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©Ferrata Storti Foundation Stem Cell Transplantation • Research Paper Rabbit-immunoglobulin G levels in patients receiving thymoglobulin as part of conditioning before unrelated donor stem cell transplantation Mats Remberger Background and Objectives. The role of serum concentrations of rabbit antithymoglob- Berit Sundberg ulin (ATG) in the development of acute graft-versus-host disease (GVHD) after allogene- ic hematopoietic stem cell transplantation (HSCT) with unrelated donors is unknown. Design and Methods. We determined the serum concentration of rabbit immunoglobu- lin-G (IgG) using an enzyme linked immunosorbent assay in 61 patients after unrelat- ed donor HSCT. The doses of ATG ranged between 4 and 10 mg/kg. The conditioning consisted mainly of cyclophosphamide and total body irradiation or busulfan. Most patients received GVHD prophylaxis with cyclosporine and methotrexate. Results. The rabbit IgG levels varied widely in each dose group. The levels of rabbit IgG gradually declined and could still be detected up to five weeks after HSCT. We found a correlation between the grade of acute GVHD and the concentration of rabbit IgG in serum before the transplantation (p=0.017). Patients with serum levels of rabbit IgG >70 mg/mL before HSCT ran a very low risk of developing acute GVHD grades II-IV, as compared to those with levels <70 mg/mL (11% vs. 48%, p=0.006). Interpretations and Conclusions. The measurement of rabbit IgG levels in patients receiving ATG as prophylaxis against GVHD after HSCT may be of value in lowering the risk of severe GVHD. Key words: ATG, GVHD, BMT, thymoglobulin, rabbit-IgG. Haematologica 2005; 90:931-938 ©2005 Ferrata Storti Foundation From the Department of Clinical he outcomes of unrelated donor and rate of T-cell depletion. Thymoglob- Immunology (MR, BS) and Center hematopoietic stem cell transplan- ulin® is produced by immunizing path- for Allogeneic Stem Cell Transplantation (MR), Karolinska Ttation (HSCT) have substantially ogen-free rabbits with fresh human thy- Institutet, Karolinska University improved during the past decade. The rea- mocytes. The g immunoglobulin (Ig) frac- Hospital, SE-141 86 Stockholm, son for this is more sophisticated tissue tion contains polyclonal antibodies Sweden. typing and perhaps also better supportive against multiple cell surface antigens.5-7 care. However, graft-versus-host disease Thymoglobulin binds to T cells and, to a Correspondence: (GVHD) is a major cause of morbidity and lesser extent, to B cells, monocytes, Mats Remberger, PhD, Clinical 1,2 5-7 Immunology, F79, Karolinska mortality using unrelated donors. The macrophages and neutrophils. It also University Hospital, SE-141 86 risk of graft failure is also higher in unre- binds to platelets and red blood cells, Stockholm, Sweden. E-mail: lated donor HSCT. By adding antithymo- albeit at low levels. It produces its effect [email protected] cyte globulin (ATG) to the pre-transplant via antibody-dependent cell-mediated conditioning protocol, acute GVHD and cytotoxicity (ADCC), induction of apop- early mortality of unrelated donor HSCT tosis and down-regulation of cell surface have been reduced to levels similar to antigens.8 The elimination half-life after 1 those of matched related HSCT.3,4 Many day of treatment has been shown to be 2- different doses and types of ATG are used 3 days.9 Since 1990, we have mainly used in transplant centers. The optimal dose of four doses of thymoglobulin as part of the the various ATG preparations, as regards pre-transplant conditioning therapy in the prevention of graft failure and GVHD, unrelated donor HSCT10 and have found a is not yet fully understood. However, the dose-dependent effect of ATG on acute dose of ATG may not be the sole cause of GVHD grades II-IV and III-IV.10 Here we the level of in vivo depletion of donor T- have analyzed rabbit IgG levels in 61 cells. The distribution in the patient’s tis- patients and compared these with the sues and elimination also affect the level dose of ATG given and GVHD. haematologica/the hematology journal | 2005; 90(7) | 931 | M. Remberger et al. Design and Methods Table 1. Patients’ and donors’ characteristics for the 61 patients who received rabbit ATG (thymoglobulin) as part of their condition- ing before unrelated donor HSCT. Patients This is a study of 61 patients who received thy- Thymoglobulin moglobulin (Genzyme, Cambridge, MA, USA) as part of their conditioning before allogeneic HSCT N61 from a matched unrelated donor. The transplants Diagnosis were performed between April 1998 and June 2003 Acute myeloid leukemia 18 at the Center for Allogeneic Stem Cell Transplant- Myelodysplastic syndrome 5 Acute lymphoid leukemia 10 ation, Karolinska University Hospital, Stockholm, Chronic myeloid leukemia 11 Sweden. Most patients had a hematologic malignan- Other malignancy 5 cy. Of the 61 patients, 35 were male and 26 female Non-malignant disease 8 The median age of the series was 35 years (range 1- Solid tumor 2 61) and 14 (23%) of the patients were less than 18 Disease stage (early/late) 34/25 years old. Table 1 shows the patients’ and donors’ Recipients’ gender (M/F) 35/26 characteristics. Recipients’ age 35 (1-61) Conditioning Donors’ gender (M/F) 39/22 The conditioning regimen in 6 patients consisted of cyclophosphamide (60 mg/kg/day for 2 days), fol- Donors’ age 38 (21-54) lowed by 10 Gy single fraction total body irradiation 8 NC dose (¥10 /kg) 6.5 (1-54.5) (TBI), with the lungs shielded to receive no more than 9 Gy.11 Another 21 patients received fractionat- SC source (BM/PBSC) 28/33 ed TBI (4 Gy/day for 3 days) combined with Conditioning cyclophosphamide. As an alternative, busulfan, 4 Cy+TBI 10 Gy 6 FTBI+Cy 21 mg/kg/day for 4 days, and cyclophosphamide was Bu+Cy 25 given to 25 patients.11 Nine patients were given RIC 9 reduced conditioning with fludarabine (30 mg/m2) GVHD prophylaxis for 3-5 days combined with 2 Gy TBI (n=2), busulfan MTX+CsA 53 total dose 8 mg/kg (n=3) or cyclophosphamide total CsA+Prednisolone 3 dose 60 mg/kg (n=4).12-14 CsA+MMF 3 Rapamycin+Prograft 2 GVHD prophylaxis G-CSF post-HSCT 31 (51%) Post-transplant immunosuppression consisted of Thymoglobulin dose cyclosporine and a short course of methotrexate in 4 mg/kg 14 53 patients.15 Other protocols included cyclosporine 6 mg/kg 21 8 mg/kg 15 combined with prednisolone (n=3) or mycopheno- 10 mg/kg 11 late mofetil (n=3). The combination of rapamycin Early disease: complete remission, chronic phase 1, Late disease: > complete and prograft was used in two patients. During the remission, chronic phase 1, NC dose: nucleated cell dose, SC: stem cells; first month, blood cyclosporine levels were kept at BM: bone marrow, PBSC: peripheral blood stem cells, Cy: cyclophosphamide; TBI: total-body irradiation; FTBI: fractionated TBI; Bu: busulfan; RIC: reduced 200-300 ng/mL. After three or six months depending intensity conditioning; MTX: methotrexate; CsA: cyclosporine; on whether the patients had malignant or non-malig- MMF: mycophenolate mofetil; G-CSF: granulocyte colony-stimulating factor. nant disease, respectively, the cyclosporine dose was lowered by 25% every alternate month and discon- tinued after six (malignant) or 12-24 months (not before the first dose of thymoglobulin. In those malignant diseases) in the absence of GVHD. patients who did develop adverse events, 100-200 mg of hydrocortisone was also given. Thymoglobulin doses The total dose of thymoglobulin ranged between 4 Rabbit IgG detection and 10 mg/kg and was given at a dose of 2 mg/kg/day Samples of serum were collected before the trans- for 2-5 days (Table 1).10 The last dose of thymoglobu- plant (n=58)(after the last dose of thymoglobulin), at lin was always given the day before transplantation. one week (n=53), two weeks (n=19), three weeks Pre-treatment with 500-1000 mg of methylpred- (n=31), four weeks (n=9) and 5 weeks (n=12) after nisolone and 2 mg of i.v. anti-histamine was given to HSCT. These samples were kept at -20°C pending most patients as prophylaxis against adverse events analysis. They were then thawed and analyzed using | 932 | haematologica/the hematology journal | 2005; 90(7) Rabbit IgG levels after HSCT an enzyme-linked immunosorbent assay (ELISA). the initial response. In more severe cases, ATG, Each serum sample was tested in duplicate in three 5- methylprednisolone, methotrexate or psoralen and fold dilution steps. Samples and controls were dilut- ultraviolet A light (PUVA) was used. Chronic GVHD ed in phosphate-buffered saline containing 0.05% was initially treated with cyclosporine and steroids. If Tween 20 (Polysorbatum 20, Apoteksbolaget, no response occurred, some patients were given total Sweden) and 0.1% human AB serum. Polystyrene lymph node irradiation, PUVA or extracorporeal microtiter plates (Nunc polysorp, Denmark) were PUVA. coated overnight with 2 mg/mL swine anti-rabbit IgG antibodies (Z0400, DAKO Cytomation, Denmark) at Definitions +4°C in 0.1 M carbonate-bicarbonate buffer (pH 9.6). Bone marrow aspirates were performed and the We used rabbit serum as a positive control, human morphology analyzed 3, 6, and 12 months and then AB serum as a negative control. Thymoglubulin was yearly after HSCT. Leukemia relapse was defined as used as a standard at 1000, 100, 50, 25, 12.5 and 6 more than 5% blasts in bone marrow aspirates or the ng/mL; 100 mL of standards, controls and samples presence of extramedullar leukemia cells i.e., were added and incubated for 3 hours at room tem- extramedullar relapse. Bacteremia was defined by perature. Between the various incubation steps, the the first positive blood culture related to a febrile wells were rinsed four times with saline containing episode during the first 30 days after transplantation.
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