Volume 17, Issue 5 , Pages 717-722, May 2011
Fludarabine-Based Conditioning Chemotherapy for Allogeneic Hematopoietic Stem Cell Transplantation in Acquired Severe Aplastic Anemia
Article Outline
Thirty-eight patients who met the diagnostic criteria for severe aplastic anemia underwent allogeneic hematopoietic stem cell transplantation (HSCT). The median patient age was 20 years (range, 14-36 years). Twenty-four patients were treatment-naïve, 11 had failed one or more previous courses of immunosuppressive therapy, and 3 had failed a previous HSCT. The conditioning regimen included fludarabine 30 mg/m2/day for 3 days (days −9, −8, and −7) and cyclophosphamide 50 mg/kg/day for 4 days (days −5, −4, −3, and −2). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and short-course methotrexate. All patients underwent transplantation with unmanipulated bone marrow as the stem cell source. The median total nucleated cell (TNC) dose was 2.43 × 108/kg (range, 0.60-6.7 × 108/ kg). The conditioning regimen was well tolerated, with minimal treatment-related mortality. Engraftment was observed in all patients after transplantation; the median time to engraftment of neutrophils and platelets was 18 and 23 days, respectively. Twenty-five of the 27 patients with available chimeric studies at day 180 maintained donor chimerism. Acute GVHD grade ≥II was diagnosed in 4 patients (11%). Extensive chronic GVHD was observed in 8 patients (25%) who survived beyond day +100, at a median observation time of 43 months. Graft rejection with relapse of aplais was observed in one patient. The overall survival (OS) for the whole group was 79%. A trend toward improved OS was observed in the treatment-naïve patients (83% vs 71%), but this was statistically insignificant (P = .384). The fludarabine-based conditioning regimen used in this study with relatively young cohort of patients was well tolerated, with a low rate of rejection and treatment outcomes comparable to those seen in other, more intense and potentially more toxic conditioning regimens. Our results await validation in a larger study, optimally in a randomized controlled manner.
Key Words: Aplastic anemia, Transplantation, Fludarabine
Introduction
Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available curative treatment for severe aplastic anemia (SAA). The outcome of allogeneic HSCT has improved significantly over the last decade [1]. In patients with AA, a nonneoplastic hematologic disorder, the main goal of transplantation is to achieve successful engraftment, ideally without acute or chronic graft-versus-host disease (aGVHD, cGVHD).
Despite significant progress in the structure of conditioning regimens, graft failure remains a significant concern, particularly in patients who have undergone previous transplantation. Adequate lymphoablative and immunoablative action of the preparative regimen is critical in these patients [1].
Currently, a combination of antithymocyte globulin (ATG) and cyclophosphamide (Cy) is considered the standard conditioning regimen [2]. Several programs have recently used fludarabine (Flu) as the main immunoablative/lymphoablative component, with encouraging outcomes comparable to those from ATG-containing conditioning but lower toxicity 3, 4.
In this report we summarize our single-institution experience with the use of Flu in combination with Cy (Flu/Cy) for conditioning before HSCT in 38 consecutive patients with SAA. To the best of our knowledge, this is the largest series to date examining the use of Flu-based conditioning in prospectively and uniformly treated SAA patients.
Methods
A total of 38 consecutive patients (all aged >14 years) with a diagnosis of SAA underwent HSCT with grafts from fully matched sibling donors (6/6 HLA-matched siblings, as determined by low-resolution molecular typing for the HLA-A and -B loci and high-resolution molecular typing for the -DR locus). Eligibility criteria included left ventricular ejection fraction >50%, serum creatinine <130 μmol/L, serum bilirubin <35 μmol/L, liver enzymes <2 times the upper limit of normal value, serum albumin >30 gm/L, pulmonary function test values >50% predicted, Karnofsky score >70%, signed informed consent for both donor and recipient, and no contraindication for bone marrow (BM) donation in the sibling donor. Exclusion criteria included failure to meet any of the foregoing eligibility criteria, absence of donor or recipient consent form, human immunodeficiency virus positivity, major psychiatric disorder, and positive pregnancy test. The hospital’s Institutional Review Board approved the study design.
Conditioning Chemotherapy and Transplantation
All patients received Flu 30 mg/m2/day for 3 days (days −9, −8, and −7) and Cy 50 mg/kg/day for 4 days (days −5, −4, −3, and −2). Doses were calculated based on adjusted ideal body weight [ideal body weight + 0.25(actual-ideal)]. GVHD prophylaxis consisted of cyclosporine (CsA) and short-course methotrexate (15 mg/m2 on day +1 and 10 mg/ m2/day on days +3 and +6). Unmanipulated BM was the stem cell source in all grafts. Chimerism was evaluated using probes specific for polymorphic DNA sequence short-tandem repeats. CsA taper was started on day +120 using a 10% dose reduction every 2 weeks, with the goal of maintaining a linear taper and a careful follow-up of blood counts with every step reduction in dose.
Supportive Care
Each patient was admitted to a neutropenic isolation room with HEPA filters. All patients underwent cytomegalovirus surveillance using peripheral blood PP65 antigenemia testing. Prophylaxis for Pneumocystis jiroveci, herpes simplex, and Candida spp was provided to all patients.
All patients received irradiated and filtered blood products. Red blood cell transfusions were given to patients with a hemoglobin level <80 mg/dL. Platelet transfusions were given to patients with a platelet count <10 × 109/L, or <15 × 109/L in those with fever or had clinical signs of bleeding. Febrile episodes were treated with broad-spectrum antibiotics, which were subsequently modified as required according to the HSCT unit protocol. Detailed information on institutional HSCT supportive care policies and procedures is provided at http://bportal.kfshrc.edu.sa/wps/portal/bportal/KFCC.
Evaluation of Response and Definitions
Myeloid engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count (ANC) >0.5 × 109/L. Platelet engraftment was defined as an unsupported platelet count maintained above 20 × 109/L. Primary graft failure was defined as failure to achieve an ANC >0.5 × 109/L for 3 consecutive days. Secondary graft failure was defined as subsequent development of persistent ANC <0.5 × 109/L after achieving adequate engraftment, with compatible BM hypocellularity. GVHD was graded according to the clinical assessment using modified Seattle criteria [5]. Regimen-related toxicity was graded according to National Cancer Institute’s Common Toxicity Criteria, version 3 [6].
Statistical Analysis
Overall survival (OS) included all patients who were alive at the time of last evaluation. Survival analysis was conducted using the Kaplan-Meier method; with the log-rank test used for statistical significance [7]. A two-sided P value <.05 was considered statistically significant. All statistical analyses were performed using SAS version 9.1 (SAS Institute, Cary, NC).
Results
Patient Characteristics
Between August 2004 and September 2008, a total of 38 patients who met the criteria of acquired SAA underwent HSCT on the foregoing protocol (Table 1). Median patient age was 20 years (range, 14-36 years). The male:female ratio was 1.2:1, and the median time from diagnosis to transplantation was 50 days (range, 24-4507 days). A paroxysmal nocturnal hemoglobinuria (PNH) clone was detected in 4 patients. Eleven patients had failed one or more previous courses of immunosuppressive therapy, and 3 patients had failed a previous HSCT (2 of whom had also received previous immunosuppressive therapy before HSCT). The average number of blood units transfused before HSCT was not accurately available, because most of the patients were referred from outside hospitals; however, 4 patients had a high titer of alloreactive antibodies before transplantation.
Table 1. Patient Characteristics
| Number of patients | 38 |
| Age, years, median (range) | 20 (14-36) |
| Male: female ratio | 1.2:1 |
| Time from diagnosis to transplantation, days, median (range) | 50 (24-4507) |
| Failed previous immune suppression, n | 11 |
| Failed previous HSCT, n | 3 |
| Alloreactive antibodies, n | 4 |
| PNH clone, n | 4 |
| Donor–recipient sex mismatch, n | 14 |
| Donor–recipient major ABO mismatch, n | 9 |
| 6/6 HLA matching, n | 38 |
| Graft TNC content, × 108/kg, median (range) | 2.43 (0.60-6.7) |
Total Nucleated Cell Dose, Engraftment, and Chimerism
The median TNC dose was 2.43 × 108/ kg (range, 0.60-6.7 × 108/ kg). Engraftment was observed in all patients after transplantation. The median time to neutrophil engraftment was 18 days (range, 12-28 days), and that for platelet engraftment was 23 days (range, 16-104 days).
At day +120, a donor pattern of hematopoiesis was detected in all 32 living patients. Twenty-five of the 27 patients with available chimeric study data at day +180 still exhibited a stable or rising donor pattern. One patient experienced a total loss of donor pattern, but then regained donor chimerism after a booster dose of stem cells and immune suppression with mycophenolate mofetil, and another patient with decreasing donor pattern responded well to reinitiation of immune suppression.
Clinical Outcome
Clinical outcomes are summarized in Table 2.
Table 2. Clinical Outcomes
| Clinical Outcome | Frequency, n (%) |
|---|---|
| Mucositis ≥ grade 1 | 29 (76) |
| Febrile neutropenia | 36 (95) |
| Hepatic veno-occlusive disease (VOD) | 1 (3) |
| Hemorrhagic cystitis | 2 (5) |
| Hemorrhagic complications | 2 (5) |
| Post Transplantation Lymphoproliferative Disorder | 1 (3) |
| Acute GVHD grade ≥II | 4 (11) |
| Chronic GVHD (extensive) | 8 (25) |
| Graft rejection with aplasia (secondary) | 1 (3) |
Toxicity
Bearman grade ≥I mucositis was observed in 29 patients (76%), and febrile neutropenia was seen in 36 (95%). Hepatic veno-occlusive disease (VOD) was observed in one patient. Clinically significant hemorrhagic cystitis occurred in 2 patients.
GVHD
aGVHD grade ≥II was diagnosed in 4 patients (11%). Grade IV steroid-refractory aGVHD was the cause of death in one patient. Extensive cGVHD was observed in 8 (25%) of the patients who survived beyond day +100.
Graft Rejection and Survival
At a median observation time of of 43 months post-HSCT, only one patient (3%) demonstrated graft rejection. This patient showed a complete loss of donor chimerism with relapse of aplasia and required an additional stem cell boost to restore donor hematopoiesis. Currently, he is maintaining a stable mixed chimerism status, is off immune suppression therapy, and is transfusion-independent.
The 43-month OS of the study population was 79% (Figure 1). When patients were stratified by previous treatment(s), the 43-month OS was 83% for the treatment-naive group and 71% for the previously treated group (P = .384) (Figure 2).
Figure 1
Kaplan-Meier OS curve for all patients.
Figure 2
Kaplan-Meier OS survival curves according to previous treatment.
Day +100 treatment-related mortality (TRM) was 16% (6/38 patients). Five patients suffered an early pre-engraftment death, most due to infectious complications. One patient died because of uncontrolled grade IV aGVHD.
Two patients died beyond day +100 post-HSCT. One patient died from infectious complications while receiving immunosuppressive therapy for extensive cGVHD, and the other died from procedure-related hemorrhagic complications after liver biopsy.
Discussion
Historically, several immunoablative/lymphoablative treatments have been combined with Cy for pretransplantation conditioning of SAA in the setting of a fully matched sibling donor 1, 8. Radiation therapy in the form of total body irradiation (TBI) or total nodal irradiation (TNI) is a potent immunoablative therapy known to promote efficient engraftment 1, 8. Radiation therapy in transplantation for SAA has been associated with significant late toxicity, however, related mainly to the development of secondary malignancies [9].
The addition of ATG to Cy given at total dose of 200 mg/kg has been demonstrated to promote excellent engraftment and long-term outcome. Presently, this combination is considered more or less the standard conditioning regimen for SAA in the setting of matched related HSCT [2]. However, ATG is associated with significant side effects including, but not limited to, immediate allergic reaction, hemolysis, increased risk of infection, serum sickness–like reaction, and even secondary renal dysfunction. ATG toxicity also tends to be more pronounced with age. Moreover, the prohibitive cost of ATG therapy makes it unaffordable in many transplant centers, particularly in some developing countries where SAA may occur at higher frequencies [10].
Prevention of graft failure can be addressed with other, more intense conventional conditioning regimens. However, this benefit can be offset by higher treatment-related morbidity and TRM in a disease for which the primary goal of transplantation is only to secure adequate and durable engraftment and there is no malignancy requiring eradication by high-dose chemoradiotherapy or a graft-versus-malignancy effect [1].
Flu is a purine analog with potent lymphoablative and immunoablative effects. It works synergistically with cyclophosphamide by inhibiting alkylation-induced DNA repair [11]. Flu is known to induce protracted lymphopenia with significant depression of CD4 and CD8 lymphocytes and occasional development of transfusion-associated GVHD when nonirradiated blood products are administered [11]. Compared with ATG and TBI, it is well tolerated, with minimal short- and long-term side effects [11].
The use of Flu as an engraftment-promoting immunoablative/lymphoablative conditioning component is increasing because of the drug’s wide availability, high tolerability even at older ages, and affordability. Flu is particularly useful when an effective immunoablative and lymphoablative component is needed, such as in reduced-intensity conditioning (RIC) and in patients with a nonneoplastic hematologic disorder who are heavily pretransfused, such as those with acquired AA, other BM failure syndromes, and hemoglobinopathies 3, 4. The Flu-based conditioning regimen used in the present study was well tolerated, with very limited toxicity.
Unmanipulated BM provided the stem cell source for all patients, based on retrospective data from the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation (EBMT) suggesting inferior outcomes with the use of peripheral blood stem cells (PBSCs) in this disease. In addition, the current consensus of the EBMT’s Severe Aplastic Anemia Working Party recommends using BM as the stem cell source in the matched related transplantation setting [1].
Overall transplantation complications and morbidity in this study were acceptable, with no unusual complications or toxicities observed. Of note, our subjects were of younger median age than the AA patients reported in previous studies from Western Europe and North America, which possibly could have contributed to a more favorable outcome.
On the other hand, although data on pretransplantation transfusions were not available for our study patients, the median time from diagnosis to transplantation in our study is much longer than that in the studies from Western Europe and North America. Intuitively, reducing and possibly almost eliminating pretransplantation transfusions would markedly improve patient outcomes. Moreover, the long delay with cytopenia before transplantation could have contributed to an increased susceptibility to infection and the increased number of sepsis-related deaths in our patients.
The number of patients who developed aGVHD or cGVHD was limited, and the incidence was comparable to that reported in previous studies, including studies that used Cy and ATG for conditioning. We have previously reported on the lower likelihood of GVHD in our region of the world, presumably related to the high racial and genetic homogeneity of our population. The possibility that this factor contributed to the limited GVHD observed in the present study cannot be excluded [12].
Engraftment rates were satisfactory even for those patients who had failed previous immunosuppressive therapy or HSCT. Such patients have been consistently reported to be at high risk for graft failure in previous studies.
Numerous reports from single-institution studies have described the use of Flu-based conditioning in different schedules and combinations for HSCT for aplastic anemia with favorable outcomes 3, 4, 13, 14, 15, 16, 17, 18, 19, 20, 21 (Table 3). Retrospective data from an EBMT study also suggested favorable outcomes with the use of Flu-based conditioning for HSCT for AA in older patients [22].
Table 3. Reports on Fludarabine-Based Conditioning with Different Outcomes
| Present Study | Mahmoud et al. [13] | Kumar et al. [14] | Ambulkar et al. [15] | Gómez-Almaguer [16] | Resnick et al. [4] | Srinivasan et al. [17] | |
|---|---|---|---|---|---|---|---|
| Age, years, median (range) | 20 (14-36) | 18 (17-36) | 25 (4-65) | 20 (9-55) | 30 (11-65) | ||
| Number of patients | 38 | 17 | 5 | 24 | 23 | 13 | 26 |
| Flu, mg/m2 | 30/day × 3 | 30/day × 3 | 30/day × 6 | 30/day × 6 | 30/day × 3 | 30/day × 6 | 25/day × 5 |
| Cy, mg/m2 | 1000/day × 2 | 350/day × 3 | |||||
| Cy, mg/kg | 50/day × 4 | 60/day × 2 | 60/day × 2 | 60/day × 2 | 60/day × 2 | ||
| Others | ATG 30-40/kg/day × 4 days | Bu | ATG 10/kg/day × 4 days | ATG 40/kg/day × 4 days | |||
| 4 mg/kg/day × 2 | |||||||
| Acute GVHD, % | 11 | 23.5 | 20 | 29 | 17.3 grade I-II | 8.3 | 65 |
| Chronic GVHD, % | 25 | 0 | 40 | 32 | 26 | 12.5 | 56 |
| OS, % | 79 | 88.2 | 100 | 82.8 | 88 at 1500 days | 84 at 5 years | 77 |
| Type | Matched sibling | Matched sibling | Matched sibling | Matched sibling | Matched related | Matched related = 22; one antigen mismatch = 4 | |
| Source | Unmanipulated BM | Unmanipulated PBSCs | Unmanipulated PBSCs | Unmanipulated PBSCs | PBSCs = 9; BM = 4 | Unmanipulated PBSCs |
| George et al. [3] | Kang et al. [18] | Gupta et al. [19] | Bacigalupo et al. [20] | Chan et al. [21] | |
|---|---|---|---|---|---|
| Age, years, median (range) | 20.8 (5-43) | 7-18 | 13(8-35) | 14 (3-37) | 0.7-11.5 |
| Number of patients | 35 | 5 | 7 | 38 | 5 |
| Flu, mg/m2 | 30/day × 6 | 30/day × 4 | 30/day × 4-5 | 30/day × 4 | 30/day × 4-5 |
| Cy, mg/m2 | 300/day × 4 | 300/day × 4 | |||
| Cy, mg/kg | 60/day × 2 | 50/day × 4 | 60/day × 2 or 50 mg/kg × 4 | ||
| Other | ATG 10/kg/day × 4 (n = 11) | ATG 2.5/kg/day × 3 | Pediatric: Campath 0-2/kg/day × 5 | ATG 3.75 mg/kg × 4 | ATG 30 mg/kg × 4 |
| Adult: Campath 20/day × 5 | |||||
| Acute GVHD, % | 29 | 20 | 42.8 | 11 | 40 |
| Chronic GVHD, % | 32 | 0 | 16.6 | 27 | 80 |
| OS, % | 82.8 at 22 months | 80 at 566 days | 71.4 | 73 at 2 years | 100 |
| Type | Matched related | Matched unrelated | Matched unrelated | Matched unrelated = 33, related mismatched = 5 | Related one locus mismatched = 3; matched unrelated = 2 |
| Source | Granulocyte colony-stimulating factor PBSCs = 28; BM = 7 | Unmanipulated BM | Unmanipulated BM | Unmanipulated BM = 36; unmanipulated PBSCs = 2 | Unmanipulated BM |
Our institution is a tertiary care referral institution for HSCT and BM failure disorders in particular and we cannot exclude the possibility of some selection bias based on patient referral. Obviously, our finding of favorable outcomes from a Flu-based conditioning regimen for HSCT to treat SAA in a relatively young cohort of patients, as well as the findings of other single-center studies, await validation in a larger study, ideally in a randomized controlled manner.
Acknowledgments
Financial disclosure: The authors have no conflicts of interest to disclose.
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Financial disclosure: See Acknowledgments on page 722.
PII: S1083-8791(10)00352-6
doi:10.1016/j.bbmt.2010.08.013
© 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.
Volume 17, Issue 5 , Pages 717-722, May 2011
