Bone Marrow Transplantation (2005) 35, 463–466 & 2005 Nature Publishing Group All rights reserved 0268-3369/05 $30.00
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Stem cell transplantation for patients with Fanconi anemia with low-dose cyclophosphamide and antithymocyte globulins without the use of radiation therapy M Ayas1, A Al-Jefri1, M Al-Mahr1, S Rifai1, A Al-Seraihi1, A Tbakhi2, M Mustafa3, A Khairy1, E Moussa1, A Iqbal2, L Shalaby1 and H El-Solh1 1
Department of Pediatric Hematology-Oncology, King Faisal Specialist Hospital and Research Center (KFSHRC), Riyadh, Saudi Arabia; 2Department of Pathology and Laboratory Medicine, KFSHRC, Riyadh, Saudi Arabia; and 3Department of Pediatrics, King Hussein Cancer Center, Amman, Jordan
Summary: In all, 22 patients with confirmed Fanconi anemia (FA) underwent stem cell transplantation (SCT) from HLAmatched, related donors at KFSHRC. Median age at SCT was 7.6 years (range, 2.5–14.6 years). Conditioning regimen consisted of cyclophosphamide (CY) 15 mg/kg/ day intravenously (i.v.) for 4 consecutive days, in addition to equine antithymocyte globulins (ATG) given i.v. at 40 mg/kg/day for four doses pre-SCT. No radiation therapy was given. For graft-versus-host disease prophylaxis, we used cyclosporin at the standard doses; ATG was added at 20 mg/kg/dose i.v. on days 2, 4, 6, 8, 10, and 12 post-SCT (total of six doses). All patients engrafted and are alive and transfusion independent with a median follow-up time of 20.2 months (range, 3.3–59 months). One patient however developed a decrease in her WBC and platelet count. Her work-up revealed slightly hypocellular bone marrow, and a series of chimerism studies over 1 year confirmed that she has stable mixed chimerism; she remains transfusion independent. We conclude that low-dose CY without radiation therapy can be used satisfactorily in the conditioning of patients with FA undergoing related SCT. Bone Marrow Transplantation (2005) 35, 463–466. doi:10.1038/sj.bmt.1704787 Published online 17 January 2005 Keywords: Fanconi anemia; cyclophosphamide; antithymocyte globulins; stem cell transplantation
Fanconi anemia (FA) is an autosomal recessive disorder belonging to the group of chromosomal instability syn-
Correspondence: Dr M Ayas, Department of Pediatric HematologyOncology, King Faisal Specialist Hospital and Research Center (KFSHRC), MBC 53, PO Box: 3354, Riyadh 11211, Saudi Arabia; E-mail:
[email protected] Received 21 June 2004; accepted 21 October 2004 Published online 17 January 2005
dromes. Clinically, FA is characterized by congenital malformations, progressive marrow failure, and predisposition to acute myelogenous leukemia1–4 and solid tumors.5,6 Although allogeneic stem cell transplantation (SCT) has been established as the only treatment modality that can definitively restore normal hematopoiesis in these patients, recent data suggest that the risk of solid tumors may be increased by transplantation,5,6 and that radiation therapy may be a risk factor for the development of such malignancies post-SCT.7,8 The use of low-dose cyclophosphamide (Cy) as a conditioning regimen has yielded excellent results. However, the question of the optimal dose to be used remains difficult to resolve as many of these regimens employ additional means such as radiation therapy and antithymocyte globulins (ATG), and because the number of patients available for a prospective randomized study is small.9–13 In an effort to determine whether low-dose CY without the use of radiation therapy is adequate for conditioning of FA patients, we launched a prospective study in May 1999, using CY 60 mg/kg in addition to ATG, and report here our results.
Patients and methods Patients From May 1999 to February 2004, 22 patients (14 females and eight males) with confirmed FA underwent allogeneic SCT at KFSHRC; the diagnosis was confirmed by chromosomal breakage studies with mitomycin-C and diepoxybutane. All patients had to have had fewer than 12 blood product transfusions in the year before SCT to be accepted on the study. The median age at the time of SCT was 7.6 years (range, 2.5–14.6 years). All patients underwent bone marrow examination within 2 months prior to SCT and all were required not to have any evidence of myelodysplasia or leukemia, or any clonal abnormality on cytogenetic analysis of their bone marrows to be accepted on the study.
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Donors
Table 1
In all, 20 patients received their SCT from fully HLAmatched siblings and two received their SCT from fully HLA-matched parents. All donors had documented negative chromosomal breakage studies. Harvested marrows were not manipulated, and the median CD34 dose was 6.7 106 (range, 2.2–24.3 106) per kg of recipient body weight.
STR locus
STR size
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 D5S818 FGA Amelogenin X Amelogenin Y
120–180 190–240 250–300 310–340 100–150 160–200 210–250 260–290 300–360 100–140 150–210 220–250 260–340 130–180 210–360 107 112
Preparative regimen The conditioning regimen consisted of CY 15 mg/kg/day intravenously (i.v.) for 4 consecutive days; Mesna 5 mg/kg was given i.v. over 15 min prior to each CY dose, and then 2 mg/kg i.v. every 4 h for five doses on each day of CY. Patients were hydrated with i.v. fluids at double maintenance 6 h before the first dose of CY and continued for 24 h after the last dose of CY; the duration of post-CY hydration was extended to 7 days after three cases of hemorrhagic cystitis were encountered. In addition, equine ATG was given i.v. at 40 mg/kg for four doses pre-SCT. No radiation therapy was given. The study protocol was approved by the research advisory council as well as the ethics committee at KFSHRC. Informed consents were obtained from the parents according to institutional guidelines.
Graft-versus-host disease (GvHD) prophylaxis Cyclosporin was given at the standard doses; equine ATG was added at 20 mg/kg/dose i.v. on days 2, 4, 6, 8, 10, and 12 post-SCT (total of six doses).
STR loci and their molecular sizes
were analyzed on an ABI 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fragment size and peak area data were determined by GeneScan and GenoTyper software (Applied Biosystems, Foster City, CA, USA),14 and the percentage of donor and recipient DNA was quantitated from STR analysis to estimate the degree of stem cell engraftment.14 The means of ratios of the peak areas of donor and recipient alleles at all informative loci were used to calculate the DNA percentages in post transplant samples.
Results Supportive care Engraftment
All patients were treated in HEPA-filtered rooms and isolated until their absolute neutrophil count (ANC) was greater than 0.5 109/l for 3 consecutive days. Intravenous immunoglobulin (IVIG) was administered weekly at a dose of 500 mg/kg from day 7 until day þ 90. All patients received acyclovir 750 mg/m2/day from day 1 post-SCT to day 28. All patients also received prophylactic antifungal therapy with fluconazole 3–5 mg/kg/day from day 0 till engraftment. No prophylactic ganciclovir was given. All blood products were leukocyte filtered and irradiated.
Engraftment was defined as an increase in the ANC X0.5 109/l for 3 consecutive days. All patients engrafted; median time to engraftment was 13.5 days (range, 9–28 days) and median time to self-sustained platelet count of 20 109 was 27.5 days (range, 12–139 days). Chimerism was documented according to the method described above; full chimerism was defined as 495% donor cells. The patients’ chimerism ranged from 29 to 100% in the lymphoid line and 23 to 100% in the myeloid line.
Engraftment analysis
Graft-versus-host disease
STR analysis was carried out on peripheral blood DNA samples from the donor and the recipient before SCT to determine the genotype at 16 loci (Table 1). STR analysis was also performed on peripheral blood samples from the recipient after engraftment. The analysis was performed on lymphocytes and granulocyte fractions using the AmpFlSTR Identifiler PCR amplification kit (PE Applied Biosystems, Foster City, CA, USA).14 Briefly, 10 ml of the genomic DNA were added to 15 ml of the ABI Identifiler kit master mix. The PCR protocol was as follows: 11 min incubation at 951C, 28 cycles of 1 min at 941C, 1 min at 591C, and 1 min at 721C, followed by 1 h incubation at 601C and then held at 41C. After amplification, samples
Acute GvHD, grade 2 or higher, developed in four patients (18%); all patients responded favorably to steroids. No patient thus far developed chronic GvHD.
Bone Marrow Transplantation
Toxicity All patients received broad-spectrum antibiotic coverage after SCT for febrile episodes. Bacteremia was documented in eight patients only, all of whom responded appropriately to therapy. One patient developed CMV infection (positive CMV antigenemia) with no evidence of disease; the patient was treated with ganciclovir and reverted to negative. Candida krusei was isolated from the blood of one patient;
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the infection cleared after therapy with amphotericin-B. Hemorrhagic cystitis occurred in four patients (only one case occurred after extending the double maintenance hydration to 7 days after the last dose of CY). Mucositis grade X2, requiring i.v. analgesia, developed in all patients (oral mucositis was scored from 1 to 3, depending on redness, ulceration, and ability to eat). All patients also developed hypertension (blood pressure greater than 95th percentile for age) during SCT, requiring short-term antihypertensive treatment; patients suffered from no long-term renal dysfunction. Mild self-limited veno-occlusive disease of the liver developed in three patients. Chronic hemolytic anemia requiring steroid therapy developed in one patient about 8 months post-SCT; the patient responded well to therapy and is now on minimal maintenance steroid dose, doing well, and enjoys normal counts 3.5 years after transplant.
Follow-up All patients are alive and transfusion independent at a median follow-up time of 20.2 months (range, 3.3–59 months). Only five patients had a decrease in their chimerism studies to below 50% donor cells. Four of these patients continue to enjoy normal WBC, hemoglobin, and platelet levels. However, one patient developed neutropenia and thrombocytopenia not requiring transfusion about 11 months post-SCT; the work up revealed a slightly hypocellular bone marrow with mixed chimerism, and patient was started back on CSA. She is now 22 months post-SCT; her neutropenia resolved but the platelet count continues to be around 30–50 109/l, and her chimerism levels are stable. The last analysis was carried out 18 months post-SCT and it showed 60% donors cells in the T-lymphoid line and 82% donor cells in the myeloid line.
Discussion Allogeneic SCT has been proven to correct the hematological abnormalities in patients with FA. Low-dose CY has been used effectively in the conditioning of such patients,9–13 but the optimal dose of CY (ie the lowest dose that can ensure sustained engraftment with minimal toxicity) remains undecided. Using low-dose CY at 20 mg/kg i.v. in addition to 500 cGy thoracoabdominal irradiation (TAI), Socie´ et al10 reported a survival of 74.4% at 54 months and 58.5% at 100 months in 50 FA patients at a median follow-up of more than 6 years, acute GvHD grade XII occurred in 55% of the patients, and chronic GvHD occurred in 69.95% of the patients at risk. Similarly, Kohli-Kumar et al reported sustained engraftment in 17 of 18 FA patients who underwent allogeneic SCT using the same regimen with the addition of ATG pre- and post-SCT (median follow-up of 27 months); chronic GvHD only was reported in 16% of the patients.11 Our group reported comparable findings in 19 FA patients using the same protocol of low-dose CY, TAI, and ATG; the actuarial survival at 4 years was 66%, acute GvHD Xgrade 2 developed in three patients, and no chronic GvHD was observed.12 On the other hand,
Medeiros et al reported an actuarial survival of 88% at 37 months in 16 FA patients using only CY at 100 mg/kg; acute and chronic GvHD developed in 13 and 7% of the patients, respectively.13 Thus, the role of radiation therapy in the conditioning of FA patients remains uncertain, and alternatives should be sought especially in light of the data suggesting an increased risk of cancers after transplantation of patients with aplastic anemia who received radiation containing conditioning regimens.7,8 In this series, we demonstrated that using CY at a dose of 60 mg/kg may favorably circumvent the need to use radiation, thus decreasing potential early and late transplantation-related complications in these patients while maintaining the ability of restoring normal hematopoiesis. However, lower CY doses may be sufficient for sustained engraftment without the use of radiation; one of our patients was excluded from the analysis because she received a modified dose of CY (30 mg/kg total dose) due to a significant elevation of her bilirubin. She engrafted and continues to enjoy normal counts at 44 months post transplant; chimerism studies indicated 100% donor cells in both the myeloid and lymphoid cells. The addition of other agents such as fludarabine has also allowed further reduction of the CY doses with still favorable outcomes.15–18 The role and the optimal dose of ATG in preparing FA patients for SCT are equally unclear; however, the addition of ATG after graft infusion as an in vivo T-cell depletion method has been shown to dramatically reduce the incidence of acute GvHD, and subsequently chronic GvHD.19–21 A major concern associated with the use of ATG (pre- and post-SCT) has been the profound immune suppression, which may increase the susceptibility to EBVrelated post transplant lymphoproliferative disorders (PTLD); in our series, no such cases were observed, nor were there other secondary malignancies noted thus far. Of note is that the majority of the reported ATG-associated PTLD cases occurred with the use of ATG for the treatment of acute GvHD, frequently in the presence of other major risk factors such as transplants from unrelated donors and ex vivo T-cell depletion.22–24 In conclusion, this study provides evidence that low-dose CY without radiation therapy can be used satisfactorily in the conditioning of patients with FA undergoing related SCT; however, the use of more aggressive therapy including total body irradiation maybe warranted in patients who present with myelodysplasia and/or clonal abnormality in order to eradicate the abnormal clone.25,26
References 1 Auerbach AD, Allen RG. Leukemia and preleukemia in Fanconi anemia patients. A review of the literature and report of the international Fanconi anemia registry. Cancer Genet Cytogenet 1991; 51: 1–12. 2 Alter BP, Young NS. The bone marrow failure syndromes. In: Nathan DG, Oski FA (eds). Hematology of Infancy and Childhood, Vol 1. Saunders: Philadelphia, PA, 1998, pp 259–272. Bone Marrow Transplantation
SCT for FA patients with low-dose CY and ATG only M Ayas et al
466 3 Alter B. Fanconi’s anemia, current concepts. Am J Pediatr Hematol Oncol 1992; 14: 170–184. 4 Alter B. Fanconi’s anemia and its variability. Br J Haematol 1993; 85: 9–14. 5 Rosenberg P, Greene M, Alter B. Cancer incidence in persons with Fanconi anemia. Blood 2003; 101: 822–826. 6 Alter B, Greene M, Velazquez I, Rosenberg P. Cancer in Fanconi anemia. Bone Marrow Transplant 2003; 101: 2072–2073 (letter). 7 Socie´ G, Henry-Amar M, Cosset JM et al. Increased incidence of solid malignant tumors after bone marrow transplantation for severe aplastic anemia. Blood 1991; 74: 277–279. 8 Deeg HJ, Socie´ G, Schoch G et al. Malignancies after marrow transplantation for aplastic anemia and Fanconi anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood 1996; 87: 386–392. 9 Gluckman E. Allogeneic bone marrow transplantation in Fanconi anemia. Bone Marrow Transplant 1996; 18 (Suppl. 3): 33–35. 10 Socie´ G, Devergie A, Girinski T et al. Transplantation for Fanconi’s anaemia: long term follow-up of fifty patients transplanted from a sibling donor after low-dose cyclophosphamide and thoraco-abdominal irradiation for conditioning. Br J Haematol 1998; 103: 249–255. 11 Kohli-Kumar M, Morris C, DeLaat C et al. Bone marrow transplantation in Fanconi anemia using matched sibling donors. Blood 1994; 84: 2050–2054. 12 Ayas M, Solh H, Mustafa MM et al. Bone marrow transplantation from matched siblings in patients with Fanconi’s anemia utilizing low dose cyclophosphamide, thoracoabdominal radiation and antithymocyte globulins. Bone Marrow Transplant 2001; 27: 139–143. 13 Medeiros CR, Zanis-Neto J, Pasquini R. Bone marrow transplantation for patients with Fanconi anemia: reduced doses of cyclophosphamide without irradiation as conditioning. Bone Marrow Transplant 1999; 24: 849–852. 14 Schichman SA, Suess P, Vertino AM et al. Comparison of short tandem repeat and variable number tandem repeat genetic markers for quantitative determination of allogeneic bone marrow transplant engraftment. Bone Marrow Transplant 2002; 29: 243–248. 15 De la Fuente J, Reiss S, McCloy M et al. Non-TBI stem cell transplantation protocol for Fanconi anaemia using HLAcompatible sibling and unrelated donors. Bone Marrow Transplant 2003; 32: 653–656.
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16 McCloy M, Almeida A, Daly P et al. Fludarabine-based stem cell transplantation protocol for Fanconi’s anaemia in myelodysplastic transformation. Br J Haematol 2001; 112: 427–429. 17 De Medeiros CR, Silva LM, Pasquini R. Unrelated cord blood transplantation in a Fanconi anemia patient using fludarabinebased conditioning. Bone Marrow Transplant 2001; 28: 110–112. 18 Kapelushnik J, Or R, Slavin S, Nagler A. A fludarabine-based protocol for bone marrow transplantation in Fanconi’s anemia. Bone Marrow Transplant 1997; 20: 1109–1110. 19 Ayas M, Al-Mahr M, Al-Jefri A et al. Does adding ATG to the GVHD prophylaxis regimen help reduce its incidence? Bone Marrow Transplant 2003; 31: 311. 20 Guardiola P, Socie G, Li X et al. Acute graft-versus-host disease in patients with Fanconi anemia or acquired aplastic anemia undergoing bone marrow transplantation from HLAidentical sibling donors: risk factors and influence on outcome. Blood 2004; 103: 73–77. 21 Bacigalupo A, Oneto R, Lamparelli T et al. Pre-emptive therapy of acute graft-versus-host disease: a pilot study with antithymocyte globulin (ATG). Bone Marrow Transplant 2001; 28: 1093–1096. 22 Lynch BA, Vasef MA, Comito M et al. Effect of in vivo lymphocyte-depleting strategies on development of lymphoproliferative disorders in children post allogeneic bone marrow transplantation. Bone Marrow Transplant 2003; 32: 527–533. 23 Peggs KS, Banerjee L, Thomson K, Mackinnon S. Post transplant lymphoproliferative disorders following reduced intensity conditioning with in vivo T cell depletion. Bone Marrow Transplant 2003; 31: 725–726. 24 Micallef IN, Chhanabhai M, Gascoyne RD et al. Lymphoproliferative disorders following allogeneic bone marrow transplantation: the Vancouver experience. Bone Marrow Transplant 1998; 22: 981–987. 25 Ayas M, Al-Jefri A, Al-Mahr M et al. Allogeneic stem cell transplantation in patients with Fanconi’s anemia and myelodysplasia or leukemia utilizing low-dose cyclophosphamide and total body irradiation. Bone Marrow Transplant 2004; 33: 15–17. 26 Ikushima S, Hibi S, Todo S et al. Successful allogeneic bone marrow transplantation in a case with myelodysplastic syndrome which developed following Fanconi’s anaemia. Bone Marrow Transplant 1995; 16: 621–624.
