医学交流课件：Biomarkers and phenotyping in asthma

Biomarkersandphenotypinginasthmaf.chung@imperial.ac.uk

15thAstraZenecaRespiratoryWeeksummit&2ndChinaRespiratoryLeadershipForum(CRLF)UnbiasedBiomarkersforthePredictionofRespiratoryDiseaseOutcomesInnovativeMedicinesInitiativecalltopic:UnderstandingSevereAsthma2010-2015UniversityofAmsterdam,UniversityofSouthampton,ImperialCollegeLondon,UniversityofManchester,UniversityofNottingham,FraunhoferInstituteHannover,CNRS-EISBMLyon,UniversitédeMéditerraneeMontpellier,KarolinskaInstituteStockholm,UniversityHospitalUmea,UniversityTorVergataRome,UniversitàCattolicadelSacroCuoreRome,UniversityofCatania,HvidoreHospitalCopenhagen,UniversityHospitalCopenhagen,HaukelandUniversityBergen,SemmelweisUniversityBudapest,JagiellonianUniversityKrakow,UniversityHospitalBern,UniversityofGhentEFPIAPartnersNovartisAlmirallAmgenAstraZenecaBoehringerIngelheimChiesiGlaxoSmithKlineJohnson&Johnson/JanssenMerckUCBRoche/GenentechSME’sAerocrineBioSciConsultingSynairgenPhilipsResearchPatientorganisationsAsthmaUKEuropeanLungFoundationEFAIntPrimaryCareRespiratoryGroupLegaItalianoAntiFumoNetherlandsAsthmaFoundationwebsitehostedbytheELF:www.ubiopred.euFundedbytheEuropeanUnionBarcelona2014Severeasthma:non-smoking(308)Severeasthma:smoking&ex-smoking(110)Mild/ModerateAsthma(98)Non-asthma(101)P-valueAge(yr)50.954.542.438.92.9E-17Female(%)65.9150.9150.0038.615.16E-06BMI(kg/m2)29.0829.5625.8825.312.02E-10Exacerbationsinpastyr2.482.550.3702.51E-26IgE(IU/ml)119.512689.423.455.40E-15Atopy(%)695880386.1E-066Nasalpolyps(%)34.71.33E-06FEV1(%pred)67.4267.2588.37101.761.81E-44Oralcorticosteroids(%)45.844.7009.73E-17Sputumeosinophils(%)2.75

4.131.050.002.69E-12ExhaledNO2723.525.5019.003.00E-04Total:617participantsDemographicsofUBIOPREDcohortShawetal:ClinicalandinflammatorycharacteristicsoftheEuropeanU-BIOPREDadultsevereasthmacohort.EuropeanRespiratoryJournal46:1308(2015)Severeasthma:non-smoking(308)Severeasthma:smoking&ex-smoking(110)Mild/ModerateAsthma(98)Inhaledcorticosteroids(%)99.710098.9Leukotrienemodifier(%)46.642.50LAMA(%)22.927.90Antihistamines24.114.60Xanthine(%)20.4210NebulisedSABA(%)28.924.72.3Oralcorticosteroids(%)45.844.70Prednisonedose(mg/day)13.2±0.8514.8±1.810Omalizumab(%)17.416.30Mucolytics(%)10.818.00Macrolide(%)10.311.80Immunosuppresants(%)2.93.60Total:617participantsDrugtherapyinUBIOPREDcohortShawetal:ClinicalandinflammatorycharacteristicsoftheEuropeanU-BIOPREDadultsevereasthmacohort.EuropeanRespiratoryJournal46:1308(2015)Th2-highvsTh2-lowinmild-moderateasthmaA:Mild-moderateasthmaB:HealthyFeaturesofTh2-highasthma●MorebloodandBALeosinophils●↑serumIgE●↑mucinMUC5AC●↑IL5andIL13inbiopsies●↑bronchialhyperresponsiveness●FEV1increasewithICSTranscriptomicanalysisofepithelialbrushings:expressionofTh2cytokinesWoodruffetalAJRCCM2009;180:388MolecularphenotypingHowcommonisaTh2(IL-13)highinsevereasthma?CohortNonSmokingSevereAsthmaSmokingSevereAsthmaMild/ModerateAsthmaIL13Th2high%37%(18/49)17%(3/18)25%(9/36)Non-smokingSevereasthmaSmokingSevereasthmaMild-ModerateasthmaHealthyEnrichmentscoreforIL-13IVSsignatureGeneSetVariationAnalysisU-BIOPREDBronchialBrushingsDefine“Th2(IL-13)High”as>95th%ileofHealthycontrolsSteliosPavlidis,MatthewLoza,FredBaribaudforUBIOPREDTranscriptomeanalysisofbronchialbrushingsforTh2signaturefromepithelialcellsactivatedbyIL-13invitro(IL13IVSdefinition)(11%)(13%)(10%)(13%)(7%)(8%)(5%)101patients(32%)werelowforall3biomarkersBloodEOS≥300/ulDistributionofhighFeNO,highserumperiostinandhighbloodeosinophilcountin418severeasthma(UBIOPRED)SerumPeriostin≥55pg/mlFeNO≥30ppb101patients(32%)werelowforall3biomarkersBloodEOS≥300/ulHighFeNO,highserumperiostinandhighbloodeosinophilcountlinkedtoexacerbations,FEV1andTh2signatureSerumPeriostin≥55ug/lFeNO≥30ppbTh2ES0.10Exacerbations2.17Th2ES0.36Exacerbations2.83Th2ES0.08Exacerbations2.2Th2ES0.05Exacerbations2Th2ES0.29Exacerbations2.2Th2ES0.00Exacerbations3.25Th2ES0.13Exacerbations1Th2ES-0.11Exacerbations3.07Th2expressionscore:UsinggenesetvariationanalysiswithIL-13/epithelialcellgeneexpressioninepithelialbrushingsHowtoapproachphenotyping/endotyping/clustering/fingerprint/handprint?Biased:definitionofseverity,basedontreatment,onclinicalfeatures‘Unbiased’onclinicalandinflammatoryfeaturesSubphenotyping

severeasthmaeg

oralsteroiddependencyStartfromOmicsclusteringHandprint(UBIOPRED):severalomicsplatformsSputumBloodClinicalfeaturesTranscriptomicsProteomicsMicrobiomePARAMETERSUSED-Ageofasthmaonset-Pack-yearsofsmoking-BodyMassIndex(BMI)-FEV1%predicted-FEV1/FVC-AsthmaControlQuestionnaire:ACQ-5-Exacerbationsinpastyear-MedicationOralCorticosteroid(OCS)dailydoseUnsupervisedclusteringinUBIOPREDUBIOPREDasthmaclustersbasedonunsupervisedclusteringof8clinicalfeaturesCluster1:Moderate-to-severeasthma;well-controlled;mediumtohighdoseICSCluster2:Severeasthma;Lateonsetasthma;smokerorex-smoker;airflowobstructionCluster3:Severeasthma;Oralcorticosteroid-dependent;airflowobstructionCluster4:Severeasthma;Female;Obese;frequentexacerbationsPARAMETERSUSED-Ageofasthmaonset-Pack-yearsofsmoking-BodyMassIndex(BMI)-FEV1%predicted-FEV1/FVC-AsthmaControlQuestionnaire:ACQ-5-Exacerbationsinpastyear-MedicationOralCorticosteroid(OCS)dailydoseUBIOPRED:smoking(cluster2)vsnon-smoking(cluster3)ofairflowobstruction●PathwayanalysisofcelltranscriptomicsRegulationofactincytoskeleton(ITGB1,FN1,ACTN2)Fibronectinmatrixformation(ITGB1,FN1)●Differentially-expressedproteinsinsupernatantsLYN:srcnon-receptorlyntyrosinekinaseFUT5:Fucosyltransferase5Lefaudeuxetal2016Wheelocketel.ERJ2013;42:80220132015UBIOPREDPROCESSOFSYSTEMSMEDICINETissuesamplesAnalysisofsputuminflammatorycellstranscriptomicsandproteome:‘sputumfingerprint’ChristosRossios,SteliosPavlidis,ChihhsiKuo,UrujHodaAJRCCMDistributionofneutrophilicandeosinophilicinflammationinsputumNeu>73.6%;Eos>1.49%Neu>73.6%;Eos<1.49%Neu<73.6%;Eos>1.49%Neu<73.6%;Eos<1.49%UrujHoda(PAUCI-GRANULOCYTIC)IL-1receptorassociatedgeneexpressionGeneSymbolSevereasthma(A+B)Vsnon-asthmaSevereasthma(A+B)Vsmild-moderateGeneTitleIL18R16.94013.5744interleukin18receptor1IL1RL16.06215.2603interleukin1receptor-like1CLC5.56293.4012Charcot-LeydencrystalproteinIL1RL15.26763.4201interleukin1receptor-like1IL1R24.35632.3219interleukin1receptor,typeIIIL18RAP3.87032.0532interleukin18receptoraccessoryproteinEDN13.63832.2799endothelin1SAMSN13.58982.1454SAMdomain,SH3domainandnuclearlocalizationsignals1LOC4006803.58052.1705UncharacterizedLOC400680CD243.53793.1411CD24moleculeSERPINB93.37722.3908serpinpeptidaseinhibitor,cladeB(IL1R23.32932.2053interleukin1receptor,typeIISLAMF73.32851.7347SLAMfamilymember7HIST1H2AC3.32842.036histonecluster1,H2acIRAK33.30551.7196interleukin-1receptor-associatedkinase3RHOH3.2861.7955rashomologfamilymemberHRHOH3.19632.2478rashomologfamilymemberHDifferentially-expressedgenesinsputumcellsenrichedinsevereasthmavsmild-moderateasthmaRossios,Pavlidisetal2016FC1.62,FDR0.007FC1.59,FDR0.0058FC1.31,FDR0.0963IL-1bprotein(Somalogic)elevatedinsputumofneutrophilicasthmaticsIL-1βHierarchicalClusteringNumberofdifferentiallyexpressedgenes478DEGsDefining‘diseasedrivers’fromsputuminflammatorycellpattern:eosinophilicvsnon-eosinophilicEOSvsHCnon-EOSvsHCnon-EOSvsEOSEosinophilicphenotype:

SputumEOS>1.5%(n=67)non-Eosinophilicphenotype:SputumEOS<1.5%(n=51)HealthyControl(n=21)Differentiallyexpressedgenefrom3setsofcomparison201genes145genes197genesKuoetal2016TAC1TAC2TAC3Eosinophilicnon-EosinophilicClustersfromdiseasedriversPhenotypefromcellproportion478DEGs478DEGsn=31n=31n=56Hierarchicalclusteringofdifferentiallyexpressedgenesbetweeneosinophilicvsnon-eosinophilicasthmainsputumcellsTAC:Transcriptome-associatedclusterKuoetal2016GTPase:RASAL3Metabolismofsteroid:UGT2B28TSLP

Receptor:CRLF2TcellReceptor:TRGC2,TRGV9,TARPProtease:PRSS33Eosinophillysophospholipase(Charcot-Leydencrystalprotein):CLCCytokinepromotingTh17differentiation:VSTM1Lactosebinding:LGALS12Suppressorofcytokinesignaling:SOCS2IL33Receptor:IL1RL1EotaxinReceptor:CCR3Phosphatase:PTPN7Tryptase:TPSB2Carboxypeptidase:CPA3Arachidonate15-Lipoxygenase:ALOX15G-proteincoupledreceptor:GPR42Transcriptionalfactor:OLIG2,SATB1IL3Receptor:IL3RABcellsurface:CD24Actin:FAM101BATPase:ATP2A3Cytokine:CSF1Multiplecytokinereceptors&Enzymes(protease,lipase..)Mechanism(genes)underlyingTAC1

TAC1TAC2TAC328genesKuoetal2016Receptor:TREML2,CLEC4D,IFIH1,SIGLEC5,LILRA5Cytokine:CXCL10(IP10,ligandofCXCR3),TNFSF10(TRAIL)GTPase:GIAMP4Adaptor:REPS2,SPATA13Transcriptionalactivator:CREB5Ubiquitination:WDFY3,UBE2D1Transcriptionalactivator:NMICaspase:CASP4Chromatinreplication:BAZ1AIFN-inducedprotein:IFIT2,IFIT3,IFI16,MNDAGTPcyclohydrolase:GCH1TNF-inducedprotein:TNFAIP3GTPase:RHOHCaspaseinhibitor:CARD16Neutrophilfunction:S100A9TNFandINFregulationPatternrecognitionreceptorMechanism(genes)underlyingTAC2

TAC1TAC2TAC327genesKuoetal2016Fattyacidmetabolism:ME1Intracellularvesicleandorganelletransport:MYO6,LMAN1

CAMP-DependentProteinKinase:PRKACBmitochondrialribosomalproteins:MRPS15,MRPS21Ubiquitination:TULP4,ZYG11B,BTBD1,RNF135Proteinaseinhibitor:CSTAToll-likereceptor:TLR7Celladhesionregulation(byinhibitingtrans-repressor):CTNND1Anti-viralenzyme:OAS1Pre-mRNAsplicing:ZCRB1,LSM5Lectinreceptor:LMAN1,CD302

CytochromeCoxydase:COA6Methyltransferase:METTL5Acetyltransferase:NAA20,ACAA2Fuctosidase:FUCA1Adenosinedeaminase:CECR1Aldehydedehydrogenase(Dendriticsignature):ALDH1A1Phospholipase:PLBD1Glycolipidmetabolism:PYURFMultiplemetabolicandubiquitinationenzymesMechanism(genes)underlyingTAC3

DDC1DDC2DDC331genesKuoetal2016Expressionofvariousgenesignaturesinthe3TACsusinggenesetvariationanalysisTAC1:T2withILC2,TSLPR,IL-33REosinophilicTAC2:Inflammasome,NeutrophilactivationNeutrophilinflammationTAC3:Th17,MitochondrialOXPHOS,AgeinggenesPaucigranulocyticoreosinophilicDDC1DDC2DDC3KuoandPavlidis2016

TAC1TAC2TAC3

Mechanisms‘T-2associated’Epithelialdriven‘Inflammasome’Macrophagedriven‘Mitochondrial’OxidativestressSputuminflammationEosinophilic/MixedNeutrophilic/MixedEosinophilic/PaucigranulocyticMicroarrayIL33R,TSLPR,CCR3,IL3RAIFN&TNFsuperfamily,CASP4MetabolicgenesGSVATh2/ILC2NLPR3/DAMP-associatedTh17;OXPHOS;ageingProtein(Somalogics)IL-16,Periostin,Serpinpeptidaseinhibitor1,ADIPOQTNFAIP6,MIF,TyrosinekinasesrcCathepsinB,GClinicalfeaturesSevereasthma;HighestnasalpolypsandOCSuse;SevereairflowobstructionModerate-to-severeasthmaMildairflowobstruction

Moderate-to-severeasthmaMildairflowobstruction

Transcriptome-associatedclusters(TAC)ofmoderate-severeasthmafromsputumanalysisKuoetal2016ClusteringapproachEosinophilcountsinbiopsyWithingroupsumofsquaresOptimalclustering4clustersonPartitioningAroundMedoids(PAM):intohigh/loweosinophilcounbtsBronchialsubmucosalprofileofhighandlowsubmucosalEOSgroupSubmucosalprofilesmean(SD)highEOS(n=33)lowEOS(n=48)p-valueCellcounts(count/mm3)Eosinophil9.8(6.1)1.1(1.1)2.2x10-9Neutrophil18.5(17.6)13.1(11.6)0.128Macrophage5.8(5.2)2.6(3.2)0.003CD4Tcell16.2(11.8)12.1(18.2)0.220CD8Tcell24.5(16.8)19.0(22.8)0.209Mastcell25.5(13.8)20.1(16.1)0.110ASMfraction(%)31.2(13.2)32.7(13.7)0.628Thickness(μm)9.6(2.0)8.2(1.7)0.006GeneexpressionprofilesinbronchialbiopsiesofasthmaassociatedwitheosinophilicinflammationKuoetal2016

Top10enrichmentfrompublicontologydatabasesofthe284differentially-expressedgenesDatabaseIDNamep-valueGO:0030198extracellularmatrixorganization2.2x10-6GO:0034341responsetointerferon-gamma1.1x10-5GO:0030334regulationofcellmigration5.2x10-5GO:0022617extracellularmatrixdisassembly5.4x10-5GO:0051270regulationofcellularcomponentmovement9.6x10-5KEGG:04512ECM-receptorinteraction1.4x10-5KEGG:04974Proteindigestionandabsorption4.1x10-5REAC:1474244Extracellularmatrixorganization9.4x10-8REAC:1650814Collagenbiosynthesisandmodifyingenzymes1.0x10-4REAC:1474228Degradationoftheextracellularmatrix8.3x10-4VOLCANOPLOTMMP10andMETaspredictiveclassifiersofbronchialeosinophilicasthmaGeneexpressionprofilesinbronchialbiopsiesofasthmaassociatedwitheosinophilicinflammationMMP10:

matrixmetallopeptidase10MET:METproto-oncogenereceptortyrosinekinase

HepatocyteGrowthfactorreceptorU-BIOPREDSputumHandprintofSevereAsthmaDianeLefaudeux1,B.DeMeulder,M.J.Loza,M.Sanak,D.W.Burg,J.P.R.Schofield,R.Djukanovic,K.F.Chung,F.Baribaud,P.J.Sterk,C.Auffray

1EISBM–CNRS-ENS-UCBL–FrancePlatformsusedforSputum‘omicsanalyses

Transcriptomic(cell)Affymetrix®HTHG-U133+PMonly(N=120)Proteomic(supernatants)Focused:SomaLogic®

SOMAscanassay(N=119)Non-Focused:UPLCMS/MS(N=246)Lipidomic(supernatants)Focused:Eicosanoidspanel(N=315)72asthmaticswiththe4platformsSimilarityNetworkFusion(SNF)Similaritynetworkfusionforaggregatingdatatypesonagenomicscale,B.Wangetal.,NatureMethods11,333–337(2014)DataIntegrationFrom:Similaritynetworkfusionforaggregatingdatatypesonagenomicscale,B.Wangetal.,NatureMethods11,333–337(2014),doi:10.1038/nmeth.2810SimilarityNetworkFusion(SNF)PatientsDNAmethylationDataPatientsmRNAexpressionPatientSimilarityMatricesPatientsPatientsPatientsPatientsPatientSimilarityNetworkFusionIterationsFusedPatientSimilarityNetworkSignificantFeaturesofhandprintsTranscriptomics(2324)SomaLogic®(258)Eicosanoids(7)UPLCMS/MS(17)Sputumhandprint:3clusters:characteristicsClusterSH1(N=22)ClusterSH2(N=27)ClusterSH3(N=23)P-valueSevereasthma2019180.4203FEV1

(%predicted)62.4±21.677.9±19.866.1±24.60.0411Exacerbation(previousyear)2(1–2)1(0–2.5)1(0–3)0.499

2ACQ51.8±1.142.08±1.411.83±1.280.733

1SinussurgerydiagnosedYes:

42.8%Yes:

3.7%Yes:18.2%0.003

3OCSuseYes:50%Yes:20.8%Yes:

40.9%0.1093Bloodneutrophils(x103/µL)5.83±1.84.15±1.984.71±2.240.0201Bloodeosinophils(x103/µL)0.3(0.132–0.53)0.2(0.14–0.3)0.26(0.115–0.445)0.1912Sputumeosinophils(%)2.42(0.40–10.5)0.39(0–3.64)29.0(1.04–52.6)<0.0012Sputumneutrophils(%)75.7±18.147.9±19.434.5±21.3<0.0011Sputummacrophages(%)9.88(4.16–16.8)48.8(29.9–63.5)26(18.3–38.3)<0.00121ANOVA 2Krukal-Wallistest 3

χ²-testFunctionalPathwayAnalysisTranscriptomics1vs2(2178probesets)TermP-valueStructuralconstituentofribosome(GO:MF)<0.001Translationalinitiation(GO:BP)<0.001Translationaltermination(GO:BP)<0.001GeneExpression(REAC)-Nonsense-MediatedDecay-Translation<0.001<0.001<0.001Ribosome(KEGG)<0.001Cellularrespiration(GO:BP)<0.001Oxidativephosphorylation(KEGG)<0.001Thecitricacid(TCA)cycleandrespiratoryelectrontransport(REAC)<0.001Membraneorganisation(GO:BP)<0.0011vs3(178probesets)TermP-valueResponsetostress(GO:BP)<0.001RegulationofBcellapoptoticprocess(GO:BP)0.0168Immuneresponse(GO:BP)0.0282ImmuneSystem

(REAC)0.0388Interferonalpha/betasignaling(REAC)0.01702vs3(68probesets)TermP-valueLinoleicacidmetabolism(KEGG)0.05TranscriptionalRegulationbyTP53(REAC)0.05ImpairedhostdefenceINFLAMMATION&REMODELLINGDisorderedphysiologySymptomsExacerbationsGeneProteinCellTissueOrganPatientHOSTIntegratedMulti-scaleApproachtoObstructiveAirwayDiseaseMechanismsEnvironmentalChallengeSusceptibilityPATHOGENSBacteria/Viruses/MouldsALLERGENSPOLLUTION&PhysicalDamageTIMET2ASMChyperresponsivenessRemodeling/repair

EosinophilicinflammationSEVEREASTHMA:Poorasthmacontrol;Recurrentexacerbations;Chronicairflowobstruction;CorticosteroidinsensitivityTh2B-cellEosinophilIgEIL-4,IL-5,IL-13EpitheliumAllergens,Virus,ParticlesPollution&oxidantsTh0MHCIITCRB7.2CD28IL-12DendriticcellsGrowthfactorse.g.TGFb

AirwaysmoothmuscleExtracellularmatrixGM-CSFEotaxinRANTESNeutrophilTSLP,IL-33,IL-25Th1Th17ILC-2TNFaIL-1ßIFNγ,TNFαIL-17A,E,FIL6TGFβIL-8MacrophageMastcellHistamineLeukotrienesNeutrophilicinflammationMitochondrialdysfunctionTLR