Use of a five-agent GVHD prevention regimen in recipients of unrelated donor marrow

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1 Bone Marrow Transplantation, (1999) 23, Stockton Press All rights reserved /99 $ Use of a five-agent GVHD prevention regimen in recipients of unrelated donor marrow AR Zander 1, T Zabelina 2, N Kröger 1, H Renges 1, W Krüger 1,C.Löliger 3,MDürken 4, M Stockschläder 5, M de Wit 6, G Wacker-Backhaus 6, S Bielack 4, N Jaburg 1,BRüssmann 1, R Erttmann 4 and H Kabisch 4 1 Bone Marrow Transplantation Center, 3 Department of Transfusion Medicine, 4 Department of Pediatric Oncology, 6 Department of Oncology/Hematology, University Hospital Eppendorf, Hamburg, Germany; 2 Department of Bone Marrow Transplantation, Pavlow University, St Petersburg, Russia; and 5 Department of Hematology, University Hospital Freiburg Summary: A five-agent GVHD prophylaxis programme consisting of cyclosporin A, methotrexate, anti-thymocyte-globulin, pentaglobin and metronidazol was given to 48 recipients of unrelated donor marrow with chronic myelogenous leukemia, acute leukemia, myelodysplastic syndromes, and familiar lymphocytic hemophagocytosis of an average age of 33.5 (0.6 56) years. GVHD grades II IV occurred in 18 patients (39%) and grades III IV in five patients (11%). Chronic GVHD developed in nine patients (23%), three limited and six extensive. Fifteen patients died. Clinical relapse was detected in eight patients. Four patients died as a consequence of the underlying disease and subsequent treatment, 11 patients died of transplant-related causes. After a median follow-up of 19 months, the overall and diseasefree survival are 67% and 62%, respectively. Survival by age is as follows: 0 19 years: 12/13 patients; years: 14/25 patients; years: 7/10 patients. The five-agent GVHD prophylaxis regimen is effective. Matched-unrelated donor transplants can be carried out safely in patients younger than 50 years of age. The results in patients younger than 20 years of age should encourage matched-unrelated donor transplants at earlier stages of the disease. Keywords: unrelated donor; anti-thymocyte-globulin; GVHD prevention Allogeneic bone marrow transplantation from an HLAidentical sibling has been established as a standard method for treatment of hematological malignancies and severe aplastic anaemias. 1,2 The use of matched-unrelated donors for bone marrow transplantation in these diseases has resulted in lower survival rates than those expected for HLA-identical siblings BMT. 3 7 An increased incidence of acute and chronic GVHD has been the major problem with matchedunrelated donor transplantation for hematological diseases, particularly in older patients We added three agents Correspondence: Prof AR Zander, Bone Marrow Tranplantation Center, University of Hamburg, Martinistraße 52, D Hamburg, Germany Received 12 September 1998; accepted 3 December 1998 which have been shown to contribute to GVHD control by different mechanisms to the standard GVHD prophylaxis with cyclosporin A and methotrexate. 12 Anti-thymocyteglobulin given prior to transplant has been shown to modify GVHD in recipients of marrow from unrelated donors. 13 Metronidazol, an antibiotic, highly effective against anaerobic bacteria, has been shown in a randomized study to ameliorate acute GVHD. 14 A previous retrospective study showed that colonization with anaerobic bacteria was highly associated with incidence and severity of acute GVHD. 15 Pentaglobin, an immunoglobulin preparation with an enriched IgM fraction has been studied in allogeneic bone marrow transplantation and has a moderate effect on acute GVHD and liver toxicity Recent studies have shown that IgM-containing immunoglobulin fractions have a significantly higher modulatory effect on lymphocyte proliferation and cytokine release during alloimmune response in vitro than do IgG-enriched preparations. 19,20 We describe the effect of these five drugs on occurrence of acute and chronic GVHD and survival in recipients of allogeneic blood and marrow transplantation. Materials and methods Study patients From February 1995 to January 1998, 48 patients with hematological malignancies received bone marrow (45) or blood stem cell transplants (three) from an unrelated donor. Patient and donor characteristics are summarized in Table 1. Standard risk were patients with AML, MDS, ALL in CR, CML in first chronic phase, and familiar lymphocytic hemophagocytosis without active disease. All others were considered high risk. Patients gave written informed consent. Evaluation was based on data available until 31 March HLA typing and donor matching HLA-A and -B antigens were typed by serologic methods, and HLA-DRB1 alleles were typed with sequence-specific oligonucleotide probes. 8,21 Donors were required to match the recipient for the serologically defined HLA-A and -B antigens as well as HLA-DRB1 alleles. A single minor

2 890 Table 1 Characteristics of 48 patients who received a hemopoietic stem cell transplant from an unrelated donor Age Patient 33.5 (0.6 56) donor 32 (19 50) Sex of patient and donor male/male 25 female/male 8 male/female 6 female/female 9 HLA-matching status matched for HLA-A, -B, DRB1 44 minor mismatch HLA-A 2 minor mismatch HLA-DRB1 2 Type of donor stem cell transplant bone marrow 45 peripheral blood 3 No. of mononuclear marrow cells transplanted ( 10 8 per kilogram of recipient s body weight) median 1.5 range No of CD34-positive cells (n = 45) ( 10 6 per kilogram of recipient s body weight) median 4.0 range CMV status of patient and donor seronegative/seronegative 25 seronegative/seropositive 6 seropositive/seronegative 8 seropositive/seropositive 9 Diagnosis chronic myelogenous leukemia 28 acute myelogenous leukemia 6 myelodysplastic syndrome 3 acute lymphoblastic leukemia 5 familiar lymphocytic hemophagocytosis 6 standard risk 25 high risk 23 mismatch at HLA-A (two patients) and HLA-DRB1 (two patients) was allowed. Pretransplantation lymphocyte cross-matches with patient sera and donor cells were negative in all cases. kilogram over 12 h on days 3, 2, 1. Pentaglobin (250 mg per kilogram i. v.) was given on days 1, 7, 14, 21 and 28. Metronidazol (Clont, Bayer, Leverkusen, Germany) at a dose of 400 mg i.v. was given three times per day from conditioning until discharge. Patients were nursed in private rooms with hepa-filtered air. Antibiotic prophylaxis was with Ofloxacin and antifungal prophylaxis with fluconazole. Acyclovir was given as herpes zoster prophylaxis. Pneumocystis prophylaxis consisted of oral or i.v. trimethoprim and sulfamethoxazol (Bactrim; Roche Grenzach-Wyhlen, Switzerland) given on 3 consecutive days each week, or inhalation with pentamidine. Only blood products from CMV-negative donors were given. Weekly monitoring of blood and urine for CMV antigen by PCR and short-term culture was carried out. In cases of repeated positivity ganciclovir treatment was initiated. Prostaglandin E1 (Prostavasine; Schwarz Pharma, Mannheim, Germany) at a dose of 500 g was given daily as a continuous infusion for patients with hepatic toxicity when the total bilirubin rose above 2.0 to prevent venoocclusive disease of the liver. 24 Engraftment Standard definitions were used, ie granulocytes 500 mm 3 for at least 3 consecutive days, and platelets Presence of donor cells was documented by polymerized chain reaction 5 and chromosome analysis by chromosomespecific probes in cases of sex mismatched transplants. Graft-versus-host disease Standard criteria were used for acute and chronic GVHD. 25 Probability of clinically extensive chronic GVHD was evaluated in patients who survived for at least 80 days with sustained engraftment, and was calculated on the population alive at that time point. Conditioning regimens Thirty-six patients received conditioning with total body irradiation, 1200 cgy given over 3 days in six fractions, followed by cyclophosphamide (120 mg/kg) with or without VP-16 (30 45 mg/kg). Twelve patients received conditioning regimens without TBI, consisting of busulfan (16 mg/kg) and cyclophosphamide and/or VP ,23 Supportive care Donor marrow or blood stem cells were not T cell depleted. GVHD prophylaxis consisted of cyclosporine (3 mg per kilogram given from day 1 up to 6 months post transplant). The cyclosporine dose was adjusted to cyclosporine levels. Prior to discharge from the hospital, patients were switched from i.v. cyclosporine to oral cyclosporine (10 mg/kg). Cyclosporine was tapered from day 84 and discontinued at day Methotrexate (10 mg/m 2 ) was given days 1, 3 and 6 post-transplant. Anti-thymocyte-globulin (Fresenius, Bad Homburg, Germany) was given at a dose of 30 mg per Relapse Relapse of the underlying disease was established by cytology of blood and marrow and by chromosome analysis. Statistical analysis Statistical analysis was performed with the WIN-STATsoftware (Kalmia, Cambridge, MA, USA). Survival curves for each event (disease-free survival and overall survival) were estimated by the Kaplan Meier method. 26 For comparisons the log-rank test was used. A P value of 0.05 was considered significant. Results One patient had primary graft failure, and was retransplanted 51 days after the first transplant from the same donor, unsuccessfully; one patient died without engraftment

3 prior to day 9. Leukocytes 1000 were reached at an average of 16 days (range 12 24); platelets were reached at an average of 23 days, ranging from 15 to 111 days. The number of CD34 cells per kilogram body weight correlated with platelet engraftment (P = 0.01), but not with leukocyte engraftment (P = 0.21); the number of mononuclear cells in the graft correlated with leukocyte engraftment (P = ), not with platelet engraftment (P = 0.27). Transplant-related toxicity and side-effects of ATG Fever occurred in 36 patients (75%), exanthema in six (12.5%), broncho-spasm in one (2%), anaphylactic shock in one (2%), and serum-sickness between 2 and 4 weeks post administration of ATG in three (6.2%). Graft-versus-host disease grades II IV occurred in 18 patients (39%) and grades III and IV in five patients (11%). Chronic GVHD developed in nine patients (23%), three limited (7.5%) and six extensive (15%). Probability Figure 1 n = 48 67% OS (months) Overall survival of all 48 patients in months. ried out beyond 2 years post first diagnosis, survival was 34% (P = 0.007). 891 Relapse Clinical relapse was detected in eight patients, a median of 318 days post transplant (range days). Causes of death Fifteen (31%) patients died. Four patients died as a consequence of the underlying disease and subsequent treatment, 11 patients died of treatment-related causes: bleeding and toxicity: two; GVHD: two; fungal: three; CMV: three; bacterial infection: one. Four of the seven patients who died of infection had concomitant GVHD. Thirteen (27%) patients had cytomegalovirus infections. One out of 25 patients in the low-risk CMV group and 12 of 23 (52%) of patients who were at high risk for developing CMV, developed CMV infections. A total of 17 bacterial infections, six fungal infections and 17 viral infections in the herpes group were diagnosed before discharge. One patient developed pulmonary tuberculosis 3 months post transplant. Bilirubin elevation above 2.0 occurred in 23 patients (48%) and was treated with Prostavasine. Mild VOD occurred in 11 (22.9%), moderate VOD in 12 (25%) of patients. No life-threatening VODs occurred. Hemorrhagic cystitis occurred in two patients, and severe hemolysis in three patients. Survival After a median follow-up of 19 months (range 2 38 months), the overall survival at 3 years is 67% (Figure 1). Disease-free survival is 62%; survival-by-two-decades shows a survival of 12/13 20, 14/25 from and 7/10 40 years of age. Twenty-eight patients were transplanted for CML, 22 in chronic phase, six beyond first chronic phase. The overall survival at 3 years was 66% and the disease-free survival 62%. If bone marrow transplantation was carried out within 2 years of first diagnosis, survival (n = 16) was 88%; if bone marrow transplantation was car- Discussion This study shows that five-agent graft-versus-host disease prophylaxis with cyclosporine, methotrexate, anti-thymocyte-globulin, pentaglobin and metronidazol is well tolerated. Most patients who received anti-thymocyte-globulin, had side-effects which could be managed with corticosteroids and histamine antagonists. The incidence of grades II IV acute GVHD of 39% and of grades III IV of 11% compares favorably with the observed 77% seen in matcheddonor recipient pairs by Hansen et al 9 and 35% of matcheddonor recipient pairs, respectively. Chronic GVHD (23%) with only six (15%) of patients having extensive chronic GVHD is surprisingly low, compared with the 67% seen by Hansen et al, 9 53% by McGlave et al 7 and 62% by Szydlo et al. 27 Acute and chronic GVHD disease remain the major causes of morbidity and mortality after allogeneic bone marrow transplantation. GVHD is largely mediated by the immunocompetent T lymphocytes present in the donor bone marrow. Despite prophylaxis with cyclosporine and methotrexate, 35 65% of patients receiving allogeneic bone marrow or blood stem cell grafts from an HLA-identical sibling develop acute GVHD, and 30 40% chronic GVHD. The incidence of acute and chronic GVHD is increased in recipients of matched-unrelated donor marrow transplants. 28 T cell depletion of donor marrow has been carried out successfully to decrease GVHD, but was unsuccessful in improving survival Anti-thymocyte-globulin has little effect on the treatment of established GVHD, and has been successfully introduced in combination with cyclophosphamide as conditioning for allogeneic transplants for patients with severe aplastic anemia. An observed effect beyond facilitation of engraftment was a decrease in acute and chronic GVHD Preliminary evidence shows that the use of ATG prior to bone marrow transplant might favorably influence the occurrence of acute and chronic GVHD in disease entities other than severe aplastic anemia, too. 13 Pharmacokinetic studies of ATG have shown that

4 892 rabbit serum can be documented 4 5 weeks post administration in the recipient, and that in the first 4 5 days post administration high enough levels are in circulation to suppress in vitro PHA-response. 36 It is therefore likely that ATG leads to an in vivo T cell purging of the donor bone marrow. An influence of the gastrointestinal flora on the occurrence and incidence of acute GVHD had been established for many years in pre-clinical models. 37 Non-absorbable and intravenous antibiotics have been incorporated into transplant regimens to decrease transplant-related mortality and GVHD. A retrograde analysis by Beelen et al 14,15 on the gastrointestinal flora of allogeneic recipients showed that anaerobic bacteria were the agents associated with acute GVHD in man rather than gram-negative bacteria. A prospective randomized study evaluating metronidazole in recipients of allogeneic bone marrow revealed a significant decrease in acute GVHD and a trend towards improved survival. Immunoglobulins have been used widely to decrease the incidence of CMV infections. Passive immunotherapy with intravenous immunoglobulins decreases not only incidence of interstitial pneumonia and the severity of infections but also the risk of acute GVHD. 38 Bacterial endotoxins can activate cells of the monocyte macrophage system and lead to subsequent release of interleukin-1 and tumor-necrosis factor (TNF), known to enhance T cell proliferation. Endotoxin lipopolysaccharide in the cell wall of gram-negative bacteria can enter the circulation after disruption of the mucosal barrier, and can accumulate in the blood in cases of impaired hepatic clearance. Administration of pentaglobin, a preparation that contains 3.8 g/dl IgG, but is also enriched for IgM 0.6 g/dl and IgA 0.6 g/dl, has been given in a randomized study to recipients of allogeneic bone marrow. The treated group showed a significantly decreased peak of endotoxemia, and a decrease in pyrexias. 18 Pentaglobin alone had been administered as a single agent for the treatment of early GVHD to 10 patients with a modest improvement in five. 16 Larger studies have not been carried out with this agent. In view of studies by Nachbaur et al 19,20 on the in vitro immuno-modulatory activity of immunoglobulins it appears likely that this might be the mechanism by which an anti GVHD effect can be achieved. Using Pentaglobin the authors demonstrated that proliferation in the mixed-lymphocyte reaction was highly suppressed by IgMcontaining Pentaglobin fractions. Equivalent suppression of MLR using 7S IVIG was achieved only at a 10-fold higher concentration, than was the case with the Pentaglobin solution. Cytokine analysis and culture supernatants provide evidence that Pentaglobin leads to a down regulation of interleukin-2, interferon-, and monocyte-derived tumornecrosis factor, as well as interleukin-6 release. 19,20 In summary, five-agent GVHD prophylaxis with cyclosporine, methotrexate, anti-thymocyte-globulin, Pentaglobin and metronidazol is an effective regimen. Whether all five components are essential for this effect cannot be stated with certainty. Our data on GVHD prevention and survival show that matched-unrelated donor transplants can be carried out safely in a patient population younger than 50 years of age and probably beyond that. Matched-unrelated donor transplants should therefore be carried out for hematological diseases with a poor prognosis, such as AML in first remission with poor-risk factors, MDS with poor-risk factors, and CML in chronic phase, if no HLA-identical family donor is available. The results in patients 20 years of age should encourage matched-unrelated donor transplant at earlier stages of the disease. Further attempts will be made to prevent CMV infection in high-risk patients where 52% (12/23) developed CMV infection and three died. This high-risk group will in the future be treated pre-emptively with ganciclovir. References 1 Thomas ED, Storb R, Clift RA et al. Bone-marrow transplantation. New Engl J Med 1975; 292: Goldman JM, Apperley JF, Jones L et al. Bone marrow transplantation for patients with chronic myeloid leukemia. New Engl J Med 1986; 104: Beatty PG, Clift RA, Mickelson EM et al. Marrow transplantation from related donors other than HLA-identical siblings. New Engl J Med 1985; 313: Anasetti C, Petersdorf EW, Martin PJ et al. Marrow transplantation from unrelated volunteer donors. In: Buckner CD, Clift RA (eds). Technical and Biological Components of Marrow Transplantation. Cancer Treatment and Research no. 76. 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