NonInvasive Rejection Diagnosis Using Urine NMR Spectra

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Non-Invasive Rejection Diagnosis Using Urine NMR
Spectra

• David Rush
• Winnipeg Transplant Group
• University of Manitoba

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Immune Monitoring for Rejection of Kidney
Transplants

• the clinical manifestations of acute rejection
  have changed with present-day immmunosuppression.
  There are usually no local symptoms, and the
  abnormalities are typically limited to insidious,
  low-level dysfunction of the graft...
• systematic and repeated urinalyses performed
  in the absence of substantial changes in graft
  function may provide a unique opportunity to
  detect subclinical episodes of rejection that may
  culminate in chronic rejection
• Soulillou (NEJM (2001) 3441006)
• Editorial comment to Li et al (NEJM (2001)
  344945)

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Surveillance for Acute RejectionStandard of
Practice Serum Creatinine
Treatment
Diagnostic Threshold
Cr
Baseline Function
Inflammation
Strengths Samples the Entire Graft Rapid
Turnaround Time Non-invasive Inexpensive Wid
ely Available
Weaknesses Lacks Specificity (Need a Biopsy to
Diagnose Rejection) Lacks Sensitivity
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Immune Surveillance Goal is to Develop a
Biomarker in the Blood or Urine
Anti-HLA Antibody
GRAFT
CTL
Capillary
Renal Tubule
Urine
Blood
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Immune Surveillance Probe for the Inflammatory
Programs of Acute Rejection
IL-2
Th
M?
TNFa
IFNg
IL-4 IL-10
anti-HLA Ab
B
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Immune Surveillance Blood and Urine Biomarkers

• Blood
• PBMC RT-PCR CTL gene transcripts ( Fas, Granzyme,
  Perforin )
• Vasconcellos et al (Transplantation 199866562)
• Urine
• Flow cytometry to detect CD3 and HLA-DR on urine
  cells
• Roberti et al (Transplantation 199559495)
• RT-PCR CTL gene transcripts ( Granzyme, Perforin
  )
• Li et al (NEJM 2001344945)

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Immune Surveillance Blood or Urine Biomarker
Development

• Limitations to the development of biomarker
• Tarnished Gold Standard (i.e. classification
  error of the biopsy)
• Lack of Specificity of any single biomarker
• Biomarkers should distinguish Acute Rejection vs.
  Drug toxicity, Infection, ATN
• Specificity could be improved by developing a
• Donor antigen specific assay
• Requires donor antigen source (e.g. donor spleen
  cells)
• Profile based on all components ( known / unknown
  ) in a blood or urine sample
• Requires strategies able to profile all
  components in a sample

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Immune Surveillance Donor Antigen Specific
Biomarkers

• Require Donor Cells for Analysis
• Flow Cross-match (anti-HLA Ab)
• OMalley et al (ITS 1998 Abstr 1370)
• ELISPOT Cytokine Assay
• Heeger et al (J Immunol (1999) 1632267)
• DTH Assay (Tolerance Assay)
• VanBuskirk et al (J Clin Invest (2000) 106145)

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Immune Surveillance Strategies to Profile all
Components in the Blood or Urine
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Immune Surveillance Can Early Allograft
Inflammation be Detected by a Distinct Urine MR
Spectral Profile?

• Study Design
• Gold Standard Protocol Biopsy (months 1, 2, 3
  and 6)
• Urine Collected at time of Protocol Biopsy and
  stored at -80C
• Study Population
• Normal Urine Spectra
• Transplant Patients with Normal Histology by
  Protocol Biopsy
• Rejection Urine Spectra
• Transplant Patients with Acute Rejection by
  Protocol Biopsy

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Developing an MR Biomarker Makes No Assumption as
to What Target is Important
Normal Spectra
Rejection Spectra
Classifier Rejection
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INFORMATICS Rate-Limiting Step is Analysis of
the Spectral Profile

• 1H MR spectra
• 0.5-4.5 and 6.5-9.5 ppm
• 1690 data points / spectra

• Multivariate classification strategy
• Optimal region selector (data reduction)
• Bootstrap cross-validation
• Linear Discriminant Analysis (LDA) classifier

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1H MR Biomarkers Developed from the Urine Spectra
Correctly Identify Allograft Histology

• Normal vs Rejection Histology

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A Biomarker for Rejection Must Be Specific
Weeks Post-Transplant
Creatinine (mmol/L)
Simulect Neoral MMF Prednisone
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The Biomarker for Rejection May Precede the
Histologic Diagnosis of Rejection
Weeks Post-Transplant
Simulect Neoral MMF Prednisone
Creatinine (mmol/L)
Steroids
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The Biomarker for Rejection can Persist After
Allograft Function Returns to Baseline
Weeks Post-Transplant
Creatinine (mmol/L)
Neoral MMF Prednisone
Steroids
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Urine 1H MR BiomarkerPrecedes and Persists
after the Diagnosis of Rejection

• 46 patients had 154 protocol biopsies
• 31/154 biopsies had diagnosis of Acute Rejection
• 24/31 had a urine sample prior to the biopsy
• 18/24 the urine MR classifier for rejection was
  present 1-2 weeks prior to the biopsy.
• 15/24 had urine samples collected after the
  biopsy
• 9/15 the urine MR classifier for rejection
  disappeared within 4 weeks and was confirmed by
  repeat protocol biopsy.
• 4/15 the urine MR classifier for rejection
  persisted at for 4 weeks and a a repeat
  protocol biopsy confirmed the persistence of
  rejection.
• 2/15 have rejection classifier at last follow up
  (not biopsied)

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Case Presentation from Yesterday
Weeks Post-Transplant
Creatinine (mmol/L)
Simulect Neoral MMF Prednisone
Steroids
Steroids
14
N
i1t0
i2t2 (SC)
i2t2 (SC)
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Conclusions

• Subclinical renal allograft rejection appears to
  have a distinct urine 1H MR spectrum
• Resolution of subclinical rejection may correlate
  with the disappearance of the spectrum and vice
  versa
• Repeated, frequent urine spectral analysis may
  establish whether there is a link between
  subclinical acute rejection and the development
  of chronic rejection
• Monitoring of urine 1H MR spectra may assist in
  drug withdrawal and tolerance protocols

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Collaborators
UNIVERSITY OF MANITOBA
Peter Nickerson John Jeffery Sylvia Dancea
NRC INSTITUTE FOR BIODIAGNOSTICS
Roxanne Deslauriers Raymond Somorjai Miriam
Glogowski Tony Shaw