Fibrosis & Structural Decline Of Liver Allografts: What .

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Fibrosis & Structural Decline of Liver Allografts:what and how to measure & potential underlyingcausesCarla Venturi Monteagudo MD, PhD

THE NORMAL LIVER ARCHITECTUREParenchymal Cells-Hepatocytes-CholangiocytesNon-parenchymal Cells-Endothelial Cells-Kupffer Cells-Hepatic Stellate Cells-Myofibroblasts-Natural Killer Cells- B LymphocytesExtra Cellular NTRODUCTION

THE NORMAL LIVER ARCHITECTUREPortal Flow DistributionCentral VeinPortal TriadSinusoidsthe zone 3 receives less oxygen and nutrients than zone 1, where the blood flow ofthe hepatic artery branch and portal vein is poured to conform the sinusoids.INTRODUCTION



FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONHigh proportion of fibrosis described in the long-term, mainlyassociated to inflammation, chronic hepatitis & chronic rejection-Evolutive process? Patient predisposing condition? Could be related to posttransplant persistent injuries?INTRODUCTION

FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONASSESSMENT&MONITORINGInvasive Approach-Liver Biopsy-“GOLD C ANALYSISCollagen I-II-IIICounterstainedQuantify the fibrosis area found inthe liver biopsy specimen stainedby PicroSirius-Red.SEMIQUANTITATIVEHISTOLOGIC SCORINGSYSTEMSPathologists review the liver biopsyclassifying fibrosis in mildmoderate or severe according thescoresMasson s TrichromeCollagenNucleiCytoplasmINTRODUCTION

Fibrosis Semiquantitative AssessmentHistological Scoring SystemsDESIGNED TO STAGE CHRONIC HEPATITIS NO FOR TRANSPLANTEDLIVERSSCHEUER system (1991)Combines Necroinflammation and Fibrosis grade 0-4Portal inflammation & necrosisLobular inflammation & necrosisPortal FibrosisMETAVIR system (1994)Combines piecemeal and lobular necrosis with inflammation and fibrosisActivity & Necroinflammation A 0 -3Portal Fibrosis 0-4ISHAK system (1995)Periportal or periseptal interface hepatitis 0-4Confluent necrosis0-6Focal (spotty) lytic necrosis, apoptosisand focal inflammationPortal inflammation0-40-4Portal and Bridging Fibrosis0-6INTRODUCTION

LIVER BIOPSYFibrosis at the Three Main Areas of the Liver ParenchymaPortal FibrosisSinusoidal fibrosisCentrilobullar fibrosisConventional systems used to stage fibrosis in the native liver fail torecognize these patterns of graft fibrosis.INTRODUCTION

FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONASSESSMENT & MONITORINGNon-Invasive ApproachHepatic ImagingMultiparametric MRI No Ped TXTransient Elastography (AUROC 0.8-0.9)Acoustic radiation force impulse (AFRI) (AUROC 0.8)Magnetic Resonance Elastography (MRE) (AUROC 0.92) No Ped TXEvaluation area 1-3 cm3Transient Elastography Equipmentexpensive, range of probes are needed,influenced by obesity & inflammation.Reproducible measurements are notpossible in 20% of patients. More difficultin split or reduced grafts.Less accurate in middle fibrosis.INTRODUCTION

FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONASSESSMENT & MONITORINGNon-Invasive ApproachSerum markers of fibrosisHyaluronic Acid (HA)Animo-terminal propeptide of type III collagen (PIIINP)Tissue inhibitor of matrix metalloproteinase 1 (TIMP1)APRI: AST/ platelet ratio indexType 4 collagen S, Fibronectin & LamininELF panel*HA: appeared to be a fair predictor of liver allograft fibrosis (Hartley JL, et al. JPGN 2006;43 217-21)ELF panel*: accurate in pediatric NAFLD (AUROC 0.92); no correlation with the degree of pediatric allograftfibrosis. (Goldschmidt I, et al Ped Transpl. 2013; 17:525-34)MicroRNAs: Intrahepatic microRNAs arepredictive of inflammation, rejection &proliferation.(need LB)Markers of cell Death: CK18, sensitivemarker of fibrosis in ped NAFLD.INTRODUCTION

FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONASSESSMENT & MONITORINGNon-Invasive ApproachImmunological investigationAutoantibody positivity (SMA- ANA), reflect cause of graft injury; related tochronic hepatitis & fibrosisClass II donor-specific human leukocyte antigen antibodies (DSAs), mostly DQ,has been associated with graft inflammation, fibrosis, De novo AIHDonor-specific T cells have been shown to predict the risk of acute rejectionfollowing pediatric TXINTRODUCTION

FIBROSIS IN PEDIATRIC LIVER TRANSPLANTATIONPATHOGENESIS& EVOLUTIONASSESSMENT &MONITORING-Howis the evolutionof activatedHSCsexpressin pediatricliverThe ActivatedHepatic StellateCells whichmyofibroblasticphenotype,are thesource of collagen in the injured liverallograftalongthemaintime?Activated HSCs areidentified by ASMAimmunoreactivity inthe liver biopsyASMA immunoreactivityPREVENTION& REVERSION-Could the activated HSCs predict high fibrosisdevelopment in the long term?INTRODUCTION


Patients & MethodsRetrospective analysis 1999-2005 of 170 Pediatric LT recipientsExclusion Criteria: Re-transplantation; inadequate LB; incomplete follow-up( 3 LB) 31Clinical -Biochemical & Serologic AssessmentPre-LT factors:Donor AgeDonor typeIschemia TimeRecipient age-gender-weightheight- blood pressureLiver Transplant indicationCMV - EBV statusPost-LT factors:Vascular and biliary complicationsInfections (0-6 months)Autoantibodies & gammaglobulins %History of Post-transplantlymphoproliferative diseaseAvailable data of Doppler ultrasound- TEAdequate and available protocol liver biopsyyearsLT6 months1235710

Patients & Methods- Histologic Assessment170 Pediatric LT recipients - 31 Excludedyears139 patients6 months1595 Liver Biopsies 112 11523571011096815724Available & adequate protocol liver biopsyNormal Histology : absent or minimal non-specific portal infiltrate.Acute & Chronic rejection*Portal inflammationCentrilobular dropoutSteatosisDuctal proliferationCholestasisDe Novo autoimmune hepatitis**Necroinflammatory activityFibrosis staging*AR Episodes: increased liver enzymes ([AST] [ALT] [GGT]:NR 5–50 IU/L, histological features (Banff )and treatment with i.v Steroid.**De novo AIH: progressive graft dysfunction, increased autoantibodies and serum gamma-globulin levels, with histologic features of chronicactive hepatitis (portal inflammation with limiting plate disruption, and lobular hepatitis with or without plasma cell infiltration)

Design of a new histological fibrosis scoringLiver Allograft Fibrosis Score (LAFSc)

Histologic Features and Staging definitions of the LiverAllograft Fibrosis Score 0-9 (zones 1, 2)0-3IIIIINon-expanding fibrosis in less than 50% of portaltracts.Fibrosis in more than 50% of portal tracts and/orexpansion into short fibrous septa into the periportalparenchyma.Marked expansion of most or all portal tracts withbridging fibrosis expanding to other portal tracts orcentral areas with or without occasional nodules.Little fibrosis with thin focal collagen depositsinvolving less than 50% of sinusoids.Little fibrosis with thin diffuse collagen depositsinvolving more than 50% of sinusoids, or thicker butfocal fibrosis in less than 50% of sinusoids.Thick, marked, diffuse sinusoidal fibrosis.Circular perivenular fibrosis involving less than 50%of central veins without invasion into the perivenularparenchyma.Circular perivenular fibrosis in more than 50% ofcentral areas and/or expansion into short fibroussepta into the perivenular parenchyma.Marked centrolobular fibrosis with bridging to othercentral areas and/or portal tracts.NoFibrosis0-3CentrolobularVein(zone 3)INoFibrosisDesign of a new histologic fibrosis scoring

Patients & MethodsClinical, biochemical and serological dataPOPULATION INCLUDEDAvailable & Adequate LB at 6 months and 7 yearsData of Non-invasive methods (TE & APRI index) at 7 years38 patients/ 76 LBLTProtocol liver biopsies12338 LB at 6 monthsLiver TransplantindicationDonor TypeImmunosupressionreceived at LTyears10538 LB at 7 yearsDemographic DataMedian LT- age (yrs)Biliary AtresiaMetabolic DiseasesCholestasisTumorsLiving Related Donor (n)Deceased Donor (n)TAC SteroidesTAC BasiliximabTAC monotherapy1.6 (r: 0.4 - 14)21 (55%)8 (21%)8 (21%)1 (3%)23 (60%)15 (40%)18 (47%)14 (37%)6 (16%)Validation of the new semi-quantitative scoring systemDesign of a new histologic fibrosis scoring

Patients & Methods- Histologic AssessmentLTProtocol liver biopsies1238 LB at 6 months3years10538 LB at 7 years176 New tissue sections cut & stained for Hematoxilin & Eosin (inflammationactivity) Masson’s Trichrome (fibrosis scored by the New Score, METAVIR- Ishak)2COMPUTER-ASSISTED MORPHOMETRIC ANALYSIS used as reference PATTERN forthe new score validation (PicroSirius-Red stain), that measure the proportion ofcollagen found at the digitalized image of each liver biopsy.3Morphometric analysis results were correlated with the New Score, METAVIR, Ishak& TE – APRI index4Correlation between Pathologists (intra/inter observers agreement)H&E and Masson’s trichrome-stained samples evaluated by external pathologistDesign of a new histologic fibrosis scoring

Results IFibrosis staged by LAFSc- METAVIR & Ishak systems6 MONTHS7 YEARSCorrelation among morphometric analysis with LAFSc- METAVIR & IshakLAFSc was the most accurate semi-quantitative score for evaluating fibrosisDesign of a new histologic fibrosis scoring

Results ICorrelation between collagen deposits (morphometric analysis) & Fgl1gl2p y of Liver allograft fibrosis score analysed by observersHigh intra-observer agreement 0.97, p 0.0001Inter-observer agreement: and 0.79, p 0.0001Intraclass correlation coefficientDesign of a new histologic fibrosis scoring

Results ICorrelation among morphometric analysis and semi-quantitative scoringwith non-invasive methods for fibrosis assessment (n 38)No correlation was found among TE or APRI index with morphometric analysis,METAVIR, Ishak & LAFScDesign of a new histological fibrosis scoring

Dynamics Of Allograft Fibrosis In Pediatric LiverTransplantation

Patients & MethodsPOPULATIONINCLUDED139Pediatric LTreceptors-Clinical, biochemical, serological data,-Immunosuppression-Doppler Ultrasound-Available & adequate LB at 6 months, 3 and 7 yrs.Protocol liver biopsies- long-term follow-up54154 LB 6months210 years554 LB 3 years54 LB 7 yearsDemographic DataMedian age at LT (years, range)Median Weight at LT (kg, range)Liver TransplantBiliary AtresiaIndicationP.I.F. CholestasisMetabolic diseasesTumorsAlagille SyndromeDonor TypeLiving Related Donor/ Deceased Donor (n,%)Median donor age (years, range)Median Ischemia time (minutes, range)Immunosuppression TAC Steroidsreceived at LTTAC BasiliximabTAC monotherapy1.28(0.2-15.7)7.66(3.8-53.7)30 (55%)9 (16%)8 (15%)5 (9%)2 (4%)29 (53%) - 25 (47%)30.1 (0.4- 50.3)169.5 (68- 892)24 (44%)23 (43%)7 (13%)Dynamics of liver allograft fibrosis

Patients & MethodsProtocol liver biopsies54Pediatric LTreceptors154 LB 62 LB254 LB 3 years510 years54 LB 7years-Normal vs increased liver enzymes along the time (NV 5-50 AST, ALT, GGT)-Patients who did not received Steroids anytime.-Tacrolimus monotherapy 4ng/ml with normal liver enzymes ( prope-T)-Two or more immunosuppressors or Tacrolimus monotherapy 4 ng/ml.-New tissue sections stained for H&E , Masson s Trichrome, PicroSirious-Red& Activated Hepatic Stellate Cells (ASMA immunostaining)-Pathologist Review & Fibrosis scoring: METAVIR (F0- F4) & Liver AllograftFibrosis Score (LAFSc 0-9)- Fibrosis & ASMA-positive area quantified by morphometric analysis1-Correlation among fibrosis with clinical variables, IS and histologic features associated2-Correlation among ASMA-positive area with fibrosis (LAFSc & PSR%) at same period/long-termStatistical Methods: SPSS 18.0 Chicago. IL. Results expressed as percentage, median, mean and SD;statistical significance for p-values 0.05. Relation among variables evaluated by Pearsoncorrelation. Linear and quadratic regressions were fitted to analyze relationship among variables.Dynamics of liver allograft fibrosis

Results II- Histologic Assessment of Allograft FibrosisProportion of Fibrosis staged by LAFSc30F0LAFScF125F220F3F415F510F6F75F8LAFScX SDIC 95%6 months2.9 (0.5)1.2- 1.83 years3.3( 1.8)1.5- 2.17 years4.3 (1.8)1.6- 2.106m%3y%7y%F9Fibrosis progressed along the time in 40 (74%) patients.Stable or reduced fibrosis was found in 14 (26%) patients.Patients with increased liver enzymes show similar amount of fibrosis than those withnormal liver functionDynamics of liver allograft fibrosis

Results II- Fibrosis evolution at parenchymalareasCentrilobular FibrosisPortal F0F190%0F3Linear progression p 0.017y2840%65F0Linear progression p 0.016m90%F33y100%392020%F0Sinusoidal Fibrosis7y40%1820%3y100%55246m7yF20F3No linear p 0.23y7y6m3y7yMeanCI 95%Dynamics of liver allograft fibrosis

Results III- Evolution of Fibrosis & Activated-HSCs (ASMA)Fibrosis progressed along the time p 0.001Activated HSCs decreased along the time p 0.01FIBRO SIS E V O LUT IO N615244 463231406 MO N T H S108403 YEAR S7 YEAR Sno fibrosis %mild %moderate %severe %Lineal (moderate %)LAFSc : mild 1- 3; moderate 4-6; severe 7- 9Increment by areas in the long-termSinusoidal33%Centrilobular 45%Portal57%Median ASMAIC 95%6 months6.9 (1.5-23.3)6.4- 9.03 years5.7 (0.4-38)6.0 - 9.87 years4.1 (0.7-18)4.3 - 6.5Activated-HSCs showed inverse evolution respect to Fibrosis in the long-termLiver Transplantation 2016 Jun;22(6):822-9Relevance of Hepatic Stellate Cells

Results III- Evolution of Fibrosis according to Activated -HSCs at 6mActivated-HSCs at 6 months 8% 20 patients 8% 34 patientsFIBROSIS E VOLUTIONActivated-HSCs 8 at 6months a risk factor forfibrosis development at 7years3025PSR%2015105PSR%r 2 0.48p 0.01LAFScr 2 0.30p 0.03Statitistical method: Mixed regression06 MON THS3 YEA R S7 YEA R STIMEASMA 8 20ASMA 8 34p-valueFibrosis 6m16.7 812.3 7 0.03Fibrosis 3 y11.9 711.4 6 0.8Fibrosis 7 y24.6 817.5 7 0.01p-value 0.001 0.04Note: p-values represent the significance between meansLiver Transplantation 2016 Jun;22(6):822-9Relevance of Hepatic Stellate Cells

Results IV Demographic data of the 139 LT recipientsNumber of patients / Liver biopsies (n)Median age at LT (years, range, range)Median Weight at LT (kg, range)LT Indication: (n, %) Biliary AtresiaMetabolic diseasesProgressive Intrahepatic Familial CholestasisTumorsAlagille SyndromeOthersLiving Related Donor/ Deceased Donor (n, %)Split Liver/ Reduced Deceased DonorMedian donor age (years, range)Median Ischemia time (minutes, range)139 (69 boys) /5951.4 (0.2- 16.8)8.4 (3.7- 63.2)75 (54%)21 (15%)17 (12%)11 (8%)11 (8%)4 (4%)66 (47 %) / 66 (47%)4 (3%) / 3 ( 2.5%)29 (0.4- 56.6)232.0 (66- 892)Immunosuppression at LT (n, %):TAC BasiliximabTAC SteroidsTAC monotherapyTAC MMf SteroidsTAC MMF DaclizumabTAC Basiliximab MMfTAC MMfTAC Steroids Daclizumab42 (30%)33 (24%)28 (20%)13 (9%)8 (6%)6 (4%)6 (4%)3 (2%)Dynamics of liver allograft fibrosis

Results IV Evolution of clinical variables studiedClinical VariablesLB:595/ Patients 1390-6m.1151 yrs112Vascular complications 12 (10%) 1 (1%)Time of follow-up2 yrs3 yrs5 yrs11096812 (2%)7 yrs5710 yrs241 (1%)1 (1%)1 (2%)--------Biliary complicationsPost-LT AAAR Steroids treatedPTLD (EBER ) n 28Gammaglobulins 15%19 (16%) 2 (2%)3 (3%)26 (23%) 30 (27%) 29 (26%)64 (56%) 8 (7%)8 (7%)8 (7%)9 (8%)7 (6%)40 (35%) 38 (34%) 48 (44%)1 (1%)15 (15%)5 (5%)2 (2%)32 (33%)1 (1%)8 (10%)4 (5%)1 (1%)59(73%)---------6 (10%)5 (9%)--------43 (75%)1 (4%)3 (12%)--------1 (4%)16 (67%)Gammaglobulins (X)13.616.217.317.216.214.316.3Abbreviations: LB, liver biopsy; LT, liver transplantation; AA, autoantibodies; AR, acute rejection; PTLD, post-transplantlymphoproliferative disease; EBER, Epstein Barr virus RNA .

Results IV Fibrosis evolution over timeN 139pts.595LB54.543.532.521.510.506 months1 yearSinusoidal2 yearsCentrilobular3 yearsPortal5 years7 yearsLAFSc10 yearsLineal (LAFSc)PortalDynamics of liver allograft fibrosis

Results IV Fibrosis evolution over timeincrement %reduction %unchanged %Lineal (reduction %)60N 139pts.50595LB403020100increment %reduction %unchanged %6mo-2y.4436203-5 y.5421257-10 y.332821LIVER ALLOGRAFT FIBROSIS IS A DYNAMIC PROCESSDynamics of liver allograft fibrosis

Results IV Association between fibrosis & clinical variablesClinical VariablesFibrosisFibrosisN 54p 0.001(46.3%)p 0.001(18.5%)p 0.01N 139p 0.02(47.5%)p 0.01(20%)p 0.03p 0.04(10%)p 0.03Centrilobular p 0.04 (7y)Gammaglobulins 15%p 0.04(11%)p 0.02Positives AutoAntibodies ( 1/40)p 0.01p 0.01Centrilobular p 0.01 (3y)Biliary complications 0-6 mp 0.01(24%)p 0.03(16%)Sinusoidal p 0.05 (6m) p 0.01 (3y)Male genderp 0.01(50%)p 0.002(50%)Sinusoidalp 0.001Centrilobular p 0.04 (7y)Deceased donor graftsLymphoproliferative diseaseIschemia time 400 minVascular complications (0-6m)Fibrosis LocationPortal p 0.001- p 0.003- p 0.01Portal p 0.01(7y)Portal p 0.06 (6m), p 0.01(3y)Centrilobular p 0.02 (7y)Dynamics of liver allograft fibrosis

Results IV Main histological features found at 595 LB Normal liver histology 5%, 3% & 1 % of LB at 6 mo. 3 & 5 years. Isolated Fibrosis 8- 19% over time. Fibrosis mild unspecific portal inflammatory infiltrate 15-33% overtime (70% NLE)Periodos of 115112110968157242%6 (5%)3 (3%)3 (3%)--------1 (1%)------------------Isolated14%FibrosisFibrosis mild 22%portal Infiltrate9 (8%)16 (14%)16 (14%)15 (16%)13 (16%)11 (19%)2 (8%)24 (21%)29 (26%)25 (23%)14 (15%)18 (22%)14 (24%)8 (33%)DuctalproliferationSteatosis44%57 (49%)51 (45%)47 (43%)44 (46%)36 (44%)20 (35%)9 (37%)21%29 (25%)29 (26%)21 (19%)17 (18%)12 (15%)11 (19%)5 (21%)InflammatoryInfiltrateCholestasis81%94 (82%)100 (89%)85 (77%)80 (83%)61 (75%)44 (77%)22 (91%)12%25 (22%)15 (13%)15 (14%)10 (10%)6 (7%)1 (2%)1 (4%)Interfacehepatitis17%18 (16%)18 (16%)19 (17%)21 (22%)11 (14%)11 (19%)2 (8%)LBNo fibrosisDynamics of liver allograft fibrosis

Results IV- Histological Features associated to fibrosisUnspecific inflammation: 1y p 0.001; 3y p 0.002; 5y p 0.001PORTAL FIBROSISDuctal proliferation: 6mo p 0.001; 1y p 0.002; 5y p 0.003; 7y p 0.02Cholestasis: 6mo p sis 5 & 10 y p 0.04Steatosis 6 mo;1y & 2 y p 0.001Ductal proliferation: 1y p 0.006; 2y p 0.005; 5y p 0.03Patients with steatosis did not show waning of itCellular drop out & interface hepatitis did not show correlation with fibrosis locationDynamics of liver allograft fibrosis

25Results IV Immunosuppression-Fibrosis evolution over timeSteroids vs Steroids-free patients[p 0.8]21(87%) kept on ST 1 yearx dose: 0.25 0.1 mg/kg13(54%) kept on ST 2 yearsx dose: 0.11 0.1 mg/kg(PSR%)24yesN 5437 vs 170Steroids at LT13 (43%)Further ST for AR6m3yx dose: 0.3 0.1 mg/kg7ySteroids-freeCI 95%SteroidsCI 95%1030noN 13997 (70%)STEROIDS-free42 (30%)0STEROIDSLAFSc[p 0.2]6m1235710Steroid therapy was not associated with reduced fibrosis in this populationDynamics of liver allograft fibrosis

Results IV Immunosuppression-Fibrosis according to PropeTolerance statusPatients at 7 yearsN 54Prope-T (n 18)Mean PSR% 19.0 9.7Mean LAFSc 3.9 1.7Non Prope-T (n 36)[p 0.5]18.8 9.4[p 0.8]4.1 1.710Fibrosis evolution in Prope-Tolerance vs Non Prope-Tolerance LB (n 595)595PROPE T175NO PROPE T 4200LAFSc[p 0.6]Total LB6mPeriod12357106 mo.1y2y3y5y7y10 yTotal12211511096815724PROPE T------13 (11%)26 (24%)44 (46%)36 (44%)39 (68%)17 (71%)122 (100%)98 (89%)84(76%)52(54%)45(56%)18 (32%)7 (29%)NO PROPE TPrope-tolerance did not contribute to increase fibrosisDynamics of liver allograft fibrosis

Discussion & Future Perspectives

Pediatric liver allograft fibrosis could be seen as a dynamic process withgradual progression over time.Fibrosis progression does not mean abnormal liver function, irreversiblecirrhosis or re-transplant indicationLAFSc identified fibrosis at portal, centrilobular and sinusoidal areas,being the most accurate score for evaluating allograft fibrosisFibrosis placed at specific areas of the liver parenchyma could be relatedto clinical complications or transplant eventsDISCUSSION

To date, the non-invasive methods for fibrosis assessment have beenunable to replace LB.The steroids could not prevent fibrosis developmentNo evidence of higher fibrosis was found in patients with lowimmunosuppressionA high proportion of activated-HSCs found at early stages of LT seems tobe a risk factor for early and long-term fibrosis development.DISCUSSION

Future PerspectivesPediatric liver allograft fibrosis need to be categorized by an accuratemethod specifically designed to stage allograft fibrosisCentralized studies are needed to confirm pediatric allograft fibrosisevolutionStudies evaluating the antifibrogenic properties of IS are mandatory, toadequate the treatment to fibrosis stage.To develop accurate non-invasive tools for fibrosis assessment to avoid theliver biopsyDISCUSSION

Thanks for your attention

Autoantibody positivity (SMA- ANA), reflect cause of graft injury; related to chronic hepatitis & fibrosis Class II donor-specific human leukocyte antigen antibodies (DSAs), mostly DQ, has been associated with graft inflammation, fibrosis, De novo AIH Donor-specific