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Treatment of b-thalassaemia major is either supportive with transfusion–chelation therapy or curative by allogeneic bone marrow transplantation. (BMT). Patients ...
Bone Marrow Transplantation (2003) 31, 1081–1087 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00

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Thalassaemia Paediatric allogeneic bone marrow transplantation for homozygous b-thalassaemia, the Dutch experience LM Ball1, AC Lankester1, PC Giordano2, MH van Weel1, CL Harteveld2, RGM Bredius1, FJ Smiers1, RM Egeler1 and JMJJ Vossen1 1 The Department of Paediatrics, Leiden University Medical Centre, Leiden, the Netherlands; and 2The Haemoglobinopathies Laboratory, Department of Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands

Summary: We reviewed the results of the Dutch paediatric bone marrow transplant (BMT) program for children receiving HLA-identical BMT for b-thalassaemia major over an 18-year period. In all, 19 patients underwent a total of 21 transplants in our treatment centre between July 1984 and February 2002. Eight females (age 0.3–12 years; median 5 years) and 11 males (age 0.8–18 years; median 6 years) were included. Information, prospectively collected, included molecular defects, donor genotype, b/a-globin expression rates, serum ferritin levels, hepato-splenomegaly, chelation history, virology screening, liver pathology together with post-transplant outcome inclusive of leucocyte chimerism. In total, 11 patients received standard busulphan/cyclophosphamide (Bu/Cy) conditioning, with or without ATG. Stable engraftment was seen in 5/11 with late rejection occurring in six patients. Of these, two children underwent a second successful SCT. For this group, overall event-free survival (EFS) and disease-free survival (DFS) were 90 (10/11) and 64% (7/11), respectively. The probability of rejection was 55%. Subsequent addition of melphalan to the conditioning regimen resulted in long-term stable engraftment in all patients with an EFS/DFS for this group of 90% (9/10). Treatment-related mortality, irrespective of conditioning, was low at 5% (1/19 patients). Veno-occlusive disease (VOD) occurred in 19% (4/21 transplants) and acute GvHD in 19% (4/21 transplants). Post-BMT b/a synthetic ratio measurement monitored donor erythroid engraftment and predicted rejection with a return to transfusion dependency. Maintained full donor chimerism is indicative of stable engraftment both for leucocyte and erythroid lineages, whereas mixed donor chimerism is not. Our results emphasise the importance of the conditioning regimen and post-transplant chimerism surveillance predictive of rejection or long-term stable engraftment. Bone Marrow Transplantation (2003) 31, 1081–1087. doi:10.1038/sj.bmt.1704066

Correspondence: Dr LM Ball, Department of Paediatrics, Leiden University Medical Centre, Albinusdreef 2, Postbus 9600, 2300 RC, Leiden, The Netherlands Received 17 July 2002; accepted 22 January 2003

Keywords: paediatrics; stem cell transplantation b-thalassaemia; melphalan; chimerism

Homozygous b-thalassaemia is a globin chain defect characterised by severe haemolytic anaemia due to functional dyserythropoiesis, with persisting hypoxia, extramedullary erythropoiesis, hepato-splenomegaly and skeletal malformations. Treatment of b-thalassaemia major is either supportive with transfusion–chelation therapy or curative by allogeneic bone marrow transplantation (BMT). Patients treated intensively with ‘state of the art’ transfusion–chelation therapy, in association with multidisciplinary treatment for the many complications, may survive more than 40 years.1 Allogeneic BMT from an HLA-identical donor is an accepted treatment modality for children with homozygous b-thalassaemia, and at present remains the only proven curative therapy for this condition.1,2 Observations over a 20-year period confirm the durability of this cure.3 Overall survival at age 25 years was found by Piga et al4 to be 99% in compliant patients conventionally treated compared to 82% for those undergoing BMT. However, noncompliance with conventional therapy resulted in poorer outcome of only 70%. Many centres have reported extensively on their experience5–7 and presently, the disease-free survival (DFS) in children undergoing matched sibling BMT prior to the aggravation of disease- and/or transfusion-related organ damage is reported at approximately 90%.7,8 Outcome has been shown to correlate with three separate risk categories (class 1–3) based upon the established criteria of hepatomegaly, inadequate chelation compliance and portal fibrosis.9 The reported probabilities of survival, rejection and of EFS were 94, 0, and 94% in class 1; 80, 9 and 77% in class 2: and 61, 16 and 53% in class 3. The standard conditioning regimen for BMT in the haemoglobinopathies is busulphan–cyclophosphamide (Bu/Cy) based with or without the use of antithymocyte globulin (ATG). Careful adaptations of this conditioning regimen have reduced the incidence of treatment-related toxicity, most notably hepatic, especially for class 3 patients.10,11 However, modifications of the conditioning therapy may impact not only on toxicities but, as

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importantly, on sustainable engraftment.11 Rejection of fully engrafted donor stem cells remains a problem, especially in class 2 and 3 patients, often occurring late and up to 12 months following initial successful engraftment. The bthalassaemia trait is rare in the indigenous Dutch population but is frequent in the country of origin of the heterogeneous immigrant populations. These are also characterised by an often consanguinous choice of partner. Patients are often the offspring of first generation immigrants. Despite their often relatively young age, they present with additional clinical complications either because of noncompliance with chelation or having received inadequate transfusion–chelation therapy in their original homeland. According to the Pesaro classification,9 most of the patients referred to our centre fall into the poorer outcome groups (class 2 or 3). We reviewed the results of the Dutch paediatric bone marrow transplant programme for children receiving HLA-identical transplantation for b-thalassaemia major. Our results highlight the importance of the conditioning regimen and post-transplant surveillance relevant to (prediction of) rejection or long-term stable engraftment.

Materials and methods Patients Between July 1984 and February 2002, 21 transplants in 19 children with homozygous b-thalassaemia were undertaken in the paediatric bone marrow transplantation unit of the Leiden University Medical Centre, the Netherlands. The patients consisted of eight females (age 4 months–12 years 3 months; median 5 years at the time of transplant) and 11 males (age 9 months–18 years 2 months; median 6 years at the time of transplant). All underwent full bone marrow transplantation from HLA identical donors. These were mainly sibling donors, but in three cases consanguinous parents were used. HLA typing was undertaken using both standard and highresolution DNA typing for HLA locus A, B, C and DR/ DQ loci. Mixed lymphocyte culture and cytotoxic T-lymphocyte precursor assays were nonreactive for each donor-recipient pair tested.

total of 15 patients additionally received rabbit ATG (total dose: 20 mg/kg Pasteur Merieuxs and in patients 462 and 487 10 mg/kg Sangstat Imtexs), related to a previous history of numerous transfusions. Irradiation (TAI thoraco-abdominal–inguinal) was only given to one patient (295-2) prior to a ‘boost’ of donor cells. Melphalan (total dose 140 mg/m2) was included in 11 transplants. Dose modification or alternative conditioning inclusive of fludarabine (total dose 120 mg/m2) was undertaken in three transplants because of previous rejection or perceived high risk of complications. Busulphan was administered orally over four consecutive days (total dose 16 mg/kg). From July 2000 (patients 451, 462 and 487), busulphan was administered intravenously (I.V.) over 4 consecutive days (total dose 12.8 mg/kg) with pharmacokinetic estimation of systemic exposure.13 No dose adjustments were, at this stage, carried out on the basis of the pharmacokinetic data. Cyclophosphamide (total dose 200 mg/kg) was administered intravenously daily for 4 days with Mesnas prophylaxis. Cell dose: A target dose of 2–4  108/kg recipient weight total nucleated cells as infused on day +0. Actual as opposed to target cell dose was achieved in all but two patients (462 and 487). Both patients were in the second group with sustained engraftment. Prophylaxis against acute GvHD: Cyclosporine 2 mg/kg recipient weight/day from day 1 given intravenously until oral medication was tolerated at an oral dose of 6 mg/kg/ day with dose adjustment according to the results of regular blood sampling. This was continued 180 days and tapered thereafter until the drug was discontinued at nine months after SCT. Methotrexate (10 mg/m2) was administered on days +1, 3 and 6.

Supportive treatment All patients were nursed in positive-pressure isolation rooms with total gut decontamination using nonabsorbable antimicrobials.14 Acute and chronic GvHD were graded according to the standard criteria.15,16 Acute GvHD was treated with methylprednisolone 2 mg/kg /day i.v.

Risk categorisation Patients were stratified as far as possible, according to the risk factors previously described by the Pesaro group.9 Liver biopsies were not undertaken in the very early transplants or in very small children (portal fibrosis has not been observed in children with thalassaemia under 3 years of age12) and in one child with a concurrent inherited coagulopathy. Donor and recipients were screened for exposure to hepatotropic viruses, inclusive of HCV by PCR analysis when serology indicated positive results.

Conditioning Conditioning regimens and marrow infusion: All but one of the patients was conditioned using Bu/Cy-based therapy. A Bone Marrow Transplantation

Haematological, biochemical and molecular analysis Characterisation of the haemoglobinopathy of patients and family was established using standard haemotological and biochemical methods. The Hb fractions were measured either manually17 or automatically using HPLC as previously described.18 b/a-chain synthesis ratios were measured pre- and serially post-transplant using a rapid modified method.19 Donor b/a-chain synthesis ratios were similarly measured pretransplant. Donor–recipient white blood cell (WBC) chimerism were analysed either by XYFISH20 or VNTR polymorphism,21 as previously described. As the patient group proved to be heterogeneous, pretransplant analysis of the b-thalassaemia genotype of both the recipient and donor was carried out by DNA direct sequencing as previously reported.22

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1083 Table 1 Patient number

Family/ ethnic origin

Sex

Donor relation

Age at BMT

1984–98 067 102

Pakistan Sardinia

F M

Sister Brother

0 year 4 months 18 years

1 2 (a3)

629 3125

104 110 133 165 208 239 243 253-1 295-1

Morocco China Morocco Morocco Morocco Morocco China Pakistan Morocco

F F F F F M M M M

Brother Sister Brother Brother Brother Father Brother Sister Sister

7 years 1 month 13 years 5 months 4 years 6 months 7 years 9 months 0 year 11 month 0 year 9 months 3 years 4 months 0 year 9 months 11 year 9 months

2 2 (a3) 2 2 1 2 1 1 1 (a2)

Turkey Turkey Sardinia Morocco

F F M M

Brother Brother Brother Sister

Morocco Iran Pakistan Afghanistan Iraq Pakistan

M F M M M M

Brother Father Sister Mother Sister Sister

12 years 3 months 4 years 11 month 17 years 11 month 14 years 1 month 14 years 2 months 5 years 3 months 8 years 6 months 8 years 6 months 6 years 6 months 7 years 8 months 3 years 1 month

3 1 3 2 2 2 3 2 3 3 2 (a3)

>1998 340 342 356 295-2 boost 393 438 253-2 451 462 487

Patient class

Ferritin pre-BMT

GvHD

Rejection

Outcome

No No

Alive Alive

2940 3125 988 1507 725 510 3362 379 2399

— Acute gr. 2 + Chr. mild — — — — — — — — —

Yes No Yes Yes No Yes No Nob Yes

Alive Alive Alive Deadc Alive Alive Alive Alive Alive

5439 1723 1898 2401 2400 2033 4363 1344 1461 762 2637

Chr. mild Gr. 1 — — — — Gr. 1 — — — Gr. 1

No No No

Alive Alive Alive

No No No No No No No

Alive Alive Alive Alive Deadd Alive Alive

a

Patient without liver biopsy in whom classification is incomplete See text c Died 8 years after transplant with disease complications d Transplant related death b

Results Patient groups and transplant outcome Patients referred to our unit originated from seven different countries. Patient characteristics are shown in Table 1. A total of eight different molecular defects were defined as either b0 or b+-thalassaemia mutation, cd39 (C-T) being the most frequent (see Table 2). The 21 transplants undertaken fall into two main groups depending upon the date of transplant. The first group comprises patients treated before 1998, after which highdose melphalan was introduced into our standard conditioning regimen of Bu/Cy (with or without ATG) in children transplanted for b-thalassaemia, because of the disappointing rates of rejection. Of the 11 children in the first group undergoing allogeneic SCT without the use of melphalan there were no failures of engraftment. The total number of nucleated cells infused ranged from 2.7 to 7.5  108/kg recipient weight (median 4.2). Four patients have long-term stable engraftment and are transfusion independent with a follow-up 7–15 years. However, six children developed a rejection episode between 2–11 months (median 3 months) after initial successful engraftment followed by autologous reconstitution and were subsequently maintained on standard transfusion–chelation therapy. One child (patient 253), although suffering from a heterozygous b-thalassaemia by haematological and biochemical analysis also became

transfusion dependent and was considered ‘quantitatively’ poorly engrafted. He and another child (patient 295) received a further allogeneic BMT from the original donors with no further documented rejection episode. Of the four remaining children, one died because of complications of transfusion-related iron overload unrelated to BMT toxicity (patient 165). In this group, the probabilities of overall survival, rejection-free survival, rejection and of DFS are 90, 45, 55 and 64% respectively, with a follow-up of 7–15 years; median 10 years. For this group, rejection, followed by autologous haematopoietic reconstitution, was the main reason for transplant failure with all but one of these children alive but transfusion dependent. In the second group of seven patients, following the introduction of melphalan to the standard conditioning, stable engraftment was seen in 6/7 children, with a followup period 6 months–6 years: median 4 years. The total number of nucleated cells infused ranged from 1.6 to 7.5  108/kg recipient weight (median 4.1). One child died in the immediate post-transplant period (day +57) following successful engraftment because of hepatic failure and pulmonary dysfunction presumed secondary to toxicity and opportunistic infection (patient 451). The two children who received second transplants 3 and 6 years after their original SCT (see above) were conditioned with melphalan. In one child, melphalan and TAI were added to Bu/Cy after which sustained engraftment was observed with a follow-up of 4 years (patient 295-2). The second patient underwent conditioning with Bone Marrow Transplantation

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Bone Marrow Transplantation

Table 2 b/a ratio pre-BMT

b/a ratio post-BMT

Molecular Defects (i/ii)

Defect type

Defect donor

067 102 104 110

(i, ii) IVS 1-5 (G-C) (i, ii) cd39 (C-T) (i, ii) FS cd5 (-CT) (i) cd 41-42 (-4) (ii) IVS 1I-654 (C-T) (i, ii) cd39 (C-T) (i, ii) FS cd5 (-CT)

b+/b+ b0/b0 b0/b0 b+/b+

IVS 1-5 (G-C) het Normal FS cd5 (-CT) het Normal

0.02 0.05 0.08 0.04

0.54 NA 0.03 1.1

b0/b0 b0/b0

cd39 (C-T) het Normal

0.00 0.02

NA 1.09 0.00 0.51 0.48

133 165 208

239 243 253-1

295-1 340 342 356 295-2/3 (boost) 393 438 253-2 451 462 487

(i, ii) cd39 (C-T)

(i, ii) IVS 1I-745 (C-G) (i) cd 41-42 (-4) (ii) IVS 1I-654 (C-T) (i, ii) IVS 1-5 (G-C)

(i, ii) cd39 (C-T) (i, ii) FS cd8 (-AA) (i, ii) IVS 1-110 (G-A) (i, ii) cd39 (C-T) (i, ii) cd39 (C-T) (i) cd39 (C-T) (ii) cd5 (-CT) (i) cd8 (-AA) (ii) ? (i, ii) IVS 1-5 (G-C) (i, ii) cd 8/9 (+G) (i, ii) IVS 1-110 (G-A) (i, ii) IVS 1-110 (G-A)

b0/b0

b+/b+ +

+

b /b

b+/b+

b0/b0 0

0

b /b

cd39 (C-T) het

IVS 1I-745 (C-G) het IVS 1I-654 (C-T) het IVS 1-5 (G-C) het

Normal or a thal FS cd8 het

0.03

NA 0.03 0.01

0.03 0.00

b+/b+ b0/b0 b0/b0 b0/b0

IVS 1-110 (G-A) het cd39 (C-T) het Normal or a thal Normal

0.06 NA 0.03 0.03

b0/b?

cd8 (-AA) het

NA

b+/b+ b0/b0 b+/b+/b+ b+/b+

IVS 1-5 (G-C) het cd 8/9 het IVS 1-110 (G-A) het IVS 1-110 (G-A) het

0.02 NAw 0.06 0.02

(+24months)

(+3months) (+11months)

(+3months) (+12months) (+2months)

% HbA2 post-BMT

% HbF post-BMT

Phenotype post-BMT

Leucocyte chimerism

5.8 2.2 3.6 2.9

3.9 0.7 NA 0.8

heterozyg. b-thal homozyg. normal homozyg. b-thal homozyg. normal

NA NA NA NA

NA 2.9 2.6; 3.0 2.6; 3.0

NA NA

homozyg. b-thal homozyg. b-thal

0.7; 2.2

heterozyg. b-thal

NA MC MC FC FC

2.9; 3.1

1.2; 3.3

homozyg. b-thal

NA

3.5; 6.4

0.9; 2.2

heterozyg. b-thal

NA

3.9; 3.2; 8.3

2.5; 11.5; 0.5; 0.5

? heterozygg. b-thal See text (transfusions required)

MC MC MC

(no analysis)

(+3months) (+13months) (+28months)

(+48months)

0.7 0.1 0.52 0.6 0.42 0.23 0.4 0.42 0.03

(+1months)

(+3months)

3

19

homozyg. b-thal

NA

0.5 0.44 0.5 0.56 NA NA

(+6months)

5.2

2.2

heterozyg. b-thal

FC

4.6 6 2.1 NA

3.4 5.7 10.7 NA

heterozyg. b-thal heterozyg. b-thal 2nd heterozyg a/normal homozyg normal

FC MC FC FC

5.3; 5.2

15.7; 8.7

heterozyg. b-thal

FC

(+4months)

5.7 NAw 3.3 5.1

2.5 NAw 4 2.4

2nd heterozyg b-thal heterozyg. b-thal heterozyg. b-thal heterozyg. b-thal

MC FC FC FC

(+8months)

(+5months) (+3months) (+10months) (+6months) (+8months) (+24months)

(+3months) (+10months) (+48months)

(+48months)

(+48months)

(+12months) (+3months) (+41months)

0.38 (+6months) 0.49 (+12months) 0.42 (+6months) NAw 0.42 (+3months) 0.6 (+3months)

Superscipts in columns 6 and 10 refer to analysis+months post BMT MC: mixed chimerism; FC: full donor chimerism; NA: not analysed; +: died before analysis; Heterozyg: heterozygous; homozyg: homozygous

(+40months) (+41months) (+16months) (+5months)

(+1months) (+2months) (+4months)

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Patient no.

Paediatric allogeneic BMT for b-thalassaemia LM Ball et al

fludarabine, melphalan and Campath-1H, and now has sustained engraftment 2 years later (patient 253-2). Both patients received bone marrow cells from their original sibling donors. At the time of second transplant, despite their compliance with chelation therapy in the period between transplants both had become class 2 patients (based upon results of a liver biopsy). There was no hepatic toxicity during their repeated transplantation attempts and GvHD did not occur. One final patient (patient 462) successfully engrafted with modified conditioning administered because of active hepatitis C infection with liver cirrhosis (grade III) and class 3 stratification. A combination of TAI, reduced cyclophosphamide, ATG and melphalan was used and full engraftment has been maintained for 16 months. Acute GvHD grade 2, which responded completely to oral steroid therapy, was documented with no long-term sequelae. Since the introduction of high-dose melphalan to our conditioning regimen we have, by chance, only transplanted children stratified as either class 2 or 3. Nevertheless, our EFS/DFS is 90%. Transplant-related mortality was low (1/11) and to date no late rejection episodes have been seen. All surviving patients have a Kanofsky/Lansky score of 100%. To date, the probabilities of overall survival, rejectionfree survival, rejection and of DFS are 90, 90, 0 and 90%.

Chimerism b/a synthetic ratios were measured in the reticulocytes of 13/ 19 patients pre- and post-transplant. Haematological and biochemical data were collected from patients as well as their donors. Serial estimations post-transplant in those patients in whom rejection occurred was predictive in one case and confirmatory in another two, rejection occurring late (46 months after SCT). In recent years sequential samples have been collected but no rejection episodes have occurred. All patients without rejection episodes have maintained donor levels of b/a ratios post-SCT, consistent with heterozygous or homozygous normal ratios depending upon the status of the individual’s donor. The haematological and biochemical parameters correlated also with the heterozygous (elevated HbF and HbA2) or normal Hb expression of the graft. Chimerism was successfully analysed and documented in 13/21 transplant episodes. In patient 356, continued mixed chimerism was evident by standard analysis of circulating WBCs in the presence of donor levels of b/a synthetic ratios now stable 4 years after SCT. Conversely, in patient 165, mixed chimerism, as analysed in circulating WBCs, remained stable at 3 months and 1 year after SCT. However, donor levels of b/a synthetic ratios did not, returning to recipient type 11 months later. These findings were compatible with the clinical details of the patient, that is, requirement of a reinstated transfusion regimen The fluctuating heterozygous ratios, measured in patient 253-1 at a low 3H-leucine label incorporation and in parallel with fluctuating HbA2 levels, show an initial engraftment process followed by a clinical requirement for blood transfusion therapy. The mixed chimerism, present in the leucocytes of the patient seems either absent or suppressed in the reticulocytes of the poorly active graft, expressing the expected b/a globin chains but at an insufficient level.

The results of molecular analysis and chimerism are summarised in Table 2.

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Toxicities The addition of melphalan has increased the occurrence of mucositis from 12 to 100%. Not only has the frequency but also the severity of mucositis increased. Mucositis occurred between +5 and +12 days without evidence of GvHD. Opiate analgesia and parenteral feeding were required in the most severe cases. Irrespective of the conditioning regimen used 4/19 children developed veno-occlusive disease (VOD). Rates of acute and chronic GvHD have been low in our experience with acute GvHD grade 1 occurring in only 3/ 21 (14%) and grade 2 in 2/21 (9%) of transplant episodes and mild chronic GvHD occurring in 2/21 (9%) of transplant episodes. No deaths because of these complications were recorded.

Discussion The reported outcome of BMT for young children with thalassaemia includes mainly class 1 patients, in whom survival rates are among the highest and transplant related morbidity and mortality among the lowest. However, data for young children in class 2 or 3 risk group, as is the case of our patient group, reported separately from mixed age populations are less common. Our initial rejection rate was not anticipated23 in light of the fact that we were using identical treatment strategies as published by other centres. This could not be explained by differences of stem cells infused as the range and median number of nucleated cells infused (the actual dose) showed no significant differences between groups treated with or without melphalan or those with or without late rejection. Similar outcomes, however, were seen by other paediatric BMT units,24–27 initially causing some major centres to question the validity of the procedure in children25 and leading us to modify our approach in an attempt to overcome this phenomenon. High-dose melphalan has been considered an effective myelo-ablative conditioning agent, especially in a paediatric setting, given the fact that it has minimal organ specific toxicity. Our strategy of including melphalan in our conditioning regimens has consistently prevented the late relapses that were the main cause of previous transplant failure. Severe mucositis required supportive care but has not resulted in any transplant-related mortality.28 Most importantly, the use of melphalan did not aggravate solid organ toxicity because of the overall treatment. The late effects of this strategy are yet to be established. Interestingly, the use of high-dose melphalan in the transplant setting for paediatric malignant disease does not seem to have led to an increase in secondary tumours, although it may be argued that this is not strictly comparable. Reduced intensity conditioning, or so called ‘mini transplants’, have been proposed for patients with haemoglobinopathies, in order to reduce transplant Bone Marrow Transplantation

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procedure-related risks, especially in class 2 or 3 patients.29 It has yet to be determined if a reduction in myelo-ablation will increase the risk of rejection in these at risk patients. We believe melphalan offers a potentially useful alternative to Bu/Cy-based regimens in the setting of future clinical studies. We have successfully utilised this approach by substituting a melphalan/fludarabine combination for Bu/ Cy in a class 3 patient (patient 462), resulting in full donor chimerism and sustained engraftment for over 2 years. It was once considered that ablation of all host stem cells was required to establish conditions for complete marrow engraftment of donor cells (full donor chimerism). Mixed chimerism was thought to predict rejection of the donorgraft.30 Chimerism evaluation following allogeneic bone marrow transplant in long-term survivors of ex-thalassaemia patients has been undertaken by analysis on wholeblood samples or bone marrow. It has become apparent that mixed chimerism is not uncommon after BMT for thalassaemia, although the rate may vary with the conditioning regimen used.31–33 Mixed chimerism often persists and is compatible with long-term cure, that is, transfusion independence.34,35 What is evident is that the biology of engraftment in patients undergoing BMT for thalassaemia is complex and further evaluation of haematopoietic stability following transplant is required. Our results further illustrate this point and highlight that sustained erythropoietic engraftment of donor origin may occur despite mixed donor leucocyte chimerism. That full leucocyte engraftment is not necessary for sustained donor origin erythropoiesis, may explain the reports of transfusion independence in patients with mixed chimeric grafts.36 We also found that maintained mixed donor WBC chimerism may mask a loss of erythroid engraftment. Importantly, no patient with full donor WBC chimerism has, in our experience, suffered a loss of donor erythroid engraftment. We have studied our patients at the molecular level to assess the impact (if any) of donor genotype with transplant outcome. The number of cases is too small for statistical significance but, as expected, we did not find the outcome of BMT to be influenced by the molecular genotype (b0 or b+ thalassaemia defect) and/or by the normal or heterozygous state of the donor. Pretransplant factors such as previous chelation therapy, age, HCV status and pretransplant blood transfusion had no apparent influence on outcome in this group of children, perhaps because most of the children had similar poor quality standard management in their countries of origin. No patients or donors had been exposed to hepatitis B and only three patients had been exposed to HCV (one with active disease), again possibly reflecting that the majority of our patients were irregularly transfused prior to their arrival in The Netherlands. In addition to hepatic dysfunction caused by iron overload, hepatitis may result from hepatotropic viral infection, for example, hepatitis C virus (HCV). Pretransplant active HCV infection associated with elevated serum aspartate transaminase levels, predicts the development of veno-occlusive disease following allogeneic bone marrow transplantation.37 However, the prognosis of chronic HCV infection appears more benign than expected, especially in

Bone Marrow Transplantation

children,38 although in adults transplanted for thalassaemia, chronic active hepatitis remains a significant cause of mortality.11 The occurrence of de novo HCV infection does not correlate with the severity of liver disease observed in the first 6 months post-BMT.39 Our patient transplanted with chronic active hepatitis C (patient 462) had no additional toxicities, albeit his conditioning was modified, and confirms that active hepatitis C infection is not an absolute contraindication to BMT. As reported by other groups, serum ferritin was a poor indicator of hepatic damage as determined by pathological classification of hepatic biopsies.40,41 Of those patients in whom liver biopsies were obtained, the fact that portal fibrosis was common is a further indication of the lack of (quality) chelation in their countries of origin. The survival, rejection and DFS rates in our second group of patients compares with other reported outcomes in paediatric patients.6–8 However, a significant element in our recent data is the more favourable outcome for our patients with class 2 and 3 risk stratification. The relatively low frequency of patients and the required experience in the management and follow-up of this disease, both at the clinical and laboratory level, justify the centralisation of this type of complex treatment. A high rate of success and cure can be offered to children with b-thalassaemia major, in particular to those with a less favourable prognosis owing to a history of prior suboptimal management. Further evaluation of the long-term effects of BMT and management of pretransplantation-related complications associated with b-thalassaemia after BMT is a continuing programme of our unit in collaboration with the Haemoglobinopathies Laboratory at Leiden University Medical Centre.

Acknowledgements We thank the medical, nursing and ancillary staff of the IHOBA unit together with Mrs H. Bakker, data manager, Department of Paediatrics, LUMC and the staff of the Europdonor, Leiden, The Netherlands, for their help in the management of patients undergoing bone marrow transplantation. We thank Dr M.J.D van Tol of the Paediatric Immunology Laboratory, LUMC and Dr J Wijnen of the Sylvius Laboratory, Leiden for XY FISH and VNTR analysis, respectively.

References 1 Apperley JF. Bone marrow transplant for the hemoglobinopathies: past, present and future. Clin Haematol 1993; 6: 299–325. 2 Weatherall D. The treatment of thalassaemia, slow progress and new dilemmas. N Engl J Med 1993; 329: 877–879. 3 Mentzer WC. Bone marrow transplantation for hemoglobinopathies. Curr Opin Hematology 2000; 7: 9–17. 4 Piga A, Longo F, Voi V et al. Late effects of bone marrow transplantation for thalassemia. Ann NY Acad Sci 1998; 850: 294–299. 5 Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990; 322: 417–421.

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