Graft rejection Busulfan concentration and graft rejection in ... - Nature

Bone Marrow Transplantation (2002) 30, 167–173  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt

Graft rejection Busulfan concentration and graft rejection in pediatric patients undergoing hematopoietic stem cell transplantation JS McCune1,2, T Gooley1, JP Gibbs2, JE Sanders1,2, EW Petersdorf1,2, FR Appelbaum1,2, C Anasetti1,2, L Risler1, D Sultan1 and JT Slattery1,2 1

Fred Hutchinson Cancer Research Center, Seattle, WA, USA; and 2University of Washington, Seattle, WA, USA

Summary: We retrospectively analyzed the relationship between busulfan average steady-state plasma concentration (CSS) and graft rejection in 53 children receiving busulfan/cyclophosphamide (BU/CY) preparative regimens prior to hematopoietic stem cell transplantation (HSCT). Patients received a total oral busulfan dose of 11 to 28 mg/kg followed by a total cyclophosphamide dose of 120 to 335 mg/kg in preparation for allogeneic grafts (HLA-matched or HLA partially matched sibling, parent or unrelated donor). Graft rejection occurred in eight (15%) patients. Busulfan CSS (P = 0.0024) was the only statistically significant predictor of rejection on univariate logistic regression analysis, with the risk of rejection decreasing with an increase in busulfan CSS. Severe (grade 3 or 4) regimen-related toxicity (RRT) occurred in four patients. Ten patients (19%) had a busulfan CSS higher than 900 ng/ml, one of whom had severe RRT. Higher and variable doses of cyclophosphamide may explain the lack of a relationship between busulfan CSS and RRT in children. It may be possible to improve the outcome of HSCT in pediatric patients receiving the BU/CY regimen through optimization of busulfan CSS and better definition of the contribution of activated cyclophosphamide metabolites to toxicity. Bone Marrow Transplantation (2002) 30, 167–173. doi:10.1038/sj.bmt.1703612 Keywords: busulfan; pediatrics; rejection; hematopoietic stem cell transplantation.

The success of busulfan/cyclophosphamide (BU/CY) preparative regimens for hematopoietic stem cell allografts is due, in part, to the remarkable complementary nature of the two agents demonstrated in preclinical studies.1–5 The promise of BU/CY preparative regimens was quickly realized in clinical studies. The first regimen used extensively for adults receiving allografts was 16 mg/kg busulfan with 200 mg/kg cyclophosphamide.6 The dose of cyclophosCorrespondence: JS McCune, University of Washington, Box 357630, Seattle, WA 98195, USA Received 28 September 2001; accepted 28 February 2002

phamide was later decreased to 120 mg/kg for adults to diminish regimen-related toxicity.7 Achieving consistent engraftment has been a greater challenge in children and the dose of cyclophosphamide in general has remained at 200 mg/kg.8 It is now well established that children treated with highdose busulfan, based on either a mg/kg or a mg/m2 basis, achieve lower plasma concentrations, measured as area under the plasma-concentration time curve (AUC) or steady-state concentration (Css, which is AUC/dosing interval) than adults.9–15 Children below the age of 4 years are most at risk of having low busulfan plasma concentrations. The lower plasma levels of busulfan are apparently due to an enhanced ability to metabolize the drug through an upregulation of busulfan-glutathione conjugase (glutathione Stransferase A1–1).16,17 We15 and others18 have observed a relationship between low busulfan Css and the incidence of graft rejection in patients receiving BU/CY. In a population of 15 adults (⬎18 years ) and 28 children (0.6–16 years) receiving BU/CY, there was a difference in the busulfan Css needed to retain HLA-matched sibling grafts (⬎200 ng/ml) in comparison to that needed for partially matched related or matched unrelated donor grafts (⬎600 ng/ml).15 Bolinger et al18 did not observe a difference in threshold busulfan Css between the different graft sources in 31 children receiving BU/CY ⫾ ATG, although few patients received grafts from partially matched related or matched unrelated donors (n = 6).18 However, a busulfan Css ⬍600 ng/ml was correlated with rejection.18 Further definition of the relationship between busulfan Css and graft rejection in children should lead to the development of more effective conditioning regimens with a lower incidence of graft rejection. This retrospective data analysis was undertaken to identify the relationship between busulfan Css and graft rejection in children. The relationship between busulfan Css and regimen-related toxicity (RRT), which has been found in adults,19,20 could not be assessed due to the low incidence of severe RRT in these patients.

Pediatric transplants – busulfan levels JS McCune et al

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Methods Patients Records collected as part of routine clinical busulfan monitoring between April 1991 and September 1998 at the Fred Hutchinson Cancer Research Center were examined retrospectively. Inclusion required that the patients were less than 18 years old, were receiving a preparative regimen containing busulfan and cyclophosphamide, had not received a previous hematopoietic stem cell transplant and were enrolled in treatment protocols that stipulated busulfan monitoring. These patients represent approximately 52% of the pediatric patients treated with a preparative regimen including both busulfan and cyclophosphamide during that time period. Records were examined for age, gender of recipient and donor, disease, prior chemotherapy, donor– recipient HLA matching, height, weight, body surface area, preparative regimen, donor cell source and dose, medications used and toxicity up to day 28, and rejection and relapse to day of last follow-up. Patients or their guardians provided informed consent for the pharmacokinetic analysis. The final database contained information on 53 patients, 17 of whom were included in an earlier report.15 Patient pre-transplant demographics are described in Table 1. The median age was 6 years (range 0.25–16), with 21 patients (39%) ⬍4 years of age. Fifty-one percent of the patients were male. Forty-two percent of the patients had received chemotherapy weeks to months prior to administration of the preparative regimen for HSCT. Patients received busulfan every 6 h for a total of 16 doses, for a total oral busulfan dose of 11–28 mg/kg. The oral busulfan dose was less than 16 mg/kg in four patients, 30 received 16 mg/kg and 19 received greater than 16 mg/kg. In very young children unable to co-operate with oral administration of busulfan, a nasogastric tube was inserted for the administration of Table 1

Patient demographics

n Age Male:Female Disease Acute myeloid leukemia (AML) Myelodysplastic syndrome/refractory anemia Severe combined immunodeficiency syndrome (SCID) Thalassemia Wiskott-Aldrich syndrome Chronic myelogenous leukemia Metachromatic leukodystrophy Chediak-Higashi Niemann-Pick disease Sickle cell anemia Acute lymphoblastic leukemia Myeloproliferative disorder Leukocyte adherence defect Blackfan-Diamond anemia Hemophagocytic lymphohistiocytosis Bare lymphocyte syndrome Chronic granulomatous disease Received prior chemotherapy

Bone Marrow Transplantation

53 6 years (range 0.25–16) 27:26 19 7 5 3 3 2 2 2 2 1 1 1 1 1 1 1 1 22

busulfan as a slurry of crushed tablets or later as a suspension. All other patients received 2 mg tablets with doses rounded to the nearest 1 mg of the dose calculated from body weight. Food was withheld for 1 h before and after busulfan administration. For control of emesis, patients weighing ⭐40 kg received either granisetron 10 ␮g/kg intravenously (i.v.) 20–30 min before the first busulfan dose and then every 12 h until completion of busulfan or ondansetron 0.15 mg/kg i.v. or orally (to nearest 2 mg) 20–30 min before each busulfan dose. Lorazepam 0.05 mg/kg i.v. or orally every 6 h and/or diphenhydramine 1 mg/kg i.v. or orally every 4–6 h were used as needed for breakthrough nausea and vomiting. For patients weighing ⬎40 kg, either granisetron 1 mg orally 20–30 min before the first dose of busulfan and every 12 h until completion of busulfan or ondansetron 8 mg orally 20–30 min before each busulfan dose was administered. Prochlorperazine 10 mg i.v. or orally every 4 h; lorazepam 0.5–2 mg i.v. or orally every 4 h and/or diphenhydramine 25–50 mg i.v. or orally were used as needed for breakthrough nausea and vomiting. The various characteristics of the HSCT treatment are listed in Table 2. Cyclophosphamide was administered after busulfan as part of their transplant preparative regimen. The total dose of cyclophosphamide varied from 120 to 335 mg/kg depending on disease and the treatment protocols in which a patient was enrolled. Ten (19%) of the children received cyclophosphamide 120 mg/kg, 35 (66%) received 200 mg/kg and the eight remaining patients (15%) received ⬎200 mg/kg. Nine (17%) patients received antithymocyte globulin (ATG) as part of the preparative regimen. The target population for the different dose levels of busulfan and cyclophosphamide are listed in Table 3. No other antineoplastic agents or irradiation were given immediately before or concomitantly with busulfan. Forty-two percent of patients received phenytoin for seizure prophylaxis. Bone marrow was used in 50 patients, with two patients receiving peripheral blood stem cells and one cord blood stem cells. The median cell dose from allogeneic bone marrow donors was 4.21 ⫻ 108/kg, with a range of 1.53 ⫻ 108 to 2.15 ⫻ 109 cells/kg. Twenty-six children received a graft from an HLA-matched sibling. The remaining 27 patients received grafts from HLA-matched unrelated (n = 12), partially HLA-matched parent (n = 5), partially HLA-matched unrelated donor (n = 5), HLA-matched parent (n = 3) and HLA partially matched sibling (n = 2). Using serologic methods, 11 of these 27 donor–recipient pairs were mismatched at the A, B, or C class I antigens. Of the remaining 16 donor–recipient pairs, 14 were matched at the A, B and C class I antigens and two pairs were HLA-matched at the A, B antigens. DNA sequencing of the HLA-A, B, and C genes could not be performed for 15 of the 16 serologically matched pairs because donor and/or recipient cells or DNA were not available.21 Three of these 16 donor–recipient pairs were serologically mismatched at the DRB1. Dosing and pharmacokinetic methods for oral busulfan Blood samples were collected immediately before and 60, 120, 180, 240 and 360 min after the fifth and ninth doses of busulfan. Plasma busulfan concentrations were determined by gas chromatography with mass spectrometry detec-


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Table 2 Transplant characteristics Preparative regimen

44 (83%) BU/CY 9 (17%) BU/CY with ATG 702 (range 120–1371) 22 (42%) 5301 (range 2692–8276)

Busulfan Css (ng/ml)a Received phenytoin CY dose (mg/m2)a CY dose (mg/kg) 120 mg/kg 200 mg/kg ⬎200 mg/kg Donor

10 35 8 26 12 5 5 3 2 26 13 2 12 50 2 1

Donor–recipient matchb

Stem cells GVHD prophylaxis Methotrexate and cyclosporine Methotrexate alone Other

(19%) (66%) (15%) HLA-matched sibling HLA-matched unrelated HLA-partially matched parent HLA-partially matched unrelated HLA-matched parent HLA-partially matched sibling HLA-matched sibling HLA-matched at A, B, C antigens HLA-matched at A, B antigens HLA-mismatched at A, B or C antigens Bone marrow Peripheral blood Cord blood

39 (74%) 9 (17%) 5 (9%)

a

Median (range). Serological class I HLA.

b

Table 3

Preparative regimen based on diagnosis

Target population

Busulfan Total dose

Myelodysplastic syndrome and myeloproliferative disorders AML AML Sickle cell anemia, thalassemia SCID Inborn errors of metabolism

16 mg/kg 16 mg/kg 160 mg/m2 14 mg/kg 16 mg/kg ⭓19 mg/kg

Inborn errors of metabolism

16 mg/kg

Cyclophosphamide total dose

Additional agents

Targeting Post 1995 Unrelated donors Mismatched related or matched unrelated graft

120 mg/kg 120 200 200 200 200

mg/kg mg/kg mg/kg mg/kg mg/kg

Radiolabeled BC8a ATG ATG ATG

5–8 g/m2

n = 1.

a

tion as previously described.15,22 The samples are assayed immediately to calculate busulfan Css. The AUC is determined by summation of successive trapezoids with extrapolation using apparent elimination half-life determined by regression of the log-transformed, first-order decline data. The busulfan Css is calculated by dividing the AUC by the dosing interval.15,22 A busulfan AUC of 875 ␮m-min is equivalent to a busulfan Css of 600 ng/ml, assuming a 6 h dosing interval. The busulfan Css after each of these two doses was determined and the average of the two values was used in the outcome analyses. The Css after the fifth dose could not be determined in 3/53 (5%) patients and, in those cases, the Css after the ninth dose only was used. The busulfan Css was obtained after the ninth dose in all patients.

Assessment of rejection and toxicity All patient medical charts submitted from referring physicians and from research data files were reviewed to the day of last follow-up. Median follow up was 542 days (range 15–3092 days) with date of last clinic visit or correspondence from patient, second transplant or death recorded as last follow-up. Post-transplant donor engraftment was determined by the presence of progressively rising neutrophil counts after the post-transplant nadir and bone marrow examination. When appropriate, additional evidence of engraftment was determined by in situ hybridization studies and/or HLA serological typing and/or DNA chimerism studies. Graft rejection was defined as failure of donor engraftment accompanied by return of Bone Marrow Transplantation


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host lymphocytes. Patients had bone marrow samples examined on days 28, 80 and 365 post transplant. Other samples were collected at other times based on clinical indication. Regimen-related toxicity (RRT) was evaluated as described by Bearman et al.23 All charts were reviewed (by JSM, grade 4 confirmed by JES) and the overall grade assigned was the maximum RRT grade for cardiac, bladder, renal, pulmonary, hepatic, central nervous system, mucositis, and gastrointestinal during the first 28 days after transplant. Statistical analysis All statistical analyses were performed using SPSS version 8.0 (SPSS, Chicago, IL, USA). The relationships between individual patient variables and rejection were characterized using stepwise logistic regression. Categorical patient variables included HLA-class I match (matched sibling vs serological match vs serological mismatched), prophylaxis for graft-versus-host disease (single agents vs combination), gender match between donor–recipient (matched vs male donor–female recipient vs female donor– male recipient), prior chemotherapy (yes vs no), disease (cancer vs not cancer) and administration of ATG (yes vs no). Continuous patient variables included age, marrow cell dose per kg of actual body weight, busulfan steady-state concentration (Css) from doses 5 and 9, cyclophosphamide dose expressed relative to body weight (mg/kg) and cyclophosphamide dose expressed relative to body surface area (mg/m2).

Results Busulfan pharmacokinetics The median busulfan clearance in younger children 0.25– 4 years of age was 5.91 (range 2.80–29.10) ml/min/kg and 140 (range 88.3–598) ml/min/m2. In older children (5–16 years of age), the median (range) was 3.56 (1.65–12.18) ml/min/kg and 116 (59.5–310) ml/min/m2 (P = 0.024 for ml/min/m2). The median value for bias, calculated by subtracting day 5 from day 9 busulfan clearance and subsequently dividing by the day 5 clearance, was ⫺2% (range ⫺62% to +84%). The median value for precision, the absolute value of this ratio, was 19% (range 0.18–84%). The precision was less than 10% for 24 of the 53 (48%) patients. Twenty-three of 29 patients (79%) treated on protocols that specified adjustment of dose to achieve a desired busulfan Css required dose adjustment based on pharmacokinetic analysis of the first dose. Outcome of HSCT The median day of the ANC nadir was 7, and the median day to engraftment as measured by the return of ANC to ⬎0.5 ⫻ 109/l was 19 days. The median time for platelet count to rise above 50 ⫻ 109/l was 21 days. Graft rejection occurred in eight of the 53 (15%) patients, with four of Bone Marrow Transplantation

the rejections occurring after day 80 (Table 4). These eight patients had the following diseases (n/disease): thalassemia (2), acute myeloid leukemia (AML, n = 1), hemophagocytic lymphohistiocytosis (1), myeloproliferative disorder (1), bare lymphocyte syndrome (1), myelodysplastic syndrome (1), and metachromatic leukodystrophy (1). Two of these eight patients who had rejected their grafts had received chemotherapy prior to receiving their preparative regimen (for AML and hemophagocytic lymphohistiocytosis). On logistic regression analysis, busulfan Css was the only statistically significant parameter associated with the observed probability of rejection in univariate model (P = 0.0024). (Busulfan Css was the only variable associated with rejection (P = 0.0059) in subset analysis excluding the three children without busulfan clearance data after dose 5. Therefore, the results are presented for all children.) Other variables that were of borderline statistical significance, based on the criteria of P ⬍ 0.10 on univariate analysis, were also identified. Serological HLA-class I match (P = 0.0964) and the cyclophosphamide dose (mg/m2) (P = 0.0703) were marginally related to graft rejection, while cell dose, graft-versus-host disease prophylaxis, cyclophosphamide dose (mg/kg), gender match between recipient and donor, age and ATG administration were unrelated to graft rejection (P ⬎ 0.1). Figure 1 shows the relationship between busulfan Css and rejection (with 1 indicating rejection). The solid line represents the predicted probability of graft rejection in relation to busulfan CSS. At low busulfan Css (⬍200 ng/ml) the predicted probability of rejection was 55%, whereas, at higher busulfan Css (⬎600 ng/ml), the predicted probability of rejection was less than 12%. Graft rejection occurred in five of 20 patients with a busulfan Css ⬍600 ng/ml and in three of 33 patients with a busulfan Css ⬎600 ng/ml (Table 5). No relapses occurred in the 14 children transplanted for AML in first complete remission. The median busulfan Css in these patients was 837 ng/ml (range 246–1371 ng/ml). Relapse occurred in one AML patient transplanted in refractory relapse, one in second complete remission and one with biphenotypic leukemia who had a busulfan Css of 681, 719 and 720 ng/ml, respectively. The other two patients who relapsed were patients with acute lymphoblastic leukemia and one with chronic myelogenous leukemia. Forty (75%) and nine (17%) patients experienced a maximum toxicity of grade 1 and 2, respectively, in any organ system. Four of the 53 (8%) patients experienced severe RRT (grade 3 or 4), three of whom experienced grade 4 hepatotoxicity. The relationship between RRT and individual patient variables could not be assessed by logistic regression analysis because of the limited number of severe RRT. One of the four patients with severe RRT had a busulfan Css ⬎900 ng/ml, the previously defined concentration associated with severe RRT in predominantly adolescent and adult populations.15,19,20 However, 10 of the 53 patients (19%) had a busulfan Css higher than 900 ng/ml.15,19,20 Nine of the 10 patients with a Css ⬎900 ng/ml had no toxicity greater than grade 2 in various organ systems.


Table 4 UPN

171

Description of pediatric patients experiencing rejection Day of rejection

7657 8137 6384 9717 8437c 6280 6330d 6163

Prior chemotherapy

92 53 33 48 97 110 22 101

Yes Yes No No No No No No

Busulfan CSS (ng/ml)

246 291 120 639 676 123 249 856

CY dose

Class I serological

mg/kg

mg/m2

Recipient–donor match

Donor

Gender recipient: donora

200 200 200 120 200 288 200 120

5238 3946 4533 3920 4186 6785 4923 3145

Matched Matched Matched Matched Matched Matched Matched Matched at HLA-A Mismatched at B, C

Sibling Sibling Parent Parent Unrelated Unrelated Unrelated Unrelated

F:F M:M F:M F:F M:F M:M M:F F:F

TNC cell doseb

6.95 1.02 1.45 2.10 6.28 4.24 3.59 2.81

⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻

108 109 109 108 108 108 108 108

a

Female (F); Male (M). Total nucleated cell dose/kilogram. c Not typed for C antigen. d Received ATG.

Probability of rejection

b

1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.10 0.20

0

200

400

600 800 1000 1200 Busulfan Css (ng/ml)

1400

1600

Figure 1 The relationship between busulfan Css and graft rejection in pediatric patients undergoing HSCT. The solid line represents the predicted probability of rejection in relation to busulfan Css determined by univariate logistic regression (P = 0.0024).

Table 5 Incidence of graft rejection in relation to busulfan Css and allogeneic graft sourcea Source of graft

Overall Matched sibling Serologically matched at HLA A, B, Cb Serologically mismatched at HLA A, B, or C

Busulfan CSS (ng/ml) ⬍600

⬎600

5/20 2/10 3/5 0/5

3/33 0/16 2/10 1/7

a

Number of rejections/Number of patients. Class C data not available in two patients, both of whom had busulfan Css ⬎600 ng/ml.

b

Discussion The major finding of this study was that busulfan Css is a determinant of graft rejection in children undergoing HSCT following the BU/CY preparative regimen. We observed a decreasing risk of rejection with increasing busulfan Css.

The other variables examined did not appear to influence the probability of rejection; however, the analysis is limited because of the relatively low number of patients who experienced rejection (n = 8, Table 4). In these eight patients, the busulfan Css ranged from 120 to 856 ng/ml. The graft source was HLA class I serologically matched unrelated (3), HLA class I serologically matched parent (2), matched sibling (2), and HLA class I serologically mismatched unrelated (1). Busulfan Css was less than 300 ng/ml in the two patients who rejected grafts from a matched sibling donor. Of the remaining six patients experiencing rejection, five were transplanted from a serologically matched donor, and one received a graft from a class I serologically mismatched donor. There were 16 serologically matched donor–recipient pairs in which the class I allele data was pursued. However, lack of cells or DNA from donors or recipients precluded sequencing analysis in 15 of the 16 donor–recipient pairs, of which five experienced rejection. Hence, it is not known whether the five graft failure cases encoded undetected class I allele disparities that might have contributed to their risk of graft failure.21 Future studies of graft failure in children conditioned with busulfan will require definition of the potential role of allele disparities. The results from this analysis qualitatively agree with our earlier observations.15 In a report from Bolinger et al,18 a busulfan Css ⬍600 ng/ml was associated with graft rejection in 31 children with allogeneic grafts from various sources (25 HLA matched sibling; 4 HLA matched unrelated donor; 1 matched parent; 1 mismatched parent) receiving BU/CY ⫾ ATG. In addition, these investigators reported in abstract form that adjusting the busulfan dose to achieve a busulfan Css of 600 to 900 ng/ml increased the rate of engraftment from 74% to 96% in 24 children.24 In contrast, no relationship between busulfan Css and graft rejection was observed in children with ␤-thalassemia receiving a graft from a genotypically HLA-matched sibling, even though six of 64 patients had busulfan Css ⬍200 ng/ml and 53 had busulfan Css ⬍600 ng/ml.25 However, only five (7.5%) of the children experienced rejection, sugBone Marrow Transplantation


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gesting that these children received grafts better matched in minor histocompatability antigens than our patients or those in Bolinger et al’s study. The relationship between relapse in patients diagnosed with AML and individual patient variables, including busulfan Css, could not be assessed using logistic regression because a limited number (3) of relapse events occurred. No relapses occurred in the 14 children transplanted for AML in first complete remission, who had a median busulfan Css of 837 ng/ml. We have previously demonstrated that busulfan Css ⬎900 ng/ml has been associated with a decreased incidence of relapse in adults with chronic myelogenous leukemia (CML) receiving the BU/CY preparative regimen.26 Baker et al27 recently reported that relapse was not related to busulfan Css in 52 patients with AML in first complete remission who received BU/CY followed by autologous (n = 25) or allogeneic (n = 27) grafts. Sixteen (31%) of the patients relapsed after an unspecified time to last follow-up. The low number of patients receiving grafts from one type of allograft may have limited this analysis, as exemplified by the regression analysis showing that graft source was not associated with the risk of relapse (relative risk = 2.7 with 95% confidence interval of 0.9–8.0; P = 0.08), which is in contrast to other reports in this patient population.28 Severe RRT did not appear to be related to busulfan Css in these children. The incidence of severe RRT increases as busulfan Css exceeds 900 ng/ml in studies primarily involving adults receiving a BU/CY preparative regimen.15,19,20 However, in adults with CML receiving BU/CY, elevated busulfan Css does not increase the risk for severe RRT, possibly because of less intense chemotherapy in the period prior to HSCT.26 Ten of 53 children in the present study achieved busulfan Css ⬎900 ng/ml, one of whom had severe RRT. Three children with Css ⬍900 ng/ml had grade 4 hepatotoxicity. Bolinger and coworkers24 did not observe a relationship between busulfan Css and RRT in 31 children receiving BU/CY ⫾ ATG, with a cyclophosphamide dose of 200 mg/kg. However, only one of the 31 patients studied had a busulfan Css ⬎900 ng/ml. These results lead to at least two hypotheses. Specifically, young age may protect from severe RRT or the higher doses of cyclophosphamide received by children may obscure any relationship between busulfan Css and toxicity. The majority (81%) of the children in this study received 200 mg/kg or more cyclophosphamide while only 19% received 120 mg/kg, the dose most often used in adults. Each of the three children who experienced grade 4 hepatotoxicity received at least 200 mg/kg of cyclophosphamide. Hepatic veno-occlusive disease has recently been related to the AUC of the acid metabolite of 4-hydroxycyclophosphamide in 105 adult CML patients receiving a bone marrow graft from an unrelated donor following a CY/TBI preparative regimen.29 In co-cultures of hepatocytes and sinusoidal endothelial cells, acrolein, a toxin formed by ␤elimination from aldophosphamide (a tautomer of 4hydroxycyclophosphamide) is a hepatotoxin.30 The suggestion that cyclophosphamide contributes to hepatotoxicity in HSCT is also suggested by the apparent lower incidence of VOD in patients receiving busulfan in combination with melphalan and thiotepa.31 As doses of a second hepatotoxin


(cyclophosphamide) increase and are varied within a study, one would expect the relationship between busulfan Css and the incidence of VOD to become more difficult to detect. In conclusion, busulfan Css is a determinant of graft rejection in children receiving a BU/CY ⫾ ATG preparative regimen. Severe regimen-related toxicity to this preparative regimen does not appear to be related to busulfan Css, in contrast to what has been observed in adults. Higher doses of cyclophosphamide in children may have obscured any relationship between busulfan Css and RRT in children. It is likely that the outcome of HSCT in pediatric patients receiving the BU/CY regimen could be improved through optimization of busulfan Css.

References 1 Tutschka PJ, Santos GW. Bone marrow transplantation in the busulfan-treated rat. III. Relationship between myelosuppression and immunosuppression for conditioning bone marrow recipients. Transplantation 1977; 24: 52–62. 2 Santos GW, Tutschka PJ. Marrow transplantation in the busulfan-treated rat: preclinical model of aplastic anemia. J Natl Cancer Inst 1974; 53: 1781–1785. 3 Storb R, Weiden PL, Graham TC et al. Hemopoietic grafts between DLA-identical canine littermates following dimethyl myleran. Evidence for resistance to grafts not associated with DLA and abrogated by antithymocyte serum. Transplantation 1977; 24: 349–357. 4 Storb R, Buckner CD, Dillingham LA, Thomas ED. Cyclophosphamide regimens in rhesus monkey with and without marrow infusion. Cancer Res 1970; 30: 2195–2203. 5 Storb R, Prentice RL, Thomas ED. Marrow transplantation for treatment of aplastic anemia. An analysis of factors associated with graft rejection. New Engl J Med 1977; 296: 61–66. 6 Santos GW, Tutschka PJ, Brookmeyer R et al. Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. New Engl J Med 1983; 309: 1347–1353. 7 Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood 1987; 70: 1382–1388. 8 Woods WG, Kobrinsky N, Buckley JD et al. Timed-sequential induction therapy improves postremission outcome in acute myeloid leukemia: a report from the Children’s Cancer Group. Blood 1996; 87: 4979–4989. 9 Hassan M, Oberg G, Bekassy AN et al. Pharmacokinetics of high-dose busulphan in relation to age and chronopharmacology. Cancer Chemother Pharmacol 1991; 28: 130–134. 10 Hassan M, Ljungman P, Bolme P et al. Busulfan bioavailability. Blood 1994; 84: 2144–2150. 11 Grochow LB, Krivit W, Whitley CB, Blazar B. Busulfan disposition in children. Blood 1990; 75: 1723–1727. 12 Regazzi MB, Locatelli F, Buggia I et al. Disposition of highdose busulfan in pediatric patients undergoing bone marrow transplantation. Clin Pharmacol Ther 1993; 54: 45–52. 13 Vassal G, Gouyette A, Hartmann O et al. Pharmacokinetics of high-dose busulfan in children. Cancer Chemother Pharmacol 1989; 24: 386–390. 14 Vassal G, Deroussent A, Challine D et al. Is 600 mg/m2 the appropriate dosage of busulfan in children undergoing bone marrow transplantation? Blood 1992; 79: 2475–2479. 15 Slattery JT, Sanders JE, Buckner CD et al. Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics [published erratum appears in


16

17 18

19 20 21

22 23 24

Bone Marrow Transplant 1996 Oct; 18(4): 829]. Bone Marrow Transplant 1995; 16: 31–42. Gibbs JP, Murray G, Risler L et al. Age-dependent tetrahydrothiophenium ion formation in young children and adults receiving high-dose busulfan. Cancer Res 1997; 57: 5509– 5516. Gibbs JP, Liacouras CA, Baldassano RN, Slattery JT. Upregulation of glutathione S-transferase activity in enterocytes of young children. Drug Metab Dispos 1999; 27: 1466–1469. Bolinger AM, Zangwill AB, Slattery JT et al. An evaluation of engraftment, toxicity and busulfan concentration in children receiving bone marrow transplantation for leukemia or genetic disease. Bone Marrow Transplant 2000; 25: 925–930. Grochow LB. Busulfan disposition: the role of therapeutic monitoring in bone marrow transplantation induction regimens. Semin Oncol 1993; 20: 18–25. Dix SP, Wingard JR, Mullins RE et al. Association of busulfan area under the curve with veno-occlusive disease following BMT. Bone Marrow Transplant 1996; 17: 225–230. Petersdorf EW, Gooley TA, Anasetti C, et al. Optimizing outcome after unrelated marrow transplantation by comprehensive matching of HLA class I and II alleles in the donor and recipient. Blood 1998; 92: 3515–3520. Slattery JT, Risler LJ. Therapeutic monitoring of busulfan in hematopoietic stem cell transplantation. Ther Drug Monit 1998; 20: 543–549. Bearman SI, Appelbaum FR, Buckner CD et al. Regimenrelated toxicity in patients undergoing bone marrow transplantation. J Clin Oncol 1988; 6: 1562–1568. Bolinger AM, Zangwill AB, Slattery JT et al. Target dose adjustment of busulfan using pharmacokinetic parameters in

25

26

27 28

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pediatric patients undergoing bone marrow transplantation for malignancy or genetic disease. Blood 1999; 94: 145a. Pawlowska AB, Blazar BR, Angelucci E et al. Relationship of plasma pharmacokinetics of high-dose oral busulfan to the outcome of allogeneic bone marrow transplantation in children with thalassemia. Bone Marrow Transplant 1997; 20: 915– 920. Slattery JT, Clift RA, Buckner CD et al. Marrow transplantation for chronic myeloid leukemia: the influence of plasma busulfan levels on the outcome of transplantation. Blood 1997; 89: 3055–3060. Baker KS, Bostrom B, DeFor T et al. Busulfan pharmacokinetics do not predict relapse in acute myeloid leukemia. Bone Marrow Transplant 2000; 26: 607–614. Woods WG, Neudorf S, Gold S et al. A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission: a report from the Children’s cancer group. Blood 2001; 97: 56–62. McDonald GB, Ren S, Bouvier ME et al. Venoocclusive disease of the liver and cyclophosphamide pharmacokinetics: a prospective study in marrow transplant patients. Hepatology 1999; 17: 314A. DeLeve LD. Cellular target of cyclophosphamide toxicity in the murine liver: role of glutathione and site of metabolic activation. Hepatology 1996; 24: 830–837. Lee JL, Gooley T, Bensinger W et al. Veno-occlusive disease of the liver after busulfan, melphalan, and thiotepa conditioning therapy: incidence, risk factors, and outcome. Biol Blood Marrow Transplant 1999; 5: 306–315.

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