dmdr maria hdhernandez-fuentes - sefh · p-value q-value genes with highest enrichment scores...

Bi dBiomarcadoresde utilidadde utilidadclínica enclínica en TrasplanteTrasplante

D M H d FDr Maria Hernandez-Fuentes

Guy’s and St Thomas’ NHS Foundation Trust and King’s College London’scomprehensive Biomedical Research Centre

The Translational ‘fever’The Translational feverScientificScientific discovery

Useful ClinicalClinical Event


Insuficiencia funcional de un órgano www.getbetterhealth.comórgano ....

Transplante

=> inmunosupresión de por vida

Biomarcadores pueden indicar como utilizarmantener los pacientes en las mejoresmantener los pacientes en las mejorescondiciones posibles


Necesitamos biomarcadores en transplante?p

• predecir la evolución natural del trasplante. Supervivencia del paciente y el injertoinjerto• predecir el riesgo de cada individuo de sufrir eventos de importancia

• Acute rejection (AR)j ( )• Chronic rejection (CR)• Developing donor-specific tolerance

• evaluar el grado de actividad/deterioro de la función del injerto• evaluar el grado de actividad/deterioro de la función del injerto• high-performance techniques:

• genómicaó• microarrais expresión mRNA

• microarrais microRNA• metabolómica

t ó i• proteómica• cytómica

gran capacidad de detectar biomarcadores múltiples


gran capacidad de detectar biomarcadores múltiples

Rejection vs ToleranceRejection vs Tolerance

Regulation

RejectionTissue damage:

eGFR eGFRCRT


Transas

Consequence for patientsConsequence for patients

Cardio-Vascular events Rejection:

Acute

Cancer

AcuteChronic

Infections CellularAb mediatedAb-mediated

Nephrotoxicity Alive!


Alive!

Cuándo analizar biomarcadoresPERI-TRANSPLANTPRE-TRANSPLANT POST-TRANSPLANT

Humoral sensitization

Drug toxicity

RejectionRecurrentprimarydisease Pharmacological

immunosuppression

1 year

Heterologousimmunity

immunosuppression

Ischemiaf i

Cellularalloimmunity

reperfusion

Surgicaldamage

Drug

Immune system

Graft response to injuryDrug

toxicity

Rejection

Pharmacological

Systemic effects of organdamage

Patient compliance


Pharmacologicalimmunosuppression

Biomarcadores de utilidad clínicaBiomarcadores de utilidad clínica

Bi d d R h A d• Biomarcadores de Rechazo Agudo en trasplante renaltrasplante renal

• Biomarcadores de rechazo agudo en trasplante cardiaco

Bi d d t l i t l t•Biomarcadores de tolerancia en trasplantesde hígado y riñóng y

• Perspectivas para el futuro


M l lMolecular

Cellular / HistologicalCellular / Histological

Clinical

Time

Tx Rationale: earlyTx Rationale: early intervention

GWAS microRNAmRNA

proteins

Molecular and cellular surveillance t t iproteins strategies

anticipate histological and


histological and clinical rejection.

Rúbricas moleculares en biopsiasRúbricas moleculares en biopsiasCriterios Histopatológicos BANFF

ion

of r

ejec

tob

abili

tyig

ned

pro

Anglicheau D, et al Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5330-5.PA

M a

ssi

Famulski KS, et al American Journal of Transplantation 2010; 10: 810–820.


A Molecular classifier for predicting future graft lossEinecke G, et al J of Clinical Investigation 2010; 120 (6): doi:10.1172/JCI41789

Biomarcadores en Orina / SangreBiomarcadores en Orina / Sangre

Muthukumar et al

CD154 ICOS CTLA4 PD1

Muthukumar, et al Alakulppi et al, Transplantation 2007;83: 791–798


Kidney ALograft Immune Biomarkers of REjection& Immune Monitoring for AR (KALIBRE & IMAR)& Immune Monitoring for AR (KALIBRE & IMAR)

Study cohort: 200 consecutive kidney transplant patients y y p p

(adults) performed at Guy’s Hospital

1 2 3 4 5 6 7 8 9 10 11 12 Time(months)

Frequency

W kl Fortnightly Monthly + any ARWeekly Fortnightly Monthly

4ml peripheral blood EDTA 3 3 l bl d U iRecruitment 4ml peripheral blood EDTA 3x3 ml blood

Cryopreserved PBMCs mRNA

Urin

e

Recruitment

Biobank

Flow cytometry RT-PCRBRC facilities


Preliminary Results 1Preliminary HTqPCR data:

10 i

y

10 muestras por pacientecada 2 semanas en 13 estables and 8 con ARestables and 8 con AR diagnosticado con BiopsiaIP−10

Semana 2 post-Tx

C f

Granzyme B

Results completos en TGF−b

C6orf25

p2012

PF4


Sta

ble

Sta

ble

Sta

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Sta

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Sta

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Sta

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Sta

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Sta

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Sta

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Rej

ecto

r

Sta

ble

Rej

ecto

r

Sta

ble

Rej

ecto

r

Sta

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Rej

ecto

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Sta

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Sta

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Rej

ecto

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ecto

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Integrando información de dominio úblipúblico

3 biopsy-based microarray studies of AR frompediatric renalpediatric renal, adult renal &adult cardiac transplantationadult cardiac transplantation identified 45 genes upregulatedin all three.

chose 10 proteins for serum ELISA assays => 3 high in AR

PECAM1 is increased in AR in hepatic, renal and cardiac Chen R, ... Butte A. PLoS


hepatic, renal and cardiac Chen R, ... Butte A. PLoSComput Biol. 2010 Sep 23;6(9).


Bi d d R h A d• Biomarcadores de Rechazo Agudo en transplante renaltransplante renal

• Biomarcadores de rechazo agudo en trasplante cardiaco

Bi d d t l i t l t•Biomarcadores de tolerancia en trasplantesde hígado y riñóng y

• Perspectivas para el futuro


CARGO studyCARGO study

n= 63 (31 AR, 32 quiescent)

n= 184 (62 AR, 122 quiescent)122 quiescent)



Bi d d R h A d• Biomarcadores de Rechazo Agudo

• Biomarcadores de rechazo agudo en• Biomarcadores de rechazo agudo en trasplante cardiaco

• Biomarcadores de tolerancia en trasplantesd hí d iñóde hígado y riñón

• Perspectivas para el futuroPerspectivas para el futuro


Transplantation tolerance would addressTransplantation tolerance would address major problems that limit transplants half-life:

transplants would last longer; less patients would transplants would last longer; less patients would return to waiting list

chronic rejection would be reducedj

i i ld b i i i d/ ithd immunosuppression could be minimised/withdrawn


... “bespoke immunosuppression”

ObjetivosObjetivos Definir “Rúbrica molecular” asociada tolerancia Definir Rúbrica molecular asociada toleranciaoperativa

Desarrollo de tests in vitro específicos, reproducibles y fiables asociados a toleranciareproducibles y fiables asociados a toleranciaoperativa

Transferencia a la clínica: desarrollo de herramientas que permitan predecir a quéherramientas que permitan predecir a quépacientes se les puede disminuir la inmunosupresión


inmunosupresión.

Identificación de Biomarcadores de t l i t l t d hí dtolerancia en trasplante de hígado

SerumLeukocytesWh l bl d Liver tissue

Immunosuppressedstable recipient

Graft dysfunction Non-tolerantLiver

Whole blood Liver tissue

Drug Withdrawal Stable graft

Graft dysfunctionand biopsy-proven

rejectionrecipients

>3

Transplantation

Withdrawal Operationaltolerance

Stable graftfunction and no

signs ofhistological

damage

years

SerumLeukocytesWhole blood

Liver tissue SerumLeukocytesWhole blood

Liver tissue SerumLeukocytesWhole blood

Liver tissue


Prof Alberto Sanchez-Fueyo, Hosp Clinic, Barcelona y Cols

497 Patients were screened

395 Patients were excluded

497 Patients were screened

160 had been transplanted for < 3 years58 had medical disorders incompatible with the safe conduct of the study

and/or interpretation of the results. 35 declined to participate30 were included in another clinical trial29 had history of autoimmune liver disease19 h d b l li f ti t t di ifi d it i19 had abnormal liver function tests exceeding pre‐specified criteria19 exhibited abnormalities in basal liver biopsy exceeding pre‐specified criteria18 could not be closely followed‐up14 had history of rejection in the previous 12 months9 had no side effects of IS drugs4 were already receiving no immunosuppressive drugs

102 Patients were enrolled

4 P ti t ithd f t d d i d d ti4 Patients withdrawn from study during dose reduction classified as Non‐TOLERANT in the ITT analysis

41 Patients were TOLERANT 57 Patients REJECTED


Peripheral blood transcriptional and cellular markers of operational tolerance

Enriched in tolerant recipients p-value q-value Genes with highest enrichment scores Geneset

database

Natural Killer (CD56) cell lineage

0.000 0.000 KLRC3, NCAM1, CD160, BNC2, KIR3DL2, LAIR2, SH2D1B, KIR3DL1,KLRF1, CLIC3, AKR1C3, FEZ1, PDGFRB, GZMB, IL2RB

Haematlas

Graft versus host disease

0.000 0.000 KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL1, GZMB, KIR2DL5A, KLRD1 KEGG

disease

Antigen processing and presentation

0.000 0.000 KLRC4, KLRC3, KIR3DL2, KIR3DL1, KIR2DS3, KIR2DS4, KIR2DL5A,KIR2DL1, KIR2DS1, KLRD1

KEGG

N t l Kill ll0.000 0.001 KLRC3, KIR3DL2, SH2D1B, KIR3DL1, KIR2DL2, GZMB, KIR2DL1, TNF,

KLRD1 KIR2DL3Biocarta

Natural Killer cell mediated cytotoxicity

KLRD1, KIR2DL3

VIP pathway0.000 0.001 EGR3, EGR2, PRKAR1B, NFKBIA, PRKAR2B, NFATC2, PPP3CC KEGG

DARPP32 events0.000 0.006 PDE4D, PRKAR2B, PRKAR2B, PPP3R1, PDE4B, PPP3C. Biocarta

Immunoregulatory 0.000 0.013 CD160, KIR3DL2, KIR3DL1, KIR2DL2, KIR2DS2, KIR2DL3, CD226,KIR2DL1, ICAM4

Reactome


interactions

Intra‐graft expression differences between tolerant and non‐tolerant grafts: a role for iron metabolism genesnon‐tolerant grafts: a role for iron metabolism genes

Non-TOL TOL TFRC 0,000 -2,029HAMP 0,000 3,737MYO19 0,000 1,356

FDR Fold changeGene symbol

FTHL12 0,000 -1,414FTHL8 0,000 -1,430TANK 0,000 -1,249GHSR 0,000 -1,261UNG 13,663 -1,394UPK3A 13,663 -1,245C1ORF61 13 663 1 219C1ORF61 13,663 -1,219TPPP3 13,663 -1,229MUTED 13,663 -1,258TSPAN2 13,663 -1,256FDXR 13,663 -1,451RTP2 13,663 -1,222EPHX1 13 663 -1 380

GO identification Ontology P-value Pathway/Function Genes

GO:0006879 BP 0,00004 cellular iron ion homeostasis CP, FTH1, HAMP, TFRCEPHX1 13,663 -1,380

G3BP1 13,663 -1,220TAS2R50 13,663 -1,283HSD17B11 13,663 -1,297FAM162A 13,663 -1,295RNASE13 13,663 -1,215DPP4 13,663 -1,285

TFRC

GO:0055072 BP 0,00008 iron ion homeostasis CP, FTH1, HAMP, TFRC

GO:0016724 MF 0,00010oxidoreductase / ferroxidase activity, oxidizing metal ions, oxygen as acceptor

CP, FTH1DPP4 13,663 1,285CCDC46 13,663 -1,184FBXL4 13,663 -1,238ANKRD5 13,663 -1,225UMODL1 13,663 -1,213TUBA8 14,802 -1,205SYNE2 14,802 -1,216PLXNA4B 14,802 -1,263CCNJ 14,802 -1,306RAG2 14,802 -1,195CHD3 14,802 -1,301ADSSL1 14,802 -1,227FTHL3 14,802 -1,281TRPV1 14 802 1 450


176 genes; FDR <25%TRPV1 14,802 -1,450WAC 14,802 -1,328SLC39A4 14,802 -1,261MCOLN1 19,031 1,295

Kidney: Indices of Tolerance, y ,King’s College London, UK

Robert LechlerMaria Hernandez Fuentes

Oxford University, UKKathryn WoodStephanie ChapmanPiotr Trzonkowski

BerlinCologne

BerlinCologne

Miltenyi Memorec, GermanyUwe JanssenBettina PetersStefan TomiukMaria Hernandez Fuentes

Pervinder Sagoo Esperanza Perucha Elvira JimenezFlavia RovisSaskia Stevenson

Gregor WarneckeIan RobertsLondon

OxfordLondonOxford

Imperial College London, UK

Charité University Medicine, Germany

Hans Dieter Volk

Stefan Tomiuk

Saskia StevensonSharon Hughes

London, UKAnthony WarrensAmany BallowRuhena SergeantJan WatersJackie and Katie

Hans-Dieter VolkBirgit Sawitzki

Guy’s Hospital, UK

Rachel Hilton

Immune Tolerance Network

Irene Rebollo Mesa

Jackie and KatieRachel HiltonRobert VaughnFred ComptonLiz, Ola and Diane

McGill University Montreal, CanadaDavid Stephens

Network

Institute for Medical Immunology, ULB, Belgium Michel Goldman

Ligia Cracium

Brussels

NantesNantes

ITERT - INSERM, FranceJean-Paul Soulillou

Sophie Brouard

Vicki SeyfertLarry Turka

Kenneth NewellKasia BourcierLigia Cracium

Alain Le MoineMyriam Libin

NantesNantesSophie BrouardCecile BraudeauMagali Giral Patrick Miqueu

Kasia BourcierIgnacio Sanz

Wei ChungwenJames Roger


Participating PhysiciansParticipating PhysiciansUKPoland

Dr Magdalena KrajewskaIreland

Dr Richard BakerSt. James's University Hospital

Dr Sue CarrLeicester General Hospital

Dr Rachel HiltonFrance

Dr Herve Le Monies de S.

Dr Magdalena KrajewskaMedical University of Wroclaw, Wrocław

Dr Alan WatsonSt Vincent’s

Hospital, Dublin

Dr Rachel Hilton Guy’s &St. Thomas’ NHS Trust London

Dr Phillip MasonJohn Radcliffe Hospital, Oxford

Dr Will McKaneSheffield Kidney Institute

Hôpital Victor Provo, Roubaix

Dr Legendre& Sophie Lechaton

Hôpital Necker-Enfants

SwitzerlandDr Markus Mohaupt

Inselspital, Universitätsspital Bern

Sheffield Kidney InstituteDr Jo TaylorDorset County Hospital

Dr Sui Phin KonGuy’s &St. Thomas’ NHS Trust London

D Ad Sh if

pMalades, Paris

Dr Magali Giral& Dr Jean Paul Soulillou

CHU-HôtelDieu, NantesDr Evangeline Pillebout Dr Adnan Sharif

Univesity Hospital Wales, Cardiff

Spain

BelgiumDr Alain Lemoine

Dr Evangeline PilleboutHôpital Saint Louis, Paris

Czech RepublicD O d j Vikli kýSpain

Dr Josep Campistol& Dr Fritz Dickman

Hospital Clínic de Barcelona Dr Juan Bravo

& Dr Martin WissingCUB Hôpital Erasme

Bruxelles

Italy

Dr Ondrej ViklickýKlinika Nefrologie TC IKEM

Prague


Hospital Universitario Virgen de las Nieves, Granada

yDr Giuseppe Orlando

Ospedale S. Eugenio, Rome

Patient sets to search for biomarkers of Tolerance:

EU cohort• Tolerants

USA cohort• Tolerants

• n=11• Stable function

L P d

• Tolerants• n=24

• Stable function• Low Pred• CNI-free• Triple therapy

Stable function• Monotherapy• Triple therapy

• Triple therapy• Chronic rejection

• CAN


Lymphocyte subsetsLymphocyte subsetsEU USA


Microarray assaysMicroarray assaysAgilent customized 8 x 15 K format with 60 li l id b60-mer oligonucleotides as probes. “RISET 2.0” microarray consists of 5,069 diff b i i li Si l ldifferent probes in triplicates Single-color microarray experiments


MicroarrayTraining set T t t

Custom microarray analysis (5069 probes, 4607 genes)

Microarray analysis

Training set Test set

CNI

Tol-DF Tol-DF

s-CNIs-nCNI s-LP

CR CANs-CNIMono

Four-class analysisusing a Kruskal-Wallis nonparametric test.

CD79BTCLA1HC HC

pProbes were rankedwithin the training set based on their P values with adjustment

HS3ST1SH2D1BMS4A186

174

1204for 1% FDR. Top 10 ranked probes that overlapped with genes

MS4A1 TLR5FCLR14identified

in the test set were subsequently used for ROC

l i

PNOCSLC8A1FCRL2


Top 10 ranked probes/genes selected for ROC analysisanalysis. FCRL2

Set of Biomarkers 1Set of Biomarkers 1

Lower percentage of recently activated T cells Lower percentage of recently activated T cells

Expansion of peripheral blood B cells. High p p p gB/T cell ratio.

Absence of anti-donor Ab

Lower direct pathway anti donor responses Lower direct pathway anti-donor responses

High Foxp3/a1,2-mannosidase High Foxp3/a1,2 mannosidase

Differential expression of a set of 10 genes


Cross-platform biomarkerCross platform biomarker ROC curve

EU USAEU USA

Sensibility = 1 Sensibility = 0.903Specificity = 0 923Specificity = 1 Specificity = 0.923


Probability of beingProbability of being tolerant

Training set Test setEU USA

oler

ant

3

10-2

10-1

100

oler

ant

1

100

of B

ein

g To

10-11

10-10

10-9

10-8

10-3

f B

eing

To

10-2

10-1

obab

ility

o

10 16

10-15

10-14

10-13

10-12

10

babi

lity

of10 710-610-510-410-3

Patient groups

Pro

10-17

10-16

Tol-DF s-LP s-CNI/-nCNI CR

Patient groups

Pro

10-1010-910-810-7

Tol-DF Mono s-CNI CAN


Patient groups Patient groups

Validación extensiva:Validación extensiva: GAMBIT

• Prevalencia del fenotipo de tolerancia: Recipientescon función estable > 3 5 yrs post Tx econ función estable, > 3-5 yrs post-Tx, e inmunosupresión standard, BioM of Tol is ~ 5 - 15%. n = 200

• Repetitividad: > 80% de pacientes tolerantesRepetitividad: > 80% de pacientes tolerantesidentificados de novo expresan los BioM of Tol. n = 20

t l+ controls

• Estabilidad: BoM of Tol permanecen estables


pdespues de disminución de inmunosupresión

ConclusionesConclusiones Se estan desarrollando bioensayos y biomarcadoresy yque ayudan determinar si lo pacientes tienen un riesgo mas alto de sufrir rechazo si lo pacientes tienen un riesgo mas alto de sufrir rechazo agudo => oportunidad de incrementar el tratamiento si los pacientes estan desarrolando características de si los pacientes estan desarrolando características de tolerancia => oportunidad de disminuir el tratamiento

El futuro del tratamiento del post transplante se El futuro del tratamiento del post-transplante se basará en regímenes terapeúticos basados en

dBiomarcadores Medicina personalizada


p

The GAMBIT ConsortiumKing’s College London, UKMaria Hernandez Fuentes

Sonia NorrisEstefania No a Lampe ti

Cardiff and Vale University Health Board

Hull Royal InfirmaryDr Sunil BhandariKaren James

Tracy Cathcart

St Jame’s HospitalLeedsDr Richard Baker

Estefania Nova-LampertiPaula MobilloYogesh KamraMano Runglall

Tom LewisFlorence Delany

Health BoardDr Sian Griffin

Tracy Cathcart

Royal Free HospitalDr Alan Salama

Leicester General HospitalDr Sue CarrRachel Westacott

Dr Aravind Cherukuri

St Geo ge’s HospFlorence DelanyIrene Rebollo-Mesa

Rosalynn MillerDarlene Catalan

Jude GreenRobert Lechler

Roy Marshall King’s College HospitalDr Sui Phin KonBeatriz Tucker

Nicolene Atkinson

Queen Alexandra Hospital,Postmouth

Dr Judith Stevens Manchester Royal

St George’s HospDr Iain McPhee

Dr Joyce PopoolaRaj Ramkhelawon

Robert LechlerGraham Lord

Guy’s Hospital, UKRachel HiltonRobert Vaughn Transplantační laboratoř

Kent and CanterburyDr Chris FarmerGillian Eaglestone

Hazel Broad

Dr Judith StevensLynn Watkins

Frances Williams

Manchester Royal InfirmaryDr Hany Riad

Dr Nick Simon Clare Griffing

Hospital Universitari Valld'Hebrón, Barcelona

Dr Daniel SeronMaria Sarria

Transplantační laboratořIKEM, Prague

Dr Ondrej VicklikyPetra Hribova

INSELSPITAL, Universitätsspital BernDr Markus Mohaupt

Petra Hribova

Evelina Children’s HospDr Manish Sinha

Liz Reus, Joy & Paula

Hospital de las Cruces, Bilbao

Dra Sofia Zarraga

Petra Hribova

G t O d St t

Salford RoyalDr Phillip KalraLesley Haydock

Northern General Hospital,SheffieldDr William McKane

Faith OkuhoyaGlasgow General HospitalDr Patrick Mark


Great Ormond StreetDr Stephen Marks

Dr Patrick MarkDonna Kelly

Lorraine McGregor

Guy’s and St Thomas’ NHS Foundation Trust and King’s College London’sComprehensive BiomedicalComprehensive Biomedical Research Centre

Dr Paramit Chowdhury

D Rachel HiltonDr Rachel Hilton

Prof Steve Sacks

Prof Robert LechlerProf Alberto Sanchez-

Prof Robert Lechler

Prof Graham LordFueyo

Hospital Clinic Barcelona


dmdr maria hdhernandez-fuentes - sefh · p-value q-value genes with highest enrichment scores...

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