Introduction

Thalassemia major is an inherited disease requiring chronic life-long red blood cell transfusion support to treat the anemia caused by ineffective intramedullary erythropoiesis and enhanced red blood cell destruction in the peripheral circulation.¹ During the last three decades, conventional treatment of thalassemia major, based primarily on hypertransfusion programs and intensive iron chelation with desferrioxamine, has markedly improved the survival of patients.² However, despite the significant improvements, therapy related complications mostly associated with iron overload such as liver fibrosis, heart disease, endocrinological dysfunction and so on progress with time. Conventional treatment can postpone but cannot totally prevent these complications resulting in significant morbidity and mortality.³ Moreover, conventional therapy, although prolonging survival, does not offer a high quality of life, requiring strict compliance with treatment, and is associated with significant psychological problems.⁴ The prognosis and survival of patients with thalassemia major supported by conventional treatment is highly dependent on compliance and the socioeconomic status of the country of origin. Thalassemia is more frequent in developing countries, where the mortality due to the disease remains extremely high because of inadequate medical and pharmaceutical support.⁵

The only curative modality for thalassemia major patients today is the replacement of the defective hematopoietic system by allogeneic stem cell transplantation (allo-SCT).⁶ One major limitation of allo-SCT is the lack of suitable donors, since only 30% of potential transplant candidates have HLA-identical family members.⁷ Because of the limited availability of matched family members, attention has turned to alternative donors such as mismatched family members, and/or matched unrelated donors (MUD), but the outcome of these transplants has been confounded by increase of transplant-related complications including early and late toxicity, mortality and rejection.⁸ The major obstacles for the application of allo-SCT even from matched family members have been the lack of durable engraftment resulting from rejection by multiple transfused patients or autologous stem cell reconstitution.^{8, 9} The outcome of allo-SCT from HLA-identical family donors is largely dependent on the age of the recipient as well as on pretransplant parameters reflecting the degree of organ damage from iron overload. With the use of a busulfan–cyclophosphamide preparative regimen, the long-term overall and thalassemia-free survival after allo-SCT from matched family members ranges from 60 to 95%, and from 50 to 90% respectively, depending on the disease risk-status.⁶

From 1982 until present, 69 patients with TM underwent allo-SCT at our department. Our initial approach was based on an irradiation-containing regimen. Recently, we introduced fludarabine-based conditioning for both malignant and non-malignant indications with promising efficacy and minimal toxicity.¹⁰ In the present study we report our experience in allo-SCT from matched related and unrelated donors, using a reduced toxicity fludarabine-based regimen in a cohort of 20 patients with thalassemia major.

Patients and methods

Patient characteristics

Sixty-nine patients with severe thalassemia major (TM) underwent allo-SCT at the department of Bone Marrow Transplantation and Cancer Immunotherapy of the Hadassah University Hospital in Jerusalem from 1982 until 2005. In 1996 we introduced a new reduced toxicity fludarabine-based conditioning regimen. For the following 10 years until October 2005, 20 patients received transplants with this regimen from matched related or unrelated donors. There were 12 women and 8 men with a median age of 5.6 years (range: 2.4–23.3 years). The study protocol was approved by the ethical committee of our hospital. Each participant or his or her parents signed a written informed consent. Minor donors were approved for donation by the court. The characteristics of these patients are shown in Table 1. All these patients were previously transfused. Chelation was defined as adequate when desferrioxamine treatment was initiated within 18 months from the first transfusion and administered subcutaneously for 8–12 h continuously daily for at least 5 days each week. Any deviation from the above program was considered as inadequate chelation treatment. As we did not include routine liver biopsy in our pretransplant evaluation, we cannot accurately stratify our patients according to Pesaro risk classification system. However, 11 of 20 patients had hepatomegaly (at least 2 cm below the right costal margin), nine patients had inadequate iron chelation and all of these nine patients also had hepatomegaly. Abnormal liver function tests defined as elevation of liver enzymes at least twice the upper normal limit, was observed in nine patients. Among nine patients with at least two risk factors, six had abnormal liver function tests. Therefore, we can conclude that 11 of 20 patients (55%) could be stratified to at least class II risk group, while we cannot rule out that at least some of the nine patients with both risk factors belonged to a higher-risk category. Only one patient was below the age of 3 years at the time of transplant, and had no hepatomegaly. As liver fibrosis has rarely been observed in TM patients below this age, we consider that the risk status of this patient can be safely classified as class I. In this cohort, 4 of 20 patients were above the age of 17 years at the time of transplantation, and represented a high-risk group. All these patients had hepatomegaly and inadequate iron chelation: 15 of 20 patients were CMV-positive, while one patient was HCV-positive (Table 1).

Table 1 - Patient characteristics.

Full table

Conditioning regimen

Conditioning regimen consisted of a combination of fludarabine, busulfan and rabbit antithymocyte globulin (ATG-Fresenius). All 20 patients received fludarabine 30 mg/kg/day 6 from days -9 to -4. The first two patients received low total dose of busulfan (8 mg/kg), and one of them had a late rejection. Therefore all 18 subsequent patients received high-dose busulfan. Among them, six received oral busulfan 4 mg/kg/day 4 from days –7 to -4, while 12 patients received the intravenous formulation, busulfex 3.2 mg/kg/day 4 days. All 20 patients received ATG-Fresenius 10 mg/kg/day 4 from days –4 to day –1.

Donors

Donors were HLA-A, B, C and high resolution DR fully matched siblings in 17 cases, another matched family member (grandfather) in one case, while two patients underwent transplantation from MUD. PBSC donors were injected subcutaneously with granulocyte-colony-stimulating factor (G-Neupogen, 5 g/kg two times daily for 5 days) and mobilized PBSC were collected on days 5 and 6. Bone marrow aspiration was performed under local or general anesthesia from the posterior ileum region using standard BM aspiration needles. Unmanipulated PBSC and BM served as the source of stem cells in 8 and 12 patients, respectively. Characteristics of donor–recipient pairs and of the graft inoculum are shown in Table 2.

Table 2 - Donors and graft characteristics.

Full table

GVHD prophylaxis and treatment

GVHD prophylaxis consisted of CyA 3 mg/kg i.v. starting from day 4. The duration of CyA administration was dependent on the results of chimerism analysis and the presence of clinical or laboratory signs of GVHD. In general, patients without signs of GVHD and without complete donor chimerism were gradually tapered off CyA, at 2 months after transplant. For patients with complete donor chimerism, and/or signs of GVHD, CyA administration was continued for at least 6 months and was gradually reduced thereafter.

Acute and chronic GVHD were graded according to the International Bone Marrow Transplantation Registry (IBMTR) severity indices.¹¹ Immediately, upon the appearance of signs and symptoms of GVHD, i.v. methylprednisolone (2 mg/kg) and CyA were administered.

Chimerism analysis

To assess engraftment, degree of chimerism and residual thalassemic haemopoiesis, patients were monitored at regular intervals by cytogenetic analysis, and by donor and host-specific DNA markers, using male and female amelogenine gene PCR bands, and by VNTR-PCR assay.^{12, 13}

Supportive treatment

Before transplantation, all patients received trimethoprim/sulfamethoxazole (10 mg/kg/day trimethoprim) on days -10 to -2, acyclovir (500 mg/m² 3/day) from days –10 to +100, and allopurinol (300 mg/day) on days -10 to -1. Administration of trimethoprim/sulfamethoxazole (twice weekly) was reinstituted after recovery from neutropenia as a preventive measure against pneumocystis carinii infection. Neutropenic patients with culture-negative fever received a combination of gentamicin, cefazolin and piperacillin, as a first-line antibiotic protocol. Persisting fever was treated with amikacin and tazocin as a second-line protocol, while meropenem and vancomycin were used as the third-line protocol. In cases of persistent fever not responding to antibiotic therapy within 5 days, amphotericin B (0.7 mg/kg/day) was added until the neutropenia resolved.

Starting on day 10, patients were monitored with a DNA-PCR test and later during the study period pp65 antigenemia was carried out on a weekly basis to detect CMV. Two consecutive positive PCR results or positive antigenemia with more than one cell positive for pp65 served as an indication for replacing acyclovir with ganciclovir 10 mg/kg/day until a minimum of two negative tests was obtained.

Patients were treated with reverse isolation in rooms equipped with HEPA filters, and received a regular diet. Additional supportive measures, such as parenteral nutrition and blood component transfusion, were administered as necessary.

Definitions

Day of neutrophil engraftment was defined as the first of 3 consecutive days with ANC>0.5 10⁹/l. Day of platelet engraftment was defined as the first of 3 days with platelets (PLT) >20 10⁹/l without any transfusion support. The overall peak grade of acute and chronic GVHD was reported in our study for all the statistical evaluations. All patients who had a successful engraftment and survived for at least 4 weeks were evaluable for acute GVHD (aGVHD) analysis; for chronic GVHD (cGVHD) analysis, all patients who survived for 100 days were evaluable. Transplant-related mortality (TRM) was defined as mortality from any cause not directly associated with rejection. Overall survival (OS) was calculated from the time of transplant until death from any cause. Thalassemia-free survival (TFS) was calculated from the time of the transplant until thalassemia recurrence with transfusion dependence or death from any cause.

Statistical analysis

For statistical analysis and graphics, data were put to Microsoft Office Excel 2003. Assessments of median (M), average (X) and standard deviation (s.d.) were used for parametric criteria, while Fisher's exact test for 2 2 tables were used for categorical data using online interaction calculation tools.¹⁴ For survival analysis Kaplan–Meier curves were used.

Results

Engraftment

All patients except one achieved primary engraftment. One patient who underwent matched unrelated transplantation had primary graft failure. This patient received autologous backup and he is currently alive but with thalassemic hemopoiesis. Engraftment of neutrophils was observed in a median of 15 days, (range: 10–27 days) while engraftment of platelets was achieved in a median of 15 days (range: 9–35 days) after transplantation. Six patients never required any platelet transfusion support, while all the patients required red cell transfusions. The median number of transfused PLT units was 2 (range: 0–32), while the median number of transfused packed red cells units was 3 (range: 2–9), respectively. No episodes of hemolysis or pure red cell aplasia were observed among ABO-mismatched donor–recipient pairs.

Chimerism and rejection

During the first month post-transplant, 7 of 20 patients achieved complete donor chimerism in the peripheral blood, 12 patients had mixed chimerism, while one patient had primary graft failure and got back-up of his own marrow on day +31. During the first 2 months, 12 of 20 patients achieved complete donor chimerism, while seven had mixed chimerism. With a median follow-up period of 9 months, (range: 5–112 months), 11 of 12 patients with complete donor chimerism at 2 months, never rejected the graft and all of them remain fully converted to donor type DNA, while only 1 of 12 rejected the graft 8 months post-transplant, with spontaneous autologous recovery. Two of seven patients with mixed chimerism at 2 months rejected the graft at 4 months post-transplantation and both of these patients had spontaneous autologous recovery. Two patients among the seven with mixed chimerism at 2 months subsequently achieved complete donor chimerism (19 and 4 months post-transplant, respectively), while three patients remain in a state of stable mixed chimerism at 17, 25 and 29 months post-transplantation (Table 3). For patients with mixed chimerism at 1–2 months we decreased the dose of CyA rapidly and kept them on suboptimal CyA blood levels. One patient received donor lymphocyte infusions.

Table 3 - Graft-vs-host disease, chimerism status and final outcome.

Full table

Acute and chronic GVHD

Five out of twenty patients (25%) developed aGVHD. However, the cumulative incidence of aGVHD grade >1 and of severe aGVHD grade >2 were 20% (4 of 20 patients) and 10% (2 of 20 patients), respectively. None of our patients experienced aGVHD grade 4. Acute GVHD was limited to the skin in one patient, while intestinal and liver involvement was observed in four and two patients, respectively. Resolution of aGVHD after treatment was observed in four patients, while one patient progressed to chronic GVHD.

Five of twenty (25%) patients developed cGVHD, which was extensive in two cases. One patient developed progressing cGVHD, one developed de novo cGVHD, while the quiescent type was observed in the remaining three patients. Chronic GVHD resolved or improved in all affected patients, and currently none of these patients are under immunosuppressive treatment.

Among 12 patients with complete donor chimerism at 2 months, five developed aGVHD, while none of the seven patients with mixed chimerism had any evidence of aGVHD (P=0.07; Fisher's exact test for one-tailed probability). Conversion to complete donor chimerism preceded the appearance of the clinical manifestations of aGVHD in all the observed cases. Among eight patients who received PBSC, four developed aGVHD, while only 1 out of 12 patients who received BM developed aGVHD (P=0.058). None of the 11 patients who received BM from matched sibling donors developed any sign of GVHD.

Regimen related toxicity

Overall, the conditioning was well-tolerated and the post-transplant outcome was satisfactory. None of our patients developed hemorrhagic cystitis. Two of twenty (10%) patients developed veno-occlussive disease of the liver (VOD). One patient had mild VOD, which resolved without any specific intervention except fluid restriction. One patient developed VOD of moderate severity, which was treated with fluid restriction, diuretics and defibrotide. Mild mucositis was observed in most of our patients (Table 3).

Infectious complications

Seventeen of twenty patients developed fever during the neutropenic phase. Among nine patients, nine episodes of bacteremia were observed: one case of Acinetobacter baumanii, one case of Klebsiella, one case of Stenotrophomonas maltophilia, three cases of Staphylococcus coagulase negative, one case of Enterococcus faecium, one case of undefined Streptococcus species, one case of Enterobacter cloaca and one case of Staphylococcus aureus. None of the patients developed septic shock, and all these cases of bacteremia were treated successfully with an appropriate antimicrobial regimen. CMV reactivation was observed in nine patients and in all cases was treated successfully with gancyclovir. None of our patients developed CMV disease. One patient with cGVHD developed pneumonocystis carinii pneumonia that was treated successfully with trimethoprim-sulfomethoxazole. No systemic fungal disease was observed among our patients.

Overall and thalassemia-free survival

With a median follow-up period of 39 months, (range: 5–112 months), 16 of 20 patients have sustained engraftment, and are transfusion independent. Among them, 13 patients have complete donor chimerism, while three remain mixed chimeras at 17, 25 and 29 months post-transplantation, respectively. The Kaplan–Meier estimated thalassemia-free survival for this cohort of patients is 80%, while the overall survival is 100%. Fifteen patients are currently off any immunosuppressive medication, while one patient is still on GVHD prophylaxis, 9 months post transplantation (Figure 1).

Figure 1.

Disease-free survival, 20 thalassemia patients after HSCT.

Full figure and legend (11K)

Discussion

The most extensive experience to date in the treatment of thalassemic patients with allo-SCT has been reported by the Pesaro group in Italy. The initial preparative regimen used in their studies was planned two decades ago, and consisted of the combination of busulfan 14–16 mg/kg and cyclophosphamide 200 mg/kg (Bu14Cy200). The concept behind the design of Bu14Cy200 was that busulfan in high doses would be sufficient for the eradication of host marrow, while high-dose cyclophosphamide would be sufficiently immunosuppressive to prevent rejection of the infused marrow. Soon after the initial studies it was documented that the outcome of allo-SCT was mostly influenced by pretransplant parameters reflecting the degree of organ damage due to iron overload. Based on the presence or absence of hepatomegaly, liver fibrosis and adequate iron chelation, the Pesaro group classified transplant candidates into three categories: class I, II and III.⁶ Analysis of their data showed that although Bu14Cy200 was effective in the treatment of class I and II patients, it was associated with significant transplant-related mortality in class III patients.^{6, 15, 16} Namely, the OS rates were 95 and 85%, while the TFS rates were 90 and 80% for class 1 and 2 patients, respectively. Early results indicated that the transplant outcome was worse for class 3 patients with probabilities of OS, TFS and rejection of 60, 53 and 16%, respectively.¹⁵ Reduced survival rates in patients with high-risk disease status was mainly associated with increased TRM. There is strong evidence that cyclophosphamide metabolites such as acrolein can lead to depletion of liver glutathione, a potent antioxidant factor, resulting in endothelial cell damage of the small liver venules and sinusoids.¹⁷ Oral busulfan in contrast to intravenous busulfex is also associated with higher probability of liver toxicity due to first pass of the absorbed drug into the liver. Moreover, busulfan potentiates cyclophosphamide toxicity, making the combination of BuCy a significant risk factor for the development of sinusoidal obstructive syndrome.¹⁸ This observation is particularly important for thalassemic patients with liver iron overload that are more prone to post-transplant liver toxicity. To improve survival of class III patients, the Pesaro group reduced the dose of Cy from 200 to 120–160 mg/kg. Although the overall survival of high-risk patients improved, the thalassemia-free survival remained unaffected because of an increase in graft failure rates.¹⁵ In addition, early experience suggested that the results of transplantation from HLA-identical siblings for thalassemia were particularly poor for patients older than 16 years, mainly because of a high proportion of class III risk disease status above this age limit.¹⁶ As the possibility of the existence of a matched sibling donor is around 30%, attention has turned during recent years to the use of alternative donors such as partially mismatched family members or matched unrelated donors. The efficacy of such transplantations remains largely unsatisfactory due to the observed high transplant-related mortality and rejection rates.⁸ Because of these limitations many clinicians are reluctant to offer such an option to their patients. However, even with the best supportive treatment, severe thalassemia major still remains a progressive disease, which not only limits the life expectancy but also has an adverse impact on the quality of life of these patients. In contrast, in view of the significant improvements in supportive treatment of thalassemic patients, TRM rates above 5% seem rather unsatisfactory. Since allo-SCT is currently the only available curative procedure for TM patients, development of new regimens that will further reduce TRM without compromising engraftment rates, especially for class II and III patients, is warranted.

Over the recent years, reduced-intensity allogeneic stem cell transplantation (RIC) was designed to induce host-versus-graft tolerance by engraftment of donor stem cells, and establishing conditions for donor anti-host allo-reactivity to displace residual hematopoietic cells of host origin by donor T and NK cells. This approach is currently used successfully for treatment of malignant and non-malignant disorders.¹⁹ Use of RIC for non-malignant diseases appears tempting because of reduced short- and long-term toxicity, thus avoiding growth retardation and sterility. In non-malignant hematopoietic disorders such as thalassemia major, graft-versus-tumor effect is not required, and thus clinical GvHD should be minimized, or better, avoided. However, the preparative regimen should be sufficiently myelo-suppressive to ensure sustained engraftment of donor stem cells and to prevent recurrence of the thalassemic hematopoiesis by displacement of residual hematopoietic cells of host origin by donor T cells, which in a way resembles graft-versus-malignancy in patients with hematological malignancies. This is particularly important because patients with thalassemia have greatly expanded marrow volume and graft failure, except for cases involving immune mediation, may be also the result of the successful competition between donor and the remaining host stem cells that survive the conditioning regimen. The ultimate goal of transplantation in thalassemia as well as other non-malignant diseases is the creation of a state of mutual tolerance between donor and recipient. Host-versus-graft tolerance will minimize the possibility of immune-mediated graft rejection, while graft-versus-host tolerance will prevent undesirable overt GVHD in this setting.¹⁹ Our original protocol designed mainly for the treatment of malignant diseases, consisted of the combination of fludarabine and busulfan. The FLU–BU combination proved to be most effective in preparation for allo-SCT in high-risk patients with hematological malignancies otherwise ineligible for conventional conditioning due to age limitations or concurrent co-morbidities.¹⁰ Previous studies exploring the feasibility and efficacy of a non-myeloablative conditioning regimen in allo-SCT for patients with hemoglobinopathies including thalassemia were unsatisfactory. Although the regimen-related toxicity was minimal, almost all of the patients lost their graft and had full autologous recovery after discontinuation of post-transplant immunosuppression.^{20, 21} Similar to these results, one of our first two patients who received our standard NST protocol, containing half of the busulfan dose, had late graft rejection. Since eradication of a significant percentage of host hematopoiesis is mandatory to prevent recurrence of thalassemia, all subsequent patients, received a standard dose of busulfan (total dose: 16 mg/kg p.o.) or busulfex (total dose: 12.8 mg/kg i.v.) instead of the reduced dose that is part of our standard NST protocol. In this new protocol we substituted cyclophosphamide with fludarabine and ATG, potent immunosuppressive agents, with the aim to reduce regimen-related toxicity such as hemorrhagic cystitis and veno-occlusive disease of the liver, without compromising engraftment.^{22, 23} Although no liver biopsy was performed for proper classification, 11 of 20 patients had at least one risk factor (hepatomegaly), while nine of them had at least two risk factors (hepatomegaly and inadequate iron chelation). Moreover, in this study we included four patients above the age of 17 years, while two patients were transplanted from matched unrelated donors.

All our patients achieved fast engraftment and only one experienced primary graft failure. In accordance with previous studies the chimerism status at 2 months post-transplant seems to be the most important prognostic parameter for late graft failure.²⁴ In this study, only 1 of 12 patients with complete donor chimerism rejected the graft, whereas two of seven patients with mixed chimerism at 2 months had a late graft rejection.

In our study, despite the use of reduced toxicity conditioning, we observed a very low incidence of early or late graft failure suggesting that fludarabine can successfully replace cyclophosphamide. In this regard, CyA withdrawal or gradual tapering based on the results of chimerism analysis, may also augment the graft-versus-host hematopoiesis effect that can result in an immune-mediated elimination of residual thalassemic stem cells and thus further reduce the incidence of recurrence of disease. In this study, seven patients were mixed chimeras at 2 months post-transplantation, and all of them were gradually tapered off CyA. Among these seven patients, two converted to complete donor chimeras, three remained mixed chimeras but with increased percentage of donor chimerism, while two rejected the graft later. None of these seven patients developed acute or chronic GVHD. The inclusion of ATG in our regimen is another factor that may improve the outcome by minimizing graft rejection on the one hand and by reducing GVHD, on the other. In this regard, the successful application of ATG was also documented in patients with severe aplastic anemia.²⁵

Transplant-related mortality in TM patients is largely dependent on the presence of clinical or subclinical organ dysfunction that is associated with the degree of iron overload. Pretransplant organ involvement renders patients less tolerant to the high intensity regimens required to ensure engraftment and to prevent rejection of the infused marrow. TRM can be as low as 5% in good risk patients (Pesaro class I), but increases to almost 40% in high-risk patients (Pesaro class III). TRM, even at low levels, is the most significant argument against allo-SCT for patients with non-malignant disorders, such as thalassemic patients especially in the view of the relatively long life expectancy with intensive supportive treatment. In our study, we observed minimal regimen-related toxicity, while the TRM rate was 0%. These results look promising if we take into consideration that a significant proportion of our patients were in a moderate to high-risk status.

The low incidence of GVHD observed in our study may be related to the combination of reduced toxicity conditioning on the one hand, and to the inclusion of ATG in the conditioning on the other. Pretransplant administration of ATG was shown to be effective in reducing GVHD incidence and TRM, even in patients undergoing allo-SCT from unrelated donors.^{26, 27} Of note is the significant association between GVHD and the use of PBSC as a source of stem cells. Indeed, we did not observe any case of acute or chronic GVHD among 11 patients who underwent allogeneic bone marrow transplantation from matched siblings. Therefore, if this observation can be confirmed, the use of bone marrow rather than PBSC as a source of stem cells may be preferable for future transplant procedures. Also of interest is the observation of the association between chimerism status and GVHD. None of our patients with mixed chimerism at 2 months developed GVHD, while in all cases conversion to complete donor chimerism preceded the manifestations of acute GVHD. Monitoring of the chimerism status at weekly intervals seems to be a powerful tool in our approach. Patients with complete donor chimerism at 2 months post transplant, have a very low probability of late graft failure, while they are at risk for GVHD development. Therefore, for such patients, we recommend continuation of intensive immunosuppression for GVHD prevention. In contrast, patients with mixed chimerism at 2 months post transplant are at risk for subsequent graft rejection, while the probability of GVHD seems to be very low. Our results suggest that for such patients, gradual tapering of immunosuppression may represent a safe approach for prevention of late graft failure.

In conclusion, it seems that substitution of high-dose cyclophosphamide by fludarabine and ATG is effective, and results in minimal regimen-related toxicity without compromising the rate of engraftment. The use of BM as a source of stem cells may be safer than PBSC, although this must be confirmed in a prospective study. Weekly monitoring of the chimeric status and gradual withdrawal of immunosuppression are most important parameters for evaluation of post transplant care that need to be further investigated. Larger cohorts of patients and prospective clinical trials are required to confirm the benefits of our promising approach for the treatment of TM and other genetic disorders treatable by allo-SCT.

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Acknowledgements

Work was carried out at the Danny Cunniff Leukemia Research Laboratory and we are thankful for the continuous support of our ongoing basic and clinical research. This research was partially supported by an unrestricted grant from ESP Pharma, Edison, NJ, USA.