These authors have contributed equally to this work
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Antibody-mediated rejection (AMR) is caused by antibodies that recognize donor human leukocyte antigen (HLA) or other targets. As knowledge of AMR pathophysiology has increased, a combination of factors is necessary to confirm the diagnosis and phenotype. However, frequent modifications to the AMR definition have made it difficult to compare data and evaluate associations between AMR and graft outcome. The present paper was developed following a Broad Scientific Advice request from the European Society for Organ Transplantation (ESOT) to the European Medicines Agency (EMA), which explored whether updating guidelines on clinical trial endpoints would encourage innovations in kidney transplantation research. ESOT considers that an AMR diagnosis must be based on a combination of histopathological factors and presence of donor-specific HLA antibodies in the recipient. Evidence for associations between individual features of AMR and impaired graft outcome is noted for microvascular inflammation scores ≥2 and glomerular basement membrane splitting of >10% of the entire tuft in the most severely affected glomerulus. Together, these should form the basis for AMR-related endpoints in clinical trials of kidney transplantation, although modifications and restrictions to the Banff diagnostic definition of AMR are proposed for this purpose. The EMA provided recommendations based on this Broad Scientific Advice request in December 2020; further discussion, and consensus on the restricted definition of the AMR endpoint, is required.
Although biopsy-proven acute rejection (BPAR) remains widely used as a primary efficacy variable in the clinical trial setting (
Advances in the development of sensitive assays for DSA identification have improved our understanding of AMR histopathology (
The detrimental impact of AMR on kidney transplantation outcome has been known for decades, as illustrated by the early routine implementation of crossmatching to avoid this rejection phenotype (
The next advancement followed the introduction of C4d staining, which documented histopathogenetic links between circulating DSA and organ damage, by detecting complement activation by DSA fixed to surface antigens on the endothelial cell (
Chronic (c)AMR subtypes were first recognized in the Banff 2005 update, as chronic active (ca)AMR, with transplant glomerulopathy (TG) and/or severe peritubular capillary basement membrane multilayering (PTCML) and/or simple interstitial fibrosis and tubular atrophy and/or arterial fibrous intimal thickening, with C4d positivity (
Antibody-mediated changes (
Diagnosis | Diagnostic criteria groups |
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|
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• Lesion Score C4d > 1 (immunofluorescence on fresh frozen tissue) OR C4d > 0 (immunohistochemistry on paraffin-embedded tissue) AND | • Banff Lesion Score g > 0 in the absence of glomerulonephritis and/or Banff Lesion Score ptc>0 in the absence of TCMR or borderline changes |
• Banff Lesion Scores t0, v0, no arterial intimal fibrosis with mononuclear cell inflammation in fibrosis and formation of neointima, no criterion from Group 1 (AMR Banff activity), no criterion from Group 4 (histologic features of AMR chronicity), no increased expression of thoroughly validated gene transcripts/classifiers in the biopsy tissue strongly associated with AMR | • Banff Lesion Score v > 0 |
• Acute thrombotic microangiopathy in the absence of any other cause | |
• Acute tubular injury in the absence of any other apparent cause | |
|
|
• No criterion from Group 4 (histologic features of AMR chronicity) AND | • At least moderate microvascular inflammation (g + ptc>1) in the absence of borderline changes (Diagnostic Category 3) or acute TCMR (aTCMR; Diagnostic Category 4). If borderline changes or aTCMR are present, Banff Lesion Score g + ptc>1 is not sufficient; g ≥ 1 is required |
• ≥1 criterion from Group 1 (AMR activity) AND | • Increased expression of thoroughly validated gene transcripts/classifiers in the biopsy tissue strongly associated with AMR |
• ≥1 criterion from Group 2 (antibody interaction with tissue) AND | |
• ≥1 criterion from Criteria Group 3 (DSA or equivalent) AND | |
|
|
• ≥1 feature from Group 4 (histologic features of AMR chronicity) AND | • DSA (anti-HLA or other specificity) |
• ≥1 criterion from Group 2 (antibody interaction with tissue) AND | • Banff Lesion Score C4d > 1 (IF on fresh frozen tissue) or C4d > 0 (IHC on paraffin-embedded tissue) |
• ≥1 criterion from Group 3 (DSA or equivalent) | • Increased expression of thoroughly validated gene transcripts/classifiers in the biopsy tissue strongly associated with AMR |
|
|
• Banff 2017 permits the use of this term for biopsy specimens showing TG and/or PTCML in the absence of criterion of current/recent antibody interaction with the endothelium (Criteria Group 2) but with a prior documented diagnosis of active or chronic active AMR or documented prior evidence of DSA | • Banff Lesion Score cg > 0 (by light microscopy or EM, if available), excluding biopsies with evidence of chronic thrombotic microangiopathy |
• ≥7 layers in 1 cortical peritubular capillary and ≥5 in 2 additional capillaries, avoiding portions cut tangentially by EM, if available (severe PTCML); arterial intimal fibrosis of new onset, excluding other causes; leukocytes within the sclerotic intima favor chronic AMR if there is no prior history of biopsy-proven TCMR with arterial involvement, but are not required |
AMR, antibody-mediated rejection; aTCMR, acute T cell-mediated rejection; DSA, donor-specific antibody; EM, electron microscopy; HLA, human leukocyte antigen; IF, immunofluorescence; IHC, immunohistochemistry; PTCML, peritubular capillary basement membrane multilayering; TCMR, T cell-mediated rejection; TG, transplant glomerulopathy.
Adapted from Roufosse et al., 2018 (
Currently, AMR diagnosis within Banff Classification category 2 is based on four partially overlapping components: histological features of AMR activity; evidence of antibody interaction with graft vascular endothelium; histological features of AMR chronicity; and DSA or equivalents (
Updates to the Banff Classification of AMR over time make it difficult to maintain long-term follow-up registries or compare literature. Moreover, the interobserver agreement (κ-statistic) of the most important lesion scores for AMR was quoted as 0.39 for Banff Lesion Score g, 0.38 for ptc, and 0.48 for cg—at best a fair-to-moderate agreement, even among very experienced transplant nephropathologists (
Several problems arise when reviewing evidence of an association between AMR and allograft outcome. Firstly, AMR definitions have changed very frequently since 2001, as outlined above, making it difficult to compare data from studies conducted over the last 2 decades. Secondly, Banff diagnostic criteria and categories are adjusted based on antecedent literature, and as they arise as a synthesis of several different studies, rarely fully align with literature on which they are based. Conversely, outcomes of AMR diagnoses according to their strict definitions in the Banff Classification have rarely been investigated for either their association with outcome, or their success in delineating which patients are eligible for a specific therapy; where this has been done, results show improved prediction of outcome with the 2013 version compared to the 2003/2007 Classification, and with the 2013 version compared to the 2017 Classification (
To our knowledge, no method of transcriptome analysis has been formally recognized as thoroughly validated by Banff. No transplant centers have obtained adequate clinical validation to use transcript analysis for defining AMR, as required by Banff consensus. In addition, although the Banff Classification makes no distinction between AMR in patients with preformed DSA (high-risk) and non-sensitized (low-risk) transplant recipients, the diagnosis and treatment pathways for both groups might be quite different, as are the underlying biology and clinical phenotypes. Consequently, AMR classification may need to include more than histology, because identical histological diagnoses in the kidney (such as TG or TMA) can be the consequence of different disease entities (
Thus, although the basic principles of diagnosing AMR have generally remained constant, given the considerable changes to Banff definitions of AMR, longitudinal comparison of literature findings is more challenging for AMR than for aTCMR. Interpretation of the AMR literature must be undertaken cautiously, taking account of these limitations. In reviewing evidence that could serve as background for the definition of AMR, first it is important to evaluate studies that have assessed outcomes associated with various combinations of biopsy features. In the following sections, we divide this information into subcategories broadly based on the Banff classification. After evaluating the literature on allograft outcome, we consider data relating to associations between outcome and individual biopsy features that are components of AMR (
This subcategory is discussed in conjunction with C4d positivity with acute tubular injury (ATI) in the absence of other apparent cause.
Much of the evidence for an association between aAMR and outcome (i.e., graft loss) comes from publications that only include components of aAMR (e.g., MVI, C4d, and/or DSA) (
Studies investigating associations between Banff diagnostic category “active AMR” and outcome (
References | Endpoint | Definition of aAMR | Study type | Cohort | Findings | Level of evidence (grade) |
---|---|---|---|---|---|---|
Solar-Cafaggi et al. ( |
Graft loss | Mixed (unseparated) aAMR and caAMR (Banff 2007 or 2017 criteria); indication and protocol biopsies; incomplete DSA data | Single-center retrospective | N = 201 | Increased graft loss ( |
Very low |
Sai et al. ( |
sCr × 1.5; cAMR and graft loss | Banff 2013 | Retrospective | N = 627 protocol + indication biopsies; C4d+ AMR ( |
Significantly more cAMR and graft loss on follow-up (between-group analysis [Mann−Whitney], not outcome analysis) | Very low |
Orandi et al. ( |
Graft loss | Banff 2013 Methods do not clarify whether cases with cg are included | Retrospective | N = 217 patients with AMR; (142 clinical AMR, 77 subclinical) + controls (426 clinical, 231 subclinical); high proportion (63%) of HLA-incompatible transplants, so may not apply to conventional transplantation | Graft loss in subclinical AMR 2.15-fold (95% CI 1.19–3.91; |
Low +1 (RR > 5 for clinical AMR) |
Orandi et al. ( |
TG and graft loss | Banff 2013 aAMR and/or caAMR likely both included | Single-center, retrospective; all biopsies in 1st year post transplant (indication + protocol) | 51 C4d− and 156 C4d+ cases of AMR | TG risk same in C4d− and C4d+ but not vs. controls; 1-year and 2-years post-AMR graft survival: C4d− vs. controls: 2.56-fold (95% CI 1.08–6.05; |
Low |
Everly et al. ( |
Graft loss | Altered Banff definition used: Banff 2003 AMR including suspicious (i.e., if ≥ 2 of the following present: DSA, histopathologic changes consistent with AMR and C4d+ staining in PTC ± other structures); do not specify active or chronic active | Retrospective | Patients with acute cellular rejection ( |
Significantly worse survival in AMR ( |
Low |
Loupy et al. ( |
TG; 1/sCr and eGFR | Altered Banff definition used (Banff 1997 + addition of C4d− AMR); contains some cg/chronic cases at baseline | Retrospective | Pre-sensitized patients ( |
Subclinical AMR at 3 months associated with interstitial fibrosis and tubular atrophy, TG and worse function at 1 year | Low |
a, acute/active; AMR, antibody-mediated rejection; c, chronic; ca, chronic active; CI, confidence interval; DSA, donor-specific antibody; eGFR, estimated glomerular filtration rate; HLA, human leukocyte antigen; PTC, peritubular capillaries; sCr, serum creatinine; TG, transplant glomerulopathy.
Given the heterogeneity of definitions, overall, the quality of evidence that strictly defines the association between aAMR and increased graft loss is low; in recipients of HLA-incompatible grafts, quality of evidence is higher. However, if one evaluates the literature with less stringency about the exact AMR definition, there is consensus that aAMR is an important risk factor for graft failure (
In the absence of dysfunction (i.e., subclinical AMR in protocol biopsies), the outcome used for features of aAMR is usually TG rather than graft loss, although a retrospective study indicated that subclinical AMR in 1-year protocol biopsies had a detrimental impact on graft survival (
Our proposal is that aAMR, exactly as defined by the current Banff classification, cannot be adopted as a surrogate endpoint for future cAMR and graft loss in low-risk situations, i.e., non-sensitized graft recipients, without DSA against the graft. Conversely, in high-risk patients, i.e., sensitized patients with DSA against the graft, evidence supports features of aAMR as a surrogate for graft loss, especially if associated with graft dysfunction. Future research should aim to establish outcome (graft loss, graft function, future cAMR, or caAMR) in patients with aAMR, strictly defined according to Banff criteria and specifically excluding cases with features of chronicity. Such research should involve retrospective and prospective studies, and high- and conventional-risk transplantations. Data from randomized controlled trials investigating aAMR treatment regimens would also be particularly valuable. Although further data are awaited, there is broad consensus on the clinical relevance and impact of aAMR after kidney transplantation. Since aAMR leads to therapeutic interventions, treatment burden, associated morbidity, and increased cost, features of aAMR represent a key endpoint for interventional studies.
According to the 2017 Banff Classification, a diagnosis of cAMR or caAMR can only be established based on presence of TG (Banff Lesion Score cg > 0) and/or severe PTCML. For caAMR, this must be accompanied by “evidence of antibody interaction with tissue” and “DSA or equivalent”; for cAMR, this must be in conjunction with “a prior documented diagnosis of aAMR or caAMR or documented prior evidence of DSA.” The ill-defined transplant vasculopathy is no longer considered a chronicity parameter for these diagnoses (
Studies investigating associations between Banff diagnostic categories cAMR/caAMR and outcome; and/or investigating TG (
References | Endpoint | Parameter investigated | Findings | Level of evidence (grade) |
---|---|---|---|---|
Lesage et al. ( |
Graft loss | GBM splitting | ∼50% graft loss within 24 months after index biopsy; HR even after adjustment for sCr and proteinuria >5 vs. controls | Moderate |
Wavamunno et al. ( |
Death-censored transplant survival | Ultrastructural | No difference in death-censored transplant survival between 7 patients with Banff cg ≥ 1 (Banff 1997) within the first 5 years post transplantation vs. 8 controls | Very low |
Perkowska-Ptasinska et al. ( |
Transplant survival in subgroups of TG | Banff cg ≥ 1 Banff 2007 = Banff 1997 | 38/158 patients with TG lost transplant within 98 months (range, 3–215 months); no control cohort; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Shimizu et al. ( |
Graft loss in patients with a diagnosis of TG | Banff cg ≥ 1 Banff 2009 = Banff 1997 | 22% graft loss within observation time (time unclear); includes ABO-incompatible transplants; Unclear whether Banff chronic AMR fulfilled; no comparison with controls regarding outcome | Not applicable |
Hayde et al. ( |
Death-censored transplant survival | cAMR compared to IFTA and TG (authors’ own definitions; invalid criteria for TG: “by electron microscopy … electron-lucent widening of the subendothelial zone of the GBM, subendothelial accumulation of flocculent material, with or without a new subendothelial basement membrane layer”) | cAMR associated with significantly lower graft survival compared with IFTA ( |
Low |
Pefaur et al. ( |
Transplant survival | Unclear criteria for TG | Retrospective study, 3 patients; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Shimizu et al. ( |
Transplant survival | Banff cg ≥ 1 Banff 2007 = Banff 1997 | Retrospective study, 13 patients, no control group; 2/13 grafts lost; unclear observation time; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
John et al. ( |
Death-censored transplant survival | Banff cg ≥ 1 Banff 1997 | Retrospective study, 36 patients with TG, 5-years death-censored graft loss 16.7%; no control group; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Nair et al. ( |
Graft loss | Unclear criteria for TG that do not necessarily involve GBM splitting | Three patients with TG within first 6 months post transplantation; no control group; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Lopez Jimenez et al. ( |
Graft loss | Banff cg ≥ 1 Banff 1997 | Retrospective study, 30 patients with TG; 50% graft loss mean 25 ± 20 months post biopsy; no control group; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Kamal et al. ( |
Graft loss | Banff cg ≥ 1 Banff 2007 + 2009 = Banff 1997 | Retrospective study, 52 patients with TG, 17 (32%) with graft loss, median time to graft loss 16 months, no control cohort; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Dobi et al. ( |
Transplant survival | Banff cg ≥ 1a Banff 2013 | Retrospective analysis, 57 patients with TG; no control cohort; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Halloran et al. ( |
Death-censored transplant survival | Banff cg ≥ 1a Banff 2013 | Retrospective analysis, 27 patients with TG; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Toki et al. ( |
No outcome data (eGFR at time of biopsy) | Banff cg ≥ 1b Banff 2013 | Retrospective analysis, 127 patients with TG; extraction of outcome data on c/caAMR/TG not possible; no outcomes data | Not applicable |
Courant et al. ( |
Death-censored transplant survival | cAMR according to Banff 2013 with DSA+ | Retrospective data, 9 patients with cAMR; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Lubetzky et al. ( |
Transplant survival | Banff cg ≥ 1 Banff 1997; patients with TG and DSA+/MVI− | Retrospective analysis, 24 patients 50% graft loss in 3 years; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Sablik et al. ( |
Transplant survival | Banff cg ≥ 1b Banff 2015 | Retrospective analysis, 41 patients with caAMR; no control cohort; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
Aubert et al. ( |
Transplant survival | Banff 2009, 2011, 2013. Unclear if cg1a included | Retrospective analysis, 385 patients with TG; different immunological, functional and histological TG subtypes described; no comparison to control group without TG; extraction of outcome data on c/caAMR/TG not possible | Not applicable |
a, acute/active; AMR, antibody-mediated rejection; c, chronic; ca, chronic active; DSA, donor-specific antibody; eGFR, estimated glomerular filtration rate; GBM, glomerular basement membrane; HR, hazard ratio; IFTA, interstitial fibrosis and tubular atrophy; MVI, microvascular inflammation; sCr, serum creatinine; TG, transplant glomerulopathy.
A retrospective study of 44 patients with TG examined the outcome of graft loss, with TG defined as Banff cg > 0 (glomerular basement membrane splitting of >10% of the entire tuft in the most severely affected glomerulus) (
To investigate associations between TG and other parameters, as well as with outcomes, using archetypal analysis, a retrospective study of 385 patients with TG identified five distinct immunological, histological, and functional profiles of TG that were associated with allograft failure (
Because of repeated revisions to Banff criteria (including gene transcripts and the requirement for EM, to detect PTCML and early TG lesions), the incidence of caAMR is under-reported. No studies fulfill all criteria for this diagnosis according to Banff 2017 or have a sufficient follow-up to use strictly defined Banff caAMR as an endpoint (
We therefore recommend that clinical trials in kidney transplantation using caAMR as an endpoint or an inclusion criterion strictly adhere to Banff consensus criteria and report granular histological features of Banff Lesion Scores, to allow between-trial comparisons. Additional research is needed, in high- and conventional-risk transplantation scenarios that consider the effect of treating aAMR earlier, equally defined according to the strict Banff Classification.
A noteworthy change to the Banff Classification in 2017 was its omission of ‘suspicious for aAMR’ and ‘suspicious for caAMR’ categories (
Until 2019, no publication presented outcomes for patients with “suspicious for aAMR.” Then, the evidence appeared, with a caveat, because the 123 DSA− patients with AMR included six patients with TG (Banff Lesion Score cg ≥ 1); irrespective of C4d status, outcomes for patients with histological features of AMR but without DSA were no different than for controls without AMR (
The literature offers even less information about the diagnostic subcategory of “suspicious for caAMR,” eliminated from Banff in 2017. One study involving 21 DSA− patients showed an average transplant survival after diagnosis of 3.7 years (
Some DSA− cases “suspicious for AMR” could be explained by non-HLA antibodies. Without any hard evidence, standardized tests, or validated assay and cut-off value to screen for non-HLA-DSA, we do not recommend that non-HLA-DSA be considered in the diagnosis of AMR. Further research is needed before non-HLA antibodies can be included in the definition of endpoints for registration studies.
Overall, we do not recommend using cases in the “suspicious for” categories as endpoints.
This section reports on two category 2 diagnoses that are separated in the Banff 2017/2019 Classification (
Technically, according to the Banff classification, a biopsy that is C4d+ with DSA but with a reasonable other cause of ATI (e.g., ischemia/reperfusion injury) is not AMR, yet publications have not assessed for (or reported on) other causes of ATI. Some early reports on C4d staining date from before widespread recognition of the full spectrum of histological features of aAMR, therefore descriptions of poor outcomes for C4d+ cases “without features of rejection” must be handled cautiously.
The 2001 Banff meeting recognized a form of AMR with no or little inflammation, included in the list of category 2 diagnoses as “acute tubular necrosis-like minimal inflammation, C4d+.” It was stated that “acute humoral rejection may be manifested only by ATI without other evidence of rejection (seen in 10% of cases).” The evidence cited (
The 2007 Banff meeting (
Additional publications investigating the link between ATI-AMR and C4d+ without evidence of rejection, with outcome data, are presented in
Studies investigating ATI-AMR and C4d+ without evidence of rejection (
References | Endpoint | Definition of Banff phenotype | Findings | Level of evidence (grade) |
---|---|---|---|---|
Haas et al. ( |
AMR | C4d+ in early post-reperfusion biopsies | Predicts future AMR ( |
Low |
Djamali et al. ( |
AMR | C4d+ in early post-reperfusion biopsies; mild to moderately sensitized transplant recipients | Predicts future AMR | Low |
Kikic et al. ( |
Graft loss | Biopsies with C4d; 42% of patients in the C4d+ group were pre-sensitized; mean time to biopsy in C4d+ group 0.75 mo | C4d associated with graft loss independently of presence of histological features of AMR; HR 1.85 ( |
Low |
Nickeleit et al. ( |
Benefit from antirejection therapy | C4d+ with mild allograft dysfunction and no histological evidence of rejection | C4d+ with mild allograft dysfunction and no histological evidence of rejection does not benefit from antirejection therapy | Low |
Dickenmann et al. ( |
Improved function after treatment | C4d+ biopsies without other histopathological features of AMR | Function improves in this group after treatment | Low |
Dominy et al. ( |
AMR | C4d+ without evidence of rejection; mild ATI at most | Rather than histological features or DSA, transcript analysis for AMR signature distinguishes minority at risk of subsequent AMR | Low |
AMR, antibody-mediated rejection; ATI, acute tubular injury; DSA, donor-specific antibody; HR, hazard ratio.
It is impossible to give a guideline recommendation because of inconsistent findings in the argument that C4d+ ATI without evidence of rejection is associated with increased risk of graft loss. In addition, there is no recent consensus definition of ATI, or of degrees of severity of ATI, or of what reasonably constitutes exclusion of other causes of ATI. Therefore, we recommend that the C4d+ ATI-only form of AMR and C4d+ without evidence of rejection subcategory of AMR should not be used as an efficacy measure in clinical trials. We also recommend that future research incorporates definitions of ATI and assessments of its severity, based on definitions agreed in the context of international collaborations (e.g., Banff Working Group for Rules and Dissemination). Such research should include both patients with preformed antibodies (sensitized) and non-sensitized patients, with representation of early and late post-transplant periods.
Endarteritis is also a feature of aTCMR that initially was not included in AMR definitions; this makes findings from early studies difficult to interpret for the given purpose (Banff Lesion Score v in “acute TCMR,” “mixed acute TCMR-AMR,” “pure AMR”). Although endarteritis was described as a risk factor for graft loss (
Banff acknowledges that TMA can have a variety of causes in kidney transplant recipients (e.g., recurrent disease, infection, antiphospholipid antibodies, medication toxicity). During the 2015 Banff meeting, a working group was formed (
We are unaware of sufficient published data about the outcomes of adequately investigated cases of AMR-associated TMA. The largest case series describes 33 patients with TMA and C4d positivity, 40% of whom experienced transplant failure within 2 years of diagnosis (
MVI is the main histological feature indicating activity in aAMR and caAMR. The Banff criteria for AMR use cut-off values of MVI >0 and >1, respectively, to establish C4d+ and C4d− aAMR; these values were established by consensus, based on published evidence (
Studies investigating the association between MVI and outcomes (
References | Endpoint | Predictor | Findings | Level of evidence (grade) |
---|---|---|---|---|
Haas and Mirocha ( |
TG | MVI + endothelial lesions on EM | Indication biopsies (DSA at time of biopsy): MVI + endothelial lesions on EM associated with TG | Low |
Bagnasco et al. ( |
TG | g | Patients with pre-transplant DSA (deceased donors including ABO-incompatible donors): g in protocol + indication biopsies associated with TG ( |
Low |
Einecke et al. ( |
Graft loss | MVI | In multivariate analysis (indication + protocol biopsies, DSA post-transplant, living + deceased donors), graft failure correlated with MVI and scarring, but C4d staining was not significant | Low |
Sis et al. ( |
Graft loss | MVI | Indication biopsies (anti-HLA antibodies at time of biopsy, living + deceased donors): g + ptc predicted graft failure independently of time, C4d and transplant glomerulopathy ( |
Low |
Verghese et al. ( |
Graft loss | MVI | Retrospective, no data on DSA, includes mixed TCMR-AMR indication biopsies. In indication biopsies carried out <1 year post-transplant, MVI associated with decreased death-censored graft survival, independent of the presence of C4d ( |
Low |
de Kort et al. ( |
Graft loss | MVI | Retrospective cohort study indication biopsies of patients with |
Low |
AMR, antibody-mediated rejection; CI, confidence interval;
Based on the low-quality evidence that MVI is an independent predictor of graft loss, we cautiously recommend that the MVI score is used as an efficacy marker for clinical trials in kidney transplantation. We also recommend that further research is undertaken to confirm the effect of MVI on outcome, in prospective randomized controlled trials, with granular histological data for Banff Lesion Scores and DSA.
There are caveats to the prognostic value of C4d status: thresholds for positivity scoring differ, depending on antibody and study. For example, the monoclonal antibody used on frozen tissue is particularly sensitive; therefore >10% of PTC must be positive, whereas for the polyclonal anti-C4d antibody used on formalin-fixed paraffin-embedded tissue samples, any percentage and intensity of PTC positivity sufficiently describes a biopsy as C4d+ (
Literature findings related to the potential prognostic value of C4d status are varied, likely because of the dynamic process (
Transplantation studies that feature C4d and outcomes (
References | Endpoint | Predictor | Findings | Level of evidence (grade) |
---|---|---|---|---|
Naesens et al. ( |
Graft loss | Composite | Retrospective (indication biopsies, no info on DSA): C4d, TG, ongoing interstitial inflammation, dn/recurrent glomerular disease, IFTA significantly and independently associated with post-biopsy graft survival (MVI highly significant in univariate, but not retained in final multivariate model) | Low |
Kikic et al. ( |
Graft loss | C4d | C4d associated with graft loss independently of the presence of histological features of AMR | Low |
Sapir-Pichhadze et al. ( |
Graft loss | C4d | Systematic review (3492 abstracts: 3485 indication and 868 protocol biopsies). C4d+ associated with inferior allograft survival compared with DSA or histopathology alone | Low |
Matas et al. ( |
Graft loss | C4d | Cross-sectional (retrospective) cohort (indication biopsies, DSA at time of biopsy, living + deceased donors): C4d−/DSA- recipients had significantly better (and C4d+/DSA+ worse) death-censored graft survival than other groups. C4d+/DSA− and C4d−/DSA+ had similar intermediate death-censored graft survival | Low |
AMR, antibody-mediated rejection;
As TG is the main feature indicating chronicity in the diagnosis of caAMR and cAMR, much of the evidence indicating that this feature is an indicator of outcome has been covered above (
In 55 patients with TG (Banff Lesion Score cg ≥ 1b) (
Applying Racusen’s criterion for defining glomerular basement membrane splitting, mentioned above (
Normal peritubular capillaries have a single basement membrane under the endothelial cell, and PTCML is characterized by an increase in basement membrane layers. Low levels of PTCML are seen in several conditions, whereas severe PTCML is a defining feature of chronicity in the Banff definition of AMR. Severe PTCML is characterized as seven or more layers of basement membrane in at least a single cortical peritubular capillary, and five or more layers in at least two additional capillaries.
Currently, PTCML is only diagnosed in transplantation biopsies by EM evaluation, which limits its use to centers sufficiently resourced to undertake such examinations. Even within EM-capable centers, this diagnostic method may be reserved for cases for which there is an indication [defined in Banff 2013 (
The limited number of observational studies investigating the link between PTCML and outcome (
Studies that feature PTCML assessment and outcomes (
References | Endpoint | Definition of PTCML | Findings | Level of evidence (grade) |
---|---|---|---|---|
Einecke et al. ( |
Graft loss | One PTC with ≥5 basement membrane layers | In non-selected transplant population, 1 PTC with ≥5 basement membrane layers predictive of graft loss in multivariate analysis (HR 1.98, |
Low |
Roufosse et al. ( |
TG | Numbers of PTC with ≥3 and ≥5 basement membrane layers | Risk of TG increases with increasing numbers of PTC with ≥3 and ≥5 basement membrane layers | Low + 1 (‘dose–response’ gradient) |
de Kort et al. ( |
Graft loss | Three PTC with ≥5 basement membrane layers | In patients with |
Low |
de Kort et al. ( |
TG | Mean basement membrane layer count >2.5 | Mean PTCML count >2.5 associated with increased risk of TG ( |
Low |
Dobi et al. ( |
Graft loss | PTC circ score ≥3 | In patients with cAMR, PTC circ ≥3 predicts graft loss | Low |
AMR, antibody-mediated rejection; c, chronic;
The definition of transplant vasculopathy as evidence of AMR chronicity remains ambiguous in the Banff Classification (
Increased expression of thoroughly validated gene transcripts/classifiers in biopsy tissue strongly associated with AMR provides evidence of current/recent antibody−tissue interactions, according to the Banff 2017/2019 definition of AMR (
Studies that feature ‘Evidence of gene transcripts/classifiers strongly associated with AMR’ and outcomes (
References | Endpoint | Definition of molecular marker | Findings | Level of evidence (grade) |
---|---|---|---|---|
Sellares et al. ( |
Graft loss | AMR classifier | Retrospective cohort, 315 patients; AMR classifier predicts graft loss in Cox multivariate analysis | Low |
( |
Graft loss and progression of chronic injury | AMR molecular score and endothelial DSA-selective transcripts | 2 cohorts (principal |
Low |
Loupy et al. ( |
||||
Yazdani et al. ( |
Graft loss | Differential expression of 503 unique genes in AMR, with significant enrichment of NK cell pathways | Retrospective cohort, with validation in external cohort for outcome analysis; microarray transcriptomic data from case–control study ( |
Low |
Sis et al. ( |
Graft loss | ENDATs | Retrospective cohort of indication biopsies validated in independent set; microarray analysis for ENDATs. Many individual ENDATs were increased in AMR and predicted graft loss; high ENDAT score in patients with DSA predicts graft loss (but no increase in graft loss if DSA− and ENDAT+) | Low |
Dominy et al. ( |
Graft loss |
|
Retrospective cohort of 57 biopsies from patients with AMR or normal surveillance biopsies; 2-gene signature predicts graft loss in whole group and within DSA+ group | Low |
Kamal et al. ( |
Graft loss; no formal outcome analysis | Various gene expression levels | Retrospective cohort of patients with TG; significantly increased levels endothelial cell–associated transcripts, gene transcripts associated with complement cascade, interleukins and their receptors, and granulysin in patients with graft loss | Very low |
AMR, antibody-mediated rejection; CI, confidence interval; DSA, donor-specific antibody; ENDAT, endothelial cell–associated transcript; HR, hazard ratio; NK, natural killer; TG, transplant glomerulopathy.
Although there is consistent evidence that gene transcripts/classifiers strongly associated with AMR are associated with graft loss (and in some cases, the evidence comes from multivariate analyses with validation groups), it is impossible to give a guideline recommendation or consensus-based statement. Different gene sets/classifiers are used across the studies, with no unifying set of genes agreed on for future validation in prospective research. Also, to our knowledge, no transplant centers have clinical validation for use of transcript analysis for AMR, especially for improving the prediction of graft outcome. Consequently, we recommend that gene transcripts/classifiers strongly associated with AMR are not used as efficacy measures for clinical trials. Future research in the context of international collaborations on agreed gene sets/classifiers (e.g., Banff Working Group for Molecular Pathology) should assess non-selective populations of transplant biopsies and determine clinically meaningful molecular scoring systems that predict cAMR development and graft failure. These studies should include multivariate analyses in combination with traditional clinical, histopathological, or immunogenetic parameters.
Studies investigating outcomes in cases with subclinical AMR, including incomplete phenotypes (
References | Endpoint | Definition of predictor | Findings | Level of evidence (grade) |
---|---|---|---|---|
Loupy et al. ( |
GFR, TG, IFTA | Subclinical AMR | Patients with subclinical AMR at 3 mo had at 1 year: Higher rate of IFTA (100% vs. 33.3%; |
Low |
Lerut et al. ( |
PTCML, cAMR | PTC | Protocol biopsies with ptc at 3 mo associated with PTCML ( |
Low |
Haas et al. ( |
CAN score (cg + ci + ct + cv) | Subclinical AMR | Subclinical AMR (stable SCr, PTC, diffuse PTC C4d, positive DSA) during 1st year post transplantation associated with higher increase in CAN score in follow-up biopsies 335 ± 248 (SD) days later (3.5 ± 2.5 vs. 1.0 ± 2.0; |
Low |
|
cAMR | MVI + class II DSA | Multivariate analysis demonstrated that presence of MVI and anti-HLA class II DSA at 3 mo was associated with a 4-fold increased risk of progression to cAMR independently of C4d ( |
Low |
Cosio et al. ( |
Graft loss | ‘cAMR’ (= cg > 0 or MVI≥2) | 4.2% of protocol biopsies at 1 year showed cAMR; risk of death-censored graft survival HR 12.6 (95% CI 6.58–24.3; |
Moderate |
Gloor et al. ( |
GFR, proteinuria | TG | Prognosis of subclinical TG was equally poor as TG diagnosed with graft dysfunction, with progressive worsening of histopathologic changes and function | Low |
Papadimitriou et al. ( |
NR | Indication + protocol biopsies (concurrent DSA): More incomplete phenotype in protocol than in indication biopsies Persistence/worsening of AMR in a subsequent biopsy occurred in 38.2% of cases independently of strength of AMR findings in 1st biopsy (e.g., progression to cAMR occurred also in cases with suspicious or non-diagnostic findings) | Low | |
Tsuji et al. ( |
cAMR | MVI | MVI in protocol biopsies at 3 mo correlates with later development of cAMR ( |
Low |
AMR, antibody-mediated rejection; c, chronic; DSA, donor-specific antibody; GFR, glomerular filtration rate; HLA, human leukocyte antigen; IFTA, interstitial fibrosis and tubular atrophy; m, mean; MVI, microvascular inflammation; NR, not reported; PTC, peritubular capillary; PTCML, peritubular capillary basement membrane multilayering; sCr, serum creatinine; SD, standard deviation; TG, transplant glomerulopathy.
A literature search for studies evaluating the frequency of subclinical AMR management showed that ∼60% of patients received treatment, usually with antibody-targeted therapies. Again, national variations were observed. In Paris, 57% of patients with subclinical AMR received antirejection therapy (
Based on the evidence presented above, we propose a restricted definition of the Banff phenotypes of AMR, if used as an endpoint in interventional trials (
Restricting the Banff classification for AMR for the purpose of endpoints in clinical trials, based on the evidence reviewed (
Banff 2017 Category 2: Antibody-mediated changes | Restricted definition of AMR for use as primary endpoint |
---|---|
|
|
1. Histologic evidence of acute tissue injury, including ≥1 of the following | 1. Histologic evidence of acute tissue injury |
• MVI (g > 0 and/or ptc>0), in the absence of recurrent or |
• At least moderate MVI (g + ptc≥2 with g ≥ 1 in the presence of aTCMR, caTCMR, or borderline changes, or infectious disease of the transplant) |
• Intimal or transmural arteritis (v > 0) | • — |
• Acute TMA the absence of any other cause | • — |
• Acute tubular injury, in the absence of any other apparent cause | • — |
2. Evidence of current/recent antibody interaction with vascular endothelium, including one or more of the following | 2. Evidence of current/recent antibody interaction with vascular endothelium |
• Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d > 0 by IHC on paraffin-embedded sections) | • At least MVI g + ptc≥2 with g ≥ 1 in the presence of aTCMR, caTCMR or borderline changes, or infectious disease of the transplant), identical to criterion 1 for aAMR |
• At least moderate MVI ([g + ptc]≥2) in absence of recurrent/ |
• With or without C4d positivity (C4d ≥ 1 on paraffin tissue or ≥2 on frozen tissue) |
• Increased expression of gene transcripts/classifiers in the biopsy tissue strongly associated with AMR, if thoroughly validated | • — |
3. Serologic evidence of donor-specific antibodies (DSA to HLA or other antigens). C4d staining or expression of validated transcripts/classifiers as noted above in criterion 2 may substitute for DSA; however thorough DSA testing, including testing for non-HLA antibodies if HLA antibody testing is negative, is strongly advised whenever criteria 1 and 2 are met | 3. DSA positivity (anti-HLA antibodies) |
|
|
1. Morphologic evidence of chronic tissue injury, one or more of the following | 1. Morphologic evidence of chronic tissue injury, including ≥1 of the following |
• TGA (cg > 0) if no evidence of cTMA or chronic recurrent/ |
• cg ≥ 1 according to Banff 2011 (≥10% of the glomerular capillary walls in the most severely affected glomerulus involved) |
• Severe peritubular capillary basement membrane multilayering (requires EM) | • — |
• Arterial intimal fibrosis of new onset, excluding other causes; leukocytes within the sclerotic intima favor cAMR if there is no prior history of TCMR, but are not required | • — |
2. Identical to criterion 2 for aAMR, above | 2. Identical to criterion 2 for aAMR, above |
3. Identical to criterion 3 for aAMR, above, including strong recommendation for DSA testing whenever criteria 1 and 2 are met | 3. DSA positivity (anti-HLA antibodies) |
|
|
1. Morphologic evidence of chronic tissue injury, including ≥1 of the following | 1. Morphologic evidence of chronic tissue injury |
• Transplant glomerulopathy (cg > 0) if no evidence of cTMA or chronic recurrent/ |
• Cg ≥ 1 according to Banff 2011 (≥10% of the glomerular capillary walls in the most severely affected glomerulus involved) if no evidence of TMA of any other cause or recurrent or |
• Severe PTCML (requires EM) | • — |
2. Absence of evidence of current/recent antibody interaction with the endothelium (criterion 2 for active AMR, above) | 2. Absence of evidence of current/recent antibody interaction with the endothelium (criterion 2 for aAMR, above) |
3. Prior documented diagnosis of a or caAMR or documented prior evidence of DSA | 3. Prior documented diagnosis of a or caAMR or documented prior evidence of DSA |
a, acute/active; AMR, antibody-mediated rejection; ca, chronic active;
• Evidence relating to the relationship between AMR and outcomes is largely based on retrospective analyses that do not utilize the strict, most recent Banff categories of AMR, but instead investigate individual features of AMR, combinations of individual features of AMR, or combined Banff categories (such as combining aAMR and caAMR). ○ Strongest evidence for associations between individual features and impaired graft outcome is noted for MVI score ≥2 (if borderline changes, aTCMR or infection are present, g + ptc>1 is not sufficient and g > 1 is required) and cg>10% (>10% of the most severely affected glomerulus). ○ Together with presence of HLA-DSA, these parameters should be the basis for AMR endpoints, acknowledging their limitations (lack of specificity, between-study heterogeneity in definitions used, and high interobserver variability). • Based on evidence for association between individual features of AMR and outcome, AMR diagnosed in indication or protocol biopsies should be considered as a primary endpoint in clinical trials for kidney transplantation. • However, based on available evidence, we suggest refinement of the Banff 2017 definition for AMR diagnosis to the following three AMR-related endpoints: ○ Restricted aAMR, defined by the conservative threshold of at least moderate MVI (g + ptc≥2 with g ≥ 1 in the presence of aTCMR, caTCMR, or borderline changes) and DSA positivity (anti-HLA antibodies) with or without C4d positivity (C4d ≥ 1 on paraffin tissue or ≥2 on frozen tissue). ○ Restricted caAMR, defined by the conservative threshold of cg ≥ 1 according to Banff 2011 (≥10% of the glomerular capillary walls in the most severely affected glomerulus involved) plus at least moderate MVI (g + ptc≥2 with g ≥ 1 in the presence of aTCMR, caTCMR, or borderline changes) and DSA positivity (anti-HLA antibodies) with or without C4d positivity (C4d ≥ 1 on paraffin-embedded tissue; ≥2 on frozen tissue). ○ Restricted cAMR, defined by the conservative threshold of cg ≥ 1 according to Banff 2011 (≥10% of glomerular capillary walls in most severely affected glomerulus involved) and current or past DSA positivity (anti-HLA antibodies) with or without C4d positivity (C4d ≥ 1 on paraffin-embedded tissue; ≥2 on frozen tissue). • Other features of AMR used in Banff AMR definitions (ATI in the absence of any other cause; TMA; Banff Lesion Score v ≥ 1; increased transcripts associated with AMR; cg<10%; PTCML; arterial intimal fibrosis of new onset; DSA− cases) show less-robust evidence than MVI score ≥2 and cg>10%. ○ In isolation, without the other features of AMR described above, these features should not be considered as efficacy endpoints for clinical trials. • The use of histology as endpoint for studies after kidney transplantation needs to consider that histological scoring reproducibility is at best moderate. • There is a clear need for additional investigations of outcomes for all features and all categories of AMR. ○ Any such studies should follow the Banff 2017 recommendations on best practice for pathology endpoints in clinical trials (
• The CHMP recognized the issues in defining AMR. • The CHMP welcomed and endorsed the suggestion to initiate a discussion on the use of the Banff classification as a tool to define AMR as an endpoint in clinical trials, in addition to its diagnostic and research value. • The rationale behind the restricted definitions of aAMR and caAMR for use as primary endpoints was well received by the CHMP. ○ For this to happen, evidence-based classification and state-of-the-art, transparent, and standardized review processes of scientific data are required to demonstrate the usefulness of the restricted Banff definitions for AMR.
This article is one of several papers developed from the Broad Scientific Advice request, submitted to the EMA/CHMP by ESOT in 2020: interactions between the EMA and ESOT regarding this request began in 2016. For the present article, through discussions, working groups on histological and functional endpoints in kidney transplantation developed the ESOT position on the core question ‘Does CHMP agree with the updated definitions of rejection and their potential use as primary endpoints in studies of kidney transplantation?’. The Center for Evidence in Transplantation provided support with data extraction requests: these literature searches formed the basis of evidence used in the advice request and the present article. Input into the working groups’ outputs was provided from all ESOT members involved in the advice request process.
The present article was adapted by MN from the final Broad Scientific Advice request submission (June 2020), documentation from the meeting between ESOT and the CHMP Scientific Advice Working Party (SAWP) (September 2020), and the final response from the SAWP (December 2020). The first draft of the article was developed by JUB and CR and reviewed by DS and MR by e-mail. The article was finalized and approved by all co-authors before submission.
This initiative was supported by the European Society for Organ Transplantation.
CR is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. CR’s research activity is made possible with generous support from Sidharth and Indira Burman. JUB consults for Sanofi. MR has received lecture fees from Astellas and Chiesi; and research grant support (paid to institution) from Astellas and Chiesi for investigator-initiated studies. GB has received honoraria and/or research funding from Astellas, CareDx, CSL Behring, Fresenius, Hansa, Neovii, and Vitaeris. KB has received honoraria and/or research funding from Alexion, Astellas, Bristol Myers Squibb, Chiesi, Fresenius, Hansa, Hexal, Merck, Novartis, Otsuka, Pfizer, Roche, Sandoz, Siemens, and Veloxis. DG reports consultancy agreements and scientific boards for AstraZeneca, BMS, Hansa, and Sanofi. LH reports speaker fees from Astellas, consultancy and research support from Chiesi, consultancy for Novartis, and research support from Sandoz. RO has received grants/research support from Amgen, Astellas, and Chiesi; and speakers’ bureaux/honoraria from Amgen, Astellas, Chiesi, Hansa, Neovii, Novartis, and Teva. SS has received grants/research support from Bridge to Life, Chiesi, Neovii, Novartis, Organ Recovery Systems, and Sandoz; speakers’ bureaux/honoraria from Astellas, BMS, Chiesi, Novartis, OrganOx, and Sanofi; and consulting fees from Astellas, Atara, Merck, NefroHealth, Novartis, Sandoz, and Teva.
The remaining authors declare that the work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the experts involved with the European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP) who participated in the Broad Scientific Advice request. The published information is based on EMA feedback received during the Broad Scientific Advice request. EMA/CHMP have not been involved in the drafting or review of the manuscript to be published. This publication does not constitute a formal EMA/CHMP endorsement of the manuscript. Medical writing support was provided by Linda Edmondson, independent medical writer, funded by ESOT.