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patient, aged 3 years, had not started chelation therapy but the other 19 patients had good compliance with desferriox- amine treatment. The mean serum ferritin ...
Bone Marrow Transplantation, (1999) 23, 307–310  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Busulphan level and early mortality in thalassaemia patients after BMT CK Li1, PMP Yuen1, R Wong1, CP Pang2, WK Lai2, E Law2, MMK Shing1, KW Chik1 and TF Leung1 Departments of 1Paediatrics and 2Chemical Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

Summary: The aim of the study was to correlate busulphan (BU) levels of thalassaemia patients with outcome of allogeneic transplant. BU levels were measured by gas chromatography mass fragmentography. All patients received a standardised dose of BU 16 mg/kg, and cyclophosphamide 150 or 200 mg/kg. For area-under-thecurve analysis (AUC), blood samples were obtained at 0, 1, 2, 3, 4 and 6 h after the first and fifth dose for all patients, and additional levels were measured after ninth and/or 13th dose in most patients. Outcome parameters examined included veno-occlusive disease of liver (VOD), idiopathic interstitial pneumonitis, chimerism, and day 90 survival. Twenty consecutive thalassaemia patients who underwent haematopoietic stem cell transplantation were studied. The median age at transplant was 11.2 years (range 3–21 years). Mean BU AUC levels were correlated with age at transplant (r ⴝ 0.58, P ⴝ 0.007). Nine patients developed VOD and six had mixed chimerism, but these did not correlate with mean BU AUC level. Four patients died before day 50 from VOD and interstitial pneumonitis. Patients with BU AUC levels greater than the median (908 ␮mol ⴛ min/l) had significantly lower probability of survival at day 90 (60%), whereas patients with BU AUC level less than the median all survived beyond day 90. No patient had graft rejection. In conclusion, a high BU AUC level was associated with a higher treatment-related mortality in thalassaemia patients after transplant. Keywords: busulphan; early mortality; thalassaemia

Thalassaemia major is the most common non-malignant disease being treated by haematopoietic stem cell transplant. Conditioning with busulphan (BU) and cyclophosphamide has been adopted by most centres. The long-term disease-free survival has been reported to be in the range of 60 to 90% depending on risk factors which include age, hepatomegaly, iron chelation and hepatic fibrosis.1 Early results have shown a high incidence of treatment-related mortality (TRM) in the high risk patients. Reduction of Correspondence: Dr CK Li, Department of Paediatrics, Prince of Wales Hospital, Shatin, Hong Kong, China Received 23 June 1998; accepted 23 September 1998

intensity of conditioning reduces the TRM but at the expense of higher rejection rate.2 It has been suggested that use of BU in conditioning is associated with a higher incidence of veno-occlusive disease (VOD) and interstitial pneumonitis. BU kinetics based on area-under-the-curve or concentration at steady state has been studied to correlate with treatment complications, graft rejection, relapse of leukaemia and transplant-related mortality.3–6 We analysed the plasma BU levels in 20 thalassaemia patients and the relationship with outcome parameters.

Materials and methods Patients From January 1996 to February 1998, 20 consecutive thalassaemia major patients underwent allogeneic haematopoietic stem cell transplantation. Donors were HLA-identical siblings in 19, and one was the phenotypically identical father. Three received PBSC while the remainder received BM. The median age at transplant was 11.2 years, ranging from 3 to 21 years. All except one patient received regular monthly blood transfusions before 18 months of age. One patient, aged 3 years, had not started chelation therapy but the other 19 patients had good compliance with desferrioxamine treatment. The mean serum ferritin before transplant was 4243 pmol/l (s.d. 2187, range 970–8950 pmol/l). Two patients had a mild increase in liver alanine transferase (ALT) but both were less than twice the upper limit of normal. No patient was hepatitis B antigen-positive, but one was hepatitis C antibody-positive. Liver biopsy was performed in all patients. None had features of hepatitis but five had evidence of hepatic fibrosis. Treatment regimen Conditioning consisted of BU 1 mg/kg p.o. every 6 h for 16 doses (total 16 mg/kg), and cyclophosphamide 50 mg/kg i.v. for 3 days if the patient was older than 10 years, and for 4 days if 10 or younger (total 150 or 200 mg/kg). Antithymocyte globulin was given for immunosuppression at 10 mg/kg/day i.v. from day ⫺5 for 11 days (total 110 mg/kg) in the first six patients, and was later changed to 30 mg/kg/day i.v. for 3 days from day ⫺4 (90 mg/kg) for the other 14 patients. GVHD prophylaxis was methotrexate 15 mg/m2 i.v. on day 1 and 10 mg/m2 i.v. on days 3, 6 and

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11. Cyclosporin was started from day ⫺1 at 1.5 mg/kg i.v. 12 hourly; tapering was started from day 50 if there was no acute GVHD, and discontinued on day 180 if no chronic GVHD.

Busulphan level assay Plasma BU levels were measured by gas chromatography– mass fragmentography. Non-isotopic busulphan was used as the internal standard. After extraction into ethyl acetate BU and busulphan were iodinated into 1, 4-diiodobutane and 1, 5-diiodiopentane respectively. GC-MS analysis was carried out on HP5890II gas chromatograph with a 30 m 100% methyl silicon narrow-bore fused-silica capillary column interfaced to a HP5972 mass spectrometer. Helium was the carrier gas, and the sample molecules were identified by total ion monitoring and quantified by selective ion monitoring of m/z 183 and 197. The calibration curve was linear up to 4 ␮g/ml. The lowest detection limit was 0.04 ␮g/ml and the analytical recovery rate about 97%. BU levels were checked after the first and fifth doses for all patients. Seventeen patients had additional levels checked after ninth and/or 13th dose. Three had levels checked for two doses, three for three doses and 14 for four doses. Blood samples were collected at 0, 1, 2, 3, 4 and 6 h after a dose of BU, and the BU level using area-under-the-curve (AUC) analysis was calculated for each dose.

Treatment related complications and outcome parameters VOD was diagnosed clinically if two of three criteria were present: tender hepatomegaly, unexplained weight gain or ascites, and increase in serum bilirubin of more than 35 ␮mol/l. Idiopathic interstitial pneumonitis (IP) was diagnosed if there were radiological changes but no infective organisms were identified on bronchoalveolar lavage or post-mortem examination. Chimerism was studied by X and Y chromosome FISH if sex mismatch was present. In the situation of sex match, DNA from peripheral blood was amplified by several variable number tandem repeat (VNTR) loci and informative markers were identified. Day 90 survival was used as cut-off day for TRM.

Statistical analysis The mean AUC was tested for correlation with age, pretransplant liver function and ferritin by Pearson’s correlation. The mean AUC was tested with outcome parameters (VOD, IP, chimerism, day-90 survival) by t-test and Mann– Whitney test as appropriate. Patients were divided into two groups, below and above the BU median level. Outcome parameters were examined between these two groups by the ␹2 or Fisher exact test. Probability of survival up to day 90 was estimated by Kaplan–Meier product limit method with patients censored at death or last contact for survival.

Results Busulphan levels BU levels are shown in Table 1. The first dose AUC was significantly less than those on the subsequent 3 days (P ⬍ 0.005), and on the average 30% less. The AUC after first dose was thus not included in the analysis. The levels on day 2, 3 and 4 (fifth, ninth and 13th dose) were closely correlated and there was no significant change over the 3 days. The mean AUC was taken as the mean of AUC for the 2nd to 4th days. Mean BU level of day 2 to 4 was significantly correlated with age at transplant (r ⫽ 0.58, P ⫽ 0.007). Mean BU and median BU levels were 914 ␮mol ⫻ min/l and 908 ␮mol ⫻ min/l, ranging from 508–1278 ␮mol ⫻ min/l. Mean BU levels did not correlate with pretransplant ALT, bilirubin and serum ferritin levels. Treatment-related complications VOD occurred in nine patients (45%) and two had bilirubin levels in excess of 500 ␮mol/l. The group with VOD had higher mean BU levels than the group without VOD (1001 ⫾ 198 vs 842 ⫾ 180 ␮mol ⫻ min/l), but this did not reach statistical significance (P ⫽ 0.077). Five patients had resolution of VOD, and the three patients with mean BU levels less than the median all recovered. Only two of the six patients with mean BU level greater than the median recovered from VOD. Idiopathic IP occurred in four patients and three of them occurred before day 30 after transplant (early IP). One patient had IP after day 90 during tapering of immunosuppressive treatment for acute GVHD. Postmortem examination did not reveal any infectious agents in these patients. The three patients with early IP had mean BU levels greater than the median BU level, and no patients with BU levels less than the median developed early IP. Acute graft-versus-host disease occurred in eight patients and in all cases this was limited to skin. Two had grade II GVHD and the others had grade I. No patient developed life-threatening infection. One patient had a generalised seizure on day 21 after transplant. Outcome parameters Neutrophil engraftment was defined as neutrophils ⬎0.5 ⫻ 109/l for 3 consecutive days. All 20 patients achieved engraftment at a median of 18 days. No patient developed graft rejection at a median follow-up of 15 months (range 6 to 30 months). Chimerism tested by FISH or VNTR revealed mixed chimerism in six patients (30%), and all were transfusion independent. There was no difference in mean BU level between the mixed and complete chimerism groups. Four patients died before day 50 because of treatment related complications: two from severe VOD, and two from IP with associated VOD. One patient died on day 101 because of late IP. The four patients who died before day 50 had mean BU levels greater than the median. The probability of surviving up to day 90 was significantly higher in the group with BU levels less than the median (100% vs 60%, P ⫽ 0.029) (Table 2). Presence of VOD was associated with a higher chance of treatment-related mortality

Busulphan-related mortality in thalassaemia CK Li et al

Table 1

No. of patients Median Mean s.d. Min Max a

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Busulphan level (␮mol ⫻ min/l) using AUC First dose

Fifth dose

Ninth dose

13th dose

Mean levela

20 718 729 155 435 993

20 916 925 236 478 1507

16 879 892 154 626 1207

15 956 895 229 539 1397

20 908 914 200 508 1278

Mean level is the mean BU levels of fifth/ninth/13th doses.

Table 2

Outcome parameters according to median BU level

⬎Median (n ⫽ 10) ⬍Median (n ⫽ 10) P value

VOD

Early IP

Mixed chimerism

Survival to day 90

6 3 ⬎0.05a

3 0 ⬎0.05a

1 5 ⬎0.05a

6 10 0.029b

a

Fisher exact test. Kaplan–Meier product limit method. VOD ⫽ veno-occlusive disease; IP ⫽ interstitial pneumonitis.

b

(P ⫽ 0.026). Patients older than 10 years of age at transplant had lower probabilities of surviving up to day 90 (P ⫽ 0.029), and all four early deaths were in the age group older than 10 years. Pretransplant liver function and hepatic fibrosis had no impact on day 90 survival. The early death group however had higher serum ferritin levels, 6588 vs 3657 pmol/l (P ⫽ 0.012). GVHD did not have an adverse effect on survival. Discussion Over 1000 thalassaemia major patients treated by haematopoietic stem cell transplant have been reported. Several risk factors have been identified to be associated with poorer disease-free survival, namely hepatomegaly, hepatic fibrosis and poor compliance of iron chelation.1 Rejection of bone marrow graft and treatment-related mortality are the most common causes of transplant failure. Thalassaemia patients receive multiple blood transfusions before transplant which may lead to sensitisation of patients to non-HLA antigens, and thus rejection of the bone marrow graft. Intensification of conditioning has been shown to reduce rejection rate, but at the expense of higher treatmentrelated mortality.2 BU is used commonly in conditioning regimens for various malignant and non-malignant diseases. BU has a potent marrow ablative effect, but also has significant toxicity to organs other than bone marrow. Our centre has used a uniform conditioning regimen for all thalassaemia patients. The dose of BU is standardised to 1 mg/kg every 6 h for 16 doses. The dose of cyclophosphamide is reduced in patients older than 10 years, aimed at reducing TRM. We have previously reported a 100% survival of our thalassaemia patients transplanted before the age of 10 years.7 Despite a reduction in cyclophosphamide, there is still significant TRM in the older age group. The higher TRM in

older patients is due to VOD and IP, both of which are reported to be associated with use of BU.8 The metabolism of BU has been shown to be faster in young patients, and thus a lower BU concentration at steady state is achieved.4,9 The present study analysed the BU level with transplant outcome. Our result confirms the age effect on BU metabolism, with higher BU levels in older patients. Severe VOD and early IP occurred only in patients with BU levels above the median. Early death occurred only in patients older than 10 years, although age and BU level were closely correlated. Since these patients had received a lower dose of cyclophosphamide, the higher TRM is likely due to higher exposure to BU. BU damage to the liver and lung may be aggravated by more severe iron overload in these older patients. Using a lower BU dose empirically may be associated with a higher rejection rate.2 Rejection rates have been reported in the range of 20–30% in various studies, but BU levels were not monitored in most studies.10,11 BU levels by concentration at steady state (BU Css) greater than 200 ng/ml are suggested to prevent graft rejection in matched sibling transplant.4 None of our patients had BU Css less than 200 ng/ml and no rejection occurred. The safe upper limit for BU levels was however not established. Factors other than BU level will affect TRM including previous chemotherapy or chelation therapy in thalassaemias. Therefore, the BU level recommended for one disease may not be applicable to other diseases. It has been shown that BU pharmacokinetics can be influenced by the underlying disease.12 Thalassaemia patients who have iron overload due to repeated transfusion may be predisposed to VOD and IP. The current study was of a homogeneous group of patients with the same disease who received the same busulphan dosage for conditioning. This showed that a BU level less than the median, 908 ␮mol ⫻ min was free from TRM. It may be possible to monitor BU levels and to adjust the BU dose to maintain a safe but effective BU level.

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A recent study from Italy reported that there was no association between BU phamacokinetics and patient age.13 VOD was only 4.5%, IP was 11% and the graft rejection rate 7.5%. The authors could not find any association between BU levels and post-transplant mortality, and suggested that BU pharmacokinetics were not predictive for transplant outcome. The AUC levels in that study were lower than those in our current study, and the dosing schedule of BU was also different. BU was given at 8 hourly intervals, rather than the usual 6 hourly interval. Methotrexate was not given as GVHD prophylaxis in most patients. Methotrexate is hepatotoxic and has been suggested to be one of the risk factors for VOD.14 These differences may explain the lower incidence of VOD in the Italian study as compared to our study. However, the lower BU level might be a contributing factor to a higher graft rejection rate. In our study none of our patients, including the older age group patients, had graft rejection. Dose adjustment according to BU level has been suggested by some workers; however, whether this may result in a lower TRM is still not proven.15 In view of a high TRM in high risk thalassaemia patients, dose adjustment may be a reasonable approach to reduce TRM and at the same time maintain stable engraftment. In conclusion, our study showed that high BU levels are associated with a higher TRM in thalassaemia patients. Graft rejection appears to be prevented by our current conditioning regimen.

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Acknowledgements We thank the BMT Unit nursing staff for their assistance in the study, Kent Tsang and Henry Pong for performing the FISH and VNTR. This work was supported in part by the Hong Kong Paediatric Bone Marrow Transplant Fund of the Chinese University of Hong Kong and the Children’s Thalassaemia Foundation.

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