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 Rejection Standard of Practice: Serum Creatinine Diagnostic Threshold Baseline Function Treatment Inflammation Cr Strengths Samples the Entire Graft Rapid Turnaround Time Non-invasiveInexpensive Widely Available Weaknesses Lacks Specificity (Need a Biopsy to Diagnose Rejection) Lacks Sensitivity

4. Capillary Immune Surveillance Goal is to Develop a Biomarker in the Blood or Urine GRAFT CTL Renal Tubule UrineBlood Anti-HLAAntibody

5. Immune Surveillance Probe for the Inflammatory Programs of Acute Rejection Immune Surveillance Probe for the Inflammatory Programs of Acute Rejection Th IL-2 CTL Granzyme B PerforinFas Th MM TNF  IFN  B anti  HLA Ab IL-4IL-10 APC Th Costimuli B7:CD28 B7:CD28 CD40-CD40L CD40-CD40L IL-2Allorecognition Direct Direct Indirect IndirectIL-15

6. Immune Surveillance Blood and Urine Biomarkers Blood: »PBMC RT-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) Li et al (NEJM 2001;344:945)

7. Immune Surveillance Blood or Urine Biomarker Development Limitations to the development of biomarker 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 Biomarkers should distinguish Acute Rejection vs. Drug toxicity, Infection, ATN Specificity could be improved by developing a: Specificity could be improved by developing a: »Donor 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 sample Requires strategies able to “profile” all components in a sample Requires strategies able to “profile” all components in a sample

8. 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) Immune Surveillance Donor Antigen Specific Biomarkers APC Th Costimuli B7:CD28 B7:CD28 CD40-CD40L CD40-CD40L IL-2Allorecognition Direct Direct Indirect IndirectIL-15

9. DNAmRNAProteinacgtaccaaggtaacgcggtttttcgtgtatctccctt GenomeTranscriptomeProteome 30,000 – 50,000 Genes > 100,000 mRNAs > 1,000,000 Proteins Immune Surveillance Strategies to Profile all Components in the Blood or Urine

10. Immune Surveillance Can Early Allograft Inflammation be Detected by a Distinct Urine MR Spectral Profile? Study Design: 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: 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. INFORMATICS Rate-Limiting Step is Analysis of the Spectral Profile 1 H MR spectra 1 H MR spectra »0.5-4.5 and 6.5-9.5 ppm »1690 data points / spectra Multivariate classification strategy: Multivariate classification strategy: » Optimal region selector (data reduction) » Bootstrap cross-validation » Linear Discriminant Analysis (LDA) classifier

13. 1 H MR Biomarkers Developed from the Urine Spectra Correctly Identify Allograft Histology Spectral Regions SensitivitySpecificityPPVNPVCrispness 1 st Generation (33 vs 35) 688%93%93%96%75% 2 nd Generation (70 vs 41) 6 + 5 98%96%98%96%96% 3 rd Generation (81 vs 46) 6 + 6 91%95%95%91%94% Normal vs Rejection Histology

14. A Biomarker for Rejection Must Be Specific Weeks Post-Transplant Creatinine (  mol/L) BiomarkerNNNNNNNNBiopsyi0t0(ATN)i1t0i1t0i0t0 Simulect ™ Neoral ™ MMF ™ Prednisone

15. The Biomarker for Rejection May Precede the Histologic Diagnosis of Rejection Weeks Post-Transplant Creatinine (  mol/L) BiomarkerRjRjNRjNNNBiopsy (SC) i2t3i0t0i0t0 Steroids Simulect ™ Neoral ™ MMF ™ Prednisone

16. The Biomarker for Rejection can Persist After Allograft Function Returns to Baseline Weeks Post-Transplant Creatinine (  mol/L) BiomarkerRjRjRjRjRjRjRj Biopsyi2t2(SC)i3t2(CL) RjRjRjRj 18N 19N 20N Steroids Neoral ™ MMF ™ Prednisone

17. Urine 1 H MR Biomarker Precedes and Persists after the Diagnosis of Rejection Urine 1 H MR Biomarker Precedes and Persists after the Diagnosis of Rejection 46 patients had 154 protocol biopsies 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. Weeks Post-Transplant Creatinine (  mol/L) BiomarkerRjRj Biopsyi1t1 Rj Steroids Simulect Neoral ™ MMF ™ Prednisone Case Presentation from Yesterday N i1t0i2t2(SC)i2t2(SC) 14 Steroids

19. Conclusions Subclinical renal allograft rejection appears to have a distinct urine 1 H MR spectrum Subclinical renal allograft rejection appears to have a distinct urine 1 H MR spectrum Resolution of subclinical rejection may correlate with the disappearance of the spectrum and vice versa 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 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 1 H MR spectra may assist in drug withdrawal and tolerance protocols Monitoring of urine 1 H MR spectra may assist in drug withdrawal and tolerance protocols

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