Risk factors for more rapid progression of severe liver fibrosis in children with cystic fibrosis-related liver disease: A multi-center study validated by liver biopsy.

BACKGROUND AND AIMS: Early identification of risk factors for the development of severe fibrosis in children with cystic fibrosis-related liver disease (CFLD) is crucial as promising therapies emerge. METHODS: This multi-center cohort study of children with a priori defined CFLD from 1999 to 2016, was designed to evaluate the clinical utility of CF-specific characteristics and liver biomarkers assessed years prior to liver biopsy-proven CFLD to predict risk of developing severe fibrosis (F3-4) over time. Fibrosis was staged by Metavir classification. RESULTS: The overall study cohort of 42 patients (F0-2 (n = 22) and F3-4 (n = 20)) was 57% male (n = 24) with median age of 7.6 years at baseline visit versus 10.3 years at biopsy. Median FEV1 % predicted was lower in F3-4 participants at baseline versus F0-2 (59% vs. 85%; p = .002), while baseline FIB-4, APRI and GGT were higher in F3-4. Median splits for FIB-4 (≥.13), APRI (≥.36), GPR (≥.09), GGT (≥25.5), and FEV1 % (<64%) were associated with more rapid progression to F3-4 (p < .01 for all). Using a combination of change/year in FIB-4, APRI, and GPR to predict F3-4, the AUROC was .81 (95% CI, .66, .96; p < .0001). For up to 5.8 years prior, thresholds for GPR were met 6.5-fold more rapidly, and those for APRI and FIB-4 were met 2.5-fold more rapidly, in those who progressed to F3-4 than those that did not. CONCLUSIONS: This study suggests mild-moderate pulmonary dysfunction and higher liver biomarker indices at baseline may be associated with faster progression of CFLD.

Growth Hormone Stops Excessive Inflammation After Partial Hepatectomy, Allowing Liver Regeneration and Survival Through Induction of H2-Bl/HLA-G.

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.

Synthetic peptides derived from the Schistosoma mansoni secretory protein Sm16 induce contrasting responses in hepatic stellate cells.

Sm16 is a 16 KDa protein released by Schistosoma mansoni that modulates inflammatory responses in host cells. Sm16 is expressed by several life cycle stages of S. mansoni, including the egg stage. Schistosome eggs are known to provoke chronic schistosomiasis pathology, which involves the development of liver fibrosis. Hepatic stellate cells (HSCs), which are responsible for this fibrosis, are susceptible to immunomodulation by S. mansoni whole egg secretions. To define the effects of Sm16 exposure on HSCs, two synthetic peptide derivatives of Sm16, coined "KS-84″ and "KS-66″, were tested against LX-2 cells, an immortalised human HSC line, and RNA sequencing was used to assess the transcriptional changes induced by each peptide. In total, 78 and 798 genes were found to be significantly differentially expressed by KS-84 and KS-66 treatment, respectively. In silico pathway analysis of these genes revealed that KS-84 reduced LX-2 cell activation and fibrotic potential, whereas KS-66 increased both processes. Reduced transforming growth factor-ß1 (TGF-ß1) signalling was identified as a potential mechanism of KS-84-induced inhibition of LX-2 activation. Taken together, these findings indicate a potential role for Sm16 in combatting fibrotic liver disease.

TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. METHODS: The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. RESULTS: Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206+ macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. CONCLUSION: These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. LAY SUMMARY: Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation.

Utility of Serum Biomarker Indices for Staging of Hepatic Fibrosis Before and After Venesection in Patients With Hemochromatosis Caused by Variants in HFE.

BACKGROUND & AIMS: Hemochromatosis that is associated with variants in the homeostatic iron regulator gene (HFE) is characterized by intestinal absorption of iron and excessive body and hepatic iron stores; it can lead to hepatic fibrosis and cirrhosis. Fibrosis has been staged by analysis of liver biopsies, but non-invasive staging methods are available. We evaluated the ability of aspartate aminotransferase:platelet ratio index (APRI), the fibrosis-4 (FIB-4) index, and gamma-glutamyl transferase:platelet ratio (GPR) to assess hepatic fibrosis staging in subjects with HFE-associated hemochromatosis, using liver biopsy-staged fibrosis as the reference standard. METHODS: We performed a retrospective, cross-sectional analysis of 181 subjects with HFE-associated hemochromatosis and hepatic fibrosis staged by biopsy analysis and available serum samples. We calculated APRI, FIB-4, and GPR at diagnosis for all 181 subjects and following venesection therapy in 64 of these subjects (7 subjects had follow-up biopsy analysis). We used area under the receiver operating characteristic curve (AUROC) analysis to assess the relationships between APRI score, FIB-4 score, and GPR and advanced (F3-F4) fibrosis and to select cut-off values. RESULTS: Hepatic fibrosis stage correlated with APRI score (r = 0.54; P < .0001), FIB-4 score (r = 0.35; P < .0001), and GPR (r = 0.36, P < .0001). An APRI score above 0.44 identified patients with advanced fibrosis with an AUROC of 0.88, 79.4% sensitivity, 79.4% specificity, and 81% accuracy. A FIB-4 score above 1.1 identified patients with advanced fibrosis with an AUROC of 0.86, 80% sensitivity, 80.3% specificity, and 81% accuracy. A GPR above 0.27 identified patients with advanced fibrosis with an AUROC of 0.76, 67.7% sensitivity, 70.3% specificity, and 69% accuracy. APRI score was significantly more accurate than GPR (P = .05) in detecting advanced fibrosis; there was no difference between APRI and FIB-4. Venesection treatment was associated with significant reductions in APRI (P < .0001) and GPR (P < .001), paralleling fibrosis regression observed in available liver biopsies. Post-venesection APRI identified 87% of subjects with advanced fibrosis that decreased to levels that indicate stage F1-F2 fibrosis. CONCLUSIONS: In a retrospective study of 181 subjects with HFE-associated hemochromatosis, we found that APRI and FIB-4 scores identified patients with advanced hepatic fibrosis with 81% accuracy. APRI scores might also be used to monitor fibrosis regression following venesection.

Cost-Effectiveness of a Serum Biomarker Test for Risk-Stratified Liver Ultrasound Screening for Hepatocellular Carcinoma.

OBJECTIVES: Risk-stratified ultrasound screening for hepatocellular carcinoma (HCC), informed by a serum biomarker test, enables resources to be targeted to patients at the highest risk of developing cancer. We aimed to investigate the cost-effectiveness of risk-stratified screening for HCC in the Australian healthcare system. METHODS: A Markov cohort model was constructed to test 3 scenarios for patients with compensated cirrhosis: (1) risk-stratified screening for high-risk patients, (2) all-inclusive screening, and (3) no formal screening. Probabilistic sensitivity analyses were undertaken to determine the impact of uncertainty. Scenario analyses were used to assess cost-effectiveness in Australia's Aboriginal and Torres Strait Islander peoples and to determine the impact of including productivity-related costs of mortality. RESULTS: Both risk-stratified screening and all-inclusive screening programs were cost-effective compared with no formal screening, with incremental cost-effectiveness ratios of A$39 045 and A$23 090 per quality-adjusted life-year (QALY), respectively. All-inclusive screening had an incremental cost-effectiveness ratio of A$4453 compared with risk-stratified screening and had the highest probability of being cost-effective at a willingness-to-pay (WTP) threshold of A$50 000 per QALY. Risk-stratified screening had the highest likelihood of cost-effectiveness when the WTP was between A$25 000 and A$35 000 per QALY. Cost-effectiveness results were further strengthened when applied to an Aboriginal and Torres Strait Islander cohort and when productivity costs were included. CONCLUSIONS: Cirrhosis population-wide screening for HCC is likely to be cost-effective in Australia. Risk-stratified screening using a serum biomarker test may be cost-effective at lower WTP thresholds.

RNA sequencing of LX-2 cells treated with TGF-ß1 identifies genes associated with hepatic stellate cell activation.

BACKGROUND: Hepatic stellate cells (HSCs) are liver-resident myofibroblast precursors responsible for the production of collagen and maintenance of the hepatic extracellular matrix (ECM). As such, they are generally associated with fibrotic liver diseases. HSCs become "activated" in response to tissue damage or pathogen invasion, a process most commonly driven by transforming growth factor-ß1 (TGF-ß1). Despite this, the full extent of TGF-ß1 signalling in these cells is poorly understood. Clarifying the range and diversity of this signalling will further improve our understanding of the process of HSC activation. METHODS AND RESULTS: RNA sequencing was used to quantitate the transcriptomic changes induced in LX-2 cells, an activated human HSC line, following TGF-b1 treatment. In total, 5,258 genes were found to be significantly differentially expressed with a false discovery rate cut-off of < 0.1. The topmost deregulated of these genes included those with no currently characterised role in either HSC activation or fibrotic processes, including CIITA and SERPINB2. In silico analysis revealed the prominent signalling pathways downstream of TGF-ß1 in LX-2 cells. CONCLUSIONS: In this study, we describe the genes and signalling pathways significantly deregulated in LX-2 cells following TGF-ß1 treatment. We identified several highly deregulated genes with no currently characterised role in HSC activation, which may represent novel mediators of fibrotic responses in HSCs or the liver macroenvironment. This work may be of use in the identification of new markers of liver fibrosis and could provide insight into prospective genes or pathways that might be targeted for the amelioration of fibrotic liver disease in the future.

The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes.

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

Regression of Fibrosis Stage With Treatment Reduces Long-Term Risk of Liver Cancer in Patients With Hemochromatosis Caused by Mutation in HFE.

BACKGROUND & AIMS: Fibrosis stage can decrease following treatment in patients with hemochromatosis caused by mutations in the homeostatic iron regulator gene (HFE), but the effects on cirrhosis are not clear. We assessed regression of severe fibrosis and the ensuing risk of liver cancer after treatment. METHODS: We performed a retrospective analysis of data from 106 patients in France or Australia who were homozygous for the C282Y mutation in HFE with F3 fibrosis (n = 40) or F4 fibrosis (n = 66) at diagnosis and from whom at least 1 liver biopsy was collected during follow up. We collected data from the time of first biopsy and during follow-up period on patient demographics, treatment, smoking habits, alcohol consumption, infection with hepatitis B or C viruses, and other diseases. The median time between first and last liver biopsy was 9.5 years (range, 3.5-15.6 years). We collected results of tests for liver function, markers of iron stores, and platelet levels. Patients were followed for a median 17.6 years (range, 9.8-24.1 years) for development of liver cancer occurrence. RESULTS: At last liver biopsy, 41 patients (38.6%) had fibrosis scores of F2 or less. Liver cancer occurred in 34 patients (52.3%) with F3 or F4 fibrosis at last liver biopsy vs 2 patients (4.8%) with fibrosis scores of F2 or less at last liver biopsy (P < .001). Liver cancer incidences were 32.8 per 1000 person-years (95% CI, 22.7-45.9 per 1000 person-years) in patients with F3 or F4 fibrosis and 2.3 per 1000 person-years (95% CI, 0.2-8.6 per 1000 person-years) in patients with fibrosis scores of F2 or less (P < .001). In multivariate analysis, male sex (hazard ratio [HR], 6.09; 95% CI, 1.21-30.4), age at diagnosis (HR, 1.16; 95% CI, 1.09-1.25), presence of diabetes (HR, 3.07; 95% CI, 1.35-6.97), excess alcohol consumption (HR, 3.1; 95% CI, 1.47-6.35), serum level of ferritin at diagnosis (P < .01), and regression to fibrosis scores of F2 or less (HR, 0.08; 95% CI, 0.01-0.62) were significantly associated with risk of liver cancer. CONCLUSIONS: In a retrospective analysis of patients with hemochromatosis caused by the C282Y mutation in HFE, we found that severe liver fibrosis can regress with treatment. In patients with fibrosis regression to a stage F2 or less, the long-term risk for liver cancer is significantly reduced.

Increased frequency of GNPAT p.D519G in compound HFE p.C282Y/p.H63D heterozygotes with elevated serum ferritin levels.

Glyceronephosphate O-acyltransferase (GNPAT) p.D519G (rs11558492) was identified as a genetic modifier correlated with more severe iron overload in hemochromatosis through whole-exome sequencing of HFE p.C282Y homozygotes with extreme iron phenotypes. We studied the prevalence of p.D519G in HFE p.C282Y/p.H63D compound heterozygotes, a genotype associated with iron overload in some patients. Cases were Australian participants with elevated serum ferritin (SF) levels ≥300µg/L (males) and ≥200µg/L (females); subjects whose SF levels were below these cut-offs were designated as controls. Samples were genotyped for GNPAT p.D519G. We compared the allele frequency of the present subjects, with/without elevated SF, to p.D519G frequency in public datasets. GNPAT p.D519G was more prevalent in our cohort of p.C282Y/p.H63D compound heterozygotes with elevated SF (37%) than European public datasets: 1000G 21%, gnomAD 20% and ESP 21%. We conclude that GNPAT p.D519G is associated with elevated SF in Australian HFE p.C282Y/p.H63D compound heterozygotes.

Phenotypic analysis of hemochromatosis subtypes reveals variations in severity of iron overload and clinical disease.

The clinical progression of HFE-related hereditary hemochromatosis (HH) and its phenotypic variability has been well studied. Less is known about the natural history of non-HFE HH caused by mutations in the HJV, HAMP, or TFR2 genes. The purpose of this study was to compare the phenotypic and clinical presentations of hepcidin-deficient forms of HH. A literature review of all published cases of genetically confirmed HJV, HAMP, and TFR2 HH was performed. Phenotypic and clinical data from a total of 156 patients with non-HFE HH was extracted from 53 publications and compared with data from 984 patients with HFE-p.C282Y homozygous HH from the QIMR Berghofer Hemochromatosis Database. Analyses confirmed that non-HFE forms of HH have an earlier age of onset and a more severe clinical course than HFE HH. HJV and HAMP HH are phenotypically and clinically very similar and have the most severe presentation, with cardiomyopathy and hypogonadism being particularly prevalent findings. TFR2 HH is more intermediate in its age of onset and severity. All clinical outcomes analyzed were more prevalent in the juvenile forms of HH, with the exception of arthritis and arthropathy, which were more commonly seen in HFE HH. This is the first comprehensive analysis comparing the different phenotypic and clinical aspects of the genetic forms of HH, and the results will be valuable for the differential diagnosis and management of these conditions. Importantly, our analyses indicate that factors other than iron overload may be contributing to joint pathology in patients with HFE HH.

Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis.

BACKGROUND & AIMS: Although transient elastography (TE) is used to determine liver stiffness as a surrogate to hepatic fibrosis, the normal range in children is not well defined. We performed a systematic review and individual participant data (IPD) meta-analysis to determine the range of liver stiffness in healthy children and evaluate the influence of important biological parameters. METHODS: We pooled data from 10 studies that examined healthy children using TE. We divided 1702 children into two groups: ≥3 years (older group) and < 3 years of age (younger group). Univariate and multivariate linear regression models predicting liver stiffness were conducted. RESULTS: After excluding children with obesity, diabetes, or abnormal liver tests, 652 children were analysed. Among older children, mean liver stiffness was 4.45 kPa (95% confidence interval 4.34-4.56), and increased liver stiffness was associated with age, sedation status, and S probe use. In the younger group, the mean liver stiffness was 4.79 kPa (95% confidence interval 4.46-5.12), and increased liver stiffness was associated with sedation status and Caucasian race. In a subgroup analysis, hepatic steatosis on ultrasound was significantly associated with increased liver stiffness. We define a reference range for normal liver stiffness in healthy children as 2.45-5.56 kPa. CONCLUSIONS: We have established TE-derived liver stiffness ranges for healthy children and propose an upper limit of liver stiffness in healthy children to be 5.56 kPa. We have identified increasing age, use of sedation, probe size, and presence of steatosis on ultrasound as factors that can significantly increase liver stiffness.

Live imaging of collagen deposition during experimental hepatic schistosomiasis and recovery: a view on a dynamic process.

Hepatic fibrosis is the central cause of chronic clinical pathology resulting from infection by the blood flukes Schistosoma japonicum or S. mansoni. Much has been elucidated regarding the molecular, cellular and immunological responses that correspond to the formation of the granulomatous response to trapped schistosome eggs. A central feature of this Th2 response is the deposition of collagen around the periphery of the granuloma. To date, traditional histology and transcriptional methods have been used to quantify the deposition of collagen and to monitor the formation of the hepatic granuloma during experimental animal models of schistosomiasis. We have investigated the dynamic nature of granuloma formation through the use of a transgenic mouse model (B6.Collagen 1(A) luciferase mice (B6.Coll 1A-luc+)). With this model and whole-animal bioluminescence imaging, we followed the deposition of collagen during an active schistosome infection with Chinese and Philippines geographical strains of S. japonicum and after clearance of the adult parasites by the drug praziquantel. Individual mice were re-imaged over the time course to provide robust real-time quantitation of the development of chronic fibrotic disease. This model provides an improved method to follow the course of hepatic schistosomiasis-induced hepatic pathology and effectively supports the current dogma of the formation of hepatic fibrosis, originally elucidated from static traditional histology. This study demonstrates the first use of the B6.Coll 1A-luc+ mouse to monitor the dynamics of disease development and the treatment of pathogen-induced infection with the underlying pathology of fibrosis.

Accuracy of Transient Elastography Data Combined With APRI in Detection and Staging of Liver Disease in Pediatric Patients With Cystic Fibrosis.

BACKGROUND & AIMS: Liver disease develops in 15%-72% of patients with cystic fibrosis, and 5%-10% develop cirrhosis or portal hypertension, usually during childhood. Transient elastography (TE) is a noninvasive method to measure liver stiffness. We aimed to validate its accuracy in detection of liver disease and assessment of fibrosis in children with cystic fibrosis. METHODS: We performed a cross-sectional study to evaluate the accuracy of TE in analysis of liver disease in 160 consecutive children who presented with cystic fibrosis (9.0 ± 0.4 years old, 53% male) at a tertiary referral pediatric center in Australia, from 2011 through 2016. Patients were classified as having cystic fibrosis-associated liver disease (CFLD) or cystic fibrosis without liver disease (CFnoLD) based on clinical, biochemical, and imaging features. Fibrosis severity was determined from histologic analysis of dual-pass liver biopsies from children with CFLD, as the reference standard. Data from healthy children without cystic fibrosis (n = 64, controls) were obtained from a separate study. Liver stiffness measurements (LSMs) were made by Fibroscan analysis, using the inter-quartile range/median ≤30% of 10 valid measurements. Children with macronodularity or portal hypertension with heterogeneous changes on ultrasound without available biopsy were assigned to the category of stage F3-F4 fibrosis. RESULTS: LSM was made reliably in 86% of children; accuracy increased with age. LSMs were significantly higher in children with CFLD (10.7 ± 2.4 kPa, n = 33) than with CFnoLD (4.6 ± 0.1 kPa, n = 105) (P < .0001) or controls (4.1 ± 0.1kPa) (P < .0001); LSMs were higher in children with CFnoLD than controls (P < .05). At a cut-off value of 5.55kPa, LSM identified children with CFLD with an area under the receiver operating characteristic (AUROC) curve of 0.82, 70% sensitivity, and 82% specificity (P < .0001). Classification and regression tree models that combined LSM and aspartate aminotransferase to platelet ratio index (APRI) identified children with CFLD with an AUROC curve of 0.89, 87% sensitivity, and 74% specificity (odds ratio, 18.6). LSMs correlated with fibrosis stage in patients with CFLD (r = 0.67, P = .0001). A cut-off value of 8.7kPa differentiated patients with stage F3-F4 fibrosis from patients with stage F1-F2 fibrosis (AUROC, 0.87; 75% sensitivity; 100% specificity, P=.0002). The combination of LSMs and APRI improved the differentiation of patients with F3-F4 fibrosis vs F1-F2 fibrosis (AUROC, 0.92; 83% sensitivity; and 100% specificity (P < .01). CONCLUSIONS: LSMs made by TE accurately detect liver disease in children with cystic fibrosis; diagnostic accuracy increases when LSMs are combined with APRI. LSMs also differentiate between children with cystic fibrosis with mild-moderate fibrosis vs advanced fibrosis.

Detection of HFE Haemochromatosis in the clinic and community using standard erythrocyte tests.

Detection of HFE Haemochromatosis (HH) is challenging in the absence of clinical features. HH subjects have elevated erythrocyte parameters compared to those without HH, but it remains unclear how this could be applied in clinical practice. Thus, we determined the sensitivity, specificity and clinical utility of erythrocyte parameters in 144 HH subjects with (n = 122) or without (n = 22) clinical and/or biochemical expression of iron overload, 1844 general population controls, and 700 chronic disease subjects. For both expressing and non-expressing HH subjects, the mean pre- and post-phlebotomy values of mean cell volume (MCV) and mean cell haemoglobin (MCH) were always significantly higher when compared to all other groups and demonstrated excellent diagnostic utility for detection of HH in men and women (AUROC 0.83-0.9; maximal sensitivity and specificity 82% and 78%) using cut-off values for MCV >91 fL or MCH >31 pg, respectively. Between 34 and 62% of all HH subjects would be detected, and <4% of all non-HH subjects would undergo unnecessary testing, if those with MCV or MCH values >94 fL or 32.2 pg, respectively, were evaluated.

MicroRNA Sequencing Identifies a Serum MicroRNA Panel, Which Combined With Aspartate Aminotransferase to Platelet Ratio Index Can Detect and Monitor Liver Disease in Pediatric Cystic Fibrosis.

Cystic fibrosis (CF)-associated liver disease (CFLD) is a hepatobiliary complication of CF. Current diagnostic modalities rely on nonspecific assessments, whereas liver biopsy is the gold standard to assess severity of fibrosis. MicroRNAs (miRNAs) regulate liver disease pathogenesis and are proposed as diagnostic biomarkers. We investigated the combined use of serum miRNAs and aspartate aminotransferase (AST) to platelet ratio (APRI) to diagnose and assess CFLD severity. This was a cross-sectional cohort study of the circulatory miRNA signature of 124 children grouped by clinical, biochemical, and imaging assessments as follows: CFLD (n = 44), CF patients with no evidence of liver disease (CFnoLD; n = 40), and healthy controls (n = 40). Serum miRNAs were analyzed using miRNA sequencing (miRNA-Seq). Selected differentially expressed serum miRNA candidates were further validated by qRT-PCR and statistical analysis performed to evaluate utility to predict CFLD and fibrosis severity validated by liver biopsy, alone or in combination with APRI. Serum miR-122-5p, miR-365a-3p, and miR-34a-5p levels were elevated in CFLD compared to CFnoLD, whereas miR-142-3p and let-7g-5p were down-regulated in CFLD compared to CFnoLD. Logistic regression analysis combining miR-365a-3p, miR-142-3p, and let-7g-5p with APRI showed 21 times greater odds of accurately predicting liver disease in CF with an area under the receiver operating characteristics curve (AUROC) = 0.91 (sensitivity = 83%, specificity = 92%; P < 0.0001). Expression levels of serum miR-18a-5p were correlated with increasing hepatic fibrosis (HF) stage in CFLD (rs  = 0.56; P < 0.0001), showing good diagnostic accuracy for distinguishing severe (F3-F4) from mild/moderate fibrosis (F0-F2). A unit increase of miR-18a-5p showed a 7-fold increased odds of having severe fibrosis with an AUROC = 0.82 (sensitivity = 93%, specificity = 73%; P = 0.004), indicating its potential to predict fibrosis severity. Conclusion: We identified a distinct circulatory miRNA profile in pediatric CFLD with potential to accurately discriminate liver disease and fibrosis severity in children with CF.

Serum MicroRNAs as Biomarkers in Hepatitis C: Preliminary Evidence of a MicroRNA Panel for the Diagnosis of Hepatocellular Carcinoma.

Early diagnosis of cirrhosis and hepatocellular carcinoma (HCC) due to chronic Hepatitis C (CHC) remain clinical priorities. In this pilot study, we assessed serum microRNA (miRNA) expression to distinguish cirrhosis and HCC, alone and in combination with the aminotransferase-to-platelet ratio (APRI), Fibrosis 4 (FIB-4), and alpha-fetoprotein (AFP). Sixty CHC patients were subdivided into 3 cohorts: Mild disease (fibrosis stage F0-2; n = 20); cirrhosis (n = 20); and cirrhosis with HCC (n = 20). Circulating miRNA signatures were determined using a liver-specific real-time quantitative reverse transcription PCR (qRT-PCR) microarray assessing 372 miRNAs simultaneously. Differentially-expressed miRNA candidates were independently validated using qRT-PCR. Serum miRNA-409-3p was increased in cirrhosis versus mild disease. In HCC versus cirrhosis, miRNA-486-5p was increased, whereas miRNA-122-5p and miRNA-151a-5p were decreased. A logistic regression model-generated panel, consisting of miRNA-122-5p + miRNA-409-3p, distinguished cirrhosis from mild disease (area under the curve, AUC = 0.80; sensitivity = 85%, specificity = 70%; p < 0.001). When combined with FIB-4 or APRI, performance was improved with AUC = 0.89 (p < 0.001) and 0.87 (p < 0.001), respectively. A panel consisting of miRNA-122-5p + miRNA-486-5p + miRNA-142-3p distinguished HCC from cirrhosis (AUC = 0.94; sensitivity = 80%, specificity = 95%; p < 0.001), outperforming AFP (AUC = 0.64, p = 0.065). Serum miRNAs are differentially expressed across the spectrum of disease severity in CHC. MicroRNAs have great potential as diagnostic biomarkers in CHC, particularly in HCC where they outperform the only currently-used biomarker, AFP.

Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction: Role in Pediatric Cystic Fibrosis Liver Disease.

Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis; however, its cause(s) and early pathogenesis are unclear. We hypothesized that a bile acid-induced ductular reaction (DR) drives fibrogenesis. The DR was evaluated by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD, and it demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction, liver progenitor cells (LPCs) were treated with taurocholate, and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation, and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin 19, connexin 43, integrin ß4, and γ-glutamyltranspeptidase), whereas the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia+ LPCs, and increased active γ-glutamyltranspeptidase enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α, and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7+ DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD.

Cirrhosis in Hemochromatosis: Independent Risk Factors in 368 HFE p.C282Y Homozygotes.

INTRODUCTION AND AIM: We sought to identify independent risk factors for cirrhosis in HFE p.C282Y homozygotes in a cross-sectional study. MATERIAL AND METHODS: We evaluated 368 p.C282Y homozygotes who underwent liver biopsy and compared characteristics of those with and without cirrhosis. We performed multivariable logistic regression on cirrhosis with: age; sex; race/ethnicity; diabetes; blood pints/units donated voluntarily; erythrocyte pints/units received; iron supplement use; alcohol intake, g/d; body mass index, kg/m2; swollen/tender 2nd/3rd metacarpophalangeal joints; elevated alanine aminotransferase; elevated aspartate aminotransferase; steatosis/fatty liver; iron removed by phlebotomy, g; and GNPAT p.D519G positivity. RESULTS: Mean age of 368 participants (73.6% men) was 47 ± 13 (standard deviation) y. Cirrhosis was diagnosed in 86 participants (23.4%). Participants with cirrhosis had significantly greater mean age, proportion of men, diabetes prevalence, mean daily alcohol intake, prevalence of swollen/ tender 2nd/3rd metacarpophalangeal joints, mean serum ferritin, elevated alanine aminotransferase, elevated aspartate aminotransferase, and mean iron removed; and significantly fewer mean blood pints/units donated. GNPAT p.D519G positivity was detected in 82 of 188 participants (43.6%). In a multivariable model for cirrhosis, there were four significant positive associations: age (10-y intervals) (odds ratio 2.2 [95% confidence interval 1.5, 3.3]); diabetes (3.3; [1.1, 9.7]); alcohol intake (14 g alcohol drinks/d) (1.5 [1.2, 1.8]); and iron removed, g (1.3 [1.2, 1.4]). There was no statistical evidence of two-way interactions between these variables. CONCLUSION: In conclusion, cirrhosis in HFE p.C282Y homozygotes is significantly associated with age, diabetes, daily alcohol intake, and iron removed by phlebotomy, taking into account the effect of other variables.