M. Suthanthiran, M.D. Stanton Griffis Distinguished Professor Chief, Nephrology and Hypertension Chief, Transplantation Medicine New York Presbyterian - Cornell Noninvasive Prediction of Acute Rejection of Renal Allografts

What Does a Transplant Clinician Want? (Besides an Outstanding Renal Pathologist) Repetitively ascertain allograft status by noninvasive means Anticipate rejection Prognosticate rejection Mechanism-based therapy Predict allograft function Immunosuppression weaning or reintroduction of therapy Individualize/Optimize immunosuppression

1993 Nobel Prize in Chemistry Kary B. Mullis Invention of The Polymerase Chain Reaction

Li et al. J Exp Med 1991 Development of Competitive Quantitative PCR Assay

Development of Urinary Cell mRNA Profiling Protocol ( Li et al. N Engl J Med 2001) 50 cc urine specimen Urine sediment, isolation of total RNA Reverse transcription to cDNA Urinary cell levels of mRNA measured using competitive quantitative PCR assays

Urinary Cell CTL mRNA Profiling Study Urine Specimens and Renal Allograft Recipients : 151 Specimens from 85 Recipients. 24 specimens from 22 recipients with biopsy confirmed acute rejection; 5 from 5 recipients with CAN; 15 from 11 recipients with Other findings; and 107 specimens from 47 recipients with stable allograft function. Design and Development of Competitive Quantitative PCR Assays: Levels of mRNA for granzyme B, perforin and cyclophylin B in urinary cells were measured using gene specific primer pairs and gene specific competitor DNA construct. Statistical Analysis: Transcript levels were expressed in femtogram of mRNA per microgram of total RNA, and log transformed to reduce skew. One- way mixed – level ANOVA and Dunnett’s test for multiple comparisons were used. Receiver-operating-characteristic curve analysis was used to determine cutoff points yielding the highest combined sensitivity and specificity for predicting an episode of acute rejection.

HYPOTHESIS Measurement of mRNA encoding cytotoxic proteins granzyme B and perforin offers a noninvasive means of diagnosing acute rejection of renal allografts.

Levels of mRNA in Urinary cells Li et al. N Engl J Med 2001 Box and whisker plots show the 10 th, 25 th, 50 th (median) and 90 th percentile values for perforin mRNA, granzyme B mRNA and cyclophilin B mRNA in urine samples from patients classified as having acute rejection, Other findings, chronic allograft nephropathy or a stable course after transplantation. The levels of perforin and granzyme B, but not those of cyclophilin B, were significantly higher in the patients with an episode of acute rejection than in the other groups (P=0.001, by one-way mixed-level analysis of variance). Values in parenthesis are the numbers of urine samples. In all cases log-transformed values are shown

Receiver-Operating-Characteristic Curves for mRNA Levels The fraction of true positive results (sensitivity) and false positive results (1 – specificity) for perforin mRNA levels, granzyme B mRNA levels, and cyclophilin B mRNA levels as markers of acute rejection are shown. Acute Rejection could be predicted with a sensitivity of 83% and a specificity of 83% with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79% and a specificity of 77% with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Li et al. N Engl J Med 2001

Levels of mRNA in Sequential Urine Samples Perforin mRNA, granzyme B mRNA, and cyclophilin B mRNA were measured in urine samples obtained during the first nine days after transplantation. The mean (±SE) levels of perforin mRNA and granzyme B mRNA but not those of cyclophilin B mRNA were higher in the 8 patients in whom acute rejection developed within the first 10 days after transplantation than in the 29 patients in whom acute rejection did not develop within the first 10 days after transplantation. The respective numbers of urine samples obtained from the patients with an episode of acute rejection and those without such an episode were as follows: 6 and 43 on day 1, 2, or 3 after transplantation; 5 and 26 on day 4, 5, or 6; and 6 and 14 on day 7, 8, or 9. Means, standard errors, and P values were estimated with use of a mixed-level two- way analysis of variance. In all cases, log- transformed values are shown. Li et al. N Engl J Med 2001

Summary of Findings Noninvasive diagnosis of acute rejection is feasible by measurement of mRNA for perforin and granzyme B in urine. Measurement of mRNAs in sequential urine specimens may predict the development of acute rejection. No significant association exists between mRNA levels and biopsy grade or the time from kidney transplantation to the development of acute rejection time. Graft dysfunction due to non-immulogical causes (e.g., ATN) is not associated with an increase in urinary cell levels of perforin or granzyme B mRNA. Noninvasive diagnosis of BKV nephropathy is feasible by measurement of mRNA for BKV VP1 mRNA in urine (Ding et al. Transplantation 2002). Bacterial urinary tract infection is not associated with an increase in urinary cell levels of perforin and granzyme B (Dadhania et al. Transplantation 2003).

Allograft Directed Immune Response Cellular Traffic (Tatapudi et al. Kidney Intl 2004) Physical Contact (Ding et al. Transplantation 2003) Target Cell Damage (Muthukumar et al. Transplantation 2003) Protective/Regulatory Response

FOXP3 A Specification and functional factor for CD25+CD4+ regulatory T cells (Tregs). (Fontenot et al. Nat Immunol 2003; Sakaguchi S. Nat Immunol 2005) Mutations in human FOXP3 gene result in an autoimmune disease characterized by polyendocrinopathy and enteropathy. (Wildin et al. Nat Genet 2001; Bennett et al. Nat Genet 2001) Tregs implicated in experimental transplantation tolerance. (Graca et al. J Exp Med 2002; Lee I et al. J Exp Med 2005)

Urinary Cell FOXP3 mRNA Profiling Study Urine Specimens and Renal Allograft Recipients: 83 urine specimens from 83 renal allograft recipients 36 specimens from 36 recipients with biopsy confirmed acute rejection; 18 from 18 recipients with CAN; and 29 from 29 recipients with stable allograft function. Development of Pre-amplification Enhanced Real Time Quantitative PCR Assays: Levels of mRNA for FOXP3, CD25,CD3ε, perforin and 18S rRNA in urinary cells were quantified using the standard curve method developed by Ding et al (Transplantation 2002). Statistical Analysis: Transcript levels were log transformed and mRNA copy numbers normalized with the use of 18S rRNA copy number. Kruskal- Wallis, Dunn’s test for multiple comparisons and Mann-Whitney test were used for group comparisons. Chi-square test for linear trend in graft failure rates.

HYPOTHESES Acute rejection is associated with a lower level of urinary cell FOXP3 mRNA levels compared to stable patients Urinary cell FOXP3 mRNA predict acute rejection outcome, and identify patients at high risk for graft loss after an episode of acute rejection.

Levels of FOXP3 mRNA in Urinary Cells Box plots show the 10th, 25th, 50th (median), 75th, and 90th percentile values for log-transformed FOXP3 mRNA, CD25 mRNA, CD3  RNA and perforin B mRNA in urine samples obtained from 36 patients with biopsy- confirmed acute rejection, 18 patients with biopsy-confirmed chronic allograft nephropathy (CAN) and 29 patients with stable graft function and normal allograft biopsies (normal biopsy). The levels of FOXP3 mRNA, CD25 mRNA, CD3  RNA and perforin mRNA were higher in the urinary cells from patients with acute rejection than in the patients with CAN or normal biopsies. P-values are based on the Kruskal- Wallis test, treating the log- transformed mRNA levels as the dependent variable. In all cases log-transformed levels, normalized by 18S rRNA, are shown. Muthukumar et al. N Engl J Med 2005 An inverse relationship was found between FOXP3 mRNA levels and serum creatinine in the acute rejection group (r=-0.38, P=0.02)

Levels of FOXP3 mRNA in Urinary Cells Predict Reversal of an Episode of Acute Rejection Levels of FOXP3 mRNA in urinary cells but not the levels of CD25 mRNA, CD3  mRNA and perforin mRNA were significantly higher in the group with successful reversal than in the group without reversal. FOXP3 mRNA level predicted rejection reversal with a sensitivity of 90% and a specificity of 73% (P=0.001). The levels of mRNA for CD25, CD3 and perforin were not predictive of reversal of acute rejection. In logistic – regression analyses predicting non-response, levels of FOXP3 mRNA in urinary cells remained significant after statistical control for serum creatinine level (P=0.04) and the time from transplantation to rejection (P=0.02). A linear combination of FOXP3 and creatinine was a better predictor of rejection reversal (90% sensitivity and 96% specificity) than FOXP3 mRNA levels alone or serum creatinine levels alone (85% sensitivity and 90% specificity). Successful reversal of acute rejection was not predicted by the subject’s age, sex, race, graft donor source, Banff grade, or initial anti-rejection treatment. Muthukumar et al. N Engl J Med 2005

Relative Risk of Graft Failure after an Episode of Acute Rejection The rates and relative risks of graft failure within six months after an episode of acute rejection for thirds of FOXP3, CD25, CD3, and perforin mRNA levels are shown. The graft-failure rate was 50 percent and the relative risk was 6 for the lowest third of FOXP3 mRNA levels; 25 percent and 3, respectively, for the middle third; and 8 percent for the highest third (P=0.02 by the chi-square test for linear trend) (Panel A). There was no relationship between graft failure after an episode of acute rejection and the thirds of mRNA levels for CD25, CD3, and perforin (Panels B, C, and D). Muthukumar et al. N Engl J Med 2005

Summary of Findings The levels of FOXP3 mRNA are higher in the urinary cells from patients with acute rejection than in the patients with CAN or normal biopsies. A significant inverse relationship exists between FOXP3 mRNA levels and serum creatinine measured during an episode of in the acute rejection. Measurement of FOXP3 mRNA in urine offers a noninvasive means of improving the prediction of outcome of acute rejection of renal transplants. A linear combination of FOXP3 and creatinine is a better predictor of rejection reversal (90% sensitivity and 96% specificity) than FOXP3 mRNA levels alone (90% sensitivity and 73% specificity) or serum creatinine levels alone (85% sensitivity and 90% specificity). A strong inverse relationship exists between levels of FOXP3 mRNA in urinary cells and the time from kidney transplantation to the development of acute rejection (r=-0.42, P=0.01).

Tolerance Signature

Intragraft levels of FOXP3 mRNA in Human Renal Allografts Total RNA was isolated from renal-allograft biopsy specimens and reverse transcribed to cDNA, and levels of mRNA were measured with the use of pre-amplification enhanced real-time quantitative polymerase-chain-reaction assays. A total of 23 biopsy specimens were examined for intragraft levels of FOXP3 mRNA, granzyme B mRNA, and housekeeping gene 18S ribosomal RNA (18S rRNA). Of the 23 biopsy specimens, 6 were obtained from four patients with stable renal-allograft function who were not receiving immunosuppressive therapy (IS) (stable IS- free group). Eight biopsy specimens were obtained from eight patients with stable renal-allograft function and normal protocol biopsy results; these patients were receiving maintenance immunosuppressive drug therapy comprising tacrolimus and mycophenolate mofetil (stable-with-IS group). Five biopsy specimens were obtained from five kidney donors (normal-kidney group), and four biopsy specimens were obtained from four patients with biopsy-confirmed acute rejection (acute-rejection group). The mRNA copies were log transformed and normalized with 18S rRNA. Data analysis using Nested ANOVA and Tukey’s HSD showed that the intragraft level of FOXP3 mRNA in the Stable IS- free group was higher compared to the Stable with IS group (P=0.007), or the Normal Kidney group (P=0.0002), but lower than in the Acute Rejection group (P<0.0001). Intragraft level of granzyme B mRNA in the Stable IS-free group was not different from that in the Stable with IS group (P=0.91), or the Normal Kidney group (P=0.36), and lower than in the Acute Rejection group (P=0.0002). Kawai et al. N Engl J Med 2008

Allograft Status: A Time-line Model Time - Lines for Diagnosis Time - Lines for Treatment Efficacy ? Molecular Histological Clinical

mRNA Expression Signatures Predictive of Allograft Status “Ceterum censeo Carthaginem esse delendam” Diagnose rejection by noninvasive means Anticipate rejection Identification of rejection prior to tissue injury Mechanism-based therapy Prognosticate rejection Predict Allograft function Informed weaning or reintroduction of therapy Individualize/Optimize immunosuppression