Allosensitization and outcomes in pediatric heart transplantation

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Allosensitization and outcomes in pediatric heart transplantation William T. Mahle, MD,a Margaret A. Tresler, MPH,b R. Erik Edens, MD, PhD,c Paolo Rusconi, MD,d James F. George, PhD,b David C. Naftel, PhD,b Robert E. Shaddy, MD,e and the Pediatric Heart Transplant Study Group From aDepartment of Pediatrics, Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia; b Department of Surgery, Division of Cardiothoracic Surgery, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama; cDepartment of Pediatrics, University of Iowa Medical School, Iowa City, Iowa; dDepartment of Pediatrics, University of Miami School of Medicine, Miami, Florida; and eDepartment of Pediatrics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania.

KEYWORDS: allosensitization; antibody-mediated rejection; pediatrics; heart transplantation

BACKGROUND: Allosensitization among children being considered for heart transplantation remains a great challenge. Controversy exists as to the best approach for those with elevated panel-reactive antibody (PRA) titers. We sought to define the association between elevated PRA and outcomes using data from the multi-institutional Pediatric Heart Transplant Study Group. METHODS: Between January 1993 and December 2008, 3,016 patients (⬎1 month of age) were listed for heart transplantation. PRA data at listing were available for 2,500 (83%) patients, and 2,237 underwent transplantation with PRA data being available for 1,904 (85%). Because various PRA assays were employed (e.g., cell-based and solid phase) we entered the highest value regardless of methodology. RESULTS: Among the factors associated with high PRA at transplant were Status 1 at listing, previous sternotomy and prior Norwood procedure. An elevated PRA at listing was associated with higher risk of death while waiting. Of subjects with PRA ⱖ50% only 57% were transplanted by 1 year on the waitlist, as compared with 76% of those with PRA ⬍10%. Waitlist mortality for the highly allosensitized subjects (ⱖPRA 50%) was 19% by 12 months. Survival at 1 year after transplantation was significantly lower in those with PRA ⱖ50% versus those with PRA ⬍10% (73% vs 90%, respectively, p ⬍ 0.0001). Those with elevated PRA who had a negative prospective crossmatch had no difference in survival compared with those without allosensitization. There was no significant association between PRA levels and time to first rejection or development of coronary allograft vasculopathy. CONCLUSIONS: Significant allosensitization is associated with more than a 2-fold increased risk of death within the first transplant year. Although prospective crossmatching abrogates the risk of post-transplant mortality, it may contribute to higher pre-transplant attrition due to longer waitlist times. There is a critical need for strategies to minimize the impact of allosensitization and antibody-mediated rejection immediately after transplantation. J Heart Lung Transplant 2011;30:1221–7 © 2011 International Society for Heart and Lung Transplantation. All rights reserved.

The presence of pre-formed anti-HLA antibodies in recipient serum has been associated with acute allograft rejection and post-transplant mortality in adults who have undergone heart transplantation.1,2 The United Network Reprint requests: William T. Mahle, MD, Sibley Heart Center, Egleston Children’s Hospital, Emory University School of Medicine, 1405 Clifton Road NE, Atlanta, GA 30322. Telephone: 404-785-1672. Fax: 404-325-6021. E-mail address: [email protected]

for Organ Sharing (UNOS) recommends routinely screening patient serum for alloreactive antibodies prior to heart transplantation.3 When the panel-reactive antibody (PRA) activity of potential cardiac allograft recipients is ⱖ10%, UNOS recommends that a prospective crossmatch be performed to lessen the risk associated with allosensitization. Studies have reported that allosensitization is associated with increased mortality after pediatric heart transplantation

1053-2498/$ -see front matter © 2011 International Society for Heart and Lung Transplantation. All rights reserved. doi:10.1016/j.healun.2011.06.005

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as well as increased waitlist attrition.4 –7 Some investigators have suggested that alterations in immunosuppression can mitigate the adverse impact of allosensitization,8,9 but these findings have engendered controversy as to how best to manage the child with elevated PRA awaiting transplantation. In fact, some have advocated heart transplantation of the allosensitized child without attempting a prospective crossmatch.5 To consider the relative benefits of such a strategy it is necessary to understand the outcomes of allosensitized children undergoing heart transplantation. In this study we analyze data from a prospective, multicenter registry to assess the relationship of allosensitization to outcome, as well as the impact of donor-specific crossmatching. Our analysis seeks to add to the emerging knowledge of allosensitization in pediatric heart transplantation by addressing some of the key procedure- and patient-related factors and analysis of other critical outcome measures, such as coronary allograft vasculopathy (CAV) and rejection.

Methods Patient population and data collection Data were collected on patients listed for heart transplantation between January 1, 1993 and December 31, 2008 at 33 institutions participating in the Pediatric Heart Transplant Study. Demographic, clinical and event data, including death, retransplantation, infection, rejection, malignancy and immunosuppression, were collected on all pediatric cardiac transplant recipients at the participating centers. The data were collected using coded event forms and sent to the data analysis center at the University of Alabama at Birmingham where the information was entered into the database, verified and corrected as needed, as described previously.10 The respective institutional review boards approved all studies. The study excluded those subjects who were listed for heart transplantation in the first 30 days of life. The rationale for excluding this population is that an elevated PRA in a neonate most likely reflects maternal allosensitization and should not pose an increased risk for antibody-mediated rejection. Follow-up on all patients was complete through December 31, 2008.

Measurement of PRA Data regarding patient sensitization obtained on the initial patient listing included percent PRA and method employed. If lymphocyte subset (i.e., percent T-cell, percent B-cell) testing was carried out these data were recorded as well. PRA data were also collected at the time of transplantation. Because various assays of PRA were employed—the complement-dependent cytotoxicity assay being the most common—we entered the highest value regardless of methodology.

Outcome measures Our study examined the impact of sensitization on pretransplant mortality and post-transplant mortality. The primary outcome measures were survival to transplantation or survival after heart transplantation. In addition, we examined the relationship between pre-transplant PRA and the time to first rejection. A rejection episode was defined as a clinical event that prompted acute augmentation of immunosuppressive therapy. Last, the risk of coronary artery disease was explored. Coronary allograft vasculopathy (CAV) was determined by coronary angiography. CAV was defined as present or absent.11

Statistical analysis We examined the data using standard descriptive statistics, including mean and standard deviation. For analysis, PRA values were stratified as follows: ⬍10%; 10% to 19%; 20% to 49%; and ⱖ50%. The stratification of PRAs was made a priori. The cut-point of 10% was logical based on UNOS recommendations. The other cut-points were defined based on clinical impression prior to undertaking analysis. We compared sub-groups using contingency tables and t-tests. Competing outcomes methods were employed to assess waitlist outcomes.12 Time to end-points, such as death, CAV or rejection, was characterized by actuarial (Kaplan–Meier) methods. Correlation between elevated PRA and patient factors was established using linear regression. We identified risk of death post-transplant by multivariate risk factor, forward, stepwise analyses in the multiphase hazard domain. Statistical significance was established at p ⫽ 0.05.

Results During the study period, 3,016 patients were listed for heart transplantation and PRA data were available for 2,500 patients (83%). Of the 2,237 patients who underwent transplantation, pre-transplant PRA data were available for 1,904 (85%). At listing, 397 (15.8%) of these patients had a PRA of ⱖ10% and 189 (7.6%) had a PRA of ⱖ50%. Analysis of patient- and procedure-related factors associated with the development of an elevated PRA at time of transplantation is shown in Table 1. A prior Norwood operation was most strongly associated with elevation of PRA. Of those subjects who were highly sensitized (PRA ⱖ50%) more than onethird had undergone a prior Norwood procedure (Table 2). Subjects with an elevated PRA at listing had a slightly longer waiting period until transplantation, as shown in Figure 1a and b. More importantly, allosensitization was associated with an increased proportion of subjects who died while on the waitlist. The waitlist mortality by 12 months was 19% in those with a PRA of ⱖ50% compared with 9% for those with a PRA of ⬍10% (p ⫽ 0.05).

Mahle et al. Table 1

Allosensitization in Pediatric Heart Transplantation

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Predictors of Higher PRA at Transplant

Variable

Coefficient ⫾ SE

Previous sternotomy Non-white Status 1 at transplant VAD at transplant Congenital etiology Previous Norwood operation

2.88 1.99 2.79 4.54 4.60 12.64

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

1.43 0.99 1.10 1.52 1.48 1.62

p-value 0.04 0.04 0.01 0.003 0.002 ⬍0.0001

PRA, panel-reactive antibody; VAD, ventricular assist device. Intercept ⫽ 1.19; R2 ⫽ 10%.

An elevated PRA was strongly associated with posttransplant mortality. Six-month survival for subjects with a PRA of ⱖ50% at transplant was 77%, compared with 93% for those with a PRA of ⬍10% (p ⬍ 0.001; Figure 2). The increased mortality risk for those subjects with PRA of 20% to 50% was less pronounced, although still statistically significant. Most of the increased risk appears to be in the first months after transplant. Among those who survived at least 1 year, there was a non-significant trend toward increased mortality for those with a higher PRA (Figure 3). Adjusting for other variables known to be associated with mortality after transplantation, an elevated PRA remained significantly associated with an increased risk of death in the early phase after transplantation (p ⬍ 0.0001; Table 3). The cause of death did not differ based on degree of allosensitization (Table 4). The percent of subjects free from rejection did not differ based on PRA levels (Figure 4). Similarly, an elevated PRA Table 2

PRA Study of Transplant Patients, 1993 to 2008

Demographics Male White Age, in years (mean) Clinical condition Congenital etiology Norwood prior to listing Sternotomy prior to listing Status at transplant Status 1 On inotropes On ventilator VAD ECMO

PRA 0 to 49 (n ⫽ 1,800)

PRA 50⫹ (n ⫽ 104)

p-value

987 (55%) 1,279 (71%) 7.7

66 (64%) 73 (70%) 6.8

0.09 0.8 0.1

724 (40%)

84 (81%)

⬍0.0001

149 (8%)

38 (37%)

⬍0.0001

741 (41%)

83 (80%)

⬍0.0001

1,362 1,055 319 177 115

(77%) (59%) (18%) (10%) (6%)

89 70 28 13 7

(86%) (67%) (27%) (12%) (7%)

Figure 1 Competing outcomes plot of waiting time to transplantation. (A) Plot for subjects with PRA ⬍10. (B) Plot for subjects with PRA ⱖ50%.

at time of transplantation was not associated with risk of developing transplant allograft vasculopathy (p ⫽ 0.8). A number of interventions can be employed to lessen the risk from allosensitization in the immediate post-transplant period. In this series, some form of non-standard immune therapy was administered in 36.8% of subjects with a transplant PRA of ⱖ20% within 30 days of transplantation (Table 5). The most commonly employed adjunctive therapy was plasmapheresis. We also sought to determine how a prospective crossmatch influences the post-transplant course for allosensi-

0.05 0.08 0.02 0.4 0.9

Data expressed as n (%), unless noted otherwise. ECMO, extracorporeal membrane oxygenation; PRA, panel-reactive antibody; VAD, ventricular assist device.

Figure 2 Survival after transplantation, stratified by recipient PRA (n ⫽ 1,904).

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The Journal of Heart and Lung Transplantation, Vol 30, No 11, November 2011 Table 4 Causes of Death After Transplantation, Stratified by PRA at Transplant PRA 0% to 9%

Cause of death

Figure 3 Percent freedom from death among patients surviving at least 1 year, stratified by recipient PRA (n ⫽ 1,354).

tized patients. Of those subjects with a PRA of ⬎20%, 16 had a negative prospective crossmatch. These subjects demonstrated survival equivalent to those with a PRA of ⬍20% (Figure 5). However, subjects with a PRA of ⱖ20% who were listed for prospective crossmatch, but received a crossmatch-positive organ, had a substantially lower 1-year survival than those not allosensitized. One may infer important relationships from the retrospective donor crossmatch data as well. A donor-specific crossmatch (either prospective or retrospective) was available for 1,255 subjects. The donor-specific crossmatch was positive in 165 (13.1%) of these subjects. A positive donorspecific crossmatch was significantly associated with higher post-transplant mortality (Figure 6).

Discussion In this study we have shown that the presence of elevated PRA in children listed for heart transplantation is associated

Table 3

Risk Factors for Death After Transplantation Early

Variable Age (younger) Age (older) African American Congenital etiology Not dilated CM ECMO Status 2 Donor age (older) Year of transplant (earlier) PRA (maximum)a

Constant

Hazard ratio

p-value

1.76

⬍0.0001

2.17

0.0001

2.53

⬍0.0001

1.09 1.73

⬍0.0001 0.008

1.15

⬍0.0001

Hazard ratio

p-value

1.04 3.42

0.004 ⬍0.0001

1.53

0.03

1.58

0.02

1.37

0.01

CM, cardiomyopathy; ECMO, extracorporeal membrane oxygenation; PRA, panel-reactive antibody. a Hazard ratio denotes 10-unit increase in PRA.

Myocardial infarction Early graft failure Fatal arrhythmia Infection Non-specific graft failure Rejection, acute Sudden cardiac death Other Total

10% to 19%

20% to 49%

ⱖ50%

36

3

1

2

16

0

2

3

7 42 11

0 2 1

1 5 1

0 4 1

68 35

1 2

2 3

4 4

104 319

3 12

8 23

21 39

PRA, panel-reactive antibody.

with longer waiting times to transplantation and increased pre-transplant mortality, presumably related to a requirement for prospective crossmatching. High PRA is also associated with a 2-fold increase in early post-transplant mortality. A negative prospective crossmatch abrogated this risk. A variety of factors have been shown to lead to sensitization in those awaiting transplantation. Exposure to HLA antigens that might elicit sensitization occurs most commonly with blood transfusions.13 We have demonstrated that a prior sternotomy, possibly simply a marker for a greater exposure to blood products, was associated with increased risk of allosensitization. Other investigators have also suggested that, when homograft material is used in reparative or palliative surgery, these materials elicit an immune response that increases the PRA titers.14,15 Such homograft material is routinely used in the management of several congenital heart lesions, such as hypoplastic left heart syndrome and pulmonary atresia. In this study, we demonstrated that patients with a prior Norwood procedure

Figure 4

Freedom from rejection, stratified by PRA.

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Table 5 Interventions to Manage Allosensitization After Transplantation in Subjects with PRA ⱖ20% (n ⫽ 185) Intervention

N (%)

Plasmapheresis at time of transplant onlya Plasmapheresis after transplant Gammaglobulin Cyclophosphamide Rituximab Any supplemental therapy to manage high PRA after transplantation

10 (5.4%) 62 12 11 5 68

(33.5%) (6.5%) (5.9%) (2.7%) (36.8%)

PRA, panel-reactive antibody. a Plasma exchange using cardiopulmonary bypass circuit.

accounted for over one third of the subjects with marked allosensitization (PRA ⱖ50%). These findings have important long-term implications as progressive heart failure is a known complication after Fontan-type palliation and may be more common in those subjects with a single right ventricle. As shown in prior pediatric studies, elevated PRA was found to be a significant risk factor for post-transplant mortality.6,7 Importantly, in our study higher PRA values seemed to confer higher risk of mortality. The risk of death was highest for those patients with a PRA of ⱖ50%. For this group of patients, the mortality at 1 year was over 2-fold greater than for those with a PRA of ⬍10%. In recent years there has been an increased understanding of the strength of antibody response. Newer solid-phase methods, such as the Luminex assay, can provide specific antibody titers to donor antigens with high sensitivity. Data forms for this study, however, did not contain this type of quantitative information. When considering the relative merits of prospective crossmatching, it may be important to consider both the breadth (i.e., higher PRA values) and strength (i.e., titer) of the antibody response. Importantly, elevated PRA appears to be a risk factor for both early and late mortality. The risk is greatest in the first few months after transplantation.

Figure 5 Survival after transplantation, stratified by prospective crossmatching. This analysis includes only those subjects who at the time of listing were specified to require a prospective crossmatch (n ⫽ 148, with 5 missing DSC).

Figure 6 Survival after transplantation, stratified by donor-specific crossmatch (n ⫽ 1,904) (obtained prospectively or retrospectively).

Presumably, patients with elevated PRA are at increased risk in part because of pre-formed alloantibodies, specific for donor allograft HLA Class I antigens, that can elicit hyperacute rejection and primary organ dysfunction. As such, one could expect that the incidence of clinically significant rejection would be higher in those with an elevated PRA. We were unable to demonstrate that patients with elevated PRA had a shorter time to first rejection episode. This lack of association is somewhat surprising. One explanation is that many centers did not routinely perform specialized staining for antibody-mediated rejection with techniques such as C4D. Hence, rejection may have been underdiagnosed in this population. One strategy to lessen the effects of allosensitization is to alter the immunosuppressive approach in the posttransplant period. These interventions included plasmapheresis, intravenous immunoglobulin, mycophenolate mofetil, rapamycin, rituximab and anti-thymocyte globulin. Several investigators have suggested that such interventions can reduce the risk of graft loss after transplantation.8,9 Pollock-BarZiv and colleagues reported 1-year survival of 71% among allosensitized patients receiving plasmapheresis with or without rituximab— essentially equivalent the survival rate noted in the pooled PHTS experience.8 One-year survival was somewhat higher in studies by Feingold et al (78%) and Holt et al (85%).5,9 However, all of these series reported 1-year survival well below the 91% for those not sensitized from the PHTS series. Therefore, it remains unclear to what degree these adjunctive therapies may improve outcomes in subjects with elevated PRA, and thus further prospective studies are warranted. In our series, 37% of subjects with a PRA of ⱖ20 received additional interventions within 30 days post-transplant to mitigate allosensitization or treat antibody-mediated rejection. The most common such intervention was plasmapheresis. Although one would like to know whether these interventions improved outcome, such an analysis would be challenging because these interventions may have been undertaken in response clinical deterioration.

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A prospective crossmatch is another method to lower the risk of acute rejection in a highly sensitized recipient.16 However, prospective crossmatching significantly reduces the availability of donor organs and prolongs the waiting time. In our study, the patients with elevated PRA at listing had a significantly longer waiting time and a significantly higher mortality while awaiting transplantation than those with a PRA of ⬍10%. This would coincide with previous series that have identified high PRA as a risk factor for pre-transplant death.6,17 Although it is beyond the scope our study, those findings raise interesting questions about the competing risks of death awaiting transplantation versus transplantation without prospective crossmatching. One could hypothesize that some patients may have a higher probability of survival with a positive prospective crossmatch than waiting many months for a negative prospective crossmatch. The introduction of the virtual crossmatch, which does not require serum from the prospective donor to be transported great distances, appears promising and may reduce the waiting times for allosensitized patients awaiting a negative prospective crossmatch.18 There have also been notable findings regarding retrospective crossmatch data. It appears that those subjects with elevated PRA and a negative retrospective crossmatch had no increased mortality compared to those with low PRA. Although one should continue to be vigilant for antibody-mediated rejection in this population, it may be reasonable to pursue a less aggressive immunosuppressive regimen. One of the major limitations of this study relates to the variable methods for determining PRA among the centers participating in the PHTS. Moreover, many institutions have changed the methods for the assay of PRA levels during the study period. The CDC assay was the most commonly used technique for determination of PRA levels. However, several investigators have shown that newer modalities, such as solid-phase assays and enzyme-linked immunoassay techniques, are more sensitive.19 One study reported that up to 25% of patients with 0% PRA by CDC were found to have positive PRA by flow-based methods.20 Those patients with 0% CDC PRA but positive flow-based PRA are at increased risk of rejection. Additional limitations need to be acknowledged in this analysis. The PHTS did not routinely collect data regarding strategies to “desensitize” subjects with high PRA awaiting transplantation. Some transplant centers have reported excellent results in lowering PRA levels before transplantation.21,22 These strategies cannot be addressed by the PTHS data. Also, it is not possible to know with certainty whether the increased waitlist mortality is due primarily to a strategy of planned prospective crossmatching. Although the transplant data form collects information regarding prospective crossmatching, the listing form does not. Finally, the PHTS data set cannot reliably distinguish between cell- and antibody-mediated rejection. In conclusion, an elevated pre-transplant PRA is significantly associated with post-transplant mortality. Because of concern for an elevated PRA, many patients may wait lon-

ger to receive a negative crossmatch organ, thereby increasing the risk of pre-transplant mortality. The role of immunologic interventions in children to mitigate the effects of allosensitization remains to be determined. Further research is critically needed in the area of allosensitization, especially among patients who have undergone palliative surgical procedures.

Disclosure statement The authors have no conflicts of interest to disclose.

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17. Wright EJ, Fiser WP, Edens RE, et al. Cardiac transplant outcomes in pediatric patients with pre-formed anti-human leukocyte antigen antibodies and/or positive retrospective crossmatch. J Heart Lung Transplant 2007;26:1163-9. 18. Zangwill S, Ellis T, Stendahl G, et al. Practical application of the virtual crossmatch. Pediatr Transplant 2007;11:650-4. 19. Mansour I, Messaed C, Azoury M, et al. Panel-reactive antibodies using complement-dependent cytotoxicity, flow cytometry, and ELISA in patients awaiting renal transplantation or transplanted patients: a comparative study. Transplant Proc 2001;33:2844-7.

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20. Tambur AR, Bray RA, Takemoto SK, et al. Flow cytometric detection of HLA-specific antibodies as a predictor of heart allograft rejection. Transplantation 2000;70:1055-9. 21. Weston M, Rolfe M, Haddad T, et al. Desensitization protocol using bortezomib for highly sensitized patients awaiting heart or lung transplants. Clin Transpl 2009;393-9. 22. Keogh A, Spratt P, Macdonald P, et al. Pilot study of low-dose azathioprine in presensitized patients awaiting heart or lung transplantation. J Heart Lung Transplant 1995;14:945-9.