Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs.

Terminology and labeling of cellular products-2: Implementation plan.

The publication of new standards for terminology and labeling marks an important step in ensuring consistency and traceability of cellular therapies at the global level. However, it is only with the widespread implementation of the standard that the benefits can be truly realized. This paper provides guidance on the practical aspects of adopting these new standards for organizations with differing current levels of computerization. It discusses project management, equipment, licensing, and validation topics.

Terminology and labeling of cellular products: 1. Standards.

The International Cellular Therapy Coding and Labeling Advisory Group was established to address the growing need for standardization of terminology and labeling for cellular therapy products as a result of increasing international transfer of these products. This paper presents new standards for terminology and labeling. These standards have been developed through a consultative process and are supported by key professional and accreditation bodies. By using these standards, together with the unique donation identification numbers and international product reference tables provided by the International Society of Blood Transfusion (ISBT) 128 Standard, consistency and traceability can be assured at the global level. A companion paper provides guidance on the implementation of the ISBT 128 system.

Voluntary accreditation of cellular therapies: Foundation for the Accreditation of Cellular Therapy (FACT).

Voluntary accreditation of cells, tissues, and cellular and tissue-based products intended for human transplantation is an important mechanism for improving quality in cellular therapy. The Foundation for the Accreditation of Cellular Therapy (FACT) has developed and implemented programs of voluntary inspection and accreditation for hematopoietic cellular therapy, and for cord blood banking. These programs are based on the standards of the clinical and laboratory professionals of the American Society of Blood and Marrow Transplantation (ASBMT), the International Society for Cellular Therapy (ISCT), and NETCORD. FACT has collaborated with European colleagues in the development of the Joint Accreditation Committee in Europe (jACIE). FACT has published standards documents, a guidance manual, accreditation checklists, and inspection documents; and has trained as inspectors over 300 professionals active in the field. All inspectors have a minimum of 5 years' experience in the area they inspect. Since the incorporation of FACT in 1996, 215 hematopoietic progenitor cell facilities have applied for FACT accreditation. Of these facilities, 113 are fully accredited; the others are in the process of document submission or inspection. Significant opportunities and challenges exist for FACT in the future, including keeping standards and guidance materials current and relevant, recruiting and retaining expert inspectors, and establishing collaborations to develop standards and accreditation systems for new cellular products. The continuing dialogue with the Food and Drug Administration (FDA) is also important to ensure that they are aware of the accomplishments of voluntary accreditation, and keep FACT membership alerted to FDA intentions for the future. Other potential avenues of communication and cooperation with FDA and other regulatory agencies are being investigated and evaluated.

Outcomes of treatment of children and adolescents with recurrent non-Hodgkin's lymphoma and Hodgkin's disease with dexamethasone, etoposide, cisplatin, cytarabine, and l-asparaginase, maintenance chemotherapy, and transplantation: Children's Cancer Group Study CCG-5912.

PURPOSE: To determine the toxicity and response rate in children treated with dexamethasone, etoposide, cisplatin, high-dose cytarabine, and L-asparaginase (DECAL) for recurrent non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). PATIENTS AND METHODS: Ninety-seven children with recurrent NHL (n = 68) or HD (n = 29) were enrolled. Treatment consisted of two cycles of DECAL, then bone marrow transplantation or up to four cycles of ifosfamide, mesna, and etoposide alternating with DECAL maintenance therapy. RESULTS: After two cycles of DECAL induction therapy, complete response (CR) or partial response (PR) was reported in 19 (65.5%; 10 CRs and nine PRs) of 29 patients with HD and 29 (41.6%; 23 CRs and six PRs) of 68 patients with NHL. When only 24 patients with HD and 58 patients with NHL who were assessable for response were considered, the response rates were 79.2% (19 of 24 patients) and 50.0% (29 of 58 patients), respectively. Five-year event-free survival was 26% +/- 9% and 23% +/- 5% in patients with HD and NHL, respectively. Five-year survival was 31% +/- 14% and 30% +/- 6%, respectively. Although median time to treatment failure was significantly longer in patients with HD (EFS, P =.002; survival, P =.011), this difference did not translate into a higher long-term survival. Grade 3 or 4 toxic effects were observed during induction in 70 (72%) of 97 patients and during maintenance in 45 (70%) of 64 courses of DECAL therapy. Pancytopenia and systemic infections in particular were frequently observed. Other toxic effects were uncommon. Although not a formal part of the therapy or the study design, 42 patients who responded to therapy who underwent bone marrow transplant did not show any benefit from this approach. CONCLUSION: DECAL is an effective and tolerable salvage regimen for treating patients with recurrent NHL and HD.

Full hematopoietic engraftment after allogeneic bone marrow transplantation without cytoreduction in a child with severe combined immunodeficiency.

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.

FAHCT accreditation: common deficiencies during on-site inspections.

BACKGROUND: The Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT) was established in 1996 to develop and implement the inspection and accreditation program of the parent organizations, the International Society for Hematotherapy and Graft Engineering (ISHAGE) and the American Society of Blood and Marrow Transplant (ASBMT). Training of inspectors began in September 1996 and the first on-site inspections were conducted in September 1997. METHODS: The process of attaining FAHCT Accreditation includes the assessment of written application materials against uniform accreditation criteria and an on-site inspection of each of the sites in the applicant hematopoietic progenitor cell (HPC) transplantation program. Observations at each inspection are recorded by the inspector on a checklist, reviewed by the FAHCT Accreditation Chairman and/or Technical staff, and presented to the FAHCT Board of Directors for review. Each observation is determined to be in compliance with Standards, a deficiency, or a variance. The deficiencies observed in these inspections were tallied following Board review and entered into an electronic database. Those deficiencies occurring in > 25% of the transplant programs reviewed are described below. RESULTS: Significant deficiencies in the clinical programs included inadequate data management; incomplete or inadequate Quality Management Plan; and incomplete or absent standard operating policies and procedures. In the collection facilities, the most common significant deficiencies were incomplete or absent standard operating procedures; inadequate management of the results of donor evaluation findings and test results; lack of documentation of adverse reactions to collection; and deficiencies in the label applied to the HPC at the end of collection. Deficiencies in HPC laboratory services included validation procedures and/or records missing; ABO/Rh testing of the HPC component and/or the comparison of the results with prior test results not done or documented; labels in use did not meet Standards; incomplete quality control documentation; HPC infusion documents not available, or in use; HPC storage policies and procedures incomplete; adverse reactions to HPC infusion not documented, or tracked; and engraftment not tracked by the laboratory. DISCUSSION: Although all programs had a few to many deficiencies noted initially at the on-site inspection, 23 have successfully completed full accreditation. A few specific standards give problems to many programs. Participants, in general, believe the changes made in preparation for FAHCT accreditation have improved their HPC transplantation programs.

Immunological properties of mononuclear cells from blood stem cell harvests following mobilization with erythropoietin + G-CSF in cancer patients.

BACKGROUND: Although the recombinant granulocyte colony-stimulating factor (G-CSF) is a good CD34 cell mobilizer, the effects of G-CSF mobilization on the immune effector function of the individual is not always optimal. We studied the functional and phenotypic properties of peripheral blood stem cells (PBSC) collected from 15 cancer patients mobilized with G-CSF plus recombinant erythropoietin (EPO). METHODS: The patients received EPO (300 U/kg) and (G-CSF 1 microg/kg) per day as mobilizing cytokines and an autologous graft product was collected with at least daily apheresis procedures until a target number of CD34 cells and mononuclear cells were obtained. Mononuclear cells from the first four PBSC collections were tested for their natural killer (NK), activated NK and lymphokine activated killer (LAK) cytotoxicity in vitro against K562 and Raji tumor target cells. RESULTS: There was a significant increase in NK, activated NK and LAK cytotoxicity in EPO + G-CSF mobilized cells when compared to mononuclear cells from premobilization blood baseline values. Although there was no increase in CD3+ T cells, there was a significant increase in myeloid cells (CD14+), B-cells (CD20+) and NK cells (CD56+) following mobilization. There was no difference in T cell response to the mitogens PHA and Con-A, but there was an increase in B-cell response to PWM following mobilization. Thus, the combination of EPO + G-CSF not only mobilized hematopoietic precursor cells but also increased the number of immune effector cells in the PBSC collections.

Comparison of monocyte-dependent T cell inhibitory activity in GM-CSF vs G-CSF mobilized PSC products.

This study compares the immune properties of peripheral blood stem cell (PSC) products mobilized with different hematopoietic growth factors (HGFs) as well as apheresis products and peripheral blood leukocytes (PBL) from normal individuals. We found that monocytes in mobilized PSC products appear to inhibit T cell function independent of whether granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was used for mobilization. In addition, the GF used to mobilize the stem cell product may be less important to the CD4:CD8 ratio than the extent of prior chemotherapy, as we found an inverse correlation between chemotherapy and the CD4:CD8 ratio. In other observations, all apheresis products, whether mobilized or unmobilized, contained significantly more monocytes compared to normal PBL. The mononuclear cells (MNC) from G-CSF or GM-CSF mobilized PSC products had a similar T cell phytohemagglutinin (PHA) mitogenic response that was significantly lower (P = 0.001 and P = 0.005, respectively) than non-mobilized apheresis products. We also examined the T cell inhibitor (TI) activity of the MNC from the PSC products for allogeneic lymphocyte proliferation and found that PSC products significantly reduced the proliferation of allogeneic PBL to PHA. A significant correlation (P = 0.001, r = 0.517) between the frequency of monocytes and TI activity also was observed.

Comparison of ISHAGE protocol CD34 cell enumeration with a lineage negative backgating technique.

BACKGROUND: CD34(+) cell enumeration in PBSC apheresis products has become the standard for assessing graft hematopoietic potential. METHODS: An in-house, three color, lineage negative-gating technique [University of Nebraska Medical Center (UNMC) protocol] for CD34 cell enumeration was compared with the ISHAGE protocol over 100 apheresis products. Cell doses determined by both methods were compared with each other and to colony-forming units-granulocyte/macrophage (CFU-GM) assay results. RESULTS: Overall, the assays compared well with each other for samples with CD34 cell doses > 0.2 2 10(6)/kg (r values > 0.8). The ISHAGE method showed a constant negative bias, with a mean of 38% in comparison to the UNMC protocol, which was more linear at lower cell doses. Both assays showed similar correlation with CFU-GM doses after log conversion (UNMC, r = 0.915; ISHAGE, r = 0.917). When comparing integer values, however, the ISHAGE method correlated with CFU-GM only in the high dose range (CFU-GM > 2 2 10(4)/kg), while the UNMC method correlated across the entire measured range of CFU-GM doses. Finally, an inter-technologist gating reproducibility study (n = 6) yielded a 23% coefficient of variation (CV) for the ISHAGE method and a 7% CV for the UNMC method, when the same two sets of CD34 histograms were analyzed to calculate cell dose. DISCUSSION: In this study the lineage negative protocol (UNMC) had a larger dynamic range, correlated better with CFU-GM results and showed better inter-technologist reproducibility than the ISHAGE method.

Selective plasminogen binding: cysteinyl-lysine-dextran protein interactions.

The dipeptide, L-cysteine-L-lysine, when coupled to a dextran matrix demonstrates a much higher binding of Glu-plasminogen than dextran alone. Plasminogen binds to the dipeptide coupled matrix with a high affinity and binding capacity and is retained by the surface for long periods. In comparison, other proteins such as bovine serum albumin, alpha2-antiplasmin, tPA, fibrinogen, and plasmin are not retained by the material demonstrating that the L-cysteine-L-lysine-dextran is highly selective for Glu-plasminogen. While fibrinogen alone does not bind to the surface, fibrinogen on surfaces to which plasminogen has been pre-adsorbed does demonstrate adsorption interactions. The nature of the interactions depend on the amount of plasminogen bound. Fibrinogen in contact with high concentrations of adsorbed plasminogen causes an increase in the desorption rate of the surface bound protein; if low amounts of plasminogen are initially bound, fibrinogen exposure causes an increase in total protein adsorbed by the surface. A higher amount of protein adsorbs from a mixed plasma solution. This does not appear to inhibit the binding of plasminogen, but rather may actually facilitate more plasminogen binding than is the case of plasminogen alone. The dipeptide coupled to the dextran matrix appears to be a preferred substrate for plasminogen binding compared to free lysine and epsilon-amino caproic acid.

Lymphocyte reconstitution after allogeneic blood stem cell transplantation for hematologic malignancies.

Forty-one patients were studied at set times after allogeneic blood stem cell transplantation (alloBSCT) for recovery of lymphocyte numbers and function. Cells were mobilized with G-CSF from HLA-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate; G-CSF was administered post-transplant. Median time to absolute lymphocyte count (ALC) >500/microl was 17 days vs 41 and 49 days in historical alloBMT patients with G-CSF (n = 23) or no cytokine (n = 29) post-transplant, respectively (P < 0.0001). CD4/CD8+ ratio was 1.9 on day 28 after alloBSCT, then gradually declined to 0.8 at 1 year due to more rapid CD8+ cell recovery. Mean phytohemagglutinin-induced T cell responses were lower than normal on day +28 (P < 0.05), then tended to recover towards normal values. Natural-killer cytotoxicity remained low from day +28 to 1 year post-alloBSCT, but considerable lymphokine-activated killer cytotoxicity was induced from cells already obtained on day +28. Faster lymphocyte recovery correlated with better survival in alloBSCT patients (median follow-up 287 days, P = 0.002), ALC recovery was not affected by acute GVHD, CMV infections or doses of infused cells. ALC recovery did not correlate with survival in either historical alloBMT group. These data suggest that after alloBSCT lymphocyte reconstitution is faster than after alloBMT, and that quicker lymphocyte recovery predicts better survival in the alloBSCT setting.

Ex vivo treatment of bone marrow with phosphorothioate oligonucleotide OL(1)p53 for autologous transplantation in acute myelogenous leukemia and myelodysplastic syndrome.

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.

Allogeneic bone marrow transplantation for children with acute leukemia: long-term follow-up of patients prepared with high-dose cytosine arabinoside and fractionated total body irradiation.

High-dose therapy and allogeneic matched sibling bone marrow transplantation (BMT) is considered to be the treatment of choice for children with relapsed acute lymphoblastic leukemia (ALL), or for children with acute myeloid leukemia (AML) in first remission. However, the rate of bone marrow relapse after transplant for either of these diseases remains high. In this study, we assessed the efficacy and toxicity of high-dose cytosine arabinoside and total body irradiation (TBI) followed by allogeneic BMT, for children with acute leukemia or myelodysplastic syndrome (MDS). Sixty-five pediatric patients underwent allogeneic related (n = 57) or unrelated (n = 8) BMT. Twenty-seven were transplanted for ALL in second remission (CR2), and 16 for AML in first remission (CR1). The other 22 were high risk patients: six were transplanted for ALL in third remission (CR3), two for AML in CR2, two for myelodysplastic syndrome (MDS) and 12 for acute leukemia in relapse. Patients were prepared with cytosine arabinoside 3000 mg/m2 per dose twice daily for 6 days followed by 12000 cGy TBI as 200 cGy fractions twice daily for 3 days. Minimum follow-up is 21 months. Five-year event-free survival (EFS) and the actuarial relapse rate is 59 and 14% for patients with ALL in second remission, and 38 and 14+% for patients with AML in first remission. Twelve patients have relapsed (three are alive in remission after testicular or marrow relapse) and 28 have died of other causes. Acute GVHD with or without infection was the cause of death in 11 patients. Ten of the 11 patients who died of acute GVHD were considered at 'high risk' for GVHD (inadequate GVHD prophylaxis, or mismatched family donor or a matched unrelated donor). Toxicities in the immediate post-BMT period included diarrhea, oropharyngeal mucositis and conjunctivitis. Significant late toxicities included short stature, avascular necrosis of bone, and poor school performance (most often in patients who had received prior cranial irradiation). Our conclusions are that high-dose Ara-C and TBI followed by allogeneic bone marrow transplantation is effective therapy for children in second complete remission of their acute leukemia. However, significant late toxicities occur, and it is clear that more effective, less toxic therapies are necessary for these patients.

Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colony-stimulating factor.

PURPOSE: The optimal dose of granulocyte colony-stimulating factor (G-CSF) for mobilization of allogeneic-blood stem cells (AlloBSC) has yet to be determined. As part of a prospective trial, 41 related human leukocyte antigen (HLA)-matched donors had blood cells mobilized with G-CSF at 5 micrograms/kg/d by subcutaneous administration. The purpose of this trial was to monitor adverse effects during G-CSF administration and stem-cell collection, to determine the optimal timing for stem-cell collection, and to determine the cellular composition of stem-cell products following G-CSF administration. PATIENTS AND METHODS: The median donor age was 42 years. Apheresis began on day 4 of G-CSF administration. At least three daily 12-L apheresis collections were performed on each donor. A minimum of 1.0 x 10(6) CD34+ cells/kg (recipient weight) and 8.0 x 10(8) mononuclear cells/kg were collected from each donor. All collections were cryopreserved in 5% dimethyl sulfoxide and 6% hydroxyethyl starch. RESULTS: Toxicities associated with G-CSF administration and the apheresis process included myalgias/arthralgias (83%), headache (44%), fever (27%), and chills (22%). The median baseline platelet count of 242 x 10(4)/ mL decreased to 221, 155, and 119 x 10(6)/mL on days 4, 5, and 6 of G-CSF administration, respectively. Median numbers of CD34+ cells in collections 1, 2, and 3 were 1.99, 2.52, and 3.13 x 10(6)/kg, respectively. The percentage and total number of CD4+, CD8+, and CD56+/CD3- cells remained relatively constant during the three collections. Median total numbers of cells were as follows: CD34+, 7.73 x 10(6)/kg; and lymphocytes, 6.93 x 10(8)/kg. CONCLUSION: Relatively low doses of G-CSF can mobilize sufficient numbers of AlloBSC safely and efficiently.

Comparison of idarubicin to daunomycin in a randomized multidrug treatment of childhood acute lymphoblastic leukemia at first bone marrow relapse: a report from the Children's Cancer Group.

The outcome of children with acute lymphoblastic leukemia (ALL) and bone marrow relapse has been unsatisfactory largely because of failure to prevent subsequent leukemia relapses. Ninety-six patients were enrolled and received vincristine, prednisone, L-asparaginase, and an anthracycline as reinduction therapy. Ninety-two patients were randomized to receive either daunomycin (DNR) or idarubicin (IDR). After achievement of second complete remission (CR2), maintenance chemotherapy included the same anthracycline, IDR or DNR, high-dose cytarabine, and escalating-dose methotrexate. Compared to DNR (45 mg/m2/week x 3), IDR (12.5 mg/m2/week x 3) was associated with prolonged myelosuppression and more frequent serious infections. Halfway through the study, the dose of IDR was reduced to 10 mg/m2. Overall, second remission was achieved in 71% of patients. Reinduction rate was similar for IDR and DNR. Reasons for induction failure differed; none of 15, 1 of 5, and 5 of 7 reinduction failures were due to infection for DNR, IDR (10 mg/m2), and IDR (12.5 mg/m2), respectively. Two-year event-free survival (EFS) was better among patients who received IDR (12.5 mg/m2) (27 +/- 18%) compared to DNR (10 +/- 8%, P = 0.05) and IDR (10 mg/m2) (6 +/- 12%, P = 0.02). However, after 3 years of follow-up, late events in the high-dose IDR group result in a similar EFS to the lower-dose IDR and DNR groups. In conclusion, IDR is an effective agent in childhood ALL. When used weekly at 12.5 mg/m2 during induction, the EFS outcome during the first 2 years of treatment appears better than lower-dose IDR or DNR (45 mg/m2), although this difference was not sustained at longer periods of follow-up. Increased hematopoietic toxicity seen at this dose might be reduced through the use of supportive measures, such as hematopoietins and intestinal decontamination.

Phase I trial of recombinant fusion protein PIXY321 for mobilization of peripheral-blood cells.

PURPOSE: Mobilization of peripheral-blood cells (PBC) with cytokines alone results in rapid hematopoietic recovery and avoids the potential morbidity associated with mobilization by chemotherapy. PIXY321, a fusion protein that consists of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), has enhanced hematopoietic colony-forming activity as compared with individual or equimolar combinations of the two cytokines. A phase I trial of PIXY321 for mobilization of PBC in patients with malignant lymphoma was performed. PATIENTS AND METHODS: Thirteen patients with malignant lymphoma who were eligible for high-dose therapy (HDT) were enrolled onto the trial. All patients were ineligible for autologous bone marrow transplantation due to overt metastatic disease in the marrow or to severe marrow hypocellularity. PIXY321 was administered at three dose levels of 250, 500, and 750 micrograms/m2/d by continuous infusion until completion of PBC collections. Collections were initiated when the WBC count was greater than 10 x 10(9)/L or 4 days after the initiation of PIXY321, whichever came first. Collections were continued until a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg patient weight were obtained. RESULTS: PIXY321 was generally well tolerated. Side effects associated with PIXY321 administration did not exceed grade 2 and included fever (85%), chills/sweats (54%), myalgias (38%), fatigue (31%), nausea/vomiting (31%), headache (31%), edema (23%), and rhinorrhea (23%). The median numbers of colony-forming units-granulocyte/macrophage (CFU-GM) in the graft products for the three dose levels were 0.31, 2.94, and 2.88 x 10(4)/kg, respectively; the median numbers of burst-forming units-erythroid (BFU-e) were 0.20, 6.94, and 12.78 x 10(4)/kg, and the median numbers of CD34+ cells were 2.30, 0.74, and 0.39 x 10(6)/kg. Following transplantation, the median times to an absolute neutrophil count (ANC) > 0.5 x 10(9)/L were 12, 15, and 12 days, respectively, and the median times to platelet transfusion independence were 30, 19, and 15 days. CONCLUSION: PIXY321 can be safely administered and effectively mobilizes PBC in patients with bone marrow defects. PIXY321-mobilized PBC autotransplants result in rapid and sustained hematopoietic recovery.

Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome.

Long-term survival and improved neuropsychological function have occurred in selected children with Hurler syndrome (MPS I H) after successful engraftment with genotypically matched sibling bone marrow transplantation (BMT). However, because few children have HLA-identical siblings, the feasibility of unrelated donor (URD) BMT as a vehicle for adoptive enzyme therapy was evaluated in this retrospective study. Forty consecutive children (median, 1.7 years; range, 0.9 to 3.2 years) with MPS I H received high-dose chemotherapy with or without radiation followed by BMT between January 27, 1989 and May 13, 1994. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients are alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute graft-versus-host disease (GVHD) was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD BMT because of graft rejection or failure. Of the 20 survivors, 13 children have complete donor engraftment, two children have mixed chimeric grafts, and five children have autologous marrow recovery. The BM cell dose was correlated with both donor engraftment and survival. Thirteen of 27 evaluable patients were engrafted at 1 year following URD BMT. Neither T-lymphocyte depletion (TLD) of the bone marrow nor irradiation appeared to influence the likelihood of engraftment. Ten of 16 patients alive at 1 year who received a BM cell dose greater than or equal to 3.5 x 10(8) cells/kg engrafted, and 62% are estimated to be alive at 3 years. In contrast, only 3 of 11 patients receiving less than 3.5 x 10(8) cells/kg engrafted, and 24% are estimated to be alive at 3 years (P = .05). The mental developmental index (MDI) was assessed before BMT. Both baseline and post-BMT neuropsychological data were available for 11 engrafted survivors. Eight children with a baseline MDI greater than 70 have undergone URD BMT (median age, 1.5 years; range, 1.0 to 2.4 years). Of these, two children have had BMT too recently for developmental follow-up. Of the remaining six, none has shown any decline in age equivalent scores. Four children are acquiring skills at a pace equal to or slightly below their same age peers; two children have shown a plateau in learning or extreme slowing in their learning process. For children with a baseline MDI less than 70 (median age, 2.5 years; range, 0.9 to 2.9 years), post-BMT follow-up indicated that two children have shown deterioration in their developmental skills. The remaining three children are maintaining their skills and are adding to them at a highly variable rate. We conclude that MPS I H patients with a baseline MDI greater than 70 who are engrafted survivors following URD BMT can achieve a favorable long-term outcome and improved cognitive function. Future protocols must address the high risk of graft rejection or failure and the impact of GVHD in this patient population.