Non-Invasive Rejection Diagnosis Using Non-Invasive Rejection Diagnosis Using Urine NMR SpectraUrine NMR Spectra

David RushDavid RushWinnipeg Transplant GroupWinnipeg Transplant Group

University of ManitobaUniversity of Manitoba

Immune Monitoring for Rejection of Immune Monitoring for Rejection of Kidney TransplantsKidney Transplants

“…“…the clinical manifestations of acute rejection have changed with the clinical manifestations of acute rejection have changed with present-day immmunosuppression. There are usually no local present-day immmunosuppression. There are usually no local symptoms, and the abnormalities are typically limited to symptoms, and the abnormalities are typically limited to insidious, low-level dysfunction of the graft...”insidious, low-level dysfunction of the graft...”

“… “… systematic and repeated urinalyses performed in the absence of systematic and repeated urinalyses performed in the absence of substantial changes in graft function may provide a unique substantial changes in graft function may provide a unique opportunity to detect subclinical episodes of rejection that may opportunity to detect subclinical episodes of rejection that may culminate in chronic rejection…”culminate in chronic rejection…”

Soulillou (NEJM (2001) 344:1006)Soulillou (NEJM (2001) 344:1006)

Editorial comment to Li et al (NEJM (2001) 344:945)Editorial comment to Li et al (NEJM (2001) 344:945)

Surveillance for Acute RejectionSurveillance for Acute RejectionStandard of Practice: Serum CreatinineStandard of Practice: Serum Creatinine

Diagnostic Threshold

Baseline Function

Treatment

InflammationCrCr

StrengthsStrengthsSamples the Entire GraftSamples the Entire Graft

Rapid Turnaround TimeRapid Turnaround Time

Non-invasiveNon-invasive

InexpensiveInexpensive

Widely AvailableWidely Available

WeaknessesWeaknesses

Lacks SpecificityLacks Specificity

(Need a Biopsy to Diagnose Rejection)(Need a Biopsy to Diagnose Rejection)

Lacks SensitivityLacks Sensitivity

CapillaryCapillary

Immune SurveillanceImmune Surveillance Goal is to Develop a Biomarker in the Blood or UrineGoal is to Develop a Biomarker in the Blood or Urine

GRAFT CTLCTL

Renal TubuleRenal Tubule

UrineUrineBloodBlood

Anti-HLA Anti-HLA AntibodyAntibody

Immune SurveillanceImmune Surveillance Probe for the Probe for the Inflammatory Programs of Acute RejectionInflammatory Programs of Acute Rejection

ThTh

Th

IL-2IL-2

CTL

Granzyme BGranzyme BPerforinPerforin

FasFas

Th

MTNFTNFIFNIFN

B antiantiHLA AbHLA Ab

IL-4IL-4IL-10IL-10

APC

Th

CostimuliCostimuli• B7:CD28B7:CD28• CD40-CD40LCD40-CD40L

IL-2IL-2

AllorecognitionAllorecognition• DirectDirect• IndirectIndirect

IL-15IL-15

Immune SurveillanceImmune Surveillance Blood and Urine BiomarkersBlood and Urine Biomarkers

Blood:Blood:» PBMCPBMC RT-PCR CTL gene transcripts ( Fas, Granzyme, Perforin )RT-PCR CTL gene transcripts ( Fas, Granzyme, Perforin )

Vasconcellos et al (Transplantation 1998;66:562)Vasconcellos et al (Transplantation 1998;66:562)

Urine:Urine:» Flow cytometry to detect CD3 and HLA-DR on urine cellsFlow cytometry to detect CD3 and HLA-DR on urine cells

Roberti et al (Transplantation 1995;59:495)Roberti et al (Transplantation 1995;59:495)

» RT-PCR CTL gene transcripts ( Granzyme, Perforin )RT-PCR CTL gene transcripts ( Granzyme, Perforin ) Li et al (NEJM 2001;344:945)Li et al (NEJM 2001;344:945)

Immune SurveillanceImmune Surveillance Blood or Urine Biomarker DevelopmentBlood or Urine Biomarker Development

LimitationsLimitations to the development of biomarkerto the development of biomarker» ““Tarnished” Gold Standard (i.e. classification error of the biopsy)Tarnished” Gold Standard (i.e. classification error of the biopsy)

» Lack of Specificity of any single biomarkerLack of Specificity of any single biomarker Biomarkers should distinguish Acute Rejection vs. Drug toxicity, Infection, ATNBiomarkers should distinguish Acute Rejection vs. Drug toxicity, Infection, ATN

SpecificitySpecificity could be improved by developing a:could be improved by developing a:» Donor antigen specific assayDonor antigen specific assay

Requires donor antigen source (e.g. donor spleen cells)Requires donor antigen source (e.g. donor spleen cells)

» Profile based on all components ( known / unknown ) in a blood or urine sampleProfile based on all components ( known / unknown ) in a blood or urine sample Requires strategies able to “Requires strategies able to “profileprofile” all components in a sample” all components in a sample

Require Donor Cells for AnalysisRequire Donor Cells for Analysis

» Flow Cross-match (anti-HLA Ab)Flow Cross-match (anti-HLA Ab)O’Malley et al (ITS 1998 Abstr #1370)O’Malley et al (ITS 1998 Abstr #1370)

» ELISPOT Cytokine AssayELISPOT Cytokine AssayHeeger et al (J Immunol (1999) 163:2267)Heeger et al (J Immunol (1999) 163:2267)

» DTH Assay (“Tolerance Assay”)DTH Assay (“Tolerance Assay”)VanBuskirk et al (J Clin Invest (2000) 106:145)VanBuskirk et al (J Clin Invest (2000) 106:145)

Immune SurveillanceImmune Surveillance Donor Antigen Specific BiomarkersDonor Antigen Specific Biomarkers

APC

Th

CostimuliCostimuli• B7:CD28B7:CD28• CD40-CD40LCD40-CD40L

IL-2IL-2

AllorecognitionAllorecognition• DirectDirect• IndirectIndirect

IL-15IL-15

DNADNA mRNAmRNA ProteinProtein

acgtaccaacgtaccaaggtaacgaggtaacg

cggtttttcgtcggtttttcgtgtatctcccttgtatctccctt

GenomeGenome TranscriptomeTranscriptome ProteomeProteome

30,000 – 50,00030,000 – 50,000GenesGenes

> 100,000> 100,000mRNAsmRNAs

> 1,000,000> 1,000,000ProteinsProteins

Immune SurveillanceImmune Surveillance Strategies to Profile all Components in the Blood or UrineStrategies to Profile all Components in the Blood or Urine

Immune SurveillanceImmune Surveillance Can Early Allograft Inflammation be Can Early Allograft Inflammation be

Detected by a Distinct Urine MR Spectral Profile?Detected by a Distinct Urine MR Spectral Profile?

Study Design: Study Design:

» Gold Standard:Gold Standard: Protocol Biopsy (months 1, 2, 3 and 6) Protocol Biopsy (months 1, 2, 3 and 6)

» Urine:Urine: Collected at time of Protocol Biopsy and stored at -80°C Collected at time of Protocol Biopsy and stored at -80°C

Study Population:Study Population:

» ““Normal” Urine Spectra:Normal” Urine Spectra:– Transplant Patients with Normal Histology by Protocol BiopsyTransplant Patients with Normal Histology by Protocol Biopsy

» ““Rejection” Urine Spectra:Rejection” Urine Spectra:– Transplant Patients with Acute Rejection by Protocol BiopsyTransplant Patients with Acute Rejection by Protocol Biopsy

Developing an MR Biomarker Makes No Assumption as Developing an MR Biomarker Makes No Assumption as to What Target is Importantto What Target is Important

““Normal”Normal”SpectraSpectra

““Rejection”Rejection”SpectraSpectra

ClassifierClassifier““Rejection”Rejection”

INFORMATICSINFORMATICS Rate-Limiting Step is Analysis of the Spectral ProfileRate-Limiting Step is Analysis of the Spectral Profile

11H MR spectraH MR spectra

» 0.5-4.5 and 6.5-9.5 ppm 0.5-4.5 and 6.5-9.5 ppm

» 16901690 data points / spectra data points / spectra

Multivariate classification strategy:Multivariate classification strategy:

» Optimal region selector (data reduction)Optimal region selector (data reduction)

» Bootstrap cross-validationBootstrap cross-validation

» Linear Discriminant Analysis (LDA) classifierLinear Discriminant Analysis (LDA) classifier

11H MR Biomarkers Developed from the Urine Spectra H MR Biomarkers Developed from the Urine Spectra Correctly Identify Allograft HistologyCorrectly Identify Allograft Histology

Spectral RegionsSpectral Regions

SensitivitySensitivity

SpecificitySpecificity

PPVPPV

NPVNPV

CrispnessCrispness

11stst Generation Generation

(33 vs 35)(33 vs 35)

66

88%88%

93%93%

93%93%

96%96%

75%75%

22ndnd Generation Generation

(70 vs 41)(70 vs 41)

6 + 56 + 5

98%98%

96%96%

98%98%

96%96%

96%96%

33rdrd Generation Generation

(81 vs 46)(81 vs 46)

6 + 66 + 6

91%91%

95%95%

95%95%

91%91%

94%94%

Normal vs Rejection HistologyNormal vs Rejection Histology

A Biomarker for Rejection Must Be SpecificA Biomarker for Rejection Must Be Specific

0

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10 11 12

Weeks Post-TransplantWeeks Post-TransplantC

reat

inin

e (

Cre

atin

ine

( m

ol/L

)m

ol/L

)

BiomarkerBiomarker NN NN NNNN NNNN NN NN

BiopsyBiopsy i0t0i0t0(ATN)(ATN)

i1t0i1t0 i1t0i1t0i0t0i0t0

Simulect ™Simulect ™Neoral ™Neoral ™MMF ™MMF ™PrednisonePrednisone

The Biomarker for Rejection May Precede The Biomarker for Rejection May Precede the Histologic Diagnosis of Rejection the Histologic Diagnosis of Rejection

0

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10 11 12

Weeks Post-TransplantWeeks Post-TransplantC

reat

inin

e (

Cre

atin

ine

( m

ol/L

)m

ol/L

)

BiomarkerBiomarker RjRj RjRj NNRjRj NNNN NN

BiopsyBiopsy(SC)(SC)i2t3i2t3 i0t0i0t0i0t0i0t0

Steroids

Simulect ™Simulect ™Neoral ™Neoral ™MMF ™MMF ™PrednisonePrednisone

0

100

200

300

400

500

0 1 2 3 4 5 6 7 8 9 10 11 12

The Biomarker for Rejection can Persist After The Biomarker for Rejection can Persist After Allograft Function Returns to BaselineAllograft Function Returns to Baseline

Weeks Post-TransplantWeeks Post-TransplantC

reat

inin

e (

Cre

atin

ine

( m

ol/L

)m

ol/L

)

BiomarkerBiomarker RjRj RjRj RjRjRjRj RjRjRjRj RjRj

BiopsyBiopsy i2t2i2t2(SC)(SC)

i3t2i3t2(CL)(CL)

RjRj RjRj RjRj RjRj

18

NN

19

NN

20

NN

Steroids

Neoral ™Neoral ™MMF ™MMF ™PrednisonePrednisone

UrineUrine 1 1H MR BiomarkerH MR BiomarkerPrecedes and Persists after the Diagnosis of RejectionPrecedes and Persists after the Diagnosis of Rejection

46 patients had 154 protocol biopsies46 patients had 154 protocol biopsies» 31/154 biopsies had diagnosis of Acute Rejection31/154 biopsies had diagnosis of Acute Rejection» 24/31 had a urine sample prior to the biopsy24/31 had a urine sample prior to the biopsy

– 18/24 the urine MR classifier for rejection was present 1-2 weeks prior 18/24 the urine MR classifier for rejection was present 1-2 weeks prior to to the biopsy.the biopsy.

» 15/24 had urine samples collected after the biopsy15/24 had urine samples collected after the biopsy

– 9/15 the urine MR classifier for rejection disappeared within 4 weeks 9/15 the urine MR classifier for rejection disappeared within 4 weeks and was confirmed by repeat protocol biopsy.and was confirmed by repeat protocol biopsy.

– 4/15 the urine MR classifier for rejection persisted at for 4 weeks and a 4/15 the urine MR classifier for rejection persisted at for 4 weeks and a a repeat protocol biopsy confirmed the persistence of rejection.a repeat protocol biopsy confirmed the persistence of rejection.

– 2/15 have rejection classifier at last follow up (not biopsied)2/15 have rejection classifier at last follow up (not biopsied)

0

100

200

300

400

500

0 1 2 3 4 5 6 7 8 9 10 11 12

Weeks Post-TransplantWeeks Post-Transplant

Cre

atin

ine

(C

reat

inin

e (

mol

/L)

mol

/L)

BiomarkerBiomarker RjRj RjRj

BiopsyBiopsy i1t1i1t1

RjRj

Steroids

SimulectSimulectNeoral ™Neoral ™MMF ™MMF ™PrednisonePrednisone

Case Presentation from YesterdayCase Presentation from Yesterday

NN

i1t0i1t0 i2t2i2t2(SC)(SC)

i2t2i2t2(SC)(SC)

14

Steroids

ConclusionsConclusions

Subclinical renal allograft rejection appears to have a distinct urine Subclinical renal allograft rejection appears to have a distinct urine 11H MR spectrumH MR spectrum

Resolution of subclinical rejection may correlate with the Resolution of subclinical rejection may correlate with the disappearance of the spectrum and disappearance of the spectrum and vice versavice versa

Repeated, frequent urine spectral analysis may establish whether Repeated, frequent urine spectral analysis may establish whether there is a link between subclinical acute rejection and the there is a link between subclinical acute rejection and the development of chronic rejectiondevelopment of chronic rejection

Monitoring of urine Monitoring of urine 11H MR spectra may assist in drug withdrawal H MR spectra may assist in drug withdrawal and tolerance protocolsand tolerance protocols

CollaboratorsCollaborators

Peter Nickerson Peter Nickerson John Jeffery John Jeffery Sylvia DanceaSylvia Dancea

UNIVERSITY OF MANITOBAUNIVERSITY OF MANITOBA

Roxanne DeslauriersRoxanne DeslauriersRaymond SomorjaiRaymond SomorjaiMiriam GlogowskiMiriam GlogowskiTony Shaw Tony Shaw

NRC INSTITUTE FOR BIODIAGNOSTICSNRC INSTITUTE FOR BIODIAGNOSTICS