Effect of two cyclophosphamide derivatives on hemopoietic progenitor cells and pluripotential stem cells.

The studies described herein were undertaken to help define the effects of certain cyclophosphamide derivatives that have been used for selective removal of leukemic cells from marrow samples used for autologous transplantation. We have tested the effect of 4-HC and another cyclophosphamide congener, ASTA-Z 7557, on pluripotent stem cells (CFU-S) and committed progenitor cells (CFU-GM) in mice. The CFU-S were evaluated by the spleen colony assay at eight days and 12 days after transplant. The eight-day colonies are transient in nature, rapidly growing, mainly erythroid, and lack pluripotential precursors. The 12-day colonies are believed to provide a measure of hemopoietic stem cells as they slowly grow and do contain primitive precursors. Our data show that at the maximum dose levels tested, both drugs caused a 100% loss of CFU-GM and about 80%-95% inhibition of early transient CFU-S. In contrast, about 70% of the pluripotent 12-day CFU-S were spared. These data appear to explain the hemopoietic recovery seen in man after transplantation with marrow cells treated with 4-HC despite their relative absence of hemopoietic progenitor cells.

Haploidentical bone marrow transplantation from mother to child with advanced leukemia.

Use of the mother as mismatched marrow donor was assessed in 19 children with advanced leukemia. Patients were homogeneous for HLA incompatibility, age, donor, and conditioning regimen, and stage of disease. All received busulfan and cytoxan, combined with unmodified donor marrow, ALG given before and after transplant, and short MTX and cyclosporine as GVHD prophylaxis. Survival, LFS, and relapse respectively were 26, 26, and 33%. Incidence of overall and severe acute GVHD was 58 and 32%, respectively. Four patients had failure of engraftment, and two of these are alive with autologous reconstitution in complete remission. Probability of rejection was 21%. Results of haploidentical transplants were compared with those of children with advanced leukemia treated at the same institution, who received marrow from HLA-identical siblings. The probability of long-term leukemia-free survival was similar in the two groups. We thus propose using the mother as an alternative marrow donor in children with advanced leukemia.

Bone marrow transplantation from alternative donors for thalassemia: HLA-phenotypically identical relative and HLA-nonidentical sibling or parent transplants.

Twenty-nine patients with thalassemia and a median age of 6 years (range 1.1-33 years) were given a BMT from an alternative donor. Six of the 29 donors were HLA-phenotypically identical and two were mismatched relatives, 13 were mismatched siblings and eight were mismatched parents. Six patients received no antigen (relatives), 15 patients one antigen, five patients two antigen and three patients three antigen disparate grafts. Twenty-three patients were in class 2 or class 3, whereas six patients were in class 1. Thirteen patients were given BUCY, nine patients BUCY plus ALG, six patients BUCY plus TBI or TLI and one patient BUCY with prior cytoreductive-immunosuppressive treatment as conditioning. As GVHD prophylaxis four patients received MTX, 22 CsA + MTX + methylprednisolone (MP) and three patients CsA + MP. Thirteen of 29 patients (44.8%) had sustained engraftment. The probability of graft failure or rejection was 55%. There were no significant differences between antigen disparities and graft failure. The incidence of grade II-IV acute GVHD was 47.3% and chronic GVHD was 37.5%. The incidence of acute GVHD was higher in patients receiving one or two antigen disparate in the GVHD direction grafts (vs no antigen) (P EQ 0.04; odds ratio 10.8; 95% CI 1.5-115). The probability of overall and event-free survival was 65% and 21%, respectively, with median follow-up of 7.5 years (range 0.6-17 years) for surviving patients. The degree of HLA disparity between patient and donor did not have a significant effect on survival. The incidence of nonhematologic toxicity was low. Transplant-related mortality was 34%. GVHD (acute or chronic) was a major contributing cause of death (50%) followed by infections (30%). We conclude that at present, due to high graft failure and GVHD rates, BMT from alternative donors should be restricted to patients who have poor life expectancies because they cannot receive adequate conventional treatment or because of alloimmunization to minor blood antigens.

An unusual DRB1*1503 haplotype without a detectable DRB5 locus in a black African family.

A DRB1*1503 allele not associated with DRB5 locus has been detected in an African family during routine HLA typing for bone marrow transplantation. PCR/SSOP analysis showed the DR2-associated alleles in all the family members but the DRB5 locus appeared to be absent in the patient and his brother. The samples were then analyzed for the presence of DRB6 pseudogenes and we found that the unusual haplotype was associated with DRB6*0101 allele. This finding strengthen the hypothesis of a recombination hot spot between DRB1 and DRB6 genes.

[The use of autologous bone marrow transplants in malignant hemopathies and solid tumors]. / L'uso del trapianto di midollo osseo autologo nelle emopatie maligne e nei tumori solidi.

Bone marrow collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to -196 degrees C were monitored immediately after thawing by the semisolid agar CFU-C assay and 2 different methods of cell reconstitution were compared. In the first way, thawed cells were plated after the removal of DMSO by washing the cell suspension, in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The number of CFU-C per 2 X 10(5) cells plated was higher washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same with both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in the AML and CML patients.

Ontogeny of granulocyte-macrophage progenitor cells in the human fetus.

The CFU-Gm content of liver, spleen and bone marrow of human fetuses was determined from the 8th to 28th week of gestational age. The study of progenitor cells from 43 fetal livers revealed that the CFU-Gm increased from 8 to 12 weeks, was relatively stable through 16 weeks and thereafter declined by 22 to 24 weeks. The total number of liver CFU-Gm rose from 10(4) to greater than 10(6) CFU-Gm by the 16th week of gestation and declined to approximately 4 X 10(5) progenitor cells by 22 to 24 weeks. Peak values of bone marrow CFU-Gm were noted at 10 to 14 weeks of gestation. Generally, the numbers of these progenitor cells declined by 16 to 18 weeks; however, several high values were obtained at later times. Splenic CFU-Gm were relatively constant in number between 12 and 28 weeks of gestation with no correlation with fetal age. Thus, these studies indicate that certain fetal tissues contain substantial numbers of hemopoietic progenitor cells. Based on the quantity of CFU-Gm obtained, it appears feasible to use fetal liver cells as a source of progenitor cells for transplantation.

Limiting-dilution analysis for the determination of leukemic cell frequencies after bone marrow decontamination with mafosfamide or merocyanine 540.

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.

Molecular genetic techniques permit sensitive assessment of host hematopoiesis after marrow transplantation for thalassemia. Information on this persistence and the cell lines in which it occurs may permit therapeutic intervention in patients at high risk for rejection and/or relapse. The objective of this study, therefore, was to determine the evolution and cell line distribution of persistent mixed chimerism detected in 55 patients treated for beta thalassemia. Our findings indicated that rejection occurred in 20 patients, the host component disappeared in 20, and mixed chimerism without transfusion need persisted for 1 to 7 years in 15. In three patients with stable mixed chimerism for 4, 5, and 7 years, host hematopoiesis fluctuated between 25% and 75%. Despite this, donor pattern beta-globin chain synthesis maintained hemoglobin levels between 10 and 13.5 g/dL without transfusion. In these three patients, the polymerase chain reaction of the VNTR and the fluorescent in situ hybridization analysis revealed the coexistence of donor and host cells in the different peripheral blood cell subpopulations and precursors studied (CD2+, CD4+, CD8+, and CD19+ granulocytes; glycophorin-A+, erythroid burst-forming units, CD33+, granulocyte-macrophage colony-forming units). We found that rejection and disease recurrence occur in approximately one third of patients with early mixed chimerism. High levels of host type hematopoiesis can be present in patients not requiring transfusion.