Survival of the patients included (n = 148) did not differ from that of patients excluded from analysis (n = 222; 100% vs. 97.8%; P = 0.2) at 1 year and at the end of follow-up (median: 3.4 years; 96.6% vs. 91.9%; P = 0.08). Graft survival censored for death with functional graft 1 year after transplantation was significantly lower in patients excluded (excluded: 95.1% vs. included: 100%; P = 0.004), but not significantly different at the end of follow-up (excluded: 90.5% vs. included: 95.9%; P = 0.07). Graft function at the end of follow-up, censored for failed grafts, was also not significantly different between the two groups (median estimated glomerular filtration rate [eGFR] excluded vs. included: 43.06 vs. 45.08 mL/min/1.73 m2; P = 0.7). Graft survival censored for death between groups 1 to 4 as defined in Figure 2, at any point in time was not significantly different.
Two hundred ninety-six biopsies could be evaluated as TSB. One hundred eighty-four (62.2%) revealed significant pathological findings, summarized in Table 2. From 3 to 6 months, the prevalence of SCR was stable (45% vs. 41%; P = 0.2), whereas unspecific lesions doubled in number (44% vs. 22%, P<0.001), which was mainly due to an increase in IF/TA (32% vs. 9%, P<0.001). The prevalence of calcineurin inhibitor (CI) toxicity and polyomavirus nephropathy remained unchanged. Patients with SCR at 3 and 6 months had significantly more often unspecific lesions at 6 months as compared with patients who had no SCR at 3 and 6 months (71% vs. 23%; P<0.001) or to patients with SCR at 3 months only (71% vs. 28%; P = 0.002). Patients with any kind of pathology in both biopsies (group 4) had a significant increase of unspecific lesions from 3 to 6 months (76.6% vs. 40.6%; P<0.001). Unspecific lesions were more common in group 4.2 than in group 4.1 (68% vs. 42%, P = 0.01).
Excluded patients with TSB at 3 months but diagnostic biopsies at 6 months (n = 40) as compared with patients included (n = 148) had significantly fewer biopsies with no pathology at 6 months and had significantly more unspecific lesions at 6 months whereas CI toxicity and SCR were not significantly different between the two groups. There was a significant increase in incidence of chronic lesions (P = 0.007) from 3 to 6 months in the group of excluded patients. The biopsy findings at 3 months did not significantly differ between the group excluded (n = 40) and the group included.
The courses of median delta eGFR from the four groups defined by the severity of morphological findings are shown in Figure 2. When comparing the four groups with each other at the same point in time after transplantation, significant differences could be found at 6 months between group 1 and 2 (P = 0.01), and between group 2 and 3 (P = 0.02); at the end of follow-up between group 2 and 4 (P = 0.03) and between group 3 and 4 (P = 0.01). The courses of median delta eGFR for group 4.1 and 4.2 in comparison with group “1/2/3” (all patients of group 1, 2 and 3 combined) are also shown in Figure 2. A significant difference in median delta eGFR could be found between group 1/2/3 and group 4.2 at the end of follow-up (P = 0.005).
Interventions After TSBs
In 93 of 148 patients (62.8%) the 3-month and/or 6-month biopsy was followed by a therapeutic intervention: 36 (24.3%) at 3 months, 31 (20.9%) at 6 months, and 26 (17.6%) at 3 and 6 months. Patients with pathology in both biopsies (group 4) had significantly more interventions compared to patients with no pathology in both biopsies (group 1) (64.3% vs. 21.4%; P = 0.001). Fifty-five patients (37.2%) never underwent any intervention.
Impact of Maintenance Immunosuppression
Patients were analyzed in three groups according to their main immunosuppressant (cyclosporine A, tacrolimus, and rapamycin). The impact of initial maintenance immunosuppression on the incidence of SCR and the therapeutic consequences can be seen in Table 3. Median delta eGFR after 1 year and at end of follow-up was not significantly different among the three groups. Patients with rapamycin had a significantly higher prevalence of SCR at 6 months and received more often antirejection therapy than patients with cyclosporine or tacrolimus.
Diagnostic Biopsies in the First Year Posttransplant
Along with the 296 TSB, a further 110 diagnostic biopsies were performed in the first year in cases of clinical signs of graft dysfunction. The number of diagnostic biopsies per patient was not significantly different among group 1 to 4 and among the three main immunosuppressants. Thirty-three percent of all diagnostic biopsies were performed within 10 days posttransplant, 50% within 24 days. Diagnostic biopsies revealed Banff type IA/B rejection in five patients in groups 1 to 3 and in none patient in group 4, whereas type IIA/B rejection was observed in six patients in groups 1 to 3 and in 12 patients in group 4. Antibody-mediated rejection was diagnosed in one case in group 1 to 3 and 4, respectively.
Protocol biopsies are assigned to fixed points in time after transplantation irrespective of renal function (7). Usually it is not known or not well described in the literature whether there is graft dysfunction at the time of biopsy. Therefore, it might be that previous studies included patients with protocol biopsies that were actually diagnostic biopsies. This study differs from previous analyzes as only repeat protocol biopsies in the absence of any clinical signs of graft dysfunction were analyzed in patients with an uneventful clinical course. These biopsies were named “true surveillance biopsies” (TSB) to underline their different character. Surprisingly, only 40%, 148 of 370 patients, with repeat protocol biopsies in our population had exclusively TSB after 3 and 6 months. More than one half of protocol biopsies were judged as diagnostic, at the time of biopsy or retrospectively. The correct judgment as a diagnostic biopsy in the context of clinical graft dysfunction is supported by the significant lower graft survival after 1 year of this group as compared with the 100% graft survival in the study group. It could be expected that significant pathologic findings in the biopsies of the selected study group were rare. But more than one half of biopsies at 3 months and two third of biopsies at 6 months revealed relevant pathological changes. The most striking finding was the high number of biopsies showing SCR and the progression of unspecific lesions from 3 to 6 months. Whether SCR persisted from 3 to 6 months or was recurrent at 6 months, cannot be answered with certainty. But the progression of unspecific lesions during this time favors an ongoing process of rejection, at least in a substantial number of patients. The high prevalence of SCR in this study, despite the pure subclinical setting is surprisingly and contradictory to certain previous publications, where it was reported as being low, especially under tacrolimus (8, 9). But it is not unique since a few years ago Nankivell et al. (10) too found SCR in 45.7% of their 3-month biopsies. The difference can be explained in part by the inclusion of borderline rejections that accounted for approximately 20% of all rejection episodes, by different inclusion criteria, and by different points in time of the biopsies. This high rate of SCR in the absence of clinical signs underlines the importance and the significance to perform TSB. SCR were equal often observed in CyA and Tac regimens and highest in the rapamycin group as already shown by others (11). But our study was not designed to show differences between different immunosuppressive regimens so data should not be overinterpreted. Nevertheless, the results show that current immunosuppressive regimens effectively prevent acute clinical rejection, but seem to be insufficient to avoid “smoldering” subclinical inflammation.
SCR, if left untreated, is known to be associated with progression to unspecific lesions (tubular atrophy and interstitial fibrosis) (12, 13), inferior graft survival, and function (14). These unspecific lesions were present in approximately one fifth of all 3-month biopsies and their number had nearly doubled after 6 months, and thus corresponding with the results of Yango et al. (8), who also reported unspecific lesions as being prevalent at 6 months after transplantation (77.5%). The degree was mild in most cases but the findings show that chronic allograft lesions begin early and develop in the absence of clinical events (e.g., clinical acute rejection). Nevertheless, its occurrence was related to sustained or repeated SCR at 3 and 6 months and its prevalence was significantly lower in patients with reversed rejection (i.e., SCR at 3 months but no SCR at 6 months). Patients with TSB at 3 months but with diagnostic biopsy at 6 months (thus being excluded from the study) showed a significant increase in incidence of chronic lesions that led to clinically apparent graft dysfunction already after half a year. These results correspond with previous observations (3, 12, 13, 15) and emphasize the importance of early treatment to prevent irreversible damage. CI toxicity was present in 17% of biopsies after 3 months and therefore, as pointed out in a recent editorial (16), can contribute to chronic graft damage.
The findings in the TSB were relevant for the treating physician and had an impact on patient management despite the pure subclinical situation. Treatment strategy was modified in more than 60% as a consequence of the biopsy results. Best treatment modality of pathological findings in TSB remains unanswered by this observational study, but their occurrence is highly relevant, as graft function significantly declined at the end of follow-up in case of repeat and severe pathology (group 4.2). Therefore, TSB are useful to early identify patients with relevant subclinical pathology, before irreversible graft damage and impairment of graft function occur. On the other hand, they allow identifying patients with no graft pathology. In these cases, the burden of immunosuppression can be reduced more safely and further biopsies can be avoided (17). Decrease of calcineurin inhibitor dosage can further contribute to the prevention of chronic graft damage.
The strength of this study is its setting, as it shows purely subclinical changes over time in repeat TSB. The time of follow-up, about 3 [1/2] years, covered a longer period than many previous analyses (3–5, 8, 18) and allowed to demonstrate a significant impact of subclinical pathologies on long-term graft function. In contrast to many other studies, 16-Gauge core needle biopsies were obtained, thus reducing the risk of sampling errors. However, the study also has its limitations: its observational character and the nonstandardized intervention with regard to the biopsy findings. It was up to the treating nephrologists to decide if and what kind of intervention was applied. In addition biopsies at the time of implantation were not included into analysis, so the impact of preexisting donor factors could not be evaluated. Therefore, further prospective randomized studies are necessary to elucidate the best treatment modality against subclinical findings.
In conclusion, repeat TSBs reveal significant subclinical changes in a high proportion of biopsies. This is mainly SCR, indicating that current immunosuppression prevents clinical rejection in most cases, but is not always sufficient to avoid subclinical inflammation. Repeated or sustained subclinical pathologies within the first 6 months are followed by a significant decline of long-term graft function. Furthermore, it must be emphasized that even the strict inclusion criteria applied in this study did not allow the clinical identification of patients without pathological changes in their biopsies. Therefore, TSB are a useful tool for the early detection of subclinical pathologies with the intention to treat and also for confirming the absence of any pathology in the graft. Yet, we suggest that the time of repeat surveillance biopsy should be adjusted individually, based on the results of the first biopsy, that is, in case of significant pathology followed by intervention or modification of maintenance immunosuppression, a protocol biopsy as reassessment is advisable after 2 months. In case of no pathology or no change in maintenance immunosuppression, a further protocol biopsy may be indicated after 6 to 9 months. The best treatment modality against subclinical findings remains unclear. Further studies will be needed to define the best type of intervention. Finally, the study shows that the term “protocol biopsy” should be better defined. Less than 50% of our patients had protocol biopsies without any evidence of graft dysfunction. We suggest using the term “TSB” in these cases.
MATERIALS AND METHODS
Since 2001 regular protocol biopsies have been performed 3 and 6 months posttransplant in all renal transplant recipients at the University Hospital of Basel with the exception of recipients of fully identical grafts. The present observational study includes all patients who met the following inclusion criteria: (1) a protocol biopsy was available at 3 and 6 months; (2) both protocol biopsies were judged as TSB at the time of biopsy by the treating physician, and (3) retrospectively by a blinded staff nephrologist. Biopsies were classified as “TSBs” if the summary of the following parameters in the clinical context raised no suspicion of graft dysfunction in the judgment of the two independent nephrologists: course of serum creatinine (increase less than 15% of baseline), course of eGFR, course of proteinuria, blood pressure, course of body weight, and blood levels of immunosuppressants. This approach was chosen as it comes closest to daily clinical practice. Patients with additional diagnostic biopsies during the follow-up were included into the analysis. Graft biopsies at the time of implantation (“zero hour biopsies”) were only performed in patients receiving a graft from a deceased donor. As 60% of the analyzed patients had a living donor, zero hour biopsies were excluded from analysis. All baseline data were collected from the patients’ medical charts. Three main immunosuppressants with four different immunosuppressive regimens were applied during the study as listed in detail in Table 1. Patients were classified as having high immunological risk if there was blood group ABO incompatibility with the donor or if there were donor-specific antihuman leukocyte antigen antibodies, detected by luminex technique before transplantation. The study was approved by the local ethic committee on April 2, 2009 (Ref. Nr. EK: 29/09).
Two ultrasound guided biopsy cores were obtained after 3 and 6 months. The larger biopsy core was used for light microscopy, the smaller one for immunofluorescence and electron microscopy, if necessary. Core biopsy needles of 16 Gauge were used, thus reducing the risk of sampling error. The biopsy working up was made by one pathologist (M.J.M.) and followed standard procedures. The morphological lesions were scored according to the Banff 2007 classification (19). C4d staining was done in all biopsies by immunofluorescence (most) or immunohistochemistry (few). All biopsies were stained for SV 40 large T antigen (polyomavirus detection) using immunohistochemistry (20, 21). The rate of biopsy related adverse events was small: pain (2.7%), macrohematuria (2.3%), bladder tamponade (0.7%), hematoma (0.1%), and shock/blood transfusion (0.1%).
A TSB was considered to have significant pathological findings if it fulfilled at least one of the 11 criteria listed in Table 2. Based on the morphological evaluation, patients and biopsies were divided into the following four groups: (1) no pathology at 3 and 6 months; (2) no pathology after 3 months but after 6 months; (3) pathology at 3 months but not after 6 months; and (4) pathology after 3 and 6 months. Group 4 was further divided into two subgroups, 4.1 and 4.2, by an arbitrarily defined score: each morphological lesion was given points (Table 2): borderline lesion (1), T-cell mediated rejection IA (2), T-cell mediated rejection IIA (3), antibody-mediated rejection (4) toxic tubulopathy, arteriolopathy, obsolescent glomeruli more than 10%, arteriolar hyalinosis, IF/TA, polyomavirus nephropathy (each 1). A maximum of 13 points per biopsy could be reached, a total of 26 for both biopsies. On the basis of the total number of points from both biopsies added together (maximum achieved: 13 points), the group was split at the median (4 points) into subgroups 4.1 (less than 4 points for both biopsies) and 4.2 (4 to 13 points for both biopsies).
An intervention was defined as a time-dependent change of therapy in consequence of the findings in the TSB. The interventions were not protocol driven; it was up to the treating nephrologists to decide if and what kind of intervention was applied. The type of intervention after the biopsy was classified as follows: (i) Acute rejection therapy (Table 3); (ii) Modulation of maintenance immunosuppression, that is, adding, discontinuation, or change of dose or trough levels of the maintenance immunosuppressants; (iii) Lowering calcineurin inhibitor levels to treat CI toxicity. Levels of cyclosporine A microemulsion or tacrolimus were lowered if the TSB showed signs of CI toxicity; (iv) Lowering maintenance immunosuppression to treat polyomavirus nephropathy as previously described in detail (22).
eGFR was calculated according to the Modification of Diet in Renal Disease formula (23). Changes in eGFR (delta eGFR) were defined as the difference between two points in time and were calculated between baseline, at 3 months, 6 months, 1 year after transplantation, and at the end of follow-up.
Statistical analyses were performed using the JMP version 8 statistics package (www.jmp.com). All values are reported as medians and ranges. For all analyses, differences were considered statistically significant for P values less than 0.05. Mann-Whitney U test was used to determine significance for numerical data, difference in median delta eGFR. Chi-square test or Fisher’s exact test was used to assess the significance for categorical data: incidence of success/failure in therapy and incidence of additional diagnostic biopsies.
The authors thank the nurses and technicians of the clinic for Transplantation Immunology and Nephrology, and the Institute for Pathology for their great support.
1. Nankivell BJ, Chapman JR. The significance of subclinical rejection
and the value of protocol biopsies. Am J Transplant 2006; 6: 2006.
2. Burdick JF, McGraw D, Bender W, et al.. Renal allograft infiltrate in the absence of rejection. Transplant Proc 1984; 16: 1580.
3. Rush DN, Jeffery JR, Gough J. Sequential protocol biopsies in renal transplant patients: Repeated inflammation is associated with impaired graft function
at 1 year. Transplant Proc 1995; 27: 1017.
4. Rush D, Nickerson P, Gough J, et al.. Beneficial effects of treatment of early subclinical rejection
: A randomized study. J Am Soc Nephrol 1998; 9: 2129.
5. Kurtkoti J, Sakhuja V, Sud K, et al.. The utility of 1- and 3-month protocol biopsies on renal allograft function: A randomized controlled study. Am J Transplant 2008; 8: 317.
6. Choi BS, Shin MJ, Shin SJ, et al.. Clinical significance of an early protocol biopsy
in living-donor renal transplantation: Ten-year experience at a single center. Am J Transplant 2005; 5: 1354.
7. Henderson LK, Nankivell BJ, Chapman JR. Surveillance protocol kidney transplant biopsies: Their evolving role in clinical practice. Am J Transplant 2011; 11: 1570.
8. Yango A, Gohh R, Wang LJ, et al.. The utility of 6-month protocol renal biopsy under modern immunosuppression. Clin Nephrol 2008; 70: 490.
9. Rush D. Can protocol biopsy
better inform our choices in renal transplantation? Transplant Proc 2009; 41 (6 suppl): S6.
10. Nankivell BJ, Borrows RJ, Fung CL, et al.. The natural history of chronic allograft nephropathy. N Engl J Med 2003; 349: 2326.
11. Thierry A, Thervet E, Vuiblet V, et al.. Long-term impact of subclinical inflammation diagnosed by protocol biopsy
one year after renal transplantation. Am J Transplant 2011; 11: 2153.
12. Rush D, Somorjai R, Deslauriers R, et al.. Subclinical rejection
—A potential surrogate marker for chronic rejection—May be diagnosed by protocol biopsy
or urine spectroscopy. Ann Transplant 2000; 5: 44.
13. Masin-Spasovska J, Spasovski G, Polenakovic M, et al.. Chronic allograft nephropathy (CAN) in early renal protocol biopsies: Does treatment of borderline and subclinical acute rejections prevent development and progression of CAN? Prilozi 2005; 26: 91.
14. Seron D, Moreso F. Protocol biopsies in renal transplantation: Prognostic value of structural monitoring. Kidney Int 2007; 72: 690.
15. Mengel M, Chapman JR, Cosio FG, et al.. Protocol biopsies in renal transplantation: Insights into patient management and pathogenesis. Am J Transplant 2007; 7: 512.
16. Chapman JR. Chronic calcineurin inhibitor nephrotoxicity—Lest we forget. Am J Transplant 2011; 11: 693.
17. Rush D. Protocol biopsies for renal transplantation. Saudi J Kidney Dis Transpl 2010; 21: 1.
18. Rush D, Arlen D, Boucher A, et al.. Lack of benefit of early protocol biopsies in renal transplant patients receiving TAC and MMF: A randomized study. Am J Transplant 2007; 7: 2538.
19. Solez K, Colvin RB, Racusen LC, et al.. Banff 07 classification of renal allograft pathology: Updates and future directions. Am J Transplant 2008; 8: 753.
20. Seemayer CA, Gaspert A, Nickeleit V, et al.. C4d staining of renal allograft biopsies: A comparative analysis of different staining techniques. Nephrol Dial Transplant 2007; 22: 568.
21. Seemayer CA, Seemayer NH, Durmuller U, et al.. BK virus large T and VP-1 expression in infected human renal allografts. Nephrol Dial Transplant 2008; 23: 3752.
22. Schaub S, Hirsch HH, Dickenmann M, et al.. Reducing immunosuppression preserves allograft function in presumptive and definitive polyomavirus-associated nephropathy. Am J Transplant 2010; 10: 2615.
23. Levey AS, Coresh J, Greene T, et al.. Using standardized serum creatinine values in the modification of diet in renal disease study equation for estimating glomerular filtration rate. Ann Intern Med 2006; 145: 247.
Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
Kidney transplantation; Protocol biopsy; Subclinical rejection; Graft function; Long-term graft survival