1. Wisit Cheungpasitporn Journal Club October 25, 2013

2. Why do renal allografts fail?7%El-Zoghby ZM, Stegall MD, Lager DJ, Kremers WK, Amer H, Gloor JM, Cosio FG. Am J Transplant. 2009;9(3):527-35. 3. Gold Standard for diagnosis of acute rejectionThe "tricorder" used by Bones McCoy to scan patients and get the right diagnosisGraft biopsy currently the GOLD STANDARD for diagnosis of acute rejection. 4. Limitations of Renal Biopsy Specimen adequacy Need for the presence of cortex Patchy distribution of disease Borderline lesions Prior treatment Chronic parenchymal scarringFurness PN et. al. Kidney Int. 2001 Nov;60(5):1998-2012. 5. A Kidney allograft biopsy Low major complication rate (including transfusionrequirement and catheterization) of between 0.4 and 1.0 percent Only one graft lost in approximately 2500 biopsiesSchwarz A, Gwinner W, Hiss M, et al. Am J Transplant 2005; 5:1992. 6. Page kidney after renal allograft biopsyHeffernan E et. Al. J Clin Ultrasound. 2009 May;37(4):226-9. 7. Timely detection and treatment of rejection: an important goalRenal Biopsy Creatinine-Decreased UO -HTN -Graft tendernessAnglicheau D, Suthanthiran M. Transplantation. 2008 Jul 27;86(2):192-9. 8. Robbins, Basic Pathology, 8th ed. Pg 131 9. Acute T Cell Rejection : Molecular events Cellular Traffic (Invitation): IP10, CXCR3 (Tatapudi et al. Kidney Intl 2004)Physical Contact: CD103 (Ding et al. Transplantation 2003)Target Cell Damage (Induced suicide): Granzyme B/ Perforin (Muthukumar et al. Transplantation 2003)Protective/Regulatory Response Collateral Protection: PI-9 Damage control: FoxP3 (Muthukumar et al. N Engl J Med. 2005)Robbins, Basic Pathology, 8th ed. 10. IP-10 PI-9Treg Cells; CD4, CD25: FOXP3 Ho J et. al. Am J Kidney Dis. 2012 Oct;60(4):629-40. 11. Gwinner W. World J Urol. 2007 Oct;25(5):445-55. 12. Gwinner W. World J Urol. 2007 Oct;25(5):445-55. 13. Li et al. N Engl J Med 2001 14. Urinary Cell CTL mRNA Profiling Study Urine Specimens and Renal Allograft Recipients : 151Specimens 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 107 specimens from 47 recipients with stable allograft function Design and Development of Competitive Quantitative PCRAssays: Levels of mRNA in urinary cells were measured. granzyme B and perforin cyclophylin BLi et al. N Engl J Med 2001 15. Levels of mRNA in Urinary cells Box and whisker plots show the 10th,25th, 50th (median) and 90th percentile values in urine samples. The levels of perforin and granzyme B,but not those of cyclophilin B, were significantly higher in the pts 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 numbersof urine samples. In all cases logtransformed values are shown.Li et al. N Engl J Med 2001 16. Receiver-Operating-Characteristic Curves for mRNA Levels Acute Rejection could be predicted with: a sensitivity of 83% and a specificity of83% with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA sensitivity of 79% and a specificity of77% 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 17. Summary of Findings Noninvasive diagnosis of acute rejection is feasible bymeasurement of mRNA for perforin and granzyme B in urine. Measurement of mRNAs in sequential urinespecimens may predict the development of acute rejection. No significant association exists between mRNA levelsand biopsy grade or the time from kidney transplantation to the development of acute rejection time.Li et al. N Engl J Med 2001 18. Summary of Findings 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 feasibleby measurement of mRNA for BKV VP1 mRNA in urine Bacterial urinary tract infection is not associated withan increase in urinary cell levels of perforin and granzyme BLi et al. N Engl J Med 2001 19. Ho J et. al. Am J Kidney Dis. 2012 Oct;60(4):629-40. 20. Limitations 1. Does the biomarker detect subclinical injury? 2. Is the biomarker detectable before the onset ofdecreased transplant function? 3. Does the biomarker correlate with response to treatment? 4. Does use of the biomarker affect clinical outcomes? 21. Hartono c. et. al. Curr Opin Organ Transplant. 2010 February; 15(1): 3541. 22. N Engl J Med Volume 369(1):20-31 July 4, 2013 23. Two primary research hypotheses Urinary cell mRNA profile at the time of a diagnosticbiopsy predicts biopsy-based diagnosis of rejection Urinary cell mRNA profile is predictive of acuteallograft rejection in the near future 24. TRIAL DESIGN Prospective observational CTOT-04 study, sponsoredby the National Institutes of Health (NIH) April 2006 - January 2008, 497 patients had beenenrolled at five clinical sites. A total of 492 patients received a kidney transplant,and 4300 urine specimens were collected from 485 25. Study design flow diagram 26. Urine Samples and mRNA Measurement TGF-1 mRNA and 18S rRNA as quality controlparameters: passing quality control if 18S rRNA 5107 copies/mcg of total RNA isolated fromthe urine pellet TGF-1 mRNA 100 copies/mcg of total RNA isolated from the urine pellet. 27. TRIAL DESIGN Seven mRNAs: Perforin Granzyme B Proteinase inhibitor-9 (PI-9) CD103 CD3 Interferon inducible protein-10 (IP-10) CXCR3 28. Stable graft function Cr 2.0 mg/dL for available assessments 6, 9, and 12 mths after transplant No graft loss or death 12 mths after transplant No Rx for acute rejection No evidence of CMV or BKV 29. Equation estimated the likelihood of A Rejection Fitting the Model A diagnostic score of 1.213, withthe use of thisequation, was the cutoff point that maximized the combined sensitivity and specificity. 30. AUC 0.85 (95% CI, 0.780.91; P