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Non-Invasive Rejection Diagnosis Using Urine NMR Spectra

Non-Invasive Rejection Diagnosis Using Urine NMR Spectra. David Rush Winnipeg Transplant Group University of Manitoba. Immune Monitoring for Rejection of Kidney Transplants.

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Non-Invasive Rejection Diagnosis Using Urine NMR Spectra

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  1. Non-Invasive Rejection Diagnosis Using Urine NMR Spectra David Rush Winnipeg Transplant Group University of Manitoba

  2. 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) 344:1006) Editorial comment to Li et al (NEJM (2001) 344:945)

  3. 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 Widely Available Weaknesses Lacks Specificity (Need a Biopsy to Diagnose Rejection) Lacks Sensitivity

  4. Immune SurveillanceGoal is to Develop a Biomarker in the Blood or Urine Anti-HLA Antibody GRAFT CTL Capillary Renal Tubule Blood Urine

  5. Fas CTL • Allorecognition • Direct • Indirect Granzyme B Perforin IL-2 Th IL-15 APC Th Th Th • Costimuli • B7:CD28 • CD40-CD40L Immune SurveillanceProbe for the Inflammatory Programs of Acute Rejection IL-2 Th M IFNg TNFa IL-4 IL-10 anti-HLA Ab B

  6. Immune SurveillanceBlood and Urine Biomarkers Blood: • PBMCRT-PCR CTL gene transcripts ( Fas, Granzyme, Perforin ) Vasconcellos et al (Transplantation 1998;66:562) Urine: • Flow cytometry to detect CD3 and HLA-DR on urine cells Roberti et al (Transplantation 1995;59:495) • RT-PCR CTL gene transcripts ( Granzyme, Perforin ) Li et al (NEJM 2001;344:945)

  7. Immune SurveillanceBlood or Urine Biomarker Development • Limitationsto 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 • Specificitycould 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

  8. Allorecognition • Direct • Indirect IL-2 Th IL-15 APC • Costimuli • B7:CD28 • CD40-CD40L Immune SurveillanceDonor Antigen Specific Biomarkers Require Donor Cells for Analysis • Flow Cross-match (anti-HLA Ab) O’Malley et al (ITS 1998 Abstr #1370) • ELISPOT Cytokine Assay Heeger et al (J Immunol (1999) 163:2267) • DTH Assay (“Tolerance Assay”) VanBuskirk et al (J Clin Invest (2000) 106:145)

  9. DNA mRNA Protein Genome Transcriptome Proteome acgtacca aggtaacg cggtttttcgt gtatctccctt 30,000 – 50,000 Genes > 100,000 mRNAs > 1,000,000 Proteins Immune SurveillanceStrategies to Profile all Components in the Blood or Urine

  10. Immune SurveillanceCan 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 -80°C • Study Population: • “Normal” Urine Spectra: • Transplant Patients with Normal Histology by Protocol Biopsy • “Rejection” Urine Spectra: • Transplant Patients with Acute Rejection by Protocol Biopsy

  11. Developing an MR Biomarker Makes No Assumption as to What Target is Important “Normal” Spectra “Rejection” Spectra Classifier “Rejection”

  12. INFORMATICSRate-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

  13. Spectral Regions Sensitivity Specificity PPV NPV Crispness 1st Generation (33 vs 35) 6 88% 93% 93% 96% 75% 2nd Generation (70 vs 41) 6 + 5 98% 96% 98% 96% 96% 3rd Generation (81 vs 46) 6 + 6 91% 95% 95% 91% 94% 1H MR Biomarkers Developed from the Urine Spectra Correctly Identify Allograft Histology Normal vs Rejection Histology

  14. Biomarker N N N N N N N N Biopsy i0t0 (ATN) i1t0 i0t0 i1t0 A Biomarker for Rejection Must Be Specific Weeks Post-Transplant Creatinine (mmol/L) Simulect ™ Neoral ™ MMF ™ Prednisone

  15. Biomarker Rj Rj Rj N N N N Biopsy i2t3 i0t0 i0t0 (SC) The Biomarker for Rejection May Precede the Histologic Diagnosis of Rejection Weeks Post-Transplant Simulect ™ Neoral ™ MMF ™ Prednisone Creatinine (mmol/L) Steroids

  16. 18 N 19 N 20 N Biomarker Rj Rj Rj Rj Rj Rj Rj Rj Rj Rj Rj Biopsy i2t2 (SC) i3t2 (CL) The Biomarker for Rejection can Persist After Allograft Function Returns to Baseline Weeks Post-Transplant Creatinine (mmol/L) Neoral ™ MMF ™ Prednisone Steroids

  17. 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)

  18. Biomarker Rj Rj Rj Biopsy i1t1 Case Presentation from Yesterday Weeks Post-Transplant Creatinine (mmol/L) Simulect Neoral ™ MMF ™ Prednisone Steroids Steroids 14 N i1t0 i2t2 (SC) i2t2 (SC)

  19. 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

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

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