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Keywords:

  • Cytomegalovirus;
  • pancreas transplantation;
  • steroid-free immunosuppression

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The impact of a prednisone-free immunosuppressive regimen was evaluated in simultaneous pancreas–kidney (SPK) recipients. Patient and graft survivals, rejection rates and the incidence of CMV disease were determined. Two hundred consecutive SPK transplant recipients received tacrolimus-based immunosuppression with (n = 100) or without (n = 100) chronic prednisone therapy. Patients were induced with lymphocyte depleting antibodies or IL-2 receptor blockers and received prophylactic antiviral therapy. Patient and graft survivals and rejection rates were not statistically significantly different between treatment groups. Two-year cumulative incidence of CMV in recipients in the prednisone-free protocol was reduced (7.2% vs. 16%; p = 0.15). Considering only recipients at highest risk (D+/R− or D+R+), incidence of CMV disease in the prednisone-free group (n = 61) compared to the steroid-treated group (n = 48) was reduced from 36% to 18% (p < 0.05). Multivariate analysis confirmed the independent effect of prednisone treatment on the incidence of CMV (RR 2.3; p = 0.04). In the prednisone-free protocol, incidence of CMV was less frequent in recipients receiving induction with Campath versus rabbit antilymphocyte globulin (2.4% vs. 12.6%; p = 0.14). Eliminating prednisone immunotherapy did not adversely affect outcomes and was associated with a reduced rate of CMV in SPK recipients of organs from sero-positive donors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Despite improvement in prophylactic therapy, cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality following simultaneous pancreas–kidney (SPK) transplantation (1–3). SPK transplant recipients appear to be at higher risk of developing CMV disease than other solid organ transplant recipients. The causes are multifactorial, but include an increase in the use of potent T-cell depleting induction agents, a higher than expected number of CMV sero-negative recipients (R−) receiving organs from sero-positive donors (D+), and the high incidence of concomitant medical comorbidities. The overall incidence of CMV disease in patients undergoing SPK using standard immunosuppression is approximately 13–17% (2,4). However, in high-risk patients (D+/R−) the incidence rises to greater than 40% (2,4,5). Furthermore, CMV is associated with increased mortality following SPK, higher rates of rejection and an increased risk of non-CMV infectious complications (5,6).

Recent improvements in immunosuppression including the use of mycophenalate mofetil (MMF) and tacrolimus have resulted in markedly improved graft survival following SPK transplantation (7,8). Furthermore, with effective induction therapy, the incidence of acute rejection has been markedly decreased. As a result, SPK transplantation can be successfully performed with a steroid-free regimen with excellent graft and patient survival (9,10). Furthermore, steroid-free regimens do not increase the incidence of rejection and, in renal transplant recipients, are associated with a reduced incidence of CMV (11).

The impact of steroid-free immunosuppression on the incidence of CMV in patients undergoing SPK has not been well characterized. In this study, we examined the incidence of CMV in a large single institution series of 200 SPK transplant recipients managed with and without the use of prednisone to assess the incidence of tissue-invasive disease and non-tissue-invasive CMV infection. Secondary end points included patient and graft survival and the incidence of rejection.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Study design

This is a single center, retrospective study of 200 consecutive SPK transplant recipients. This project was approved by the institutional review board of the Feinberg School of Medicine of Northwestern University. Transplants were performed between July 1995 and April 2003, and follow-up completed May 14, 2004. The minimum follow-up in the study cohort was 13 months and the mean 47.3 ± 24 months (range 13–106 months).

Recipient demographics and surgical procedures

Recipient demographics are shown in Table 1. The surgical issues pertaining to organ procurement and transplantation have been described elsewhere by our group, and others (12,13). The issues germane to cadaveric organ procurement impacting upon judgment to utilize the organs for transplantation included donor hemodynamics, drug and alcohol use, age, weight, history of diabetes, history of renal disease and risk of transmission of malignancy or infectious disease (e.g. HIV, hepatitis B and C) and intraoperative assessment.

Table 1.  Demographics of SPK transplant recipients
CharacteristicPrednisone + (Value or %)Prednisone-free (Value or %)
N100100
Age (years)37.2 ± 7.9; (range 15–59)41.4 ± 8.3 (range 18–59)
Gender (M:F)62:3858:42
Race
 Caucasian8287
 African American1311
 Hispanic52
Duration of diabetes (years)25.2 ± 7.525.4 ± 7.7
Cardiac revascularization pre-transplant
 CABG1314
 PTCA (±stent)78
Dialysis duration17.4 ± 10.819.4 ± 11.1
 pre-transplant (range 1–60 months) (range 1–56 months)
Dialysis modality pre-transplant
 Hemodialysis4852
 Peritoneal dialysis2622
 None2626
HLA match
 A/B match0.9 ± 0.90.8 ± 0.9
 DR match0.3 ± 0.50.4 ± 0.5
PRA (mean)
 Current3.5 ± 10.7%5.6 ± 18.1
CMV serostatus
 D+/R−40.6% (n = 33)10.3% (n = 30)
 D+/R+25.6% (n = 15)12.5% (n = 31)
 D−/R+0% (n = 16)0% (n = 14)
 D−/R−2.8% (n = 36)0% (n = 25)

Immunosuppression, therapeutic drug monitoring and rejection therapy

Two strategies for immunosuppression were employed. For patients transplanted between July 1995 and November 1999, quadruple immunosuppression including corticosteroids was used (n = 100). Recipients received induction therapy with either equine antithymocyte globulin (n = 49; ATGAM®, Pharmacia and Upjohn Co., Kalamazoo, MI), or an IL-2 receptor antagonist (daclizumab, n = 32; basiliximab, n = 2). Seventeen recipients did not receive induction therapy. All patients received mycophenolate mofetil (MMF; Cellcept, Roche, Nutley, NJ). The starting target dose was 3 g/day and was reduced to 2 g/day at 6 months post-transplant. All recipients also received tacrolimus. Target 12-h trough concentrations were 10–14 ng/mL by second generation immunoassay (IMX®, Abbott Labs, IL). The 12-h trough concentrations were constant throughout the post-transplant course. Corticosteroids were initiated intraoperatively as 500 mg of Solumedrol®, then prednisone was used and tapered over a year to 5.0–7.5 mg.

For patients (n = 100) transplanted between January 2000 and April 2003, a prednisone-free immunosuppression protocol was used. Patients received induction therapy with either antilymphocyte globulin (rabbit) [n = 58; Thymoglobulin®, Sangstat, Cambridge MA] or alemtuzumab (n = 42; Campath 1H®, ILEX Oncology, Inc., San Antonio, TX). The dose of antilymphocyte globulin (rabbit) was 6 mg/kg, and that of Campath was 30 mg. Maintenance immunotherapy was tacrolimus-based (target 12-h trough concentration of 8–12 ng/mL) and combined with either MMF (n = 62; starting target dose was 3 g/day and was reduced to 2 g/day at 6-months post-transplant) or sirolimus (Rapamune, Wyeth-Ayerst Laboratories, Madison, NJ; n = 38; target 24-h trough concentration 6–8 ng/mL). Acute rejection episodes were diagnosed in all cases by ultrasound-guided percutaneous renal or pancreas allograft biopsy. Antirejection therapy to treat acute cellular rejection of the kidney and/or pancreas allograft involved a 7–14 day course of OKT3 (Orthoclone®, Ortho Biotech, NJ) or rabbit antilymphocyte globulin.

CMV prophylaxis and treatment, diagnosis, and definition of disease

CMV antiviral prophylaxis was administered to all recipients post-transplant and after treatment of a rejection episode. Between July 1995 and July 2002, recipients received therapeutic doses of intravenous ganciclovir during the inpatient stay. Ganciclovir or valganciclovir was prescribed to all recipients for at least 3 months post-transplant. In the group of patients treated with prednisone, recipients in the high-risk subgroup (D+/R−) also received CMV hyperimmune globulin over 16 weeks. From July 2002 to April 2003 patients were treated exclusively with 3 months of oral valganciclovir.

Diagnosis of CMV was established by a combination of clinical findings and viral detection by shell vial assay or antigenemia assay. Recipients with the diagnosis of non-invasive CMV disease, sometimes referred to as 'CMV syndrome', had clinical symptoms of fever and malaise, signs of leukopenia, without evidence of tissue invasion. All recipients with the diagnosis of non-invasive CMV disease were hospitalized to initiate treatment. Recipients with tissue-invasive CMV infection had the same clinical symptoms and signs as those with non-invasive CMV disease, with the addition of tissue invasion diagnosed by tissue biopsy. All cases of CMV disease were treated with a 2-week course of parenteral ganciclovir followed by 3 months of oral administration of ganciclovir. If tissue-invasive infection occurred, CMV hyperimmune globulin was also administered.

Other medical therapy

Perioperative antimicrobial therapy was used for 7 days and included vancomycin and piperacillin/tazobactam (aztreonam and metronidazole if patient penicillin allergic) and fluconazole. Trimethoprim/sulfamethoxazole was prescribed daily. Prophylactic anticoagulation consisted of enoxaparin (Lovenox®) 30 mg SQ qd for 3 days, and enteric-coated aspirin daily (325 mg). Oral nystatin or mycelex troches, and antiulcer prophylaxis were also prescribed. For recipients of bladder drained pancreas transplants, bicarbonate supplementation was prescribed.

Statistical analyses

Continuous data are presented as mean ± standard deviation. Actuarial 2-year patient and graft survival rates were determined for the 200 recipients of SPK transplants from the time of transplantation. Kidney graft failure was defined as removal, loss of function requiring return to dialysis or death with a functioning graft. Pancreas graft failure was defined as removal of the graft, loss of endocrine function requiring return to exogenous insulin therapy or death with a functioning graft. Actuarial survival estimates were calculated using Kaplan–Meier life table analysis (14), and the series was followed through May 14, 2004.

Between-group comparisons of time to CMV disease were made using the Cox–Mantel statistic; the Bonferonni method of adjustment was used when multiple pairwise comparisons were made. Initially, univariate Cox proportional hazard models were used to assess the effects of various clinical and treatment variables on time to CMV disease. The clinical and treatment variables included: age, gender, diabetes duration, dialysis duration, coronary artery revascularization, induction therapy (IL-2RA, ATGAM, Thymoglobulin, Campath), maintenance immunosupprression (MMF, sirolimus, prednisone), acute rejection, technique of pancreas graft exocrine drainage, pre-transplant coronary revascularization, and donor and recipient CMV serologic status. Predictors associated with a p value < 0.30 in the univariate Cox models were considered as candidate predictors for the multivariate Cox model analyses. The relative risk and the associated 95% confidence interval for each predictor are presented. All statistical tests were two-tailed, and p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Graft and patient outcome

The overall patient, kidney and pancreas graft survivals were 98.5%, 95.4% and 94.5% at 1 year and 96.1%, 92.5% and 91.0% at 2 years (Figure 1). Patient survival did not vary between groups treated with and without prednisone (96.3% vs. 96.0%; p = 0.41) at 2 years. Prednisone-free treatment did not affect 2-year kidney graft survival (93.0% with prednisone vs. 91.8% without prednisone; p = 0.23) or pancreas graft survival (90.0% with prednisone vs. 91.9% without prednisone; p = 0.92). Furthermore, prednisone-free treatment with appropriate induction therapy was associated with a rate of acute rejection at 12 months of 4.0% vs. 14.0% (p = 0.12) for recipients with prednisone.

image

Figure 1. Six year actuarial patient and graft survival rates in SPK transplant recipients given tacrolimus-based immunosuppression including (n = 100) or excluding (n = 100) prednisone therapy. The actuarial 2-year survival rates (patient, kidney, pancreas) were 96%, 93% and 90% in patients with immunosuppression including prednisone and 96%, 92% and 92% in the absence of prednisone.

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Rates of CMV disease and infection

The overall (n = 200) actuarial 2-year rates of CMV were 12.8% (5.1% non-invasive, 7.7% tissue invasive). The cumulative incidence of CMV varied markedly according to donor and recipient CMV serologic status. Considering the entire study cohort (n = 200), the incidence of CMV at 2 years for each of the four subgroups was as follows: D−/R− group (0%) (N = 61); D−/R+ group (1.6%) (N = 30); D+/R+ group (17.5%) (N = 46); and D+/R− group (26.4%) (N = 63). As expected, recipients of organs from donors that were sero-positive for CMV had the highest rates of CMV post-transplant.

Rate of CMV disease in patients with and without prednisone therapy

Figure 2 shows the incidences of CMV according to donor and recipient CMV serologic status stratified by the use of prednisone therapy. Avoidance of prednisone treatment was associated with a trend toward a reduction in the overall rate of CMV (8.6% vs. 17%; p = 0.15).) The rate of non-invasive CMV disease was statistically significantly (p = 0.05) reduced in the prednisone-free group (3.1%) vs. the prednisone group (12%). There were no significant differences in the rate of tissue-invasive CMV infection (5.5% vs. 5.0%; p = 0.9). Figure 3A illustrates that among the highest risk patients, those with CMV-positive donors (D+/R− and D+/R+), the overall (non-invasive and tissue invasive) rate of CMV was statistically significantly (p = 0.05) reduced in the prednisone-free group (18%; n = 61) vs. the prednisone treated group (36%; n = 48). The rate of non-invasive CMV disease was statistically significantly (p = 0.02) reduced in the prednisone-free group (8.8%) vs. the prednisone treated group (25.6%) (Figure 3B). There were no significant differences in the rate of tissue-invasive CMV infection (9.1% vs. 10.4%; p = 0.77) (Figure 3C).

image

Figure 2. Incidence of any CMV in SPK transplant recipients receiving tacrolimus-based maintenance immunosuppression with prednisone (Pred+) (n = 100) or in the absence of prednisone (Pred−) (n = 100) according to donor (D) and recipient (R) CMV serologic status.

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image

Figure 3. Incidence of any CMV (A), non-invasive CMV disease (B), and tissue-invasive CMV infection (C), in SPK transplant recipients at high risk (D+/R+, D+/R−) receiving prednisone (Pred+) (n = 100) or in the absence of prednisone (Pred−) (n = 100) therapy.

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Univariate and multivariate analyses of predictors of CMV in all patients

The effect of clinical and treatment variables on the risk of acquiring CMV (tissue invasive and non-invasive) in the overall patient population based on the univariate analysis is presented in Table 2. Several demographic factors (male gender, diabetes duration, prior cardiac revascularization) were significant in the univariate analysis. In addition, there was a trend toward a higher rate of CMV in prednisone-free immunosuppression patients treated with MMF compared to sirolimus. However, in the multivariate Cox proportional hazard analysis, only three factors were found to be associated with an increase in the development of CMV (Table 3): positive CMV serologic status of the donor (relative risk 27.927; p = 0.001), diabetes duration (RR 1.045 [per year]; p = 0.04) and prednisone therapy (RR 2.318; p = 0.04).

Table 2.  Univariate analyses of predictors of CMV in all SPK transplant recipients
VariableRelative risk95% CI–RR p-value
Male gender2.480.99–6.230.05
Age (per year)1.030.98–1.080.21
Diabetes duration (years)1.051.00–1.110.03
Dialysis duration (mos)1.010.98–1.040.66
Cardiac revascularization1.930.86–4.400.10
Donor CMV positive25.283.34–194.90.001
IL-2 receptor antagonist2.130.93–4.860.07
ATGAM® induction1.170.50–2.770.69
Thymoglobulin® induction1.050.45–2.470.90
Campath® induction0.170.02–1.280.08
Prednisone therapy1.800.79–4.150.15
MMF (vs. Rapamune)2.120.84–5.410.11
Rejection0.340.08–1.490.14
Table 3.  Multivariate analyses of predictors of CMV disease in SPK transplant recipients
VariableRelative risk95% CI–RR p-value
Donor CMV positive27.933.64–214.090.0011
Diabetes duration (years)1.051.00–1.090.0396
Prednisone therapy2.321.01–5.300.0422

Univariate and multivariate analyses of predictors of CMV in high-risk patients

The effect of prednisone therapy was subsequently evaluated in SPK transplant recipients at highest risk for the development of CMV disease (D+/R+, D−/R+). Multivariate analysis of this cohort confirmed the association of prednisone treatment with higher rates of CMV (2.27; p = 0.05). There were no other variables that were significantly associated with a higher rate of CMV in this population.

Impact of induction therapy on prednisone-free patients

Analysis of the choice of induction therapy in the prednisone-free cohort was conducted to assess the differential effects of rabbit antilymphocyte globulin therapy (n = 58) and Campath (n = 42). When comparing the outcome following antilymphocyte globulin and Campath induction at 2 years, there were no significant differences in patient survival (96.6% vs. 97.6%; p = 0.83), kidney graft survival (93.1% vs. 92.3%; p = 0.46) or pancreas graft survival (94.8% vs. 90.5%; p = 0.12).

In the overall prednisone-free cohort (n = 100), the choice of induction therapy did not affect the rate of CMV disease. However, the incidence of tissue-invasive CMV infection in patients who received Campath induction (0.0%) was lower than those who received antilymphocyte globulin (8.8%), although this did not reach statistical significance (p = 0.85). Furthermore, in SPK transplant recipients at high risk (D+/R−, D+/R+) for CMV, Campath was associated with a reduction in the risk of any CMV, 4.0% vs. 19.4% (antilymphocyte globulin; p = 0.14) (Figure 4). Stratifying for severity of disease in the high-risk group (D+R−, D+R+), the rate of tissue-invasive CMV infection in the Campath group was less (0%) compared to the antilymphocyte globulin treated recipients (13.9%; p = 0.09). while the rate of non-invasive CMV disease in the Campath group was comparable (4% vs. 5.6% antilymphocyte globulin; p = 0.84).

image

Figure 4. Incidence of any CMV in prednisone-free SPK transplant recipients at high risk (D+/R+, D+/R−) according to induction therapy: antilymphocyte globulin (n = 58) vs. Campath (n = 42).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Advances in immunosuppression have resulted in a significant improvement in survival following kidney and pancreas transplants. Nationally, 1- and 5-year patient survival is now 95% and 84%, while pancreas allograft survival is 85% and 70% (15). This success reflects the improved results following the introduction of tacrolimus, MMF and sirolimus into the SPK immunosuppressive protocols. Steroid-free therapy is now being safely accomplished in SPK transplant recipients (9,10). In patients with diabetes, elimination of steroid therapy may have additional advantages including improved glucose tolerance, reduced levels of peripheral hyperinsulinemia and, although not yet proven, improvement in overall patient survival.

Reduction in the incidence and severity of CMV disease and infection following SPK remains an important goal in post-transplant management. CMV has been associated with higher rates of patient mortality, graft rejection and non-transplant infection (1,5). This effect is particularly evident in the highest risk patients (D+/R−). Nationally, patients who are (D+/R−) have a significant increase in 5-year mortality when compared with low risk (D−/R−) patients (RR 1.39; p < 0.001) (6).

Thus, alternative strategies are needed to reduce the risk of acquiring CMV disease. Current viral prophylaxis strategies using ganciclovir and its oral equivalent valganciclovir have been effective in reducing CMV following transplant in the renal transplant population, although disease recurrence can occur following cessation of therapy (2,3,16–19). In addition, viral prophylaxis appears to delay the onset of disease in the high-risk population, allowing patients to recover from the surgical stress of transplant prior to developing active disease. In this study, patients were treated with a series of chemopreventive strategies including ganciclovir (IV and PO) and CMV hyperimmune globulin in patients receiving prednisone. Valganciclovir alone, once available, was used in the prednisone-free group. Despite effective therapy, CMV developed in 12.8% of recipients overall.

Minimization immunosuppressive strategies may offer another avenue to reduce the incidence of CMV in SPK patients. The elimination of the use of corticosteroids appears to provide a successful new approach to the reduction of CMV disease. In the kidney population, Matas et al. (11), reported a significant reduction in the incidence of CMV using prednisone-free immunosuppression when compared with historical controls. In SPK patients, Cantarovich et al. (9) reported an incidence of 28.5% in SPK patients treated without the use of steroids, as compared to 60% reported in other groups using similar induction therapy and prednisone.

This study sought to determine the impact of immunosuppressive minimization on the incidence of CMV disease and infection in SPK transplant recipients. This series demonstrated the impact of prednisone-free immunosuppression in the SPK patients who all received T-cell depleting induction therapy, tacrolimus-based maintenance immunosuppression and prophylactic antiviral therapy. We made two important observations: (1) when the entire transplant cohort was considered, the rate of non-invasive CMV disease was statistically significantly reduced in the prednisone-free group; and (2) when the recipients at high risk (D+/R− and D+/R+) were considered, the overall (non-invasive and tissue invasive) rate of CMV was statistically significantly reduced in the prednisone-free group. Furthermore, early trend data suggested that the incidence of CMV may be reduced even further in patients receiving Campath induction when compared to patients treated with antilymphocyte globulin. Importantly, the immunosuppressive modification did not compromise patient or graft survival or result in higher rates of acute rejection.

This study has several potential limitations. Because this is a non-randomized, retrospective trial, there may be other significant alterations of patient characteristics or clinical management that had a role in reducing the incidence of CMV. The change in induction agent may have been associated with a change in the incidence of CMV. However, in the univariate analysis, there were no significant differences in CMV occurrence according to induction agent. In fact, more potent T-cell depleting agents were used routinely in the prednisone-free cohort compared to the prednisone-treated recipients. The use of the agents would be more likely to bias the study in favor of prednisone containing regimens as IL-2 receptors have previously been associated with a reduction in the incidence of CMV in renal transplant patients (20). Furthermore, a multivariate analysis was used to assess the independent impact of a variety of potential factors and only CMV serostatus, duration of diabetes and prednisone therapy were found to be significant predictors of CMV disease.

With respect to the choice of maintenance immunosuppression, all recipients received tacrolimus and either MMF or sirolimus. Univariate and multivariate analyses did not indicate that the relative risk of CMV was statistically significantly influenced by MMF or sirolimus, although there was a trend to lower risk with sirolimus. Reduced risk of CMV in liver transplant recipients given sirolimus has been reported (21). There was also a small (2 ng/mL) reduction in the goal tacrolimus level that may have contributed modestly to the reduction in the incidence of CMV disease. Thus, while the potential for a confounding effect exists, it appears that the absence of prednisone contributed significantly to the reduction in CMV in the high-risk population.

An additional potential limitation is the change in viral prophylaxis agents (from oral gancyclovir to valgancyclovir). However, patients treated in the prednisone group appeared to have been treated more aggressively using both IV and PO ganciclovir and anti-CMV immunoglobulin in high-risk patients when compared to patients in the prednisone-free group. Furthermore, valganciclovir has not been shown to be more effective than oral ganciclovir when used in appropriate dosage (16,22). Thus, the observed effects are unlikely to be due to a modification in antiviral therapy.

In summary, the elimination of prednisone was associated with a reduction in the incidence of CMV in high-risk (D+/R−, D+/R+) SPK transplant recipients without compromising transplant outcomes. The use of steroid-free protocols in addition to appropriate antiviral therapy should be strongly considered to reduce the burden of CMV in this population.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References