Fat-containing hepatocellular carcinoma in patients with cirrhosis: proposal of a diagnostic modification regarding enhancement characteristics.

OBJECTIVES: The aim of this study was to develop and validate an algorithm for the non-invasive diagnosis of these fat-containing HCCs. METHODS: Eighty-four cirrhotic patients with 77 fat-containing HCCs and 11 non-HCC fat-containing nodules were retrospectively included. All MRIs were reviewed; nodule characteristics, European Association for the Study of the Liver (EASL) and LI-RADS classifications, and survival were collected. One of the major features of LI-RADS v2018 (non-rim-like arterial phase hyperenhancement [APHE]) was changed to include different enhancing patterns at arterial phase and a new fat-LI-RADS algorithm was created for fat-containing nodules in cirrhosis. Its diagnostic performance was evaluated in both a derivation and external validation cohort (external cohort including 58 fat-containing HCCs and 10 non-HCC fat nodules). Reproducibility of this new algorithm was assessed. RESULTS: In the derivation cohort, 54/77 (70.1%) fat-containing HCCs had APHE, 62/77 (80.5%) had enhancement compared to the nodule itself at arterial phase (APE), 43/77 (55.8%) had washout, and 20/77 (26.0%) had an enhancing capsule. EASL and LI-RADS had a sensitivity of 37.7% (29/77) and 36.4% (28/77), respectively, for the diagnosis of fat-containing HCC and both had a specificity of 100% (11/11). The new fat-LI-RADS algorithm increased sensitivity to 50.6% (39/77) without decreasing the specificity of 100% (11/11). The validation cohort confirmed the increased sensitivity, with a slight decrease in specificity. The concordance for the diagnosis of HCC for fat-LR5 was 85.3% (58/68). CONCLUSION: The new fat-LI-RADS algorithm proposed here significantly improves the performance of the non-invasive diagnosis of fat-containing HCC and thus could reduce the number of biopsies conducted for fat-containing HCCs. CLINICAL RELEVANCE STATEMENT: The European Association for the Study of the Liver and LI-RADS guidelines are poorly sensitive for the diagnosis of fat-containing HCC, mainly because of the low rate of arterial phase hyperenhancement (APHE) displayed by fat-containing HCC. Using all types of enhancement instead of APHE improves sensitivity of LI-RADS. KEY POINTS: • Fat-containing HCCs on MRI account for 7.5% of HCCs and have different imaging characteristics from non-fatty HCCs. • The European Association for the Study of the Liver and LI-RADS algorithms for the non-invasive diagnosis of HCC have low sensitivity for the diagnosis of fat-containing HCC with MRI (37.7% and 36.4%, respectively). • The new fat-LI-RADS, which includes a slight modification of the "arterial enhancement" criterion, improves the sensitivity for the diagnosis of fat-containing HCC using MRI, without degrading the specificity.

Associations between perfluoroalkyl substances and the severity of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease worldwide and the determinants driving its severity remain to be elucidated. Perfluoroalkyl substances (PFAS) are synthetic chemical compounds. They are used in commonplace products and persistent in water, soil and the human body. In vitro and animal studies suggest a pathogenic role for PFAS in metabolic diseases such as NAFLD. OBJECTIVES: We aimed to evaluate the association between NAFLD severity and serum PFAS concentrations in humans. METHODS: One hundred biopsy-proven NAFLD patients were included with a well-balanced distribution between the different stages of severity: 25 patients with simple steatosis, 25 with early non-alcoholic steatohepatitis (NASH and F0-F1 fibrosis), 33 with fibrotic NASH (NASH and F2-F3 fibrosis), and 17 with cirrhotic NASH (NASH and F4 fibrosis). Liver histological features were evaluated according to the NASH Clinical Research Network classification. Seventeen PFAS were measured by high-performance liquid chromatography coupled with tandem mass spectrometry on serum samples stored at -80 °C. RESULTS: The median age was 60 years, 61 % of patients were male, 46 % had diabetes and the median body mass index (BMI) was 32 kg/m2. Long-chain PFAS were associated with steatosis grade (p = 0.03). Among the nine PFAS detected in > 50 % of the patients, Perfluoro-n-heptanoic acid (PFHpA) showed significantly higher concentrations in grade 3 steatosis versus grade 1 (p = 0.02). Perfluoro-n-dodecanoic acid (PFDoA) concentrations were higher in patients with significant fibrosis (p = 0.04) and PFHpA in patients with advanced fibrosis (p = 0.02). The association between PFHpA and steatosis grade remained significant in multivariate analysis adjusted for age, gender, BMI, diabetes presence and dyslipidemia (p = 0.004). DISCUSSION: Our study showed a significant association between PFHpA and liver steatosis in NAFLD. According to data available in the literature, PFHpA could be implicated in liver steatosis through ß-oxidation and biosynthesis of fatty acids.

Practical diagnosis of cirrhosis in non-alcoholic fatty liver disease using currently available non-invasive fibrosis tests.

Unlike for advanced liver fibrosis, the practical rules for the early non-invasive diagnosis of cirrhosis in NAFLD remain not well defined. Here, we report the derivation and validation of a stepwise diagnostic algorithm in 1568 patients with NAFLD and liver biopsy coming from four independent cohorts. The study algorithm, using first the elastography-based tests Agile3+ and Agile4 and then the specialized blood tests FibroMeterV3G and CirrhoMeterV3G, provides stratification in four groups, the last of which is enriched in cirrhosis (71% prevalence in the validation set). A risk prediction chart is also derived to allow estimation of the individual probability of cirrhosis. The predicted risk shows excellent calibration in the validation set, and mean difference with perfect prediction is only -2.9%. These tools improve the personalized non-invasive diagnosis of cirrhosis in NAFLD.

Predictive abilities comparison from multiple dynamic prediction models.

With the development of personalized medicine, the study of individual prognosis appears to be a major contemporary scientific issue. Dynamic models are particularly well adapted to such studies by allowing some potential changes in the follow-up to be taken into account. In particular, this leads to more accurate predictions by updating the available information throughout the patient monitoring. Some mathematical tools have been developed to quantify and compare the effectiveness of dynamic predictions using dynamic versions of the area under the receiver operating characteristic curve and the Brier score in the competing risks setting. Nevertheless, only two predictive abilities can be compared. This may be too restrictive in a clinical context where more and more information can be collected during patient follow-up thanks to recent technological advances. Here we propose a new procedure that allows multiple comparisons of the predictive abilities of different biomarkers, based on the dynamic area under the receiver operating characteristic curve or Brier score. Performances of our testing procedure were assessed by simulations. Moreover, a motivating application in hepatology will be presented. Finally, this work compares more than two dynamic predictive abilities of biomarkers and is available via R functions on GitHub.

Detection of arterial phase hyperenhancement of small hepatocellular carcinoma with MRI: Comparison between single arterial and multi-arterial phases and between extracellular and hepatospecific contrast agents.

PURPOSE: The purpose of this study was to compare the detection rate of arterial phase hyperenhancement (APHE) in small hepatocellular carcinoma (HCC) between single arterial phase (single-AP) and triple hepatic arterial (triple-AP) phase MRI and between extracellular (ECA) and hepato-specific (HBA) contrast agents. MATERIALS AND METHODS: A total of 109 cirrhotic patients with 136 HCCs from seven centers were included. There were 93 men and 16 women, with a mean age of 64.0 ± 8.9 (standard deviation) years (range: 42-82 years). Each patient underwent both ECA-MRI and HBA (gadoxetic acid)-MRI examination within one month of each other. Each MRI examination was retrospectively reviewed by two readers blinded to the second MRI examination. The sensitivities of triple- and single-AP for the detection of APHE were compared, and each phase of the triple-AP sequence was compared with the other two. RESULTS: No differences in APHE detection were found between single-AP (97.2%; 69/71) and triple-AP (98.5%; 64/65) (P > 0.99) at ECA-MRI. No differences in APHE detection were found between single-AP (93%; 66/71) and triple-AP (100%; 65/65) at HBA-MRI (P = 0.12). Patient age, size of the nodules, use of automatic triggering, type of contrast agent, and type of sequence were not significantly associated with APHE detection. The reader was the single variable significantly associated with APHE detection. For triple-AP, best APHE detection rate was found for early and middle-AP images compared to late-AP images (P = 0.001 and P = 0.003). All APHEs were detected with the combination of early-AP and middle-AP images, except one that was detected on late-AP images by one reader. CONCLUSION: Our study suggests that both single- and triple-AP can be used in liver MRI for the detection of small HCC especially when using ECA. Early AP and middle-AP are the most efficient phases and should be preferred for detecting APHE, regardless of the contrast agent used.

Validation of the Blood Test MACK-3 for the Noninvasive Diagnosis of Fibrotic Nonalcoholic Steatohepatitis: An International Study With 1924 Patients.

BACKGROUND & AIMS: Drug development in nonalcoholic steatohepatitis (NASH) is hampered by a high screening failure rate that reaches 60% to 80% in therapeutic trials, mainly because of the absence of fibrotic NASH on baseline liver histology. MACK-3, a blood test including 3 biomarkers (aspartate aminotransferase, homeostasis model assessment, and cytokeratin 18), recently was developed for the noninvasive diagnosis of fibrotic NASH. We aimed to validate the diagnostic accuracy of this noninvasive test in an international multicenter study. METHODS: A total of 1924 patients with biopsy-proven nonalcoholic fatty liver disease from 10 centers in Asia, Australia, and Europe were included. The blood test MACK-3 was calculated for all patients. FibroScan-aspartate aminotransferase score (FAST), an elastography-based test for fibrotic NASH, also was available in a subset of 655 patients. Fibrotic NASH was defined as the presence of NASH on liver biopsy with a Nonalcoholic Fatty Liver Disease Activity Score of 4 or higher and fibrosis stage of F2 or higher according to the NASH Clinical Research Network scoring system. RESULTS: The area under the receiver operating characteristic of MACK-3 for fibrotic NASH was 0.791 (95% CI 0.768-0.814). Sensitivity at the previously published MACK-3 threshold of less than 0.135 was 91% and specificity at a greater than 0.549 threshold was 85%. The MACK-3 area under the receiver operating characteristic was not affected by age, sex, diabetes, or body mass index. MACK-3 and FAST results were well correlated (Spearman correlation coefficient, 0.781; P < .001). Except for an 8% higher rate of patients included in the grey zone, MACK-3 provided similar accuracy to that of FAST. Both tests included 27% of patients in their rule-in zone, with 85% specificity and 35% false positives (screen failure rate). CONCLUSIONS: The blood test MACK-3 is an accurate tool to improve patient selection in NASH therapeutic trials.

Evaluation of washout using subtraction MRI for the diagnosis of hepatocellular carcinoma in cirrhotic patients with spontaneously T1-hyperintense nodules.

PURPOSE: The purpose of this study was to assess the value of subtraction imaging on post-arterial phase images (i.e., portal venous, delayed/transitional and hepatobiliary phases) for the non-invasive diagnosis of hepatocellular carcinoma (HCC) in spontaneously hyperintense nodules on T1-weighted imaging in patients with cirrhosis. MATERIALS AND METHODS: Forty-five patients with a total 55 hepatic nodules that were spontaneously hyperintense on T1-weighted images were initially retrieved. All patients underwent MRI examination of the liver using extracellular agent. Each nodule was assessed for sensitivity and specificity using LI-RADS (Liver Imaging Reporting and Data System) during two reading sessions performed first without then with subtraction images on post-arterial phase images. The final standard of reference was defined by a step-by-step algorithm previously published combining histology, typical imaging, alfa fetoprotein and follow-up. RESULTS: Forty-six nodules (26 HCC) in 39 patients with cirrhosis were analyzed. Using LI-RADS, the sensitivity and specificity for the diagnosis of HCC were 64% (95% CI: 41-83) and 67% (95% CI: 41-87) without subtraction; and 73% (95% CI: 50-89) (P > 0.999) and 33% (95% CI: 13-59) (P = 0.553) on subtraction imaging using extracellular contrast agent. Fifty-five percent (22/40) of nodules displayed a washout without subtraction and 70% (28/40) did so on subtraction imaging obtained with extracellular contrast agent. Twenty nodules out of 40 (50%) were classified LI-RADS 5 without subtraction, and 28 out of 40 nodules (70%) with subtraction. CONCLUSION: The results of this study suggest that the use of subtraction imaging on post-arterial phase images (i.e., PVP, DP/TP and HBP) is not relevant for the non-invasive diagnosis of HCC for spontaneously hyperintense nodules on T1-weighted images in patients with liver cirrhosis.

Dynamic personalized prediction of the individual liver-related risk after sustained viral response in HCV patients.

Sustained viral response (SVR) significantly improves the prognosis in patients with hepatitis C virus (HCV) chronic infection but does not totally alleviate the risk of liver-related complications (LRC). We aimed to evaluate whether the dynamics of multiple measurements of simple parameters after SVR enable the development of a personalized prediction of prognosis in HCV patients. HCV mono-infected patients who experienced SVR in two prospective cohorts (ANRS CO12 CirVir cohort: derivation set; ANRS CO22 HEPATHER cohort: validation set) were included. The study outcome was LRC, a composite criterion including decompensation of cirrhosis and/or hepatocellular carcinoma. Joint latent class modelling accounting for both biomarker trajectory and event occurrence during follow-up was developed in the derivation set to compute individual dynamic predictions, with further evaluation in the validation set. In the derivation set (n = 695; 50 LRC during the median 3.8 [1.6-7.5] years follow-up), FIB4 was identified as a biomarker associated with LRC occurrence after SVR. Joint modelling used sex and the dynamics of FIB4 and diabetes status to develop a personalized prediction of LRC. In the validation set (n = 7064; 273 LRC during the median 3.6 [2.5-4.9] years follow-up), individual dynamic predictions from the model accurately stratified the risk of LRC. Time-dependent Brier Score showed good calibration that improved with the accumulation of visits, justifying our modelling approach considering both baseline and follow-up measurements. Dynamic modelling using repeated measurements of simple parameters predicts the individual residual risk of LRC and improves personalized medicine after SVR in HCV patients.

Enhanced diagnosis of advanced fibrosis and cirrhosis in individuals with NAFLD using FibroScan-based Agile scores.

BACKGROUND & AIMS: Currently available non-invasive tests, including fibrosis-4 index (FIB-4) and liver stiffness measurement (LSM by VCTE), are highly effective at excluding advanced fibrosis (AF) (F ≥3) or cirrhosis in people with non-alcoholic fatty liver disease (NAFLD), but only have moderate ability to rule-in these conditions. Our objective was to develop and validate two new scores (Agile 4 and Agile 3+) to identify cirrhosis or AF, respectively, with optimized positive predictive value and fewer indeterminate results, in individuals with NAFLD attending liver clinics. METHODS: This international study included seven adult cohorts with suspected NAFLD who underwent liver biopsy, LSM and blood sampling during routine clinical practice or screening for trials. The population was randomly divided into a training set and an internal validation set, on which the best-fitting logistic regression model was built, and performance and goodness of fit were assessed, respectively. Furthermore, both scores were externally validated on two large cohorts. Cut-offs for high sensitivity and specificity were derived in the training set to rule-out and rule-in cirrhosis or AF and then tested in the validation set and compared to FIB-4 and LSM. RESULTS: Each score combined LSM, AST/ALT ratio, platelets, sex and diabetes status, as well as age for Agile 3+. Calibration plots for Agile 4 and Agile 3+ indicated satisfactory to excellent goodness of fit. Agile 4 and Agile 3+ outperformed FIB-4 and LSM in terms of AUROC, percentage of patients with indeterminate results and positive predictive value to rule-in cirrhosis or AF. CONCLUSIONS: The two novel non-invasive scores improve identification of cirrhosis or AF among individuals with NAFLD attending liver clinics and reduce the need for liver biopsy in this population. IMPACT AND IMPLICATIONS: Non-invasive tests currently used to identify patients with advanced fibrosis or cirrhosis, such as fibrosis-4 index and liver stiffness measurement by vibration-controlled transient elastography, have high negative predictive values but high false positive rates, while results are indeterminate for a large number of cases. This study provides scores that will help the clinician diagnose advanced fibrosis or cirrhosis. These new easy-to-implement scores will help liver specialists to better identify (1) patients who need more intensive follow-up, (2) patients who should be referred for inclusion in therapeutic trials, and (3) which patients should be treated with pharmacological agents when effective therapies are approved.

Validation of the new 2021 EASL algorithm for the noninvasive diagnosis of advanced fibrosis in NAFLD.

BACKGROUND AND AIMS: The European Association for the Study of the Liver (EASL) has recently proposed an algorithm for the diagnosis of advanced liver fibrosis. We aimed to evaluate the diagnostic accuracy of this algorithm in nonalcoholic fatty liver disease (NAFLD). APPROACH AND RESULTS: One thousand fifty-one patients with NAFLD, liver biopsy, and four noninvasive tests (NITs; Fibrosis-4 [FIB4], vibration controlled transient elastography [VCTE], FibroMeter, Fibrotest) were included. The enhanced liver fibrosis (ELF) score was available in 396 patients. A cohort of 230 patients from primary care/diabetes clinics had FIB4, VCTE, and ELF. Compared with the performance of single NITs, agreement between two NITs (FIB4 and VCTE, VCTE and patented serum tests) increased specificity and positive predictive value by 20%, thus justifying the sequential use proposed in the EASL algorithm. The FIB4/VCTE/FibroMeter and FIB4/VCTE/Fibrotest algorithms performed similarly, providing 85% diagnostic accuracy and a liver biopsy requirement rate of only 10%. The FIB4/VCTE/ELF algorithm performed similarly in the subgroup where ELF was available. Simulations of algorithm accuracies at different prevalence showed that positive predictive values rapidly increased, reaching a plateau above 75% starting at 15% prevalence. Negative predictive values remained higher than 90% up to 25% prevalence. The rate of liver biopsy requirement remained stable, increasing by only 5% between low and high prevalence settings. When the EASL algorithm was applied in the primary care/diabetes clinic cohort, liver biopsy requirement was only 3%, and the agreement among the three steps provided 75% positive predictive value. CONCLUSIONS: Our study validates the algorithm proposed by the EASL in its latest 2021 guidelines for the diagnosis of advanced fibrosis in the setting of NAFLD.

Impact of Type 2 Diabetes on the Accuracy of Noninvasive Tests of Liver Fibrosis With Resulting Clinical Implications.

BACKGROUND & AIMS: Noninvasive tests (NITs) of liver fibrosis have been suggested to be less accurate in type 2 diabetes mellitus (T2DM). We aimed to compare the accuracy of 6 NITs between patients with or without T2DM, explain any differences, and adapt diagnostic algorithms for clinical practice accordingly. METHODS: We included 1051 patients with nonalcoholic fatty liver disease with liver biopsy, blood fibrosis tests (Nonalcoholic Fatty Liver Disease Fibrosis Score, FIB4, Fibrotest, FibroMeter), vibration-controlled transient elastography (VCTE), and the combinatory elasto-blood test FibroMeterVCTE. The study endpoint was advanced fibrosis on liver biopsy. RESULTS: NIT areas under the receiver operating characteristic curve were significantly lower in patients with T2DM, mostly because of a decrease in specificity. For FIB4, this decrease in specificity was only related to the higher age of patients with T2DM enrolled. For Fibrotest, FibroMeter, and FibroMeterVCTE, the decrease in specificity was related to age but also to higher alpha2-macroglobulin level, which is known to increase in T2DM. Sensitivity was unaffected by T2DM, but it masked a doubled raw number of false negatives because of the 2-fold higher prevalence of advanced fibrosis in that setting. The sequential algorithm FIB4-vibration-controlled transient elastography had 90.3% accuracy in patients without T2DM vs 79.0% in those with (P < .001). Algorithms using first-line specialized tests maintained a low rate of false negatives and false positives in T2DM. CONCLUSIONS: The decrease in NIT accuracy observed in T2DM is partly biased by the different characteristics of the groups studied, but also linked to T2DM itself through modification of the levels of some NIT biomarkers. Specialized tests should be used first-line to diagnose advanced liver fibrosis in T2DM.

Analysis of hepatic fibrosis markers in the serum of chronic hepatitis B patients according to basal core promoter/precore mutants.

The A1762T/G1764A double mutant in the basal core promoter (BCP) region of the hepatitis B virus (HBV) is associated with severe hepatic lesions while the G1899A mutation with the double mutant is associated with a significant reduction in the risk of severe fibrosis. This study aims to measure a number of markers in the serum of patients with chronic HBV infection and to assess relationships between these markers and BCP/precore mutants with consideration of the stage of fibrosis. The serum levels of resistin, TGF-ß1, MMP-1, TIMP-1, collagen IA1 and PDGF-BB, which are markers that are known to be involved in the process of hepatic fibrosis, were assayed. The serum levels of PDGF-BB and TIMP-1, and the mutation profile were independently associated with advanced fibrosis. A higher level of TIMP-1 was associated with advanced fibrosis regardless of the mutation status, and a higher level of PDGF-BB was associated with nonsevere fibrosis in patients infected with viruses harboring the A1762T/G1764A or A1762T/G1764A/G1899A mutations. Our results suggest an impact of the A1762T/G1764A mutant on the biological pathway related to TGF-ß1 and PDGF-BB. In vitro studies are needed to understand the impact of these mutants on the serum secretion of markers involved in fibrosis severity.

Non-invasive tests accurately stratify patients with NAFLD based on their risk of liver-related events.

BACKGROUND & AIMS: Previous studies on the prognostic significance of non-invasive liver fibrosis tests in non-alcoholic fatty liver disease (NAFLD) lack direct comparison to liver biopsy. We aimed to evaluate the prognostic accuracy of fibrosis-4 (FIB4) and vibration-controlled transient elastography (VCTE), compared to liver biopsy, for the prediction of liver-related events (LREs) in NAFLD. METHODS: A total of 1,057 patients with NAFLD and baseline FIB4 and VCTE were included in a multicenter cohort. Of these patients, 594 also had a baseline liver biopsy. The main study outcome during follow-up was occurrence of LREs, a composite endpoint combining cirrhosis complications and/or hepatocellular carcinoma. Discriminative ability was evaluated using Harrell's C-index. RESULTS: FIB4 and VCTE showed good accuracy for the prediction of LREs, with Harrell's C-indexes >0.80 (0.817 [0.768-0.866] vs. 0.878 [0.835-0.921], respectively, p = 0.059). In the biopsy subgroup, Harrell's C-indexes of histological fibrosis staging and VCTE were not significantly different (0.932 [0.910-0.955] vs. 0.881 [0.832-0.931], respectively, p = 0.164), while both significantly outperformed FIB4 for the prediction of LREs. FIB4 and VCTE were independent predictors of LREs in the whole study cohort. The stepwise FIB4-VCTE algorithm accurately stratified the risk of LREs: compared to patients with "FIB4 <1.30", those with "FIB4 ≥1.30 then VCTE <8.0 kPa" had similar risk of LREs (adjusted hazard ratio [aHR] 1.3; 95% CI 0.3-6.8), whereas the risk of LREs significantly increased in patients with "FIB4 ≥1.30 then VCTE 8.0-12.0 kPa" (aHR 3.8; 95% CI 1.3-10.9), and even more for those with "FIB4 ≥1.30 then VCTE >12.0 kPa" (aHR 12.4; 95% CI 5.1-30.2). CONCLUSION: VCTE and FIB4 accurately stratify patients with NAFLD based on their risk of LREs. These non-invasive tests are alternatives to liver biopsy for the identification of patients in need of specialized management. LAY SUMMARY: The amount of fibrosis in the liver is closely associated with the risk of liver-related complications in non-alcoholic fatty liver disease (NAFLD). Liver biopsy currently remains the reference standard for the evaluation of fibrosis, but its application is limited by its invasiveness. Therefore, we evaluated the ability of non-invasive liver fibrosis tests to predict liver-related complications in NAFLD. Our results show that the blood test FIB4 and transient elastography stratify the risk of liver-related complications in NAFLD, and that transient elastography has similar prognostic accuracy as liver biopsy. These results support the use of non-invasive liver fibrosis tests instead of liver biopsy for the management of patients with NAFLD.

Validation of a Diagnostic Algorithm for Hepatocellular Carcinoma at Gadoxetic Acid-enhanced MRI.

Online supplemental material is available for this article.

New sequential combinations of non-invasive fibrosis tests provide an accurate diagnosis of advanced fibrosis in NAFLD.

BACKGROUND & AIMS: Advanced liver fibrosis is an important diagnostic target in non-alcoholic fatty liver disease (NAFLD) as it defines the subgroup of patients with impaired prognosis. The non-invasive diagnosis of advanced fibrosis is currently limited by the suboptimal positive predictive value and the grey zone (representing indeterminate diagnosis) of fibrosis tests. Here, we aimed to determine the best combination of non-invasive tests for the diagnosis of advanced fibrosis in NAFLD. METHODS: A total of 938 patients with biopsy-proven NAFLD were randomized 2:1 into derivation and validation sets. All patients underwent liver stiffness measurement with vibration controlled transient elastography (VCTE) and blood fibrosis tests (NAFLD fibrosis score, Fibrosis-4 [FIB4], Fibrotest, Hepascore, FibroMeter). FibroMeterVCTE, which combines VCTE results and FibroMeter markers in a single test, was also calculated in all patients. RESULTS: For the diagnosis of advanced fibrosis, VCTE was significantly more accurate than the blood tests (area under the receiver operating characteristic curve [AUROC]: 0.840 ±â€¯0.013, p ≤0.005). FibroMeter was the most accurate blood test (AUROC: 0.793 ±â€¯0.015, p ≤0.017). The combinatory test FibroMeterVCTE outperformed VCTE and blood tests (AUROC: 0.866 ±â€¯0.012, p ≤0.005). The sequential combination of FIB4 then FibroMeterVCTE (FIB4-FMVCTE algorithm) or VCTE then FibroMeterVCTE (VCTE-FMVCTE algorithm) provided an excellent diagnostic accuracy of 90% for advanced fibrosis, with liver biopsy only required to confirm the diagnosis in 20% of cases. The FIB4-FMVCTE and VCTE-FMVCTE algorithms were significantly more accurate than the pragmatic algorithms currently proposed. CONCLUSION: The sequential combination of fibrosis tests in the FIB4-FMVCTE and VCTE-FMVCTE algorithms provides a highly accurate solution for the diagnosis of advanced fibrosis in NAFLD. These algorithms should now be validated for the diagnosis of advanced liver fibrosis in diabetology or primary care settings. LAY SUMMARY: The evaluation of liver fibrosis is mandatory in non-alcoholic fatty liver disease (NAFLD), as advanced fibrosis identifies the subgroup of patients with impaired prognosis. FibroMeterVCTE is a new fibrosis test combining blood markers and the result of vibration controlled transient elastography (VCTE) into a single diagnostic test. Our results show that FibroMeterVCTE outperforms other blood fibrosis tests and VCTE alone for the diagnosis of advanced fibrosis in a large multi-centric cohort of 938 patients with biopsy-proven NAFLD. Sequential algorithms using a simple blood test or VCTE as a first-line procedure, then FibroMeterVCTE as a second-line test accurately classified 90% of patients.

Multifaceted Hoxa13 function in urogenital development underlies the Hand-Foot-Genital Syndrome.

Hand-Foot-Genital syndrome is a rare condition caused by mutations in the HOXA13 gene and characterized by limb malformations and urogenital defects. While the role of Hoxa13 in limb development has been extensively studied, its function during the development of the urogenital system remains elusive mostly due to the embryonic lethality of Hoxa13 homozygous mutant mice. Using a conditional inactivation strategy, we show that mouse fetuses lacking Hoxa13 function develop megaureters, hydronephrosis and malformations of the uterus, reminiscent of the defects characterizing patients with Hand-Foot-Genital syndrome. Our analysis reveals that Hoxa13 plays a critical role in Müllerian ducts fusion and in ureter remodeling by regulating the elimination of the caudal common nephric duct, eventually preventing the separation from the nephric duct. Our data also reveal a specific role for Hoxa13 in the urogenital sinus, which is in part mediated by Gata3, as well as Hoxa13 requirement for the proper organization of the ureter. Finally, we provide evidence that Hoxa13 provides positional and temporal cues during the development of the lower urogenital system, a sine qua non condition for the proper function of the urinary system.

Hoxa1 and Hoxb1 are required for pharyngeal arch artery development.

Hox transcription factors play critical roles during early vertebrate development. Previous studies have revealed an overlapping function of Hoxa1 and Hoxb1 during specification of the rhombomeres from which neural crest cells emerge. A recent study on Hoxa1 mutant mice documented its function during cardiovascular development, however, the role of Hoxb1 is still unclear. Here we show using single and compound Hoxa1;Hoxb1 mutant embryos that reduction of Hoxa1 gene dosage in Hoxb1-null genetic background is sufficient to result in abnormal pharyngeal aortic arch (PAA) development and subsequently in great artery defects. Endothelial cells in the 4th PAAs of compound mutant differentiate normally whereas vascular smooth muscle cells of the vessels are absent in the defective PAAs. The importance of Hoxa1 and Hoxb1, and their interaction during specification of cardiac NCCs is demonstrated. Together, our data reveal a critical role for anterior Hox genes during PAA development, providing new mechanistic insights into the etiology of congenital heart defects.

Hox Genes in Cardiovascular Development and Diseases.

Congenital heart defects (CHD) are the leading cause of death in the first year of life. Over the past 20 years, much effort has been focused on unraveling the genetic bases of CHD. In particular, studies in human genetics coupled with those of model organisms have provided valuable insights into the gene regulatory networks underlying CHD pathogenesis. Hox genes encode transcription factors that are required for the patterning of the anterior-posterior axis in the embryo. In this review, we focus on the emerging role of anteriorly expressed Hox genes (Hoxa1, Hoxb1, and Hoxa3) in cardiac development, specifically their contribution to patterning of cardiac progenitor cells and formation of the great arteries. Recent evidence regarding the cooperative regulation of heart development by Hox proteins with members of the TALE-class of homeodomain proteins such as Pbx and Meis transcription factors is also discussed. These findings are highly relevant to human pathologies as they pinpoint new genes that increase susceptibility to cardiac anomalies and provide novel mechanistic insights into CHD.