Biology of Blood and Marrow Transplantation
Volume 14, Issue 2 , Pages 220-228, February 2008

High Busulfan Exposure Is Associated with Worse Outcomes in a Daily i.v. Busulfan and Fludarabine Allogeneic Transplant Regimen

  • Michelle Geddes

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • Corresponding Author InformationCorrespondence and reprint requests: Michelle Geddes, MD, Foothills Medical Centre 601 South Tower, 1403-29 Street NW, Calgary, Alberta T2N 2T9, Canada.
    • ,
  • S. Bill Kangarloo

      Affiliations

    • Tom Baker Cancer Centre, Calgary, Canada
    • ,
  • Farrukh Naveed

      Affiliations

    • Tom Baker Cancer Centre, Calgary, Canada
    • ,
  • Diana Quinlan

      Affiliations

    • Tom Baker Cancer Centre, Calgary, Canada
    • ,
  • M. Ahsan Chaudhry

      Affiliations

    • Tom Baker Cancer Centre, Calgary, Canada
    • ,
  • Douglas Stewart

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • ,
  • M. Lynn Savoie

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • ,
  • Nizar J. Bahlis

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • ,
  • Christopher Brown

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • ,
  • Jan Storek

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada
    • ,
  • Borje S. Andersson

      Affiliations

    • Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas
    • ,
  • James A. Russell

      Affiliations

    • Division of Hematology and Hematologic Malignancy, University of Calgary, Calgary, Canada

Received 7 May 2007; accepted 29 October 2007.

Article Outline

Abstract 

Low plasma busulfan (Bu) area under the concentration-time curve (AUC) is associated with graft failure and relapsed leukemias, and high AUC with toxicities when Bu is used orally or i.v. 4 times daily combined with cyclophosphamide in myeloablative hematopoietic stem cell transplantation (SCT) conditioning regimens. We report Bu AUC and its association with clinical outcomes in 130 patients with hematologic malignancies given a once-daily i.v. Bu (3.2 mg/kg days −5 to −2) and fludarabine (Flu, 50 mg/m2 days −6 to –2) regimen. Total-body irradiation (TBI) 200 cGy × 2 was added for 51 patients with acute leukemias. Plasma AUC varied 3.6-fold (2184-7794 μM·min, median 4699 μM·min). Patients with an AUC >6000 μM·min had lower overall survival (OS) than those with AUC ≤6000 μM·min at 12 months (38% versus 74%) and 36 months (23% versus 68%, P < .001). This effect was apparent in patients with standard-risk and high-risk disease, and persisted when potential confounders were considered (hazard ratio 3.2, 95% confidence interval 1.7-6.3). Nonrelapse mortality (NRM) at 100 days (6% versus 19%) and progression free survival (PFS; 58% versus 16%) at 3 years were better with AUC ≤6000 μM·min. These data support a role for therapeutic dose monitoring and dose adjustment with daily i.v. busulfan.

Key Words: Hematopoietic stem cell transplantation, Transplantation conditioning, Drug toxicity, Busulfan, Pharmacokinetics, Therapeutic drug monitoring

 

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Introduction 

Busulfan (Bu) is a bifunctional DNA alkylating agent important in conditioning regimens for hematopoietic stem cell transplantation. Plasma area under the concentration-time curve (AUC) after oral Bu varies about 10-fold because of nausea and vomiting, unpredictable and variable intestinal absorption, and individual differences in hepatic metabolism 1, 2, 3. Furthermore, Bu levels are known to vary with age, disease status, obesity (unless dosing is based on body surface area or adjusted ideal body weight) and, in children, with circadian rhythmicity 2, 4, 5. When given in 4 oral doses daily with cyclophosphamide (Cy) in Bu/Cy, low plasma Bu exposure has been associated with potentially fatal outcomes including graft failure and relapse of chronic myelogenous leukemia (CML), whereas high exposure is associated with toxicities including veno-occlusive disease of the liver (VOD, a.k.a. sinusoidal obstruction syndrome, SOS) and neurotoxicity 1, 6, 7, 8, 9, 10. Targeting Bu levels after oral dosing is associated with successful engraftment and favorable outcomes 11, 12, 13.

An intravenous formulation was developed to improve dose assurance, with decreased variability in plasma AUC; however, a severalfold range in AUC remains between patients 14, 15, 16, 17. We are using once-daily i.v. Bu with Flu in a myeloablative regimen designed to be convenient, allow consistent dose-to-dose Bu levels in the same patient and complete drug clearance between doses, and minimize toxicities 18, 19, 20. This regimen combines 2 effective antineoplastic drugs that may act synergistically through multiple enzymes including Flu inhibition of DNA ligase and DNA primase, and prevention of DNA polymerization, to reduce repair of alkylator-induced damage [19]. We have previously reported the outcomes with this regimen in a variety of hematologic malignancies [20], demonstrating relatively low toxicities and nonrelapse mortality (NRM) compared to outcomes reported with Bu/Cy 21, 22, 23, 24. However, the need for therapeutic drug monitoring and the optimal Bu exposure when given once daily i.v. with Flu is unknown. Here, we report Bu exposure by AUC after once-daily i.v. dosing and associations between Bu exposure and clinical outcomes.

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Materials and Methods 

Patients 

Adult patients undergoing allogeneic stem cell transplantation (SCT) conditioning for hematologic disease at a single center between July 2000 and October 2005 were enrolled if they provided informed consent for the study and met institutional guidelines for transplant eligibility. The protocol and consent forms were approved by the University of Calgary Health Research Ethics Board.

Patients were considered standard risk if transplanted during first or second complete remission (CR) of acute leukemia or first chronic phase (CP) of CML; all other patients were high risk. High-resolution human leukocyte antigen (HLA) typing was completed in all patients and donors for DR and DQ; Class I typing was done at medium resolution for A, B, and C until 2001, and thereafter all typing was high resolution.

Treatment Regimen 

Patients received i.v. Bu (Busulfex, PDL Biopharma, Redwood City, CA) at a myeloablative dose of 3.2 mg/kg on days –5 to –2 and Flu 50 mg/m2 on days –6 to –2 before SCT. Busulfan doses were given over 3 hours, and were based on actual body weight unless the patient was ≥25% above ideal body weight (IBW, 50 kg [men] or 45.5 kg [women] + 2.3 mg/kg for each inch over 5 feet), in which case adjusted ideal body weight was used (ideal body weight + 0.4[actual – ideal body weight]). Additional total-body irradiation (TBI) 200 cGy × 2 doses was given to 51 patients with acute leukemias on day –1 or 0. Seizure prophylaxis with phenytoin started 7 days before the first dose of Bu until 24 hours after the last dose to maintain trough levels of 40-80 μmol/L.

Graft-versus-host disease (GVHD) prophylaxis comprised short-course intravenous methotrexate (MTX) 15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11, and cyclosporine A 2.5 mg/kg i.v. or 6.25 mg/kg orally twice a day titrated to a trough level of 150-400 μg/L. MTX doses were decreased according to standardized pharmacy guidelines for renal failure or severe mucositis, and folinic acid 5 mg was given every 6 hours from 24 hours after each MTX dose until 12 hours before the next dose. Rabbit antithymocyte globulin (ATG; Thymoglobulin, Genzyme, Cambridge, MA) 4.5 mg/kg was given in divided doses to all patients on days −2, −1, and 0 before transplant. Acute GVHD (aGVHD) and chronic GVHD (cGVHD) were graded according to standard criteria [25] and treated by immunosuppression with steroids, ATG, mycophenylate mofetil (MMF), monoclonal antibodies (mAbs), calcineurin inhibitors, and/or photopheresis as clinically indicated.

Standard antiinfective prophylaxis with acyclovir and trimethoprim/sulfamethoxazole was given for at least 1 year posttransplant, and routine antifungal prophylaxis was not given. Growth factors were not routinely given. All blood products were from cytomegalovirus (CMV) antibody negative donors, and pp65 antigen surveillance was instituted to allow preemptive therapy with gancyclovir for CMV reactivation.

Busulfan Plasma AUC Determination 

For the pharmacokinetic analysis patient blood samples (5 mL) were collected in heparinized tubes after the third Bu dose (on day –3), at the end of infusion and at 1, 3, 5, and 7 hours post-Bu infusion. Day –5 pharmacokinetics were also done on 8 consecutive consenting patients to ensure consistency between doses in AUC. Bu concentrations in plasma were determined by UV-HPLC as previously described, with slight modifications [26]. The modifications were mainly done in the Bu derivatization procedure, which was performed with 0.5 mL of 15% sodium diethyldithiocarbamate trihydrate (DDTC) in 0.1 N sodium hydroxide solution subsequently extracted with 1/3 volume of chloroform. Prior to the derivatization step, 0.5-mL plasma samples were deproteinized by an equal volume of acetonitrile. Derivatized Bu was extracted with 2.0 mL of ethyl acetate, followed by evaporation under nitrogen and reconstitution in 100 μL of methanol; 30 μL was injected into the HPLC system (Agilent 1100, Agilent Technologies, Santa Clara, CA). The analysis was performed on a Waters NovaPak C18 (4 μm; 4.6 × 250 mm) analytic column using 80% methanol mobile phase delivered at 1.5 mL/min. Plasma drug concentration was measured at 278 nm by a diode array detector. Intraday and interday coefficient of variation for the assay were <15%, limit of quantification was 0.05 μg/mL, and the assay was linear between 0.05 and 5.0 μg/mL.

Pharmacokinetic data were analyzed by noncompartmental analysis using WinNonlin Professional version 5.0.1 software (Pharsight Corp., Mountain View, CA). The AUC was calculated by the log-linear trapezoidal rule from time zero to the last sampling time (AUClast). The AUC from the last sampling time to infinity was extrapolated by Clast/λz, where Clast and λz are the last measured (nonzero) plasma Bu concentration and the terminal slope on the Ln scale, respectively, and this extrapolated area was added to the AUClast for the final AUC to infinity calculation (AUC∞). The fraction of the AUC∞ extrapolated from Clast/λz was <10% of the total AUC value for all patients.

Engraftment 

Engraftment was assessed by daily complete blood counts until discharge, then at least weekly until bone marrow aspiration and biopsy at 12 weeks (sooner if clinically indicated). Neutrophil engraftment was defined as the first of 3 consecutive days that neutrophils were ≥0.5 × 109/L, and platelet engraftment as the first day after platelets remained ≥20 × 109/L for 7 days without transfusion support. If engraftment did not occur by 28 days in the absence of residual marrow malignancy, the patient was deemed to have primary graft failure. Chimerism for all patients was assessed by DNA analysis of variable number of tandem repeats by PCR or, in sex-mismatched donor-recipient pairs, through FISH studies for the X and Y chromosomes.

Toxicity 

Mucositis was graded according to the Bearman scale and veno-occlusive disease (VOD) by the Jones criteria 27, 28. Alanine aminotransferase and conjugated bilirubin were monitored twice weekly during the first 4 weeks and maximum weekly measurements were recorded. Hemorrhagic cystitis was defined as macroscopic hematuria with pain on voiding. Patients were monitored for seizure activity and evidence of microangiopathic hemolytic anemia.

Statistical Analysis 

A threshold value of 6000 μM·min was chosen to evaluate toxicities and outcomes with once-daily i.v. Bu. This was extrapolated from previously reported data by multiplying times 4 the AUC previously associated with decreased survival using a 4 times daily i.v. BuCy regimen for CML (AUC >1520 μM·min) to approximate the equivalent total daily Bu exposure [15]. We also evaluated patient subgroups with different ranges of Bu exposure for NRM; this ranged between 9% and 23% in groups with exposures of 2000 to 6000 μM·min. Mortality in the group exposed to 5500-6000 μM·min (N = 11) was 9% compared to 58% for the 16 patients with exposures >6000 μM·min (P = .03), despite a higher proportion of patients with high-risk disease in the lower exposure group (91% versus 62.5%). Above this level the difference between those above and below the threshold diminished as more patients with NRM moved below the threshold; therefore, use of a 6000 μM·min threshold allows a greater margin of safety and is biologically consistent with previous data.

NRM, engraftment, and overall survival (OS) were also assessed in patients with a daily Bu exposure of <3500 μM·min daily, approximately 4 times the AUC previously associated with decreased engraftment using 4 times daily oral Bu/Cy (AUC <900 μM·min), and the group with an intermediate AUC 3500-6000 μM·min 7, 8.

Patient and disease characteristics and treatment toxicities were compared between patients with high (>6000 μM·min) and low (≤6000 μM·min) Bu exposure. Kaplan-Meier curves were constructed to examine OS and progression-free survival (PFS) and the log-rank test compared equality of the survivor functions by Bu exposure. Patients known to be alive (for OS) and relapse-free (for PFS) were censored on the last day of follow-up. Cox proportional hazards modeling was used to examine the individual impact of potential confounding variables on the crude hazard ratio for death from any cause with high versus low Bu exposure. These included age, CD34+ cell dose, high-risk or standard-risk disease status, use of TBI, stem cell source, matched related or alternative donor type, CMV positivity in donor or recipient, ≥1 HLA mismatch, ABO compatibility, and female donors with male recipients. We also estimated the cumulative incidence in the presence of competing risks of nonrelapse mortality (NRM), relapse, Grade II-IV or III-IV aGVHD and cGVHD.

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Results 

One hundred thirty patients with a variety of hematologic conditions were enrolled. One hundred fourteen patients (88%) had low Bu exposure (AUC ≤6000 μM·min), whereas 16 (12%) had high exposure (AUC >6000 μM·min). Follow-up among survivors ranged from 4-67 months (median 29 months), and was shorter among those with low Bu exposure (median 28 months) compared to high Bu exposure (median 39 months). Patient, disease, and transplant characteristics by Bu exposure are shown in Table 1.

Table 1. Patient, Disease and Treatment Characteristics of Patient Groups According to Busulfan Exposure
CharacteristicAUC <6000 μM·min (n = 114)AUC >6000 μM·min (n = 16)
Age (years)
range19-6621-63
mean (standard deviation)43.6 (11.6)42.9 (13.3)
Diagnosis (%)
AML43 (37.7)9 (56.3)
MDS6 (5.3)1 (6.3)
ALL16 (14.0)1 (6.3)
CML13 (11.4)1 (6.3)
CLL7 (6.1)0 (0.0)
Hodgkins lymphoma3 (2.6)1 (6.3)
NHL15 (13.2)1 (6.3)
Multiple myeloma6 (5.3)2 (12.5)
Other4 (3.5)0 (0.0)
Donor type (%)
Matched related67 (58.8)6 (37.5)
Alternative donor47 (41.2)10 (62.5)
Disease status (%)
Standard risk52 (45.6)9 (56.3)
High risk62 (54.4)7 (43.8)
TBI (%)45 (39.4)6 (37.5)
Stem cell source (%)
Bone marrow96 (84.2)14 (87.5)
Peripheral blood18 (15.8)2 (12.5)
CD34+ cells × 106/kg
Range0.4-16.71.2-13.3
Median5.05.6
Female donor to male recipient (%)31 (27.2)1 (6.3)
CMV + donor or recipient (%)82 (71.7)9 (56.3)

AML indicates acute myelogenous leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkins lymphoma.

Busulfan Pharmacokinetics 

Busulfan AUC ranged from 2184-7794 μM·min (mean 4716, median 4699 μM·min, Figure 1). The mean clearance was 13.1 ± 4.0 L/h, with a percent coefficient of variation of 30% as the measure of variability. To assess for dose-to-dose consistency of i.v. busulfan pharmacokinetics, 8 patients were evaluated by collection of samples on days 1 and 3 of busulfan administration (transplant days −5 and −3). PK parameters on each day were calculated by noncompartmental analysis and exposure was not significantly different between days 1 and 3 for each patient, consistent with previous studies done by our group and others 18, 20. Comparison between noncompartmental analysis and 1 and 2 compartment models did not show any significant differences in calculated AUCs.

Engraftment 

There was no difference between groups with an AUC ≤6000 versus >6000 μM·min in median number of days to stable neutrophil engraftment (15 days, range: 10-115 days versus 14.5 days, range: 11-19 days, P = .38 by Wilcoxon rank-sum) and platelet engraftment (18 days, range: 0-115 days versus median 18 days, range: 0-25 days, P = .36). Graft failure was seen in 3 patients, all with an AUC ≤6000 μM·min (P = .51); AUC values in these patients were 5124 μM·min, 2639 μM·min, and 4384 μM·min.

Regimen-Related Toxicities 

No difference in organ-specific toxicities was seen between groups, including seizures, hemolytic anemia, or hemorrhagic cystitis, and no diffuse alveolar hemorrhage was seen (Table 2). Although not statistically significant, there was a trend to less severe mucositis in the low exposure group (P = .08); all patients with Bearman grade I mucositis had an AUC <6000 μM·min. No patient had clinical evidence of VOD, and when weekly maximum bilirubin (total and conjugated) and alanine aminotransferase levels were compared between exposure groups in the first 4 weeks after transplant, no difference was seen.

Table 2. Incidence of Organ-Specific Toxicities Seen in Patient Groups According to Busulfan Exposure
AUC <6000 μM/min, median (%) n = 114AUC >6000 μM/min, median (%) n = 16P
Stomatitis grade 2-3a96 (84)16 (100).08
Hemorrhagic cystitis15 (13)3 (19).54
Microangiopathic Hemolytic anemia6 (5)0 (0).35
Seizures2 (2)1 (6).26

All tests were grouped tests of proportions (chi-square).

aBearman scale [25].

GVHD 

The total incidence of aGVHD and cGVHD was not different between groups. The cumulative incidence at 3 months of grade III-IV aGVHD was similar at 25% in the group with high exposure (95% confidence interval [CI]: 8%-47%) and 13% in the lower exposure group (95% CI: 8%-20%). Grade II-IV GVHD was experienced in 31 of 114 (27%) of our sample population with Bu AUC ≤6000 μM·min and 5 of 16 (31%) of our sample with Bu AUC >6000 μM·min. The cumulative incidence of cGVHD at 8 months for patients with high and low busulfan exposure was similar at 50% (95% CI: 24%-71%) and 52% (95% CI: 42%-60%), respectively.

Relapse 

The cumulative incidence of relapse is shown in Figure 2. The relapse rate at 12 months was not different between groups at 31% (95% CI: 11%-54%) among those with high Bu exposure compared to 20% (95% CI: 13%-28%) among those with low Bu exposure.

NRM 

There was a lower cumulative incidence of NRM in the low Bu exposure group (P = .002); at 100 days NRM was 6% (95% CI: 3%-12%) compared to 19% in the high exposure group (95% CI: 5%-40%; Figure 3). At 12 months, the cumulative incidence of NRM remained higher in the group with busulfan AUC >6000 μM·min (38%, 95% CI: 15%-60%) compared to those with AUC ≤6000 μM·min (14%, 95% CI: 9%-22%). Causes of NRM in the 2 groups are outlined in Table 3. Deaths were considered to be from GVHD if they were a direct result of GVHD or a complication of GVHD therapy (ie, infection). They were considered to be from sepsis if they were a result of infection in the absence of aGVHD or chronic extensive GVHD and its treatment.

Table 3. Causes of Nonrelapse Mortality in Transplant Recipients According to Busulfan Exposure Posttransplantation
TimingCause of Nonrelapse MortalityNumber of Deaths AUC ≤6000 μM/min, n = 114 (%)Number of Deaths AUC >6000 μM/min, n = 16 (%)
<100 days posttransplantSepsis/infection2 (2)1 (6)
GVHD3 (3)2 (13)
2 GVHD/infection1 GVHD
1 GVHD/PTLD1 GVHD/infection
Other2 (2)0 (0)
1 encephalopathy, 1 arrhythmia
100 days or more posttransplantSepsis/infection1 (1)0 (0)
GVHD4 (4)3 (19)
1 GVHD1 GVHD
2 GVHD/infection2 GVHD/infection
1 GVHD/PTLD
Other3 (3)1 (6)
2 graft failure, 1 IPS1 intracerebral hemmorhage
1 adenocarcinoma, 1 spont liver hemorrhage
Total 17 (15)7 (44)

GVHD indicates graft-versus-host disease; PTLD, posttransplant lymphoproliferative disorder; IPS, interstitial pneumonia syndrome.

OS and PFS 

Estimated survival of the entire group was 70% at 12 months (95% CI: 61%-77%) and 62% at 36 months (95% CI: 52%-70%). Patients with an AUC >6000 μM·min had worse OS than those in the low exposure group (P <.001; Figure 4). At 12 months, survival in the group with AUC ≤6000 μM·min was 74% (95% CI: 65%-82%) compared to 38% (95% CI: 15%-60%) in the group with high Bu exposure, and at 36 months, survival in these groups was 68% (95% CI: 57%-76%) compared to 23% (95% CI: 6%-46%), respectively. The survival difference was most striking in standard risk patients (P < .001), with a 3-year estimated OS of 79% (95% CI: 64%-89%) in the low exposure group and 33% (95% CI: 8%-62%) in the high Bu exposure group. The effect was also seen among patients with high-risk disease (P = .05), although the number of events in this group was small; 3-year estimated survival in the low exposure group was 59% (95% CI: 45%-71%) compared to 14% (96% CI 1%-46%) in the high Bu exposure group.

Earlier censoring of patients in the low Bu exposure group because of shorter time under observation may complicate this comparison; therefore, we also generated conservative Kaplan-Meier curves (not shown) under the assumption that all censored individuals in the low exposure group died on the last day of follow-up. Estimated OS at 12 months remained significantly lower among patients in the high exposure group (38%, 95% CI: 15%-60%) compared with those in the low exposure group (61%, 95% CI: 52%-70%).

PFS at 3 years estimated by Kaplan-Meier curves was higher in the group with Bu exposure ≤6000 μM·min (P < .001) at 58% (95% CI: 47%-67%) compared to 16% in those with high busulfan exposure (95% CI: 3%-38%; Figure 5). When the conservative analysis done above was repeated for PFS, it remained significantly worse among patients with high versus low exposure.

In a Cox proportional hazards model, the crude hazard ratio for the effect of high busulfan exposure (AUC >6000 μM·min versus ≤6000 μM·min) on OS was 3.2 (95% CI: 1.7%-6.3%). Simultaneous adjustment for all possible confounders was not possible because of the relatively small number of events, but we did explore the impact on the hazard ratio of adjustment for 1 variable at a time. Adjustment for age, CD34+ cell dose, standard versus high-risk disease group, treatment with TBI, stem cell source, donor type, ABO incompatibility, the presence of at least 1 HLA mismatch and female donor-to-male recipient did not alter the crude hazard ratio appreciably (data not shown). Adjustment for CMV positivity in either donor or recipient increased the estimated hazard ratio of high Bu exposure to 4.1 (95% CI: 2.0%-8.1%).

We also analyzed any differences in engraftment and OS between patients with AUC <3500 μM·min, and between 3500 and 6000 μM·min. No difference was seen in days to stable neutrophil or platelet engraftment or OS between groups (data not shown).

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Discussion 

Although the 3.6-fold variability in Bu AUC following once-daily i.v. dosing with Flu is less than that seen with oral Bu, this is the first study showing decreased OS and PFS, and increased NRM, among patients with Bu exposures greater than an AUC of 6000 μM·min. It was hoped that an i.v. form of Bu might allow safe administration without the need for TDM, which is often used with oral 4-times daily BuCy to keep Bu exposure within an AUC range of 900-1500 μM·min, a range that has been associated with better OS and disease-free survival in a 4-times daily i.v. BuCy2 regimen for CML [15]. High exposure to once-daily i.v. Bu given with Cy is likely to also be associated with higher toxicity. Although substitution of Flu for Cy might have been expected to allow higher exposures to Bu, our data support a threshold effect with high busulfan exposure associated with an increased risk for lethal adverse events. Although this high NRM could be attributable to drug exposure, it is also possible that the high exposure is a surrogate marker for metabolic factors contributing to both high exposure and to other factors leading to a higher risk of NRM. We may answer this in time if mortality in patients who would otherwise have been overexposed remains high despite having exposure adjusted on the basis of a test or day 1 dose.

The reason for better survival in patients with a Bu AUC ≤6000 μM·min appears to be reflected in the nonrelapse mortality. The small number of patients with adverse events in the high Bu exposure group limits the ability to determine the specific cause of increased mortality; specific toxicities were not significantly different between Bu exposure groups, however, this study is not powered to detect them. One way in which more intense conditioning may increase NRM is by increasing tissue damage and inflammation, possibly stimulating GVHD; this effect has been suggested previously with i.v. busulfan in Bu/Cy 14, 29. The finding of no difference in aGVHD and cGVHD rates between AUC groups is consistent with that seen in previous case series [9]; however, most of the deaths in our high Bu exposure patients were related to GVHD or complications of its treatment, that is, infections. The worse survival of patients with high Bu exposure was most striking in patients with low-risk disease, a group expected to do well. Our data suggest that avoiding exposures to Bu AUC >6000 μM·min may be associated with reduction of the already low NRM in this group. Because of the marked differences in outcomes between groups with high and low busulfan exposure we did not feel it was ethically justified to continue accruing patients to this study.

Patient baseline characteristics were assessed and, although some differences existed, in multiple bivariate analyses none except for CMV positivity in donor or recipient were found to have a meaningful impact on the hazard ratio for death associated with high Bu exposure. As more patients in the low Bu exposure group were CMV positive in the donor and/or recipient, this would actually be expected to decrease the survival difference between groups. The small number of patients in the high Bu exposure group precluded the construction of a multivariable Cox proportional hazards model simultaneously accounting for potential confounding variables. Addition of low-dose TBI (400 cGy) did not alter the hazard ratio associated with high Bu AUC in our analysis, and was equally represented in both groups. This is consistent with our previous studies showing that addition of TBI does not increase NRM in AML, and that the NRM in early leukemia with this combination is below 5% 30, 31.

This regimen is designed to avoid toxicities of Cy, including potentially fatal hepatic VOD. In a small study using daily i.v. Bu with Cy, 2 of 5 patients with a Bu AUC >6000 μM·min developed severe VOD, and both eventually died; in another study by Fernandez et al 32, 33, 6 patients were given i.v. Bu 3.2 mg/kg daily with Cy; 1 patient developed VOD. Following once-daily i.v. Bu with Flu, both we and de Lima et al 20, 22 have reported common elevations in bilirubin and liver enzymes but a very low incidence of VOD (0 of 70 and 2 of 96 patients, respectively). As opposed to i.v. Bu, oral Bu dosing involves a hepatic first pass effect. Metabolites of Cy itself likely contribute to VOD occurring after the Bu/Cy regimen, an effect that may be avoided with fludarabine [34].

Our data provide no evidence that a higher Bu exposure >6000 μM·min provides protection in terms of lower relapse to compensate for increased toxicity. It is difficult to comment on the relapse rate in this setting, as this study involves a heterogeneous population of patients with a variety of malignancies at different stages of disease. However, the presence of high-risk disease pretransplant was not found to impact the hazard ratio for high busulfan exposure. Further study of these findings among larger group of patients within specific diagnosis categories is warranted.

In oral Bu dosing, TDM is associated with a relatively low frequency of VOD and improved engraftment 35, 36. Deeg et al [37] have given targeted oral Bu 4 times daily (PK-guided dose adjustment to 600-900 ng/mL, equivalent to AUC of approximately 900-1350 μM·min) with Cy for elderly MDS patients and found favorable rates of OS, NRM, and relapse compared to transplantation with other regimens. Individualized dose adjustment of 4-times daily i.v. Bu targeting an AUC of 1150 μmol/L/min per dose has been well tolerated in children with Cy and thiotepa with low rates of VOD [38].

This study suggests that Bu once-daily dosing given with Flu for an AUC >6000 μM·min may be associated with increased NRM and worse OS. These data are consistent with findings in other studies using different dosing regimens, and support the role of TDM with once-daily i.v. Bu. Further study with larger patient numbers is required to establish whether patient outcomes are improved with TDM. The development of reliable test dose assays or real-time Bu dose adjustment will aid in targeting Bu exposure into the therapeutic range, and may provide a useful tool to minimize Bu toxicity and NRM [39].

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Acknowledgments 

The authors gratefully acknowledge the research nurses, nursing staff in the hematopoietic stem cell transplant program at the Foothills Hospital, Leanne Kmet, and Dr. Peter Faris for advice regarding statistical analysis, Jeanne Sheldon for work on figures, and especially the patients and families for their participation. This study was supported in part by research funding from the Alberta Cancer Foundation and PDL BioPharma Inc. The company was not involved in study design or data interpretation. The authors declare no competing financial interests. J.R. designed research, M.G. and D.M.Q. collected data, S.B.K. and B.A. designed the assay, S.B.K. and F.N. performed research, M.G., S.B.K., B.A., and J.R. analyzed and interpreted data, and M.G. and J.R. drafted the manuscript. All other authors collected data and participated in patient care.

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PII: S1083-8791(07)00557-5

doi:10.1016/j.bbmt.2007.10.028

Biology of Blood and Marrow Transplantation
Volume 14, Issue 2 , Pages 220-228, February 2008

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