1. 1 What’s New in Transplant Innovative Approaches to Detect Graft Loss and Novel Treatments to Support Graft Acceptance

2. 2 Overview Current methods of detecting graft loss in transplantation have advanced the field, but have limitations 1 Technologies that might address those limitations and create a better diagnostic gold standard are being investigated and developed 2 The pathway to get these new technologies into clinical practice has yet to be defined. 2 This presentation is a preview of future technologies New ways to preserve and treat grafts are also being investigated 3 1.Bromberg JS, et al. Am J Transplant. 2012;(12):2573-2574. 2.Bromberg JS, et al. Am. J Transplant. 2009;9:11-13. 3.Bon D, et al. Nat Rev Nephrol. 2012; 8(6):339-347.

3. 3 Getting Laboratory Technologies Into Clinical Practice Is Neither a Linear Nor Smooth Path 1.Bromberg JS, et al. Am J Transplant. 2009;9:11-13. 2.Huskey J, et al. Clin J Am Soc Nephrol. 2011;6:423-429. Developing methods to detect graft loss is challenging when the mechanisms of graft loss are not fully known 1 New methods have the double duty of 1 –Offering more precise detection –Furthering the understanding of biological mechanisms of graft loss To better gauge new approaches, the strengths and weaknesses of the laboratory science used to create the approach must be identified 2 Example: ImmuKnow TM Assay 2 Measure of intracellular adenosine triphosphate (ATP) released from CD4+ T cells Quantify cell-mediated immunity in transplant recipients Identify patients at risk for acute rejection (AR) or opportunistic infections

4. 4 New Techniques Are Exciting, But Discovery Must Be Counterbalanced With Trial Limitations If these factors are taken into account, landmark advances may be realized –Adequate sample size –Prospective vs retrospective analysis –Detection of biomarkers indicating concurrent diagnosis vs prediction of an outcome –Validation of each new biomarker –Correlating genes to biopsies may have limitations, when biopsies themselves are imperfect gold standards Bromberg JS, et al. Am J Transplant. 2012;(12):2573-2574.

5. 5 Why Go Molecular?

6. 6 Why Use Genomics? 1.Bromberg JS, et al. Am J Transplant. 2012;(12):2573-2574. 2. Li L, et al. Am J Transplant. 2012;12(10):2710-2718. 3.Anglicheau D, et al. Transplantation. 2012;93:1136-1146. 4.Flechner SM, et al. Am J Transplant. 2004;4(9):1475-1489. Current markers for rejection—serum creatinine and biopsies—are imperfect as the gold standards 1 –Biopsies are also subject to reader bias and sampling error –They are invasive procedures New assays that look at biomarkers –Are less invasive than biopsies 2,3 –May yield more precise measurements then currently used methods by virtue of being objective 2,4

7. 7 A Brief Look at How Genomic Biomarkers Are Discovered: Polymerase Chain Reaction 1.Muthukumar T, et al. N Engl J Med. 2005;353:2342-2351. 2.Anglicheau D, et al. Transplantation. 2012;93:1136-1146. 3.Ben-Dov IZ, et al. Transplantation. 2012;94:1086-1094. Polymerase chain reaction (PCR) –Method of RNA/DNA amplification used to detect or quantify whether gene expression in a sample. It can be used as a validation strategy for other detection methods 1-3 eg, if gene expression is discovered using microarray technology, it can then be independently validated using PCR 3

8. 8 A Brief Look at How Genomic Biomarkers Are Discovered: MicroRNA Anglicheau D, et al. Transplantation. 2010 ;90(2):105-112. MicroRNA (miRNA) –Thought to regulate gene expression at the posttranscriptional level –Plays a central role in cell proliferation, differentiation, apoptosis, fat metabolism, and oncogenesis –Several cancers are associated with dysregulation of miRNA expression –Messenger (mRNA) can be targeted by multiple miRNAs

9. 9 DNA Microarray Technology: How the Genes That Might Be Rejection Culprits Are Identified Principle of cDNA microarray assay for gene expression Principle of cDNA microarray assay for gene expression Make cDNA (complementary DNA) reverse transcript Label cDNAs with fluorescent dyes ControlExperimental Hybridization to microarray Laser excitation at dye-specific Hz Laser emissionComputer calculates ratio of intensity Red = up-regulation Green = down-regulation Black = constitutive expression += Reproduced with permission: © 2012 by Steven M. Carr, after Gibson & Muse 2002.

10. 10 Gene Expression Histology Strong correlations yield effective gene profiling From PBLs with analysis using DNA microarrays From pathologic biopsies Flechner SM, et al. Am J Transplant. 2004;4(9):1475-1489. Mapping DNA to Tissues to Yield Gene Profiles PBL, peripheral blood lymphocyte.

11. 11 Current Molecular Diagnostic Pursuits Overview

12. 12 Molecular Diagnostics in Current Research The following examples look at methods for detecting biomarkers in urine and peripheral blood (PB) in AR The next set of examples includes noninvasive detection for fibrosis and interstitial fibrosis tubular atrophy (IFTA), including 1study that yielded unexpected results Finally, the last section focuses on whether a molecular scorecard can be created to predict graft loss If these new detection methods pass the test of utility and accuracy in the clinical mainstream, they could offer noninvasive and precise detection methods

13. 13 Current Molecular Diagnostic Pursuits Acute Rejection

14. 14 mRNA in Urinary Cells Is a Biomarker for AR Log FOXP3 mRNA Graft Loss Within 6 Months After Acute-Rejection Episode, % P=0.02 RR=6.0 RR=3.0 RR=1.0 <3.173.17-4.84>4.84 Muthukumar T, et al. N Engl J Med. 2005;353:2342-2351. FOXP3 mRNA is a marker for regulatory T (Treg) cells and AR –High levels of FOXP3 are associated with a lower risk for graft failure after acute rejection RR, relative risk.

15. 15 A 5-Gene Signature Suggested to Identify AR in Pediatric Renal Allograft Li L, et al. Am J Transplant. 2012;12(10):2710-2718. Aim: To provide a PB diagnostic test for AR in pediatric patients Methods: 367 unique PB samples were paired with a graft biopsy for blinded phenotype classification. A logistic regression model was designed for analysis Findings: A 5-gene set classified –AR from stable (absence of AR), with 91% sensitivity and 94% specificity –AR from all other non-AR phenotypes, with 91% sensitivity and 90% specificity 5-gene set was shown to diagnose AR –DUSP1, PBEF1, PSEN1, MAPK9, and NKTR

16. 16 A Measured Response to PBL Diagnostics and Biomarker Studies: A Bit of Fair Balance PBL signature for AR is important in making microarray a clinical reality, but it has important limitations –Majority of biopsies from white children/adolescents –Correlating genes to biopsies—the latter of which are a subpar gold-standard measurement—might not be predictive –The study designs and statistical methods suitable to make a prediction are much more complex than those to make a diagnosis of concurrent rejection The study by Li et al is a landmark study because of its further ability to diagnose AR using PB; however, challenges remain in validating the signature Bromberg JS, et al. Am J Transplant. 2012;(12):2573-2574.

17. 17 Current Molecular Diagnostic Pursuits Fibrosis and Interstitial Fibrosis and Tubular Atrophy (IFTA)

18. 18 mRNA Offers a First Step Toward a Noninvasive Test for Fibrosis Urine samples from 114 patients with and without fibrosis assigned to discovery and validation sets Discovery set of 76 urine samples Fibrosis biopsy group 32 urine samples Preamplification enhanced real-time quantitative PCR of 22 mRNAs and 18 rRNA Evaluation of bivariate association of individual mRNA measures to likelihood of fibrosis Construction of diagnostic equation by logistic regression 4-gene model Normal biopsy group 44 urine samples Discovery Set Stage 1: Discovery Phase Validation set of 38 urine samples Fibrosis biopsy group 16 urine samples Preamplification enhanced real-time quantitative PCR Normal biopsy group 22 urine samples Validation Set Stage 2: Independent Validation Phase Diagnostic accuracy of the 4-gene model Reproduced with permission from Anglicheau D, et al. Transplantation. 2012;93:1136-1146. Anglicheau D, et al. Transplantation. 2012;93:1136-1146.

19. 19 Using Gene Expression to Better Understand IFTA Building on previous research that identified mRNA expression signatures in fibrosis and IFTA, investigators analyzed miRNA (which regulates mRNA) to –Understand the mechanics of pathophysiology of fibrogenesis –Determine whether IFTA is associated with altered expression of miRNA and if miRNA can be used diagnostically The miRNA transcriptome of allografts with or without fibrosis were sequenced, catalogued, and validated IFTA miRNA patterns were distinct from that of healthy biopsy specimens –Independent of clinical patient characteristics –Predicted allograft outcome Ben-Dov IZ, et al. Transplantation. 2012;94:1086-1094.

20. 20 Discovery of IFTA Biomarkers Also Yielded Unpredicted Results A gene expression ‒ based signature in PBL discovered –Predictive accuracy of 80% for mild IFTA and 92% for moderate to severe IFTA* Unpredicted for outcome Banff 1 (mild IFTA) Banff 2,3 (moderate to severe IFTA) Associated with number of genes representing different pathways connected to immune/ inflammatory and tissue injury mechanisms Significantly fewer immune/inflammatory genes expressed than in mild IFTA More genes linked to metabolic and other pathways * Chronic allograft nephropathy (CAN) is now called IFTA and has been changed here to IFTA to match the current lexicon Kurian SM, et al. PLoS One. 2009;4(7):e6212.

21. 21 Current Molecular Diagnostic Pursuits Molecular Modeling to Predict Graft Loss

22. 22 Creating a Molecular Risk Score to Predict Late Graft Loss Using Microarray Aim: Identify molecules that predict organ failure after signs of renal dysfunction or proteinuria Findings: Genes associated with graft failure were related to tissue injury, epithelial dedifferentiation, matrix remodeling, and TGF-β effects Grafts that failed had –Higher incidence of proteinuria and rapid deterioration in function before biopsy and a lower glomerular filtration rate at time of biopsy Grafts that failed had no differences in –Primary disease, time after transplantation, maintenance immunosuppression, or incidence of anti-HLA antibodies Einecke G, et al. J Clin Invest. 2010;120(6):1862-1872. HLA, human leukocyte antigen.

23. 23 Molecular risk scores in individual biopsies; each triangle is a biopsy Creating a Molecular Risk Score to Predict Late Graft Loss (cont) Kaplan-Meier plots for the 2 risk groups Einecke G, et al. J Clin Invest. 2010;120(6):1862-1872. High Risk Biopsies Orders by Risk Score Censored Graft failure Low Risk Survival Probability Time After Biopsy, days Risk Score Risk groups Low (n=53, Failed = 5) High (n=52, Failed = 25) P = 3 x 10 -7 Reproduced with permission from Einecke G, et al. J Clin Invest. 2010;120(6):1862-1872.

24. 24 Current Molecular Diagnostic Pursuits Looking at the Antibody Targets: Endothelial Cells

25. 25 Given that endothelial cells (ECs) are antibody targets, altered expression of EC genes—identified by their transcripts—may identify ABMR Expression of these transcripts, if antibodies were also present, predicted graft loss with higher sensitivity (77% vs 31%) and lower specificity (71% vs 94%) than did the presence of C4d Clinical affect –Reveals that C4d-negative stain does not rule out ABMR Sis B, et al. Curr Opin Organ Transplant. 2010;15(1):42-48. Analyzing Altered Gene Expression in Endothelial Cells Could Identify Antibody-Mediated Rejection (ABMR)

26. 26 2 Types of ABMR: C4d Positive and C4d Negative With a Common Target Complement activation Ifn-  effects Endothelial activation Leukocyte recruitment Fc receptors Platelets Ifn-  effects Endothelial activation Leukocyte recruitment Fc receptors Platelets C4d-Positive ABMRC4d-Negative ABMR Complement activation C4d deposition No detectable C4d deposition by immunofluorescence Platelets CTL NK MØ NK MØ CTL MØ NK Cytokines C1q Platelets Microcirculation is the main target of ABMR Endothelial activation Increased gene expression Sis B, et al. Curr Opin Organ Transplant. 2010;15(1):42-48. IFN, interferon. CTL, cytotoxic T lymphocytes. NK, natural killer. Reproduced with permission from Sis B, et al. Curr Opin Organ Transplant. 2010;15(1):42-48.

27. 27 Innovative Treatment Protocols

28. 28 HLA Desensitization Provides a Survival Advantage to Patients Compared rates of death between one group undergoing desensitization treatment and 2 control groups. Control groups included – Waiting list + dialysis (dialysis only) –Patients who underwent either dialysis or HLA-compatible transplantation (dialysis or transplantation group) No. at Risk Desensitization 210 170 143 11 75 58 42 28 14 treatment Dual therapy 1027 854 688 497 321 230 157 96 41 Dialysis only 1012 822 626 419 250 159 93 54 17 Survival, % Months Desensitization treatment Dialysis only Dialysis or transplantation Montgomery RA, et al. N Engl J Med. 2011;365(4):318-326. Reproduced with permission from Montgomery RA, et al. N Engl J Med. 2011;365(4):318-326.

29. 29 Innovative Treatments for Graft Preservation or Recipients May Improve Kidney Recovery Ischemic preconditioning Regional normothermic circulation Resuscitation Ischemic preconditioning Regional normothermic circulation Resuscitation Donor Kidney evaluation (metabolomic NMR) Kidney evaluation (metabolomic NMR) Graft preservation Graft preservation Recipient Preservation solution content (polyethylene glycols) Perfusion machine (oxygenation, temperature) Pharmacologic supplementation (inflammation, coagulation, energy metabolism, oxygen) Gene expression modulation (siRNA) Preservation solution content (polyethylene glycols) Perfusion machine (oxygenation, temperature) Pharmacologic supplementation (inflammation, coagulation, energy metabolism, oxygen) Gene expression modulation (siRNA) Cell therapy (adipocytes, amniocytes, stem cells) Postconditioning (erythropoietin, adenosine) Cell therapy (adipocytes, amniocytes, stem cells) Postconditioning (erythropoietin, adenosine) DiagnosticTreatments Bon D, et al. Nat. Rev Nephrol. 2012;8(6):339-347. Reproduced with permission from Bon D, et al. Nat. Rev Nephrol. 2012;8(6):339-347. siRNA, small interfering RNA. NMR, Nuclear magnetic resonance

30. 30 Banff Guidelines Molecular Understanding Is Creating Change

31. 31 Molecular Data Have Compelled Banff to Refine Current Guidelines Recognizing the limitations of the previously empirically based histologic definitions and scoring thresholds, Banff is working to refine its guidelines Isolated v-lesion working group Isolated v-lesion working group Fibrosis scoring working group Fibrosis scoring working group Glomerular lesion working group Glomerular lesion working group Molecular pathology working group Molecular pathology working group Polyomavirus nephropathy working group Polyomavirus nephropathy working group Quality assurance working group Quality assurance working group data-driven and validated refinement of Banff criteria data-driven and validated refinement of Banff criteria Sis B, et al. Am J Transplant. 2010;10(3):464-471. Permission Pending from Sis B, et al. Am J Transplant. 2010;10(3):464-471.

32. 32 In Addition to Using Working Groups, Banff Looks to the Future Compelling molecular research data led to discussion of updating Banff to utilize –“omics” technologies –New tissue markers Combine histopathology and molecular parameters within the Banff working classification Sis B, et al. Am J Transplant. 2010;10(3):464-471.

33. 33 Summary

34. 34 Summary Short-term transplant outcomes have seen remarkable improvements in the recent decade, and this seems to be a result of advances in the field, such as more targeted therapies, diagnostics, and clinical procedures However, long-term outcomes remain a concern and, as a result, different methods and techniques are being addressed to further advance the field The power of genomics, coupled with new ways of looking at treatment, may advance the field so long-term outcomes are as improved as short-term outcomes were during the past decade

35. 35 © 2012 sanofi-aventis U.S. LLC US.THY.12.12.003