Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML.

A total of 24 patients (median age 58; range, 27-71 years) with chronic myeloid leukemia (CML) in first chronic (CP1) (n=14), second chronic (n=4), or accelerated phase (n=6) who were not candidates for conventional hematopoietic cell transplantation (HCT), received nonmyeloablative HCT from HLA-matched siblings a median of 28.5 (range, 11-271) months after diagnosis. They were conditioned with 2 Gy total body irradiation (TBI) alone (n=8) or combined with fludarabine, 90 mg/m(2) (n=16). Postgrafting immunosuppression included cyclosporine and mycophenolate mofetil. All patients initially engrafted. However, 4 of 8 patients not given fludarabine experienced nonfatal rejection while all others had sustained engraftment. With a median follow-up of 36 (range, 4-49) months, 13 of 24 patients (54%) were alive and in complete remission. There were five (21%) deaths from nonrelapse mortality, one (4%) during the first 100 days after transplant. The proportions of grade II, III, and IV acute GVHD were 38, 4, and 8%, respectively. The 2-year estimate of chronic GVHD was 32%. The 2-year survival estimates for patients in CP1 (n=14) and beyond CP1 (n=10) were 70 and 56%, respectively. This study shows encouraging remission rates for patients with CML not eligible for conventional allografting.

Non-myeloablative hematopoietic stem cell transplantation.

Conventional approaches to allogeneic stem cell transplantation have used toxic high-dose conditioning therapy in attempts to eradicate underlying diseases and achieve allogeneic engraftment. Preclinical studies and clinical observations have shown that to achieve engraftment conditioning regimens could be markedly reduced in intensity with reduction in treatment toxicities. The use of potent pre- and postgrafting immunosuppression facilitated stable mixed hematopoietic chimerism in a preclinical canine model. The initial clinical experiences with attenuated conditioning regimens have shown promise as a modality to achieve human stem cell transplantation with an improved safety profile. This may allow offering potentially curative hematopoietic stem cell transplantation (HSCT) to a more representative patient population (older and sicker) who are currently not eligible for such therapy. Obtaining a state of mixed hematopoietic chimerism could prove curative of the disease phenotype of various nonmalignant disturbances of the hematopoietic and immune systems. On the other hand, patients with hematopoietic malignancy will likely require conversion to full donor hematopoeisis by virtue of graft-versus-host (GVH) reactions directed against both recipient hematopoiesis and underlying malignancy. The infusion of additional donor lymphocytes has been proposed by many groups to augment graft versus tumor responses, but most likely more specific strategies will need to be developed to improve efficacy and avoid nonspecific GVH reactions.

Nonmyeloablative hematopoietic stem cell transplantation: transplantation for the 21st century.

Conventional approaches to allogeneic stem cell transplantation have used toxic high-dose conditioning therapy to achieve allogeneic engraftment and control of underlying disease. For engraftment purposes, preclinical studies and clinical observations have shown that conditioning regimens can be markedly reduced in intensity, resulting in reduced treatment toxicities. Preclinical canine studies demonstrated that the use of potent pre- and postgrafting immunosuppression allows for reduction in conditioning regimens while facilitating development of stable mixed chimerism. If attenuated conditioning regimens can be successfully translated to human stem cell transplantation, an improved safety profile will allow potentially curative treatment to a more representative patient profile not currently offered such therapy. Mixed chimerism could prove curative of disease phenotype of various nonmalignant disturbances of the hematopoietic and immune systems. For patients with hematopoietic malignancy, spontaneous conversion to full donor hematopoeisis after stem cell transplant may prove curative by virtue of graft versus host reactions directed against the malignancy, however infusion of additional donor lymphocytes may be needed to treat persistent disease.

Randomized, placebo-controlled, double-blind study of a cytomegalovirus-specific monoclonal antibody (MSL-109) for prevention of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

MSL-109 is a monoclonal antibody specific to the cytomegalovirus (CMV) glycoprotein H with high neutralizing capacity. In a prospective, randomized, double-blind study, allogeneic hematopoietic stem cell transplantation (HSCT) recipients with positive donor and/or recipient serology for CMV before transplantation received either 60 mg/kg MSL-109 (n = 59), 15 mg/kg MSL-109 (n = 60), or placebo (n = 60) intravenously every 2 weeks from day -1 until day 84 after transplantation. CMV pp65 antigenemia, CMV-DNA load in plasma, and viremia by culture were tested weekly. Primary end points were development of pp65 antigenemia at any level and/or viremia for which ganciclovir was given. There was no statistically significant difference in CMV pp65 antigenemia or viremia among patients in the 60-mg group (pp65 antigenemia, 47%; viremia, 15%), the 15-mg group (52%; 23%), and the placebo group (45%; 17%). There was also no difference in maximum levels of pp65 antigenemia, time to clearance of pp65 antigenemia after start of ganciclovir, CMV disease, invasive bacterial and fungal infections, time to neutrophil and platelet engraftment, acute graft-versus-host disease, days of hospitalization, and overall survival rate among the 3 groups. However, a subgroup analysis of CMV-seronegative recipients with a seropositive donor (D+/R-) showed a transiently improved survival rate by day 100 in MSL-109 recipients (mortality: 60-mg group, 1/13; 15-mg group, 1/12; placebo group, 6/10 [P = .02 for 60-mg versus placebo groups; P = .08 for 15-mg versus placebo groups]); by the end of follow-up, the difference was no longer statistically significant. The improved survival rate in D+/R- patients could not be attributed to a reduction in CMV disease; however, MSL-109 was associated with improved platelet engraftment and less grade III to IV acute graft-versus-host disease in this subgroup. In a subgroup analysis of CMV-seropositive recipients of MSL-109 (D+/R+ and D-/R+), overall mortality was increased compared to that of the placebo group (P = .12 for the 60-mg versus placebo groups, P = .05 for the 15-mg versus placebo groups, and P = .04 for the dose levels combined versus placebo). MSL-109 was well tolerated and no immune response to the drug was observed. Thus, MSL-109 was safe but did not reduce CMV infection in allogeneic HSCT recipients. The transient survival advantage seen early after transplantation in CMV D+/R- patients and the negative effect on survival in seropositive patients remain unexplained. Thus, there is no evidence that MSL-109 is beneficial in CMV-seropositive HSCT recipients.

Nonmyeloablative hematopoietic cell transplantation. Replacing high-dose cytotoxic therapy by the graft-versus-tumor effect.

Conventional allografting produces considerable regimen-related toxicities that generally limit this treatment to patients younger than 55 years and in otherwise good medical condition. T cell-mediated graft-versus-tumor (GVT) effects are known to play an important role in the elimination of malignant disease after allotransplants. A minimally myelosuppressive regimen that relies on immunosuppression for allogeneic engraftment was developed to reduce toxicities while optimizing GVT effects. Pre-transplant total-body irradiation (200 cGy) followed by post-transplant immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) permitted human leukocyte antigen (HLA)-matched sibling donor hematopoietic cell engraftment in 82% of patients (n = 55) without prior high-dose therapy. The addition of fludarabine (90 mg/m2) facilitated engraftment in all 28 subsequent patients. Overall, fatal progression of underlying disease occurred in 20% of patients after transplant. Non-relapse mortality occurred in 11% of patients. Toxicities were low. Grade 2-4 acute graft-versus-host disease (GVHD) associated with primary engraftment developed in 47% of patients, and was readily controlled in all but two patients. Donor lymphocyte infusions (DLI) were not very effective at converting a low degree of mixed donor/host chimerism to full donor chimerism; however, the addition of fludarabine reduced the need for DLI. With a median follow-up of 244 days, 68% of patients were alive, with 42% of patients in complete remission, including molecular remissions. Remissions occurred gradually over periods of weeks to a year. If long-term efficacy is demonstrated, such a strategy would expand treatment options for patients who would otherwise be excluded from conventional allografting.

Rapid engraftment after allogeneic transplantation of density-enriched peripheral blood CD34+ cells in patients with advanced hematologic malignancies.

BACKGROUND: Acute graft versus host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Preclinical studies have suggested that a T-cell subset with a CD4-/CD8- double-negative (DN) T-cell phenotype is capable of suppressing GVHD. Double-negative T cells can be mobilized into the peripheral blood with granulocyte colony-stimulating factor (G-CSF) and enriched by density centrifugation. The current study was performed to study the feasibility and safety of applying a density gradient separation technique for enrichment of CD34+ and DN T cells, while depleting CD4+ and CD8+ single-positive (SP) T cells from peripheral blood progenitor cells (PBPCs) for the purpose of allogeneic transplantation. METHODS: Twenty-five patients with advanced hematologic malignancies were treated with a myeloablative preparative regimen consisting of fractionated total body irradiation, etoposide, and cyclophosphamide. Human leukocyte antigen identical donors were mobilized with G-CSF PBPC collected by apheresis. The apheresis product was applied to a single-step density gradient, and the low-density cell population was collected. The low-density cell population was infused as the sole source of allogeneic cells after myeloablative therapy. Graft versus host disease prophylaxis consisted of cyclosporine with or without prednisone. RESULTS: CD34 cell recovery was efficient with a median 72% yield, providing for a median CD34+ cell dose of 6.5 x 10(6)/kg (range,1.0- 13.9 x 10(6)/kg). CD3+CD4+ or CD3+CD8+ SP T cells were depleted by a median of 94.4% (range, 58.8- 99.2%), and the ratio of CD34+:SP T cells increased 10-fold. Double-negative T cells were depleted by 92% (range, 18.8- 99.4%), thus the ratio of DN:SP T cells increased less than 2-fold in 71% of apheresis samples tested. Hematopoietic engraftment was rapid, and there was no occurrence of graft failure in examinable patients. Median time to absolute neutrophil count greater than 0.5 x 10(9)/L and platelet count greater than 20 x 10(9)/L was 10.5 and 12 days, respectively. The incidence of Grade 2-4 acute GVHD was 26% (95% confidence interval [CI], 6-45%), although not all patients were examinable due to an unexpectedly high nonrecurrence mortality that at Day 180 was 62% (95% CI, 40-83%). CONCLUSIONS: These data suggest that T-cell subset manipulation via density gradient separation is a safe procedure and allowed rapid hematopoietic recovery. Selective enrichment of a donor DN T-cell subset was observed in only a few and was not associated with a reduced incidence of GVHD. However, the low-density selected cells still resulted in GVHD, and there was a high treatment-related mortality.

High-dose therapy and autologous hematopoietic-cell transplantation for follicular lymphoma beyond first remission: the Stanford University experience.

A retrospective analysis was performed to investigate the outcome of high-dose therapy (HDT) and autologous hematopoietic cell transplantation in patients with follicular lymphomas beyond first remission. Ninety-two patients with primary induction failure or relapsed follicular low-grade lymphoma (FLGL), follicular large cell lymphoma (FLCL), and transformed follicular lymphoma (TFL) were treated with myeloablative therapy consisting of etoposide (60 mg/kg), cyclophosphamide (100 mg/kg), and either carmustine (BCNU;15 mg/kg) or fractionated total body irradiation (FTBI; 1200 cGy) followed by transplantation of purged autologous bone marrow or peripheral blood hematopoietic cells. For the 49 patients with relapsed FLGL, the median age was 49 years and the median interval from diagnosis to HDT was 30 months. The 4-year estimate of overall survival (OS) was 60% (95% confidence interval [CI], 45%-75%) and of disease-free survival (DFS) was 44% (95% CI, 29%-59%). Treatment with the FTBI-containing HDT regimen was associated with significantly longer DFS (P = .04) and OS (P = .04) in our multivariate analysis. OS was also significantly longer among those treated with 3 or fewer chemotherapy regimens. For the 26 FLCL patients, the median age was 51 years and in 31% the indication for HDT was primary induction failure. For FLCL patients, the 4-year estimate of OS was 58% (95% CI, 37%-79%) and of DFS was 51% (95% CI, 30%-72%). Among the 17 patients with TFL, 13 (76%) transformed at first relapse, and only 6 patients (35%) achieved complete remission with salvage therapy prior to HDT. For TFL patients, the 4-year estimate of OS was 50% (95% CI, 24%-76%) and of DFS 49% (95% CI, 20%-78%). There were 3 occurrences of myelodysplasia (1 after treatment with TBI, 2 after BCNU treatment), yielding an estimated incidence of 7% (95% CI, 0%-16%) at 56 months. This analysis shows that relapsed FLGL patients treated with 3 or fewer different chemotherapy regimens show inferior survival. The HDT regimen containing FTBI appears to be superior to the BCNU-based regimen for relapsed FLGL, although longer follow-up is needed to evaluate late effects. Lastly, patients with TFL or induction failure and relapsed FLCL can achieve survival outcome comparable to those observed with the indolent follicular lymphomas.

Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects.

Toxicities have limited the use of allogeneic hematopoietic cell transplantation (HCT) to younger, medically fit patients. In a canine HCT model, a combination of postgrafting mycophenolate mofetil (MMF) and cyclosporine (CSP) allowed stable allogeneic engraftment after minimally toxic conditioning with low-dose (200 cGy) total-body irradiation (TBI). These findings, together with the known antitumor effects of donor leukocyte infusions (DLIs), led to the design of this trial. Forty-five patients (median age 56 years) with hematologic malignancies, HLA-identical sibling donors, and relative contraindications to conventional HCT were treated. Immunosuppression involved TBI of 200 cGy before and CSP/MMF after HCT. DLIs were given after HCT for persistent malignancy, mixed chimerism, or both. Regimen toxicities and myelosuppression were mild, allowing 53% of eligible patients to have entirely outpatient transplantations. Nonfatal graft rejection occurred in 20% of patients. Grades II to III acute graft-versus-host disease (GVHD) occurred in 47% of patients with sustained engraftment. With median follow-up of 417 days, survival was 66.7%, nonrelapse mortality 6.7%, and relapse mortality 26.7%. Fifty-three percent of patients with sustained engraftment were in complete remission, including 8 with molecular remissions. This novel allografting approach, based on the use of postgrafting immunosuppression to control graft rejection and GVHD, has dramatically reduced the acute toxicities of allografting. HCT with the induction of potent graft-versus-tumor effects can be performed in previously ineligible patients, largely in an outpatient setting. Future protocol modifications should reduce rejection and GVHD, thereby facilitating studies of allogeneic immunotherapy for a variety of malignancies. (Blood. 2001;97:3390-3400)

Cyclophosphamide and antithymocyte globulin to condition patients with aplastic anemia for allogeneic marrow transplantations: the experience in four centers.

This report summarizes the experience with a conditioning regimen of cyclophosphamide and antithymocyte globulin in patients with severe aplastic anemia given HLA-matched related marrow grafts at 4 transplantation centers. Enrolled were 94 consecutive patients, of whom 87 had received multiple transfusions and 38 had failed immunosuppressive therapy. Their ages ranged from 2 to 59 years. After transplantation, 89 patients received a methotrexate/cyclosporine regimen for graft-versus-host disease (GVHD) prevention. Cyclosporine with or without prednisone was given in 4 patients, and no immunosuppression was given in 1 patient. Ninety-six percent of patients had sustained grafts, whereas 4% rejected grafts between 2 and 7 months after transplantation. Of the 4 rejecting patients, 3 are alive with successful second engraftments. Acute grade II GVHD was seen in 21% of patients, grade III in 7%, and grade IV in 1% of patients. Chronic GVHD was seen in 32% of patients, most of whom responded completely to immunosuppressive therapy. With a median follow-up of 6.0 years (range, 0.5-11.6 years), the survival rate was 88%. No unusual long-term side effects have been seen with the regimen. We conclude that the cyclophosphamide/antithymocyte globulin regimen combined with methotrexate/cyclosporine after transplantation is well tolerated and effective in heavily pretreated patients with aplastic anemia.

High-dose therapy and autologous bone marrow transplantation for follicular lymphoma in first complete or partial remission: results of a phase II clinical trial.

Advanced stage follicular small cleaved and mixed cell lymphoma is characterized by relapse from remission and survival ranging from 6 to 12 years. Because young patients have the greatest compromise in longevity, the efficacy and toxicity of high-dose radiochemotherapy and bone marrow transplantation after conventional chemotherapy was evaluated in a prospective phase II clinical trial. Thirty-seven patients in a minimal disease state after conventional chemotherapy received fractionated total body irradiation and high-dose etoposide and cyclophosphamide, followed by purged autologous bone marrow. A reference sample of 188 patients of similar age, stage, and histology managed at this institution before 1988 was identified for comparison of patient characteristics and outcomes. Compared with reference patients, transplant recipients had a higher tumor burden at diagnosis. With a median follow-up of 6.5 years, the estimated 10-year survival after transplantation was 86%. There was a single lymphoma death yielding a 10-year disease-specific survival of 97%. There were 2 early transplant-related deaths and 2 late acute leukemia deaths. Ten patients relapsed, one with microscopic disease only. High tumor burden at diagnosis and incomplete response to chemotherapy adversely influenced survival in the reference but not in the transplanted patients. The estimated risk of death of 14% and relapse of 30% at 10 years in our transplanted follicular lymphoma patients, the majority of whom had high tumor burdens, compares favorably with our observations in appropriately matched reference patients.

Efficacy and toxicity of a CCNU-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory Hodgkin's disease.

High-dose CBV (cyclophosphamide, carmustine, and etoposide) in combination with autologous HCT achieves survival rates of approximately 50% at 5 years in recurrent or refractory Hodgkin's disease (HD). However, carmustine (BCNU) dose-dependent pulmonary toxicity occurs in 20% to 30% of patients. A decreased incidence of interstitial pneumonitis as well as a possible benefit in efficacy has been reported with lomustine (CCNU) compared to BCNU in the standard dose setting. In a dose-escalation study, we substituted CCNU for BCNU in the CBV regimen for 16 patients with HD (n = 12) or non-Hodgkin's lymphoma (n = 4). Based on the promising results, an additional 47 consecutive patients with HD were treated with the following regimen: CCNU (15 mg/kg) orally on day -6, etoposide (60 mg/kg) intravenously on day -4, and cyclophosphamide (100 mg/kg) intravenously on day -2. Peripheral blood progenitor cells and/or bone marrow were infused on day 0. With a median follow-up for the surviving patients of 3.2 years (range, 0.8-9.9 years), the 3-year overall survival rate was 57% (CI, +/-15%), event-free survival was 52% (CI, +/-14%), and freedom from progression was 68% (CI, +/-14%). There were 21 deaths, 10 due to HD. Six patients died due to respiratory failure. Interstitial pneumonitis occurred in 63% of patients and could not be correlated with prior chest radiotherapy. This regimen demonstrated survival rates similar to those of historical studies that used the CBV regimen. However, the incidence of interstitial pneumonitis was in excess of expected.

Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma.

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

High-dose chemotherapy and hematopoietic stem cell rescue for breast cancer: experience in California.

The role of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell rescue in breast cancer is still controversial. We analyzed the outcomes of 1111 consecutive patients with histologically proven breast cancer who underwent HDCT at 5 major California medical centers. The overall treatment-related mortality (TRM) was 2.3%. TRM was not influenced by disease stage or the HDCT regimen delivered, but it was influenced by hematopoietic graft source. The TRM was 6.1% when bone marrow with or without blood stem cells was used, but only 1.4% when blood stem cells alone were used (P < .001). With a median follow-up of 2.8 years (range, 0.1-8.2 years) after HDCT and autologous hematopoietic stem cell rescue, the estimated 5-year event-free survival (EFS) and overall survival (OS) for stage II/IIIA patients with > or =10 involved axillary lymph nodes were 67% and 76%, respectively. Patients with metastatic breast cancer (MBC) (median follow-up, 1.9 years [range, 0.03-8.3 years]) achieving a complete response (CR) to conventional-dose chemotherapy or rendered to a "no evidence of disease" status before HDCT had significantly better estimated 5-year EFS and OS (28% and 57%, respectively) than those achieving a partial response before HDCT (19% and 27%, respectively; P < or = .0001). Our data suggest that HDCT with hematopoietic stem cell rescue is safe and can be beneficial to patients with high-risk primary breast cancer and for those with MBC achieving CR/no evidence of disease.

Favorable treatment outcome in non-Hodgkin's lymphoma patients with "poor" mobilization of peripheral blood progenitor cells.

Our purpose was to evaluate the outcome and costs of high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation in patients with the inability to mobilize sufficient numbers of PBPCs to allow rapid engraftment after PBPC transplantation. We treated 172 consecutive non-Hodgkin's lymphoma (NHL) patients with cyclophosphamide and granulocyte colony-stimulating factor followed by apheresis to collect PBPCs. The cells were separated on a Percoll gradient and purged with monoclonal antibodies and complement. The patients were categorized as "good" mobilizers if a collection of > or =2 x 10(6) CD34+ cells/kg was obtained (n = 138, 80%) or "poor" mobilizers if <2 x 10(6) CD34+ cells/kg were obtained (n = 34, 20%). With a median follow-up of 3.5 years, there is no statistically significant difference in actuarial event-free survival, overall survival, or relapse for good mobilizers compared with poor mobilizers. However, there was a trend toward increasing nonrelapse, transplantation-related mortality of 11.8% for poor mobilizers versus 3.6% for good mobilizers (P = .08) and early death from all causes including relapse within 120 days (poor 20.6% versus good 8.7%, P = .06). The total cost for bone marrow transplantation-related care was significantly higher, at $140,264 for poor mobilizers versus $80,833 for good mobilizers (P = .0001). The population of patients with NHL who mobilize PBPCs poorly into the circulation have a higher cost for posttransplant support. However, there is no significant difference in relapse, event-free survival, or overall survival for such patients compared with those who mobilize PBPCs easily.

Acute safety and pharmacokinetics of intravenous busulfan when used with oral busulfan and cyclophosphamide as pretransplantation conditioning therapy: a phase I study.

The unpredictable intestinal absorption and erratic bioavailability of oral busulfan (Bu) has limited the drug's use in high-dose pretransplantation conditioning therapy. To standardize drug delivery, we solubilized Bu for parenteral use. This new intravenous (i.v.) Bu formulation was combined with oral Bu and cyclophosphamide (Cy) to evaluate (1) the human acute toxicity of i.v. Bu and its solvent system and (2) the pharmacokinetics of Bu in patients undergoing hematopoietic progenitor cell transplantation (HPCT). One dose of i.v. Bu (escalating from 0.08 to 0.8 mg/kg) was given over 2 hours by pump; 6 hours later, an oral Bu regimen was begun, consisting of 1 mg/kg every 6 hours for 15 doses, followed by Cy, 60 mg/kg daily for 2 days. After 1 day of rest, HPCT was performed. The i.v. Bu dose was well tolerated and did not produce any acute toxicity reaction that could be attributed to the solvent system of dimethylacetamide and polyethylene glycol (PEG)-400. All observed treatment-related toxicity was as would be expected after high-dose oral Bu plus Cy. When the i.v. Bu was used as reference solution, the pharmacokinetic analysis indicated an average bioavailability of oral high-dose Bu of 69%, ranging from <10% to virtually 100%. Further, the 2-hour infusion of i.v. Bu gave a time to maximum plasma concentration following drug administration similar to that of oral Bu (2 hours and 1.8 hours, respectively), and i.v. Bu had a clearance similar to that of oral Bu. Based on the data in this study, we suggest that the optimal (starting) dose of i.v. Bu (in combination with Cy) in our forthcoming phase 2 trial should be on the order of 0.8 mg/kg to target an area under the curve (AUC) of 1100 to 1200 micromol/L per minute. This would secure myeloablation and engraftment but save the vast majority of patients from the increased risk of serious hepatic veno-occlusive disease that has been reported when the AUC level exceeds 1500 micromol/L per minute. Bu administration via the i.v. route will assure complete bioavailability and reliable systemic drug exposure with more predictable blood levels and, therefore, possibly lower the risks for serious/life-threatening toxicity, graft rejection, and recurrent leukemia.