Clinical advances in pediatric hepatology


121

Genetic polyporphism of IL28B gene and spontaneous clearance of hepatitis C virus in c hildren

Giuseppe Indolfi1, Giusi Mangone2, Pier Luigi Calvo3, Elisa Bortolini1, Marta Regoli1, Daniele Serranti4, Carmelina Calitri3, Pier Angelo Tovo3, Maurizio de Martino4, Chiara Azzari2, 3, Massimo Resti1

1Paediatric and Liver Unit, Meyer Children΄s University Hospital of Florence, Italy, Florence, Italy; 2Immunology Unit and Laboratory of Meyer Children΄s University Hospital of Florence, Italy, Florence, Italy; 3Department of Pediatrics, Infectious Diseases Unit, University of Turin, Regina Margherita Children's Hospital, Turin, Italy, Turin, Italy; 4Department of Health Sciences, University of Florence, Italy, Florence, Italy

Spontaneous clearance of hepatitis C virus (HCV) occurs in 8-20% of the infected children. Over half of the children who undergo spontaneous clearance of the infection are infected by HCV genotype 3, and develop an aminotransferase peak at the onset of the infection. Recent genome-wide association studies performed in adults documented that single nucleotide polymorphisms (SNPs, rs12979860 and rs8099917) located on chromosome 19, upstream of the interleukin (IL) 28B gene which encodes the type III interferon (IFN)-λ3 were associated with spontaneous clearance of hepatitis C virus HCV and with response to treatment with pegylated interferon and ribavirin. Preliminary results on small cohorts of children seem to confirm this association. The aim of the present collaborative study was to evaluate the role of rs12979860 single nucleotide polymorphisms in predicting spontaneous clearance of hepatitis C virus in a large cohort of Italian children. One hundred and fifty three consecutive children (median age 14.6 years, IQR 10.2) regularly followed-up in two Italian pediatric hospitals, the Meyer Children΄s University Hospital of Florence and the Regina Margherita Children΄s Hospital of Turin, were enrolled and genotyped. Spontaneous clearance of HCV was defined in children older than 18 months of age when polymerase chain reaction for HCV ribonucleic acid was negative and HCV antibodies were positive in at least three blood samples taken 6 months apart; positive polymerase chain reaction for HCV ribonucleic acid and HCV antibodies defined chronic hepatitis C. One hundred and thirty children (86.7%) were chronically infected while 23 (13.3%) had spontaneous clearance of the virus. The IL28 C/C and C/TT/T genotypes were found in 57 (37.3%) and 96 (62.7%) children, respectively. Children with the C/C genotype (14/57; 24.6%) were 3 times more likely to clear hepatitis C virus than those with the C/T and T/T genotypes combined (9/96; 9.4%; odds ratio 3.15; 90% confidence intervals 1.34-7.53; p 0.01). The highest clearance rate was found among C/C children (24.6%), an intermediate clearance rate among heterozygous C/T (8/70; 11.4%; odds ratio 2.52; 90% confidence intervals 1.03-6.3 versus CC; p 0.05) and the lowest rate in the T/T homozygous children (1/26; 8.14%; odds ratio 2.52; 90% confidence intervals 1.27-132.98 versus CC; p 0.02). Results of this study carried out on 153 children with HCV infection demonstrated that IL28B rs12979860 C/C SNP was associated with spontaneous clearance of HCV also in the pediatric age.

Disclosures:

The following people have nothing to disclose: Giuseppe Indolfi, Giusi Mangone, Pier Luigi Calvo, Elisa Bartolini, Marta Regoli, Daniele Serranti, Carmelina Calitri, Pier-Angelo Tovo, Maurizio de Martino, Chiara Azzari, Massimo Resti

122

Expression of interferon-stimulated genes (IGS) in the liver - role for predicting response to antiviral therapy in chronic hepatitis B infection?

Ivana Carey1, Matthew J. Bruce1, Mary Horner1, Kate Childs1, Deepak Joshi1, Sanjay Bansal1, 2, Diego Vergani1, Giorgina MieliVergani1, 2

1Institute of Liver Studies' Kings College School of Medicine of King΄s College Hospital London, United Kingdom; 2Paediofric Liver, GI & Nutrition Centre, King΄s College Hospital, London, United Kingdom

Hepatitis B virus (HBV) is evading host immune responses by failure to induce efficient expression of interferon-stimulated genes (ISG) within the liver in early stage of infection. This mechanism might be associated with lack of response to interferon-α therapy. No data are available on ISG expression in the liver in relation to antiviral therapy response (HBsAg loss) in chronic hepatitis B (CHB). CXCL10 gene is interferon-۷ inducible gene and encoding interferon-۷ inducible protein 10kDa (IP10). Increase in IP10 levels during CHB antiviral therapy was predictive of HBsAg loss. Aims: To investigate whether there are differences in the expression of interferon-a/β inducible genes (ISG15, USP18, MxA, 〇AS2 and 〇AS3) and interferon-γ inducible gene (CXCL10) within the liver prior to therapy with lead-in lamivudine and add-on interferon-α between therapy responders (HBsAg loss) and non-responders in children with infancy-acquired CHB. Patients: 23 children (8 males, median age 10.2 yrs) with infancy-acquired CHB (all HBeAg+), treated for 52 weeks [lead-in LAM (3mg/kg/d) for 9 weeks; add-on IFN-α (5MU/m2 TIW) from week 9 for 44 weeks], were divided according to treatment response: 5 responders (R= HBsAg loss) and 18 non-responders (NR). Methods: Total RNA was extracted from pre-treatment biopsies in all patients and 3 healthy adult controls. mRNA expression of housekeeping gene HPRT1, 6 interferon-inducible genes (ISG15, USP18, MxA, OAS2, OAS3 and CXCL10) was measured by quantitative real-time RT-PCR. Plasma IP-1 0, HBsAg, AST and HBV DNA levels were measured at therapy baseline by ELISA [pg/ml], Abbott ARCHITECT® assay [log10 IU/ml], AutoAnalyser [IU/1] and real-time TaqMan PCR [log10 IU/ml]. The results were compared between responders and nonresponders. Results: mRNA expression of interferon-a/β inducible genes (ISG15, USP18, MxA, 〇AS2 and 〇AS3) was similar in between R and NR, but was lower than in healthy controls (ISG15: median 0.2 vs. 1, p<0.05). CXCL10 mRNA expression was significantly lower in R than NR (median 0.62 vs. 1.4, p<0.05). Baseline plasma IP10, AST and HBV DNA levels were similar in R and NR (median IP10: 123 vs. 99, p=0.4; AST: 29 vs. 31, p=0.6 and HBV DNA: 8.21 vs. 8.13, p=0.2). Baseline HBsAg levels were lower in R than NR (median 4.36 vs. 4.74, p=0.02). There was no correlation between baseline plasma IP10 and AST levels and mRNA CXCL10 expression in the liver. Conclusions: ISG expression in the liver was downregulated in children with infancy-acquired CHB and lower CXCL10 expression was linked with HBsAg loss. Further studies into the role of ISG in predicting therapy response are needed to elucidate mechanisms involved.

Disclosures:

Ivana Carey - Grant/Research Support: Gilead, BMS, Roche; Speaking and Teaching: BMS

The following people have nothing to disclose: Matthew J. Bruce, Mary Horner, Kate Childs, Deepak Joshi, Sanjay Bansal, Diego Vergani, Giorgina Mieli-Vergani

123

Dynamics of Allograft Fibrosis in Pediatric Liver Transplantation

Carla Venturi1, Christine Sempoux2, Etienne M. Sokal1, Xavier Stephenne1, Christophe Bourdeaux3, Raymond Reding3

1Pediatric Gastroenterology Unit, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium; 2Pathology Deporfment, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium; 3Pediatric Surgery and Transplant Unit, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium

Aims: To evaluate liver allograft fibrosis (LAF) dynamics in longterm pediatric liver transplant (LT)-recipients using a novel histologic scoring system and to study the influence of clinical variables and immunosuppression in LAF development. Methods: Clinical, biochemical data and histology were revised in 54 primary LT-recipients (median age: 1.3yrs. (r: 0.2-15.7) between 1999 and 2005.Immunosuppression: Steroids group: Tacrolimus (TAC) plus Steroids (n=24, 44%); No Steroids group: TAC plus basiliximab or TAC monotherapy (n=30, 56%). Protocol liver biopsies performed at 6 months, 3 and 7 years post- LT (n=162) were reviewed assessing LAF using METAVIR system and the Liver Allograft Fibrosis Score (LAFSc), previously designed and validated for LAF assessment, (Venturi C, et al, Am. J Transpl. 2012). Scoring evaluations were correlated with fibrosis quantification by morphometry using Sirius Red staining area (PSR %). Results: Progressive LAF was found in 40, (74%) of patients in the long-term (PSR% mean values: 6 months: 13.9 ±7.5; 3yrs: 11.6 ±6.8; 7yrs: 19.4± 9.6). Up to 70% of patients developed LAF with unaltered liver enzymes along the time. None patients developed cirrhosis or required re-LT. LAF separate assessments showed linear progression at centrolobular (p=0.003) and portal areas (p=0.004). Clinical variables multivariate analysis showed positive correlation for LAF development as follows: male gender; deceased donors; ischemia time> 400min.; biliary complications; vascular complications; gammaglobulins >15%; positive auto-antibodies (Anti-nuclear, Anti-smooth muscle and Anti-liver kidney mitochondrial antibodies) and lymphoproliferative disease. The association between clinical variables and LAF location is detailed in the Table. Deceased grafts showed higher fibrosis than living related grafts (n=29) (p=0.000). Steroid therapy was not associated with reduced fibrosis (p= 0.832). Conclusion: LAF could be viewed as a dynamic process with mostly progression along the time. Peri and post- LT associated variables may condition fibrosis development in a specific area of the liver parenchyma. LAFSc represents an accurate system to identify, evaluate and follow LAF in the long-term.

VariablesLAF correlationFibrosis Location
Gender (males, n=27)p=0.013Centrolobular 7y: p= 0.04 / Sinusoidal: p= 0.001
Deceased donor grafts (n=25)p=0.000Portal 6m p=0.001; 3y: p= 0.003; 7y: p=0.015
Ischemia time >400 minp=0.006Portal 6m: p= 0.062; 3y: p=0.006
Biliary complications 0-6 m(n=13)p=0.012Sinusoidal 6m: p=0.05; 3y: p=0.010
Vascular complications 0-6mp=0.044Centrolobular 7y: p=0.044
Gammaglobulins〉15%p= 0.020Centrolobular 7y: p=0.028
Positives Auto Antibodies (>1/40)p=0.017Centrolobular 3y: p=0.017
Lymphoproliferative disease (n=10)p=0.001Portal 7y: p= 0.012

Disclosures:

Etienne M. Sokal - Board Membership: Promethera Biosciences; Management Position: Promethera Biosciences; Patent Held/Filed: Promethera Biosciences

The following people have nothing to disclose: Carla Venturi, Christine Sempoux, Xavier Stephenne, Christophe Bourdeaux, Raymond Reding

124

Serum microRNAs as Novel Non-invasive Diagnostic Biomarkers of Liver Disease in Children with Cystic Fibrosis

Naomi L. Cook1,Tamara N. Pereira1,Peter J. Lewindon1, 2, Ross Shepherd1, 3, Grant A. Ramm1

1Hepatic Fibrosis' The Queensland Institute of Medical Research, Brisbane, QLD, Australia; 2Department of Gastroenterology, Royal Children΄s Hospital, Brisbane, QLD, Australia; 3Department of Pediatrics, Baylor College of Medicine, Houston, TX

Introduction: Liver disease causes significant fibrosis in up to 10% of children with cystic fibrosis (CF). The pathogenesis of CF-associated liver disease (CFLD) is incompletely understood, with onset and progression difficult to predict and monitor. Current measures of liver function, combined with ultrasound and clinical examination are insensitive and non-specific for detection of early liver disease and assessment of progressive fibrosis severity. Although biopsy remains the gold standard for diagnosis of CFLD, it is invasive and can be confounded by the focal nature of disease activity. Thus a sensitive, specific and non-invasive test is required to identify individuals at risk of developing CFLD prior to the advent of complications. Distinct circulating microRNAs (miRs) profiles have been identified in various adult chronic liver diseases. In this study we sought to quantify the expression of serum miRs in CFLD patients, CF patients without LD (CFnoLD) and non-CF pediatric controls. Methods: Serum was obtained with informed consent from 102 children (52 CFLD, 30 CFnoLD, 20 non-CF controls). RNA was extracted from 200μL serum and subjected to Qiagen Human Serum miRNA Real-Time RT-PCR Array, containing 84 miRs detectable in human serum. Identified candidate miRs were validated by RT to synthesise complementary DNA using the Exiqon miRCURY LNA PCR system for biofluids. Real-time PCR quantified the serum levels of miR-122, miR-25 and miR-21, identified as miRs of interest. miR expression was normalised to miR-19b and miR-93, determined by geNorm to be the most stable reference miRs in the array. Results: miR-122 was significantly upregulated in CFLD vs both CFnoLD and Controls (KW ANOVA, P<0.0001). Of interest, both miR-25 (KW ANOVA, P=0.01) and miR-21 (KW AN〇VA, P=0.05) were significantly increased in CFnoLD vs both CFLD and Controls. Using receiveroperator characteristic (ROC) curve analysis, liver disease in CF (i. e., CFLD vs CFnoLD alone) was discriminated by both miR-122 (AUROC=0.71, P=0.002) and miR-25 (AUROC=0.65, P=0.026). However, combined logistic regression including all three miRs (−122, −25, −21) showed a highly significant result for the detection of liver disease in CF (AUOC=0.78, P < 0.0001). Conclusions: This work provides the first evidence of changes to circulating miR levels in CFLD. We demonstrate the potential of miR-122, miR-25 and miR-21 as biomarkers for the early diagnosis of CFLD, perhaps in association with previously discovered discriminatory fibrosis biomarkers. The potential contribution of miRs in predicting disease severity and in the mechanisms of liver pathology in CF patients requires further evaluation.

Disclosures:

Peter J. Lewindon - Advisory Committees or Review Panels: Janssen; Speaking and Teaching: Janssen, Abbott

The following people have nothing to disclose: Naomi L. Cook, Tamara N. Pereira, Ross Shepherd, Grant A. Ramm

125

Identifying Frequency Of Inherited Metabolic Disorders In Patients With Infantile Liver Disease

Zoe Gray1, Kirsten McKay3, Carla Lloyd2, Jane Hartley2, Fiona MacDonald3, Christian J. Hendriksz5, Paul Gissen4, Deirdre A. Kelly2

1WTCRF/ Birmingham Children΄s Hospital, Birmingham, United Kingdom; 2Liver Unit, Birmingham Children΄s Hospital, Birmingham, United Kingdom; 3WMRGL, Birmingham Wome΄'s Hospital, Birmingham, United Kingdom; 4Institute of Child Health, University College London, London, United Kingdom; 5Salford Royal Hospital, Salford, United Kingdom

Introduction/Background: The frequency of liver disease due to rare inherited metabolic disorders, including Niemann Pick type C(NPC), Citrin Deficiency and Progressive Familial Intrahepatic Cholestasis (PFIC), is unknown. New sequencing methods, including next generation sequencing (NGS), permit analysis of multiple genes simultaneously, reducing time to molecular diagnosis and cost. Accurate timely diagnosis is essential to optimise clinical management, improve targeted therapy for liver disease in infants, and make decisions about liver transplantation. Objectives: To evaluate the use of NGS in identifying the frequency of inherited metabolic disorders in patients with infantile liver disease. Subjects/Methods: A prospective study from 13 centres worldwide has recruited 185 infants under 2 with cholestasis, acute liver failure or splenomegaly, who had DNA sequenced using targeted NGS for mutations in 6 genes. Results: 〇f the 185 patients recruited, 90% presented with cholestasis; 25% hepatomegaly; 8% splenomegaly; 32% hepatosplenomegaly, and 18% acute liver failure (ALF). Diagnosis was confirmed (homozygous, or compound heterozygous, pathogenic mutations) in 20 patients; NPC1 (1), PFIC 1(ATP8B1) (4), PFIC2 (ABCB11) (9), PFIC3 (ABCB4) (5) and PFIC1 and PFIC 3 (ATP8B1/ABCB4) (1). 7 patients had heterozygous pathogenic mutations, and 73 had unknown variants. The clinical presentation was similar to patients with known mutations, with 87% presenting with cholestasis, 32% hepatomegaly, 10% splenomegaly, 34% hepatosplenomegaly, and 20% presenting with ALF. Summary: This study has recruited 185 patients worldwide with infantile liver disease, and successfully identified the frequency (10%) of those with rare inherited metabolic diseases in this group of infants. A further 4% had heterozygous pathogenic mutations, and 7 3 novel variants were detected. Discussion/Conclusion: NGS provides early data for genetic diagnosis at a reduced cost, suggesting it is a promising screening tool in infants with cholestasis or ALF. However, novel variants of unknown pathogenicity were detected, suggesting these may cause susceptibility to liver disease, and highlighting the need for good communication between hepatology and genetics. Further analysis will allow correlation between phenotypes and genotypes to be established and clarify clinical indications for screening for these disorders.

Disclosures:

Christian J. Hendriksz - Advisory Committees or Review Panels: Actelion, Biomarin; Consulting: Biomarin, Niemann Pick C research Foundation; Grant/Research Support: Actelion, Biomarin, Shire HGT Speaking and Teaching: Sanofi/Genzyme, Shire HGT, MPS Society UK, International MPS Society

Paul Gissen - Advisory Committees or Review Panels: Synageva; Speaking and Teaching: Swedish orphan, Acitllion

Deirdre A. Kelly - Consulting: Sanofi Pasteur; Grant/Research Support: BMS, Astellas, Acit丨丨ion, MSD, Roche

The following people have nothing to disclose: Zoe Gray, Kirsten McKay, Carla Lloyd, Jane Hartley, Fiona MacDonald

126

Ascitic fluid infection in acute and chronic liver disease in children: Evaluation, comparative analysis and outcomes

Surender K. Yachha, Rohan Malik, Rishi Bolia, Anshu Srivastava, Ujjal Poddar

1Pediatric Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

Aim: To evaluate the etiology, clinical profile and outcome of ascitic fluid infection (AFI) in children with acute and chronic liver disease. Published literature in children is remarkably lacking in this regard. Methods: Children admitted with liver disease and high serum-ascites albumin gradient (SAAG) ascites between the years 2007 and 2012 were enrolled and classified according to the type of AFI as spontaneous bacterial perotinitis (SBP), culture negative neutrocytic ascites (CNNA), non-neutrocytic bacterascites (NNBA) and no-infection. Clinical profile, laboratory parameters, PELD/MELD scores wherever applicable and outcomes of children with or without AFI were compared. Results: 265 children, median age 84(1-240) months were enrolled. 181(68%) were cirrhotic and 84 (32%) non-cirrhotic. The non-cirrhotic group was comprised of acute hepatitis, acute/subacute liver failure and other causes. Overall AFI was present in 113 (42.6%) patients (SBP-28[24.7%], CNNA-48[42.5%] and NNBA-37[32.8%]). The incidence of AFI in cirrhotics (43%) and non-cirrhotics (41.6%) was similar. Children with no-AFI were less symptomatic (41/152, 27%) for fever, abdominal pain and diarrhea than AFI group (52/113, 56%)(p 0.002). Among all cases, renal failure (6/28 vs. 12/152, p 0.03) and in-hospital mortality (14/28 vs. 37/152, p 0.01) was higher in the SBP subgroup as compared to no-AFI group. In patients with cirrhosis (n=181), the subgroup with SBP as compared to no-AFI group had a significantly higher serum bilirubin (median 14 vs. 8.1 mg/dL, p = 0.01), ALT (median 180 vs. 79 IU, p = 0.01), INR (median 3.0 vs. 1.8, p = 0.01) and in-hospital mortality (52.2% vs. 25.2%, p = 0.02). Mean PELD score (children<12y) was similar in AFI (21.68) and non-AFI (20.64) groups, however the MELD score (children>12y) was higher in those with AFI (24.82 vs. 17.86, p 0.02). 〇n follow- up, mortality in AFI group (23%) was higher as compared to non-AFI (12%), this was not significant on Kaplan-Meier survival analysis. Conclusions: AFI occurs in 43% children with similar incidence among cirrhotics and noncirrhotics. AFI may be symptomatic or asymptomatic. Liver functions were significantly more deranged with increased frequency of renal failure and in-hospital mortality in SBP group. Outcome on follow-up in cirrhotic was not affected by AFI.

Outcomes on follow-up in cirrhotic children

No infection N =103SBPN=23CNNA N =35NNBA N =20
  1. α As compared fo no-infection group.

In-hospital mortality26(25.2%)12(52.2%) p=0.02 a6(17.1%)4(20%)
Follow-up58(75.3%)10(83.3%)23(79.3%)15(93.7%)
Duration (m)9(1-72)10.5(3-42)12(1-36)4(0.1-40)
Mortality7(12%)2(20%)5(21.7%)4(26.7%)

Disclosures:

The following people have nothing to disclose: Surender K. Yachha, Rohan Malik, Rishi Bolia, Anshu Srivastava, Ujjal Poddar

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