Prediction of acute renal allograft rejection in early post-transplantation period by soluble CD30

Prediction of acute renal allograft rejection in early post-transplantation period by soluble CD30

Transplant Immunology 16 (2006) 41 – 45 www.elsevier.com/locate/trim Prediction of acute renal allograft rejection in early post-transplantation peri...

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Transplant Immunology 16 (2006) 41 – 45 www.elsevier.com/locate/trim

Prediction of acute renal allograft rejection in early post-transplantation period by soluble CD30 Wang Dong a,b,⁎,1 , Yang Shunliang a,b,1 , Wu Weizhen b , Wang Qinghua b , Zeng Zhangxin b , Tan Jianming b , Wang He a a

Department of Urology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China b Organ Transplant Institute, Fujian Orient Hospital, Fuzhou, 350025, China Received 20 January 2006; received in revised form 23 January 2006; accepted 24 February 2006

Abstract To evaluate the feasibility of serum sCD30 for prediction of acute graft rejection, we analyzed clinical data of 231 patients, whose serum levels of sCD30 were detected by ELISA before and after transplantation. They were divided into three groups: acute rejection group (AR, n = 49), uncomplicated course group (UC, n = 171) and delayed graft function group (DGF, n = 11). Preoperative sCD30 levels of three groups were 183 ± 74, 177 ± 82 and 168 ± 53 U/ml, respectively (P = 0.82). Significant decrease of sCD30 was detected in three groups on day 5 and 10 post-transplantation respectively (52 ± 30 and 9 ± 5 U/ml respectively, P b 0.001). Compared with Group UC and DGF, patients of Group AR had higher sCD30 values on day 5 post-transplantation (92 ± 27 U/ml vs. 41 ± 20 U/ml and 48 ± 18 U/ml, P b 0.001). However, sCD30 levels on day 10 post-transplantation were virtually similar in patients of three groups (P = 0.43). Receiver operating characteristic (ROC) curve demonstrated that sCD30 level on day 5 post-transplantation could differentiate patients who subsequently suffered acute allograft rejection from others (area under ROC curve 0.95). According to ROC curve, 65 U/ml may be the optimal operational cut-off level to predict impending graft rejection (specificity 91.8%, sensitivity 87.1%). Measurement of soluble CD30 on day 5 post-transplantation might offer a noninvasive means to recognize patients at risk of impending acute graft rejection during early post-transplantation period. © 2006 Elsevier B.V. All rights reserved. Keywords: Soluble CD30; Acute rejection; Sensitivity; Specificity; Kidney transplantation

1. Introduction Newer immunosuppressive agents and regimens have achieved lower rates of acute renal rejection, however, acute rejection (AR) is still a leading cause of early graft dysfunction and late kidney graft loss [1,2]. Recognizing impending AR early will reduce irreversible graft damage and improve longterm survival. Needle biopsy, the most reliable method, is an invasive technique. Meanwhile, graft damage has occurred at the time of diagnosis of AR by needle biopsy. The reliable ⁎ Corresponding author. Organ Transplant Institute, Fujian Orient Hospital, No. 156 Xi'erhuan North Road, Fuzhou, 350025, China. Tel.: +86 591 83775816. E-mail address: [email protected] (W. Dong). 1 The two authors contributed equally to this work. 0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2006.02.005

recognition of rejection at an early stage continues to pose a problem. CD30, a member of the tumor necrosis facto superfamily, is a 120 kD membrane glycoprotein recognized originally on Reed– Sternberg cells of Hodgkin lymphoma (R–S/Hs) [3]. CD30 is preferentially expressed on T cells that secrete Th2-type cytokines [4]. After activation of CD30+ T cells, a soluble form of CD30 (sCD30) is released proteolytically, however, the biological significance of this process is still not clearly defined [5,6]. Recently, several reports have suggested that elevated preand post-transplantation levels of the sCD30 molecule might be predictive for an increased incidence of rejection and worse kidney graft prognosis [7–9]. To evaluate the feasibility of serum sCD30 for diagnosis of acute graft rejection, we have measured sCD30 levels of 231 renal graft recipients before and after operation.

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W. Dong et al. / Transplant Immunology 16 (2006) 41–45

2. Materials and methods

Table 2 sCD30 plasma levels (U/ml) of patients in different rejection time periods

2.1. Patient demographics

Time of AR (days)

Number (%)

Sampling time points Day 0 pretransplantation

Day 5 posttransplantation

Day 10 posttransplantation

b5 5–9 10–19 ≥20

4 24 18 3

173 ± 60 178 ± 79 181 ± 73 254 ± 68

79 ± 22 97 ± 23 98 ± 44 76 ± 12

8±6 11 ± 6 11 ± 6 5±1

Between January and December 2004, a total of 231 patients received kidney grafts at Organ Transplant Institute of Fujian Orient Hospital, which included 3 living-related donors and 228 cadaveric donors. Informed consent was obtained from each patient. They were divided into three groups according to the clinical course during one month post-transplantation. Fortynine patients who experienced an episode of acute rejection (AR) during the first month were categorized as group AR. One hundred and seventy-one patients with primary graft function and an uncomplicated course (UC) without acute rejection were categorized as group UC. Eleven patients who needed dialysis during the first week without evidence of acute rejection were categorized as delayed graft function group (DGF). The patients' data of demographic details are shown in Table 1, which were comparable in three groups.

2.2. Pre-transplant status

(8.2) (49.0) (36.7) (6.1)

2 h and plasma was separated from cells, collected and stored at − 70 °C until testing. Human sCD30 instant ELISA kits were obtained from Bender MedSystems (Vienna, Austria). Plasma levels of sCD30 were measured by ELISA assay according to manufacturers' instructions. Samples were also obtained from 49 age-matched healthy individuals as normal control (Age: 34 ± 10).

2.4. Diagnosis of AR

Donor–recipient blood group matching was identical in all patients. HLA crossmatch of patients were negative, which were determined by microdroplet assay of complement-dependent lymphocytotoxicity (CDC). PRA was determined by ELISA technology. Maintenance immunosuppressive regimens almost were standard triple therapy, which consisted of a calcineurin inhibitor (CsA microemulsion—Neoral or Tacrolimus), combined with prednisone and MMF. Besides the regimens mentioned above, Aza or MZR were used in a few patients instead of MMF. The number of those patients was small, so immunosuppressive regimens without MMF was defined as other regimens. The patients' data of HLA-A/B/DR mismatching, cold ischemia time, PRA scores and immunosuppressive regimens are shown in Table 1, which were comparable in three groups.

To avoid the complications of percutaneous kidney biopsy, acute rejection in the first week post-transplantation was diagnosed by a progressive elevation of serum creatinine (N20% of baseline creatinine) responded to anti-rejection therapy and clinical signs of rejection including fever (N38.0 °C), decreased urinary output, pain over an enlarged kidney graft, hypertension, and color Doppler indicating increase of kidney graft size and elevation of vascular resistance index (N0.8). After the first week post-transplantation, percutaneous kidney biopsy was carried out in cases of graft function deterioration, and kidney pathology was classified using the definitions given by the updated Banff 97. A rabbit polyclonal antibody (Biomedica, Vienna, Austria) was used to assess C4d status on frozen sections according to manufacturers' instructions.

2.5. Statistical analysis

2.3. Serum sCD30 Blood samples were obtained on day 0 before transplantation and on day 5 and 10 after transplantation. Then blood samples were centrifuged within

Analysis of variance, rank-sum test, multiple comparison Z test, correlation analysis and receiver operating characteristic (ROC) curves were used for statistical analysis. P values b 0.05 were considered significant.

3. Results

Table 1 Demographics of kidney allograft recipients Characteristic

Group AR

Group UC

Group DGF

Number Gender distribution (M:F) Age (X ± S.D.) Donor age Cold ischemia time (h) Number of previous grafts 0 1 2 PRA score (%) b10 10–20 20–50 N50 HLA mismatching 0 1 2 3 N3 Immunosuppressive regimen CsA + MMF + MP FK + MMF + MP Others

49 35:14 38 ± 11 28.5 ± 5.7 8.0 ± 2.2

171 142:29 39 ± 11 29.0 ± 5.0 7.7 ± 2.1

11 7:4 40 ± 17 29.5 ± 4.0 7.8 ± 1.7

One-month follow-up data show that 49 of 231 (21.2%) patients suffered AR episode, which were diagnosed on 10 days (mean) postGroup AR

600.00

r = 0.124 P = 0.06

47 (95.9%) 2 (4.1%) 0

164 (95.9%) 6 (3.5%) 1 (0.6%)

10 (90.9%) 1 (9.1%) 0

41 (83.7%) 3 (6.1%) 3 (6.1%) 2 (4.1%)

155 (90.6%) 8 (4.7%) 6 (3.5%) 2 (1.2%)

10 (90.9%) 0 0 1 (9.1%)

0 4 (8.2%) 10 (20.4%) 17 (34.7%) 18 (36.7%)

0 18 29 61 63

0 1 2 5 3

27 (55.1%) 20 (40.8%) 2 (4.1%)

92 (53.8%) 58 (33.9%) 21 (12.3%)

(10.5%) (17.0%) (35.7%) (36.8%)

(9.1%) (18.2%) (45.5%) (27.2%)

sCD30 level on day 0

500.00

DGF

400.00

300.00

200.00

100.00

0.00 0.00

3 (27.2%) 6 (54.6%) 2 (18.2%)

UC

50.00

100.00

150.00

200.00

250.00

sCD30 level on day 5

Fig. 1. Correlation between sCD30 levels on day 0 and 5.

W. Dong et al. / Transplant Immunology 16 (2006) 41–45 Table 3 sCD30 plasma levels (U/ml) of patients in three groups

Group AR Group UC Group DGF Total

Sampling time Day 0 Day 5 Day 10

400.00

Sampling time points Day 0 pretransplantation

Day 5 posttransplantation

Day 10 posttransplantation

P values

183 ± 74 178 ± 82 168 ± 53 178 ± 79

92 ± 27a,b 41 ± 20a 48 ± 18b 52 ± 30

10 ± 6 9±5 10 ± 4 9±5

b0.001 b0.001 b0.001 b0.001

300.00

sCD30 level

Patients group

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P values for pairwise comparisons: a, bP b 0.001. All other comparisons: P = NS.

transplantation (Table 2). In Group AR, 43 patients were diagnosed by kidney biopsies, which included 41 patients with tubulointerstitial rejection (TIR) and 2 patients with vascular rejection (VR). Seven patients were positive for C4d (2 patients with VR and five with TIR). Patients with AR in five days post-transplantation (n = 4) and those diagnosed as VR (n = 2) all were treated with ATG (Fresenius, 2 mg/kg/d × 7–14 d). Other patients with AR (n = 43) were treated with 3-day bolus of methylprednisolone (MP) (8 mg/kg/d). Three patients with AR, who were not reversed by bolus therapy of MP, were treated with ATG. All the patients with AR were reversed by anti-rejection therapy mentioned above. Eleven patients were diagnosed as DGF without evidence of AR, which included three patients diagnosed as ATN by kidney biopsies. Another 171 patients obtained primary graft function and had an uncomplicated course without AR. Average sCD30 level of 231 patients before transplantation was 178 ± 79 U/ml, which was much higher than that of healthy individuals (41 ± 13 U/ml, P b 0.001). Soluble CD30 levels in three groups before transplantation were 183 ± 74, 178 ± 82 and 168 ± 53 U/ml, respectively (P = 0.82). In three groups, significant decrease of sCD30 was detected on day 5 and 10 post-transplantation respectively (52 ± 30 and 9 ± 5 U/ml respectively, P b 0.001). Most importantly, the decrease of sCD30 levels after transplantation varied in different groups (Fig. 1). Compared with Group UC and DGF, patients of Group AR had higher sCD30 levels on day 5 posttransplantation (92 ± 27 U/ml vs. 41 ± 20 U/ml and 48 ± 18 U/ml,

200.00

100.00

0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

Rejection time (day posttransplantation)

Fig. 2. Correlation between sCD30 levels on day 5 and rejection time. A. Day 0 r = 0.16, P = 0.27; B. Day 5 r = 0.103, P = 0.48; C. Day 10 r = 0.106 P = 0.47.

P b 0.001). However, sCD30 levels on day 10 post-transplantation were virtually similar in patients of three groups (P = 0.43) (Table 3). Correlation between PRA scores and sCD30 levels was analyzed. Results show that PRA score was not related to sCD30 level (r = 0.03, P = 0.89, day 0 before transplantation; r = 0.196, P = 0.37, day 5 and r = 0.278 P = 0.20, day 10 after transplantation). When patients with PRA score b 10% were analyzed separately, higher sCD30 levels on day 5 post-transplantation were also observed in patients of Group AR (P b 0.001) (Table 4). Data also show that there was no association between rejection time and sCD30 levels (Fig. 2). The influence of HLA matching and immunosuppressive regimens on sCD30 levels was shown in Table 4. When patients were divided into well-matched group (with three or less mismatches) and poor-matched group (with more than three mismatches), no significant difference of sCD30 levels was shown between two

Table 4 Effect on sCD30 of PRA, HLA matching and immunosuppressive regimens

PRA b 10% Mismatching ≤ 3 Mismatching N 3

FK-treated

CsA-treated

Other regimen

Group

Number

Sampling time points Day 0 pre-transplantation

Day 5 post-transplantation

Day 10 post-transplantation

AR UC DGF AR UC DGF AR UC DGF AR UC DGF AR UC DGF AR UC DGF

41 155 10 31 108 8 18 63 3 20 58 6 27 92 3 2 21 2

180 ± 69 175 ± 82 167 ± 56 189 ± 67 175 ± 82 158 ± 58 182 ± 79 183 ± 83 193 ± 31 178 ± 63 173 ± 86 164 ± 66 180 ± 79 178 ± 79 153 ± 33 273 ± 112 190 ± 91 201 ± 40

91 ± 28a,b 41 ± 21a 47 ± 19b 90 ± 28c,d 41 ± 21c 46 ± 20d 92 ± 30e,f 41 ± 20e 53 ± 13f 95 ± 35g,h 43 ± 22g 55 ± 9h 90 ± 21i,j 40 ± 20i 33 ± 30j 84 ± 19 42 ± 16 48 ± 11

10 ± 6 9±5 10 ± 4 11 ± 7 9±6 10 ± 4 9±5 8±4 7±2 12 ± 7 11 ± 6 10 ± 3 9±5 8±4 10 ± 7 11 ± 1 8±3 7±2

P values for pairwise comparisons: a,

P b 0.001. All other comparisons: P = NS.

b, c, d, e, f, g, h, i, j

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W. Dong et al. / Transplant Immunology 16 (2006) 41–45

groups. However, compared within well-matched group or poormatched group separately, patients with AR of both groups had higher sCD30 levels than those without AR on day 5 post-transplantation (P b 0.001) (Table 4). Soluble CD30 levels were also independently evaluated in patients receiving cyclosporine A and FK506. There was also no significant difference of sCD30 levels between two groups. Significant difference of sCD30 levels was only observed between patients with AR and those without AR on day 5 post-transplantation (P b 0.001) (Table 4). Receiver operating characteristic (ROC) curve demonstrated that sCD30 level on day 5 post-transplantation could differentiate patients who subsequently suffered acute allograft rejection from patients with uncomplicated course (area under ROC curve 0.95) or those with delayed graft function (area under ROC curve 0.97). According to ROC curve, if Group UC and DGF were put together as one group, 65 U/ml may be the optimal operational cut-off level to predict impending graft rejection (specificity 91.8%, sensitivity 87.1%) (Fig. 3).

A 1.0

True Positive Rate (TPR)

0.8

0.6

0.4

0.2

Area under ROC curve 0.95 0.0 0.0

0.2

0.4

0.6

0.8

1.0

False Positive Rate (FPR)

B

4. Discussion

1.0

True Positive Rate (TPR)

0.8

0.6

0.4

0.2

Area under ROC curve 0.97 0.0 0.0

0.2

0.4

0.6

0.8

1.0

False Positive Rate (FTR)

C 1.0

True Positive Rate (TPR)

0.8

0.6

0.4

0.2

Area under ROC curve 0.70 0.0 0.0

0.2

0.4

0.6

0.8

1.0

False Positive Rate (FTR)

Fig. 3. ROC curve for comparisons of sCD30 levels among three groups. A. Comparison between AR vs. UC; B. Comparison between AR vs. DGF; C. Comparison between UC vs. DGF.

As reported by Pelzl [10], we have also demonstrated that kidney graft recipients had a significantly higher serum sCD30 values before transplantation than adult healthy persons. Our data show that sCD30 value is not age-related, which was not identical with that of Pelzl. An explanation to this discrepancy might be that only three patients were under eighteen-years old in our study. So further study including more young people is necessary to confirm the association between the sCD30 values and age. Different average levels of sCD30 have been reported [7,9–11]. Susal et al. [7] found pre-transplant sCD30 levels of N100 U/ml in only 23% of their nearly 4000 patients. However, sCD30 levels with an average of N 100 U/ml were reported by Slavcev and Rajakariar [9,11]. The similar results were observed in our study. Importantly, the discrepancy in pretransplant sCD30 levels did not change the conclusion that no significant differences were observed in pre-transplantation sCD30 concentrations between patients with and without acute rejection. Although significant decrease of sCD30 was detected after transplantation in all patients in our study, our data support the study of Pelzl [8], where on day 5 post-transplantation the differences in sCD30 levels between patients with and without acute rejection were highly significant. The sCD30 values of Group UC and DGF on day 5 post-transplantation were similar. Slavcev [9] reported that patients free of rejection in the first month post-transplant had lower sCD30 concentrations 2 weeks after transplantation compared to rejecting patients, however, in our study, there was not significant difference in sCD30 values on day 10 post-transplantation between patients with and without AR. Meanwhile, average sCD30 value (9 ± 5 U/ml) on day 10 post-transplantation in our study was much lower than that of Slavcev. Further study will be necessary to explain the discrepancy above and the significant decrease of sCD30 from day 5 to day 10 post-transplantation. Receiver operating characteristic (ROC) curves is frequently used in biomedical informatics research to evaluate

W. Dong et al. / Transplant Immunology 16 (2006) 41–45

computational models for decision support, diagnosis, and prognosis. ROC analysis could investigate the accuracy of a model's ability to separate positive from negative cases and define optimal operational cut-off level with better specificity and sensitivity [12]. So ROC curves were used in our study to evaluate the feasibility of post-transplantation serum sCD30 as predictor of acute graft rejection. High area under the ROC curve was obtained when the sCD30 values were compared between Group AR and Group UC or DGF. This suggests that measurement of sCD30 levels after transplantation could become a promising method to predict acute graft rejection and might allow the identification of patients with AR as early as day 5 after transplantation, 5 days (mean) before an acute rejection was diagnosed by conventional methods. Importantly, at this early time point, detection of AR in the kidney with DGF is made difficult because conventional noninvasive rejection parameters, such as rising serum creatinine and oliguria cannot be used to make the diagnosis, however, sCD30 allowed a differentiation of group AR from group DGF patients. Moreover, our data also show that sCD30 levels didn't vary in patients with different PRA scores, number of HLA matching or immunosuppressive regimens. More patients or multicenter study are desirable for definite conclusions, however, the present single-center study provides an indication that measurement of sCD30 levels after transplantation might be helpful in estimating the relative risk for the occurrence of acute rejection during the early posttransplantation period.

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