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Biomarkers to achieve personalized immunosuppression in transplantation. Michael Oellerich , MD, Hon MD FACB, FFPath (RCPI), FRCPath Lower Saxony Distinguished Professor of Clinical Chemistry Department of Clinical Chemistry George-August-University Göttingen Germany.

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  1. Biomarkers toachievepersonalizedimmunosuppression in transplantation Michael Oellerich, MD, Hon MD FACB, FFPath (RCPI), FRCPath LowerSaxonyDistinguished Professor of Clinical Chemistry Department of Clinical Chemistry George-August-University Göttingen Germany www.ndsprof.med.uni-goettingen.de

  2. Transplantsbythenumber in the US ~ 30,000 patients per year ~ 200,000 living graft recipients Shortageofdonororgans: Waiting list: US ~ 100,000 patients EU 63,800 patients; ~ 3,780 died in 2012 Numberoftransplantsin 2009 KidneyLiver Heart Lung PancreasIntestine 17,682 6,320 2,241 1,690 1,233 180 Acuterejectionat 3 y (%) Deceased Living DonorDonor 16.6 14.2 21.9 45.3 49.8 27.2 53.5 Organ Procurementand Transplantation Network and Scientific Registry of Transplant Recipients 2010 Annual Data Report Eurotransplant, Annual Report 2012

  3. Factors limiting long-term outcome in transplantation Irreversible chronic allograft dysfunction Adverse effects of standard immunosuppression (e.g. nephrotoxicity, cardiovascular disease, opportunistic infection, malignancy) > 50 % oftransplantedkidneysfailwithin 10 years • numerousattemptstodevelopbiomarkersthatwouldcomplement TDM andnewagentstoachievepersonalizedimmunosuppression Sagoo et al, J Clin Invest 2010; 120: 1848-61 Wieland et al, Ther Drug Monit 2010; 32: 560-572 Schröppel et al, J Clin Invest 2010; 120: 1803-1806

  4. Causesofchronicallograftdysfunction Chronic allograft dysfunction AMR Disease recurrence BK nephropathy Pascual M et al, N Engl J Med 2002; 346: 580-590 Nashan B, Hardenberg 2014

  5. Personalizedimmunosuppression: limitationsof traditional approaches • TDM: • More usefultopreventtoxicitythantopredictefficacy • Conventionalmarkers: • lossof 50% kidneyfunctionpossiblebeforerise in creatinine • liverfunctiontests not diagnosticforassessingacutecellularrejection Interventionsmaybetoolate Rodríguez-Perálvarez M et al, Transplant Int 2012; 25: 555-563 J Am SocNephol 2005; 16: 1886-1903 Falck P et al, Transplantation 2008; 85: 179-184

  6. Limitationsofimmunosuppressivedruglevelmonitoring TDM does not preciselypredicttheeffectsofimmuno-suppressive drugs on immune cells Intersubjectvariability in: sensitivityto immediate andlong-term suppressionof immune function intralymphocyteimmunosuppressivedruglevels Synergisticandantagonisticeffectsofconcomitantdrugs • Pre- and posttransplant immunologicalriskassessmentandtolerancepredictionbeforedrugminimizationwouldbedesirable.

  7. Influence of MDR1 3435 genotype on intralymphocyte trough CsA levels Data are given as geometric mean (95% confidence interval). * transplant recipients (renal, liver, lung) Crettol S et al, Pharmacogenet Genom 2008; 18: 307-315

  8. Intracellular CsA T-lymphocyte concentration has a potential of predicting rejection Renal transplant recipients Falck et al, Transplantation 2008; 85: 179-184

  9. The needfornewbiomarkerstopersonalizeimmunosuppression • Predictionofpredispositiontoacuterejection • Assessment ofminimal necessaryexposure • Early detectionofongoingundiagnosed graft injuryleadingtoacuterejectionorchronicallograftdysfunctionwouldallowearlyintervention • Assessment oftoleranceor „almosttolerance“ • Practicability: Short TAT (same day), reasonablecost Final goal: Improvementoflong-term outcome

  10. Biomarkers for immune monitoring Drug targetenzymes Markers of lymphocyte proliferation or activation Soluble molecules (e.g. sCD30) Functional T-cell assay (Cylex) Cytokines(e.g. IL-2 expression in CD8+ T cells, NFAT-regulated gene expression)

  11. Percentage of IL-2 producing CD8+ T-cells in liverrecipients with and without acute rejection Boleslawski et al, Transplantation 2004; 77: 1815 - 1820

  12. NFAT-regulated gene expression to assess response to tacrolimus NFAT-regulated genes: IL-2, IFN-, GM-CSF Patients with CMV disease Patients with acute rejection B p < 0.05 * p = 0.02 * No difference in PK data *NFAT RE (%): 1.5 h post Tac dose; stable renal transplant recipients Sommerer C et al, Ther Drug Monit 2011; 33: 373-379

  13. CD4+ cellularresponse in heart transplant recipients Israeli M et al, Transplantation 2010; 89: 968-976

  14. Longitudinal ImmuKnowmonitoringin a patient 4 years post-HTx Are baseline values for individual patients required? Israeli M et al, Transplantation 2010; 89: 968-976

  15. Allografttolerance in solid organtransplantation • Spontaneous operational tolerance: • long-term maintenanceofstable graft functionfollowingdiscontinuationofconventionalimmunosuppression • "Almosttolerance": • stable graft function in minimallyimmunosuppressedrecipients (low dose monotherapy) • Estimatedincidenceof operational tolerance: • livertransplantation:  20% • renal transplantation: lowfrequency Londoño et al, Am J Transplant 2012; in press Sánchez-Fueyo A et al, Gastroenterology 2011; 140: 51-64 Sagoo P et al, J Clin Invest 2010; 120: 1848-1861 Newell et al, J Clin Invest 2010; 120: 836-847 Roussey-Kesler et al, Am J Transplant 2006; 6: 736-746

  16. Potential predictorsoftolerance Natural regulatory T-cells (CD4+CD25++CD127¯FOXP3+) Signature of B-cell genes (IGKV4-1, IGLLA, IGKV1D-13) Specificperipheralbloodgenesets (NK relatedtranscripts) Indices oftolerancepotentiallyusefulfortheidentificationofrecipientswhowouldbenefitfromimmunosuppressionminimization

  17. Major subsetsofregulatory T cells Natural Tregs (Thymus): FOXP3 demethylation, TSDR* InducedTregs (Periphery): highlymethylated TSDR* * TSDR: Treg-specificdemethylatedregion Shalev I et al, LiverTranspl 2012; 18: 761-770

  18. FOXP3 demethylationasnTregcellsignature Baron et al, Eur J Immunol 2007; 37: 2378-89

  19. nTregs (% CD4+) - FACS vs. TSDR ddPCRassay % 38 LTx patients n = 60 r = 0.218 p = 0.095 nTreg (CD4+CD25++CD127-FOXP3+,FACS) % nTreg (FOXP3demethylation, TSDR) Schmitz J et al, ClinChem 2014; in press (Abstract)

  20. Effectofvariousimmunosuppressiondrugs on T-regulatorycells Immunosuppressivedrug T-regulatorycelleffect Cyclosporine/tacrolimusDecreasesTregs (calcineurininhibition) MPANoeffect Sirolimus/everolimus(mTORinhibition)ExpandsTregs Belatacept(co-stimulation blockade)Inhibitoryeffects ThymoglobulinLow dose: expandsTregs Therapeutic dose: sparesTregs Basiliximab(anti IL-2 receptorantibody)DecreasesTregs Tofacitinib(JAK3 inhibition)Proposed: functionalactivity main- tained, but numbersdecreased JAK: Janus kinase; MPA: mycophenolicacid; mTOR: mammaliantargetofrapamycin Wojciechowski D et al, CurrOpin Organ Transplant 2011; 16: 614-619 Levitsky J et al, Transplantation 2013; 96: 689-696

  21. T-cell subsets in peripheral blood from liver transplant patients with renal dysfunction CD3+CD8+ cells/nL CD3+CD4+ cells/nL CD4+CD25highFOXP3+ cells/nL MMF group* Baseline 0.613 ± 0.092 1.068 ± 0.099 0.062 ± 0.016 Month 12 0.424 ± 0.055+ 0.756 ± 0.085+ 0.056 ± 0.010 Control group** Baseline 0.474 ± 0.085 0.645 ± 0.096 0.051 ± 0.037 Month 12 0.530 ± 0.091 0.728 ± 0.089 0.034 ± 0.008† + P < 0.001 vs. baseline; †P < 0.05 vs. baseline; mean ± S.E.M. * MMF 2x1 g; CsA 25-50 µg/l or TAC 2-4 µg/l (n=22) ** CsA 80-120 µg/l or TAC 5-7 µg/l (n=14) Adapted from Cicinnati et al, Aliment Pharmacol Ther 2007; 26: 1195-1208

  22. Role of Tregs for prediction of tolerance - contradictory - • High Treglevels (> 70%) areassociatedwithbetterlong-term graft survival in kidney transplantation.1) • The overallfrequencyof foxp3+ Tregis not necessarilycorrelatedwith tolerance.2) • Antigen-specificTregmaybeconcentrated in thetransplantedorganandpoorlycorrelatedwithcirculatingTregcellfrequencies. 2) • The only potential biomarkerassaysavailablearederivedfromgeneexpressionstudies (different patterns in kidneyorliver transplant patients). 2) 1) Franzese O et al, ClinDevImmunol 2013; Art ID 852395 (7 pages) 2)Cobbold SP et al, J Cold Spring Harb PerspectMed 2013; 3: a015545

  23. Gene expression signatures predictive of tolerance Identification of a B cell signature associated with renal transplant tolerance in humans Kenneth A. Newell et al Study design: Identificationofrecipientswhichwouldbenefitfrom IS withdrawalorminimization Cohortof 25 tolerant renal transplant recipients (> 1 y, no IS) Results: Tolerant patientsexhibitedincreasednumbersof total and naive B cellsandshowedincreasedexpressionof multiple B celldifferentiation genes. Signatureof 3 genes (IGKV4-1, IGLLA, IGKV1D-13) was highlypredictiveoftolerance. Conclusion: Transitioningormaturing B cellsinvolved in toleranceinductionand/ormaintenance J Clin Invest 2010; 120: 1836-47

  24. Tolerancesignaturecomprising: • a setof 10 genes withsignificantlyalteredexpression • elevatednumbersofperipheralblood B and NK cells • diminishednumbersofrecentlyactivated CD4+ T cells • donor-specifichyporesponsivenessof CD4+ T cells (IFN-γ ELISpot) • high ratioof FoxP3/α-1,2-mannosidase geneexpression in peripheralblood Biomarker signaturetodetect renal transplant tolerance Sagoo et al, J ClinInvest 2010; 120: 1848-1861

  25. Predictorsofrejectionor graft loss • AlloMap • Donorheartandpre-transplant recipientbloodgeneexpressionpanel • Microarray-basedtestfor antibody-mediatedrejection in kidney transplant biopsies • Donor-specific HLA alloantibodies (DSA)

  26. Microarraydiagnosisof antibody-mediated rejection (ABMR) in kidney transplant biopsies • Posttransplant donor-specific HLA antibody (DSA) is a riskfactorforlate graft loss. • Diagnosticconventionalcriteriafor ABMR (Banffconsensus): • Microcirculationlesion (histology) • C4d staining (complementfactor C4d in capillaries) • DSA • Microarray-basedtestthatdiagnoses ABMR byassigning a score • Transcriptsmostlyexpressed in endothelialcellsor NK cells, orwere IFNG-inducible. • The molecular ABMR score was morestronglyassociatedwith graft failurethanconventionalassessment (includingearlyprogression). Halloran PF et al, Am J Transplant 2013; 13: 2865-2874 Sellares J et al, Am J Transplant 2013; 13: 971-983

  27. Graft survivalcurvesrelatedto ABMR score andconventionalassessment ABMR score > 0.2 positive (S+) and ≤ 0.2 negative (S-) Conventionalassessment: positive (C+) or negative (C-) Rapid progress: S+C+ S+C- Halloran PF et al, Am J Transplant 2013; 13: 2865-2874

  28. Predictingacutecardiacrejectionfromdonorheartandpre-transplant recipientbloodgeneexpression Background: Acuterejectorscouldpossiblyhave a predispositiontohavingeasily-activated immune cells. The goalofthestudy was toidentifybiomarkersforpredictionoflikelihoodof post-transplant acutecellularrejectionatthe time oftransplantation. Results: Biomarker panel: 25 probe setsfromdonorbiopsy(NRAS, PLA2G4A, EPS15L1, F2RL1, FBN1, ANXA4, EMP1, RAB38, STK32C)  biologicalpathways: VEGF-, EGFR-signaling, MAPK 18 probe setsfromrecipientblood(BSG, CHMP4B, HEBP1, TBL1XR1, WNK1, TMEM158, MMP25) Conclusions: Panel maypersonalizeimmunosuppressivetreatmentandrejectionmonitoring. Hollander Z et al, J Heart Lung Transplant 2013; 32: 259-265

  29. Molecularbiomarkersof graft injury • Graft-derived circulating cfDNA • UrinaryChrYdd-cf DNA • Urinary-cellmRNAprofile • (CD3εmRNA, IP-10 mRNA, 18S rRNA)

  30. Molecularbiomarkersofkidneyallograftinjury Aim: Noninvasivetestforacuterejection in kidneytransplants Methods: mRNAlevelsmeasured in urinarycellsof 485 kidney-graft recipients Results: A three-genemolecularsignature (CD3εmRNA, IP-10 mRNA, and 18S rRNAlevels) in urinarycellsdiagnosticandprognosticofacutecellularrejection in kidneyallografts N Engl J Med 2013; 369: 20-31

  31. Graft-derived circulating cell-free DNA Presence of donor-specific DNA in plasma of kidney and liver-transplant recipients Lo YMD, Tein MSC, Pang CCP, Yeung CK, Tong K, Hjelm NM • Study design: • Femaleliver (n=8) orkidney (n=28) recipientsof male donors • Chr Y-specific PCR • Results: • First descriptionofdonor-specific DNA sequencespresent in plasmaofliverandkidney transplant patients • Conclusion: • Plasma donor DNA is a cell-deathmarkerreleasedfromnecroticorapoptopiccells in the transplant organandmaybe a markerofrejection. The Lancet 1998; 351: 1329-1330

  32. UrinaryChr Y dd-cf DNA as a markerof kidney transplant injury AR: acuterejection STA: stable graft CAI: chronicallograftinjury BKVN: BK virusnephropathy Femalerecipients (n=63) of male donors  Limitation: ChrYdd-cf DNA requiresprecisesexmismatchpairing; in all othercases: sequencingofdonor DNA Sigdel TK et al, Transplantation 2013; 96: 97-101

  33. DonorcfDNAlevels in plasma determinedbyshotgunsequencing Snyder et al, PNAS 2011; 15: 6229-6234

  34. DonorcfDNAlevels in plasmaofheart transplant recipientswithrejection Snyder et al, PNAS 2011; 15: 6229-6234

  35. Graft-derived cfDNA determined by ddPCR ClinChem 2013; 59: 1732-1741

  36. Novel potential routineassayfor graft-derivedcirculatingcfDNA • Obtainblood sample (Streck tube) • Collectcellfree DNA fromplasma • Harvestwhitebloodcellstoidentifyrecipientspecifichomozygous SNPs. • Identify informative assaysfrom a libraryof41 preselected SNPs with MAF >40%. On average, about 5 heterologoushomozygous SNPs areobtained. At least 3 independent SNP assaysareused. • Performdroplet digital PCR andquantifytargetnucleicacidstocalculate% donorDNA (GcfDNA). Beck J et al, ClinChem 2013; 59: 1732

  37. GcfDNA determination by droplet digital PCR “X” target copies Make Droplets PCR Droplets Read Droplets Results Partition reagents and sample into 20,000 droplets Perform PCR on thermal cycler Quantify target nucleic acid by counting sample partitions with a positive PCR product (fluorescent) and a negative PCR product Digital readout provides percentage of circulating graft DNA Beck J et al, ClinChem 2013; 59: 1732 Adaptedfrom:

  38. GcfDNAincreasepreceded AST andbilirubin in liver graft rejection Beck J et al, ClinChem 2013; 59: 1732

  39. GcfDNA in a liver recipient without complications Beck J et al, AACC 2013

  40. GcfDNAin a liver recipient with infection and acute rejection Kanzow P, Doctoralthesis 2014

  41. GcfDNA to monitor marginal donor graft integrity Kanzow P et al, Transplantation 2014; in press

  42. GcfDNAin a liver recipient with cholestasis due to post-LTxcholangiopathy Kanzow P, Doctoralthesis 2014

  43. GcfDNAduring switch of immunosuppressive therapy

  44. GcfDNA after liver transplantation Kanzow P, Doctoralthesis 2014

  45. GcfDNAvs. tacrolimustrough levels during days 5 to 30 post surgery Oellerich M et al, TherDrug Monit 2014; 36: 136-140

  46. Re-examination of tacrolimus therapeutic range Oellerich M et al, TherDrug Monit 2014; 36: 136-140

  47. Early tacrolimusexposureandlong-term outcomes in liver transplant recipients Graft survival Rodríguez-Perálvarez M et al, J Hepatol 2013; 58: 262-270

  48. Monitoring graft-derivedcirculatingcfDNA – futureprospects - • Directlyinterrogateshealthofdonororgan • Allowsearlydetectionof transplant injury („liquid biopsy“) andmayenableearlyintervention • Helpfultomonitorimmunosuppressionminimization • May complementorpossiblyreplaceotherapproachesfor posttransplant monitoring • May improvechancesoflong-term graft survival • Cost-effectiveapproachwith same-dayturnaround Oellerich M et al, TherDrug Monit 2014; 36: 136-140 Kobashigawa J et al, J Heart Lung Transpl 2014; 32: S14 Beck J et al, ClinChem 2013; 59: 1732-1741 Snyder et al, PNAS 2011; 15: 6229-6234

  49. Available: Potential toolscomplementaryto TDM toidentifyrecipients atriskofacute rejection or for late graft loss withongoingundetected graft injuryleadingtochronicallograftdysfunction where TDM isinadequate (e.g. complianceproblems, especially in adolescents) forclarificationof non-specificelevationofconventionalorganfunctiontests toachievepersonalizedimmunosuppression (e.g. byminimizationormore potent immunosuppression) Biomarkers - wherearewe?

  50. Needed: Optimal biomarker/TDM combinationswhicharepracticalandcosteffective(e.g. GcfDNA, nTreg (TSDR), regulatorygenesets, DSA) Individual baselinesandtargetranges Tolerance-permissive immunosuppressiveregimensdevoidoftoxicities Carefulvalidation in prospectiveclinicaltrials Biomarkers - wherearewe?

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