Background
Kidney transplantation (KT) remains associated with suboptimal 5- and 10-year graft survival (77% and 56%, respectively [
1]), despite an excellent 1-year allograft survival (91%). Graft failure is primarily associated with antibody mediated rejection (ABMR) [
2‐
4].
Donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) drive ABMR. Development of the solid phase assay (SPA) to detect DSA was significant in the diagnosis of DSA [
5]. SPA can detect DSA at the time of transplantation even with a negative flow cytometry crossmatch. DSA is divided into two categories: preformed DSA and
de novo DSA (
dnDSA). Preformed DSA may be associated with hyperacute rejection or ABMR in the weeks following transplantation. The presence of preformed DSA is associated with graft failure [
6‐
8].
DSA development after KT is known as
dnDSA. A previous review [
9] noted that
dnDSA primarily occurred during immunosuppression [
10] in the context of poor adherence [
4,
11,
12]. Early calcineurin inhibitor minimization may also be associated with
dnDSA development [
9]. Wiebe et al. [
12] reported a trend towards an association with a history of acute rejection as an independent risk factor for
dnDSA.
DnDSA is independently associated with poor long-term allograft outcomes [
13]. SPA technique allows to further detect complement-binding DSA, and these DSAs are associated with reduced graft survival compared to non-complement-binding DSA [
14,
15].
Knowledge of the presence of DSA at the time of transplantation allows implementation of specific therapeutic strategies (e.g., desensitization) to reduce the levels of preformed DSA [
16]. Several studies demonstrated acceptable long-term patient survival compared to wait-listed patients on dialysis [
16,
17]. However, the therapeutic management of
dnDSA remains controversial. Plasma exchange, intravenous immunoglobulin (IVIG), anti-thymoglobulin antibodies (ATG), rituximab, bortezomib and treatment abstention are used in different combinations for the treatment of ABMR [
18]. Data on the long-term efficacy of these regimens and treatment strategies guided by the presence of
dnDSA (independent histology) are lacking.
The present study retrospectively analysed the diagnosis and clinical and histological evolution in patients who developed dnDSA.
Methods
Patient selection and follow up
We retrospectively analysed the stored sera available from all consecutive patients who underwent kidney transplantation in Geneva University Hospitals from 1986 to 2012. Patients with combined transplantation (kidney – liver, kidney – heart or kidney – pancreas) were excluded. The cumulative incidence of dnDSA is 15% in our centre. We identified 22 patients with known dnDSA from the 315 patients transplanted from 1986 to 2012 for whom we had available sera. DnDSA was defined as a DSA that developed within six months after kidney transplantation with a mean fluorescence intensity (MFI) > 500. All 22 patients were transplanted with a negative CDC crossmatch (B and T) and/or FACS crossmatch. Clinical data were recorded during regular follow up at our institution. Graft function was assessed using the estimated glomerular filtration rate (eGFR). We used the CKD-EPI formula for each patient. The eGFR was calculated at the time of biopsy. A patient’s return to haemodialysis defined graft failure. We analysed graft biopsies at the time of dnDSA detection (± 3 months) in these patients and therapy administration according to the histological data. We identified a positive DSA in 2012 and retrospectively evaluated the sera from these patients to identify the first positive serum and matched the biopsy at the time of first identification of the dnDSA. The biopsies matched with the dnDSA were a protocol or indication biopsy because of the retrospective nature of this study.
The ethical committee approved the study (N° 6-208) which is in accordance with the regulations of the Geneva University Hospitals.
Immunosuppression dnDSA/histological-based treatment
Induction therapy evolved over time and consisted of no induction or basiliximab on post-operative days (PODs) 0 and 4. Antithymocyte globulin (ATG) or daclizumab reflected the use of a steroid-free protocol in selected patients. The immunosuppressive regimen consisted of tacrolimus or cyclosporine A, corticosteroids, and mycophenolate mofetil, mycophenolic acid or azathioprine. We reported the identified treatment combination at the time of dnDSA and biopsy as reported in medical files. The reported combinations included high doses of steroids, ATG, anti-CD20 and/or plasma exchange. These treatments were adjusted according to histopathological findings and patient’s age and comorbidities (e.g., cancer, previous infections).
Graft biopsies
Protocol biopsies are performed in our centre at one year post-transplantation, but the present analysis focused on kidney biopsies performed at approximately the time of development of
dnDSA, regardless of kidney function. The clinician may not have known of the presence of
dnDSA at the time of the biopsy. A control biopsy at the last follow up was also included to evaluate the histological changes following treatment of the
dnDSA. The next available graft biopsy was used in patients who were not treated. Criteria from the Banff 2013 classification were used for the retrospective grading of biopsies [
19].
Determination of DSA using Luminex solid-phase assay
Patient sera were analysed for the presence of anti-HLA class I and class II antibodies using solid phase assays on Luminex and the Labscreen Mix assay for HLA class I and HLA class II following the recommendations of the manufacturer (One Lambda, Canoga Park, CA), as previously described [
20]. Sera collected before and after transplantation of all positive individuals were subsequently tested for anti-HLA class I- and class II-specific antibodies using the Luminex single antigen beads (one Lambda). Briefly, colour-coded microspheres coated with major HLA class I and II antigens were incubated with 10 μl serum for 30 min at room temperature in the dark. Samples were washed three times and incubated with 100 μL of 1:100 phycoerythrin-conjugated goat anti-human IgG (One Lambda Inc.). Samples were washed twice, and the fluorescent signal intensity for each microsphere was measured using a LABScan 100 flow analyser (One Lambda Inc.). The cut-off for positive samples was the normalized background (NBG) ratio recommended by the manufacturer, which was performed using HLA Fusion software (One Lambda). An MFI > 500 was considered positive, as recommended by the manufacturer, and clinical relevance was considered at MFI > 1000. Donor-specific antigens were classified as immunogenic HLA or non-immunogenic HLA based on the presence of specific antibodies in the recipient directed towards the donor-specific antigens.
Post-transplantation screening consisted of Luminex solid-phase assays performed at 1 month, 3 months, 6 months, 9 months, 12 months and annually. Each increase in serum creatinine > 25% required a new Luminex solid-phase assay.
Statistical analysis
Statistical analyses were performed using STATA v. 14.0 (College Station, TX). We used descriptive statistics to estimate the frequencies (%) and means (±SD) of study variables. Comparisons of two means were performed using t tests, and comparisons of the frequencies between groups were evaluated using Fisher’s exact test because of the small sample size. We did not perform further analyses because of the retrospective nature of the descriptive data.
Discussion
Our retrospective analysis reports clinical and histological data from a single centre cohort of kidney transplant recipients who developed dnDSA. No improvement in histopathological parameters was observed with any therapeutic intervention. We observed a loss of kidney function and increase in cg score ≥ 1 at follow up. Graft survival was similar in patients who received treatment at the time of dnDSA and patients who did not receive therapy.
Wiebe et al. [
12] analysed the clinical and histopathological correlations with
dnDSA in 315 consecutive renal transplants without pre-transplant DSA. The mean follow-up was shorter (6.2 ± 2.9 years). Forty-seven of the 315 (15%) patients developed
dnDSA 4.6 years post-transplant, which is earlier than our cases. They found that the two independent predictors of
dnDSA were a mismatch for HLA-DRβ1 > 0 (OR 5.66,
p < 0.006) and non-adherence (OR 8.75,
p < 0.001). The authors demonstrated that the median 10-year graft survival for patients with
dnDSA was lower than the no
dnDSA group (57% vs. 96%,
p < 0.0001). Their study found a non-significant trend with an odds ratio of 1.57 per rejection episode prior to an occurrence of
dnDSA (
p = 0.061). A high prevalence of TCMR episodes preceded the development of
dnDSA (36.3%) in our study. Less than 20% of the 22 patients who developed
dnDSA exhibited graft dysfunction at the time of diagnosis, which is consistent with Yamamoto et al., who reported a prevalence of 40% of patients with
dnDSA and biopsy-proven subclinical ABMR [
21]. Histological features at the time of
dnDSA identification were not broadly consistent with ABMR in our study, and less than 50% of the biopsies exhibited an MVI score ≥ 2. C4d was negative in 59.1% of our baseline biopsies, which is consistent with previous reports [
22,
23].
Further analyses of the incidence and impact of
dnDSA in renal transplant recipients were performed. Forty-seven of the 189 consecutive non-sensitized, non-HLA-identical patients who received a kidney transplant between March 1999 and March 2006 (25%) developed
dnDSA within 10 years [
24]. The cumulative incidence 5 years post-transplantation was 20%, and more than half of these patients developed
dnDSA in the first post-transplantation year. Patients of a younger age (< 35 years old), deceased donor, the presence of HLA antibodies (non-DSA) and DQ mismatch (as previously described [
25]) were most likely to develop
dnDSA in our study.
One strength of our analysis is the focus on the evolution of histological parameters in patients with
dnDSA. Focusing on cases with negative crossmatch and DSA identified 6 months post-KT avoided the analysis of patients with preformed DSA. Our data evaluated the effect of each component of treatment on the evolution of the primary features of the Banff score associated with subtypes of ABMR [
26]. Halloran et al. performed a recent principal component analysis of 164 indication biopsies [
26] and found 3 main ABMR phenotypes: early ABMR with MVI lesions only (called pgABMR), late ABMR with cg lesions (cgABMR) and mixed feature (pgcgABMR).
The present study has several limitations. First, it used a retrospective analysis or different therapeutic regimens. However, we provide data on a broad heterogeneous kidney transplant recipient population that reflects routine clinical practice. Second, the small number of patients, without a control non-
dnDSA group, prevents us from drawing any conclusions about the best protocol to treat
dnDSA. We did not evaluate patient adherence, which is a known cause of
dnDSA development and eventual graft failure [
4]. Finally, an obvious pitfall was the non-standardisation of our treatment following
dnDSA and biopsy results. The current evidence is insufficient to create an internationally approved protocol for the treatment of
dnDSA. Our treatment protocol for ABMR evolved over time and reflects changes in practice based on the literature and a tailoring of therapy based on patient age, co-morbidities and cancer risk. Notably, the absence of IVIG as a component of the treatment given at the time of
dnDSA was associated with MVI reduction. Current evidence is conflicting on the utility of IVIG in the treatment of antibody mediated injury. The benefit has been reported for active ABMR but its benefit on late/chronic ABMR is less straight forward [
27].
Evidence supporting the use of rituximab for MVI or cg lesion is also scant [
27,
28]. A recent randomized double-blind, placebo-controlled trial did not demonstrate a significant advantage of rituximab in biopsy-proven ABMR in creatinine level or proteinuria at 12 months [
28]. The decision to treat
dnDSA independently of histological features has not been addressed. Use of an intragraft transcript set measurement, as proposed in the 2013 Banff update [
19], may further help the reclassification of these biopsies, but this methodology is not widely implemented yet [
29].