Biology of Blood and Marrow Transplantation
Volume 14, Issue 5 , Pages 591-594, May 2008

Myeloablative Intravenous Busulfan/Fludarabine Conditioning Does Not Facilitate Reliable Engraftment of Dual Umbilical Cord Blood Grafts in Adult Recipients

Adult Stem Cell Transplant Program, Division of Cellular Therapy, Duke University School of Medicine, Durham, North Carolina

Received 25 January 2008; accepted 21 February 2008.

Article Outline

Abstract 

The efficacy of once-daily intravenous busulfan with fludarabine as a preparative regimen for partially matched umbilical cord blood transplantation has not been formally studied. We randomized 10 adult patients with myeloid malignancies to receive either concurrent or sequential administration of intravenous busulfan 130 mg/m2 once daily × 4 days and fludarabine 40 mg/m2 daily × 4 days, followed by dual umbilical cord blood transplantation. The median combined cryopreserved total nucleated cell dose was 3.6 × 107/kg recipient body weight (range: 2.8-4.5 × 107/kg). Graft-versus-host disease (GVHD) prophylaxis was provided by tacrolimus and mycophenolate mofetil (MMF). Donor-derived neutrophil recovery was observed in only 2 of 10 patients, resulting in premature closure of the study as per graft failure stopping rules. We conclude that the myeloablative conditioning regimen of once-daily intravenous busulfan with fludarabine provides insufficient immunosuppression to allow for engraftment of partially matched, dual umbilical cord blood grafts.

Key Words: Dual umbilical cord blood, Busfulfan, Fludarabine

 

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Introduction 

Since the publication by de Lima and colleagues in 2004, allogeneic stem cell transplant (SCT) conditioning consisting of once-daily intravenous busulfan and fludarabine (Bu/Flu) has gained widespread popularity [1]. Enthusiasm for this regimen is derived from the fact that it provides a myeloablative dose of Bu with exceptionally low early treatment-related mortality (TRM) (3%). Pharmacokinetic studies demonstrate predictable Bu blood levels with an area under the curve (AUC) comparable to dose-adjusted oral administration [1]. The regimen facilitates reliable engraftment of allogeneic peripheral blood or bone marrow grafts from HLA-identical siblings or matched unrelated donors. Widespread use of umbilical cord blood as a stem cell source for adult patients in need of allogeneic SCT has been hindered by TRM as high as 63% following myeloablative conditioning and high rates of graft failure because of an inadequate stem cell dose or immunologic rejection [2]. Preliminary evidence suggests that infusion of 2 umbilical cord blood grafts improves engraftment in adult recipients 3, 4, 5. This prompted us to conduct a prospective study of Bu/Flu preparation followed by dual umbilical cord blood transplantation. Given its nonspecific alkylating properties, we hypothesize that Bu could interfere with deoxycytidine kinase-mediated accumulation of intracellular F-ara-ATP, thereby attenuating the immunosuppressive properties of the Flu. We therefore randomized patients to sequential versus concurrent administration of i.v. Bu and Flu.

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Patients, Materials, and Methods 

Patients 

Ten patients, median age 41 years, with myeloid malignancies (acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], or myelodysplastic syndrome [MDS]) provided informed consent for participation in this randomized phase II study. Of the AML patients, subject #4 had 4% residual blasts at the time of transplantation. The remainder had no detectable disease in the bone marrow. All MDS-refractory anemia with excess blasts (RAEB)-2 patients were downgraded to RA or RAEB with pretransplantation therapy. The CML patients were in chronic phase at the time of transplantation. The study was approved by the institutional review board of the Duke University School of Medicine. Patient and disease characteristics are demonstrated in Table 1.

Table 1. Patient Characteristics
Patient No.Age Years/SexWeight (kg)DiseaseKaryotypeStatus at Transplantation
140M101AMLInv16CR2
221M76MDS (Hypocell.)−7Aplastic
329M96∗CML(BC)Ph+CP
449F77RAEB-2ComplexRAEB
532M95AMLNormalPR†
644F68MDS RAEB-2Inv3RA
750F93AMLComplexCR1
841M92AMLNormalCR2
954F76CMLPh-CP
1034M67RAEB-2ComplexRA

RAEB indicates refractory anemia with excess blasts; RA, refractory anemia; BC, blast crisis; CP, chronic phase; CR, complete remission—no morphologic evidence of disease.

∗Adjusted ideal body weight.

†Partial remission; 4% blasts with aberrant phenotype.

Treatment Plan 

Bone marrow conditioning consisted of Flu (Berlex Laboratories, Wayne, NJ), 40 mg/m2/day × 4 days and i.v. Bu (PDL Biopharma, Redwood City, CA) 130 mg/m2/day × 4 days. Patients were randomized to receive the drugs sequentially (Flu days −9 to −6; Bu days −5 to −2) or concurrently (days −5 to −2) [1]. Six patients received the drugs sequentially, and 4 concurrently. Bu pharmacokinetic measurements were performed on the first and fourth dose using a high-pressure chromatography assay as previously described [6]. Patients were transplanted with 2 partially matched umbilical cord blood grafts that were at least 4 of 6 HLA-matched with the recipient and 4 of 6 HLA matched between grafts (low resolution class I, high resolution class II) (Table 2). graft-versus-host disease (GVHD) prophylaxis was provided by tacrolimus dosed to achieve serum levels of 10-20 ng/mL for 6 months and mycophenolate mofetil (MMF) 1000 mg twice daily until 60 days following transplantation. Donor chimerism was determined by quantitative PCR amplification of informative microsatellites using DNA isolated from peripheral blood or bone marrow CD3+ and CD15+ cells. Primary graft failure was defined as a patient who never had detectable donor hematopoiesis. Secondary graft failure was defined as a patient who had detectable early donor hematopoiesis that was subsequently lost. Patients with early disease relapse were deemed not evaluable for graft failure.

Table 2. Graft Characteristics, Engraftment, and Outcome
Patient No.Combined Cord TNC/kg∗ (×107)HLA-Matching† (Cord #1 + Cord #2)Busulfan/Fludarabine AdministrationMaximum %Donor Myeloid Chimerism (Cord #1/#2)Maximum %Donor Lymphoid Chimerism (Cord #1/#2)Current StatusCause of Death
13.94/6 + 4/6Concurrent0/00/0CR‡ day 700—
23.44/6 + 4/6Sequential0/00/0Dead day 89‡Infection
32.84/6 + 4/6Concurrent0/00/0Dead day 134‡Infection
43.74/6 +4/6∗∗Sequential6/6§ Dead day 137Relapsed AML
53.96/6 + 6/6Sequential14/779/76Dead day 118Relapsed AML
64.54/6 + 4/6Concurrent25/525/20Dead day 85Relapsed AML
73.65/6 + 5/6Sequential0/9§ Dead day 33Relapsed AML
83.54/6 + 4/6Sequential0/00/0CR day 485⊥—
93.14/6 + 4/6Concurrent0/00/0Dead day 108‡Infection
103.94/6 + 4/6Sequential0/00/0Dead day 88Recurrent AML

AML indicates acute myelogenous leukemia.

∗Cryopreserved.

†Low-resolution Class I, high-resolution Class II.

‡Posthaploidentical second transplant.

§Unselected bone marrow sample.

Cord #1 and #2 are Class I and Class II allele-level matched (probable siblings).

⊥Postautologous bone marrow rescue.

∗∗5/6 in direction of rejection.

Statistical Methods 

A sample size of 24 for each arm of the study was expected to provide enough power to detect a difference in rate of engraftment. Separate stopping rules for primary or secondary graft failure were constructed for each treatment arm using the Bayesian approach that was based on an expected graft failure rate of ≤20%. The parameters assumed α = 0.8 and β = 3.2. Three graft failure events among the first 6 patients enrolled in each arm triggered early stopping. Early stopping rules were activated for both treatment arms.

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Results and Discussion 

All patients with AML were in remission at the time of protocol entry. Patient 3 was in the second chronic phase following induction therapy for lymphoid blast crisis. All subjects with MDS with increased blasts underwent induction chemotherapy and achieved a partial response before protocol therapy. The median combined umbilical cord blood cell dose was 3.6 × 107 total nucleated cells/kg (Table 2). All grafts were at least 4 of 6 HLA matched with the recipient and each other. The mean and median daily AUCs for Bu were 4185 and 4225 μmol-min (range: 2634-5278 μmol-min), respectively. These values are nearly identical to those reported by de Lima and colleagues [1], who, with the same preparative regimen, achieved 100% donor engraftment using peripheral blood or bone marrow grafts. Eight of 10 patients experienced primary or secondary graft failure, although 2 patients were not evaluable for engraftment because of early disease relapse. Only 2 of 10 patients achieved significant, yet incomplete donor hematopoietic chimerism (one on sequential, the other on the concurrent treatment arms). Stopping rules for graft failure were met for both arms of the trial, and thus there was no appreciable difference in the probability of engraftment between sequential or concurrent Bu/Flu administration. Hematopoiesis was reconstituted in 5 patients following a second transplant (4 using haploidentical grafts, 1 using an autologous backup graft), 2 of whom have achieved long-term disease-free survival (DFS) (Table 2). Three patients died of infection-related complications and 5 from relapsed AML.

The poor engraftment that we observed with Bu/Flu conditioning is in contrast to what has been reported in adult recipients using dual umbilical cord blood grafts and more immunosuppressive myeloablative or nonmyeloablative conditioning regimens 3, 4, 5. This occurred despite providing an umbilical cord blood graft of comparable HLA-matching and size. In the study by Ballen and colleagues [4], engraftment was achieved in 18 of 21 patients following reduced intensity (RIC) preparation with melphalan 100 mg/m2, Flu 150 mg/m2, and antithymocyte globulin (ATG). Brunstein and colleagues [5] transplanted 110 patients, most of whom required 2 umbilical cord blood units to achieve a target cell dose of 3.0 × 107/kg. With a nonmyeloablative preparative regimen of cyclophosphamide 50 mg/kg, Flu 200 mg/m2 and total-body irradiation (TBI) 200 cGy, the cumulative incidence of sustained engraftment was 85%. In an earlier study, the same group reported inferior engraftment when nonmyeloablative doses of oral Bu (8 mg/kg) were used instead of cyclophosphamide [7].

Following myeloablative, single-agent dosing of Bu (16 mg/kg total dose) with autologous stem cell support, Peters and colleagues [8] demonstrated sparing of mature lymphoid cells in the peripheral blood. No significant change was observed in CD3+, CD4+, or CD8+ T cell counts during the 26-day posttransplant monitoring period. B cell immune function was also spared. Although Gandhi and Plunkett [9] demonstrate that Flu potentiates alkylator-induced cytotoxicity, the lack of Bu-induced lymphocytotoxicity negates this potentially beneficial synergy and raises significant questions surrounding the immunosuppressive properties of the Bu/Flu regimen. This is particularly important in the setting of umbilical cord blood transplantation given the low stem cell dose and donor/recipient HLA-discordance.

Sequencing in drug administration has been shown to affect the efficacy of many combination therapies, including those with Flu and Bu 9, 10. The cytotoxicity of alkylating agents such as Bu is generally attributed to the formation of DNA crosslinks; however, these drugs are also known to react with many other biomolecules such as proteins, enzymes, and thiols 11, 12. Flu-induced lymphocytotoxicity is contingent on intracellular accumulation of F-ara ATP, which is in turn dependent on the activity of intracellular deoxycytidine kinase [9]. Bu-induced interference of deoxycytidine kinase would therefore compromise the immunosuppressive properties of Flu. It is for this reason that we constructed a 2-armed trial, establishing a cohort of patients who would receive Flu whose activity could not be affected by coadministration of Bu. Verification of Bu-induced deoxycytidine kinase inhibition is planned through measurement of F-ara ATP levels from samples collected following drug administration. However, given the lack of reliable engraftment in both arms of the trial, the clinical significance of this interaction is in question.

Limitations of the study include the small sample size and the fact that despite being in remission at the time of enrollment, half of the subjects had high-risk cytogenetic abnormalities. Although early relapse may serve to inflate the graft failure rate, it should be noted that 4 of 5 patients with standard risk cytogenetic abnormalities also experienced graft failure. The M.D. Anderson group ATG to Bu/Flu as added immunosuppression for recipients of unrelated donor grafts [1]. Because of concerns over impaired posttransplant immune recovery, susceptibility to opportunistic infections, and prolonged platelet recovery, we opted not to include ATG 13, 14.

The Bu/Flu regimen is gaining popularity as an attractive option to standard myeloablative conditioning for allogeneic SCT. However, our data suggest that given in the doses and schedules we tested, Bu/Flu does not provide adequate immunosuppression to facilitate engraftment of partially matched umbilical cord blood grafts and should not be used in this setting.

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Acknowledgments 

The study investigators wish to thank the nurse practitioners, physician's assistants, ward and clinic nurses, and staff of the Duke Adult Stem Cell Transplant Program for their outstanding care of the patients described in this report. In addition, the author is grateful to Michael Colvin, David Adams, and Susan Ludeman for their help with the construct of the clinical trial and the preparation of the manuscript. The study was supported in part by research funding from PDL BioPharma to M.H.

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References 

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PII: S1083-8791(08)00092-X

doi:10.1016/j.bbmt.2008.02.016

Biology of Blood and Marrow Transplantation
Volume 14, Issue 5 , Pages 591-594, May 2008

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