Introduction

Busulfan and cyclophosphamide (BuCy) are currently the most widely used myeloablative regimen without incorporating total body irradiation (TBI) to treat malignant and nonmalignant hematologic disorders with allogeneic stem cell transplantation (SCT). This regimen includes oral busulfan and intravenous cyclophosphamide. Many studies have shown the preference of 120 mg/kg BuCy over 200 mg/kg BuCy regarding the efficacy of treatment and its side effects.^{1, 2, 3} It was recently shown that high exposure to cyclophosphamide metabolites after SCT is associated with major toxicities and an increase in non-relapse mortality. BuCy dose-related toxicities are headache (40%), veno-occlusive disease (VOD) (32%), cardiomyopathy (22%) and mucositis (30%), which are the main causes of high transplant-related mortality (TRM) (10–20%), high morbidity and prolonged hospitalization, especially in middle aged and elderly patients.^{4, 5, 6}

Several modifications were made to the BuCy regimen to improve its efficacy and to decrease toxicity. Many studies showed the preference of intravenous versus oral busulfan, as its serum concentration can reach the target level much faster, with less transplant rejection or any other side effects related to fluctuation of serum drug concentration.^{3, 7, 8, 9} Pharmacokinetic studies of patients receiving i.v. busulfan for pre-SCT conditioning reveal that more than 85% of patients achieve and maintain the targeted therapeutic window (areas under the curves (AUCs) between 900 and 1500 Mmin). This is higher than reported in most studies of oral busulfan and particularly important for centers where routine pharmacokinetic monitoring is not available. Andersson et al.³ have reported results of an i.v. BuCy2-conditioning regimen in 61 high-risk patients with various hematologic diseases. In this study no patients experienced early and/or late graft rejection, the incidence of VOD was 8% and TRM at 100 days was 9.8%. Kashyap et al.⁸ in a study comparing i.v. vs oral busulfan as part of a BuCy2-conditioning regimen for allogeneic transplantation showed significant lower incidence of VOD and VOD-related mortality in patients receiving i.v. vs oral busulfan (4.9 vs 20% VOD and 3.3 vs 20% VOD-related mortality). The 100-day survival rate was also significantly higher in patients receiving i.v. busulfan.

The addition of etoposide and melphalan to BuCy regimen leads to an increase in toxicity and mortality. It seems that fludarabine is an acceptable alternative for cyclophosphamide in conditioning regimens. Fludarabine is a purine analogue, which has considerable efficacy in both immunosuppression and tumor cell killing. This compound has been shown to have minimal extramedullary toxicity at doses 90–250 mg/m². Fludarabine inhibits lymphocyte proliferation, promotes lymphocyte apoptosis and is the backbone regimen in the treatment of chronic lymphocytic leukemia (CLL), lymphoma and acute leukemia. Immunosuppressive properties of fludarabine have been documented in a reduced intensity conditioning (RIC) regimen and SCT.^{9, 10, 11} It has been proposed that fludarabine inhibits the repair of alkylator-induced DNA damage, thus providing more reasons to combine purine analogues and alkylating agents to promote allogeneic engraftment.¹² Furthermore, in some studies it has been shown that using fludarabine in RIC regimen promotes engraftment and graft versus leukemia (GVL) and it was well tolerated by the patients. But the major problem with RIC regimen is the high rate of disease relapse and transplant rejection in 25–50% of the patients.^{10, 11, 12, 13, 14, 15} Three studies performed by Bone Marrow Transplantation (BMT) referral centers have recently shown the promising results of fludarabine-containing myeloablative conditioning regimen regarding TRM, engraftment and efficacy in patients with a high risk of leukemia and in middle-aged patients with a related and unrelated HLA-full-matched donor in allogeneic SCT.^{16, 17, 18, 19, 20, 21}

In this study we evaluated the efficacy of fludarabine (200 mg/kg) and oral busulfan (16 mg/kg) as a myeloablative conditioning regimen over 5 days regarding engraftment, TRM, regimen-related toxicity (RRT), acute and chronic graft versus host disease (GVHD), overall survival (OS) and disease-free survival (DFS) in patients with standard and high-risk acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic phase of chronic myeloid leukemia (CML) (CPCML).

Patients and methods

A total of 70 patients with hematologic malignancies from January 2003 to January 2004 were enrolled in this follow-up study at the Hematology–Oncology and BMT Research Center/ Tehran University of Medical Sciences.

The eligibility criteria to include the patients in this study were diagnosis with acute leukemia or chronic phase of myelogenous leukemia, ages between 10 and 50 years, presence of an HLA-matched sibling donor, performance status (PS) between 0 and 1 according to ECOG criteria. All patients and their donors signed a written informed consent before enrollment. The only exclusion criterion was a hypersensitivity reaction to fludarabine. Considering these criteria, the patients' characteristics are shown in Table 1.

Table 1 - Patients characteristics.

Full table

Conditioning regimen and GVHD prophylaxis

The transplant conditioning regimen consisted of 40 mg/m² fludarabine i.v. infusion over 30 min on days -6 to -2 (total dose 200 mg/m²) and 4 mg/kg/day busulfan p.o. in divided doses (four times a day) on days -5 to -2 (total dose 16 mg/kg). All patients received 5 g/kg granulocyte colony stimulating factor (GCSF) twice daily, 24 h after stem cell infusion until engraftment occurred. GVHD prophylaxis consisted of 3 mg/kg cyclosporine-A (CSA) i.v. from day -2 to +6 and changed to oral CSA 12 mg/kg until day +60.^{22, 23, 24} The decision regarding cyclosporine dose adjustment was based on CSA serum level; we tried to maintain it between 100 and 300 ng/ml. The drug was tapered off by a 5% weekly from day +60 to +180 in cases with no GVHD. Owing to significant risk of mucositis, we did not use methotrexate in GVHD prophylaxis regimen.

Stem cell collection

All patients received GCSF-mobilized peripheral blood stem cell (PBSC) or bone marrow (BM) from their HLA-identical sibling donors. In the PBSCT group, donors received 10 g/kg/day of GCSF for five consecutive days before the stem cell collection. Stem cells were collected with Cobe Spectra system. Target doses of mononuclear cell (MNC) and CD34 were >2 10⁸ and >2 10⁶/kg, respectively.^{22, 23, 24}

Engraftment

Engraftment was defined as having the first 3 consecutive days of absolute neutrophil count (ANC) >0.5 10⁹/l and platelet >20 10⁹/l after at least 3 days of being transfusion-independent. Chimerism analysis was performed by the short tandem repeat PCR (STR-PCR) method at days +30 and +60 on T cells and polymorphonuclear neutrophils of the peripheral blood.

GVHD and RRTs

Severity of acute and chronic GVHD was defined according to the modified Glucksberg grading system.²⁵ Non hematological side effects and RRT from day 0 to day +100 were graded according to NCI common toxicity criteria.²⁶

Neutropenic fever was managed according to the Infectious Diseases Society of America (IDSA) Fever and Neutropenia guidelines.²⁷ Tests for cytomegalovirus (CMV) PP65 antigen or CMV DNA PCR were performed weekly. Positive cases were treated by preemptive therapy for 14–19 days or until antigen tests became negative. Irradiated single donor platelets and packed red cells were transfused to maintain the patients' platelet and hemoglobin levels above 20 10⁹/l and 9 g/dl, respectively.

Causes of death

Deaths related to SCT were categorized in two main groups: deaths due to relapse of the disease and deaths due to non-relapse mortalities consisting of infectious complications, RRT and GVHD.

Statistical methods

Categorical variables were summarized as frequency counts and percentages; continuous variables were summarized as medians and ranges. OS and DFS were estimated by Kaplan–Meier method. DFS was defined as the time between transplantation and the earliest occurrence of relapse or death from any cause. Differences in OS or DFS between subgroups were evaluated using the Log-rank test. All statistical tests were two-sided, and P-value <0.05 was used to indicate statistical significance.

Results

Engraftment

As shown in Figure 1 the median times to reach ANC to 0.5 10⁹/l and platelet to 20 10⁹/l were 10 and 12 days, respectively.

Figure 1.

Cumulative probability of engraftment.

Full figure and legend (12K)

Fifty-nine patients were evaluated for chimerism. Full chimerism, defined as having more than 95% donor cells in the peripheral blood, was achieved in 50 patients (85%) and nine patients remained in mixed chimerism between days +30 and +60.

RRT

Mucositis (the most common toxicity) and hepatic toxicity were developed in 93 and 16% of patients, respectively, which resolved with conservative therapy without any serious or permanent sequela. There were only two cardiac toxicity cases: one case with pericardial effusion and the second one with tachycardia.

The median of the highest serum creatinine level during hospitalization was 1.6 mg/dl (range, 0.8–3.7, 24.3% with Cr>2 mg/dl) and the serum cyclosporine level at the same time was 246 ng/ml (range, 9–814). Seven percent of the patients experienced hemorrhagic cystitis (HC) and 36.6% experienced moderate to severe headache. Thirty-six (51.4%), 29 (41.4%) and two (2.8%) of the patients experienced grade II, III and IV toxicities, respectively.

GVHD

Acute GVHD grades III-IV were detected in 11 (15.7%) patients. Forty (60.6%) patients developed chronic GVHD, which was extensive in five (7.6%) (Figure 2 and Table 2).

Figure 2.

Cumulative incidence of aGVHD grade I–IV versus grade II–IV.

Full figure and legend (11K)

Table 2 - Acute and chronic GVHD.

Full table

Survival

The OS and DFS were 71% (s.e.=5.5%) and 64% (s.e.=5.8%), respectively (Figure 3) with 17 months median follow-up for the surviving patients (ranged 6–22 months).

Figure 3.

OS and DFS.

Full figure and legend (11K)

One year OS of ALL patients vs other patients was 55.6% (s.e.=11.7%) and 76.2% (s.e.=6.0%), respectively, which was not statistically significant (P-value=0.1). But one year DFS of ALL patients was significantly lower than the other patients, 44.4% (s.e.=11.7%) and 71.2% (s.e.=6.3%), respectively (P-value=0.04).

Six (33.3%) and seven (20.6%) relapses occurred in ALL and AML patients, respectively. Median time to relapse was 107 days (ranged 28–415) after transplantation. Three relapsed patients with full chimerism received DLI and two of these patients responded to the treatment.

One-year DFS of ALL and AML patients according to the first or second CR was 61.5 (s.e.=7.9) vs 70% (s.e.=14.5%), respectively (P-value=0.74).

The patients transplanted in the first CR and CML patients in chronic phase had one-year OS similar to the patients transplanted in the second CR and a patient with non-response (both groups had one-year OS of 70.7% – s.e.=6.2% and 12.4% respectively). Also, the high-risk patients in contrast to the standard-risk patients had almost the same one-year OS rate – 65.8% (s.e.=11.4%) and 72.4% (s.e.=6.3%) respectively; P-value=0.47). The patients with low-grade aGVHD (grades I and II) had more favorable one-year OS in comparison to the patients with grades III-IV aGVHD 73.7% (s.e.=5.9%) and 54.6% (s.e.=15%) respectively; P-value=0.18.

Causes of deaths

Twenty of the patients died after transplantation; eight patients (11.4%) died of disease relapse or progression, and 12 (17.1%) died of transplant complications (non-relapse mortality). In Table 3, the causes of death are summarized according to the primary diagnosis.

Table 3 - Causes of deaths according to primary disease.

Full table

Discussion

BuCy is a standard myeloablative regimen without incorporating TBI. A high exposure to cyclophosphamide metabolites is associated with major toxicities and has an increase in non-relapse mortality and morbidity. The rationale for cyclophosphamide replacement with fludarabine is its low toxicity, while it is as effective as BuCy. Slavin et al.¹⁴ showed the efficacy of fludarabine 180 mg/m² and ATG in RIC regimen according to engraftment of PBSC transplant from HLA matched donors. However, graft failure and relapse were observed in 25–50% of the patients treated with RIC regimen.^{14, 15} Bornhauser et al.¹⁷ showed a targeted dose of intravenous busulfan and fludarabine without ATG in myeloablative regimen which included engraftment in 100% of the patients and the low incidence of RRT (7%). The probabilities of OS, DFS and non-relapse mortality were 42, 35 and 24% respectively. The incidence of relapse seemed no higher than standard BuCy in similar patients. The major causes of non-relapse mortality were idiopathic pneumonitis syndrome, infection and heart failure. Grade 3 mucositis was the most common nonfatal toxicities. The incidence of other toxicities such as VOD, hemorrhagic cystitis and increased creatinine were not noticeable and developed in a low number of patients.¹⁷

The study by Russell et al.¹⁸ showed that a high dose of i.v. busulfan (12.8 mg/kg) and fludarabine (250 mg/m²) plus ATG is a well tolerated and effective regimen. In this study, there was no clinically diagnosed VOD, also there was a low incidence of HC (13%). Grade II stomatitis occurred in 70% of the patients without any morbid sequela. The important point in this study was a very low incidence of acute GVHD grade III-IV (3%) and chronic GVHD (38%) after 2 years, although 40% of the patients received stem cells from alternatives donors. TRM at day +100 and 2 years were 2 and 5% for matched related donor SCT and 8 and 19% for alterative donor SCT, respectively. Relapse rate was 21% for CR1 AML and CPCML and 66% in high-risk AML. Two-year DFS and OS were 74 and 88% for a low-risk group and 26 and 37% for a high-risk and advanced disease group. This study showed that a cumulative level of i.v. busulfan was comparable to a target range established for oral busulfan.^{18, 19, 20} The third study which came from MD Anderson Cancer Center by de Lima et al.²¹ showed that 40 mg/m² fludarabine and 130 mg/m² i.v. busulfan for 4 days plus tacrolimus and methotrexate as GVHD prophylaxis in high-risk AML and myelodysplastic syndrome patients caused 3% TRM after 1 year. The 1-year OS and DFS were 65 and 52% for all patients, but the results were better in patients who were in CR; their 1-year OS and DFS were 81 and 75% respectively.

The above study showed that TRM and grade III-IV GVHD in high-risk patients were less than 10%. Relapse, engraftment and anti-leukemic effects were also in acceptable range and despite being high risk and having alternative donors, the patients' 2-year OS and DFS were more than 50%.^{17, 18, 19, 20, 21}

In the present work, we conducted our study with some differences with above-mentioned studies. ATG was not used in conditioning regimen in contrast to Russell's study because donors and recipients were HLA matched siblings. Methotrexate was not used in GVHD prophylaxis regimen in contrast to all the above three studies. Although i.v. busulfan has more advantages over oral busulfan, we did not use it as it is not available in our center. The aim of these two modifications was to increase anti-leukemic effects of therapy and to prevent mucositis and renal failure. In contrast to the above three studies in which the patients had myeloid malignancies and 60% of them were high-risk, 25% were ALL and 70% of them were in a standard-risk group and all of them received transplantation from matched sibling donors.

In our study, neutrophil and platelet engraftment occurred in all the patients on days 10 and 12. The most important RRT was mucositis (93%). HC and heart failure were both very rare. Mucositis and liver function test abnormality were relieved with supportive treatment and drug adjustment. The rise in creatinine level was rapidly relieved with an adjustment of cyclosporine doses. Grade III and IV GVHD were detected in 14 and 10% of the patients respectively on median day +20 post-SCT. The mortality rate due to acute GVHD was only 5.7%. The reason for the increased acute GVHD in comparison to previous studies may be due to the lack of methotrexate in our GVHD prophylaxis regimen. Forty patients developed chronic GVHD whose patterns were limited in 80% and extensive in the other 20%. A probable reason for the high rate of chronic GVHD is in the use of PBSCT in the majority of our patients. About 19% of our patients had relapse on median day of +107 and 80% of them relapsed before day +150. Four out of 13 relapses were extramedullary that mostly occurs in ALL. The relative high incidence of relapse may be due to the transplantation in ALL patients, in which TBI may be a better choice than busulfan and an enrolment of high-risk AML patients.

Apart from the 70 patients, one patient with mantle cell lymphoma (post-autologous SCT relapse) and the other one with secondary CML after autologous SCT for anaplastic large cell lymphoma treated with allogeneic SCT as salvage therapy and received similar conditioning regimen. Both are quite well and are in complete remission without any significant GVHD or morbidity.

We conclude that FluBu (with or without ATG) is an acceptable regimen because of its low TRM and morbidity. But, to enable increase anti-leukemic effects especially in ALL patients it needs some modifications. According to our results, FluBu may substitute BuCy with an aim to decrease transplant adverse effects without compromising its efficacy. Further comparative studies on FluBu and standard regimens in acute leukemia, CML and class-3 thalassemia are necessary.

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Acknowledgements

This research has been supported by Hematology- Oncology and Bone Marrow Transplantation / Tehran University of Medical Sciences grant.