C4d Deposits in Acute “Cell-Mediated” Rejection: A Marker for Renal Prognosis?

C4d Deposits in Acute “Cell-Mediated” Rejection: A Marker for Renal Prognosis? M. Neves, P. Cotovio, S. Machado, L. Santos, F. Macário, R. Alves, J. Pratas, M. Xavier da Cunha, A. Mota, and M. Campos ABSTRACT Background. Accumulation of C4d along peritubular capillaries (PTC) of renal allografts is normally attributed to antibody-mediated rejection. The prognostic implication of these deposits associated with “cell-mediated” rejection on graft survival remains uncertain. Our study aims to evaluate the impact of C4d deposits along PTC of patients with acute cell- mediated rejection on graft function and survival. Methods. We retrospectively analyzed patients transplanted between 2005 and 2010 with histopathologic diagnosis of acute rejection (AR). Eleven patients with “pure” antibodymediated rejection were excluded. The remaining 79 patients were divided into two groups according to type of AR by Banff 2003 criteria: type I (69.6%) versus type II (30.4%). In each group, comparisons were made between C4d-negative (⫺) and C4d-positive (⫹) biopsies. Results. Fifty-five patients presented with type I AR: 35 (63.6%) C4d⫺ and 20 (36.4%) C4d⫹. Twenty-four patients presented with type II AR: 13 (54.2%) C4d⫺ and 11 (45.8%) C4d⫹. In the type I AR group, graft survival at the first and second years was similar in C4d⫺ and C4d⫹ patients (94% and 91% versus 75% and 75%, respectively, log-rank P ⫽ .26). No differences were encountered in estimated glomerular filtration rate (eGFR) between subgroups at the first, second, and final years of follow-up. Graft loss occurred in 14.7% of C4d⫺ patients versus 25% in C4d⫹ patients (P ⫽ NS). In the type II AR group, graft survival at the first and second years was similar in C4d⫺ and C4d⫹ patients (85% and 85% versus 72% and 61%, respectively, log–rank P ⫽ .50). No differences were encountered in eGFR between subgroups at the first, second, and final years of follow-up. Graft loss occurred in 30.8% of C4d⫺ patients versus 45.5% in C4d⫹ patients (P ⫽ NS). Conclusion. Our results suggest that detection of C4d staining in acute “cell-mediated” rejection does not imply a worse renal prognosis. HE GOLD STANDARD for the diagnosis of acute rejection (AR) is the histological evaluation of the renal allograft biopsy.1 Correlation of findings on light microscopy with immunofluorescence and clinical history is essential to determine the nature and pathogenesis of the lesion.2 Complement 4d (C4d) often suffers deposition along the peritubular capillary (PTC) walls in kidney allografts undergoing AR and is considered to be a useful and sensitive diagnostic marker for antibody-mediated rejection.3,4 C4d is the degradation product of the activated complement factor C4, a component of the classical complement pathway that is typically initiated by binding of

T

antibodies to specific target molecules5 and can be covalently bound to the endothelium at the site of activation.6 From the Department of Nephrology (M.N., S.M., L.S., F.M., R.A., J.P., M.C.), Hospitais da Universidade de Coimbra; Department of Nephrology (P.C.), Centro Hospitalar de Coimbra; Department of Pathology (M.X.d.C.), Hospitais da Universidade de Coimbra; and Department of Urology and Renal Transplantation (A.M.), Hospitais da Universidade de Coimbra, Coimbra, Portugal. Address reprint requests to Marta Neves, Department of Nephrology, Hospitais da Universidade de Coimbra, Praceta Professor Mota Pinto, 3000-075 Coimbra, Portugal. E-mail: [email protected]

0041-1345/12/$–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.07.029

© 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

2360

Transplantation Proceedings, 44, 2360 –2365 (2012)

C4D DEPOSITS IN ACUTE REJECTION

Its detection seems “transplant specific.”7 C4 can also be activated via the mannose-binding lectin cascade;8,9 therefore, C4d may potentially be deposited without prior antibody binding.5 The clinical relevance of C4d staining along PTC in patients with a histological pattern of “cellmediated” AR remains uncertain. The aim of the present study was to evaluate the impact of C4d deposition along PTC in patients with biopsy-proven “cell-mediated” AR on overall kidney allograft function and graft survival. METHODS Through a retrospective, registry-based study, we gathered information on all ABO-compatible kidney transplant recipients between January 1, 2005, and December 31, 2010, at our Renal Transplantation Unit with biopsy-proven AR of the allograft. Of the 810 patients transplanted during that period, 90 (11.1%) had a histopathologic pattern of AR on kidney allograft biopsy. Of these, 11 patients who met criteria for “pure” antibody-mediated rejection (acute allograft dysfunction, documented donor-specific antibodies [DSA], positive C4d staining along PTC, and characteristic histological findings, including glomerulitis, polymorphonuclear infiltration, and/or fibrinoid necrosis) were excluded from our study, leaving 79 patients (87.8%) with evidence of cell-mediated AR on the allograft biopsy. All studied biopsies were episode graft biopsies obtained for an elevated serum creatinine and/or oligo- or anuria. The patients included in the study were further divided into two groups according to the type of AR, as classified by the Banff 2003 criteria: type I in 69.6% versus type II in 30.4% of patients. No patients were identified as having type III AR. In each group, comparisons were made between C4d-negative (⫺) and C4dpositive (⫹) staining subgroups. We gathered demographic and clinical data including gender, age at the time of transplantation, race and etiology of chronic kidney disease; transplant-specific data including graft type, classified as either cadaveric or living donor, number of HLA matches (zero to six— between donor and recipient HLA types A, B, and DR), patient’s peak serum panel reactive antibody (PRA) level (as determined in standard lymphocytotoxic assays), cold ischemic time, use of monoclonal or polyclonal antibodies as induction therapy and incidence of delayed graft function, defined as the need for dialysis during the first week after transplantation, and information at the time of diagnosis of AR (number of days until biopsy-proven diagnosis; estimated glomerular filtration rate [eGFR] at the time of AR and at the end of the follow-up period; qualitative determination of DSA against HLA types A, B, and DR; and specific antirejection therapy instituted). eGFR was calculated by the four-variable Modified Diet in Renal Disease formula, where eGFR ⫽ 175 ⫻ (serum creatinine)⫺1.154 ⫻ (age)⫺0.203 ⫻ (0.742 if female) ⫻ (1.212 if black). Two needle core renal biopsy specimens were analyzed: one specimen by light microscopy and the other by direct immunofluorescence of cryosections with a panel of fluorochrome-labeled antibodies against C4d. For light microscopy, renal allograft tissue was fixed in formalin, cut at 3 ␮m and stained with hematoxylineosin, periodic acid-Schiff, Masson’s trichrome, and periodic acidsilver methenamine. All lesions in allograft biopsies were graded according to the Banff 2003 classification. For immunofluorescence, renal allograft tissue was cut at 4 ␮m. Scoring considered focal positivity for C4d when staining was seen in more than 25% of PTC and diffuse positivity when staining was found in more than 50% of PTC.

2361 The qualitative determination of DSA against HLA types A, B, and DR became protocol in our unit only after 2008. Therefore, due to the reduced number of determinations present in our registers and the fact that all identified antibodies were non-donorspecific antibodies, no statistical analysis was performed. All statistical calculations were performed using the commercially available software program SPSS (version 17.0, SPSS, Chicago, Ill, USA) for Windows. Continuous variables are presented as means ⫾ standard deviation, and categorical variables as a percentage of the number of studied cases. Comparisons between quantitative variables were performed using the t test or the Mann-Whitney U test, and nominal variables were compared by using the ␹2 test. Graft survival curves were calculated using the Kaplan-Meier method. P values ⬍.05 are reported as statistically significant.

RESULTS

The 79 patients enrolled presented with an age ranging from 14 to 72 years, with a mean age of 46.0 ⫾ 13.8 years and median age of 48 years. There were proportionately more male patients (63.3%) than female, and 4% of the population was black. The mean follow up period was 31.4 ⫾ 24.0 months, with a median of 26.9 months. Further statistical analyses did not reveal statistical differences between focal and diffuse C4d positivity with graft outcome in either group; therefore, all focal and diffuse positive cases were referred to only as positive throughout the study. Nineteen patients (24.1%) suffered graft loss during the study period, five of these due to death with a functioning graft. Overall 1-year graft survival was 91.6% in all C4d⫺ cases and 73.9% in all C4d⫹ cases (log-rank P ⫽ .14; Fig 1). Type I AR

The general characteristics and statistical comparisons of the C4d⫺ and C4d⫹ subgroups of patients with type I AR are presented in Table 1. The mean follow-up period was 38.1 ⫾ 24.8 months versus 31.6 ⫾ 22.8 months (P ⫽ .51). Baseline characteristics of the recipients were similar between the subgroups, with regards to gender, age, etiology of chronic kidney disease, history of previous renal transplant, graft type, proportion of extended criteria donors, peak serum PRA level, number of donor-recipient HLA matches, cold ischemic time, use of mono- or polyclonal antibodies as induction immunosuppression therapy, and incidence of delayed graft function (P ⫽ NS). No statistical difference between the C4d⫺ and C4d⫹ subgroups was found in relation to the time until diagnosis of AR (40.2 ⫾ 64.8 days versus 27.6 ⫾ 44.9 days, P ⫽ .87) and allograft function at the time of AR (eGFR of 18.7 ⫾ 10.7 mL/min/1.73 m2 versus 20.2 ⫾ 12.4 mL/min/1.73 m2, P ⫽ .69), as well as choice of antirejection therapy administered. Allograft function remained similar between the subgroups at 6, 12, and 24 months after AR (eGFR of 45.7 ⫾ 13.6, 48.9 ⫾ 15.8 and 54.9 ⫾ 19.6 mL/min/1.73 m2 versus 47.6 ⫾ 16.7, 47.8 ⫾ 18.1, and 45.4 ⫾ 13.9 mL/min/1.73 m2, respectively, P ⫽ NS). At the end of the follow-up period, there was also no verified statistical difference in eGFR between

2362

NEVES, COTOVIO, MACHADO ET AL

Fig 1. Cumulative graft survival in C4d⫹ versus C4d⫺ cases.

the C4d⫺ and C4d⫹ subgroups (50.9 ⫾ 18.6 mL/min/1.73 m2 versus 47.1 ⫾ 18.9 mL/min/1.73 m2, P ⫽ .25). Overall graft survival at the first and second years after AR was 94% and 91%, respectively, in the subgroup of C4d⫺ patients, while in the subgroup of C4d⫹ patients, it was 75% and 75%, respectively, which, although showing a trend toward lower survival in the C4d⫹ subgroup, was not statistically significant (log-rank P ⫽ .26; Fig 2). Even after censoring for graft loss due to death with a functioning graft, no statistical difference was encountered between the subgroups (log-rank P ⫽ .29). Type II Acute Rejection

The general characteristics and statistical comparisons of the C4d⫺ and C4d⫹ subgroups of patients with type II AR are presented in Table 2. The mean follow-up period was 22.7 ⫾ 22.1 months versus 20.0 ⫾ 21.0 months (P ⫽ .77). Baseline characteristics of the recipients were similar between the two subgroups, with regards to gender, age, race, etiology of chronic kidney disease, history of previous renal transplant, graft type, proportion of extended criteria donors, peak serum PRA level, number of donor-recipient HLA matches, cold ischemic time, use of mono- or polyclonal antibodies as induction immunosuppression therapy and proportion of patients suffering delayed graft function (P ⫽ NS). No statistical difference between the C4d⫺ and C4d⫹ subgroups was verified in relation to the time until diagnosis of AR (13.1 ⫾ 6.1 days versus 57.5 ⫾ 76.6 days, P ⫽ .20) and allograft function at the time of AR (eGFR of

12.5 ⫾ 9.3 mL/min/1.73 m2 versus 8.7 ⫾ 3.7 mL/min/1.73 m2, P ⫽ .23), as well as choice of antirejection therapy administered. Allograft function remained comparable between the subgroups at 6, 12, and 24 months after AR (eGFR of 54.9 ⫾ 21.5, 54.9 ⫾ 14.2, and 58.7 ⫾ 21.9 mL/min/1.73 m2 versus 56.4 ⫾ 28.5, 50.6 ⫾ 22.5, and 55.7 ⫾ 30.7 mL/min/1.73 m2, respectively, P ⫽ NS). At the end of the follow-up period, no statistical difference in eGFR between the C4d⫺ and C4d⫹ subgroups was encountered (43.3 ⫾ 18.7 mL/min/1.73 m2 versus 38.1 ⫾ 32.6 mL/min/ 1.73 m2, P ⫽ .25). Overall graft survival at the first and second years after AR was 85% and 85%, respectively, in the subgroup of C4d⫺ patients, while in the subgroup of C4d⫹ patients it was 72% and 61%, respectively. Although there appears to be a trend toward lower survival in the C4d⫹ subgroup, this was not statistically significant (logrank P ⫽ .50; Fig 3). Subsequently, when graft loss due to death with a functioning graft was censored, no statistical difference was encountered between the subgroups (logrank P ⫽ .10). DISCUSSION

C4d deposits in PTC were first described by Feucht et al10 in biopsies performed on patients with high PRA levels, and were thus considered to be at high immunologic risk. Twenty years after their practice-changing work, doubts still arise about the clinical significance that these deposits represent in cell-mediated AR of the kidney allograft. It should be noted that it is not uncommon for findings of

C4D DEPOSITS IN ACUTE REJECTION

2363

Table 1. General Characteristics of Patients With Type I Acute Rejection

Recipient male gender, n (%) Recipient age (y) Recipient Caucasian, n (%) Previous renal transplant, n (%) Graft type, n (%) Cadaveric Living Extended criteria donor, n (%) PRA level before transplantation, n (%) 0–24% ⬎25% Number of HLA matches Cold ischemic time (h) Induction therapy, n (%) Thymoglobulin Basiliximab Delayed graft function, n (%) Antirejection therapy, n (%) No treatment Methylprednisone Methylprednisone ⫹ thymoglobulin Thymoglobulin Plasmapheresis ⫹ IVIG ⫹ rituximab Methylprednisone ⫹ IVIG Thymoglobulin ⫹ IVIG Plasmapheresis ⫹ IVIG Graft loss, n (%)

All Patients (n ⫽ 55)

C4d⫺ Patients (n ⫽ 35)

C4d⫹ Patients (n ⫽ 20)

37 (67.3%) 45.7 ⫾ 12.5 52 (94.5%) 5 (9.1%)

23 (65.7%) 45.7 ⫾ 11.8 35 (100%) 2 (5.7%)

14 (70%) 45.7 ⫾ 13.9 17 (85%) 3 (15%)

50 (90.9%) 5 (9.1%) 39 (70.9%)

33 (94.3%) 2 (5.7%) 25 (71.4%)

17 (85%) 3 (15%) 14 (70%)

51 (92.7%) 4 (7.3%) 2.1 ⫾ 1.0 16.4 ⫾ 6.7 30 (54.5%) 7 (12.7%) 23 (41.8%) 27 (49.1%)

34 (97.1%) 1 (2.9%) 2.2 ⫾ 0.9 17.1 ⫾ 6.4 17 (48.6%) 4 (11.4%) 13 (37.1%) 18 (51.4%)

17 (85%) 3 (15%) 1.8 ⫾ 1.0 15.2 ⫾ 7.4 13 (65%) 3 (15%) 10 (50%) 9 (45%)

1 (1.8%) 42 (76.4%) 6 (10.9%) 2 (3.6%) 1 (1.8%) 1 (1.8%) 1 (1.8%) 1 (1.8%) 10 (18.2%)

0 (0%) 29 (82.9%) 5 (14.3%) 1 (2.9%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 5 (14.7%)

1 (5%) 13 (65%) 1 (5%) 1 (5%) 1 (5%) 1 (5%) 1 (5%) 1 (5%) 5 (25%)

P Value

.49 .99 .04 .34 .34

.99 .09

.16 .33 .27

.78 .36 .11 .40 .55 .35 .35 .35 .35 .26

PRA, panel-reactive antibody; IVIG, intravenous immunoglobulin.

Fig 2. Cumulative graft survival of type I acute rejection (C4d⫹ versus C4d⫺ cases).

2364

NEVES, COTOVIO, MACHADO ET AL Table 2. General Characteristics of Patients With Type II Acute Rejection

Recipient male gender, n (%) Recipient age (y) Recipient Caucasian, n (%) Previous renal transplant, n (%) Graft type, n (%) Cadaveric Living Extended criteria donor, n (%) PRA level before transplantation, n (%) 0–24% ⬎25% Number of HLA matches Cold ischemic time (h) Induction therapy, n (%) Thymoglobulin Basiliximab Delayed graft function, n (%) Antirejection therapy, n (%) Methylprednisone Methylprednisone ⫹ thymoglobulin Thymoglobulin Thymoglobulin ⫹ IVIG ⫹ Plasmapheresis Graft loss, n (%)

All Patients (n ⫽ 24)

C4d⫺ Patients (n ⫽ 13)

C4d⫹ Patients (n ⫽ 11)

P Value

12 (50%) 47.8 ⫾ 16.6 24 (100%) 1 (4.2%)

7 (53.8%) 51.1 ⫾ 16.8 13 (100%) 1 (7.7%)

5 (45.5%) 43.9 ⫾ 16.4 11 (100%) 0 (0%)

.50 .30 .99 .99 .99

24 (100%) 0 (0%) 15 (62.5%)

13 (100%) 0 (0%) 7 (53.8%)

11 (100%) 0 (0%) 8 (72.7%)

23 (95.8%) 1 (4.2%) 2.4 ⫾ 0.9 18.4 ⫾ 4.6 8 (33.3%) 2 (8.3%) 6 (25%) 9 (37.5%)

12 (92.3%) 1 (7.7%) 2.4 ⫾ 0.9 17.8 ⫾ 3.8 5 (38.5%) 1 (7.7%) 4 (37.1%) 4 (37.1%)

11 (100%) 0 (0%) 2.5 ⫾ 1.0 19.1 ⫾ 5.5 3 (27.3%) 1 (9.1%) 2 (50%) 5 (45.5%)

2 (8.3%) 13 (54.2%) 8 (33.3%) 1 (4.2%) 9 (37.5%)

2 (15.4%) 8 (61.5%) 3 (23.1%) 0 (0%) 4 (30.8%)

0 (0%) 5 (45.5%) 5 (45.5%) 1 (9.1%) 5 (45.5%)

PRA, panel-reactive antibody; IVIG, intravenous immunoglobulin.

Fig 3. Cumulative graft survival of type II acute rejection (C4d⫹ versus C4d⫺ cases).

.42 .78

.91 .48 .68

.70 .48 .68 .39 .46 .68

C4D DEPOSITS IN ACUTE REJECTION

cell-mediated AR to occur in allograft biopsies that also show evidence of an antibody-mediated reaction.11 The association between C4d deposits in PTC and morphological signs of cell-mediated AR has been found to be statistically significant.7 In a series of published articles, C4d is found in 24% to 43% of type I rejection episodes, in 45% of type II rejection, and in 50% of type III rejection,5,7,12 findings similar to what we came across (in 36.4% of type I AR and in 45.8% of type II AR). Some published studies corroborate our results by showing that differences in serum creatinine levels between C4d⫺ and C4d⫹ cases with morphological evidence for Banff I, II, or III AR did not achieve statistical significance.7,13–16 The main drawbacks of this study remain its retrospective nature, the small case numbers, and the fact that no correlation of C4d deposition with DSA determination was performed routinely in all patients. Some published articles found significantly higher renal allograft survival rates in subgroups of patients without C4d deposits,12,17,18 while others are concordant with ours by showing that although there was a trend toward a less favorable graft outcome in C4d⫹ cases, these differences were not significant.7,16,19 Within the Banff 2003 type I AR subgroup with positive C4d staining, one quarter of the patients lost their grafts, although this value did not attain statistical difference when compared to the negative C4d staining subgroup (14.7%). With regards to the Banff 2003 type II AR subgroup with positive C4d staining, 45.5% of patients lost their grafts, but no statistical difference was encountered when results were compared to the negative C4d staining subgroup (30.8%). We are confident that had the groups enrolled a larger number of patients, a statistical difference would be found with regards to graft survival, as the trends appear to indicate. Further investigation is warranted in order to verify whether the deposition of C4d by itself in “cellmediated” AR deserves a more aggressive form of antirejection therapy to modify long-term renal outcomes.

REFERENCES 1. Colvin RB: Renal transplant pathology. In Jennette JC, Olson JC, Schwartz ML, et al (eds): Heptinstall’s Pathology of the Kidney, 5th ed. Philadelphia, PA: Lippincott-Raven; 1998, p 1409

2365 2. Lajoie G, Silva FG: Approach to the interpretation of renal biopsy. In Silva FG, D’Agati VD, Nadasy T (eds): Renal Biopsy Interpretation. New York, NY: Churchill Livingstone; 1996, p 31 3. Böhmig GA, Exner M, Habicht A, et al: Capillary C4d deposition in kidney allografts: a specific marker of alloantibodydependent graft injury. J Am Soc Nephrol 13:1091, 2002 4. Imai N, Nishi S, Alchi B, et al: Immunohistochemical evidence of activated lectin pathway in kidney allografts with peritubular capillary C4d deposition. Nephrol Dial Transplant 21:2589, 2006 5. Nickeleit V, Mihatsch MJ: Kidney transplants, antibodies and rejection: is C4d a magic marker? Nephrol Dial Transplant 18: 2232, 2003 6. Wagrowska-Danilewicz M, Zeromski J: Immunofluorescent evaluation of renal biopsy: current point of view. Pol J Pathol 61:83, 2010 7. Nickeleit V, Zeiler M, Gudat F, et al: Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 13:242, 2002 8. Petersen SV, Thiel S, Jensen L, et al: An assay for the mannan-binding lectin pathway of complement activation. J Immunol Methods 257:107, 2001 9. Thiel S, Vorup-Jensen T, Stover CM, et al: A second serine protease associated with mannan-binding lectin that activates complement. Nature 386:506, 1997 10. Feucht HE, Felber E, Gockel MJ, et al: Vascular deposition of complement—split products in kidney allografts with cellmediated rejection. Clin Exp Immunol 86:464, 1991 11. Watschinger B, Pascual M: Capillary C4d deposition as a marker of humoral immunity in renal allograft rejection. J Am Soc Nephrol 13:2420, 2002 12. Herzenberg AM, Gill JS, Djurdjev O, et al: C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol 13:234, 2002 13. Regele H, Exner M, Watschinger B, et al: Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection. Nephrol Dial Transplant 16: 2058, 2001 14. Magil AB, Tinckam KJ: Focal peritubular capillary C4d deposition in acute rejection. Nephrol Dial Transplant 21:1382, 2006 15. Demirci C, Sen S, Sezak M: Incidence and importance of C4d deposition in renal allograft dysfunction. Transplant Proc 40:174, 2008 16. Botermans JM, de Kort H, Eikmans M: C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome. Clin J Am Soc Nephrol 6:1207, 2011 17. Valente M, Furian L, Marchini F: C4d-positive renal allograft rejection biopsies in cyclosporine-treated patients: singlecenter incidence and outcome. Transplant Proc 42:2214, 2010 18. Fior F, Nacchia F, Minicozzi A: Focal C4d staining in peritubular capillaries and kidney graft survival: results of a retrospective study. Transplant Proc 42:1095, 2010 19. Kedainis RL, Koch MJ, Brennan DC: Focal C4d⫹ in renal allografts is associated with the presence of donor-specific antibodies and decreased allograft survival. Am J Transplant 9:812, 2009