Busulfan/cyclophosphamide in volunteer unrelated donor (VUD) BMT: excellent feasibility and low incidence of treatment-related toxicity

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1 Bone Marrow Transplantation, (1997) 19, Stockton Press All rights reserved /97 $12.00 Busulfan/cyclophosphamide in volunteer unrelated donor (VUD) BMT: excellent feasibility and low incidence of treatment-related toxicity H Bertz, K Potthoff, R Mertelsmann and J Finke Department of Hematology/Oncology, Freiburg University Medical Center, Freiburg, Germany Summary: described in 1980 by Hansen et al. 1 Due to the rapidly increasing number of HLA-typed volunteers with a present An increasing number of volunteer unrelated donor number of more than 3.5 million potential donors world bone marrow transplantations (VUD-BMT) are perfor- wide in the donor registries, VUD transplants are performed med every year for hematological malignancies due to frequently. However, transplant-related toxicity (TRM) is the availability of a large donor pool. Here we show the presently high in VUD BMT as compared to matched results of 36 VUD transplants from our institution using sibling donor BMT. Predominant problems include a chemotherapy-only conditioning regimen comprising engraftment failure, acute and chronic graft-versus-host disease busulfan 4 4 mg/kg and cyclophosphamide 2 60 (GVHD) and opportunistic infections. Total body mg/kg. All patients received heparin 200 IU/kg bw con- irradiation (TBI) in combination with cyclophosphamide is tinuous i.v. infusion starting the day before conditioning regarded to be the most efficient immunosuppressive regi- until day +30. Thirty-four of 36 patients (94%) men for inducing tolerance for engraftment. 2 Disadvantages engrafted and no secondary graft failure was observed. of radiation-containing regimens include ARDS or long- The two non-engraftments occurred in patients with term pulmonary dysfunction, cataracts and second malignancies CML in blast crisis with extensive myelofibrosis. All 34 in addition to difficulties due to rigid time sched- engrafted patients (100%) were in complete remission ules of the therapy. Chemotherapy-only regimens have on day +30 as shown by bone marrow biopsy and cytogenetic been developed to achieve more flexibility in allogeneic examinations. No life-threatening treatment- BMT and busulfan has been introduced as a substitute for related morbidity or mortality (TRM) were observed, TBI by Santos and Tutschka. 3,4 Busulfan is an alkylating in particular, no severe veno-occlusive disease (VOD) of agent with high activity against progenitor cells and has the liver and no fatal pulmonary complication. Use of excellent penetration into the CNS. 5,6 Presently it is only G-CSF significantly shortened the time of neutropenia available in tablet form and because of its variation in by 5 days. GVHD prophylaxis consisted of bioavailability 7 it was thought to have lower immuno- CsA/methylprednisolone with or without MTX. Acute suppressive activity. 8 However, in combination with GVHD grade II IV was observed in 18/34 patients cyclophosphamide or VP16 it has been shown to be highly (53%) and cgvhd in 12/27 patients (45%), who sur- active in autologous and allogeneic transplantation. 6,9 11 vived to day In seven patients (four with HLA Randomized and comparative studies have demonstrated class I or II mismatch) anti-t-lymphocyte globulin results as good as those seen with TBI/cyclophosphamide (ATG) was added for acute GVHD prophylaxis. One of in allogeneic sibling BMT for AML, CML and MDS 4,12 15 seven had agvhd grade II and none developed grade except for two randomized trials in patients with good risk III to IV GVHD or graft failure. We conclude that hematological disease which showed better results with Bu/CY is a feasible, save and sufficiently immunosup- TBI. 16,17 Only few data are available about Bu/CY in VUDpressive regimen for VUD transplantation. Severe acute BMT with only small patient numbers Previously, the GVHD might be avoided by additional use of ATG in main toxicity of busulfan involved the liver, and resulted GVHD prophylaxis. in a high incidence of fatal veno-occlusive disease (VOD) Keywords: busulfan/cyclophosphamide; VUD-BMT; and pneumonitis. Since the introduction of prophylactic mismatch transplants; feasibility; ATG heparin severe VOD is avoidable. 21 Here we present our single center experience of 36 consecutive adult patients who underwent VUD-BMT after Bu/CY conditioning. Stable engraftment and lack of severe Allogeneic bone marrow transplantation with volunteer pulmonary or liver toxicity resulted. unrelated donor (VUD)-derived bone marrow was first Correspondence: Dr J Finke, Department of Hematology/Oncology, Freiburg University Medical Center, Hugstetterstr. 55, D Freiburg, Germany Received 20 November 1996; accepted 27 February 1997 Patients and methods Patient characteristics are shown in Table patients (20 male/16 female) with AML, ALL, CML or MDS were

2 1170 Table 1 Patient characteristics (n = 36) was identical and in 17 there was a gender difference (eight female donors for male recipients). Gender: Male/female 20/16 Volunteer unrelated donors (VUD) were matched by Median age (years) 35.7 serotyping (n = 36) for class I and high resolution SSOP (range 20 50) DNA testing for class II (n = 32). 28 patient/donor pairs Diagnosis were HLA identical for HLA A, B and DR, two patients AML 10 had an HLA-A micromismatch, four patients had an HLA- CML 16 DR minor mismatch, and in two patients, a DP allele differ- ALL 3 MDS/sec AML 7 ence existed. MLC was performed in 33 donor/patient pairs and was nonreactive in all. Remission CR1/CP1 13 CR2/CP2 5 AP/BC 5 Supportive care no remission 13 The patients were housed in a single room with HEPA- Patient/donor gender filter conditioned air and all received a 3.2 dual lumen M/M 12 M/F 8 Quinton catheter (Quinton, Seattle, WA, USA). Microbio- F/M 9 logical prophylaxis consisted of trimethoprim-sulfamethox- F/F 7 azole (320 mg/1600 mg/day) until day 1, itraconazol 400 Patient/donor CMV status mg/day or fluconazole 200 mg/day until day +35. Acyclovir neg/neg 15 was given for prophylaxis mg/day from day +1 neg/pos 5 until day +14. All patients received heparin 200 IE/kg pos/neg 12 pos/pos 4 bw/day c.i.v. to prevent VOD and central line clotting start- ing before conditioning. The dose was adjusted to avoid HLA identical 28 mismatch 8 prolongation of the aptt and was kept throughout the (HLA-A (2), DR (4), thrombocytopenia period except in cases of bleeding. The DP allele difference (2)) target cell dose for transplant was ANC/kg bw. The Blood group marrow was transfused without prior freezing on day 0, identical 14 without manipulation except for HAES separation in cases minor mismatch 10 of blood group incompatibility patients received G- major mismatch 12 CSF (Neupogen; Amgen, München, Germany) which was G-CSF yes 21 given c.i.v. 5 g/kg bw until leukocytes counts reached no / l on 3 consecutive days. 15 patients did not receive GVHD prophylaxis hematopoetic growth factors post-transplant. In the event CsA/MP (regimen A) 4 of fever 38 C, positive blood cultures or rapid increase CsA/MP/MTX (regimen B) 25 of C-reactive protein, empiric broad spectrum antibiotics CsA/MP/MTX/ATG (regimen C) 7 were given according to standard procedures. Where fever persisted after 72 h, microbiological therapy was escalated with amphotericin B; 10 g immunoglobulin was given until day +100 starting day 1 and every 10 days post-transplant. Table 2 Graft-versus-host disease according to the prophylaxis regimen Conditioning Grade 0 I II IV (%) III IV (%) According to Tutschka 4 busulfan 4 mg/kg was administered Acute GVHD in n = 34 orally four times a day from days 7to 4, and cyclophosphamide 60 mg/kg was given day 3 and 2 i.v. over 1 h. Regimen A 2/4 2/4 (50) 2/4 (50) Twelve patients received busulfan in 25 mg tablets pro- Regimen B 8/23 15/23 (65) 5/23 (22) vided by Wellcome, Dartford, UK. For seizure prophylaxis Regimen C 6/7 1/7 (15) 0/7 (0) phenytoin mg p.o. was started on day 8 until day Chronic GVHD 12/27 patients (45%) 3 and then reduced until day 1. Mesna 100 mg/kg bw extensive 6/12 patients (50%) was administered c.i.v. on days 3, 2 and 1 and forced limited 6/12 patients (50%) diuresis without bladder irrigation was given for prophy- laxis of cystitis. Acute GVHD as described in Ref. 32 and chronic GVHD according to Ref. 33. GVHD prophylaxis In four patients GVHD prophylaxis consisted of cyclosporin transplanted between August 1993 and August The (CsA) 5 mg/kg twice a day starting day 3, and methyltransplanted median age was 35.7 years (range 20 50). Blood groups prednisolone (MP) 0.5 mg/kg/day divided in two doses were identical in 14 donor/recipient pairs. A major ABOmismatch was present in 12, and minor mismatch in 10 transplants. In 19 transplants, recipient and donor gender starting day +7 which was increased to 1 mg/kg/day on day +14 (regimen A). 23 Since January 1994, short course methotrexate (MTX) (day mg/ 2 and day +3 and +6

3 10 mg/m 2 ) 24 was added in 25 patients (regimen B) and the Engraftment dose was adjusted according to direct bilirubin and creatinine levels. CsA blood levels were measured three times a Engraftment defined as the first of 3 consecutive days with week and the target level was ng/ml blood as mealeukocytes 1000/ l was achieved in 34 patients (92%). sured by a fluorescence polymerization immunoassay Patients not receiving G-CSF (n = 14) engrafted with a (Abbot, Wiesbaden, Germany). All 25 patients received median on day (range 13 32) and patients who 100% of the target dose of MTX. Since November 1995 received G-CSF (n = 20) engrafted with a median on day seven patients received additional anti-t-lymphocyte +14 (range 12 18). Patients receiving GVHD prophylaxis globulin (ATG; Fresenius, Bad Homburg, Germany) regimen B and C (n = 10), who did not receive hemato- (regimen C). ATG 30 mg/kg/day was administered day 3 poietic growth factors recovered on a median of day until day 1 as a 12-h infusion as GVHD prophylaxis. Four (range 14 32) and the 20 patients on regimen B and C and of these seven patients had micro or minor HLA class I G-CSF recovered on a median of day (range 12 18). and II mismatches (Tables 1 and 2). This difference is statistically significant with P (Table 3). Two patients transplanted in blast crisis of CML CMV prophylaxis did not engraft. In both cases, pretransplant bone marrow biopsy revealed extensive marrow fibrosis. In one patient a Fifteen recipient and donor pairs were CMV negative. In second bone marrow infusion from the same donor without five transplants, recipient and donor were both positive, in further conditioning was given, but again no engraftment five cases only the donor was positive and in 11, only the was achieved. The patient died due to invasive aspergillosis recipient was positive (Table 1). After engraftment, weekly day +65. The second patient received unselected G-CSF CMV-PCR was performed and CMV-antigenemia was mobilized PBSC after leukapheresis twice from her hapmeasured in the blood. CMV prophylaxis was started when loidentical son, but did not engraft and died of pneumonia CMV-PCR was positive on two consecutive assays or on day There is no correlation between nonen- CMV-antigenemia was detectable. Depending on the platenine of 34 patients who engrafted exceeded a platelet count graftment, transplantation cell count or CFU-GM. Twentylet count and the blood creatinine, patients received either ganciclovir 2 5 mg/kg/d for 7 days and then once a day, of / l with a median on day +29 (range 8 95) with- or foscarnet 2 60 mg/kg/day for 14 days and afterwards out further transfusion. A median of 96 units (range 15 once a day until the PCR was again negative. 246) was transfused to achieve a platelet count above / l during aplasia. Five patients died before they Statistical analysis were transfusion-independent due to the reasons mentioned below. Packed red cells were given to keep the hemoglobin The median difference was evaluated with the Student s t- above 8.5 g/l, and a median of 12.2 packed red cells (range test for unpaired data in the two groups without G-CSF. 0 56) were transfused GVHD Results Transplantation Twenty-two of 36 transplants were performed after separ- ation by HAES sedimentation because of blood group mis- matches. The target ANC count for engraftment was /kg and this was reached in 29 patients (82%). The median transplanted nucleated cell count was /kg (range ). Bone marrow cultures showed a median growth of 104 CFU-GM/ cells (range, no growth to 228) after 14 days (Table 3). Table 3 Graft characteristics and engraftment Acute GVHD grade II IV occurred in 19/34 patients (56%), grade II in 12/34 patients (35%) and grade III IV in seven of 34 patients (21%) and was fatal in five of seven patients. According to GVHD prophylaxis regimen, we observed agvhd grade III and IV with regimen A in two of four patients (50%), with regimen B in five of 25 patients (20%) and with regimen C in none of seven patients (0%). Twenty-seven patients survived to day +100 and chronic GVHD occurred in 12/27 (44%) with a median follow-up of 329 days. Six of 27 patients (22%) suffered from limited disease and six of 27 patients (22%) suffered from extensive disease (Table 2). CFU-GM/ cells median 104 (range 0 228) Patient outcome and survival Nucleated cells/kg recipient bw median /kg bw (range ) All patients (n = 34) who engrafted had histologically pro- Neutrophils 1000/ l median day +16 (range 12 32) ven complete remission in the bone marrow biopsy on day (n = 34 patients) +30. Cytogenetic examination showed complete donor Regimen A without G-CSF (range 15 21) chimerism in all 16 patients transplanted from a donor with (4/34 patients) a different gender and in all 14 patients with Ph-positivity Regimen B and C without G-CSF (range 14 32) pretransplant. Eight of 36 patients (22%) died up to day (10/34 patients) P Causes of death were agvhd in five patients (two Regimen B and C with G-CSF (range 12 18) in group A, three in group B), systemic toxoplasmosis (1), (20/34 patients) clinical signs of pulmonary embolism (1) and nonengraftment (1).

4 1172 Busulfan/cyclophosphamide in VUD-BMT The median survival for all patients is 258 days (range ). Sixteen (45%) patients are alive in complete remission with a median follow-up of 455 days ( ). Five of 34 (14%) patients relapsed; three patients transplanted with AML in second refractory relapse died on day +124, +142 and +164 respectively, one patient with ALL transplanted in second relapse relapsed and died on day + 512, and one patient with CML transplanted in 2nd AP after one blast crisis on day The latter patient was treated with donor buffy coat cells as adoptive immunotherapy without any response. Two patients died due to chronic GVHD on day +205 and day +267 and five patients died of infectious problems: Pneumocystis carinii pneumonia (PCP) day +341 (1), toxoplasmosis (2) day +380 and +780, pneumonia of unknown origin day +104 (1), and +122 due to graft failure (1). During the time on our transplant ward we did not observe any severe pulmonary problems such as interstitial pneumonitis or respiratory failure requiring ventilation support. No severe VOD was observed and no seizures occurred. Although reactivation of CMV was detectable by PCR and CMV antigenemia, only one organ manifestation of CMV disease was observed as CMV colitis in a patient with agvhd grade IV of the gut. Hemorrhagic cystitis was observed in six of 36 patients (17%) which resolved without any late complications. duplex sonography. This is most likely due to our heparin prophylaxis regimen which was used in all patients without major bleeding complications. Furthermore, no fatal pulmonary complications were observed in contrast to reports in the literature with an incidence of up to 21% for busulfan 4,25,26 and 28% for TBI. 29 A longer follow-up is necessary to estimate the incidence of chronic pulmonary problems including fibrosis. In the future, i.v. preparations of BU 30 could facilitate its use, with reliable blood levels. Furthermore, tablets with a higher content of busulfan will make therapy more acceptable for patients. Bu/CY or other busulfan combinations using melphalan, VP16 or cytarabine 10,28,31 should be randomly compared to TBI-containing regimens in VUD transplants. We conclude that Bu/CY is a safe and sufficiently immunosuppressive conditioning protocol for HLA- identical and nonidentical VUD transplants. This combination presents a valuable alternative to TBI containing regimens with few side-effects using our prophylaxis protocol. Acknowledgements We are grateful to Barbara Sauer and Elisabeth Lenartz for excellent technical assistance and transplant coordination. Discussion References In our single center study, 36 adult patients were transplanted after conditioning with Bu/CY with marrow from 1 Hansen JA, Clift RA, Thomas ED et al. Transplantation of volunteer unrelated donors. Thirty-four patients engrafted marrow from an unrelated donor to a patient with acute leuke- without any delay on a median of day +16. No secondary mia. New Engl J Med 1980; 303: graft failure occurred. Graft failure is a frequent problem 2 Appelbaum FR. The influence of total dose, fractionation, dose rate and distribution of total body irradiation on bone in patients with CML transplanted in blast crises 19 marrow transplantation. Semin Oncol 1993; 20 (Suppl): especially with myelofibrosis and splenomegaly. The low 3 Santos GW, Tutschka PJ, Brookmeyer R et al. Marrow transincidence of primary graft failure (6%) in our study, which plantation for acute nonlymphocytic leukemia after treatment is less than described by other institutions, 25,26 occurred in with busulfan and cyclophosphamide. New Engl J Med 1983; two patients with CML in blast crises with biopsy-proven 309: excessive marrow fibrosis pretransplant. Failure to engraft 4 Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplanis most likely due to myelofibrosis and not to rejection in tation for leukemia following a new busulfan and cyclophos- these patients. It can be speculated that ATG might have phamide regimen. Blood 1987; 70: facilitated engraftment in these patients, neither of whom 5 Hassan M, Öberg G, Ehrsson H et al. Pharmacokinetic and received ATG. metabolic studies of high-dose busulphan in adults. Eur J Clin Pharmacol 1989; 36: We have demonstrated that Bu/CY is a highly effective 6 Copelan EA, Deeg HJ. Conditioning for allogeneic marrow regimen for VUD transplantation. The addition of ATG as transplantation in patients with lymphohematopoietic maligpretransplant GVHD prophylaxis showed promising results nancies without the use of total body irradiation. Blood 1992; with minor, manageable side-effects. Although the number 80: is small we did not see any severe GVHD, in particular not 7 Hassan M, Ljungman P, Bolme P et al. Busulfan bioavailability. in four out of seven patients transplanted with HLA class Blood 1994; 84: I and II micro or minor differences. Use of G-CSF postmarrow 8 Mehta J, Powles RL, Mitchell P et al. Graft failure after bone transplant shortened the time of neutropenia significantly. trasnplantation from unrelated donors using busul- In our patients, TRM was comparable or reduced compared phan and cyclophosphamide for conditioning. Bone Marrow to that in the literature for patients conditioned with VUD- Transplant 1994; 13: Ballester OF, Agaliotis DP, Hiemenz JW et al. Phase I II BMT. 18,25 The incidence of hemorrhagic cystitis was only study of high-dose busulfan and cyclophosphamide followed 17% compared to up to 30% in other reports. Severe VOD by autologous peripheral blood stem cell transplantation for as a major complication of high-dose busulfan has been hematological malignancies: toxicities and hematopoietic reported in up to 56% of patients. 15,25,27,28 However, it was recovery. Bone Marrow Transplant 1996; 18: not clinically detected in any of our patients and this was 10 Chao NJ, Stein AS, Long GD et al. Busulfan/etoposide confirmed in 15 patients by measuring liver venous flow by initial experience with a new preparatory regimen for autolog-

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