations of NAFLD (nonalcoholic fatty liver disease) Fibrosis Score (NFS) and liver stiffness measurement (LSM) by vibration controlled transient elastography (VCTE), and Fibrosis- 4 Index (FIB- 4) and LSM by VCTE tests to diagnose patients with advanced fibrosis FIB- 4 & LSM by VCTE (n=5159) NFS & LSM by VCTE (n=3094) FIB- 4 & LSM by VCTE (n=5159) NFS & LSM by VCTE (n=3094) FIB- 4 & LSM by VCTE (n=5159) NFS & LSM by VCTE (n=3094) Advanced fibrosis, % 30 28 30 28 30 28 Thresholds for bloodbased NIT <0.88, ≥2.31* <−2.55, ≥0.28* <1.3, ≥2.67† <−1.455, ≥0.676† <1.3, ≥2.67† <−1.455, ≥0.676† Thresholds for LSM by VCTE, kPa <7.4, ≥12.1* <7.4, ≥12.1* <7.9, ≥9.6† <7.9, ≥9.6† <8.0, ≥10.0† <8.0, ≥10.0† Sensitivity, % 80 (77–83) 77 (74–81) 67 (64–69) 65 (62–68) 66 (63–68) 64 (62–67) Specificity, % 81 (79–83) 83 (81–85) 85 (84–87) 86 (84–88) 86 (84–87) 86 (84–88) PPV, % 62 (60–65) 61 (58–64) 66 (64–68) 63 (61–67) 66 (64–68) 64 (61–67) NPV, % 91 (90–92) 91 (89–93) 86 (85–87) 87 (85–88) 86 (85–87) 86 (85–88) Indeterminate, % 18 (17–19) 20 (18–21) 5 (4–5) 5 (5–6) 5 (4–6) 5 (5–6) Misclassification, % 16 (14–17) 15 (13–17) 19 (18–21) 19 (17–21) 19 (18–20) 19 (17–21) Patients undergoing LSM by VCTE, % 51 (50–53) 56 (54–59) 34 (32–35) 38 (36–40) 34 (33–35) 38 (37–40) 95% CIs were estimated with 500 bootstrap replicates. *Thresholds were determined from the individual patient data study group as corresponding to 90% sensitivity (lower value) and 90% specificity (upper value) †Threshold were determined from the literature. For LSM by VCTE, a threshold pair yielding the highest sensitivity and specificity while having the smallest proportion of indeterminate cases in diagnosing advanced fibrosis was chosen. NIT, noninvasive test; NPV, negative predictive value; PPV, positive predictive value. copyright. on November 3, 2022 at USE/Fac Medicina Biblioteca FME. Protected byhttp://gut.bmj.com/Gut: first published as 10.1136/gutjnl-2021-324243 on 17 May 2021. Downloaded from