Transplantation • Research Paper Successful engraftment following allogeneic stem cell transplantation with busulfan as a single agent in very high-risk patients

Menachem Bitan Background and Objectives. Busulfan is the most commonly used myeloablative alkylat- Reuven Or ing agent, but is considered a poor anti- agent. Since engraftment of allogeneic Michael Y Shapira stem cells depends not only on adequate immunosuppression but also on successful Igor B Resnick hematopoietic competition, and considering the fact that residual of host ori- Aliza Ackerstein gin may play a beneficial role in preventing graft-versus-host disease (GVHD), we used low Simcha Samuel doses of oral busulfan as a single agent for conditioning prior to stem cell transplantation Sharon Elad (SCT) in recipients of transplants from a variety of donors. Shimon Slavin Design and Methods. Fifteen heavily pretreated high-risk patients (age 25-66, median 42 years) with hematologic malignancies were conditioned with busulfan alone, 4mg/kg/day for 2, 3, or 4 consecutive days. No additional pre- or post-transplant immunosuppressive agents were used in order to exploit the capacity of donor lymphocytes to induce graft-ver- sus-malignancy (GVM) effects. Results. Conditioning was well tolerated, trilineage engraftment was documented in all patients and none exhibited immune-mediated rejection. Time to recovery of absolute neu- trophil count >0.5 109/L and 1.0 109/L was 12 - 38 (median 15) days and 12 - 41 (medi- ¥ ¥ 9 an 15) days, respectively. The time to platelet recovery ≥20 and ≥50¥10 /L ranged from 0 to 26 (median 11) days, and from 0 to 83 (median 14) days, respectively. Acute GVHD (£grade I) occurred in 13/15 patients. Three patients benefited from long-term survival. Interpretations and Conclusions. We suggest that using busulfan alone for the preparation of patients for SCT may be sufficient for engraftment, in very high-risk heavily pre-treated patients. Key words: stem cell transplantation, graft-versus-malignancy effects, busulfan, reduced intensity conditioning, hematologic malignancies, leukemia. Haematologica 2005; 90:1089-1095 ©2005 Ferrata Storti Foundation

All authors from the Department of Transplantation & usulfan, a sulfonic acid ester, is an hepatic toxicity, mainly veno-occlusive dis- Cancer , alkylating agent with potent toxicity ease, especially in patients undergoing allo- Hadassah-Hebrew University for hematopoietic and sinusoidal geneic SCT.5-6 Other toxicities of the drug Medical Center 91120, B . endothelial cells. The drug was initially included electroencephalographic abnor- introduced in 1952, as a treatment modali- malities and an increased risk of convul- Correspondence: ty for patients with chronic myeloid sions due to the drug penetrating into the Shimon Slavin, MD, Department leukemia.1 It was only around 20 years later central nervous system,7-8 inhibition of stro- of Bone Marrow Transplantation that this agent was first used as part of the mal cell function,9-11 occasional pulmonary & Hadassah University Hospital conditioning regimen in bone marrow toxicity and an association with increased Jerusalem 91120, Israel. transplantation, substituting total body transplant-related mortality when used at E-mail: [email protected]/ irradiation.2 Considering the fact that high doses for allogeneic SCT.12 In recent [email protected] busulfan was considered as a potent mye- years, busulfex, an intravenous preparation loablative rather than immunosuppressive of busulfan, has gradually been replacing agent, most transplant centers used the oral busulfan in SCT regimens. Its main combination of busulfan and cyclophos- advantages are reduced liver toxicity and phamide or other alkylating agents or total dosage assurance with predictable pharma- body irradiation with the aim of accom- cokinetics.13-15 Given its known myeloabla- plishing maximal myeloablation as well as tive properties, busulfan was considered in optimal immunosuppression prior to trans- many centers as the treatment of choice for plantation of allogeneic stem cells. Since myeloid disorders, however, busulfan, was then, the use of busulfan spread widely and never recommended as a suitable single it was also used in conjunction with autol- agent in a preparative regimen for allogene- ogous stem cell transplantation (SCT).3-4 ic SCT because of its limited toxicity to However, it was soon discovered that the mature lymphocytes. Combinations of use of busulfan could be associated with busulfan with cyclophosphamide or other

haematologica/the hematology journal | 2005; 90(8) | 1089 | M. Bitan et al. agents were used not only to control mature lympho- Table 1. Characteristics of patients undergoing stem cell trans- cytes in order to prevent rejection, but also to exploit plantation following conditioning with busulfan as a single agent. synergistic activity against target tumors or genetical- ly abnormal stem cells. The use of busulfan, or nowa- UPN Age Basic Disease No. Survival Cause and (years) disease status of Tx. time of death days busulfex, avoiding the need for ionizing irradia- at Tx. post Tx. tion, is considered the preferred treatment for malig- (days) nant and non-malignant indications especially in the pediatric age group. However, there are situations in 977 33 CML – Blastic Resistant 2nd No Infection which heavily pre-treated patients cannot safely transformation blast and acute receive extensive immunosuppressive and myeloabla- to AML-M7 Crisis s/p GVHD (83) SCT tive conditioning; furthermore, in patients with resist- ant disease, it seems unlikely that all tumor cells can 1170 47 MM Resistant 2nd No Disease be eliminated by alone. We, therefore, Disease s/p SCT progression (12) investigated the feasibility of accomplishing durable 1242 26 AML-M2 Resistant 1st 1st Yes Survived and consistent engraftment of donor stem cells with Relapse after PR only low doses of busulfan alone, avoiding the use of post- transplant immunosuppression as GVHD prophylaxis 1251 50 AML-M4 Resistant 1st 1st Yes Survived in order to maximize induction of graft-versus-malig- Relapse after PR only nancy effects following tumor debulking with busul- fan alone. Indeed, O’Brien and Goldman described the 1253 35 CML 2nd resistant 1st No acute GVHD (16) use of busulfan as a sole preparative agent in a group blast crisis of patients with chronic myelogenous leukemia, but 1283 59 AML* 2nd resistant 1st No Disease this was in the setting of autologous stem cell trans- relapse progression (76) 16 plantation. The present report summarizes our 1323 28 T-cell ALL Resistant 1st No Disease cumulative experience in a cohort of 15 patients who 1st relapse progression (95) received allogeneic stem cell allografts after prepara- 1354 25 AML-M5 Resistant 1st No Infection and tion with busulfan as a single agent. 1st relapse acute GVHD (34)

1358 44 Biphenotypic Resistant 1st No acute GVHD (43) disease – Design and Methods refractory to therapy

Fifteen high-risk patients with advanced and fully 1362 52 CML 2nd CP 1st No acute GVHD (75) resistant disease, most of whom had been heavily 1374 46 AML-M5 Resistant 1st No Disease pre-treated, were enrolled in this study between disease - progression December 1997 and June 2000. Indications for trans- refractory (120) plantation and disease status are shown in Table 1. to therapy Patients were included if they had an absolute indica- 1378 36 CML Resistant 1st Yes Survived tion for allogeneic SCT but were not eligible for a blast crisis standard transplant procedure, or if their potential to 1380 42 CML 2nd CP 1st No Disease survive the standard protocol was judged to be low progression due to poor performance status, prior SCT or relapse (278) after SCT, provided they had not previously been 1381 66 NHL Resistant 1st No acute GVHD (47) given busulfan. The series comprised 10 males and 5 1st Relapse females, ranging in age from 25-66 (median 42) years after PR only (Table 1). The mean Karnofsky performance status 1382 31 AML-M4 2nd resistant 2nd No Infection score was 88 (range 60-100). Each patient signed relapse s/p SCT and acute GVHD informed consent to the protocol which had been (110) approved by the Institutional Review Board. Twelve UPN: unique patient number; Tx: transplantation; AML: acute myeloid leukemia; CML: chronic myeloid leukemia; MM: multiple myeloma; patients were transplanted from fully matched HLA NHL: Non-Hodgkin’s lymphoma; HD - Hodgkin’s disease; ALL: acute class I and II family members, two patients were lymphoblastic leukemia; CP: chronic phase; PR: partial remission; transplanted from mismatched sibling donors (one GVHD: graft-versus-host disease; *:sub-classification not defined. from a haploidentical sibling and the other from a one-locus mismatched sibling). One patient received a marrow allograft from a fully matched unrelated Pre-transplant conditioning consisted of oral busul- donor (MUD); this graft was non-reactive in mixed fan 4 mg/kg/day in four divided daily doses. The lymphocyte culture, as shown in Table 2. decision about the exact amount of busulfan each

| 1090 | haematologica/the hematology journal | 2005; 90(8) Conditioning for transplantation with busulfan alone

Table 2. Characteristics of donors, donations and conditioning reg- transplant immunosuppression was administered. imen. Fourteen patients were reconstituted with mobilized peripheral blood stem cells and one with bone mar- UPN Sex Matching Origin of Dose of CNS Day* of Day* of row cells. Donors of the peripheral blood stem cells Recip./ donation busulfan prophylaxis ANC 100% 9 were injected subcutaneously with granulocyte- Donor >1.0¥10 chimerism colony stimulating factor (G-CSF; Neupogen 5 mg/kg twice daily for 5 days) and the mobilized peripheral 977 M/M Full PBSC 4 mg¥4 No 15 49 match blood stem cells were collected on days 5 and 6. Bone marrow collection was done under epidural anesthe- 1170M/MUD Full PBSC 4 mg¥2 No 12 NA sia. Prior to transplantation, all patients received match trimethoprim/sulfamethoxazole (10 mg/kg/day 1242 F/F Full PBSC 4 mg¥4 No 17 17 trimethoprin) on days -8 to -2, acyclovir (500 match 2 mg/m ¥3/day) from day –8 until day +100, and allop- 1251 F/F Full PBSC 4 mg¥4 ARA-C 12 13 urinol (300 mg/day) on days -8 to -1. Administration match of trimethoprim/sulfamethoxazole (twice weekly) was reinstituted after recovery from neutropenia as a 1253 M/M Haplo. PBSC 4 mg¥4 No 13 13 preventive measure against Pneumocystis carinii infec- 1283 F/M Full BM 4 mg¥4 No 32 50 tion. Neutropenic patients with culture-negative match fever received a combination of gentamicin, cefa- 1323 M/F Full PBSC 4 mg¥4 No 13 22 zolin and mezlocillin, as a first-line antibiotic proto- match col. Persisting fever was treated with amikacin and 1354 F/M Full PBSC 4 mg¥3 No 14 18 tazocin as a second-line protocol, while meropenem match and vancomycin were used as the third-line protocol. In cases of persistent fever not responding to antibi- 1358 M/F Full PBSC 4 mg¥4 ARA-C 12 18 match &MTX otic therapy within 5 days, amphotericin B (1 mg/kg every other day) was added until the neutropenia 1362 M/F Full PBSC 4 mg¥4 ARA-C 21 49 match &MTX resolved. Starting on day -8, a DNA-polymerase chain reac- 1374 M/F 1 locus PBSC 4 mg¥4 No 21 21 mismatch ton (PCR) test and, later during the follow-up period assay of pp65 antigenemia, was done weekly to 1378 M/M Full PBSC 4 mg¥4 ARA-C 16 43 detect cytomegalovirus. Two consecutive positive match PCR results or one antigenemia assay with more 1380 F/F Full PBSC 4 mg¥2 ARA-C 41 52 then one cell positive for pp65 served as an indication match for replacing acyclovir with ganciclovir (10 1381 M/M Full PBSC 4 mg¥2 No 15 21 mg/kg/day), until a minimum of two negative tests match were obtained. Patients were treated with reverse isolation in rooms equipped with HEPA filters, and 1382 M/M Full PBSC 4 mg¥2 No 23 41 match received a regular diet. Additional supportive meas-

*Following transplantation; UPN: unique patient number; Recip.: recipient; ures, such as blood components were administered F: female; M: male; Allo.: allogeneic transplantation; Auto.: autologous as necessary. transplantation; BM: bone marrow; PBSC: peripheral blood stem cell; Haplo.: haploidentical mismatch; CNS: central nervous system; ARA-C: cytosar; Acute and chronic GVHD were graded according MTX: methotrexate; NA: not achieved. to Glucksberg’s criteria.17 Upon the appearance of signs and symptoms of acute GVHD >grade I, treat- ment was immediately started with methylpred- patient was administered was made taking into nisolone 2 mg/kg/day i.v. and cyclosporine 3 account the treatment history and estimating each mg/kg/day i.v. in two divided doses for patients in patient’s ability to stand the protocol. Ten patients the hospital and 6 mg/kg twice daily orally for outpa- were treated for four consecutive days (total dose 16 tients. mg/kg); one patient for three consecutive days (total In order to assess engraftment, degree of dose 12 mg/kg); and four patients for only two con- chimerism, minimal residual disease and early secutive days (total dose 8 mg/kg). Five of the relapse, patients were monitored at regular intervals patients also received central nervous system pro- by cytogenetic analysis, and by donor and host-spe- phylaxis with 30 mg/m2 cytosine arabinoside, cific DNA markers as previously described, including administered intrathecally. Two of these patients male and female amelogenine gene PCR bands,18 and were also given methotrexate intrathecally (12.5 mg by variable number of tandem repeats-PCR assay in and 15 mg) (Table 2). No additional pre- or post- patients with no sex mismatch.19

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Five of the 15 patients died of disease progression Results (median time to death 95 days [range 12-278 days]). Four patients died from acute GVHD (median time to During the conditioning the protocol used was death 45 days [range 16-75 days]). Three patients well tolerated by all patients. All patients were fully died from a combination of infection and GVHD mobile throughout the conditioning period. Two (median time to death 83 days [range 34-110 days]). patients out of 15 died peri-transplantation, one on Acute GVHD (≥grade I) occurred in 13/15 patients the day of engraftment due to progressive multiple and in nine patients progressed to grade III-IV. In one myeloma disease, and the second, two days after patient acute GVHD progressed to chronic GVHD. documentation of engraftment, on day +16 because As mentioned above, GVHD was the direct cause of of acute GVHD. No patient had severe oral mucosi- death in four patients. tis, facilitating maintenance by normal oral intake. Moderate or severe hepatic veno-occlusive disease did not occur in any of the patients. Neutropenic Discussion fever was noted in 13/15 patients (median of febrile episodes 1, range 0-2), with a median of four days of We summarize the outcome of the first cohort of fever (range 0-15 days). allogeneic SCT recipients conditioned with busulfan All patients displayed evidence of engraftment as a single agent, with an attempt to maximize GVM shortly after transplantation and none exhibited effects induced by alloreactive donor lymphocytes immune-mediated rejection. A transient period of post transplantation. No anti-graft-versus host pro- mixed chimerism was detected in 11 patients. Of phylaxis was given, thus intentionally maximizing these patients, one developed 100% donor GVM effects as the only possible modality to elimi- chimerism after receiving donor lymphocyte infusion nate tumor cells resistant to all conventional anti-can- (UPN 1283), a second patient (UPN 1170) died too cer agents. O’Brien and Goldman already described early from progression of his basic disease, and all the use of busulfan as a sole preparative agent in a the others converted spontaneously to full 100% group of patients with chronic myeloid leukemia, but donor chimerism shortly thereafter. In the other four this was in the setting of autologous stem cell trans- patients, rapid engraftment of 100% donor cells was plantation, in which no additional immunosuppres- confirmed without evidence of transient mixed sion was indicated and no GVM effects were antici- chimerism (Table 2). The median nadir white cell pated.16 Myeloablative conditioning alone in prepara- 9 9 count was 0.3¥10 /L (range 0.1 – 1.5¥10 /L). Time to tion for allogeneic SCT, especially in heavily pretreat- recovery of absolute neutrophil count (ANC) above ed patients with poor performance status, carries a 9 9 0.5¥10 /L and 1.0¥10 /L was 12-38 (median 15) days substantial risk of procedure-related toxicity and and 12 - 41 (median 15) days, respectively. The medi- mortality.20 Reduced intensity conditioning can 9 an nadir platelet count was 8¥10 /L (range decrease the incidence of some of these early compli- 9 21-22 2–37¥10 /L). Time for platelet recovery to values ≥20 cations. Moreover, over the years aggressive 9 9 ¥10 /L and ≥50¥10 /L ranged from 0 to 26 (median preparatory regimens have not proven advantageous 11) days, and from 0 to 83 (median 14) days, respec- and have not improved disease-free survival in any of tively. In three patients the platelet count never the disease categories in which SCT is indicated.23-24 9 dropped below 20¥10 /L throughout their hospital- Therefore, when testing the feasibility of using ization, whereas four of the patients did not reach a busulfan alone for conditioning stem cell allograft 9 platelet count above 50¥10 /L before dying. Two of recipients we used low doses of busulfan in order to the patients did not need any platelet support during identify the minimal dose that would allow durable the entire transplantation course. engraftment and, subsequently, development of The overall mortality rate was high for a number of maximal GVM effects by alloreactive donor lympho- reasons, the first being the intentional selection of cytes unperturbed by immunosuppressive agents poor-risk patients who were not eligible for a stan- used for anti-GVHD prophylaxis. It was also dard transplant procedure but rather qualified for an assumed that once engraftment had occurred in the experimental protocol. In addition, no anti-GVHD absence of post-transplant immunosuppression, prophylaxis was used in order to exploit the capacity residual hematopoietic cells of host origin as well as of donor lymphocytes to induce GVM effects, thus leukemia, would likely be eliminated by donor lym- exposing the patients to the risk of uncontrolled phocytes due to induction of optimal GVM effects in GVHD. Consequently, only three patients benefited the absence of cyclosporine or any other post-graft- from long-term survival (UPN 1242, 1251, 1378, ing anti-GVHD prophylaxis. The high incidence of median follow-up 79 months [range 67-80 months]). relapse in recipients of T-cell-depleted allografts fol-

| 1092 | haematologica/the hematology journal | 2005; 90(8) Conditioning for transplantation with busulfan alone lowing myeloablative conditioning,25 as well as in reduced intensity conditioning, as well as the effica- patients transplanted after myeloablative condition- cy of busulfan in preventing rejection.34 Furthermore, ing with no development of GVHD,26-28 or recipients it demonstrates the feasibility of overcoming fatal of stem cells obtained from an identical twin,28-29 sup- GVHD when non-myeloablative conditioning is port the major role of alloreactive donor T cells on used, which may be the consequence of a transient outcome following SCT. Furthermore, the negative stage of mixed chimerism.33 effects of cyclosporine on GVM effects inducible by Interestingly, consistent engraftment of donor stem alloreactive donor lymphocytes has been well docu- cells was confirmed in all patients, including in one mented in preclinical animal models30 and in patient reconstituted with haploidentically mis- humans.31-32 Based on the above, the present study matched stem cells and in another who received an was designed with the aim of investigating the role allograft from a matched unrelated donor, while early of conditioning with a single myeloablative agent, marrow aplasia and pancytopenia were avoided or busulfan, given in graded increments, according to minimized, thus reducing the immediate transplant- the clinical condition of the poor-risk patients eligible related complications. However, late complications for the study. The goal was to determine whether related primarily to uncontrolled GVHD were engraftment can be achieved with this alkylating unavoidable. Our data suggest that busulfan, even at agent, since once early engraftment has been accom- a dose of 8 mg/kg, can induce a sufficient degree of plished, donor lymphocytes can be expected to con- immunosuppression to ensure engraftment in tinuously ablate residual hematopoietic cells of host patients who had already been heavily pretreated origin enabling the development of 100% donor cell with chemotherapy. It is unclear whether durable chimerism. Considering the rule of balanced equilibri- engraftment could also be accomplished in previous- um,33 concerning the association between the intensi- ly untreated patients, since both prior treatment of ty of pre-transplantation immunosuppression and the underlying leukemia as well as the malignant the incidence and severity of GVHD, it was also process itself could result in some degree of immuno- interesting to investigate whether a protocol which suppression that could facilitate engraftment follow- consisted of a primarily myeloablative agent could ing subsequent conditioning with busulfan alone. also reduce the incidence of GVHD. Indeed, although Taken together, our data suggest that reduced inten- fludarabine, cyclophosphamide and anti-lymphocyte sity conditioning, either using low doses of busulfan as antibodies, commonly used in reduced intensity con- shown here, or using escalated doses of cyclophos- ditioning protocols, were not used in the present phamide and antithymocyte globulin,35 may be suffi- study, no graft rejection was observed in any recipi- cient to enable engraftment of HLA compatible stem ent of a minimum of two doses of busulfan 4 cell allografts, at least in heavily pretreated patients. As mg/kg/day. Moreover, all four patients who had a pioneered in Jerusalem more than 18 years ago,36,37 and rapid, full engraftment of donor cells received four supported by Kolb and colleagues,38,39 successful doses of busulfan 4 mg/day. On the other hand, all engraftment of matched bone marrow allografts can the other 11 patients, including 5 patients who also enable subsequent induction of effective GVM effects received all four doses of busulfan 4 mg/day, experi- by donor lymphocyte infusions, mediated by alloreac- enced a period of mixed chimerism before achieving tive donor lymphocytes.36-39 More recently, in agree- 100% donor chimerism. Thus, it is not clear whether ment with the aforementioned conclusions, it has the dose of busulfan is of importance for chimerism. shown that low doses of cyclophosphamide or The same holds true concerning engraftment. The busulfan with fludarabine with no myeloablative data (Table 2), are inconclusive on whether doses of conditioning may be sufficient to eliminate all tumor busulfan between 8 mg to 16 mg are relevant to cells in patients with hematologic malignancies,21,22 engraftment outcome. Our data from a small cohort thus paving the way to the successful use of stem cell of 15 patients show that durable engraftment and transplantation following non-myeloablative condi- occasionally cure of the underlying malignancy can tioning. These data confirmed the efficacy of quality be accomplished with minimal conditioning based over quantity, or the need to improve immune regu- on the use of busulfan as a single oral agent, with a lation rather than to increase the intensity of condi- relatively low procedure-related toxicity, considering tioning. Furthermore, as shown earlier in a preclinical the severity of disease of this heavily pre-treated animal model of B- cell leukemia, mismatched allo- cohort of patients with a poor performance status, grafts may induce curative graft-versus-leukemia although exposing the patients to the risk of severe effects with no GVHD following non-myeloablative GVHD-related toxicity. From a theoretical point of conditioning.40 view, this study seems to reiterate the importance of Although the overall outcome of the patients treat- hematopoietic competition in engraftment following ed with busulfan alone did not appear to be impres-

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sive, it should be remembered that the protocol was for standard or non-myeloablative stem cell trans- offered exclusively to patients with very advanced plantation procedures. Further investigations in larg- diseases not considered eligible for any alternative er cohorts of patients will be required to investigate transplant program. Based on our data, it seems rea- the efficacy of conditioning based on a single drug in sonable to assume that much better outcomes would comparison with that of combinations of non-mye- be achieved in patients with minimal residual disease loablative agents. or with a better performance status who could be MB: writing the article; follow-up of patients; RO, MYS: treat- conditioned with a similar protocol on an outpatient ing patients, revision of article; IBR: revision of article; basis, with no need for expensive components or We wish to thank the Danny Cunniff Leukemia Research radiation facilities. Such patients could benefit for a Laboratory; the Gabrielle Rich Leukemia Research Foundation; the Cancer Treatment Research Foundation; the Novotny Trust; the similar approach with additional anti-GVHD pro- Szydlowsky Foundation; the Fig Tree Foundation; Ronne & phylaxis to minimize the risk of uncontrolled GVHD. Donald Hess; and the Silverstein family for their continuous sup- In conclusion, our observations on a small cohort port of our ongoing basic and clinical research. This research was partially supported by an unrestricted grant of non-consecutive, poor risk patients may justify a from ESP Pharma, Edison, NJ, USA. larger study using busulfan (or busulfex for more Manuscript received February 15, 2005. Accepted June 9, consistent bioavailability of the drug), as a single 2005. agent for patients needing SCT who do not qualify

11. Guest I, Uetrecht J. Drugs toxic to the Variable number of tandem repeat References bone marrow that target the stromal (VNTR) markers for human gene map- cells. Immunopharmacology 2000; 46: ping. Science 1987;235:1616-22. 1. Haddow A, Timmis G. Myleran in 103-112. 20. Snyder DS. Ethical issues in hemato- chronic myeloid leukemia. Lancet 12. Ljungman P, Hassan M, Bekassy AN, poietic cell transplantation. In: Thomas 1953;1:207. Ringden O, Oberg G. High busulfan ED, Blume KG, Forman SJ, eds. Hema- 2. Santos GW, Tutschka PJ, Brookmeyer concentrations are associated with topoietic Cell Transplantation. Mass- R, Saral R, Beschorner WE, Bias WB, et increased transplant-related mortality achusetts USA, Blackwell Science Inc. al. Marrow transplantation for acute in allogeneic bone marrow transplant 1999. p. 396. nonlymphocytic leukemia after treat- patients. Bone Marrow Transplant 21. Slavin S, Nagler A, Naparstek E, Kape- ment with busulfan and cyclophos- 1997;20:909-13. lushnik Y, Aker M, Cividalli G, et al. 13. Russell JA, Tran HT, Quinlan D, Non-myeloablative transplantation and phamide. N Engl J Med 1983;309:1347- Chaudhry A, Duggan P, Brown C, et al. cell therapy as an alternative to conven- 53. Once-daily intravenous busulfan given tional bone marrow transplantation 3. Tutschka PJ, Copelan EA, Klein JP. Bone with fludarabine as conditioning for with lethal cytoreduction for the treat- marrow transplantation for leukemia allogeneic stem cell transplantation: ment of malignant and non-malignant following a new busulfan and cyclo- study of pharmacokinetics and early hematologic diseases. Blood 1998; 91: phosphamide regimen. Blood 1987;70: clinical outcomes. Biol Blood Marrow 756-63. 1382-8. Transplant 2002;8:468-76. 22. Champlin R, Khouri I, Kornblau S, 4. Gutierrez-Delgado F, Holmberg L, 14. Kashyap A, Wingard J, Cagnoni P, Roy Molldrem J, Giralt S. Reinventing bone Hooper H, Petersdorf S, Press O, Ma- J, Tarantolo S, Hu W, et al. Intravenous marrow transplantation. Non-mye- ziarz R, et al. Autologous stem cell versus oral busulfan as part of a busul- loablative preparative regimens and transplantation for Hodgkin’s disease: fan/cyclophosphamide preparative reg- induction of graft-vs-malignancy effect. busulfan, melphalan and thiotepa com- imen for allogeneic hematopoietic Curr Opin Oncol 1999;13:621-8. pared to a radiation-based regimen. stem cell transplantation: decreased 23. Clift RA, Buckner CD, Appelbaum FR, Bone Marrow Transplant 2003;32:279- incidence of hepatic venoocclusive dis- Bryant E, Bearman SI, Petersen FB, et al. 85. ease (HVOD), HVOD-related mortali- Allogeneic marrow transplantation in 5. Grochow LB, Jones RJ, Brundrett RB, ty, and overall 100-day mortality. Biol patients with chronic myeloid leu- Braine HG, Chen TL, Saral R, et al. Blood Marrow Transplant 2002;8:493- kemia in the chronic phase: a random- Pharmacokinetics of busulfan: correla- 500. ized trial of two irradiation regimens. tion with venoocclusive disease in 15. Shimoni A, Bielorai B, Toren A, Hardan Blood 1991;77:1660-5. patients undergoing bone marrow I, Avigdor A, Yeshurun M, et al. Intra- 24. Alyea E, Neuberg D, Mauch P, Marcus transplantation. Cancer Chemother venous busulfan-based conditioning K, Freedman A, Webb I, et al. Effect of Pharmacol 1989;25:55-61. prior to allogeneic hematopoietic stem total body irradiation dose escalation 6. Bearman SI. The syndrome of hepatic cell transplantation: myeloablation on outcome following T-cell-depleted venoocclusive disease after marrow with reduced toxicity. Exp Hematol allogeneic bone marrow transplanta- transplantation. Blood 1995;85:3005- 2003;31:428-34. tion. Biol Blood Marrow Transplant 20. 16. O’Brien SG, Goldman JM. Busulfan 2002;8:139-44. 7. Marcus R, Goldman JM. Convulsion alone as cytoreduction before auto- 25. Goldman JM, Gale RP, Horowitz MM, due to high dose busulfan. Lancet 1984; grafting for chronic myelogenous Biggs JC, Champlin RE, Gluckman E, et 2:1463. leukemia. Blood 1998;91:1091-2. al. Bone marrow transplantation for 8. Sureda A, Perez de Oteyza J, Garcia 17. Glucksberg H, Storb R, Fefer A, chronic myelogenous leukemia in Larana J, Odriozola J. High-dose busul- Buckner CD, Neiman PE, Clift RA, et chronic phase: Increased risk for fan and seizures. Ann Intern Med 1989; al. Clinical manifestations of graft-ver- relapsed associated with T-cell deple- 111:543-4. sus-host disease in human recipients of tion. Ann Intern Med 1988;108:806-14. 9. Fried W, Kedo A, Barone J. Effects of marrow from HLA-matched sibling 26. Weiden PL, Flournoy N, Sanders JE, cyclophosphamide and busulfan on donors. Transplantation 1974;18:295- Sullivan KM, Thomas ED. Anti- spleen colony forming units and on 304. leukemic effect of graft-versus-host dis- hematopoietic stroma. Cancer Res 18. Pugatsch T, Oppenheim A, Slavin S. ease contributes to improved survival 1977;37:1205-9. Improved single-step PCR assay for sex after allogeneic marrow transplanta- 10. Hays EF, Hale L, Villarreal B, Fitchen JH. identification post-allogeneic sex-mis- tion. Transplant Proc 1981; 13:248-51. “Stromal” and hemopoietic stem cell matched BMT. Bone Marrow Trans- 27. Weiden PL, Sullivan KM, Fluornoy N, abnormalities in long-term cultures of plant 1996;17:273-5. Storb R, Thomas ED. Antileukemic marrow from busulfan-treated mice. 19. Nakamura Y, Leppert M, O'Connell P, effect of chronic graft-vs-host disease: Exp Hematol 1982;10:383-92. Wolff R, Holm T, Culver M, et al. contribution to improved survival after

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allogeneic marrow transplantation. N with high-dose cyclosporine A after 37. Slavin S, Naparstek E, Nagler A, Eng J Med 1981;304:1529-33. allogeneic marrow transplantation for Ackerstein A, Samuel S, Kapelushnik J, 28. Horowitz MM, Gale RP, Sondel PM, acute leukemia. Blood 1991;77:1423-8. et al. Allogeneic cell therapy with Goldman JM, Kersey J, Kolb HJ, et al. 33. Prigozhina T, Gurevitch O, Slavin S. donor peripheral blood cells and Graft-vs-leukemia reactions after Non-myeloblative conditioning to recombinant human interleukin-2 to bone marrow transplantation. Blood induce bilateral tolerance after allo- treat leukemia relapse post allogeneic 1990;75:555-62. geneic bone marrow transplantation in bone marrow transplantation. Blood 29. Boranic M, Tonkovic I. Time pattern mice. Exp Hematol 1999;27:1503-10. 1996;87:2195-204. of the antileukemia effect of graft-vs- 34. Rao SS, Peters SO, Crittenden RB, 38. Kolb HJ, Mittermuller J, Clemm C, host reaction in mice. I. Cellular Stewart FM, Ramshaw HS, Quesen- Holler E, Ledderose G, Brehm G, et al. events. Cancer Res 1971;31:1140-7. berry PJ. Stem cell transplantation in Donor leukocyte transfusions for 30. Weiss L, Reich S, Slavin S. Effect of the normal nonmyeloablated host: treatment of recurrent chronic myel- cyclosporine A and methylpredni- relationship between cell dose, sched- ogenous leukemia in marrow trans- solone on the GVL effect across major ule, and engraftment. Exp Hematol plant patients. Blood 1990;76:2462-5. histocompatibility barriers in mice fol- 1997;25:114-21. 39. Kolb HJ, Schattenberg A, Goldman lowing allogeneic bone marrow trans- 35. Bitan M, Or R, Shapira MY, Ackerstein JM, Hertenstein B, Jacobsen N, Arcese plantation. Bone Marrow Transplant A, Samuel S, Slavin S. Non-myeloabla- W, et al. Graft-versus-leukemia effect 1990;6:229-33. tive stem cell transplantation using of donor lymphocyte transfusions in 31. Higano CS, Brixey M, Bryant EM. lymphoablative rather than myeloab- marrow grafted patients: European Durable complete remission of acute lative conditioning in the pre-fludara- Group for Blood and Marrow non-lymphocytic leukemia associated bine era by ATG and limiting doses of Transplantation Working Party with discontinuation of immunosup- cyclophosphamide. Bone Marrow Chronic Leukemia. Blood 1995; 86: pression following relapse after allo- Transplant 2005;35:953-8. 2041-50. geneic bone marrow transplantation. 36. Slavin S, Naparstek E, Nagler A, 40. Slavin S, Weiss L, Morecki S, A case report of a probable graft-vs- Ackerstein A, Kapelushnik J, Brautbar Weigensberg M. Eradication of murine leukemia effect. Transfusion Trans- C, et al. Allogeneic cell therapy for leukemia with histoincompatible mar- plant 1990;50:175-7. relapsed leukemia following bone row grafts in mice conditioned with 32. Bacigalupo A, Van Lint MT, Occhini marrow transplantation with donor total lymphoid irradiation (TLI). D, Gualandi F, Lamparelli T, Sogno G, peripheral blood lymphocytes. Exp Cancer Immunol Immunother 1981; et al. Increased risk of leukemia relapse Hematol 1995;23:1553-62. 11:155-8.

Provence, France ROBIN FOÀ

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