Post-Transplant Events

Bone Marrow Transplantation (2004) 34, 1071–1075. doi:10.1038/sj.bmt.1704694 Published online 18 October 2004

Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection

R K Avery1, B J Bolwell2, B Yen-Lieberman3, N Lurain4, W J Waldman5, D L Longworth1, A J Taege1, S B Mossad1, D Kohn3, J R Long6, J Curtis2, M Kalaycio2, B Pohlman2 and J W Williams7

  1. 1Department of Infectious Disease, Cleveland Clinic Foundation, Cleveland, OH, USA
  2. 2The Bone Marrow Transplantation Program, Cleveland Clinic Foundation, Cleveland, OH, USA
  3. 3Department of Clinical Virology, Cleveland Clinic Foundation, Cleveland, OH, USA
  4. 4Department of Immunology/Microbiology, Rush Medical College, OH, USA
  5. 5Department of Pathology, Ohio State University Medical Center, OH, USA
  6. 6Department of Pharmacy, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland, OH, USA
  7. 7Department of Surgery, University of Chicago, Chicago, USA

Correspondence: Dr RK Avery, Department of Infectious Diseases, Cleveland Clinic Foundation, Desk S-32, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: averyr@ccf.org

Received 1 December 2003; Accepted 13 July 2004; Published online 18 October 2004.

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Abstract

Ganciclovir-resistant cytomegalovirus (CMV) infection is an emerging problem in transplant recipients. Foscarnet resistance and cidofovir resistance have also been described, but no previous reports have suggested treatment regimens for patients with CMV refractory to all three of these drugs. Leflunomide, an immunosuppressive drug used in rheumatoid arthritis and in rejection in solid-organ transplantation, has been reported to have novel anti-CMV activity. However, its clinical utility in CMV treatment has not been described previously. We report an allogeneic bone marrow transplant recipient who developed CMV infection refractory to sequential therapy with ganciclovir, foscarnet, and cidofovir. The patient was ultimately treated with a combination of leflunomide and foscarnet. Both phenotypic and genotypic virologic analysis was performed on sequential CMV isolates. The patient's high CMV-DNA viral load became undetectable on leflunomide and foscarnet, but the patient, who had severe graft-versus-host disease (GVHD) of the liver, expired with progressive liver failure and other complications. We concluded that leflunomide is a new immunosuppressive agent with anti-CMV activity, which may be useful in the treatment of multiresistant CMV. However, the toxicity profile of leflunomide in patients with underlying GVHD remains to be defined.

Keywords:

leflunomide, cytomegalovirus, antiviral resistance

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Patient history

A 47-year-old, cytomegalovirus (CMV)-seropositive woman received an allogeneic, matched-sibling donor bone marrow transplant for chronic myelogenous leukemia from a CMV-seronegative donor (D-/R+), using a preparative regimen of busulfan and cyclophosphamide. Despite prophylaxis with methotrexate and tacrolimus, she developed severe graft-versus-host disease (GVHD) of the liver, skin, and mouth. She also developed hemorrhagic cystitis, chronic pancytopenia, hypogammaglobulinemia, renal insufficiency, and intolerance to tacrolimus. GVHD was treated with steroids, cyclosporine, and mycophenolate; thalidomide was administered at months 5–6 and rituximab at months 7–8.

Ganciclovir prophylaxis was initiated at 5 mg/kg/day i.v. from the time of platelet engraftment at 1 month post-transplant. CMV viremia was first noted in the third month, and the ganciclovir dose was increased to 5 mg/kg i.v. twice daily. Ganciclovir was discontinued at month 4. CMV viremia recurred in month 5 and ganciclovir was restarted. From months 5–13, the patient was on i.v. ganciclovir almost continuously, at doses of 5 mg/kg i.v. twice daily with adjustment for renal function, but CMV viremia persisted at low levels. At month 13, her CMV isolate was found to be resistant to ganciclovir by plaque reduction assay (IC50=50 muM) and by genotypic analysis. She developed fever and a rising CMV viral load, and was treated with foscarnet from months 13 to 15. CMV viremia cleared twice on treatment doses of foscarnet, but recurred twice on maintenance doses. Multiple doses of CMV hyperimmune globulin and i.v. immunoglobulin were administered in conjunction with this antiviral therapy, but viremia persisted (Figure 1).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CMV-DNA viral load and antiviral therapy over time.

Full figure and legend (16K)

In month 15, therapy was changed to cidofovir, but after three doses she developed worsening renal insufficiency, and the CMV-DNA viral load rose to 226 000 copies/ml. Resistance to foscarnet and cidofovir was demonstrated by plaque reduction assay (IC50 of 440 and 15 muM, respectively). At this point, the patient had fevers, wasting, elevated LFTs, and severe HSV oral ulceration. As all other options had been exhausted, and newly available information indicated that there was a novel anti-CMV effect of leflunomide,1 she was started on a combination of leflunomide and foscarnet. This combination was chosen since her best prior clinical response had been to foscarnet. Her induction dose of leflunomide was 200 mg/day for 7 days, followed by a maintenance dose of 50 mg/day, targeting a serum level of 60–80 mug/ml of the active metabolite A77 1726. Serum levels of A77 1726 were maintained between 70 and 90 mug/ml. The CMV viral load decreased from 226 000 copies/ml to undetectable by 6 weeks after the initiation of therapy and improvement in the HSV oral ulceration was noted; however, leflunomide was discontinued after the 3rd week of therapy due to progressive hyperbilirubinemia. She received an infusion of donor leukocytes but pancytopenia persisted. After multiple complications including enterococcal bacteremia and progressive wasting, the patient expired at 18 months post-transplant. The final CMV-DNA again turned positive 4 weeks after discontinuation of leflunomide.

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Virologic analysis

CMV-DNA monitoring throughout the patient's course was performed with the quantitative CMV-DNA hybrid capture assay.2 The initial isolate obtained while the patient was receiving ganciclovir was subjected to phenotypic analysis by plaque reduction assay by two different laboratories (Cleveland Clinic Foundation and Rush–Presbyterian–St Luke's Medical Center, Table 1) and was found to be resistant to ganciclovir. Subsequent testing of this initial isolate by one of the laboratories indicated susceptibility to both foscarnet and cidofovir. DNA sequencing analysis of this first isolate showed a deletion of codon 601. Deletions in the UL97 codon range 591–607 have been confirmed by marker transfer to confer ganciclovir resistance, although deletion of codon 601 alone has not been reported previously.3 Methods of phenotypic and genotypic analysis were performed as described previously.4


A second isolate was obtained while the patient was receiving foscarnet. The plaque reduction assay performed by one of the laboratories showed resistance to both foscarnet and cidofovir. A similar assay by the second laboratory showed foscarnet resistance but sensitivity to cidofovir and ganciclovir. Genotypic analysis of this latter isolate showed wild-type sequences with none of the proven UL97 ganciclovir resistance mutations. There was a mutation in the DNA polymerase gene at codon 756 (E–D substitution), which has been confirmed by marker transfer to confer foscarnet resistance (S Chou, personal communication). There were no mutations in the DNA polymerase that were consistent with cidofovir resistance.

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Discussion

Although ganciclovir has been the mainstay of CMV treatment in the past, ganciclovir-resistant CMV in solid organ and bone marrow transplant recipients has been reported with increasing frequency over the past several years.3, 4, 5, 6, 7, 8, 9, 10, 11 Ganciclovir-resistant CMV has been seen frequently in AIDS patients with CMV retinitis, particularly those receiving lengthy courses of ganciclovir therapy.12 It is thought that prolonged exposure to ganciclovir, particularly at concentrations that do not fully suppress viral replication, may lead to resistance.7, 11, 13, 14 Ganciclovir resistance may be assessed by phenotypic analysis by plaque reduction assay4, 15 and by genotypic analysis, which demonstrates characteristic mutations in the UL97 region or the DNA polymerase gene.4, 16, 17, 18 In the past, foscarnet has been the main agent utilized for therapy for ganciclovir-resistant CMV in transplant recipients,19 but its usefulness in transplant recipients is often limited by nephrotoxicity and electrolyte disturbances. Combination ganciclovir and foscarnet therapy has been utilized in AIDS patients with retinitis20 and in allogeneic stem cell recipients.21 Recent work suggests that a combination of reduced-dose ganciclovir and foscarnet may be effective and less toxic for ganciclovir-resistant CMV in solid organ transplant recipients.22 Cidofovir, the third FDA-approved drug for the treatment of CMV,23 has been used primarily for retinitis in AIDS patients and for CMV in allogeneic stem cell recipients.24 However, cidofovir is also potentially highly nephrotoxic, and crossresistance between ganciclovir and cidofovir has been described18, 25 when mutations occur in the UL54 region.17 Resistance to foscarnet, to cidofovir, and multiple resistance have been described.25, 26, 27, 28 No previous reports have described systemic therapy for CMV refractory to all three standard drugs.

The patient described above had protracted chronic progressive CMV infection after bone marrow transplant with wasting, weakness, fatigue, intermittent fevers and pancytopenia in the setting of severe GVHD. Ganciclovir resistance was documented by both plaque reduction assay and DNA sequencing. Similar phenotypic and genotypic assays, performed on a later isolate obtained while the patient was receiving foscarnet, revealed foscarnet resistance by plaque reduction assay and DNA sequencing. The plaque reduction assay results for cidofovir were discordant between the two testing laboratories, and cidofovir resistance was not demonstrated by genotyping, although the patient's CMV was clinically refractory to cidofovir. The 756 E–D mutation has been shown to confer foscarnet resistance, but has not been fully evaluated for cidofovir susceptibility. However, foscarnet resistance mutations that have been confirmed by marker transfer do not confer crossresistance to cidofovir.3, 18 It is possible that the cidofovir resistance observed by one of the laboratories was associated with a second circulating strain.

It was clear, however, that the patient's CMV was clinically refractory to all three drugs used in a sequential manner. Other therapeutic options which were considered included combination ganciclovir and foscarnet, but her pancytopenia was thought to preclude further ganciclovir therapy. The possibility of adoptive immunotherapy with expansion of anti-CMV lymphocyte clones from the donor was considered but not pursued as the donor was CMV seronegative; ultimately, she did receive unexpanded donor leukocytes without improvement. Multiple infusions of both CMV hyperimmune globulin and unselected IVIG were given without improvement. After much discussion, the decision to utilize leflunomide and foscarnet in combination was reached since no other treatment, either licensed or investigational, was available. Foscarnet was chosen as the second agent in the combination since she had had the best prior clinical response to foscarnet of the three standard agents. Since leflunomide was an FDA-approved drug, albeit for a different indication, and no other therapy was available, an official of the Cleveland Clinic Institutional Review Board (IRB) determined that a formal protocol need not be submitted to the IRB for the use of leflunomide in this patient.

Leflunomide is a new disease-modifying antirheumatic drug approved in 1998 in the United States for the treatment of rheumatoid arthritis.29 Among its proposed mechanisms of action are inhibition of T- and B-cell proliferation by interference with the de novo pathway of pyrimidine synthesis via inhibition of the enzyme dihydroorotate dehydrogenase and inhibition of phosphorylation of specific tyrosine kinases involved in T- and B-cell activity.30 Leflunomide is increasingly recognized as a potent immunosuppressive agent for the prevention and treatment of rejection in solid organ transplant recipients.31 It has also been reported to have novel anti-CMV activity in vitro, utilizing a mechanism different from that of other antiviral agents.1, 32 The mechanism of leflunomide antiviral activity is a topic of active research, but it does not appear to affect transcription of immediate-early or late viral genes or to inhibit viral DNA polymerase activity. It appears to inhibit virion assembly at a late stage by preventing viral nucleocapsids from acquiring their tegument.1 As leflunomide does not affect DNA polymerase or other aspects of DNA replication of CMV, it would be expected not to show crossresistance with other agents. One previous anecdotal report suggested a response to leflunomide and cidofovir in a transplant patient with CMV resistant to ganciclovir and foscarnet (J Williams, personal communication). Leflunomide also appears to have antiviral activity against HSV as well,33 and this patient's refractory oral HSV ulceration did improve on leflunomide therapy.

This patient's CMV DNAemia did clear transiently on leflunomide and foscarnet. Since foscarnet resistance was subsequently genotypically confirmed, it is likely that at least some of this anti-CMV effect was due to leflunomide. More recently, Mylonakis et al22 have presented evidence that a combination regimen of ganciclovir and foscarnet was efficacious in six solid-organ transplant recipients with ganciclovir-resistant CMV with less toxicity than full-dose foscarnet. However, this information was not available at the time this patient was treated, and her severe pancytopenia made coadministration of ganciclovir problematic.

The patient's progressive liver dysfunction may have reflected her underlying GVHD of the liver, and her liver function tests showed predominantly hyperbilirubinemia, a pattern which is not typical for leflunomide in solid-organ transplant recipients.31 Liver toxicity has been reported in rheumatoid arthritis patients receiving leflunomide, generally in the form of transient liver function test abnormalities, although more serious liver disease has been reported in a patient receiving leflunomide and methotrexate.34 In addition, the doses of leflunomide utilized in this patient were higher than those used in rheumatoid arthritis patients, and were based on the doses utilized in studies; of leflunomide, and as an agent for rejection in solid-organ transplantation.31 This patient did not have a liver biopsy performed due to severe thrombocytopenia. This complex clinical course does raise the question of whether leflunomide might have different toxicities when utilized in allogeneic bone marrow transplant recipients with GVHD of the liver, as compared with other groups of patients.

In addition, the minimal effective dose range for leflunomide used as an anti-CMV agent has yet to be determined. More recent experience suggests that renal compromise may alter the efficacious blood level target31 and patients with elevated serum creatinine may have substantially reduced protein binding; thus the effective (and toxic) level might be reduced. This has been cited as an explanation for significant variation in active metabolite pharmacokinetic features in a series of patients with chronic renal allograft dysfunction treated with leflunomide.31

The patient's CMV DNAemia did reappear close to the time of her death, after a period of undetectability, but she had had leflunomide discontinued 4 weeks previously. It is unknown whether or not she might have achieved a durable remission from CMV if leflunomide could have been administered for a longer period of time.

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Conclusions

Reports of antiviral-resistant CMV are increasing, but there is no previously reported effective therapy for CMV, which is clinically refractory to all three standard anti-CMV drugs. Leflunomide, an immunosuppressive agent utilized in rheumatoid arthritis and in solid-organ transplantation, has novel anti-CMV activity by a mechanism different from standard anti-CMV drugs. As such, it has the potential to be used for the treatment of multiresistant CMV, and was utilized in this case, in combination with foscarnet, with a resultant clearing of high-level CMV viremia. However, the patient's ultimate demise with progressive liver failure raises questions about possible enhancement of pre-existing liver dysfunction. Leflunomide is a promising agent for multiresistant CMV, but further work needs to be carried out to define its efficacy, toxicity, and optimal dose, as well as whether it is best utilized alone or in combination therapy for CMV.

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