NEWS & VIEWS TRANSPLANTATION
Molecular diagnosis of kidney transplant rejection Christian Morath and Martin Zeier
Acute or chronic antibody-mediated rejection (ABMR) of kidney allografts is currently diagnosed by the presence of donor-specific alloantibodies and distinct pathological findings in biopsy samples. A new study highlights the potential of molecular diagnostics incorporated into standard criteria for acute ABMR to help identify patients at risk of graft loss. Morath, C. and Zeier, M. Nat. Rev. Nephrol. 10, 429–430 (2014); published online 17 June 2014; doi:10.1038/nrneph.2014.106
In the past 5 years, several groups have studied molecular markers as a means to improve the accuracy and specificity of diagnosis of allograft pathologies. However, a standardized and validated approach to the molecular diagnosis of allograft injury is lacking. Molecular assays and platform technologies, such as proteomics, metabo‑ lomics and mRNA or microRNA expres‑ sion profiling of urine, blood and whole or microdissected allograft biopsy samples, have been used to study acute tubular injury, T‑cell-mediated and antibody-mediated rejection (ABMR; Table 1). In their recent article in the Journal of the American Society of Nephrology, Loupy et al.1 propose a ‘mol ecular microscope strategy’ to improve risk stratification for allograft loss in kidney recipients with early ABMR (30 years after kidney transplantation. In the study by Loupy et al.,1 only patients with acute antibody-mediated graft injury during the first year after surgery were investigated (74 patients in the principal cohort and 54 patients in the independent validation cohort). Finally, only sensitized graft recip‑ ients were included—92% of patients had DSAs before transplantation as opposed to the mostly unselected ‘standard risk’ patients in previous studies. In the current study, the ABMR molecular score correlated with the presence of DSAs, interstitial fibrosis or tubular atrophy score, mesangial matrix expansion score, C4d deposition in graft capillaries, the eDSAST dataset and the expression of natural killer (NK) cell DNA transcripts. The correlation between the three molecular scores is not surprising because when these scores were validated for the diagnosis of ABMR, the respective gene expression datasets showed a degree of overlap.4-6 The eDSAST dataset correlated with the presence of DSAs, Banff transplant glomerulop athy (cg) score,
Table 1 | Selected studies of molecular diagnostics in kidney allograft rejection Study
Patients
Result
Loupy et al. (2014)1
128 patients from two centres with ABMR up to 1 year after transplantation
ABMR score improves stratification of patients at high risk of graft loss
Halloran et al. (2013)7
264 patients from six centres with biopsy 3 days to ~27 years after transplantation
ABMR score is more strongly associated with graft failure than is conventional assessment; ABMR score is associated with future graft loss
Sellares et al. (2013)6
315 patients from four centres with biopsy 6 days to ~35 years after transplantation
ABMR score can be used for diagnosis of ABMR; ABMR score is associated with future graft loss
Hidalgo et al. (2010)5
145 patients from one centre with biopsy 6–31 years after transplantation
23 DSASTs are expressed predominantly in natural killer cells and endothelial cells; DSASTs are increased in ABMR
Sis et al. (2009)4
169 patients from two centres* with biopsy 6 days to 31 years after transplantation
ENDATs can be used for the diagnosis of ABMR, including C4d-negative ABMR; ENDAT levels are associated with future graft loss
All studies assessed mRNA expression in whole graft-biopsy samples (gene chip). *Only four biopsy samples from patients in the second centre were included. Abbreviations: ABMR, antibody-mediated rejection; DSA, donor-specific antibody; DSAST, DSA-selective transcript; eDSAST, endothelial DSAST; ENDAT, endothelial-associated transcript.
NATURE REVIEWS | NEPHROLOGY
VOLUME 10 | AUGUST 2014 © 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS mesangial matrix expansion score, ABMR molecular score and the presence of NK-cell DNA transcripts.
‘‘
Whether or not molecular tests should be implemented in routine clinical practice ... is debatable...
’’
In contrast to earlier studies of stand‑ ard risk patients with late graft loss, Loupy et al. 1 report that, in sensitized patients with acute ABMR, neither the ABMR molecular score nor the eDSAST dataset correlated with histological signs of acute antibody-mediated allograft damage—that is, microvascular inflammation (glomeru‑ litis [g] score and peritubular capillary [ptc] score). The two molecular scores instead correlated with chronic allograft pathology scores, indicating their possible association with graft loss. In the univari‑ ate analysis, the ABMR molecular score, eDSAST dataset, donor age >60 years, esti‑ mated glomerular filtration rate (eGFR) 60 years, humoral histologic score, ABMR molecular score and the eDSAST dataset were indepen‑ dently associated with an increased risk of this outcome. This finding is in agree‑ ment with previous studies that also found an independent association between these molecular scores and (mainly late) graft loss in kidney transplant recipients.4,6,7 Loupy et al.1 demonstrated that the addi‑ tion of the ABMR molecular score or the eDSAST dataset to a reference model of
AUGUST 2014 | VOLUME 10
conventional features improved the strati‑ fication of patients with biopsy-proven early ABMR who are at greatest risk of graft failure. The introduction of a further vari‑ able (such as the ABMR molecular score) into a model with only a few variables (such as donor age and humoral histologic score) inevitably leads to an improved predictive value for the risk of graft failure. The clini‑ cal relevance of an increase in the value of the C statistic (the area under the receiver operating characteristic curve) for discrimi‑ nation between patients who had lost their graft and those who had not is unclear. Molecular approaches are urgently needed to solve the current dilemma in the diagnosis and management of antibodymediated allograft injury. The study by Loupy et al.1 adds an important piece to this puzzle. Knowing which patients are at greatest risk of graft failure in the pres‑ ence of HLA alloantibodies might provide the basis for targeted therapies to amelio‑ rate antibody-mediated allograft injury. Whether or not molecular tests should be implemented in routine clinical practice (as proposed in the latest Banff classifica‑ tion)3 is debatable, as only a few retrospec‑ tive studies of patients with different types of antibody-mediated allograft injury at various time points after transplantation have been carried out. Major issues still need to be addressed, including the valida‑ tion of molecular tests as the basis for clini‑ cal decisions.8 For example, a test that uses a set of 21 genes to predict recurrence of oestrogen-receptor-positive breast cancer has now been validated in >10 studies that included several thousand patients from all over the world. Nevertheless, the proof of a survival benefit for patients with breast cancer in whom therapy is guided by results obtained using molecular testing is still pending.9
University of Heidelberg, Department of Nephrology, Im Neuenheimer Feld 162, Heidelberg 69120, Germany (M.Z., C.M.). Correspondence to: M.Z. [email protected] Acknowledgements The authors thank Dr Geraldine Rauch at the Institute of Medical Biostatics and Professor Burkhard Tönshoff at the University Children’s Hospital, University of Heidelberg, Germany, for helpful discussions. Competing interests The authors declare no competing interests. 1.
2.
3.
4.
5.
6.
7.
8.
9.
Loupy, A. et al. Molecular microscope strategy to improve risk stratification in early antibodymediated kidney allograft rejection. J. Am. Soc. Nephrol. http://dx.doi.org/10.1681/ ASN.2013111149. Hidalgo, L. G. et al. De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure. Am. J. Transplant. 9, 2532–2541 (2009). Haas, M. et al. Banff meeting report: inclusion of C4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am. J. Transplant. 14, 272–283 (2013). Sis, B. et al. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am. J. Transplant. 9, 2312–2323 (2009). Hidalgo, L. G. et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am. J. Transplant. 10, 1812–1822 (2010). Sellares, J. et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. Am. J. Transplant. 13, 971–983 (2013). Halloran, P. F. et al. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM). Am. J. Transplant. 13, 2865–2874 (2013). Volk, H. D., Sawitzki, B. & Reinke, P. Molecular analysis of renal allograft biopsies—more than a nice toy for researchers? Am. J. Transplant. 13, 539–540 (2013). Sinn, P. et al. Multigene assays for classification, prognosis, and prediction in breast cancer: a critical review on the background and clinical utility [German]. Geburtshilfe Frauenheilkd. 73, 932–940 (2013).
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
Molecular diagnosis of kidney transplant rejection Christian Morath and Martin Zeier
Acute or chronic antibody-mediated rejection (ABMR) of kidney allografts is currently diagnosed by the presence of donor-specific alloantibodies and distinct pathological findings in biopsy samples. A new study highlights the potential of molecular diagnostics incorporated into standard criteria for acute ABMR to help identify patients at risk of graft loss. Morath, C. and Zeier, M. Nat. Rev. Nephrol. 10, 429–430 (2014); published online 17 June 2014; doi:10.1038/nrneph.2014.106
In the past 5 years, several groups have studied molecular markers as a means to improve the accuracy and specificity of diagnosis of allograft pathologies. However, a standardized and validated approach to the molecular diagnosis of allograft injury is lacking. Molecular assays and platform technologies, such as proteomics, metabo‑ lomics and mRNA or microRNA expres‑ sion profiling of urine, blood and whole or microdissected allograft biopsy samples, have been used to study acute tubular injury, T‑cell-mediated and antibody-mediated rejection (ABMR; Table 1). In their recent article in the Journal of the American Society of Nephrology, Loupy et al.1 propose a ‘mol ecular microscope strategy’ to improve risk stratification for allograft loss in kidney recipients with early ABMR (30 years after kidney transplantation. In the study by Loupy et al.,1 only patients with acute antibody-mediated graft injury during the first year after surgery were investigated (74 patients in the principal cohort and 54 patients in the independent validation cohort). Finally, only sensitized graft recip‑ ients were included—92% of patients had DSAs before transplantation as opposed to the mostly unselected ‘standard risk’ patients in previous studies. In the current study, the ABMR molecular score correlated with the presence of DSAs, interstitial fibrosis or tubular atrophy score, mesangial matrix expansion score, C4d deposition in graft capillaries, the eDSAST dataset and the expression of natural killer (NK) cell DNA transcripts. The correlation between the three molecular scores is not surprising because when these scores were validated for the diagnosis of ABMR, the respective gene expression datasets showed a degree of overlap.4-6 The eDSAST dataset correlated with the presence of DSAs, Banff transplant glomerulop athy (cg) score,
Table 1 | Selected studies of molecular diagnostics in kidney allograft rejection Study
Patients
Result
Loupy et al. (2014)1
128 patients from two centres with ABMR up to 1 year after transplantation
ABMR score improves stratification of patients at high risk of graft loss
Halloran et al. (2013)7
264 patients from six centres with biopsy 3 days to ~27 years after transplantation
ABMR score is more strongly associated with graft failure than is conventional assessment; ABMR score is associated with future graft loss
Sellares et al. (2013)6
315 patients from four centres with biopsy 6 days to ~35 years after transplantation
ABMR score can be used for diagnosis of ABMR; ABMR score is associated with future graft loss
Hidalgo et al. (2010)5
145 patients from one centre with biopsy 6–31 years after transplantation
23 DSASTs are expressed predominantly in natural killer cells and endothelial cells; DSASTs are increased in ABMR
Sis et al. (2009)4
169 patients from two centres* with biopsy 6 days to 31 years after transplantation
ENDATs can be used for the diagnosis of ABMR, including C4d-negative ABMR; ENDAT levels are associated with future graft loss
All studies assessed mRNA expression in whole graft-biopsy samples (gene chip). *Only four biopsy samples from patients in the second centre were included. Abbreviations: ABMR, antibody-mediated rejection; DSA, donor-specific antibody; DSAST, DSA-selective transcript; eDSAST, endothelial DSAST; ENDAT, endothelial-associated transcript.
NATURE REVIEWS | NEPHROLOGY
VOLUME 10 | AUGUST 2014 © 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS mesangial matrix expansion score, ABMR molecular score and the presence of NK-cell DNA transcripts.
‘‘
Whether or not molecular tests should be implemented in routine clinical practice ... is debatable...
’’
In contrast to earlier studies of stand‑ ard risk patients with late graft loss, Loupy et al. 1 report that, in sensitized patients with acute ABMR, neither the ABMR molecular score nor the eDSAST dataset correlated with histological signs of acute antibody-mediated allograft damage—that is, microvascular inflammation (glomeru‑ litis [g] score and peritubular capillary [ptc] score). The two molecular scores instead correlated with chronic allograft pathology scores, indicating their possible association with graft loss. In the univari‑ ate analysis, the ABMR molecular score, eDSAST dataset, donor age >60 years, esti‑ mated glomerular filtration rate (eGFR) 60 years, humoral histologic score, ABMR molecular score and the eDSAST dataset were indepen‑ dently associated with an increased risk of this outcome. This finding is in agree‑ ment with previous studies that also found an independent association between these molecular scores and (mainly late) graft loss in kidney transplant recipients.4,6,7 Loupy et al.1 demonstrated that the addi‑ tion of the ABMR molecular score or the eDSAST dataset to a reference model of
AUGUST 2014 | VOLUME 10
conventional features improved the strati‑ fication of patients with biopsy-proven early ABMR who are at greatest risk of graft failure. The introduction of a further vari‑ able (such as the ABMR molecular score) into a model with only a few variables (such as donor age and humoral histologic score) inevitably leads to an improved predictive value for the risk of graft failure. The clini‑ cal relevance of an increase in the value of the C statistic (the area under the receiver operating characteristic curve) for discrimi‑ nation between patients who had lost their graft and those who had not is unclear. Molecular approaches are urgently needed to solve the current dilemma in the diagnosis and management of antibodymediated allograft injury. The study by Loupy et al.1 adds an important piece to this puzzle. Knowing which patients are at greatest risk of graft failure in the pres‑ ence of HLA alloantibodies might provide the basis for targeted therapies to amelio‑ rate antibody-mediated allograft injury. Whether or not molecular tests should be implemented in routine clinical practice (as proposed in the latest Banff classifica‑ tion)3 is debatable, as only a few retrospec‑ tive studies of patients with different types of antibody-mediated allograft injury at various time points after transplantation have been carried out. Major issues still need to be addressed, including the valida‑ tion of molecular tests as the basis for clini‑ cal decisions.8 For example, a test that uses a set of 21 genes to predict recurrence of oestrogen-receptor-positive breast cancer has now been validated in >10 studies that included several thousand patients from all over the world. Nevertheless, the proof of a survival benefit for patients with breast cancer in whom therapy is guided by results obtained using molecular testing is still pending.9
University of Heidelberg, Department of Nephrology, Im Neuenheimer Feld 162, Heidelberg 69120, Germany (M.Z., C.M.). Correspondence to: M.Z. [email protected] Acknowledgements The authors thank Dr Geraldine Rauch at the Institute of Medical Biostatics and Professor Burkhard Tönshoff at the University Children’s Hospital, University of Heidelberg, Germany, for helpful discussions. Competing interests The authors declare no competing interests. 1.
2.
3.
4.
5.
6.
7.
8.
9.
Loupy, A. et al. Molecular microscope strategy to improve risk stratification in early antibodymediated kidney allograft rejection. J. Am. Soc. Nephrol. http://dx.doi.org/10.1681/ ASN.2013111149. Hidalgo, L. G. et al. De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure. Am. J. Transplant. 9, 2532–2541 (2009). Haas, M. et al. Banff meeting report: inclusion of C4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am. J. Transplant. 14, 272–283 (2013). Sis, B. et al. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am. J. Transplant. 9, 2312–2323 (2009). Hidalgo, L. G. et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am. J. Transplant. 10, 1812–1822 (2010). Sellares, J. et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. Am. J. Transplant. 13, 971–983 (2013). Halloran, P. F. et al. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM). Am. J. Transplant. 13, 2865–2874 (2013). Volk, H. D., Sawitzki, B. & Reinke, P. Molecular analysis of renal allograft biopsies—more than a nice toy for researchers? Am. J. Transplant. 13, 539–540 (2013). Sinn, P. et al. Multigene assays for classification, prognosis, and prediction in breast cancer: a critical review on the background and clinical utility [German]. Geburtshilfe Frauenheilkd. 73, 932–940 (2013).
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
