Louise E. Ramm

Serum hyaluronic acid with serum ferritin accurately predicts cirrhosis and reduces the need for liver biopsy in C282Y hemochromatosis

Diagnosing the presence of cirrhosis is crucial for the management of patients with C282Y hereditary hemochromatosis (HH). HH patients with serum ferritin >1,000 μg/L are at risk of cirrhosis; however, the majority of these patients do not have cirrhosis. Noninvasive markers of hepatic fibrosis may assist in determining which patients with a serum ferritin >1,000 μg/L have cirrhosis and require liver biopsy. This study evaluated the utility of current diagnostic algorithms for detecting cirrhosis, including serum ferritin concentration, platelet counts, and aspartate aminotransferase (AST) levels, in combination with serum markers of fibrosis, hyaluronic acid and collagen type IV (CLIV), in predicting cirrhosis in HH patients. Stage of fibrosis, serum hyaluronic acid and CLIV levels, were measured in 56 patients with HH. No patient with a serum ferritin <1,000 μg/L had cirrhosis, but only 40% of patients with serum ferritin >1,000 μg/L were cirrhotic. A combination of platelet count (<200 × 109/L), elevated AST, and serum ferritin >1,000 μg/L did not detect 30% of cirrhotic subjects. Serum hyaluronic acid was increased in HH compared with controls (42.0 ± 9.8 ng/mL versus 19.3 ± 1.8 ng/mL; P = 0.02). A hyaluronic acid concentration >46.5 ng/mL was 100% sensitive and 100% specific in identifying patients with cirrhosis. In patients with serum ferritin >1,000 μg/L, hyaluronic acid levels were significantly elevated in patients with cirrhosis versus those without cirrhosis (137 ± 34.4 ng/mL versus 18.6 ± 1.5 ng/mL, respectively; P = 0.006). CLIV >113 ng/mL was 100% sensitive but only 56% specific for cirrhosis (area under the curve = 0.78; P = 0.01). Conclusion: In HH, the measurement of hyaluronic acid in patients with serum ferritin >1,000 μg/L is a noninvasive, accurate, and cost‐effective method for the diagnosis of cirrhosis. (HEPATOLOGY 2009;49:418–425.)

Aspartate aminotransferase to platelet ratio and fibrosis-4 as biomarkers in biopsy-validated pediatric cystic fibrosis liver disease

Up to 10% of cystic fibrosis (CF) children develop cirrhosis by the first decade. We evaluated the utility of two simple biomarkers, aspartate aminotransferase to platelet ratio index (APRI) and FIB‐4, in predicting degree of fibrosis in pediatric CF liver disease (CFLD) validated by liver biopsy. In this retrospective, cross‐sectional study, 67 children with CFLD had dual‐pass liver biopsies and 104 age‐ and sex‐matched CF children without liver disease (CFnoLD) had serum to calculate APRI and FIB‐4 collected at enrollment. CFLD was defined as having two of the following: (1) hepatomegaly ± splenomegaly; (2) >6 months elevation of ALT (>1.5× upper limit of normal ULN); or (3) abnormal liver ultrasound findings. Biopsies were staged according to Metavir classification by two blinded pathologists. Receiver operating characteristic (ROC) analysis and continuation ratio logistic regression were performed to assess the predictability of these biomarkers to distinguish CFLD from CFnoLD and determine fibrosis stage‐specific cut‐off values. The AUC for APRI was better than FIB‐4 (0.75 vs. 0.60; P = 0.005) for predicting CFLD and severe CFLD (F3‐F4) (0.81). An APRI score >0.264 demonstrated a sensitivity (95% confidence interval [CI]) of 73.1% (60.9, 83.2) and specificity of 70.2% (60.4, 78.8) in predicting CFLD. A 50% increase in APRI was associated with a 2.4‐fold (95% CI: 1.7, 3.3) increased odds of having CFLD. APRI demonstrated full agreement with histology staging 37% of the time, but was within one stage 73% of the time. Only FIB‐4 predicted portal hypertension at diagnosis (area under the receiver operator characteristic curve [AUC] = 0.91; P < 0.001). Conclusion: This is the first liver biopsy‐validated study of APRI and FIB‐4 in pediatric CFLD. APRI appears superior to FIB‐4 in differentiating CFLD versus CFnoLD. APRI also exhibited a high AUC in predicting severe liver fibrosis with specific cutoffs for lower stages. (Hepatology 2015;62:1576–1583)

Should HFE p.C282Y homozygotes with moderately elevated serum ferritin be treated? A randomised controlled trial comparing iron reduction with sham treatment (Mi-iron).

Introduction HFE p.C282Y homozygosity is the most common cause of hereditary haemochromatosis. There is currently insufficient evidence to assess whether non-specific symptoms or hepatic injury in homozygotes with moderately elevated iron defined as a serum ferritin (SF) of 300–1000 µg/L are related to iron overload. As such the evidence for intervention in this group is lacking. We present here methods for a study that aims to evaluate whether non-specific symptoms and hepatic fibrosis markers improve with short-term normalisation of SF in p.C282Y homozygotes with moderate elevation of SF. Methods and analysis Mi-iron is a prospective, multicentre, randomised patient-blinded trial conducted in three centres in Victoria and Queensland, Australia. Participants who are HFE p.C282Y homozygotes with SF levels between 300 and 1000 μg/L are recruited and randomised to either the treatment group or to the sham treatment group. Those in the treatment group have normalisation of SF by 3-weekly erythrocytapheresis while those in the sham treatment group have 3-weekly plasmapheresis and thus do not have normalisation of SF. Patients are blinded to all procedures. All outcome measures are administered prior to and following the course of treatment/sham treatment. Patient reported outcome measures are the Modified Fatigue Impact Scale (MFIS-primary outcome), Hospital Anxiety and Depression Scale (HADS), Medical Outcomes Study 36-item short form V.2 (SF36v2) and Arthritis Impact Measurement Scale 2 short form (AIMS2-SF). Liver injury and hepatic fibrosis are assessed with transient elastography (TE), Fibrometer and Hepascore, while oxidative stress is assessed by measurement of urine and serum F2-isoprostanes. Ethics and dissemination This study has been approved by the Human Research Ethics Committees of Austin Health, Royal Melbourne Hospital and Royal Brisbane and Womens Hospital. Study findings will be disseminated through peer-reviewed publications and conference presentations. Trial registration Trial identifier: NCT01631708; Registry: ClinicalTrials.gov

The CD4+ T-cell response to protein immunization is independent of accompanying IFN-gamma-producing CD8+ T cells.

By virtue of their strong bias towards production of interferon‐γ (IFN‐γ), CD8+ T cells have the potential to promote the development of type 1 immune responses. We have previously shown that the CD4+ T‐cell response to immunization with the protein antigen keyhole limpet haemocyanin (KLH) has a mixed interleukin‐4 (IL‐4)/IFN‐γ production profile. Here we show that this immunization regimen also stimulates accumulation in the draining lymph nodes of CD8+ T cells, which preferentially contain IFN‐γ mRNA ex vivo and secrete IFN‐γ protein in vitro. This provides a model to test whether CD8+ cell‐derived IFN‐γ participates in the normal control of the immune response to a non‐viable exogenous antigen. To investigate regulation of the anti‐KLH response by the CD8+ population or IFN‐γ produced by this or other cell types, mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on the frequency of clonogenic KLH‐specific CD4+ T cells, the IL‐4/IFN‐γ profiles of their progeny, or the isotype profiles of the serum antibody response to KLH. In contrast, IFN‐γ neutralization diminished cell accumulation in the lymph nodes and reduced both the frequency of KLH‐specific CD4+ T cells that gave rise to IFN‐γ‐producing clones and serum titres of KLH‐specific IgG2a and IgG3. Therefore, despite the potential for cross‐regulation, the CD4+ T‐cell response to this immunogen is independent of the IFN‐γ‐skewed CD8+ response.

Reduction of body iron in HFE-related haemochromatosis and moderate iron overload (Mi-Iron): a multicentre, participant-blinded, randomised controlled trial

BACKGROUND The iron overload disorder hereditary haemochromatosis is most commonly caused by HFE p.Cys282Tyr homozygosity. In the absence of results from any randomised trials, current evidence is insufficient to determine whether individuals with hereditary haemochromatosis and moderately elevated serum ferritin, should undergo iron reduction treatment. This trial aimed to establish whether serum ferritin normalisation in this population improved symptoms and surrogate biomarkers. METHODS This study was a multicentre, participant-blinded, randomised controlled trial done at three centres in Australia. We enrolled people who were homozygous for HFE p.Cys282Tyr, aged between 18 and 70 years, with moderately elevated serum ferritin, defined as 300-1000 μg/L, and raised transferrin saturation. Participants were randomly assigned, via a computer-generated random number, to undergo either iron reduction by erythrocytapheresis (treatment group) or sham treatment by plasmapheresis (control group). Randomisation was stratified by baseline serum ferritin (<600 μg/L or ≥600 μg/L), sex, and study site. Erythrocytapheresis and plasmapheresis were done every 3 weeks, the number of procedures and volume of red cells or plasma removed determined on the basis of each patients haemoglobin, haematocrit, and serum ferritin concentration, as well their height and weight. In the erythrocytapheresis group, the target was to reduce serum ferritin to less than 300 μg/L. The number of procedures for the control group was based on the initial serum ferritin and prediction of decrease in serum ferritin of approximately 120 μg/L per treatment. The primary outcome was patient-reported Modified Fatigue Impact Scale (MFIS) score, measured at baseline and before unblinding. Analyses were by intention to treat, including the safety analysis. The trial is registered with ClinicalTrials.gov, number NCT01631708, and has been completed. FINDINGS Between Aug 15, 2012, and June 9, 2016, 104 participants were randomly assigned to the treatment (n=54) and control (n=50) groups, of whom 94 completed the study (50 in the treatment group and 44 in the control group). Improvement in MFIS score was greater in the treatment group than in the control group (mean difference -6·3, 95% CI -11·1 to -1·4, p=0·013). There was a significant difference in the cognitive subcomponent (-3·6, -5·9 to -1·3, p=0·0030), but not in the physical (-1·90 -4·5 to 0·63, p=0·14) and psychosocial (-0·54, -1·2 to 0·11, p=0·10) subcomponents. No serious adverse events occurred in either group. One participant in the control group had a vasovagal event and 17 participants (14 in the treatment group and three in the control group) had transient symptoms assessed as related to hypovolaemia. Mild citrate reactions were more common in the treatment group (32 events [25%] in 129 procedures) compared with the control group (one event [1%] in 93 procedures). INTERPRETATION To our knowledge, this study is the first to objectively assess the consequences of iron removal in individuals with hereditary haemochromatosis and moderately elevated serum ferritin. Our results suggest that serum ferritin normalisation by iron depletion could be of benefit for all individuals with hereditary haemochromatosis and elevated serum ferritin levels. FUNDING National Health and Medical Research Council (Australia).

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.

miRNA‐Seq identifies a serum miRNA panel, which combined with APRI can detect and monitor liver disease in paediatric 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.This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/hep.30156 This article is protected by copyright. All rights reserved. DR. GRANT A. RAMM (Orcid ID : 0000-0001-7058-6201)

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.