Department of Hematology and Oncology, Hannover Medical School, Germany

Correspondence to: Dr B Hertenstein, Hannover Medical School, Department of Hematology and Oncology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

Mycophenolate mofetil (MMF) is increasingly used for prophylaxis and therapy of GVHD in allogeneic stem cell transplantation. In some recent reports of use of MMF in solid organ transplantation a high incidence of CMV disease has been described. We evaluated the frequency and course of active CMV infection in patients who received MMF compared to those who did not receive MMF after allogeneic stem cell transplantation. We retrospectively analyzed 48 adult patients who consecutively underwent unmanipulated allogeneic bone marrow (n = 15) or peripheral stem cell transplantation (n = 33) from HLA-compatible family donors (n = 30) or unrelated donors (n = 18) from February 1997 to September 2000 at our institution. Only patients who were evaluable for the first 100 days were included in this analysis. Sixteen patients received MMF post transplant (MMF+). CMV-antigenemia was monitored by CMV-pp65 antigen. CMV-antigenemia occurred in 14 patients and was virtually only observed in CMV-IgG+ recipients (13/23, 56%). CMV-IgG+/MMF+ patients developed a higher incidence of CMV-antigenemia (8/9, 89%) compared to the CMV-IgG+/MMF- patients (5/14, 35%; P < 0.05). Moreover, five of six patients with persistent or recurrent CMV-antigenemia received MMF. No patient in either group developed CMV disease or died of CMV-related complications. In multivariate analysis including MMF treatment, unrelated vs related donor, GVHD, CMV-serostatus of the donor and stem cell graft type, only MMF treatment was found to be a significant risk factor for both overall and complicated CMV infection.

Bone Marrow Transplantation (2002) 29, 903-906. DOI:10.1038/sj/bmt/1703583

CMV infection; mycophenolate mofetil; allogeneic stem cell transplantation

Mycophenolate mofetil (MMF) is a potent immunosuppressive drug which is now frequently used in solid organ transplantation, especially renal transplantation. MMF is also increasingly used for prophylaxis and therapy of GVHD in allogeneic stem cell transplantation.^1,2 MMF is a potent, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), leading to preferential suppression of lymphocyte proliferation.³ In some recent reports on the use of MMF in solid organ transplantation a trend towards more severe CMV infections and CMV disease has been described.^4,5,6 The impact of MMF on CMV infection in patients undergoing allogeneic stem cell transplantation is not clear. In our institution, MMF has been used for primary GVHD prophylaxis or prevention of GVHD relapse since 1997. We made the clinical observation that problems with CMV infection seemed to be increased in patients receiving MMF treatment. We therefore evaluated the frequency and course of active CMV infection in patients who received MMF for GVHD prophylaxis or therapy after allogeneic stem cell transplant compared to those not receiving MMF at our institution.

Patients and methods

Patients and transplant procedure

Forty-eight adult patients who consecutively received a transplant of unmanipulated bone marrow or peripheral stem cells from an HLA-identical donor at the bone marrow transplantation unit of the Hannover Medical School in the period between February 1997 and September 2000 were analyzed. Only patients who were evaluable for the first 100 days were included in this analysis (observation time: 6 months after transplant). Patient characteristics and transplant procedure are presented in Table 1. Sixteen patients (35%) received MMF at a median dose of 3 g/day (range 2-4 g/day) post transplant. Median application time was 111 days (range 36-180). MMF was given as part of the initial GVHD prophylaxis protocol in 11 patients (starting at the day of transplantation) and five patients received MMF for prevention of GVHD reactivation (start: median day 30, range 8-35). Acute GVHD was graded according to the Glucksberg score⁷. Fifteen of 18 patients undergoing stem cell transplantation from matched unrelated donors (MUD) received anti-thymocyte globulin (ATG, Fresenius, Bad Hamburg, Germany) during conditioning. Intravenous immunoglobulin (Gammagard SD, Baxter, Heidelberg, Germany) was given if either recipient or donor or both were CMV-seropositive prior to transplant. All patients received aciclovir (3 ´ 5 mg/kg) in the first month after transplantation. All blood products were leukocyte-depleted by cell separation or filters. CMV-negative patients with CMV-negative donors received CMV-negative blood products.

CMV monitoring

CMV antigenemia was monitored by CMV-pp65 antigen twice weekly during the hospital stay and at least once weekly after discharge. CMV antigenemia assays were carried out as previously described,⁸ with slight modification. In short, PMNL were isolated by dextran separation and resuspended in PBS to give a final cell density of 1 ´ 10⁶ leukocytes/ml. Cytospins were prepared, fixed in acetone/methanol 1:1 and incubated with a mix of the two mouse monoclonal antibodies C10 and C11 (Clonab CMV, Biotest, Dreieich, Germany) directed against the pp65 antigen. Staining was performed using the alkaline phosphatase-antialkaline phosphatase method (dilution 1:50; DAKO Diagnostika, Hamburg, Germany).

Pre-emptive treatment with ganciclovir (5 mg/kg twice daily i.v.) was given in cases of CMV-pp65 positivity of more than 1 of 4 ´ 10⁵ PBMC. Complicated CMV infection was defined as persistent (CMV-pp65 antigen still positive after 14 days of ganciclovir) or recurrent CMV antigenemia. CMV disease was defined as the occurrence of clinical symptoms plus detection of CMV in the involved organ.

Statistics

Statistical calculations were done with the Statistical Program for Social Science (SPSS, version 10.0). Differences between groups were assessed with Fisher's exact test. Multivariate analysis was calculated by unconditional logistic regression. A P value <0.05 was considered statistically significant.

Results

CMV antigenemia occurred in 14 of the 48 patients (29%). CMV antigenemia was first detected at day 21 (median day 45, range 21-160) after transplant with no difference between patients receiving MMF and those not receiving MMF (Table 2). Thirteen of 14 patients received pre-emptive treatment with ganciclovir 5 mg/kg twice daily i.v. for at least 14 days (median 14 days, range 14-23) starting at the first positive pp65 test until at least two pp65 tests were negative. In one patient, ganciclovir treatment was stopped after 10 days (with no recurrence of CMV infection). No patient developed CMV disease or died of CMV-related complications. CMV antigenemias were almost exclusively observed in CMV-IgG+ recipients (13/23, 56%). In CMV-seronegative patients, only one case of CMV antigenemia was observed (1/25, 4%) (Table 3). Eight of 14 patients with CMV antigenemia were receiving MMF.

In the CMV-IgG+ subgroup, patients who received MMF developed CMV antigenemias more frequently (8/9, 89%) compared to those who did not receive MMF (5/14, 35%; P < 0.05). The percentage of overall and complicated CMV infections was higher in patients with MUD compared to those with family donors, and in patients with acute GVHD II-IV compared to those with acute GVHD 0-I, but without reaching statistical significance. Patients with CMV-negative donors (vs CMV-positive donor) or those who received bone marrow (vs PBSC) had less overall but more complicated CMV infections (Table 3).

CMV infection was complicated in six of 14 patients by persistence (n = 1) or recurrence (n = 5: 3 ´ 2, 1 ´ 3 and 1 ´ 4 recurrences). All complicated CMV infections were observed in IgG+ patients. Five of the six complicated CMV infections were observed in patients receiving MMF. Treatment for recurrent CMV infection consisted of retreatment with ganciclovir in all patients, with an initial response in all patients. Persistent CMV infection was successfully treated with foscarnet.

In the subgroup of IgG+ patients, multivariate analysis including the use of MMF, donor type, occurence of acute GVHD, CMV serostatus of the donor and type of the stem cell graft was performed. Only the use of MMF was significantly associated with CMV infection (Table 4) and complicated CMV infection (Table 5).

Discussion

Our clinical observation that problems with CMV infection seemed to be increased in patients receiving MMF treatment, was confirmed by the results of this retrospective study. We observed a higher incidence of CMV-infection in those patients receiving MMF which were additionally complicated by persistence or recurrence. In contrast to previous studies on the use of MMF in solid organ transplantation, which reported no increase of CMV infections, but an increase of CMV disease,^4,5,6 we did not observe any cases of CMV disease independent of immunosuppression. In accordance with previous reports,^9,10,11 CMV seropositivity of the recipient was the most striking risk factor for overall and complicated CMV infections. In the subgroup of CMV-IgG+ patients, MMF was an independent risk factor for both CMV infection and complicated CMV infection on multivariate analysis in our study. It has to be considered that the analysis has been performed with a limited number of patients. The results of the multivariate analysis may thus only be interpreted as a trend. Patients with previously described risk factors, such as MUD or GVHD^9,10,12,13 had a higher incidence for overall and complicated CMV infections. However, this finding did not reach statistical significance. These results would suggest a high incidence of CMV infection after dose-reduced conditioning regimens, in which MMF is used in combination with CsA for prophylaxis of GVHD and graft rejection. However, a recent study on dose-reduced transplants¹⁴ did not reveal a higher incidence of CMV infections, although complicated infections were not analyzed separately. A possible reason might be the relatively short period of MMF treatment of 28 days in this study vs a median of 111 days in our analysis. Use of conventional vs dose-reduced regimens had no influence on the incidence of overall or complicated CMV infection in our study (data not shown).

The potential mechanisms by which MMF may increase the incidence of complicated CMV infections in both organ and stem cell transplantation is most probably intensified immunosuppression. MMF potently inhibits both T and B lymphocyte proliferation, thus impairing both the cellular and humoral immunity directed towards CMV. This might lead to a higher CMV virus replication rate and viral burden. In our study, treatment for recurrent CMV infection consisted of retreatment with ganciclovir, with response in all patients, arguing against the presence of ganciclovir-resistant CMV strains in these cases (as previously reported by Bienvenu et al¹⁵ in a renal transplant patient treated with MMF).

In conclusion, we found MMF treatment to be a significant risk factor for both CMV infection and complicated CMV infections after allogeneic stem cell transplantation. Although our data still require confirmation in a larger prospective study, the impact of MMF on hospitalization should be considered when designing future transplant strategies, until more effective methods for prophylaxis of CMV infection become available.

Acknowledgements

We are grateful to Dr Martin Stanulla (Department of Pediatric Hematology and Oncology, Hannover Medical School) for critical reading of the manuscript and to Dr Heinz Geerlings (Department of Biometry, Hannover Medical School) for his support in the statistical analysis.

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Table 1 Patient characteristics

Table 2 Onset of CMV infection in CMV-IgG pos patients

Table 3 CMV infections in different suspected risk groups

Table 4 Multivariate analysis of suspected risk factors for CMV infection in CMV-IgG pos patients

Table 5 Multivariate analysis of suspected risk factors for complicated CMV infection in CMV-IgG pos patients