Objectives: Posttransplant lymphoproliferative disorder is a known complication of solid-organ transplant. The use of depleting induction agents has demonstrated varying associations with incidence of posttransplant lymphoproliferative disorder. Alemtuzumab, a de­pleting induction agent for kidney transplant patients, has shown promising results in reducing the risk of acute rejection and graft loss in the first year. Its unique mechanism of depleting both T-cell and B-cell populations may be beneficial in preventing the occurrence of posttransplant lymphoproliferative disorder.

Materials and Methods: We examined the known risk factors for posttransplant lymphoproliferative disorder in the setting of alemtuzumab induction to determine whether incidence increases with this induction agent. We reviewed medical records of all alemtuzumab-induced kidney transplants from March 2006 to November 2015.

Results: Of the 675 transplant patients who received alemtuzumab induction, 10 developed posttransplant lymphoproliferative disorder, with a cumulative inci­dence rate of 1.5%. All diagnosed patients had several known risk factors associated with post­transplant lymphoproliferative disorder: 7 with advanced age over 60 years, 5 being cytomegalovirus-negative recipients, and all 10 donor kidneys being male patients and Epstein-Barr virus positive before transplant.

Conclusions: The incidence rate seen in our patient population was within the range of the average in the United States but far lower than the incidence rates associated with other induction agents. Alemtuzumab is associated with a lower cumulative incidence rate of posttransplant lymphoproliferative disorder compared with published reports of other induction treatments.

Key words : Campath, Kidney transplant outcomes, Transplantation

Introduction

Posttransplant lymphoproliferative disorder (PTLD) is a well-documented complication of solid-organ transplant procedures.^1,2 Most occurrences of PTLD originate from uncontrolled Epstein-Barr virus (EBV) activation, which causes an unregulated trans­formation and proliferation of nodal and extranodal lymphocytes.² Within populations of transplant patients, the disorder usually has an incidence rate that ranges between 1% and 2%; however, it can have a higher incidence in pediatric populations.³ According to the Organ Procurement and Transplantation Network data of 2012, the risk of developing PTLD after kidney transplant had a cumulative risk of 1.3%.⁴ Mortality has been shown to be roughly 50%, with some variations associated with time of diagnosis since transplant, multiple- versus single-site cancer location, B-cell-predominant malignancy, and the continued increased risk after the first year of diagnosis.^3,4,5

A number of factors have been associated with the development of PTLD, including transplant from EBV-seropositive donors to EBV-seronegative reci­pients, coexisting cytomegalovirus (CMV) infection, and the use of certain immunosuppressive agents such as muromonab-CD3 (OKT3) or tacrolimus.^6,7 Depleting induction agents have also been shown to have varying associations with PTLD incidence.¹

Alemtuzumab is a CD52-specific monoclonal antibody that has primarily been used to treat B-cell chronic lymphocytic leukemias.⁸ Because of its deleterious effects on B cells, it has been effective as a depleting induction agent for kidney transplant. Alemtuzumab has been shown to reduce the risk of acute rejection and graft loss in the first year after transplant and is unique in that it depletes both T-cell and B-cell populations.¹ Kirk and associates suggested that this property of alemtuzumab would be beneficial toward preventing PTLD, as B-cell depletion is a known treatment modality for PTLD.¹ Our study sought to prove that alemtuzumab can be used safely as an induction agent while decreasing or having no effect on the incidence rate of PTLD.

Materials and Methods

This is a single-center, Internal Review Board-approved retrospective study from March 2006 to November 2015 that compared 10 kidney transplant patients diagnosed with PTLD versus 665 kidney transplant patients without PTLD, all of whom received alemtuzumab induction. All patients received an immunosuppression induction therapy of methylprednisone (500 mg intravenously) and alemtuzumab (300 mg intravenously or 0.5 mg/kg if less than 60 kg). Postoperatively, patients were given a steroid taper of methylprednisone (250 mg intravenously on postoperative day 1 and 125 mg intravenously on postoperative day 2) and oral prednisone (60 mg on postoperative day 3, 40 mg on postoperative day 4, and 20 mg on postoperative day 5). Tacrolimus at 1.5 mg by mouth twice daily and mycophenolic acid at 540 mg twice daily were both started on postoperative day 1.

Data were collected by analyzing clinical records from the hospital databases of all alemtuzumab-induced transplant patients over a 9-year study period. We confirmed PTLD through follow-up notes and pathology reports. Recipient variables were age, sex, ethnicity, blood type, EBV and CMV status, number of transplants, transplant date, graft failure, and graft rejection. Donor variables included donor type (living vs deceased) and EBV and CMV status. Graft and patient survival rates were defined as the time between transplant and November 3, 2015, the time at which our data were collected.

Both PTLD and non-PTLD cohorts were analyzed for differences in their demographic and transplant-related data, as described below. Statistical analyses were performed using the IBM SPSS statistics software, version 21 (IBM Corp., Armonk, NY, USA). Continuous variables were presented as means for cases of normal distribution or medians for cases of nonnormal distribution. Discrete variables are presented as rates. Continuous variables were compared by t tests or nonparametric tests, whereas discrete variables were compared by chi-square tests. Survival was estimated using Kaplan-Meier analysis. The alpha error was set at 0.05, and all P values indicated two-sided tests. All protocols, experimental studies, and clinical trials involving human participants were approved by the ethics committee of the institution before the study began. Protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration. Informed consent was obtained from all patients or their guardians.

Results

Between 2006 and 2015, our records showed that 675 transplant patients received alemtuzumab induction therapy. Of these, 10 developed PTLD, resulting in a cumulative incidence rate of 1.5%, with 6 patients having pathology records confirming diagnoses and 4 being diagnosed at outside institutions where pathology records were not available. Of the 6 who had pathology reports, 3 had kappa-restricted B-cell lymphoproliferative disease, 1 had extramedullary plasmacytoma-like type PTLD, 1 had high-grade B-cell lymphoma, and 1 was positive for a lambda light-chain monoclonal serum protein PTLD. Only 3 malignant tissue samples were positive for EBV, and the remaining were not stained for EBV.

All PTLD patients were males, 9 were White, and 1 was Hispanic. Of the 665 patients who did not develop PTLD, 419 (63%) were males and 246 (37%) were females. The ethnicities of the non-PTLD patients were as follows: 69.4% White (448 patients), 22.9% African American (152 patients), 4.5% Hispanic (30 patients), 2% Asian (13 patients), and 3.3% designated as other (22 patients) (Table 1).

Seven of the 10 PTLD patients were CMV negative. In the non-PTLD group, 40.8% were negative for CMV before transplant. When CMV donor/recipient matching was compared, we found that 30% of PTLD patients were CMV negative and received a kidney from a CMV-positive donor, whereas 16% of non-PTLD patients were donor positive/recipient negative. In the PTLD group, 40% were donor negative/recipient negative, 10% were donor negative/recipient positive, and 20% were donor positive/recipient positive. In the non-PTLD patient group, 24.8% were donor negative/recipient negative, 19.7% were donor negative/recipient positive, and 39.5 % were donor positive/recipient positive (Table 1).

In our analysis of EBV status of both donors and recipients in the PTLD group, 4 patients were EBV negative before transplant, 5 patients were EBV positive, and 1 patient had unknown EBV status. All PTLD patients received EBV-positive kidneys (Table 1). In the non-PTLD group, 5.6% of patients had an EBV-negative serostatus before transplant, with the remaining having an EBV-positive serostatus. In the non-PTLD group, 90% of patients received kidneys from EBV-positive donors; thus, 4.8% were donor positive/recipient negative, 86.6% were donor positive/recipient positive, 8% were donor negative/recipient positive, and 0.6% were donor negative/recipient negative.

After transplant, grafts failed in 8 patients (80%) in the PTLD group (6 died with a functioning graft). In the non-PTLD, only 25% of patients had graft failure (166 patients; 72 died with a functioning graft). Graft rejection rate was 20% in PTLD-positive patients and 27.1% in the non-PTLD patients (Table 2).

After diagnosis of PTLD, 2 patients were treated with rituximab + cyclophosphamide-hydroxydaunorubicin-vincristine-prednisone, 1 patient received only rituximab, 2 received "other chemotherapy," 1 received fluorouracil, and 1 received carfilzomib, lenalidomide, and dexamethasone. Two patients received no treatment for PTLD, and 1 patient's treatment regimen was unknown. At last follow-up, 6 of the 10 PTLD patients had died, 3 due to PTLD, 2 because of bacterial septicemia, and 1 of unknown causes. The remaining 4 patients are still being treated and monitored (Table 2).

In a further analysis, we analyzed patients before the current alemtuzumab induction protocol, during which 759 patients received kidney transplants from March 1997 to March 2006. Seven of these patients developed PTLD. These 7 patients were all male patients with average age at diagnosis of 57 years and with 3 patients diagnosed at above 65 years old. Five patients were tested for CMV status, and 2 were positive. Two patients were tested for EBV status, and both were positive.

Discussion

Although it is often used to treat refractory chronic lymphocytic leukemia, alemtuzumab can be effective as a depletion induction agent, acting as a CD52 monoclonal antibody.8 Kirk and associates1 postulated that alemtuzumab is associated with lower incidence rates of PTLD versus other induction agents. Alemtuzumab has unique selective effects on B-lymphocyte populations that differs from other induction agents. These other agents have been shown to increase the incidence rate of PTLD, such as with thymoglobulin or OKT3.1 Our patient population had an overall PTLD incidence rate of 1.5%, falling within our national average of 1% to 10%. Alemtuzumab did not negatively affect the incidence rate of PTLD, allowing us to utilize the beneficial properties of induction therapy without increasing the rates of PTLD, matching previous findings.1

We had pathologic results for 6 of the 10 PTLD patients in our study, 4 of which were monomorphic PTLD subtypes (3 having kappa restrictive B-cell lymphoproliferative disorders and 1 having high-grade B-cell lymphoma). The remaining 2 patients could not be classified using the World Health Organization classification (1 demonstrated an extramedullary plasmacytoma-like PTLD and the other demonstrated lambda light-chain PTLD associated with a monoclonal serum protein). In the literature, approximately 85% of PTLD cases are monoclonal and of B-cell origin, with the remaining 15% made up of the rarer subtypes.1 There has been an increase in the number of these rarer subtypes; however, monoclonal B-cell-based PTLD continues to be the most predominate form.⁹ B-cell predominance has been shown to be associated with lower survival rates.⁵ In our study, half of the known patients with B-cell PTLD were deceased when our data were collected. Trofe and associates demonstrated that patients with a B-cell-predominant PTLD had a 33% survival rate versus 49% in those without this form.⁵

Our patient demographic results followed trends seen in PTLD patients. Nine of the 10 PTLD-positive patients were White males. In the non-PTLD patient population, 63.2% were White males. The literature complements our findings, with higher incidence of PTLD in male patients, especially those with White ethnicity.^6,10 Kirk and associates showed that kidney transplant recipients of White ethnicity have a higher relative risk of PTLD versus the general population (1.957).¹ Trofe and associates showed that neither ethnicity nor sex had any associated risk with mortality.⁵

The average age of our PTLD patients at transplant was 61.9 years old, with the youngest being 50 years and oldest being 75 years. The average age of patients in our non-PTLD group was 52 years. Pediatric patients are at the highest risk of developing PTLD, with a relative risk of 3.672; however, being over 60 years old at time of transplant has been found to be an independent risk factor for PLTD.^1,11 Age-associated mortality is not related to a specific age; however, the time between transplant and diagnosis of PTLD is associated with mortality.⁵ Patients who were diagnosed within 1 year after transplant had a greater risk of dying.5 In our study, half of the patients who succumbed to their illness were diagnosed with PTLD within 1 year of transplant.

Posttransplant lymphoproliferative disorder was located in the extranodal tissue in 5 patients, histologically seen in lymph nodes in 2 patients, and uncharted in 4 patients. Studies have shown that the most common location of PTLD is in the extranodal tissue, with the second most common being extranodal and nodal, and the least common site being in the lymph node.¹⁰ Mortality related to PTLD location is not associated with a specific tissue; however, having a single versus multiple site location of the malignancy does play a role.⁵ In our study, no data were collected on whether the patient had single versus multiple site locations.

Few studies have included both the EBV serostatus of the transplant recipients and donors.^11-13 In our analysis of patients who developed PTLD, 50% were EBV positive, 40% were negative before transplant, and 10% had an unknown EBV status. All 10 of our PTLD patients received kidneys from donors who were EBV positive. In addition, 40% of PTLD patients were mismatched as EBV donor positive/EBV recipient negative, compared with 4.8% in the non-PTLD group. Pretransplant EBV status is possibly the single most important risk factor for PTLD development.^3,5,6 Dharnidharka and associates stated that an EBV-positive kidney transplanted to an EBV-seronegative recipient can cause a 20-fold increase in a patient's risk of developing PTLD.¹² Kirk and associates found a relative risk of 5.255 for EBV-negative versus EBV-positive recipients at transplant.¹ McDonald and associates documented that the relative hazard ratio increased from 4.7 to 6.1 when an EBV seronegative recipient was paired with a seropositive donor.¹⁴ Curiously, Trofe and associates showed that patient mortality was not affected by EBV status.⁵ In our PTLD cohort, half of the patients who died were EBV negative before transplant.

Seven of 10 patients (70%) in our PTLD group had a negative CMV test before transplant, and 50% received kidneys from seronegative CMV donors. Recipient CMV-negative serostatus has been reported to cause a 6- to 7-fold increase in patient risk.^3,15 Studies have shown that CMV-negative recipients who receive CMV-positive transplants have a relative risk of 1.489, and CMV-negative recipients who receive seronegative CMV donor organs have a relative risk of 2.036.6,16 Four of the 10 patients in our study had this donor-negative/recipient-negative relation. However, if we focus on recipient CMV status alone, 7 of our patients had a CMV-negative status before transplant. Grundy and associates demonstrated that posttransplant CMV infection is common and possibly a significant contributor to PTLD development in CMV-negative recipients.¹⁷

After transplant, 20% of PTLD patients had graft rejection before development of the malignancy. In the non-PTLD cohort, 27.1% had some form of rejection posttransplant. No correlations were shown between PTLD and rejection; however, one can postulate that higher rates of pre-PTLD graft rejection would be associated with higher rates of PTLD. Initial treatment of rejection is immuno­suppression, which would cause a depression in a patient's immune system, thus allowing uncontrolled EBV activation and proliferation.

In the PTLD group, 6 of 10 patients had died at the time of study secondary to malignancy or bacterial septicemia. In the United States, the 1-year mortality rate after the diagnosis of PTLD is 30% to 60%, with the overall mortality rate due to malignancy being roughly 50%.^3,5 A number of factors can affect the mortality rate, including time from transplant surgery to diagnosis, B-cell predominant malignancy, and single site versus multiple site malignancy; in addition, risk of death increases by 2% each year after diagnosis.⁵ In our patient group, 3 of 6 patients who died were diagnosed within 1 year after transplant. Three patients died more than 1 year after transplant surgery, at 2628 days, 462 days, and 759 days posttransplant (Table 1). Two of the patients who died within 1 year of diagnosis also had the only known pathology showing B-cell-predominant PTLD. Single site versus multiple site malignancy was unknown in our patients. Only 1 patient survived for more than 1 year after diagnosis; this patient was the same patient who developed PTLD the latest after their transplant surgery (at 2628 days). This patient died 534 days after diagnosis. At last follow-up, 2 patients who were diagnosed > 2000 days after transplant remain alive. Further follow-up could demonstrate that there are varying growth rates associated with PTLD.

All 6 of the patients who died had functioning grafts, bringing into question their transplant rejection regimen. Treatment of PTLD is centered around the reduction of immunosuppression, which may place patients at a higher risk of developing graft failure. If all 6 patients had functioning grafts at time of death, their immunosuppression could have been decreased, treating the progression of PTLD and possibly preventing bacterial septicemia. Information is scarce about the exact treatment of PTLD; however, we agree that there is a deliate balance between PTLD treatment and graft failure. In addition, there are also varying regimens to treat PTLD. In our study, 5 different medications were used, making it hard to show any trends between specific treatments and patient outcomes. Future studies should specifically examine differing treatment modalities and their clinical outcomes.

Our study's strengths include it being a single center study where all patient information was collected from a large database of electronic medical records over a large time period, thus allowing us to identify donor and recipient variables and pathologies. In addition, all patients had received the same induction protocol before transplant surgery. The weaknesses of our study include the small number of patients diagnosed with PTLD, preventing any statistically significant analyses, database entry errors, missing diagnostic pathology of 4 of 10 patients, 1 patient lost to follow-up, no record of single versus multiple site location of PTLD, and no standardized treatment of PTLD done for our patients, which can be evident in many retroactive clinical studies. In addition, we were not able to compare our patients versus those treated with other induction agents. This is similar to other single center studies on alemtuzumab and its impact on PTLD, such as the study from Supe-Markinovina and associates, in which all patients received induction with single-dose alemtuzumab and a steroid-free protocol with tacrolimus and mycophenolate mofetil.18 Unfortunately, it is impractical to conduct a randomized trial with a control group at a health institution, which often requires a standardized, singular protocol for the treatment of patients.

In our single-center analysis, we found that alemtuzumab can be used as an effective induction agent for kidney transplant without increasing the incidence of PTLD. Although we are unable to make any statistically significant associations, we demonstrated that our patients had many of the known risk factors associated with PTLD, including male sex, White ethnicity, being CMV negative before surgery, and receiving EBV-positive donor kidneys. Although there are small variations in the literature, we agree that ethnicity, sex, and EBV and CMV statuses are the greatest risk factors associated with PTLD. Physicians can use these risk factors to screen and match patients with the best donors to decrease the chances of developing PTLD. Additionally, physicians can utilize the trends that we found associated with mortality and treatment to maximize the health of patients. Six of our patients who died had functioning grafts, demonstrating that we could have reduced their immunosuppression regimen. Future studies can standardize the treatments of PTLD to find more definitive risk factors associated with mortality.

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