Before the Banff schema was introduced, the histology of very early renal transplant biopsies in patients with normal function treated with azathioprine (AZA) and cyclosporine (CsA) was reported.1-3 Burdick et al1 reported interstitial cellular infiltrate at 1 and 4 weeks posttransplant in 4 renal transplant patients with normal function, in whom “good” graft function was maintained for more than a year. An additional observation in this study was that arteritis was always associated with clinical rejection.1 In the second study, d’Ardenne et al2 biopsied patients receiving either CsA or AZA and prednisone at 7, 21, 90, and 365 days. Diffuse interstitial infiltrates were found in >50% of both CsA- and AZA-treated patients at 7 days and about 20% at 90 days. In many of these biopsies, there were no clinical criteria for rejection. Patients with persistent diffuse infiltrates developed interstitial fibrosis in later biopsies and some lost their grafts.2 Neild et al3 biopsied 60 patients on CsA and prednisolone at 1 week, 1 month, and 1 year as well as for episodes of graft dysfunction. Rejection, responsive to methylprednisolone, was found in 35 biopsies and was characterized by interstitial infiltrates and/or arteritis; CsA toxicity, responsive to a lowering of the drug dose, was seen in 30 biopsies, of which 24 had arteriolar hyalinosis and 18 had infiltrates, while in 20 biopsies in patients with stable function, there were infiltrates and arteriolar hyalinosis in approximately half of the biopsies, 1 of which had arteritis.3 Notably, in none of the above studies is there a clear description of lymphocytic tubulitis, now recognized as the most specific lesion in acute T-cell–mediated rejection (TCMR).
The standardization of renal allograft histopathology began in 1991 as a result of the first Banff conference. The initial focus of Banff was to establish the diagnostic criteria for tubulo-interstitial (“cellular”) rejection, which were proposed in the first Banff publication in 1993.4 The merging of acute lesion scoring from the Banff schema with that of the National Institutes of Health Cooperative Clinical Trials in Transplantation (CCTT) system5 was published in 19996 and remains the standard for the diagnosis of TCMR. The histological threshold for Banff grade A TCMR was arbitrarily set at “i2t2”: a mononuclear cell infiltrate present in at least 25% of nonscarred parenchyma and >4 mononuclear cells within the tubular basement membrane of nonatrophic tubules. Infiltrates below this threshold were referred to as “borderline” or “suspicious for rejection.” Notably, the CCTT classification was much less stringent than Banff, requiring only infiltrates in >5% of the parenchyma and tubulitis in 3 tubules in 10 serial high-power fields for the diagnosis of (type I) rejection5 and did not have a “borderline” category. It is also important to note that the degrees of renal dysfunction associated with the histological criteria in Banff were never specified, whereas in CCTT, the threshold for graft dysfunction was arbitrarily set at a rise in serum creatinine of 20% above the baseline, although the criteria for rejection were equally valid at increases in creatinine of 5%, 10%, and 30%. In fact, patients without clinical criteria for rejection had biopsy criteria for rejection by CCTT criteria in 28% of cases,5 as was the case in 38% of patients in a study using Banff criteria.7
Transplant renal biopsies are usually obtained to establish a diagnosis in a patient with graft dysfunction. However, starting in 1990, our center performed “routine” biopsies at 1, 2, and 3 months after renal transplantation, because a patient with stable graft function was found to have severe interstitial fibrosis and tubular atrophy (IFTA) in a late “for cause” biopsy prior to graft loss. Our initial paper (submitted before the Banff criteria existed) was rejected, but was subsequently accepted after the first Banff paper had been published.4 In our original publication, in 29 biopsies of stable patients, 3 biopsies were “borderline” rejections, 5 Banff 1A, and 1 Banff 2A. The term “subclinical” referred to our stringent criteria of the requirements of a stable creatinine with a change of <10% in the 2 weeks prior to the biopsy and no change in immunosuppression. In the article, we speculated that “subclinical acute rejection” (SAR) might be pathogenic and that the chronic lesions attributed to CsA toxicity may be due to persistent subclinical inflammation and noted that the infiltrates persisted in many cases despite treatment.8
The use of protocol biopsies and finding of SAR were also reported subsequently by Legendre et al9 in France and Serón et al10 in Spain.
We later conducted a randomized study in patients treated with CsA, AZA, and prednisone, in which we demonstrated that patients randomized to protocol biopsies at 1, 2, and 3 months and treatment of SAR (Banff 1A or greater) had fewer early and late rejections, less IFTA at 6 months, and better graft function at 24 months than those randomized to biopsies at 6 months only.11 In 2007, a Canadian multicenter, randomized study assessed the prevalence of SAR in patients treated with tacrolimus (TAC), mycophenolate mofetil (MMF), and prednisone. Patients had implantation biopsies and were then randomized to a biopsy arm with protocol biopsies at 1, 2, 3, 6, and 24 months, or a control arm with biopsies at 6 and 24 months only. The overall prevalence of Banff 1A histology between 1 and 6 months in the biopsy arm patients was only 4.9%, with a maximum prevalence of 9% at 6 months and was 6% at 6 months in the controls. It was concluded that there was no benefit to the procurement of early protocol biopsies in patients on TAC and MMF. Notably however, the protocol ignored the prevalence of borderline rejection in protocol biopsies, which in retrospect, is a weakness of the study, although borderline rejection was treated if associated with graft dysfunction.12 However, in 2008 a randomized study was done in recipients of living donor kidney grafts receiving predominantly CsA microemulsion, MMF, and prednisone. Patients were randomized to protocol biopsies at 1 and 3 months or to no biopsy. SAR (Banff 1A) was present in 17.3% of patients at 1 month and in 12% at 3 months and was treated with corticosteroids. The serum creatinine levels at 6 months and 1 year were significantly better (by ~30 µmol/L) in the biopsied group, confirming that early protocol biopsies were beneficial in patients treated with CsA.13
Four subsequent randomized studies with protocol biopsies were done after corticosteroid withdrawal. The first study was published in 2008 by Anil Kumar et al.14 In this single-center study, 200 patients were randomized to 4 different immunosuppressive protocols: CsA plus either MMF or sirolimus (SRL) or TAC plus either MMF or SRL. Patients received anti-IL2R antibody and 2 doses of intravenous (IV) steroids after surgery, after which time steroids were discontinued. Protocol biopsies were done at 1 and 6 months, and then yearly for up to 5 years. Clinical rejections and SAR were treated with 4 days of IV steroids. After 5 years, clinical rejections, SAR, and IFTA were more frequent in the patients receiving MMF. Notably, patient survival was about 80% and graft survival about 60% for each of the 4 patient groups.14 In another study, Heilman et al15 reported on 256 patients that received either anti-IL2R, or depleting antibodies, TAC and MMF with steroid withdrawal after 4 days. Patients were biopsied at either 1 or 4 months and were found to have no inflammation (n = 172), borderline rejection (n = 50), SAR (n = 19), or clinical rejection (n = 15). Patients with no inflammation or borderline rejection remained steroid free, patients with SAR were given 1 dose of 500 mg IV Solumedrol and no further steroids, and patients with clinical rejection received 3 doses of IV Solumedrol and remained on steroids. At 1 year IFTA and IFTA + inflammation (IFTA + i) were more prevalent in the borderline SAR patients.15 In the third study, Rostaing et al16 reported on 153 patients on CsA, mycophenolate sodium (MPS), and prednisone (discontinued after 8 days) that were randomized at 3 months to switch to everolimus (EVR) or remain on CsA until 12 months; patients were further categorized by their perceived risk of developing IFTA, as determined by the presence or absence of histological changes of epithelial-mesenchymal transition. Those patients randomized to EVR had an increased incidence of clinical rejections, SAR, and de novo donor-specific antibody (dnDSA) than those that remained on CsA.16 In the fourth study, Gatault et al17 conducted a multicenter randomized study in which patients that received anti-IL2R, extended-release TAC (TACER), MMF, and prednisone (discontinued after 10 wk) were randomized at 4 months to continuation of standard doses of TACER or a reduction in TACER dose of 50%. This resulted in TAC levels of 6.0 ± 3.7 and 4.3 ± 2.7 μg/L at 12 months, in the standard versus reduced TACER dose groups, respectively. Clinical rejections, SAR, and borderline inflammation in a 12-month protocol biopsy and dnDSA (6 vs 0) were more frequent in patients on reduced TACER.17
More recently, an ongoing multicenter Canadian study was reported in which 281 renal transplant patients were randomized to high- or low-dose TACER with a further randomization to angiotensin-converting enzyme (ACE) inhibitors (or angiotensin II receptor blockers) versus other antihypertensives. Biopsies were obtained at implantation and 6 and 24 months. The addition of renin-angiotensin system (RAS) blockade to low-dose TACER resulted in a decreased incidence of acute rejection, inflammation on protocol biopsy, and less IFTA, IFTA progression, and IFTA + i, compared to the low TACER group without RAS blockade. This study will continue for another 3 years.18
A summary of the randomized studies discussed is shown in Table 1.
The results of the randomized studies shown above illustrate 2 important points. First, that the adequacy of an immunosuppressive regimen can be determined by the frequency by which such intermediate outcomes as graft inflammation (clinical or subclinical), graft functional deterioration, the development of chronic histological changes, and the development of DSAs, as a surrogate for later graft loss occur. Second, they allow for the possibility to avoid one or more of these intermediate outcomes by the timely adjustment of the immunosuppressive regimen in an attempt to prevent or at least delay late graft outcomes. New assays of pretransplant immunological risk assessment and noninvasive tests for monitoring allograft inflammation and fibrosis (discussed later in this article) may eventually replace the protocol biopsy, but in our view, we have gained valuable knowledge from their use.
RISK FACTORS FOR SAR
Class II Human Leukocyte Antigen (HLA) Matching
The association of SAR with HLA-DR mismatching has been reported many years ago. Our group showed that in the first 3 months posttransplant, in recipients of deceased donor grafts treated predominantly with CsA, the prevalence of SAR was 0%–20%, 30%–32%, and 30%–63% of patients with 0 DR, 1 DR, and 2 mismatches, respectively.19 A similar relationship between DR antigen mismatches and early subclinical tubulitis (23% in 0 and 31%–46% in <0 mismatches) was reported from Pittsburgh in patients with kidney or kidney-pancreas transplants receiving TAC and biopsied around 7 days posttransplant.20 Similarly, Choi et al21 reported the prevalence of SAR in protocol biopsies performed at 14 days posttransplant to be 2.7%, 15.4%, and 20% in 0, 1, and 2 DR mismatched recipients of living-donor transplants receiving mostly TAC.21
HLA-DQ mismatching has also been associated with cellular rejection in kidney transplants. Lim et al22 reported rejection rates of 15% in 0 DQ and 25% in 1–2 DQ mismatched in 778 recipients from the Australia and New Zealand Dialysis and Transplant Registry.22 In a more recent and larger study in over 90 000 patients using the United Network Organ Sharing database, Leeaphorn et al23 reported rejection rates of 7% in 0 DQ mismatched patients, whereas it was 11% in those with 1 or 2 DQ mismatches.23 The authors are unaware of any study that has reported a correlation of HLA-DQ mismatching and SAR independent of DR mismatches.
In 2001, our group described SAR in 3 patients with AHG-CDC negative crossmatches and retrospective positive flow cytometry crossmatches in the first 3 months posttransplant.24 More recently, Kraus et al25 reported SAR in the first 2 months in 40% of 50 positive crossmatch recipients of kidneys from living donors. These biopsies showed Banff 1A or greater histology, often in the absence of C4d deposits and circulating DSA.25 In contrast, at the time of first detection of dnDSA in stable kidney transplant recipients, our group found SAR in only 4% of patients (unpublished observations).
Baseline Immunosuppression, Conversion, and Minimization
SAR is less prevalent in immunosuppressive protocols that use TAC over CsA. For example, at 3 months the prevalence of SAR was 8% in the Canadian study that used no induction12 and 2.6% in the Mayo Clinic study, where two-thirds of patients received induction.26 In contrast, at 3 months, the prevalence of SAR was about 30% in CsA microemulsion and MMF-treated patients.27 Conversion from calcineurin inhibitors to mammalian target of rapamycin (mTOR) agents has usually resulted in an increase in the prevalence of SAR. Thierry et al28 reported on 121 patients on CsA, MMF, and prednisone randomized to conversion to SRL or to remain on CsA at 3 months post transplant. At 12 months, protocol biopsy showed a 45% prevalence of SAR in the SRL arm and 15% SAR in the CsA arm.28 Similarly, de Sandes-Freitas et al29 reported on 110 patients treated with anti-IL2R, TAC, MPS, and prednisone, 55 of whom were converted to SRL at 3 months. At 24 months, SAR was present in 14.6% of the SRL and 4.4% of the TAC patients and dnDSA was present in 17.3% and 7.3% of the SRL and TAC patients, respectively.29 An increased prevalence of SAR in patients switched to EVR from CsA has been alluded to previously.16
CELL PHENOTYPES IN SUBCLINICAL REJECTION
The phenotype of infiltrating cells in biopsies with normal histology, IFTA, SAR, and SAR plus IFTA, was compared by Moreso et al.30 The principal findings of this study were that CD3, CD45, and CD68 cells were increased in SAR with or without IFTA compared to the other groups and that in the SAR plus IFTA group there was an increase in CD20 cells over SAR alone, that was associated with a greater risk of graft loss.30 Of special interest is the infiltration of regulatory FoxP3+ cells, which although present in high numbers in biopsies with clinical rejection and worse graft survival,31 appear to predominate over other T-cell phenotypes in borderline rejection or SAR that have better graft survival.32-34 Our group reported that monocyte activation was characteristic of clinical rejection but not SAR,35 a finding which was subsequently confirmed by Girlanda et al.36 More recently, Toki et al37 reported the presence of M2 macrophages (that coexpress CD68 and CD206) in protocol biopsies to be associated with the onset of interstitial fibrosis,37 as have Wang et al,38 in biopsies obtained for graft dysfunction.
GENE TRANSCRIPTS IN SAR
Using reverse transcriptase–polymerase chain reaction (RT-PCR), our group showed that T-effector cell and cytokine transcripts (intermediate between borderline and clinical rejection biopsies) were found in SAR.39 A more comprehensive analysis of 72 RT-PCR transcripts was reported by Hoffmann et al,40 who showed that the transcripts in SAR were qualitatively similar, but expressed to a lesser degree than in clinical rejection. Notably, the effector T-cell transcripts for FASL and T-bet and that of CD152 were increased by one log or greater in clinical rejection.40 The transcriptome has also been investigated with RT-PCR in blood and with high-throughput microarrays in renal tissue by several groups.41-44 In the k-SORT study a 17 gene-set obtained by RT-PCR from blood in over 500 renal transplant patients was reported to predict the development of acute rejection up to 3 months prior to biopsy.41 A subsequent study combining the k-SORT gene biomarker and the interferon (IFN)-γ enzyme-linked immune absorbent spot assay (ELISPOT) was reported to increase the predictive probability for the diagnoses of SAR and subclinical antibody-mediated rejection over that of either test alone.42 The Edmonton group reported that a combination of 30 genes from “for cause biopsies” in renal transplant patients was more accurate than histological features to predict allograft outcome.43 The predictive genes were associated with tissue injury and remodeling, and with dedifferentiation of the epithelium, but not with IFN-γ effects or inflammatory cell infiltration. This finding has also been recently reported in a National Institutes of Health-sponsored study using 3- and 12-month protocol biopsies, in which 13 genes present in the 3-month biopsy predicted the development of graft fibrosis at 12 months. Moreover, the results of the earlier Edmonton study were validated in this work.44
SAR AND THE DEVELOPMENT OF dnDSA
Our group reported that in patients that developed dnDSA there was a trend toward more SAR in early protocol biopsies,45 and associations between SAR and dnDSA have been reported subsequently by Moreso et al46 and Chemonie et al.47
BANFF “BORDERLINE” INFLAMMATION: A REAPPRAISAL OF ITS SIGNIFICANCE
Borderline inflammation is the most common Banff category of inflammation in the modern era in both clinically indicated and protocol biopsy specimens.48 There are however inconsistencies in the degree of tubular and the extent of interstitial inflammation in the current definitions of “borderline” rejection,49 and there has been a suggestion of eliminating the category altogether.50 However, the authors believe that the category of borderline rejection is important (see below) and should be maintained while we await the results of an ongoing multicenter Banff working group on TCMR.
In 1995, our group reported on 25 patients on CsA, AZA, and prednisone that had protocol biopsies at 1 month and 2, 3, 6, and 12 months. Clinical rejections and SAR were treated with high-dose steroids, but borderline was not. Normal histology and function at 12 months was present in 50% of patients and borderline inflammation was associated with worse histology and function than in the normal group.51 In 2003, a landmark study of serial protocol biopsies performed in 120 patients, all but one of whom had a kidney-pancreas transplant, was published by Nankivell et al.52 Biopsies were performed at the time of or shortly after transplant, at 1 week, 1 month, 3 months, 6 months, 12 months, and almost yearly for up to 10 years. Most patients received CsA and AZA, but later TAC and MMF were introduced. At 3 months, the prevalence of SAR was 30%, and mean scores for interstitial fibrosis increased from 0.7 ± 0.53 at 3 months to 1.3 ± 0.67 at 2 years, more often in patients with previous SAR or borderline rejection. In 2005, the Mayo Clinic group in Rochester reported that stable kidney transplant recipients that had inflammation and fibrosis in a 1-year protocol biopsy had a greater decline in kidney function compared to those patients with fibrosis alone. Notably, the degree of inflammation was below the threshold for Banff borderline rejection.53 The finding of a worse outcome in patients with mostly borderline inflammation and fibrosis was confirmed by Moreso et al.54 More recently, Ortiz et al55 reported a retrospective analysis of 948 protocol biopsies, obtained between 3- and 7-months posttransplant, in renal transplant patients followed for up to 15 years. Patients were mostly on CsA, MMF, and prednisone, and 22% had received induction. The biopsies had normal histology in about 53%, IFTA in 30%, “borderline” in 12%, and SAR in 5%. Approximately one-third of the normal and half of the IFTA biopsies had some infiltrates in nonatrophic parenchyma. A decrease in graft survival at 15 years was observed in those patients with inflammation scores <0. Moreover, if the biopsies showing normal histology or IFTA were split into those with and without inflammation, there was a significant decrease in graft survival within those subgroups in patients that had inflammation.55
Infiltrates below the Banff threshold for rejection have also been associated with the development of dnDSA. El Ters et al56 reported 797 patients with a 1-year protocol biopsy, 15% of whom had previous rejection (50% of which were SAR) in the first-year. Pathology at 1 year included 3 lesions, severe interstitial fibrosis, fibrosis plus inflammation, and transplant glomerulopathy, all of which occurred more frequently in patients with previous acute rejection, and were associated with decreased graft survival. Moreover, patients that had rejection in the first year had double the incidence of dnDSA, compared to those that had not.56 In addition, García-Carro et al57 have reported that subclinical inflammation alone or IFTA + i in protocol biopsies obtained at 6 weeks are associated with an increased risk of dnDSA at 1 year.57 More recently, Mehta et al58 reported on 200 patients that received induction, TAC, and MMF that had protocol biopsies at 3 and 12 months. Corticosteroids were withdrawn after 7 days. There were 129 patients with and 71 patients without inflammation on the 3-month protocol biopsy. Those patients with inflammation developed more clinical rejections, and more IFTA at 12 months. In addition, dnDSA. at 12 months (9% vs 1%), and decreased renal function, and graft losses at 24 months were more common in the patients with inflammation at 3 months.58 Lastly, Nankivell et al59 have recently presented compelling evidence that patients with borderline histology, particularly if associated with graft dysfunction, can develop progressive IFTA, graft dysfunction and loss, as well as develop dnDSA.59
THE I-IFTA LESION
Nankivell et al60 described the time-dependent changes in the tubulo-interstitial compartment in the first 3 months posttransplant highlighting the rapid rate of fibrosis associated with subclinical inflammation, in which the scarred and atrophic areas were also infiltrated by mononuclear cells,60 a finding incorporated into the Banff schema in its most recent iteration.61 The comments on this lesion are discussed here because i-IFTA can be associated with borderline inflammation in nonscarred parenchyma. The poor prognosis conferred by inflammation in areas of scarring was noted by Mengel et al62 and confirmed by Mannon et al,63 Nankivell et al,64 and Lefaucheur et al.65 Inflammation in areas of scarring is now called i-IFTA by Banff61 and is felt to be due to chronic TCMR.61,64,65 However, Halloran et al66 have recently reported that the i-IFTA lesion is associated with acute kidney injury transcripts and is seen more commonly with antibody-mediated rejection than TCMR.66
THE END OF THE BEGINNING
The decrease in clinical rejection episodes in renal transplant patients has not resulted in a commensurate reduction in late graft losses, which are primarily due to alloimmunity.67-69 Moreover, subclinical alloimmunity, the result of inadequate immunosuppression, can lead to IFTA, dnDSA, graft dysfunction, and loss, as reviewed above. Clearly, therefore, to improve outcomes in renal transplantation, the risk of alloimmunity overall, and that of subclinical alloimmunity in particular, has to be defined, preferably at the individual, more so than at the population level (“precision” or “personalized” medicine).
The Sensitization in Transplantation: Assessment of Risk (“STAR”) initiative is a collaborative effort between the American Society of Histocompatibility and Immunogenetics and the American Society of Transplantation who were tasked with providing recommendations for the assessment of risk for organ transplants.70 This is indeed timely, as it is now possible to evaluate more precisely both the preexisting donor-specific B- and T-cell alloimmunity by using the IFN-γ-ELISPOT assay71,72 and the risk of primary immune responses by assessing donor and recipient HLA Class II eplet mismatches73 pretransplant. In addition, posttransplant monitoring for subclinical allograft inflammation can now be performed noninvasively by measuring urine chemokine levels74,75 and, most recently, a promising blood-based molecular biomarker has been reported in renal transplant patients that appears to identify with good negative predictive value the diagnosis of SAR, and that is positively correlated with a composite of renal function, rejection, and severe fibrosis, as well as the development of dnDSA.76 It is hoped that the clinical validation of these new tests may facilitate the transition from empiricism to personalized medicine.77
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