Inflammatory activity affects the accuracy of liver stiffness measurement by transient elastography but not by two‐dimensional shear wave elastography in non‐alcoholic fatty liver disease

Abstract Background In patients with non‐alcoholic fatty liver disease (NAFLD), the impact of the severity of steatosis and inflammatory activity on the accuracy of liver stiffness measurement (LSM) by transient elastography (TE) and by two‐dimensional shear wave elastography (2D‐SWE) in staging liver fibrosis is still debated and scarce. We aimed to focus on this aspect. Methods We prospectively studied 104 patients requiring biopsy for the assessment of NAFLD. We used ordinary least squares regression to test for differences in the association between fibrosis and LSM by TE and 2D‐SWE when other factors (steatosis and inflammatory activity) are considered. Results Among 104 patients, 102 had reliable LSM by TE, and 88 had valid LSM by 2D‐SWE. The association between fibrosis based on histology and LSM was significantly stronger when 2D‐SWE assessed LSM compared to TE (Spearman's correlation coefficient of .71; P < .001 vs .51, P < .001; Z = 2.21, P = .027). Inflammatory activity was an independent predictor of LSM by TE but not of LSM by 2D‐SWE. After controlling for fibrosis, age, sex and body mass index, the inflammatory activity and the interaction between inflammatory activity and fibrosis independently explained 11% and 13% of variance in LSM by TE respectively. Steatosis did not affect the association of fibrosis and LSM by either method. Conclusion Inflammatory activity on histology significantly affects LSM by TE, but not LSM by 2D‐SWE in NAFLD. LSM by 2D‐SWE reflects liver fibrosis more accurately than LSM by TE. Furthermore, the severity of steatosis on histology did not influence the association of LSM and fibrosis by either elastography method.


| INTRODUC TI ON
Liver fibrosis is the most important prognostic factor in patients with non-alcoholic fatty liver disease (NAFLD). 1 Since NAFLD is rapidly increasing and is already the most common cause of chronic liver disease worldwide 2,3 and one of the main indications for liver transplantation, 4,5 reliable methods to stage liver fibrosis in NAFLD are an urgent need in clinical practice. Liver biopsy remains the reference standard to characterize non-alcoholic steatohepatitis (NASH). 6 Nevertheless, liver biopsy is an invasive and expensive procedure associated with patient discomfort and sampling variability. 7,8 Elastography techniques, such as transient elastography (TE) and two-dimensional shear wave elastography (2D-SWE), provide a physical measure of liver stiffness which is closely related to fibrosis in chronic liver disease, 9 and have emerged as an alternative to liver biopsy.
Although TE has been validated in NAFLD as a method to identify and stage fibrosis, it has been shown that steatosis and inflammatory activity might influence the accuracy of liver stiffness measurements (LSM) to predict fibrosis. 9 However, the data regarding the impact of steatosis on LSM are still controversial. Some studies show no association, [10][11][12] one study showed that severe steatosis leads to an overestimation of liver fibrosis by LSM assessed using TE, 13 and another one showed that steatosis leads to an underestimation of liver fibrosis by LSM assessed by TE. 14 To date, only one study evaluated the influence of steatosis and inflammation on LSM using 2D-SWE in NAFLD, 15 and no head-to-head study compared TE and 2D-SWE vs histology with the aim of addressing which of the two methods is mostly influenced by confounders (steatosis and inflammation).
This study aimed to assess whether histological steatosis and inflammatory activity in NAFLD patients affects the accuracy of LSM by two different ultrasound elastography methods (TE and 2D-SWE) in predicting fibrosis.

| Study population
We prospectively included consecutive adult patients with NAFLD who underwent liver biopsy from August 2018 through September 2020 who had LSM performed using TE and 2D-SWE within 2 months from liver biopsy at an academic tertiary centre. Exclusion criteria were as follows: liver disease of other aetiology (chronic hepatitis B or C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, genetic hemochromatosis, drug-induced hepatotoxicity, a-1 antitrypsin deficiency, Wilson's disease, etc); exposure to drugs that can cause secondary NAFLD (corticosteroids, amiodarone and tamoxifen); significant alcohol consumption (>3 standard drinks/day in men and >2 drinks/day in women, or binge drinking defined as >5 standard drinks in men and >4 in women over a 2-hour period) 3 ; ALT >5 times the upper limit of normality; and refusal of consent to further use of personal health-related data for research.
The study was performed according to the principles of the Declaration of Helsinki, and approval was obtained from the local ethics committee (KEK BE 2018-00487).

| Clinical and laboratory assessment
Demographic, clinical, anthropometric and laboratory data were collected at the time of the biopsy. Obesity was defined as body mass index (BMI) ≥30 kg/m 2 . Diabetes mellitus was defined as a fasting glycaemia ≥7 mmol/L or an HbA1c ≥6.5%, or current antidiabetic treatment. The presence of arterial hypertension and dyslipidaemia was recorded from the clinical charts.

| LSM by two-dimensional shear wave elastography
LSM by 2D-SWE was performed using the Aixplorer ultrasound system (SuperSonic Imagine SA, Aix-en-Provence, France).
Patients were placed in a supine position, with the right arm in extension. The operator selected a region of the right lobe of the

Key points
In patients with non-alcoholic fatty liver disease (NAFLD), the impact of steatosis and inflammatory activity on the accuracy of liver stiffness measurement is still debated. Therefore, we compared two non-invasive tests, transient elastography (Fibroscan ® ) and two-dimensional shear wave elastography (2D-SWE), in this population. We found that steatosis on histology did not affect liver stiffness by Fibroscan and 2D-SWE. Whereas the inflammatory activity significantly affects liver stiffness by Fibroscan but not by 2D-SWE. Furthermore, liver stiffness by 2D-SWE was better correlated with fibrosis stage. Thus, liver stiffness using 2D-SWE appears to reflect liver fibrosis more accurately than Fibroscan in this population of NAFLD patients. liver with good spatial resolution for B-mode ultrasound imaging, free of large vascular structures and at least 15 mm below the capsule through a right intercostal space, and during breath hold activated 2D-SWE. Once a colour map with complete and homogeneous filling was obtained in the assessment area, a region of interest 15 mm in diameter was positioned in the centre of the colour map to measure stiffness using the Q box tool. We obtained three successful and valid measurements for each patient and used the mean value and the standard deviation of these measurements as liver stiffness measure. 16 Variability (SD) over 30% of the average liver stiffness value was considered as unreliable measurement. 17 When the operator obtained little or no signal in the region of interest for all acquisitions, the measurements were defined as failures. and lack of major fragmentation.

| Characteristics of the study population
One hundred and four patients undergoing liver biopsy to grade and stage NAFLD were evaluated using liver histology, relevant laboratory parameters and LSM by TE and 2D-SWE. One hundred and two had reliable LSM by TE and 88 had reliable LSM by 2D-SWE. The differences between the 88 patients with reliable LSM by 2D-SWE and the 16 patients without reliable LSM by 2D-SWE are shown in Table S1. Eighty-six patients had reliable LSM using both TE and 2D-SWE methods; a flowchart of patient inclusion and exclusion is provided in Figure S1. Baseline characteristics of the whole population and a comparison of the cohorts using TE and 2D-SWE are shown in Table 1.
The M probe and XL probe were used in 38.4% and 61.5% of patients respectively. Among XL probe patients, the BMI >30 kg/m 2 criteria was used in 92%, and the skin-to-capsule distance criteria ≥25 mm was used in 8%. Technical failure to measure liver stiffness occurred in none of the cases with TE and in 15 of the cases (13%) with 2D-SWE because of the inability to obtain an adequate signal for the TA B L E 1 Baseline features of these 104 patients and the comparison of the cohorts using transient elastography (TE) and twodimensional shear wave elastography (2D-SWE) Insulin -mU/L 31.7 ± 29.1 31.7 ± 29.6 31.9 ± 31 Platelet count -g/L 220. 8

TA B L E 1 (Continued)
F I G U R E 1 Spearman's correlations for patient characteristics, liver histology, laboratory parameters and liver stiffness by two non-invasive tests (2D-SWE and TE). Of note, the correlation of histological fibrosis stage with liver stiffness was stronger for 2D-SWE than for TE. *P < .05; **P < .01 False negative rate of LSM by TE for significant fibrosis (≥F2) was 16%. The diagnostic performance of LSM by 2D-SWE using the cut-off proposed for NAFLD patients 20 is also detailed in Table 2.
It showed good diagnostic performance for fibrosis stages F ≥ F2 and F = F4. The discriminative ability of LSM by 2D-SWE was numerically higher than by TE for distinguishing F0-F2 vs F3 with an AUROC of 0.84 (0.76-0.92) at a threshold of 9.2 kPa.
The AUROC and false positive rate for LSM by TE and 2D-SWE for diagnosing histological fibrosis stage according to the histological inflammatory activity stage are summarized in Table S2.

| Potential confounders of the association between fibrosis and liver stiffness measured by TE and 2D-SWE
We calculated a baseline linear regression model of the fibrosis stage on the LSM. To assess whether the histological steatosis, inflammatory activity, NAS score and laboratory parameters would influence the association between fibrosis and LSM by TE or 2D-SWE, we compared all further models against this baseline model.

| Influence of inflammation and steatosis on LSM by TE
The fibrosis stage on histology explained 35% of the variance of LSM by TE (P < .001). The variance explained by the model did not increase significantly compared to the baseline model after including the main effect of steatosis percentage and the interaction of steatosis percentage and fibrosis stage (R 2 cha = .04; P = 1) ( Table 3). When including inflammatory activity and the interaction between inflammatory activity and histological fibrosis stage, the model explained 25% more variance in LSM by TE (P < .01). After controlling for fibrosis, age, sex and BMI, both the main effect of inflammatory activity (P < .001) and the interaction effect between inflammatory activity and fibrosis (P = .01) remained significantly associated with LSM by TE, and independently explained 11% and 13% of variance in LSM by TE respectively ( Table 4).  Tables S3 and S4.

| Influence of inflammation and steatosis on LSM by 2D-SWE
The same analysis was applied to LSM by 2D-SWE as the dependent variable. In the baseline model, fibrosis stage on histology explained 52% in variance of LSM by 2D-SWE (P < .001). Including steatosis percentage and its interaction with fibrosis stage into the model did not significantly improve the model (R 2 cha = .00; P = 1). Similarly, including inflammatory activity and its interaction with fibrosis stage did not explain significantly more variance than the baseline model (R 2 cha = .06; P = 1) ( Table 3). We tested the same additional models as for LSM by 2D-SWE. None of these models explained significantly more variance in the dependent variable, indicating that none of the variables altered the effect of the fibrosis stage on LSM by 2D-SWE (Table S5).

| Sensitivity analysis in patients with reliable LSM using both TE and 2D-SWE methods
The analysis of the 86 patients with reliable LSM on both TE and 2D-SWE showed similar results. Inflammatory activity and its TA B L E 2 Diagnostic performance of LSM by TE and 2D-SWE for each fibrosis stage interaction with fibrosis stage explained 27% in the variance of LSM by TE (P < .01). Fibrosis stage, inflammatory activity and its interaction with liver fibrosis remained significant when controlling for age, BMI and sex. Similar to the larger data set, none of the models in which LSM by 2D-SWE was tested as the dependent variable improved the baseline model significantly. Table S6 contains the model comparisons for this sensitivity analysis.

| DISCUSS ION
In this prospective cohort study, we found that inflammatory activity assessed on histology significantly affects liver stiffness measured by TE, but not liver stiffness measured by 2D-SWE in patients with NAFLD. The severity of steatosis on histology did not affect the association between LSM and fibrosis by any of the two elastography methods used. In our cohort, LSM by 2D-SWE reflects liver fibrosis more accurately than LSM by TE in NAFLD patients. However, LSM failures were more frequent using 2D-SWE than TE owing to the technical limitations -mostly because of obesity likely impeding an efficient transmission of the ultrasound waves to the liver.
It has been reported that 2D-SWE has a higher rate of failure in patients with high BMI. 21 This is likely the reason why we experienced a relatively high rate of 2D-SWE failures in our cohort composed mostly of overweight and obese patients. Nevertheless, the 13% failure rate of LSM by 2D-SWE is in agreement with that reported in the study by Cassinotto et al. 15 In addition, in our study, we found a lower unreliable/failure rate of LSM by TE than previously reported (1.9% vs 14%), likely because the XL probe was available for patients with obesity and/or skin-to-capsule distance ≥25 mm.
Our findings are in keeping with studies using XL probe, which have reported a high rate of LSM success with TE (97% of patients could be evaluated successfully). 11,22 The diagnostic performance of LSM by TE and by 2D-SWE for fibrosis stages in this study is also in line with data from large cohorts. 11,20 Our results are in agreement with the previous data by This study has limitations. Some of them are inherent to liver biopsies, such as sampling error and interobserver variability. 8 We There are several strengths of this study. The population is a wellcharacterized prospective cohort in whom the use of the optimal TE probe (either M or XL) was used to characterize NAFLD. Moreover, the statistical analysis is done using a principled approach where predictors are tested against a meaningful baseline model rather than a simple null model. This has the advantage of adding explanatory weight to observed phenomena, while fully utilizing the limited available data.
In summary, we found that in NAFLD patients, the association between fibrosis and liver stiffness using 2D-SWE is not altered by steatosis and inflammatory activity. Furthermore, LSM by 2D-SWE was better correlated with the fibrosis stage. On the other hand, liver fibrosis assessment by LSM using TE was also not affected by steatosis, but it was affected by the histological degree of inflammatory activity and its interaction with fibrosis, which would result in an over interpretation of liver fibrosis. According to our data, we conclude that liver stiffness by 2D-SWE appears to reflect fibrosis more accurately in patients with NAFLD when technically feasible.

ACK N OWLED G EM ENT
The authors thank Prof. Jaime Bosch for his kind support. Open access funding provided by Universitat Bern. Open Access Funding provided by Universitat Bern within the CRUI-CARE Agreement

CO N FLI C T O F I NTE R E S T
The authors do not have any disclosures to report.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.