Combination Of Busulfan Research Articles

High Dose Intravenous Busulfan (BU) and Melphalan (MEL) Followed by Bortezomib (BTZ) as Conditioning with Autologous Peripheral Blood Stem Cell Transplantation (ASCT) for Patients with Multiple Myeloma (MM)

AbstractAbstract 1355 Background:Overall survival for patients with MM has improved significantly over the past 10 years primarily as a result of novel therapeutics. High dose therapy with ASCT continues to be an effective modality but the progression-free survival following ASCT has improved minimally over the same period of time due to a continued reliance on single agent melphalan. Many chemotherapy/chemoradiotherapy regimens have been used in preparation for stem cell transplantation. However, no regimen has proven superior to high dose melphalan 200 mg/ m2 (MEL 200) which is considered the standard conditioning for patients with MM with relatively predictable results. In order to improve progression free survival (PFS) and overall survival (OS), continued efforts in developing effective preparative regimens are needed. Busulfan is an alkylating agent that affects cells in an AUC-dependent manner. Recent data suggests that the combination of BU and MEL delivers better PFS compared to MEL 200 alone (Grande and Lahuerta; PETHEMA, 36th EBMT meeting. Vienna 2010). Moreover, the addition of bortezomib following MEL 200 appears to increase the VGPR rate when compared to historical cohort receiving MEL 200 alone (Lonial, et al; Blood. ASH Annual Meeting Abstracts, Nov 2008; 112: 3332). Furthermore, the recently published IFM trial of MEL 200 plus BTZ also demonstrated superior response rates and PFS compared to historical controls (Roussel et al Blood. 2010;115:32-7). Rationale:The combination of intravenous BU and MEL followed by BTZ (BuMelVel) will be an effective preparative regimen with acceptable toxicity for patients with MM. Methods:Patients received intravenous BU 130 mg/kg over 3 hours daily × 4 days from D-6 to D-3. Based on the pharmacokinetics of the first two doses, the subsequent 2 doses were adjusted to achieve an AUC of 20,000 mMol-min (5,000 mMol-min/day). MEL was administered at 140 mg/ m2 IV over 15–30 minutes on day -2. Bortezomib was administered IV push over 3–5 seconds on day -1. Bortezomib administration followed a phase I dose escalation design starting at 1.0 mg/m2 for the first cohort; then, 1.3 mg/m2 for the second cohort; then, 1.6 mg/m2/kg for the 3rd cohort and subsequent subjects at the 3rd dose level if no limiting toxicity was observed. Autologous stem cell infusion occurred on day 0. Results:20 patients have been enrolled to date, with 19 evaluable for toxicity and 13 for response assessment at D +100. The median age is 61 (47 - 69). The median time for engraftment for ANC ≥ 500 and plts ≥ 20, was 10 days. The median length of stay (LOS) was 18 days (16 – 21 days). 12 patients required a median of 2 units of PRBC and 16 patients required a median of 1 platelet transfusion. PRBC and PLT transfusions as well as LOS are not significantly different to what has been observed at our institution with MEL 200 (2 PRBC, 2 RD PLT transfusions, and 15 days LOS, respectively). All patients responded including: 77% ≥ VGPR and 54% nCR, CR, or sCR. The most common grade ≥ 3 toxicities were neutropenic fever (n = 11), mucositis (n = 9), and hypophosphatemia (n = 7). Three patients developed reversible transaminitis. One patient developed Clostridium difficile colitis and Klebsiella pneumonia UTI. Another patient developed E. coli UTI. No cases of VOD or interstitial pneumonitis were observed. There were no treatment related deaths and no re-admissions post discharge. Conclusion:BuMelVel proved to be an effective preparative regimen with ≥ VGPR and nCR/CR/sCR of 77% and 54%, respectively, as compared to responses reported with MEL/BTZ (70% and 32%, respectively); and to MEL 200 alone (43% and 11%, respectively) by Roussel et al. Furthermore, BuMelVel had an acceptable toxicity profile without any cases of VOD or interstitial pneumonitis, and mucositis/infections incidence and severity are comparable to our institutional experience with MEL 200. Therefore, the regimen of BuMelVel may lead to improvements in PFS and OS, critical criteria to improve the outcomes of patients with MM. These promising results will be further evaluated in a planned Phase III clinical trial with MEL 200. Disclosures:Rodriguez:Otsuka: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. Kaufman:Celgene, Millenium: Consultancy; Celgene, Merck: Research Funding. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Speakers Bureau.

Successful Reduced-Intensity Stem Cell Transplantation From Unrelated Cord Blood In Three Patients with X-Linked Severe Combined Immunodeficiency.

AbstractAbstract 4571Severe combined immunodeficiency (SCID) is the most severe form of the primary immunodeficiencies (PID), the most frequent type of which is X-linked SCID (X-SCID; T-B+NK-SCID). Hematopoietic stem cell transplantation (SCT) is the only curative treatment for these high-risk patients. Recently, some PID cases treated with unrelated cord blood stem cell transplantation (CBT) have been reported. To avoid severe infection during SCT and late complications, such as mental and growth retardation, reduced-intensity conditioning (RIC) regimens have been used for unrelated SCT for PID patients. However, reports of X-SCID patients receiving CBT with reduced-intensity stem cell transplantation (RIST) are quite limited. We report successful allogenic RIST from unrelated cord blood (CB) for treatment of X-SCID in our single centre. Three patients with X-SCID who had no HLA-matched sibling received unrelated CBT. Mutations in the common gamma chain gene were detected in all patients. Pre-transplantation conditioning for all patients consisted of fludarabine (FLU) (30 mg/m2/day) from day -7 to day -2 (total dose 180 mg/m2) and busulfan (BU) 4 mg/kg/day from day -3 to day -2 (total dose 8 mg/kg). All CB units were serologically matched HLA-A, B, and DR loci. Though two of the patients suffered from fungal or bacterial pneumonia before transplantation, there were no additional infectious complications during transplantation. All patients achieved engraftment with an absolute neutrophil count > 500/μ l with a mean of 22 days (range, 19–27 days), and they showed 100% donor chimerism by fluorescence in situ hybridization of their peripheral mononuclear cells using × and Y chromosome probes at one-year post transplantation. Full donor chimerism of both T and B cells was also confirmed. Only one patient (patient 2) developed grade III acute graft versus host disease (GVHD), which resolved with increased oral corticosteroid. None of the patients have developed chronic GVHD, received intravenous immunoglobulin replacements after the transplantations, or showed delayed psychomotor development during 21 to 77 months of follow-up. Only one patient (patient 3) had short stature (-2.4SD) at 21-month follow-up. The most commonly used RIC regimens worldwide have been the FLU/melphalan (LPAM) and the FLU/BU regimens. LPAM has broad stem cell toxicity to both primitive and committed stem cells, while BU may spare committed stem cells, resulting in early onset and a prolonged duration of neutropenia with the FLU/LPAM regimen. Moreover, it has been reported that the LPAM-containing conditioning regimen is a risk factor for hepatic veno-occlusive disease. Although conditioning regimens including standard-dose BU may be associated with a high rate of treatment-related complications due to organ toxicity, reduced-dose BU in combination with FLU is less myelosuppressive and less toxic than the FLU/LPAM regimen. Moreover, CBT has some advantages with regard to incidence and intensity of GVHD and availability compared with other alternative stem cell sources. Patients 1 and 2 had overcome pre-existing fungal and bacterial pneumonias, respectively, and recovered after transplantation. Thus, immediate SCT using an RIC regimen could be indicated for patients with X-SCID to reconstitute the immune system regardless of pre-existing infections. Since pre-existing infections are the principal risk factors for poor SCT outcomes, early diagnosis and transplantation before any infectious episodes is necessary for X-SCID patients. In conclusion, CBT is a suitable alternative to bone marrow transplantation for X-SCID patients requiring urgent SCT with no sibling donors. RIC consisting of FLU and BU is an effective and safe treatment for such cases. Disclosures:No relevant conflicts of interest to declare.

The synergistic cytotoxicity of clofarabine, fludarabine and busulfan in AML cells involves ATM pathway activation and chromatin remodeling

DNA alkylating agents alone or with ionizing radiation have been the preferred conditioning treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In search of less toxic alternatives, we hypothesized that combination of busulfan (Bu), fludarabine (Flu) and clofarabine (Clo) would provide superior efficacy. At low concentrations, these drugs show synergistic cytotoxicity in Bu-resistant AML KBM3/Bu250 6 cells. Similar molecular responses were observed in other AML cell lines and in primary explanted AML cells. The [Clo + Flu + Bu] combination activates an intense DNA damage response through the ATM pathway, leading to cell cycle checkpoint activation and apoptosis. Phosphorylations of SMC1 and SMC3, and methylations of histones 3 and 4, are much more pronounced in cells exposed to [Clo + Flu + Bu] than [Clo + Flu], suggesting their relevance in the efficacy of the triple-drug combination. A possible mechanism for these observed synergistic effects involves the capability of [Clo + Flu] to induce histone methylations and subsequent chromatin remodeling, which may render the genomic DNA more accessible to Bu alkylation. The Bu-mediated DNA cross-linking may provide a feedback loop which perpetuates the DNA damage response initiated by [Clo + Flu] and commits the cells to apoptosis. Our results provide a conceptual mechanistic basis for exploring this triple-drug combination in pretransplant conditioning therapy for allo-HSCT.

Pharmacokinetic targeting of intravenous busulfan reduces conditioning regimen related toxicity following allogeneic hematopoietic cell transplantation for acute myelogenous leukemia

Optimal conditioning therapy for hematopoietic cell transplantation (HCT) in acute myelogenous leukemia (AML) remains undefined. We retrospectively compared outcomes of a consecutive series of 51 AML patients treated with oral busulfan (1 mg/kg every 6 hours for 4 days) and cyclophosphamide (60 mg/kg IV × 2 days) - (Bu/Cy) with 100 consecutive AML patients treated with pharmacokinetic targeted IV busulfan (AUC < 6000 μM/L*min per day × 4 days) and fludarabine (40 mg/m2 × 4 days) - (t-IV Bu/Flu). The Bu/Cy and t-IV Bu/Flu groups significantly differed according to donor relation, stem cell source, aGVHD prophylaxis, remission status, primary vs. secondary disease, median age, and % blasts prior to HCT (p < 0.01 for each). Conditioning with t-IV Bu/Flu reduced early toxicity including idiopathic pneumonia syndrome (IPS) and hepatic veno-occlusive disease (VOD). Additionally, the trajectory of early NRM (100 day: 16% vs. 3%, and1 year: 25% vs. 15% for Bu/Cy and t-IV Bu/Flu, respectively) favored t-IV Bu/Flu. Grade II-IV aGVHD (48% vs. 82%, p < 0.0001), as well as moderate/severe cGVHD (7% vs. 40%, p < 0.0001) differed between the Bu/Cy and t-IV Bu/Flu groups, due to the predominance of peripheral blood stem cells in the t-IV Bu/Flu group. Pharmacokinetic targeting of intravenous busulfan in combination with fludarabine is associated with reduced conditioning regimen related toxicity compared to oral busulfan and cyclophosphamide. However, multivariable analysis did not demonstrate significant differences in overall survival (p = 0.78) or non-relapse mortality (p = 0.6) according to conditioning regimen delivered.

Fludarabine-based myeloablative regimen as pretransplant conditioning therapy in adult acute leukemia/myelodysplastic syndrome: comparison with oral or intravenous busulfan with cyclophosphamide

BackgroundA combination of busulfan (Bu) and cyclophosphamide (Cy) has been used as a standard myeloablative regimen for allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies postulate that fludarabine (Flu) is a less toxic substitute for Cy.MethodsForty-two patients who were diagnosed with acute leukemia or myelodysplastic syndrome and received BuFlu (n=17) or BuCy (n=25) from August, 1999 to July, 2009 at Dong-A University Medical Center were retrospectively analyzed.ResultsThe median follow-up duration was 39.75 months. The BuFlu group showed a lower incidence of mucositis (P=0.005), but there was no significant intergroup difference in the time of engraftment, nausea/vomiting, acute/chronic graft-versus-host disease, hepatic veno-occlusive disease, or hemorrhagic cystitis. Moreover, the 2 groups showed no significant difference in the cumulative risk of relapse, event-free survival, or overall survival.ConclusionBuFlu administration can be employed as a preparative regimen for allogeneic HSCT and shows efficacy and transplant-adverse effects comparable to those of BuCy. However, randomized prospective studies in more patients are warranted.

Low Toxicity and Mortality with Reversed-Order Conditioning (Cyclophosphamide Followed by Targeted Intravenous Busulfan) in Allogeneic Hematopoietic Cell Transplantation: Preliminary Results of a Prospective Clinical Trial.

Abstract 1175Poster Board I-197 BACKGROUND:Regimen-related toxicity is a significant cause of morbidity and mortality after myeloablative allogeneic hematopoietic cell transplantation (HCT). The widely-used conditioning regimen of busulfan followed by cyclophosphamide (BU/CY) has been associated with mortality rates of 12–13% at 100 days after HCT (BBMT 2007 13:355–65, Blood 2002 100:1201–7). Additionally, regimen-related liver injury in the form of moderate or severe hepatic sinusoidal obstruction syndrome (SOS) occurs in 9% of allogeneic HCT recipients conditioned with targeted BU/CY. Preclinical evidence suggests that BU, when given first in order, may deplete hepatic glutathione and potentiate the toxicity of CY, and that reversing the sequence of conditioning agents might reduce toxicity (BBMT 2007 13:853–62). We report the preliminary clinical and pharmacokinetic findings of an ongoing prospective Phase I/II clinical trial designed to test this hypothesis by reversing the standard order of conditioning agents, using CY followed by targeted IV BU. PATIENTS AND METHODS:As of June 17, 2009, 25 patients with myelofibrosis (n=14) or acute myeloid leukemia (AML)/myelodysplasia (MDS) (n=11) were enrolled and observed through day +100 after HCT. Median age was 55 (range, 34–62) years. Twelve patients (48%) had related donors, while 13 had unrelated donors. Among the 13 unrelated donors, 12 were HLA-matched while 1 was mismatched at one HLA allele. Patients received IV CY 60 mg/kg/day on days -7 and -6 before HCT, followed by IV BU (Busulfex, Otsuka Pharmaceutical) 4 mg/kg/day on days -5 through -2. After the first BU dose, subsequent doses were adjusted based on pharmacokinetic monitoring to achieve plasma BU concentrations at steady state (Css) of 800–900 ng/mL. Prophylactic phenytoin was administered on days -6 through -1, and prophylactic ursodiol was administered starting 2 weeks before HCT and continuing through day +90. All patients received G-CSF-mobilized peripheral blood stem cell grafts. Graft-vs.-host disease (GVHD) prophylaxis consisted of tacrolimus combined with methotrexate 10 mg/m2 on days +1, +3, +6, and +11. Endpoints included engraftment, liver and renal toxicity, day +100 mortality, overall survival, and pharmacokinetics of CY and its metabolites 4-hydroxycyclophosphamide (HCY) and O-carboxyethyl-phosphoramide mustard (CEPM). RESULTS:All patients initially engrafted, with neutrophil engraftment at a median of 17 (range, 11–26) days after HCT. One patient (4%) with an HLA-allele-mismatched unrelated-donor allograft had late graft failure approximately 3 months after HCT. Bilirubin values in the first 20 days after HCT peaked at a median of 1.9 (range, 0.5–12.4) mg/dL. SOS occurred in 1 patient (4%), who had a 10 kg weight gain, peak bilirubin of 2.3, and tender hepatomegaly with eventual resolution. Median peak serum creatinine in the first 20 days after HCT was 1.0 (range, 0.6–3.7) mg/dL. One patient (4%) experienced acute renal insufficiency, defined as a doubling of serum creatinine from pre-transplant baseline, within the first 20 days after HCT in the setting of septic shock. Day +100 survival was 96%; one patient died of sepsis at day +42. Kaplan-Meier estimated survival at the median follow-up of 8 months was 91% (myelofibrosis, 100% and AML/MDS, 80%). In the 6 patients who had died at last follow-up, causes of death included graft-vs.-host disease (n=2), sepsis (n=2), relapsed AML (n=1), and metastatic prostate cancer (n=1). Peak plasma concentrations of CY, HCY, and CEPM were 365.3 ± 58.2 μM, 9.5 ± 5.0 μM, and 12.7 ± 5.9 μM respectively after dose 1 of CY. After dose 2 of CY, peak plasma concentrations of CY, HCY, and CEPM were 312.9 ± 54.7 μM, 21.7 ± 8.6 μM, and 27.7 ± 11.0 μM respectively. The areas under the curve from time 0 to 48 hours for CY, HCY, and CEPM were 4737.0 ± 1361.3 μM • h, 182.4 ± 51.3 μM • h, and 508.5 ± 185.6 μM • h respectively. CONCLUSIONS:Reversing the sequence of conditioning agents in allogeneic HCT, from BU/CY to CY/BU, produced engraftment with low rates of regimen-related toxicity and 96% survival at day +100 after HCT. Our preliminary findings lend clinical support to the hypothesis that reversing the sequence of these agents may lessen exposure to toxic CY metabolites and improve clinical outcomes. Disclosures:Rezvani:Otsuka: Research Funding. Off Label Use: Describes intravenous busulfan in combination with cyclophosphamide as conditioning for allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia, myelodysplasia, and myelofibrosis.. McCune:Otsuka: Consultancy, Research Funding. Deeg:Otsuka: Research Funding.

Busulfan and Cyclophosphamide (Bu/Cy) as a Preparative Regimen for Autologous Stem Cell Transplantation in Patients with Non-Hodgkin Lymphoma: A Single-Institution Experience

High-dose chemotherapy with autologous stem cell transplantation (ASCT) has been established as a standard form of therapy for patients with non-Hodgkin lymphoma (NHL). While many high-dose chemotherapy combinations are used, no single regimen has proved superior over another. Here, we report our single center's experience in patients with NHL undergoing ASCT with the combination of busulfan and cyclophosphamide (Bu/Cy). This study is a retrospective analysis of 78 consecutive patients with NHL who underwent ASCT with Bu/Cy at Massachusetts General Hospital Cancer Center. Data were collected through review of electronic medical records. A total of 78 patients with NHL underwent ASCT with Bu/Cy preparative therapy between 1996 and 2006. Median follow-up for survivors was 5.0 years (range, 6 months to 12 years). Significant transplantation-associated complications included 9 documented bacterial infections, 4 cases of engraftment syndrome, 3 cases of hepatic veno-occlusive disease (VOD), 6 cases of cardiac complications, and 2 cases of pulmonary fibrosis. The 100-day treatment-related mortality (TRM) was 1%. At 3 years, progression-free survival (PFS) was 48% (95% confidence interval [CI]=37% to 59%) and overall survival (OS) was 65% (95% CI=53% to 74%). Our data indicate that in patients with NHL undergoing ASCT, Bu/Cy has efficacy and toxicity comparable to that of other reported regimens.

Germ Cell Transplantation Using Sexually Competent Fish: An Approach for Rapid Propagation of Endangered and Valuable Germlines

The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25°C) treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2–13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.

Experimentally induced depletion of germ cells in sub-adult Patagonian pejerrey ( Odontesthes hatcheri)

Germ cell (GC) transplantation (GCT) is a novel reproductive technology with application in seed production and conservation of endangered species. This study examined the suitability of treatment with Busulfan, a cytotoxic agent, and warm water, known to cause GC degeneration, for depletion of endogenous GCs in sub-adult Patagonia pejerrey Odontesthes hatcheri intended as hosts in GCT. In two experiments, fish were treated with six combinations of temperature (intermediate and high, 20 and 25 °C, respectively) and Busulfan (0, 20, and 40 mg/kg body weight), given intraperitoneally (ip) as a single (0 week) or repeated (0 and 4 week) dose. The effectiveness of the treatments was assessed by gonado-somatic index, histology, and (germ cell-specific) vasa gene expression after 8 weeks. Fish were allowed to recover at 17 °C for 4–8 weeks after the treatments to ascertain the permanency of the effects. The high temperature (25 °C) alone induced only incipient gonadal degeneration and germ cell loss, but was highly effective in combination with double administration of 40 mg/kg Busulfan. Males tolerated Busulfan better and were more easily depleted of germ cells than females. Animals treated for 8 weeks were severely devoid of germ cells, but were still capable of gametogenesis. Thus, the combination of Busulfan and high water temperature appeared to be efficient for depletion of GCs in adult fish; and the treated gonads retained the ability to support GC proliferation and differentiation. Furthermore, quantitative analysis of vasa transcript levels was found to be an useful to monitor the degree of gonad sterility during treatment.

Hemorrhagic Cystitis and BK Virus Infection in Children after Hematopoietic Stem Cell Transplantation.

Hemorrhagic cystitis (HC) is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of HC in adults is not fully understood and may be influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Little is known about the development of HC in children after HCST, especially about the association with BK virus infection. Therefore, we retrospectively analyzed the incidence, risk factors and BK virus association of HC in 165 consecutive children (median age, 12 years) who underwent peripheral blood stem cell (n=97; T-cell depleted: n=48) or bone marrow transplantation (n=68) between 1/2000 and 12/2006 in a single center. Fifty nine patients received autologous HSCT and 106 patients underwent allogeneic HSCT. Nineteen of the 165 patients (11.5%) developed HC after a median of 33 days (range, 1–98 days). All 19 patients with HC underwent allogeneic HSCT and showed BK viruria after transplantation. An acute graft-versus-host disease was significantly more frequent in children with HC (P < .001). Significant risk factors in univariate binary logistic analyses were age > 12 years (OR, 3.275; P < .031), use of busulfan (OR, 3.514; P < .013), use of busulfan and cyclophosphamide in combination (OR, 4.935; P < .002), and an unrelated donor (OR, 3.309; P < .043). Independent risk factors in multivariate binary logistic analyses were age > 12 years and the combination of busulfan and cyclophosphamide. We suggest that cyclophosphamide is toxic to the urinary bladder and busulfan enhances this effect. Furthermore, we analyzed the BK virus load in urine by real-time polymerase chain reaction. We found in patients without HC a significantly increased number of BK virus copies in urine in children older than 12 years (P < .009) and in children who received antithymocyte globulin (P < .001). In addition, BK virus load in urine was significantly increased in children who suffered from HC. Thirteen of 14 children with HC had a BK virus load in urine >107 copies/mL (P < .001). We observed in individual BK virus profiles an increase of BK virus copies in urine before the onset of HC. We conclude that HC in children is a disease of multiple etiologies. Allogeneic HSCT, the combination of busulfan and cyclophosphamide, age > 12 years, and an unrelated donor are risk factors for the development of HC in childhood. Increased BK virus load in urine of more than 107 copies/mL may lead to HC. Therefore, it is useful to quantify BK virus in urine in those children with above mentioned risk factors to initiate early treatment or to prevent the development of HC.

Successful unrelated mismatched cord blood transplantation in a child with malignant infantile osteopetrosis

Allogeneic hematopoietic stem cell transplantation represents the only curative option for malignant infantile osteopetrosis (MIOP), a rare disease of infants and young children, characterized by excessive accumulation of mineralized bone and abnormal hematopoiesis. We report a case of successful engraftment and stable full-donor chimerism in a patient with MIOP who underwent unrelated donor cord blood transplantation (CBT). The donor was 2-loci human leukocyte antigen (HLA)-mismatch. After a conditioning regimen based on the combination of busulfan, cyclophosphamide, total body irradiation, and antithymocyte globulin, the patient received a dose of 3.85 x 10(7)/kg of nucleated cells. Neutrophil and platelet engraftment had been achieved by day +33 and +82, respectively, and the patient was discharged home on day +89. A successful engraftment of donor hematopoiesis was demonstrated and the child experienced grade II acute graft-vs.-host disease (GVHD) involving the skin only. A remarkable but non-progressive decrease in lumbar spine bone mineral density was observed in the first nine months post-transplant. This case suggests that unrelated donor CBT may be a feasible option in case of unavailability of a fully HLA-matched related or unrelated donor.

Low treatment related mortality and improved survival in allogeneic peripheral blood stem cell transplant: Targeted intravenous Busulfan combined with Fludarabine (tBuFlu) as conditioning regimen

Myeloablative doses of intravenous busulfan in combination with fludarabine have been employed as conditioning before hematopoietic cell allografts with reduced treatment-related toxicity and mortality. In this report, we describe the early results of a targeted busulfan pharmacokinetic dosing strategy (tBuFlu) used in combination with fludarabine before either related or unrelated grafts We treated 61 pts with tBuFlu prior to allogeneic peripheral blood stem cell transplantation. The median patient age was 48 (range 22-68) years. Patient diagnoses included AML (13 de novo, 7 with prior MDS, and 7 treatment related), MDS (7 pts), MF (5), NHL (6 pts), ALL (6 pts), CML (5 pts), CLL (2), MM (2) and PNH (1). Five patients had received a prior autologous HCT. Donors were HLA-A, B, C, DRB1, DQB1 matched siblings (29), matched unrelated donors (22), or unrelated donors mismatched for one HLA antigen (6), homozygous mismatch (1), one HLA allele (2), or two HLA alleles (1). Fludarabine 40 mg/m2 was given intravenously daily for four days, with each infusion followed immediately by intravenous busulfan. The dose of busulfan for days 1 and 2 was 130 mg/m2. Pharmacokinetic analysis was performed after the first infusion of busulfan; in 59 pts, the goal was to adjust busulfan doses for days 3 and 4 to achieve an average targeted Css level of 800-1000 ng/ml. Levels were drawn incorrectly in 4 of these pts and doses were not changed. Thirty-five (59%) pts had their doses adjusted, increased in 27 and decreased in 8, while 20 pts had Css within the desired range without adjustment. Patients received tacrolimus and standard doses of methotrexate for GVHD prophylaxis, with the exception of five patients. Engraftment occurred in 58 (95%) pts. Thirty (64%) of 47 pts followed for at least 100 days experienced acute GVHD requiring treatment. Six pts have died of transplant-related complications and 7 pts have failed to achieve remission or have relapsed. Median follow-up is 174 days (range 26-448 days). The 100-day K-M estimate of survival for the whole cohort is 92%, and event-free survival 88%. The 100-day mortality in this study compares well with the 100-day mortality reported to the IBMTR for patients with AML, ALL, MDS, and CML transplanted from either HLA-matched siblings or unrelated donors. These preliminary results indicate that tBuFlu is a promising myeloablative regimen that can be utilized in older patients with low early treatment-related mortality. Myeloablative doses of intravenous busulfan in combination with fludarabine have been employed as conditioning before hematopoietic cell allografts with reduced treatment-related toxicity and mortality. In this report, we describe the early results of a targeted busulfan pharmacokinetic dosing strategy (tBuFlu) used in combination with fludarabine before either related or unrelated grafts We treated 61 pts with tBuFlu prior to allogeneic peripheral blood stem cell transplantation. The median patient age was 48 (range 22-68) years. Patient diagnoses included AML (13 de novo, 7 with prior MDS, and 7 treatment related), MDS (7 pts), MF (5), NHL (6 pts), ALL (6 pts), CML (5 pts), CLL (2), MM (2) and PNH (1). Five patients had received a prior autologous HCT. Donors were HLA-A, B, C, DRB1, DQB1 matched siblings (29), matched unrelated donors (22), or unrelated donors mismatched for one HLA antigen (6), homozygous mismatch (1), one HLA allele (2), or two HLA alleles (1). Fludarabine 40 mg/m2 was given intravenously daily for four days, with each infusion followed immediately by intravenous busulfan. The dose of busulfan for days 1 and 2 was 130 mg/m2. Pharmacokinetic analysis was performed after the first infusion of busulfan; in 59 pts, the goal was to adjust busulfan doses for days 3 and 4 to achieve an average targeted Css level of 800-1000 ng/ml. Levels were drawn incorrectly in 4 of these pts and doses were not changed. Thirty-five (59%) pts had their doses adjusted, increased in 27 and decreased in 8, while 20 pts had Css within the desired range without adjustment. Patients received tacrolimus and standard doses of methotrexate for GVHD prophylaxis, with the exception of five patients. Engraftment occurred in 58 (95%) pts. Thirty (64%) of 47 pts followed for at least 100 days experienced acute GVHD requiring treatment. Six pts have died of transplant-related complications and 7 pts have failed to achieve remission or have relapsed. Median follow-up is 174 days (range 26-448 days). The 100-day K-M estimate of survival for the whole cohort is 92%, and event-free survival 88%. The 100-day mortality in this study compares well with the 100-day mortality reported to the IBMTR for patients with AML, ALL, MDS, and CML transplanted from either HLA-matched siblings or unrelated donors. These preliminary results indicate that tBuFlu is a promising myeloablative regimen that can be utilized in older patients with low early treatment-related mortality.

High Dose Busulfan and Fludarabine with Anti-Thymocyte Globulin (ATG) Followed by Unmodified Marrow Grafts from HLA-Matched Siblings for the Treatment of High Risk Hemoglobinopathies (HGBpathies).

The standard cytoreduction for bone marrow transplantation (BMT) of pts with HGBpathies has been the combination of Busulfan (BU) and Cyclophosphamide However, for pts with advanced disease, high rates of graft rejection and toxicity were reported. For such high risk pts, a BU and Fludarabine (FLU) cytoreductive combination was used at low dose in the context of non-myeloablative BMT and was associated with a poor outcome with high rates of graft rejection. In view of the low toxicity associated with the BU-FLU combination, we used both agents at higher, potentially myeloablative doses for the cytoreduction of six pts with high risk HGBpathies. Between 12/00 and 04/05, 4 pts with thalassemia (Thal) and 2 pts with sickle cell disease (SCD), including 2 males and 4 females aged 4.6–15.3 years were transplanted using this regimen. All 4 pts with Thal had advanced Lucarelli Class 2 disease, while the 2 pts with SCD had stroke, recurrent vaso-occlusive crises (VOC), sickle lung disease and alloimmunization (n=1) and recurrent VOC, acute chest syndrome and osteomyelitis (n=1). Cytoreduction included intravenous BU (0.8–1 mg/Kg/dose x 14), FLU (30 mg/m2/day x 5) and Rabbit ATG (2.5 mg/Kg x 2). GvHD prophylaxis consisted of Tacrolimus (n=3) or cyclosporine (n=3) and methotrexate (n=5) or Steroids (n=1). Pts received an unmodified BMT from their HLA-identical sibling with total nucleated cell doses of 0.7–5.7 x 108 cells/Kg. The regimen was well tolerated with minimal toxicity. With a median follow-up of 21.5 mo (range 3–55 mo), all 6 pts are disease- and transfusion- free. There was no graft rejection and no GvHD. Chimerism status for pts with Thal was 98–100% donor, 5 mo to 2 years post BMT, while for the 2 pts with SCD, it was mixed with 50 and 80% donor cells at 3 mo and 2 years post BMT. The pt with 50% donor cells received a low graft cell dose (0.7 x 108 nucleated cells/Kg). One additional pt with SCD and a history of 2 strokes and moya-moya disease received BU FLU + melphalan (70 mg/m2 x 2). This pt had minimal post BMT toxicity, and is engrafted with 100% donor cells at 1 year post BMT. In summary, the combination of high dose BU and FLU for pts with high risk HGBpathies was well tolerated and induced full engraftment for pts with Thal, but mixed chimerism for pts with SCD. Nevertheless, all pts so treated survive with normal hematologic function. The addition of melphalan to BU FLU was also well tolerated and could be beneficial in attaining complete myeloablation and full chimerism in pts with SCD.

Targeted Intravenous Busulfan Combined with Fludarabine (tBuFlu) as Conditioning Prior to Allogeneic Peripheral Blood Stem Cell Transplant: Early Results of Low Treatment Related Mortality.

Myeloablative doses of intravenous busulfan in combination with fludarabine have been employed as conditioning before hematopoietic cell allografts with reduced treatment-related toxicity and mortality. In this report, we describe the early results of a targeted busulfan pharmacokinetic dosing strategy (tBuFlu) used in combination with fludarabine before either related or unrelated grafts We treated 43 pts with tBuFlu prior to allogeneic peripheral blood stem cell transplantation. The median patient age was 48 (range 22–67) years. Patient diagnoses included AML (8 de novo, 6 with prior MDS, and 2 treatment related), MDS (6 pts), MF (5), NHL (5 pts), ALL (4 pts), CML (3 pts), CLL (2), MM (1) and PNH (1). Three patients had received a prior autologous HCT. Donors were HLA-A, B, C, DRB1, DQB1 matched siblings (24), matched unrelated donors (13), or unrelated donors mismatched for one HLA antigen (3), one HLA allele (2), or two HLA alleles (1). Fludarabine 40 mg/m2 was given intravenously daily for four days, with each infusion followed immediately by intravenous busulfan. The dose of busulfan for days 1 and 2 was 130 mg/m2. Pharmacokinetic analysis was performed after the first infusion of busulfan; in 41 pts, the goal was to adjust busulfan doses for days 3 and 4 to achieve an average targeted Css level of 800–1000 ng/ml. Levels were drawn incorrectly in 4 of these pts and doses were not changed. Twenty-five (61%) pts had their doses adjusted, increased by 42% (± 28%) in 18 and decreased by 27% (± 14%) in 7, while 12 pts had Css within the desired range without adjustment. Patients received tacrolimus and standard doses of methotrexate for GVHD prophylaxis, with the exception of three patients treated with tacrolimus and mycophenolate mofetil or cyclosporine and prednisone. Engraftment occurred in 41 (95%) pts. Median day to ANC > 500/uL was 16 days, range (range 12–28); median day to platelet count > 20,000/uL untransfused was 18 days (range 8–45). Thirteen (52%) of 25 pts followed for at least 100 days experienced acute GVHD requiring treatment. Two pts have died of transplant-related complications (aplasia and suspected fungal pneumonitis), and 4 pts have failed to achieve remission or have relapsed. Median follow-up is 115 days (range 17–361 days). The 100-day K-M estimate of survival for the whole cohort is 94%, and event-free survival 86%. The 100-day mortality in this study compares well with the 100-day mortality reported to the IBMTR for patients with AML, ALL, MDS, and CML transplanted from either HLA-matched siblings or unrelated donors. These preliminary results indicate that tBuFlu is a promising myeloablative regimen that can be utilized in older patients with low early treatment-related mortality. We plan to escalate the busulfan dose based on targeted Css to explore a potential dose-response relationship and improve control of malignancy.

Matched-unrelated-donor bone marrow transplantation for children with leukemia.

This report describes the results of matched-unrelated-donor transplant for leukemia or myelodysplasia in the first 23 recipient children at a single medical center in Taiwan. Between August 1994 and February 2003, 23 consecutive children with leukemia or myelodysplasia underwent matched-unrelated-donor bone marrow transplantation (BMT). The preparative regimen consisted of fractionated total body irradiation and cyclophosphamide in 6 patients; busulfan in combination with etoposide and cyclophosphamide in 4 patients who received cranial irradiation before transplantation; and busulfan and cyclophosphamide in 13 patients. Engraftment was achieved in 91.3% of cases. Acute graft-versus-host disease (GVHD) occurred in 18 of 21 patients who engrafted (85.7%). Event-free survival for all patients was 24.46 +/- 9.24%. The 12 children with standard-risk disease had better event-free survival than the 11 children with high-risk disease (46.88 +/- 15.03% vs 0%, p < or = 0.001). The major obstacles to successful matched-unrelated-donor BMT are acute GVHD, relapse and infection. Early transplantation and patient selection, prophylactic and therapeutic maneuvers for GVHD, as well as appropriate donor selection and virus prophylaxis may improve the results.

Multivariate analysis of risk factors for hemorrhagic cystitis after hematopoietic stem cell transplantation.

To establish the most appropriate prophylactic therapy and risk factors for predicting hemorrhagic cystitis (HC) after stem cell transplantation (SCT), we retrospectively analyzed the clinical records of 450 transplant patients treated from 1982 to 2002. In all, 81 patients developed early- and/or late-onset HC (early=29, late=48, both=4). For the incidence of early-onset HC, administration of cyclophosphamide (CY) (p=0.0079, odds ratio (OD)=5.109, 95% confidence interval (CI)=1.533-17.030), busulfan (BU) (p=0.0015, OD=3.336, 95% CI=1.584-7.027), BU+CY (p=0.0001, OD=4.369, 95% CI=2.055-9.292), antithymocyte globulin (p=0.0009, OD=3.368, 95% CI=1.642-6.911), nonradiation (p=0.0163, OD=2.564, 95% CI=0.181-0.841), 2-mercaptoethane sodium sulfonate (Mesna) (p=0.0001, OD=7.519, 95% CI=2.847-19.858), and bladder irrigation (p=0.0001, OD=4.950, 95% CI=2.328-10.523) were risk factors. By Fisher's exact test, the combination of BU and Mesna was a more significant risk factor (P<0.001) than Mesna alone (p=0.008) compared to the administration of neither agent. By multivariate analysis, prophylactic administration of Mesna (p=0.0105, OD=5.301, 95% CI=1.477-19.026) and bladder irrigation (p=0.0001, OD=9.469, 95% CI=3.872-23.156) were significant risk factors of early-onset HC. We conclude that (i). high-dose BU as well as CY is a cause of HC, (ii). protective bladder irrigation has an opposite effect, and (iii). Mesna possibly has a toxic effect on bladder mucosa.

An assay for long-term engrafting human hematopoietic cells based on newborn NOD/SCID/beta2-microglobulin(null) mice.

Of various types of xenograft assays, the use of NOD/SCID mice has been the most popular method for quantitating candidate human stem cells. Limitations of the assay include low levels of engraftment, except when large numbers of cells are injected, and the development of thymic lymphoma, which precluded observation of long-term engraftment. In order to establish an assay that allows long-term in vivo engraftment and higher engraftment levels by a reasonable number of human cells, we tested a model based on "conditioned newborn" NOD/SCID or NOD/SCID/beta2-microglobulin(null) (BMG(null)) mice. Using human cord blood mononuclear cells, we tested various nonradiation conditioning regimens and cell injection routes. Conditioning with a combination of 5-fluorouracil (5FU) and anti-mouse c-kit blocking antibody (Ack-2) or a combination of busulfan (BU) and cyclophosphamide (CY) and the use of facial vein for the cell injection route yielded the highest levels of multilineage engraftment. At 4-5 months posttransplantation, the median of engraftment level in bone marrow with 5FU/Ack-2 and BU/CY regimens were 0.9% (range: 0.2-40.5%) and 2.1% (range: 0.3-2.4%) in NOD/SCID mice, and 11.3% (range: 0.7-38%) and 14.1% (range: 0.8-52%) in NOD/SCID/BMG(null) mice, respectively. Multilineage engraftment was demonstrated by flow cytometry and by the growth of multilineage colonies in methylcellulose culture. Secondary transplantation of the isolated human CD45(+) cells, also performed at 4-5 months posttransplantation, revealed engraftment at the levels of 1.5 +/- 0.42% at 2 months after secondary transplantation. Our assay may provide a quantitative method for analysis of human hematopoietic stem cells.

Allogeneic hemopoietic stem cell transplantation in patients with myelodysplastic syndrome or myelofibrosis

Myelodysplastic syndrome (MDS) and myeloproliferative disorders associated with myelofibrosis (MF) are stem cell disorders, and hemopoietic stem cell transplantation (HSCT) is currently the only therapy with curative potential. Among patients with less advanced MDS, 3 year survivals of 65% to 70% are achievable with HLA-identical related and HLA-matched unrelated donors. The probability of relapse is < 5%. Among patients with advanced disease (> or = 5% marrow blasts), about 35 to approximately 45% and 25 to approximately 30%, respectively, are surviving in remission after transplantation from related or unrelated donors. The incidence of post-transplant relapse is 1035%. Criteria of the International Prognostic Scoring System (IPSS), originally developed for nontransplanted patients, also predict survival following transplantation. Patients with MF, either idiopathic or on the basis of pre-existing disorders, are also transplanted successfully with stem cells from related or unrelated donors. Transplants early in the disease, before leukemic transformation, are successful in 60 to approximately 80% of patients. Success rates are lower in patients who have developed MDS or leukemia. New conditioning regimens have permitted successful HSCT even in patients in the seventh decade of life. Results with a regimen using a combination of busulfan (targeted to predetermined plasma levels) and cyclophosphamide are particularly encouraging. Improved survival with transplants from unrelated volunteer donors may, in part, reflect selection of donors on the basis of high resolution (allele-level) HLA typing. Nevertheless, transplant-related morbidity and mortality, including graft- vs. -host disease, remain challenges that need to be addressed with innovative approaches.

Secondary failure of platelet recovery after hematopoietic stem cell transplantation.

After primary recovery of platelet counts after transplantation, there can be a late persistent decline called secondary failure of platelet recovery (SFPR), which may occur although the counts of other cell lineages remain within the normal range. SFPR was defined as a decline of platelet counts below 20,000/microL for 7 consecutive days or requiring transfusion support after achieving sustained platelet counts > or = 50,000/microL without transfusions for 7 consecutive days after hematopoietic stem cell transplantation (HSCT). The study population consisted of 2871 consecutive patients receiving transplants from January 1990 to March 1997. After primary recovery of platelet counts, SFPR not due to relapse of the underlying disease was observed in 285 of 1401 (20%) patients undergoing allogeneic transplantation and 36 (8%) of 444 patients undergoing autologous transplantation, with a median time of onset after transplantation at day 63 (range, day 21-156) and day 44 (range, day 24-89), respectively. Concomitant neutropenia was seen in 57 (20%) of 285 patients undergoing allogeneic HSCT and 7 (19%) of 36 patients undergoing autologous HSCT with SFPR. By multivariable analysis, the following were factors significantly associated with SFPR after allogeneic HSCT: a transplant from an unrelated donor; a graft-versus-host disease (GVHD) prophylaxis other than methotrexate and cyclosporine; development of grade 2 through 4 acute GVHD; impaired renal or liver function; conditioning with the combination of busulfan, cyclophosphamide, and total body irradiation; stem cell dose; and infections. Cytomegalovirus infection after engraftment and source of stem cells were the only significant risk factors after autologous HSCT. The hazard rate of death was significantly higher in patients who experienced SFPR (hazard ratio = 2.6 for allogeneic HSCT; hazard ratio = 2.2 for autologous HSCT). SFPR was associated with serious complications and poor outcome after transplantation. The identification of the characteristics and risk factors for SFPR could improve patient counseling and management and lead to the design of effective treatment strategies.

Stage IV neuroblastoma in patients over 1 year of age at diagnosis: consolidation of poor responders with combined busulfan, cyclophosphamide and melphalan followed by in vitro mafosfamide-purged autologous bone marrow transplantation.

In an attempt to improve the poor prognosis of poor responders with stage IV neuroblastoma, a new combined high-dose chemotherapy conditioning regimen was tested. Event-free and overall survival, as well as the incidence of complications, were analysed. Twenty-five children aged 12-146 months at diagnosis entered this study. All were in complete remission (CR) at the time of high-dose chemotherapy. Two or three different protocols had been necessary for them to achieve a CR. High-dose chemotherapy consisted of a combination of busulfan (600 mg/m2), cyclophosphamide (4400 mg/m2) and melphalan (140 mg/m2). It was followed by autologous bone marrow transplantation (ABMT). The bone marrow graft was purged in vitro with mafosfamide. The probability of event-free survival (EFS) at 5 years post-ABMT was 34%, compared to < 8% in a historical series. Toxicity was severe but manageable and 2 complication-related deaths were observed. Veno-occlusive disease was the most frequent extrahaematopoietic complication encountered, but its outcome was always favourable. By using a very intensive conditioning regimen consisting of a combination of three alkylating agents, the EFS of poor responders with metastatic neuroblastoma was improved and similar to that of good responders. When compared with a previously published similar series of patients, the improvement in survival appears probably related to intensification of the conditioning regimen.