Ganciclovir and Acyclovir Reduce the Risk of Post-Transplant Lymphoproliferative Disorder in Renal Transplant Recipients

Authors


Corresponding author: Donnie Funch, dfunch@epidemiology.com.

Abstract

Given its association with Epstein-Barr virus (EBV), there is considerable interest in assessing the impact of prophylactic anti-viral therapy on post-transplant lymphoproliferative disorder (PTLD). A recently completed multi center case–control study assessed the impact of immunosuppressive therapy on PTLD risk among renal transplant patients and collected information on the use of anti-viral therapy. Biopsy-confirmed PTLD cases (n = 100) were matched to 375 controls by center, date of transplant, and age. Data were collected on immunosuppression and rejection therapies, demographics, pre-transplant viral status, number of rejections, and anti-viral use. With adjustment for known risk factors, prophylactic anti-viral use was associated with up to 83% reduction in the risk of PTLD, depending on the anti-viral agent. These results were stronger for the first year post-transplant. For every 30 days of ganciclovir treatment, risk of PTLD during the first year was lower by 38% (Odds Ratio [OR]= 0.62; 95% confidence interval [CI]= 0.38–1.0); acyclovir effects were less striking (OR = 0.83; 95% CI = 0.59–1.16). Anti-viral therapy appears to play a role in reducing the risk of PTLD in renal transplant patients. Ganciclovir may be more potent than acyclovir.

Introduction

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of transplantation closely associated with the Epstein−Barr virus (EBV). The majority of PTLD cases are non-Hodgkin's lymphomas, particularly of B−cell origin, and can range in severity from an infectious mononucleosis-like syndrome, to polyclonal lymphoproliferation, to malignant monoclonal lymphoma (1–3). Patients who are EBV-seronegative at the time of transplant are at markedly increased risk of developing PTLD (4–9). As a result, some transplant centers have modified their immunosuppression protocols to include some form of anti-EBV viral prophylaxis (10). Studies designed to assess the effectiveness of anti-viral prophylaxis have had variable results, with some suggesting an association between the use of these modalities and a reduction in PTLD incidence (11–13); others have noted no effect (4,14). Furthermore, some of these agents require phosphorylation by viral enzymes that are only expressed during the lytic phase of infection. Since PTLD is currently thought to represent latent infection, such phosphorylation should not occur and the agents should not be effective (15).

Many of these studies have relied on historical control series at single centers or have been based on relatively small numbers of PTLD cases, making it impossible to adjust for potential confounders. The low incidence of PTLD makes randomized clinical trials and cohort studies prohibitive in terms of both time and cost, and the increasing use of anti-viral therapy as standard practice considerably reduces the potential for a prospective 'no prophylaxis' comparison group. This multi center case–control study examines the impact of anti-viral therapy on the development of PTLD.

Patients and Methods

A description of the design for this case–control study is available elsewhere (16). Briefly, PTLD cases were identified by direct query at 20 out of 23 participating United Network for Organ Sharing (UNOS) centers; 3 centers had no diagnosed PTLD during the study period. All participants obtained approval from their local institutional review board. All cases received a renal-only transplant on July 1, 1995, or later and developed polymorphic or monomorphic (lymphomatous) PTLD following renal transplantation, no later than December 31, 2001. All PTLD cases but one were identified by the centers; UNOS records added one additional case. Overall, 149 potential cases were reported retrospectively or prospectively during the study data collection period, September 1997 through December 2001.

Controls identified by UNOS in cooperation with the centers were matched on center, age category (pediatric, adult), and date of transplant. Adults were matched within a month of transplant date and pediatric cases (≤18 years) were matched within 6 months. Controls were required to have no clinically recognized disease that would be consistent with PTLD and to have survived for a period at least equal to the time between transplant and PTLD onset for the matched case. Up to four controls were selected for each case in order to increase the power of the study. If more than four controls were available, the four with the closest fit on the matching variables were selected.

Research nurses from each center were trained in the use of a common, standardized abstract form and obtained data for all patients from their medical records. Variables collected included age and sex, medical history relevant to the transplant, HLA typing, risk factors for rejection, number of rejection episodes, prior history of nonlymphoma malignancies, cause of renal failure and viral serology at time of transplant. A risk score was also included that represented a count of the number of risk factors for rejection (cadaver donor; prior renal transplant; three or more HLA mismatches; black race; dialysis prior to transplant) present for each patient. Risk factors were selected based on individual relative risks associated with rejection.

Detailed data were collected for each patient on initial, maintenance and rejection therapies. Anti-virals (ganciclovir and acyclovir) administered at the time of transplant through the development of PTLD or at the end of the study period for controls, were noted. The duration of each anti-viral and the indication for use (prophylactic, treatment) were recorded, but dosages were not. Anti-viral regimens starting within the first 21 days post-transplant were considered prophylactic unless the indication was specifically noted as for treatment. Only anti-viral use beginning at least 30 days prior to the PTLD diagnosis, or a comparable time for controls, was counted to avoid including anti-viral use associated with the PTLD diagnosis or treatment. There was a concern that anti-viral use might not vary within centers if it was regulated as part of each center's protocol. To check for this possibility, protocols were requested from all participating centers and were available for 17 centers. Only three contained recommendations for prophylactic anti-viral therapy based on the donor/recipient cytomegalovirus (CMV) status. The remainder left the decision up to the discretion of the treating physician.

The effects of anti-viral use were examined using conditional logistic regression, which took account of the case–control matching, and which permitted control for other predictors of PTLD. Both acyclovir and ganciclovir were included in the model so that the independent effects of each could be assessed. The coefficient of the logistic regression yields an estimate (and the corresponding 95% confidence interval [CI]) of the odds ratio (OR) of PTLD associated with the presence or absence of each categorical predictor, and of the relative increase in risk associated with each continuously measured predictor.

PTLD cases and their matched controls were also divided into two groups on the basis of the time to PTLD and the conditional logistic regression was performed separately for early versus late PTLD. While ‘early’ PTLD has not been defined consistently in the literature, some recent studies have used 1 year as the cut-point, and we have accepted that convention (17–19). We examined the association between use of this therapy and specific patient and treatment characteristics known at transplant. Chi-squared tests were used to evaluate differences.

Results

One hundred thirteen reported cases met inclusion criteria. Five cases were dropped because of insufficient treatment data, and eight cases had no available pathology, leaving 100 biopsy-confirmed cases. Sixty-two patients (62%) developed PTLD within the first year following transplant (median = 185 days), while 38 were diagnosed more than 1 year following transplant (median = 769 days); time to diagnosis for all cases ranged from 45 days to more than 5 years.

Pre-transplant assessment of EBV serology was reported for 58% of the patients; by contrast, 96% of the patients were tested for CMV. The proportion of missing laboratory data did not differ between cases and controls (CMV, cases 7% and controls 3%; EBV, cases and controls 42%). Among those with EBV testing, 75% were EBV-positive (54% of the cases and 81% of the controls). CMV status presented differently, with 61% testing CMV-positive pre-transplant (62% of the cases and 61% of the controls). The risk of PTLD associated with negative CMV status was similar to that for positive CMV status (Relative Risk [OR]= 0.96; 95% CI = 0.60–1.54) and was essentially identical for both pediatric (OR = 0.96) and adult (OR = 0.95) patients. There was a marked difference in EBV status by age; 50% of the pediatric patients compared with 92% of the adults were EBV-positive. Controlling for age, patients who had negative EBV serology were more than five times more likely to develop PTLD compared to patients who had positive serology (OR = 5.5; 95% CI = 2.5–11.7). Negative EBV status was a significant predictor of PTLD risk for both pediatric (OR = 7.0) and adult (OR = 4.8) patients.

Prophylactic anti-viral use varied significantly by several variables and these are displayed in Table 1. Pediatric patients were less likely to receive anti-viral prophylaxis than adults. Individuals who tested positive on EBV pre-transplant were significantly more likely to get prophylactic anti-virals than individuals who tested negative. And patients who received triple therapy were more likely to receive prophylactic anti-virals than patients receiving double therapy; within the triple therapy group, those receiving mycophenolate mofetil (MMF) were more likely to receive anti-viral prophylaxis than those without MMF, suggesting that the stronger the perceived level of immunosuppression, the greater the likelihood of receiving prophylaxis. There was no association between anti-viral use and any of the following characteristics: race, prior transplant, donor type (cadaver, living), rejection, risk score, use of induction therapy, pre-transplant CMV serology or history of prior nonlymphoma malignancy. Use of anti-viral prophylaxis overall was stable across the study years but the use of ganciclovir increased from 25% prior to 1998 to 44% from 1998 onward, with a corresponding decrease in the use of acyclovir.

Table 1.  Patient and therapy characteristics associated with prophylactic anti-viral status
Prophylactic
anti-viral
status
Age
Adult
(>18 years)
Pediatric
(≤18 years)
Pre-transplant Epstein-Barr
virus serology
Initial immunosuppressionaTotal
PositiveNegativeUnknownDoubleTriple/MMFTriple/no MMF
  1. aDouble immunosuppression is defined as a steroid plus a single additional immunosuppressant; triple immunosuppression is a steroid plus two additional immunosuppressants. Triple is divided into regimens with and without mycophenolate mofetil (MMF).

Acyclovir only126 (34%)20 (20%)47 (23%)11 (16%)88 (44%)6 (11%)102 (35%)38 (29%)146 (31%)
Ganciclovir only91 (25%)20 (20%)64 (31%)15 (22%)32 (16%)15 (28%)68 (23%)28 (22%)111 (23%)
Both37 (10%)16 (15%)25 (12%)10 (14%)18 (9%)7 (13%)35 (12%)11 (8%)53 (11%)
Neither117 (32%)48 (46%)70 (34%)33 (48%)62 (31%)25 (47%)87 (30%)53 (41%)165 (35%)
Total patients3711042066920053292130475
p-value 0.006 <0.0001 0.01 

Table 2 displays the number of cases and controls receiving acyclovir and ganciclovir as part of their initial therapy and the duration of use. Also shown are the crude ORs comparing the rate of PTLD for each category of anti-viral use with the rate for no anti-viral use. A total of 310 (65%) study participants received at least one prophylactic anti-viral at time of transplant (54% of cases, 68% of controls). The overall OR for PTLD comparing any prophylactic anti-viral with no anti-viral was 0.56 (95% CI = 0.35–0.86), a 44% reduction in PTLD risk. Use of acyclovir was more common than ganciclovir. Individuals receiving only ganciclovir were at less than half the risk (OR = 0.47; 95% CI = 0.25–0.87) of developing PTLD as individuals who received no anti-viral prophylaxis. Acyclovir use was associated with 39% protective effect (OR = 0.61; 95% CI = 0.36–1.05). Average duration of acyclovir use was 86 days overall, although there was considerable variability. Ganciclovir was used, on average, 28 days overall, also varying markedly between individuals. Patients receiving both had a risk-estimate intermediate between acyclovir and ganciclovir. Cases receiving ganciclovir tended to have received it for less time than controls but the difference was not significant.

Table 2.  Prophylactic anti-viral use (therapy starting ≤21 days after transplant)
 Cases (n = 100)
Average durationa
Controls (n = 375)
Average duration
Crude ORb95% CI
N%DaysSDRangeN%DaysSDRange
  1. aAverage duration is mean time for prophylactic regimen.

  2. bCrude odds ratio (OR) of PTLD for each anti-viral group compared with no prophylactic anti-viral use; OR is unadjusted and does not take matching into account.

  3. SD = standard deviation.

No prophylactic anti-viral464611932 
Acyclovir only282890751–2291183185741–4180.610.36–1.05
Ganciclovir only171715252–92942531561–3300.470.25–0.87
Both acyclovir and ganciclovir9912162–53441212182–710.530.31–1.17
Any prophylactic anti-viral545453681–2292566853681–4180.560.35–0.86

Table 3 presents the individual risk associated with each variable in the model, taking only the matching factors into account. Case and control counts for each category are presented along with the OR and 95% CI for each variable or variable category. Patients who received ganciclovir were significantly less likely to develop PTLD than patients who received no prophylaxis (OR = 0.32). Patients who tested negative for EBV prior to transplant were more than five times (OR = 5.39) more likely to develop PTLD than individuals who tested positive. There was no such association for CMV. Other significant predictors were rejection experience (OR = 2.63), risk score for rejection (OR = 1.73) and prior nonlymphoma malignancy (OR = 4.07).

Table 3.  Conditional logistic regression model: univariate associations between prophylactic anti-viral use, risk factors and post-transplant lymphoproliferative disorder
VariableCasesControlsOdds ratio95% CI
LowHigh
  1. aATG also includes anti-Tac, anti-LFA-1 and CHI.

Prophylactic anti-viral (none)46119 
 With acyclovir281180.610.331.13
 With ganciclovir17940.320.150.69
 With both acyclovir and ganciclovir9440.540.221.29
EBV status pre-transplant (positive)32174 
 Pre-transplant EBV unknown411592.070.845.12
 Pre-transplant EBV negative27425.392.4911.70
CMV status pre-transplant (positive)57221 
 Pre-transplant CMV unknown8132.830.918.81
 Pre-transplant CMV negative351410.970.591.60
Rejection (none)65303 
 Rejection35722.631.504.63
Gender (male)60229 
 Female401461.030.661.62
Rejection risk factor score (0–2)56221 
 Three or more441541.731.012.96
Prior nonlymphoma malignancy (none)89363 
 One or more11124.071.719.69
Initial therapy (triple without MMF)25105 
 Double9440.850.332.22
 Triple with MMF662261.280.622.62
Induction therapy (none)41140 
 With ATGa381720.630.321.25
 With OKT321631.230.622.42

Table 4 presents the results of the multivariable conditional logistic regression model for each variable, adjusting for matching and for all other variables in the model. Results are generally consistent with the univariate model. The risk estimates for prophylactic anti-virals become more protective for both acyclovir and ganciclovir; patients receiving ganciclovir or a combination of the two anti-virals are significantly different in risk from individuals who received no prophylaxis. Adjusting for other variables markedly increases the OR for EBV-negative serostatus (OR = 12.58); it remains a highly significant predictor of PTLD. Rejection experience is significant but the risk score for rejection is not. The OR for prior nonlymphoma malignancies more than doubled with adjustment for other factors (0R = 4.07 vs. 8.88) and was also highly significant. Induction therapies remained nonsignificant in the adjusted model but the ORs for both, particularly OKT3, were in the direction of increased risk, as suggested in the literature (20).

Table 4.  Multivariate conditional logistic regression model: association between prophylactic anti-viral use and post-transplant lymphoproliferative disorder

Variable
Odds
ratio
95%
CI
  1. aATG also includes anti-Tac, anti-LFA-1 and CHI.

  2. ATG = anti-thymocyte globulin; CI = confidence interval; EBV = Epstein-Barr virus; MMF = Mycophenolate mofetil.

Prophylactic anti-viral (none)1.00
 With acyclovir0.460.14–1.52
 With ganciclovir0.170.05–0.56
 With both acyclovir and ganciclovir0.180.04–0.80
EBV status pre-transplant (positive)1.00
 Pre-transplant EBV unknown1.820.54–6.10
 Pre-transplant EBV negative12.583.51–45.06
CMV status pre-transplant (positive)1.00
 Pre-transplant CMV unknown3.770.73–19.38
 Pre-transplant CMV negative0.890.44–1.79
Rejection (none)1.00
 Rejection2.510.98–6.44
Gender (male)1.00
 Female0.970.52–1.79
Rejection risk factor score (0–2)1.00
 Three or more1.640.69–3.88
Prior nonlymphoma malignancy (none)1.00
 One or more8.881.97–40.06
Initial therapy (triple without MMF)1.00
 Double0.430.10–1.94
 Triple with MMF0.640.22–1.82
Induction therapy (none)1.00
 With ATGa1.610.56–4.67
 With OKT32.270.68–7.52

Because pre-transplant EBV status was associated with both PTLD and anti-viral use, this variable is a potential confounder in the regression analyses. To examine for this, we conducted additional analyses dropping those individuals who were known to be EBV-seronegative at time of transplant. The conditional logistic regression model again found ganciclovir to be protective (OR = 0.28; 95% CI = 0.08–0.96), while acyclovir and the combination of both had ORs less than 1 but were not significant. OR estimates appeared to be stronger for early PTLD (≤1 year) than for late-onset PTLD. After adjusting for known risk factors, transplant patients experienced a 38% reduction in risk of early PTLD for every 30 days of ganciclovir use during the first year. Figure 1 displays the results when a multiple logistic regression equation using a continuous measure of anti-viral use is used to predict reductions in early PTLD based on differing durations of acyclovir and ganciclovir use. The corresponding decline with 30 days of acyclovir was less than half, and not significant. There was no protective effect of either anti-viral on late PTLD, nor was there evidence of a compensatory increase in late PTLD, as would happen if anti-virals simply delayed the onset of PTLD past the 1-year mark. Additional survival analyses found no association between anti-viral use and 1-year survival following PTLD.

Figure 1.

Risk of post-transplant lymphoproliferative disorder with days on anti-viral therapy during the first year following transplant.

Discussion

Prophylactic anti-viral therapy formed an inconsistent part of the treatment of renal-only transplant patients in the United States during the end of the last decade and the beginning of this. Those patients who did receive anti-viral prophylaxis appear to have experienced a substantially reduced risk of PTLD, compared to those who did not. This finding is unaffected by correlations between anti-viral treatment and any identified patient characteristic, including pre-transplant EBV status.

There were few associations noted between anti-viral use and patient characteristics, risk factors for rejection or immunosuppression agents. Patients' pre-transplant viral status was examined but donor serology was not collected. Of interest was the large number of patients with no EBV testing pre-transplant. Despite the high risk associated with EBV-negative serostatus pre-transplant, these patients were less likely to receive any type of anti-viral prophylaxis to reduce the risk of EBV. CMV status was also not associated with prophylaxis although having viral serology for donors might have helped explain some of these situations. There was no preference among the CMV-seropositive patients for ganciclovir use despite the efficacy of ganciclovir for CMV prophylaxis. The fact that two thirds of the patients in the study received anti-viral prophylaxis at the time of transplant supports the relatively widespread use of this approach. The more common use of acyclovir together with longer administration time for this agent might be partially explained by the time frame for the study (1995–2001) and the lower cost of acyclovir. There was a modest shift toward more ganciclovir use in general in the latter half of the study.

The strength of the anti-viral effect appears to be a function of which anti-viral was used and the duration of anti-viral prophylaxis. Both acyclovir and ganciclovir had ORs in the protective direction although only ganciclovir was significant, despite its having been administered for a relatively shorter period of time. Ganciclovir is a potent inhibitor of intra-cellular CMV replication. CMV disease predicts the development of PTLD in liver transplant patients with primary EBV infection (17). Ganciclovir may be more effective than acyclovir in preventing release of infectious EBV from irradiated B lymphoblastoid cell-lines at concentrations that do not severely inhibit B−cell growth (21). Green and colleagues found that the addition of oral acyclovir to prophylaxis with 2 weeks of intravenous (i.v.) ganciclovir did not decrease the frequency of CMV or EBV disease after pediatric liver transplantation when compared with 2 weeks of i.v. ganciclovir alone (10). Using historical data, Darenkov and colleagues examined PTLD incidence in two groups of transplant recipients, one with no prophylactic anti-viral therapy and a second that received either ganciclovir (if either the donor or the recipient was CMV seropositive) or acyclovir (12). The number of patients was relatively small, with a total of eight PTLD cases; however, there was only a single episode of PTLD among the group receiving prophylactic anti-viral therapy, suggesting the effectiveness of this approach. In another historical cohort study, where high-dose acyclovir was used for prophylaxis, there was a negative association between this prophylaxis and the incidence of primary or reactivated EBV infection, which, in turn, was associated with PTLD (9).

As alluded to in the introduction, the mechanism of action of ganciclovir and acyclovir in preventing PTLD is not clear. Both of these drugs require phosphorylation to become active and this occurs because enzymes are present only during the lytic phase of infection (17). While there have been reports suggesting that high levels of lytic infection can be seen in transplant recipients (22), others have not found such evidence (23,24). With in vitro EBV cell lines, viral particles are released from otherwise latently infected proliferating cells, and such release can be decreased by ganciclovir (21). Therefore, while not proven, it is hypothesized that the efficacy of the anti-viral prophylaxis seen in this and other studies results from the effect on lytic infection early during the EBV infection or reactivation. This then prevents expansion of the EBV infected pool, the source of the malignant B cells (25,26). The fact that no effect was seen with prophylaxis on late PTLD is not surprising, since any potential effect on reducing early expansion would be gone. The reduction in immunosuppression that typically occurs as the patients are further from transplant would allow the expansion of cellular immunity to further control late EBV infection after prophylaxis has waned (27).

A major limitation of the present study is the absence of dosage data for the anti-virals. It is possible that high-dose acyclovir may be associated with a protective effect while low-dose acyclovir is not. The absence of this information could explain the mixed results in other studies.

The measures representing total days on each anti-viral included all indications (prophylaxis and treatment). We conducted other analyses restricting the population to individuals who had anti-viral prophylaxis only, and the results were essentially unchanged. This may be due to the relatively small percentage of patients who received anti-virals for treatment only (5%). During the post-transplant period, 65% of the patients received anti-viral prophylaxis. The current standard recommended dosing interval for CMV prophylaxis post-transplant with ganciclovir or valacyclovir is approximately 3 months (28–30). Because ganciclovir was given to most patients for substantially less time (1 month or less), it is possible that increasing the dosing interval for prophylactic use could result in further reduction in PTLD rates. The general increase in the use of anti-viral prophylaxis could possibly explain the decreasing rate of PTLD in recent reports. One study reported 218 PTLD cases in 38,519 primary kidney transplant recipients transplanted between January 1, 1997, and December 31, 2000. This translates into an incidence of 0.57% (95% CI = 0.49–0.64), significantly different from the 1% rate noted in an earlier study (31).

In summary, anti-viral therapy, particularly with ganciclovir, was associated with significantly reduced rates of early PTLD in this study. The strongest risk factor for PTLD was negative EBV serostatus at time of transplant, yet 42% of the patients were never tested for EBV. This suggests that efforts should be made to assess EBV serology prospectively in all donors and transplant recipients and anti-viral therapy considered for those at risk. The strong association between prior nonlymphoma malignancies and PTLD is also worth noting. This association has not been reported before and should be evaluated in other studies.

Conflict of interest statement

This study was funded by a research contract with F. Hoffmann La-Roche Ltd, Basel, Switzerland. Dr Funch had full access to all the study data and final responsibility for the decision to submit this manuscript for publication. Dr Pescovitz receives grant support and speaker's honoraria, and serves as a consultant for Roche.

Acknowledgments

We wish to thank all the collaborating investigators and staff at the participating transplant centers who contributed data to this study, including Edward Alfrey, Milton Hershey Medical Center; Roy Bloom, Hospital of the University of Pennsylvania; Dan Brennan, Barnes-Jewish Hospital; William Bennett and Angelo DeMattos, Oregon Health & Science University; Robert Gaston, University of Alabama at Birmingham; Sundaram Hariharan, Froedert Memorial Lutheran Hospital; David Klassen, University of Maryland; Alan Leichtman and Akinolu Ojo, University of Michigan; Jimmy Light, Washington Hospital Center; Art Matas, University of Minnesota; Mark Pescovitz, Indiana University Hospital; John Pirsch, University of Wisconsin; P. Rajagopalan, Medical University of South Carolina; Charles Sanders, Lifelink Transplant Institute; Minnie Sarwal and John Scandling, Stanford University; Steve Tomlanovich, University of California at San Francisco; Brad Warady, Children's Mercy Hospital; Francis Wright, Methodist Specialty and Transplant Hospital; and Carlos Zayas at Emory University. We also with to thank Michael Cecka, UNOS, for providing data and serving as an advisor; Nancy Dreyer, Ingenix, for her guidance in the early phases of the study; and Hilal Maradit Kremers and Susan Sacks, Roche Pharmaceuticals, for facilitating the study by providing access to information and resources.

We are grateful to Dr Michael Nalesnik and Dr Clifton Kew, who served as central pathologist and nephrologist, respectively. We would also like to acknowledge the work of Joanne Brady, the data coordinator for the study and Hnin Hnin Ko, who served as the analyst for a portion of this study. This study was supported by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Ancillary