Allografting

Bone Marrow Transplantation (2005) 36, 971–975. doi:10.1038/sj.bmt.1705173; published online 3 October 2005

Unrelated donor stem cell transplantation in adult patients with thalassemia

G La Nasa1, G Caocci1, F Argiolu2, C Giardini3, F Locatelli4, A Vacca1, M G Orofino2, E Piras1, M C Addari2, A Ledda1 and L Contu1

  1. 1Unità Operativa Centro Trapianti di Midollo Osseo, Centro Regionale Trapianti, P.O. 'R. Binaghi', Cagliari, Italy
  2. 2Dipartimento di Scienze Biomediche e Biotecnologie, Centro Trapianti di Midollo Osseo, Università di Cagliari, Italy
  3. 3Unità Operativa di Ematologia, Centro Trapianti di Midollo Osseo, Ospedale San Salvatore, Pesaro, Italy
  4. 4Oncoematologia Pediatrica, IRCCS Policlinico San Matteo, Pavia, Italy

Correspondence: Dr G La Nasa, Centro Trapianti di Midollo Osseo, P.O. 'R. Binaghi', Via Is Guadazzonis, 3 09126 Cagliari, Italy. E-mail: lanasa@tiscali.it

Received 1 July 2005; Accepted 16 August 2005; Published online 3 October 2005.

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Abstract

Allogeneic SCT remains the only potential cure for patients with thalassemia. However, most BMT candidates lack a suitable family donor and require an unrelated donor (UD). We evaluated whether BMT using UDs in high-risk adult thalassemia patients can offer a probability of cure comparable to that reported employing an HLA-compatible sibling as donor. A total of 27 adult thalassemia patients (15 males and 12 females, median age 22 years) underwent BMT from a UD selected by high-resolution HLA molecular typing. The conditioning regimen consisted of Busulphan (BU, 14 mg/kg) plus Cyclophosphamide (CY, 120 or 160 mg/kg) in 12 cases and BU (14 mg/kg), Thiotepa (10 mg/kg) and CY (120–160 mg/kg) in the remaining 15 cases. Cyclosporine-A and short-term Methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. In all, 19 patients (70%) are alive and transfusion-independent after a median follow-up of 43 months (range 16–137). A total of 10 patients (37%) developed grade II–IV acute GVHD and six (27%) chronic GVHD. Eight patients (30%) died from transplant-related causes. UD-BMT can cure more than two-thirds of adult thalassemia patients, and is a particularly attractive option for patients who are not compliant with conventional treatment.

Keywords:

unrelated BMT, adult class 3, thalassemia

Optimization of erythrocyte transfusion support and regular iron chelation therapy has produced a remarkable improvement in the life expectancy of patients with thalassemia major.1, 2 However, complications secondary to iron overload cannot be completely avoided by chelation therapy and compliance with a chronic transfusion regimen may be difficult to maintain with advancing age.3

Allogeneic bone marrow transplantation (BMT) is currently the only treatment available to cure patients with thalassemia major.4 When the donor is an HLA-identical sibling, the probability of disease-free survival (DFS) for thalassemia patients belonging to class 1 and class 2 of the Pesaro classification is in the order of 85–90%.5 Worse results have been obtained in the highest risk category (ie class 3 group), particularly in adult patients, where the probability of survival with transfusion independence after the allograft has been reported to be 58%.6

No more than one-third of patients have an HLA-identical donor within the family.7 The remaining patients must identify a suitable, HLA-compatible donor within the registries now enrolling more than 9 million volunteers worldwide. Recent reports have demonstrated that a more precise characterization of HLA alleles using high-resolution molecular typing for both HLA class I and class II molecules can reduce the risk of developing immune-mediated complications and fatal events.8, 9 In view of this observation, the Italian cooperative group for BMT (GITMO) started a pilot study on transplantation from unrelated donors (UDs) for patients with thalassemia major. The results of the first patients included in this trial have been recently reported, documenting the feasibility of this approach.10

During recent years, prenatal diagnosis has dramatically reduced the number of new thalassemia patients.11 On the other hand, advances achieved in the management of thalassemia patients have led to an increase in the life expectancy of the affected population, now predominantly represented by adult patients, who often have many clinical problems related to iron overload and/or transfusion-acquired viral infections. Today, the majority of requests for starting a search for a suitable, HLA-compatible, volunteer donor are received from this subgroup of patients.

In this report, we focus on the outcome of high-risk adult thalassemia patients, transplanted from a UD selected through high-resolution molecular typing for both HLA class I and II alleles.

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Methods

Patients

From November 1992 to August 2003, 27 consecutive Italian adult thalassemia major patients (15 males and 12 females) were transplanted from a UD in four different GITMO BMT centers and are included in this analysis. All patients were older than 16 years of age, the median age being 22 years (range 17–37). Prior to transplantation, all patients underwent a complete check-up and liver biopsy and were assigned to risk class 3 according to the criteria proposed by Lucarelli et al.12 Risk factors for being included in class 3 were (1) presence of hepatomegaly; (2) liver biopsy revealing the presence of portal fibrosis and (3) irregular pre-transplantation iron chelation. Patient characteristics are detailed in Table 1. The classification of liver iron overload was based on the scheme of Sciot, and portal fibrosis was defined in each patient as mild, moderate or severe.12 The median serum ferritin level was 2601 ng/ml (range 1668–4012). Of 27 patients, 10 (37%) were hepatitis C virus positive (ie they have HCV-RNA in peripheral blood). A total of 19 patients (70%) had positive cytomegalovirus (CMV) serology (Table 1), and 11 of these patients had been included in the previously published pilot study, performed to evaluate the impact of high-resolution HLA-matching and the identity for HLA-extended haplotypes on the outcome of thalassemia patients given UD-BMT.10 This study was approved by the local institutional review board, and each patient or one of the parents (for those below the age of 18 years) signed a written, informed consent. All patients included in the study were willing to be transplanted after an exhaustive discussion of the risk/benefit ratio with the physicians, but lacked an HLA-identical family donor. Exclusion criteria included the presence of liver chirrosis, a left ventricular ejection fraction lower than 40% before transplantation and positive serology for HIV.


Donors

In total, 13 female and 14 male donors were selected through the international UD registry network, median age being 35 years (range 19–50 years). Donor/recipient sex combinations are reported in Table 1. The median interval between start of the donor search and transplantation was 5 months (range 2–9 months). A positive result for the donor search ranged from 36% for BMT centers in Sardinia to 25% for the other BMT centers.

HLA typing and donor/recipient matching

In all donor/recipient pairs, the alleles at the HLA-A, B, Cw, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 and DPB1 loci were identified by polymerase chain reaction-single-strand polymorphism (PCR-SSP) (Dynal, Oslo, Norway) and sequence-based typing. Amplification and sequencing of HLA Class I and Class II genes were performed as previously described,13, 14, 15 using standard big dye terminator cycle-sequencing chemistry supplied with the ABI sequencing kit. The alleles were assigned according to DNA sequences.16 HLA extended haplotypes were identified and defined as previously described.10 A total of 24 donor/recipient (D/R) pairs were matched at the HLA-A, B, Cw, DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1 loci. In two pairs, there was a disparity for an allele at the HLA-Cw locus (Cw1402 vs Cw0202 for D/R pair #2; Cw0602 vs Cw0701 for D/R pair #4) and in other two pairs there was a minor mismatch at the HLA-B locus (B3503 vs B3501 and B3501 vs B3502 in pairs #6 and 11, respectively). Nine patients were matched with their donors for both HLA-DP alleles, a single allelic disparity was present in 11 donor/recipient pairs, while the remaining seven patients differed from their donors for both HLA-DP alleles. Four pairs were identical for two extended haplotypes, 11 pairs shared one extended haplotype, and 13 pairs did not share any extended haplotype (Table 1).

Transplantation procedure

A total of 15 patients received Busulphan (BU) 3.5 mg/kg/day in four divided doses for 4 consecutive days (total dose 14 mg/kg) followed by Thiotepa (TT) 10 mg/kg, administered i.v. in two doses on the same day, and Cyclophosphamide (CY) at either 40 mg/kg/day i.v. for 4 consecutive days (total dose 160 mg/kg) in nine cases, or at 60 mg/kg/day i.v. on days -3 and -2 (total dose 120 mg/kg) in six cases.10 In view of the high incidence of death in the first series of transplanted patients, the conditioning regimen in the remaining 11 patients was changed: in particular, patients were given BU at 14 mg/kg followed by CY at 120 mg/kg. Only one patient, the first of this study, was conditioned with the same dosage of BU and an increased dose of CY (namely 160 mg/kg). Marrow was infused 36 h after the last dose of CY. The median bone marrow nucleated cell dose infused was 3.6 times 108/kg (range 1.8–11.6) of recipient weight (Table 1).

All patients were treated in positive-pressure isolation rooms and received nonabsorbable oral antibiotics and a low-bacteria diet. Supportive therapy, as well as prophylaxis and treatment of infections, was homogeneous among participating centers. Prophylaxis for Pneumocystis Jiroveci pneumonia was started after engraftment, with oral Cotrimoxazole. CMV reactivation was monitored either by expression of the pp56 antigen or by direct PCR.

All patients received Cyclosporine-A (CSP 3 mg/kg day i.v. from day -2 to day +30) and short-term Methotrexate for graft-versus-host disease (GVHD) prophylaxis. Three patients were also given Anti-Thymocyte Globulin (ATG) 3.5 mg/kg on days -3 and -2, in order to reduce the risk of both graft rejection and GVHD (Table 1).

The CSP schedule was switched to 6–9 mg/kg/day orally as soon as oral administration could be tolerated; from day +60, the dose was tapered until discontinuation at 1 year.

Engraftment was documented by in situ Y chromosome hybridization of bone marrow or blood samples in sex-mismatched donor/recipient pairs and by analysis of variable number of tandem repeat (VNTR) polymorphisms and microsatellite analysis of bone marrow and/or blood samples.

Myeloid engraftment was defined as the first of 3 consecutive days when the neutrophil count was greater than 0.5 times 109/l and platelet engraftment as the first of 7 consecutive days with an unsupported platelet count greater than 20 times 109/l. Graft failure was defined as the absence of hematopoietic reconstitution of donor origin on day +45 after the allograft.

Acute and chronic GVHD were graded according to the Seattle criteria.17, 18 Patients were considered evaluable for acute and chronic GVHD if they survived for at least 7 and 100 days after BMT, respectively.

Statistical analysis

For continuous variables with a symmetric distribution, results are expressed as medians and ranges. Comparison between groups was performed by Fisher's exact test. Probabilities of survival and survival with transfusion independence of this cohort of patients were estimated by the product-limit method of Kaplan and Meier19 and expressed as percentage and 95% confidence intervals (95% CI). The probability of the transplant-related mortality (TRM) was estimated as cumulative incidence.

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Results

In 19 patients (70%), transplantation was successful with complete, donor-derived, hematological and immunological reconstitution. Figure 1 shows the Kaplan–Meier probability of event-free survival (70%), of rejection (4%) and the cumulative incidence of TRM (30%) of the 27 patients studied. The median follow-up of surviving patients is 43 months (range 16–137).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Kaplan–Meier probabilities of survival, thalassemia-free survival, mortality and rejection in 27 adult class 3 thalassemia patients transplanted from HLA-matched unrelated donors (between parenthesis: 95% confidence limit).

Full figure and legend (15K)

Eight patients died at a median of 153 days (range 17–470) after transplantation; causes of death were: hepatic veno-occlusive disease (day +17), heart failure in a patient without any sign of engraftment (day +63), cerebral hemorrhage due to acute GVHD (day +95), CMV-related interstitial pneumonia (day +119), cerebral hemorrhage related to liver failure (day +125), intestinal obstruction occurring as a complication of intestinal GVHD (day +269), and liver failure due to chronic GVHD (day +470). Four of the patients who died were HCV-RNA positive and had severe portal fibrosis. Six out of the eight dead patients had been conditioned with BU-TT-CY, while two patients had received the combination BU-CY. One of these two patients died while in profound aplasia.

The median time to granulocyte recovery was 17 days (range 7–46 days), while the median time for a self-sustained platelet recovery was 28 days (range 14–86 days). Of the 27 evaluable transplanted patients, 10 (37%) developed grade II–IV acute GVHD (Table 1). Four of these patients (15%) had grade III–IV acute GVHD. Among the 22 patients at risk, six (27%) developed chronic GVHD, which was limited in four cases and extended in two. In the 16 evaluable patients with a donor identical for at least one extended haplotype, the incidence of grade II–IV acute GVHD was 31% (5/16). By contrast, among the nine recipients who did not share extended haplotypes with the donor, five patients (56%) experienced grade II–IV acute GVHD. None of the patients who experienced grade III–IV acute GVHD, and/or extensive chronic GVHD shared one or two extended haplotypes with the donor. Most of the HLA disparities found in our cohort of donors concerned the DPB1 locus. No statistically significant correlation was found for disparity at this locus and the development of either acute or chronic GVHD.

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Discussion

From the patient's point of view, BMT still represents the only strategy able to guarantee a definitive cure for thalassemia, providing independence from both transfusions and iron chelation, as well as prevention/reversion of disease-induced organ damage. Adult patients, especially when suffering from liver complications, are less than 'ideal' candidates for an allograft, as the risk of death has been reported to be considerable.6 However, whenever the transplant is successful, they are also those who receive the maximum benefit from BMT, given the reduced life expectancy associated with continuing transfusions in the presence of reduced compliance with iron chelation.

Our study demonstrates that the outcome of UD-BMT in adult, class 3 thalassemia patients is similar to that reported in the literature for patients belonging to the same class of risk transplanted from an HLA-identical sibling donor.6 The probability of thalassemia-free survival was 70%, whereas the cumulative incidence of TRM was 30%. One patient died due to graft failure (case # 27), two cases of death were due to organ impairment (cases # 5, 9) and one death occurred for CMV infection (case # 16). The other four deaths (cases # 3, 7, 10, 15) were attributable to the occurrence of acute or chronic GVHD. A higher number of deaths was observed in patients conditioned with the protocol including TT (6/15=40%). For this reason, the conditioning regimen in the second series of patients only consisted of BU-CY. In this series of patients, we had two deaths (2/11=17%) and although the difference between the two groups of patients was not statistically significant, a conditioning regimen with three drugs (BU-TT-CY) seems to be too toxic for patients with organ impairment due to iron overload or hepatitis infection. Although the only patient who experienced graft failure was prepared with the combination of BU-CY, this regimen seems to be better indicated in adult patients with heavy iron overload.

Hongeng et al20 used the BU-CY combination with the addition of ATG in a group of 11 low-risk thalassemic children, all successfully transplanted from a UD. A recent report on a group of 33 class 3 thalassemia patients, aged less than 17 years and transplanted from HLA-identical siblings, has demonstrated the efficacy of a new conditioning regimen adding a pre-treatment with Hydroxyurea, Azathioprine, Fludarabine, to the BU-CY, starting 45 days before BMT.21 It may be that this innovative approach contributed to further improving the outcome also in adult thalassemia patients given UD-BMT.

The incidence of grade II–IV acute GVHD and of chronic GVHD in our cohort was 37 and 27%, respectively. This relatively low incidence of GVHD can probably be attributed to the careful immunogenetic selection of the donor/recipient pairs.8, 9 As the quality of life of patients with extensive chronic GvHD may be worse than that of patients with hemoglobinopathies treated with supportive therapy, all efforts must be spent in preventing the occurrence of this complication. Overall, GVHD, particularly in the severe form, was reduced in patients who shared at least one extended HLA haplotype with the donor. In a previous study, we stressed the positive impact of matching for HLA extended haplotypes and the importance of selecting donors resulted to be compatible using high-resolution molecular typing for both class I and II loci.10 These stringent criteria for compatibility also represent a significant limitation of this experience as, using this approach, only approximately one-third of thalassemia patients for whom a search was started found a suitable donor in a median time of 3–4 months. In fact, the rigorous criteria established for considering a donor as suitable imply that a number of possible donors, who would have been acceptable for patients with other, more life-threatening diseases, were discarded.

In conclusion, our results suggest that UD-BMT in adult class 3 thalassemia patients, with donors selected through high-resolution molecular typing, may offer a success rate similar to that historically reported in patients, with similar prognostic characteristics, transplanted from an HLA-identical sibling and it seems to be a feasible therapeutic approach, especially for patients who are not compliant with conventional treatment.

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References

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Acknowledgements

We thank Professor Guido Lucarelli for his precious comments and critical revision, Anna Maria Koopmans for her assistance in preparing the manuscript and the GITMO (Gruppo Italiano Trapianto Midollo Osseo) for supporting this study.