Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation

In an attempt to decrease the relapse rate after bone marrow transplantation (BMT) for advanced acute leukemia, we initiated studies using 131I-labeled anti-CD45 antibody (BC8) to deliver radiation specifically to hematopoietic tissues, followed by a standard transplant preparative regimen. Biodistribution studies were performed in 23 patients using 0.5 mg/kg trace 131I-labeled BC8 antibody. The BC8 antibody was cleared rapidly from plasma with an initial disappearance half-time of 1.5 +/- 0.2 hours, presumably reflecting rapid antigen- specific binding. The mean radiation absorbed doses (cGy/mCi131I administered) were as follows: marrow, 7.1 +/- 0.8; spleen, 10.8 +/- 1.4; liver, 2.7 +/- 0.2; lungs, 2.1 +/- 0.1; kidneys, 0.7 +/- 0.1; and total body, 0.4 +/- 0.03. Patients with acute myelogenous leukemia (AML) in relapse had a higher marrow dose (11.4 cGy/mCi) than those in remission (5.2 cGy/mCi; P = .001) because of higher uptake and longer retention of radionuclide in marrow. Twenty patients were treated with a dose of 131I estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver, with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by 120 mg/kg cyclophosphamide (CY) and 12 Gy total body irradiation (TBI). Nine of 13 patients with AML or refractory anemia with excess blasts (RAEB) and two of seven with acute lymphocytic leukemia (ALL) are alive disease-free at 8 to 41 months (median, 17 months) after BMT. Toxicity has not been measurably greater than that of CY/TBI alone, and the maximum tolerated dose has not been reached. This study demonstrates that with the use of 131I-BC8 substantially greater doses of radiation can be delivered to hematopoietic tissues as compared with liver, lung, or kidney, which may improve the efficacy of marrow transplantation.     

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.     

Interindividual conservation of T-cell receptor beta chain variable regions by minor histocompatibility antigen-specific HLA-A*0201- restricted cytotoxic T-cell clones

Minor histocompatibility antigens (mHags) are involved in the induction of graft-versus-host disease (GVHD) after HLA-identical bone marrow transplantation. Previously, we isolated a series of HLA-A*0201- restricted cytotoxic T-cell (CTL) clones specific for the same mHag HA- 1 from peripheral blood of three unrelated patients who were suffering from GVHD. We have now analyzed the composition of the T-cell receptor (TCR) V regions of 12 of these mHag HA-1-specific HLA-A*0201-restricted CTL clones by DNA sequencing of the alpha and beta chains. Of these 12 clones, derived from three unrelated individuals, five independent TCR alpha V- and beta V-region sequences were established. The TCR alpha chains were composed of varying TCR alpha V and TCR alpha J genes with no obvious similarities in structure in the N regions. However, the TCR beta chains all used the TCR beta V6S9 gene segment, and showed remarkable similarities within the N-D-N regions; ie, three independent beta-chain sequences (originating from donors Ha and Gy) shared a leucine/valine amino acid pair, whereas the other two (originating from donors Ha and Wi) shared a serine/threonine pair, all at positions 99 and 100 of the TCR beta V region. In conclusion, the TCR analysis of HA- 1 mHag-specific CTL clones has shown that the HA-1 mHag/HLA-A*0201 complex selects for highly similar TCR beta V regions.     

Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status

Bone marrow from animals treated with 5-fluorouracil (5FU) competes equally with normal marrow when assessed in vivo in an irradiated mouse, but shows markedly defective engraftment when transplanted into noncytoablated hosts. Using Southern Blot analysis and a Y-chromosome specific probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrow harvested 6 days after 5FU (FU-6) and transplanted into unprepared hosts and shown that this defect is transient; by 35 days after 5FU (FU-35), engraftment has returned to levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells, and we hypothesize that the cycle status of the stem cell may relate to its capacity to engraft in the nonirradiated host. Accordingly, we have evaluated the cycle status of engrafting normal and FU-6 marrow into normal hosts using an in vivo hydroxyurea technique. We have shown that those cells engrafting from normal marrow and over 70% of the cells engrafting from FU-6 marrow were quiescent, demonstrating no killing with hydroxyurea. We have also used fluorescent in situ hybridization (FISH) analysis with a Y-chromosome probe and demonstrated that normal and post-5FU engraftment patterns in peripheral blood were similar to those seen in bone marrow, spleen, and thymus. Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these cells to transit the cell cycle.     

The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation

Despite matching for serologically defined HLA-A, B, DR antigens, acute graft-versus-host disease (GVHD) is a major complication contributing to increased morbidity and mortality in patients who undergo marrow transplantation from unrelated donors. The extent to which unrecognized mismatching for alleles that encode DR1-DR18 contribute to the increased risk of acute GVHD and overall survival is unknown. We analyzed 364 patients and their HLA-A, B, DR serologically matched donors to determine whether molecular typing of DRB1 alleles can allow more accurate donor/recipient matching and thereby improve clinical outcome after marrow transplantation. DRB1 alleles were typed by sequence-specific oligonucleotide probe hybridization methods. Selected alleles were confirmed by DNA sequencing. Of the 364 pairs, 305 were matched and 59 were mismatched for DRB1. The probability of moderate to severe acute GVHD was .48 for the matched and .70 for the mismatched patients. Compared with mismatched patients, the estimated relative risk (RR) of GVHD for matched patients was .58 (95% confidence interval [CI], .40 to .85). DRB1 matching decreased the risk of transplant- related mortality (RR, .66; 95% CI, .44 to .97) and was associated with decreased overall mortality (RR, .71; 95% CI, .51 to 1.0). Therefore, matching DRB1 alleles of the donor and recipient decreases the risk of acute GVHD and improves survival after unrelated marrow transplantation. These results indicate that prospective matching of patients and donors for DRB1 alleles is warranted.     

Second marrow transplants in patients with aplastic anemia rejecting the first graft: use of a conditioning regimen including cyclophosphamide and antithymocyte globulin

Sixteen (11%) of 146 consecutive patients with severe aplastic anemia prepared for engraftment with cyclophosphamide (200 mg/kg) rejected marrow grafts from their HLA-identical siblings. They were given a second marrow transplant from either the same (n = 13) or a second (n = 3) HLA-identical sibling between 23 and 743 (median 86) days after the first transplant. The preparation for the second transplant included cyclophosphamide, 50 mg/kg, on each of four successive days. Twelve hours after each of the first three doses of cyclophosphamide, antithymocyte globulin, 30 mg/kg/dose, was infused. One of the 16 patients died from infection too early after the second transplant to be evaluated, two had failure of engraftment and died with infection, one rejected the second graft and is surviving almost 5 years later with full autologous marrow recovery, and 12 had successful and sustained second grafts. Of these 12, six are surviving between 11 months and 7 3/4 years. Four of the six have no graft-v-host disease (GVHD), while two have chronic GVHD requiring treatment. Five have Karnofsky scores of 100% and one of 90%. Six of the 12 patients with sustained grafts died between 63 days and 38 months after transplantation, four with infections (related in two patients to chronic GVHD), one with acute GVHD, and one with hemorrhage. The average interval from first to second transplant was 308 days during the past five years, compared to 61 days in earlier patients. Five of seven recent patients are surviving, compared to two of nine earlier patients. In conclusion, successful second transplants after cyclophosphamide and antithymocyte globulin are possible in most patients with aplastic anemia who have rejected their first marrow grafts; however, mortality remains high, with only 40% of the patients becoming long-term survivors.     

Pseudosickling of hemoglobin Setif

Hemoglobin Setif produces pseudosickling of red cells in vitro; the nature of the process and the conditions that "trigger" it are unknown. Studies of red cells, hemolysates, purified hemoglobin solutions, and artificial mixtures of Hb A and Setif suggest that pseudosickling is produced by intracellular crystallization of insoluble hemoglobin. Increased tonicity of the suspending medium accentuates the process, probably by causing a rise in intracellular hemoglobin concentration. If precipitates from A/Setif mixtures are analyzed, they always contain Hb A, suggesting an unusual mechanism for the process. Despite the fact that osmolality in the renal medulla is similar to that which produces pseudosickling in vitro, carriers do not have renal dysfunction of the type found in patients with sickle cell disease.     

Bone marrow transplantation for patients with Philadelphia chromosome- positive acute lymphoblastic leukemia

We report the treatment outcome of allogeneic bone marrow transplantation in ten patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Six patients are alive and well for 6 to 30 months (median 19 months) after transplantation. Four patients died with transplant related complications. In view of the poor prognosis associated with this disease, marrow ablation followed by allogeneic or syngeneic marrow grafting may be the preferred treatment modality if a suitable marrow donor is available.     

The effect of lithium on growth factor production in long-term bone marrow cultures

We have previously reported that lithium chloride (LiCl) stimulates the production of granulocyte-macrophage colony-forming cells (GM-CFC), pluripotent stem cells (CFU-S), and differentiated granulocytes, macrophages and megakaryocytes in murine Dexter marrow cultures and that this effect appears to be mediated indirectly by a radioresistant adherent marrow cell. In this study we have established that exposure of murine Dexter cultures to LiCl (4 mEq/L) causes an increase of colony-forming cell megakaryocytes (CFU-meg) over 1 to 6 weeks of culture in both supernatant (188% to 611%) and stromal phases (123% to 246%). Moreover, we have shown that lithium treatment of either irradiated (1,100 rad) or unirradiated stromal cells increased production of activities stimulating formation of megakaryocyte, granulocyte, macrophage, and mixed lineage colonies and proliferation of the factor-dependent cell line, FDC-P1. This FDC-P1 stimulatory activity was completely blocked by an antibody to purified recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF). The baseline or lithium-induced--stromal-derived bone marrow colony stimulating activity was partially blocked by the antibody to rGM-CSF and by an antibody to purified colony stimulating factor I (CSF-1); the two antibodies combined resulted in greater than 90% inhibition of the lithium-induced marrow stimulatory activity. In addition, radioimmunoassay (RIA) showed that although CSF-1 was detectable in supernatants of these cultures, exposure to lithium did not increase CSF-1 levels. These data indicate that Dexter stromal cells produce CSF- 1 and GM-CSF and that lithium appears to exert its stimulatory effects on in vitro myelopoiesis by inducing production of GM-CSF.