Graft-versus-host disease (GVHD) is a severe disorder and despite therapeutic efforts to decrease its distressing clinical manifestations, treatment is still not optimal. Here we report the results of studies, in which the purine analogue, fludarabine phosphate, was used in an attempt to modify and decrease GVHD after stem cell transplantation, across major histocompatibility barriers for murine leukemia. B-cell leukemia (BCL-1) bearing (BALB/c×C57BL/6) F1 mice received two cycles of fludarabine (0.8 mg/kg) for 5 days every 2 weeks, followed by 400 mg/kg cyclophosphamide i.p. Animals were then transplanted with C57BL/6 precursor cells and the development of leukemia and extent of GVHD was monitored both clinically and histopathologically. In the fludarabine-treated group, only nine of 28 (32%) mice developed leukemia, compared to 25 of 33 (76%) of control animals (P=0.0006 ). Mice treated with fludarabine-containing regimens prior to transplantation also had much less GVHD both clinically and at autopsy, while graft-versus-leukemia appeared to be augmented in the same animals.
Fludarabine used as a single agent or in combination with other drugs has been extensively used in recent years for the treatment of a variety of hemato-oncologic disorders, and has been shown to be most effective in indolent lymphoproliferative disorders, particularly chronic lymphocytic leukemia (CLL) and follicular lymphoma.1,2,3 Furthermore, fludarabine-containing combination regimens have also been used in the treatment of aggressive lymphomas as well as in acute leukemia.4,5,6,7, Most recently, it has been incorporated into nonmyeloablative cytoreduction regimens and used as for immunosuppression in the novel mini-allogeneic transplant procedure used in recent years for a variety of hematological disorders.8,9,10 In fact, fludarabine is the anchor drug in these regimens. The cytotoxic potential of fludarabine against T lymphocytes, coupled with its immunosuppressive qualities, has made it an attractive choice for both chemotherapy and immunomodulation, during allogeneic transplantation for leukemia.11,12 Indeed, the use of fludarabine-containing regimens has altered the incidence, and the degree of graft-versus-host disease (GVHD) in these patients and this regimen is used currently in a number of transplant centers.8,9,10,11,12,13
Despite the impressive results in human hematopoietic stem cell transplantation, fludarabine has as yet not been adequately tested in animal models. In this respect, there is some recent experimental evidence showing that it may be effective in transplantation across histocompatibility barriers in mice.14 Furthermore, it is also able to induce bilateral tolerance or stable chimerism after marrow or skin transplantation,15 thereby altering the incidence of graft rejection and the extent of GVHD encountered. In earlier pilot murine studies, we were also able to show an anti-GVHD prophylactic effect after marrow transplantation in mice.16
In the present study, we report the results of a series of experiments using the combination of fludarabine and cyclophosphamide as cytoreduction during allogeneic stem cell transplantation for murine leukemia. In these studies, we attempted to mimic human leukemias clinically and determine whether fludarabine could decrease the intensity and incidence of acute GVHD, while preserving or even enhancing the simultaneous graft-versus-leukemia (GVL) effect in this experimental model.
Material and methods
All animal procedures utilized in the present study were approved by the Institutional Committee for Animal Experimentation.
Male and female mice 2–6 months old (BALB/c×C57BL/6) F1 (H-2d/b) were transplanted with C57BL/6 (H-2b), bone marrow or spleen cells. The mice purchased from the Harlan Breeding Facility (Jerusalem, Israel) were kept in a standard animal facility with top-filtered cages. Cages, sawdust and water bottles were autoclaved once a week. Neomycin sulfate, at a concentration of 0.5%, was given in the drinking water for 2 weeks post-transplantation. Fludarabine monophosphate (Schering, Berlin, Germany) and cyclophosphamide (ASTA medica Ig, Frankfurt/Main, Germany) were given intraperitoneally.
Stem cell transplantation
Bone marrow cells were prepared by flushing RPMI medium through the femora and humeri of donors with a 9–25-gauge needle. Spleens were removed aseptically from donor mice, teased through a nylon mesh into RPMI 1640 medium (GIBCO, Grand Island Biological Co., Grand Island, NY, USA) and washed twice. Spleen cells (20×106 per recipient mouse) were injected intravenously into the lateral tail vein.
The t-test was used for comparison between fludarabine-treated and control animals.
Murine B-cell leukemia (BCL-1)
BCL-1, as previously described in BALB/c female mice,17 was maintained in vivo by intravenous (i.v.) passage of 106–107 peripheral blood lymphocytes (PBLs) obtained from tumor-bearing mice. All untreated recipients of BCL-1 cells (⩾100 cells) consistently develop splenomegaly followed by marked lymphocytosis in the peripheral blood and all animals die from leukemia.17 PBL counts of all experimental groups were carried out weekly. Clinical onset of leukemia was defined as PBL counts exceeding 20 000/mm3.
Monitoring of GVHD
Mice were observed daily for survival and clinical signs of GVHD, manifested as diarrhoea, weight loss and ruffled skin.
Basically, three different protocols were used.
Experiment 1: This attempted to assess the effect of the fludarabine-containing regimens on BCL-1 leukemia in F-1 mice without transplant.
Mice were injected with 104 BCL-1 leukemia cells and 1 day later received 3 doses of fludarabine (0.3, 0.5, or 0.7 mg/kg) i.p. for 3 days. A day after the administration of fludarabine, mice also received 100 mg/kg cyclophos-phamide. The mice were followed for 2 months and the incidence of leukemia and survival were recorded.
Experiment 2: Designed to assess the effect of fludara-bine followed by cyclophosphamide and stem cell transplantation given as treatment for BCL1 1eukemia in F-1 mice. This protocol attempted to mimic the clinical situation of partially responsive disease in humans with leukemia where therapy is followed by allogeneic stem cell transplantation.
F-1 mice received 105 BCL-1 leukemia cells and 1 day later 0.8 mg/kg fludarabine was given for 5 consecutive days. After 2 weeks, the mice received another 5 days cycle of 0.8 mg/kg fludarabine, followed by 400 mg/kg i.p. cyclophosphamide 2 weeks later. A day after the above treatment with cyclophosphamide, animals were transplanted with 10×106 C57BL/6 bone marrow cells and 2×106 spleen cells. Control animals received saline instead of fludarabine. The experiments performed with this schedule were repeated on four separate occasions.
Experiment 3: This experiment tried to evaluate the effect of fludarabine and allogeneic transplantation compared to syngeneic transplantation in leukemic mice. F-1 mice were treated with 0.8 mg/kg fludarabine or saline twice a week for 2 weeks. The mice were injected with 400 mg/kg cyclo i.p. 10 days later. The mice were transplanted with 20×106 C57BL spleen cells or F-1 spleen cells 24 h later.
The effect of fludarabine on GVHD across major histocompatibility (MHC) barriers was assessed in all these experiments.
Experiment 1: Four of the 10 mice receiving 0.3 and 0.5 mg/kg fludarabine and cyclophosphamide survived more than 2 months. Mice that received 0.5 mg/kg fludarabine only developed leukemia within 45 days. Two of the five mice treated with cyclophosphamide only survived more than 2 months. In the group of six mice receiving 0.7 mg/kg fludarabine and the same dose of cyclophosphamide, three (50%) survived, all control mice developed leukemia within 34 days (Figure 1).
Experiment 2: Figure 2 summarizes the results of four different experiments. A total of 25 out of 33 (76%) control animals treated with saline and transplanted with allogeneic stem cells (C57) developed leukemia and died within 60 days, while in the fludarabine-treated group only nine out of 28 mice (32%) had leukemia within this period (P=0.006).
In 12 surviving mice treated with fludarabine regimens, autopsy was performed. In these animals without clinical evidence of leukemia, histopathology revealed that one had obvious leukemia in the spleen and liver, four had minor minimal residual disease, while seven of 12 had no signs of leukemia at all. GVHD was either totally absent or minor in the examined group (Figures 3, 4 and 5.
Of the 33 control mice, only eight had no clinical evidence of leukemia. Six of these underwent autopsy, which showed that five had obvious leukemia and one had tumorous extramedullary hematopoiesis. Five of the six controls had GVHD.
Experiment 3: All six control mice without any conditioning regimens developed leukemia within 19–31 days. In addition, nine out of 10 mice (90%) transplanted with syngeneic (F1) spleen cells post-fludarabine treatment developed leukemia within 22–59 days. However, after allogeneic transplantation with C57 spleen cells (post-fludarabine treatment and Cy) only three of the 11 (27.3%) mice developed leukemia (Figure 6) (P=0.0034).
The results of the above studies provide the first experimental data in a murine leukemia model, which support the human observations recorded in leukemic patients after allotransplantation using fludarabine-containing regimens. Clinical data in humans have shown that fludarabine in combination with other agents is highly effective as cytoreductive treatment for lymphoid leukemias, and can also cause host immunomodulation as well as sufficient immunosuppression for successful allotransplan-tation.15,18,19 Indeed fludarabine has already become accepted as the anchor drug in these combination regimens for successful non-myeloablative stem cell transplantation in leukemias, lymphoma and in other malignancies.19,20,21,22 Despite the successful use of fludarabine in humans, until recently there is very little experimental evidence-based available data in animals that can be used as a parallel for the successful experience in human leukemia and transplantation.
In the studies reported here, fludarabine used in combination with cyclophosphamide achieved longer survival in allotransplanted mice with lymphocytic leukemia compared to syngeneic-transplanted animals or controls transplanted without fludarabine. In addition, these experimental animals also had less GVHD while the GVL effect was still maintained. These findings support our earlier observations with nonleukemic mice, showing that flu-darabine impressively altered the incidence of GVHD transplanted across MHC barriers.16 These earlier studies also showed that fludarabine can produce bilateral tolerance and stable chimerism after transplantation.
To the best of our knowledge, this experience is the only murine experimental data available providing information on the successful use of fludarabine, combined with cyclophosphamide for allogeneic transplant of murine leukemia. Most recently, Petrus et al14 have shown that a fludarabine-containing regimen prevents mismatched murine marrow graft rejection, while Luznik et al23 have also shown convincingly that a fludarabine-containing regimen can result in durable engraftment in incompatible murine donor transplants.
GVHD and its sequelae still remain a major complication in allotransplantation and the results of our study are indeed encouraging. The results of the present study and those of our earlier experiments16 imply that effective and successful immunomodulation and immunosuppression can be achieved in allogeneic transplantation when fludarabine is used together with cyclophosphamide. These properties of fludarabine have already been successfully exploited not only in the treatment of indolent lymphocytic leukemias/lymphomas, but also as a major part of the nonmyeloablative conditioning regimen used for stem cell transplant in humans. After using fludarabine-containing regimens, a potential state of host-versus-graft tolerance is achieved with the induction of partial chimerism. The latter serves as a basis for more successful engraftment, more effective GVL and less clinical and histopathological manifestations of GVHD.13,19,20,21,22 Experimental data certainly justify the use of fludarabine-containing regimens in human transplantation.
Sorensen JM, Vena DA, Fallavollita A et al. Treatment of refractory chronic lymphocytic leukemia with fludarabine phosphate via the group C protocol mechanism of the National Cancer Institute: five year follow-up report. J Clin Oncol 1997; 15: 458–465.
Foran JM, Oscier D, Orchard J et al. Pharamacokinetic study of single doses of oral fludarabine phosphate in patients with “low-grade” non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia. J Clin Oncol 1999; 17: 1574–1579.
Johnson S, Smith AG, Loffler H et al. Multicentre prospective randomised trial of fludarabine versus cyclophosphamide doxorubicin, and prednisone (CAP) for treatment of advanced stage chronic lymphocyte leukemia The French Cooperative Group on CLL. Lancet 1996; 347: 1432–1438.
Ghandi V, Estey E, Keating MJ, Plunkett W . Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia during therapy. J Clin Oncol 1993; 11: 116–124.
Ghandi V, Plunkett W, Weller S et al. Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics in leukemia cells. J Clin Oncol 2001; 19: 2142–2152.
Yalman N, Sarper N, Devecioglu O et al. Fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) for the treatment of relapsed or poor risk childhood acute leukemia. Turk J Pediatr 2000; 42: 198–204.
Koller CA, Kantarjian HM, Feldman EJ et al. A phase I–II trial of escalating doses of mitoxantrone with fixed doses of cytarabine plus fludarabine as salvage therapy for patients with acute leukemia and the blastic phase of chronic myelogenous leukemia. Cancer 1999; 86: 2246–2251.
Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998; 91: 756–763.
Aversa F, Tabilio A, Velardi A et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. New Engl J Med 1998; 339: 1186–1193.
Borhnauser M, Thiede C, Schuler U et al. Dose-reduced conditioning for allogeneic blood stem transplantation: durable engraftment without antithymocyte globulin. Bone Marrow Transplant 2000; 26: 119–125.
Priebe T, Platsoucas CD, Seki H et al. Purine nucleoside modulation of functions of human lymphocytes. Cell Immunol 1990; 129: 321–328.
Chun HG, Leyland-Jones B, Cheson BD . Fludarabine phosphate: a synthetic purine antimetabolite with significant activity against lymphoid malignancies. J Clin Oncol 1991; 9: 175–188.
Khouri IF, Keating M, Korbling M et al. Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. J Clin Oncol 1998; 16: 2817–2824.
Petrus MJ, Williams JF, Eckhaus MA et al. An immuno-ablative regimen of fludarabine and cyclophosphamide prevents fully MHC-mismatched murine marrow graft rejection independent of GVHD. Biol Blood Marrow Transplant 2000; 6: 182–189.
Goodman ER, Fiedor PS, Fein S et al. Fludarabine phosphate: a DNA synthesis inhibitor with potent immunosuppressive activity and minimal clinical toxicity. Am Surg 1996; 62: 435–442.
Or R, Weiss L, Amir G et al. The prophylactic potential of fludarabine monophosphate in graft-versus-host disease after bone marrow transplantation in murine models. Bone Marrow Transplant 2000; 25: 263–266.
Slavin S, Strober S . Spontaneous murine B-cell leukemia. Nature 1978; 272: 624–626.
Briz M, Cabrera R, Sanjuan I et al. Diagnosis of transfusion-associated graft-versus-host disease by polymerase chain reaction in fludarabine-treated B-chronic lymphocytic leukemia. Br J Haematol 1995; 91: 409–411.
Wijermans PW, Gerrits WB, Haak HL . Severe immunodeficiency in patients treated with fludarabine monophosphate. Eur J Haematol 1993; 50: 292–296.
Nagler A, Slavin S, Varadi G et al. Allogeneic peripheral blood stem cell transplantation using a fludarabine-based low intensity conditioning regimen for malignant lymphoma. Bone Marrow Transplant 2000; 25: 1021–1028.
Childs R, Chernoff A, Contentin N et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral blood stem-cell transplantation. New Engl J Med 2000; 343: 750–758.
Wasch R, Reisser S, Hahn J et al. Rapid achievement of complete donor chimerism and low regimen-related toxicity after reduced conditioning with fludarabine, carmustine, melphalan and allogeneic transplantation. Bone Marrow Transplant 2000; 26: 243–250.
Luznik L, Jalla S, Engstrom LW et al. Durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide. Blood 2001; 98: 3456–3464.
About this article
Cite this article
Weiss, L., Abdul-Hai, A., Or, R. et al. Fludarabine in combination with cyclophosphamide decreases incidence of GVHD and maintains effective graft-versus-leukemia effect after allogeneic stem cell transplantation in murine lymphocytic leukemia. Bone Marrow Transplant 31, 11–15 (2003) doi:10.1038/sj.bmt.1703775
- graft-versus-host disease incidence
- hematopoietic stem cell transplant
- murine lymphocytic leukemia
Fludarabine as a cost-effective adjuvant to enhance engraftment of human normal and malignant hematopoiesis in immunodeficient mice
Scientific Reports (2018)
Molecular Therapy (2018)
Comparable outcomes between younger (⩽40 years) and older (>40 years) adult patients with severe aplastic anemia after HLA-matched sibling stem cell transplantation using fludarabine-based conditioning
Bone Marrow Transplantation (2016)
Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control
Biology of Blood and Marrow Transplantation (2013)
Reduced intensity conditioning for hematopoietic stem cell transplantation: has it achieved all it set out to?