Introduction

Fludarabine-based conditioning (FBC) is increasingly used for allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematological malignancies, especially in older and debilitated individuals.^{1, 2, 3} FBC is immunosuppressive and enables donor cell engraftment with minimal toxicity. HSCT is an established therapy for several non-malignant disorders, including hemoglobinopathies, aplastic anemia, severe immunodeficiencies and metabolic disorders.^{4, 5, 6} There is a considerable risk of graft failure in these patients, especially hemoglobinopathies and aplastic anemia, because of no prior chemotherapy and the risk of immunization by repeated transfusions.^{6, 7} Doctors are reluctant to use FBC for non-malignant disorders because it increases risk of graft failure compared to conventional myeloablative conditioning (MC).⁸ In a few patients, FBC has been used in non-malignant diseases.^{9, 10, 11, 12, 13, 14, 15} This created a debate about whether FBC or MC should be used.^{16, 17} For children with severe combined immunodeficiencies, encouraging results were obtained using FBC.¹⁵ In contrast, only one out of four patients with hemoglobinopathies had stable long-term engraftment after FBC.¹³ The aim of this study was to compare disease-specific FBS and MC regarding transplant-related complications and outcome.

Patients and methods

Patients

Table 1 shows the characteristics for 23 patients with non-malignant disorders who were undergoing HSCT and receiving FBC and 32 patients treated with MC. HSCT was performed from 1998 to April 2006. Follow-up was performed on 4 January, 2007. Two patients, not included in the table, died during conditioning. These two patients were included in the survival analysis (Figure 1). Survival rates were determined using the life-table method. The study was approved by the Ethics Committee at Karolinska Institutet.

Figure 1.

Probability of survival among patients with non-malignant diseases treated with FBC (n=24) or conventional MC (n=33) before HSCT. This was intention to treat, and one patient in each group who died before transplantation is included in the analysis.

Full figure and legend (48K)

Table 1 - Patient and donor characteristics undergoing hematopoietic stem cell transplantation for non-malignant disorders.

Full table

Donors

Human leukocyte antigen (HLA) typing was carried out using high-resolution polymerase chain reaction (PCR) single-stranded polymorphism.¹⁸ Bone marrow grafts were preferred, but peripheral blood stem cells were collected after granulocyte colony-stimulating factor mobilization in 11 unrelated donors.¹⁹ All donors were consecutive HLA-A, -B, DRB1-identical siblings (n=20) or unrelated (n=35).

Conditioning

Fludarabine (Flu) (30 mg/m² for 3–6 days) was combined with cyclophosphamide (Cy) (30–60 mg/kg/day for 2 days (n=3) and 10 mg/kg/day for 2 days) in patients with Fanconi anemia (n=6),²⁰ melphalane 140 mg/m² (n=1)¹⁵ or busulfan (Bu) 8 mg/kg (n=5).³ Patients with severe aplastic anemia (SAA), in addition to Flu and Cy received 2 Gy fractionated total body irradiation (fTBI) for 3 days to prevent rejection (n=8). One SAA patient with graft failure was conditioned with Flu for 3 days and antithymocyte globulin (ATG).

Immunosuppression and chimerism

MC for SAA using a sibling donor consisted of Cy 200 mg/kg (n=14). Using unrelated donors, Cy 120 mg/kg was combined with fTBI 6 Gy (n=2) or single dose 10 Gy (n=1). The patients with familial hemophagocytic lymphohistiocytosis were treated with Bu 16 mg/kg, Cy 120 mg/kg and Vepesid 300 mg/m² (n=7). The remaining patients received Bu 14 mg/kg/day and Cy 2 g/m²/day for 4 days (n=9).²¹

Immunosuppression was a calcineurine inhibitor combined with methotrexate, mycophenolate mofetil, sirolimus or prednisolone (Table 1).^{2, 7, 22} Prophylactic immunosuppression was continued for 1.5–2 years. Supportive care was reported previously.²³

Chimerism analysis

PCR amplification of variable number of tandem repeats were used to evaluate donor/recipient chimerism in CD3+, CD19+ and CD33+ cells.²⁴ Chimerism analysis was performed from week 3 after transplantation every other week until 3 months after transplant. Patients who became complete donor chimeras (DC) were thereafter followed at yearly controls. Patients who were mixed chimeras were followed every month until 6 months after HSCT and depending on chimerism development and the clinical outcome.

Results

Engraftment

Graft failure occurred in two patients treated with Flu and Cy and in three patients treated with MC. All five were successfully regrafted using FBC. Time to absolute neutrophil count >0.5 10⁹/l was a median of 17 (range 11–35) days among the FBC patients, compared to 15 (10–29) days in the MC group (NS). A platelet count >50 10⁹/l was reached after a median of 20 days (0–42) in the FBC group and 30 (9–115) days in the MC patients (P<0.05). The median number of erythrocyte transfusions was 1 (0–8) in the FBC group and 3 (0–14) in the MC group (P<0.05). Platelet transfusions were given to three (0–24) FBC patients and five (0–20) MC patients in the two groups (P=0.03). Median time to discharge was 22 (12–96) days in the FBC group and 27 (15–88) days in the MC group (NS).

Donor chimerism

At last follow-up, complete donor chimerism (DC) for CD3+ cells was achieved in 9/22 (41%) patients in the FBC group and 19/29 (66%) patients in the MC group (P=0.01). For CD33+ cells, DC was found in 15/22 (68%) patients in the FBC group and 16/32 (50%) (P=0.03) patients in the MC group. Among the patients with Fanconi anemia, one became DC within 3 months and four remained mixed chimeras at the last follow-up.

Fever and infections

Median days with fever (>38.5°C) were 1 (0–45) in the FBC group and 5 (0–15) in the MC group (P=0.003). Bacteremia occurred in six (26%) patients in the FBC group and 16 (50%) patients in the MC groups (P=0.1). Cytomegalovirus (CMV)-PCR positivity treated with pre-emptive therapy was seen in nine patients in the FBC group and 10 in the MC group.²⁵ No patient experienced CMV disease.

Toxicity

One patient each in the two groups developed veno-occlusive liver disease. Maximum levels of bilirubin were significantly higher in the MC group (Table 2). Liver enzymes, serum creatinine and mucositis did not differ significantly between the two groups of patients. Spirometry was more often normal after HSCT in the FBC patients, compared to the MC patients (P=0.04). However, only minimal spirometric abnormalities were observed, for instance minimal obstruction or restriction. A 7-year-old girl with thalassemia major who received conditioning with Bu and Cy, developed permanent alopecia.

Table 2 - Evaluation of toxicity in patients receiving FBC or myeloablative conditioning.

Full table

Graft-versus-host disease

Acute graft-versus-host disease (GVHD) grade II was seen in three patients in the FBC group and eight in the MC group. No patient had severe acute GVHD. Chronic GVHD occurred in four (one moderate) in the FBC group and five patients (all mild) in the MC group.

Death and survival

An 18-year-old male with SAA died of acute respiratory distress syndrome during FBC. A 5-month-old boy with advanced familial hemophagocytic lymphohistiocytosis died of pneumonia during MC. A patient with Fanconi anemia treated with FBC had a tongue cancer at transplant and died from this 2.5 years after transplantation. In the MC group, a 52-year-old male with SAA died of fungal infection 9 days after transplant and two children died of pneumonia (1.5 and 4 months after transplantation). A 12-year-old boy with Kostmann's disease who was well, died mors subita during night at home 11 months after HSCT. Autopsy and cultures could not verify the cause of death. Treatment-related mortality was 4% in the FBC group and 15% in the MC group, including one patient in each group who died during conditioning. Four-year survival was 89% in the FBC group and 85% in the MC group (Figure 1). A Karnofsky score of 100% was seen in 18 of the FBC patients and 19 of the MC patients (NS, Table 2). Low Karnofsky scores (<80) were seen in two adults in the MC group with metachromatic leukodystrophy (50) and adrenoleukodystrophy (50), both with progressive disease.

Discussion

There has been a debate if reduced intensity conditioning may replace MC for patients with non-malignant disorders.^{16, 17} This prompted us to compare the outcome in patients with non-malignant disorders who received conventional MC or FBC. Because of the different disorders and previous treatment, our patients received different FBC conditioning. In patients with non-malignant disorders, the reason for conditioning is only to be immunosuppressive and pave the way for the donor hematopoietic system. Patients with Fanconi anemia got the mildest conditioning, consisting only of Flu and low-dose Cy.²⁰ Patients with SAA, who received unrelated grafts, have been heavily transfused and have also received two cycles with ATG combined with cyclosporine. Because SAA patients may be immunized by multiple transfusions in addition to Flu and Cy, they received fTBI. This conditioning for SAA may be considered as borderline reduced, although it was Flu-based. Non-myeloablative conditioning was not given to any of these patients, because it was considered to be weak in the absence of previous cytotoxic therapy. Non-myeloablative conditioning was also reported to increase the risk of graft failure, compared to reduced intensity conditioning in patients with malignancies.^{1, 2, 3, 8} All patients in the MC group received conventional well-established conditioning protocols.

In this comparison, we found that engraftment was faster and fewer transfusions were required in FBC patients compared to the MC group. This agrees with findings in patients who have undergone HSCT for hematological malignancies.^{1, 2, 3, 7} A shorter neutropenic period may be the reason for less fever in the FBC group. Furthermore, bacteremia had a trend to be less common in the FBC group, compared to MC group. Fewer infections in the FBC group also agrees with previous reports.^{1, 2, 3, 7}

There was only slightly more toxicity in the MC group (Table 2). This was evident by a higher maximum bilirubin and less often a normal post-transplant spirometry in the patients receiving MC. However, abnormal spirometry only showed marginal changes in the patients after HSCT. Otherwise, liver enzymes, serum creatinine and mucositis did not differ significantly between the groups. The reason for the overall low toxicity is that these patients have not previously been treated with cytotoxic drugs, in contrast to patients with hematological malignancies. Furthermore, all patients in both groups were treated with ursodiol for the first 3 months after transplant, which decreases the risk of liver toxicity.²⁶ Veno-occlusive disease of the liver, obstructive bronchiolitis and permanent alopecia is associated with full-dose Bu treatment.²⁷ However, these complications were rare in both groups. Only one patient in each group had veno-occlusive disease of the liver and one patient in the MC group had permanent alopecia.

None of the patients in any of the groups had severe acute GVHD. Using well-HLA-matched-related or -unrelated donors and combinations of immunosuppressive drugs, severe GVHD may be prevented as shown in this study. ATG also decreased the risk of severe acute GVHD.²⁸ With prolonged immunoprophylaxis, chronic GVHD was rare. These patients do not need GVHD to induce any anti-tumor effect. The probability of acute and chronic GVHD was similar in the FBC patients and the MC group. It may be expected that with less toxic conditioning, the incidence of GVHD should be lower. Such an effect was not seen in this material, which may be too small in this context.

Survival in both groups was almost 90%. These encouraging results were obtained using established therapies for HSCT. Because the patients received well-matched grafts and were young, the outcomes were positive. The FBC patients were older and received a lower CD34 cell dose, factors associated with worse outcome.²⁹ There may also be a selection bias with more disabled patients selected for FBC. Still, survival was similar in the two groups. Most patients in both groups had a Karnofsky score of 100. Only two patients had a low Karnofsky score (<80), because of progression of metachromatic leukodystrophy and adrenoleuko dystrophy, respectively. A detailed report of patients undergoing HSCT for inborn errors of metabolism at our unit has recently been published.³⁰ The study showed that early transplantation before disease progression is of utmost importance for a positive clinical effect in many metabolic disorders.

Encouraging results using Flu conditioning was reported in 12 patients with Fanconi anemia receiving cord blood transplants.³¹ Another study showed DC in 7/7 patients with Fanconi anemia, in contrast to only 1/5 in our patients, despite similar conditioning.⁹ Survival was encouraging in both studies.

It is expected that Flu will be increasingly used also in patients with non-malignant disorders. Even if a survival advantage was not achieved in our small series, there may be several other advantages using reduced conditioning, such as less toxicity, less side effects and better quality of life. One potential risk with a milder conditioning may be an increased incidence of graft failure and less complete DC. The number of patients with graft failure was similar in the two groups. We found that complete donor T-cell engraftment was more common in the MC group, but myeloid engraftment was better with FBC. Performing retransplantation, it is more attractive to use FBC because of less toxicity. In the longer perspective, there is also the risk of secondary cancer, which may be reduced using a milder treatment.³²

To conclude, although the material is small and heterogenous, it encourages further exploration of FBC as an alternative to MC in HSCT for non-malignant diseases.

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Acknowledgements

We thank the staff for compassionate and competent patient care. Inger Hammarberg is acknowledged for skilful preparation of this paper. Supported by grants from the Swedish Cancer Society (0070-B05-19XAC), the Children's Cancer Foundation (03/039), the Swedish Research Council (K2006-32X-05971-26-1), the Cancer Society in Stockholm, the Cancer- and Allergy Foundation, and Karolinska Institutet.