Treatment of poor-prognosis, newly diagnosed acute myeloid leukemia with ara-C and recombinant human granulocyte-macrophage colony- stimulating factor

We administered recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (120 micrograms/m2/d by continuous intravenous [IV] infusion) to 12 patients with newly diagnosed acute myeloid leukemia (AML) at relatively high risk of early death during remission induction. GM-CSF began 3 days after completion of induction chemotherapy (ara-C 1.5 g/m2 d x 4 days by continuous IV infusion after a 3 g/m2 bolus). Rates of fatal infection (42%), pneumonia and/or sepsis (83%), and CR (50%) did not differ significantly (P less than .05) from those observed after administration of the identical chemotherapy without GM-CSF to 53 historical controls with newly diagnosed AML at similarly high risk of early death. There were no significant differences between the GM-CSF-treated and the historical groups in the time required to reach neutrophil counts of 500 or 1,000/microL after administration of chemotherapy. Four patients died of infection before they could have benefited from the earliest recovery of neutrophil count observed in patients who entered CR. Growth of leukemia after GM-CSF administration was observed in only 1 of the 8 patients who survived long enough for response to induction therapy to be fully evaluated. This observation suggests that it might be safe to undertake larger, randomized studies, perhaps using earlier administration of GM-CSF, to definitively determine the role of GM-CSF added to chemotherapy in patients with newly diagnosed AML.     

Platelet transfusion therapy for alloimmunized patients: selective mismatching for HLA B12, an antigen with variable expression on platelets

There can be wide variation in the expression of the HLA B12 antigen and its "splits," B44 and 45, on the platelets and lymphocytes from the same individual. One hundred sixty-two single donor platelet transfusions mismatched only for this antigen group were administered to 54 alloimmunized patients who were refractory to random donor platelets. Satisfactory increments (one-hour post-transfusion corrected- count increment [CCI] greater than 7,500) were seen following 111/162 transfusions (69%). In 31 patients (57%), all transfusions (n = 85) produced CCI greater than 7,500, and 76% of patients received some transfusions that were satisfactory. Of note is that ten patients had excellent increments despite either preformed lymphocytotoxic antibody against the mismatched antigen or positive lymphocytotoxic cross- matches with the donor. In contrast, poor increments were seen in ten recipients under similar circumstances, implying disparities in antigen expression on the platelets and lymphocytes of different donors. There was no obvious pattern of other donor HLA antigens which could be correlated with these differences. The HLA B12 antigen group is relatively common (found in approximately 25% of the population), and these data indicate that selective mismatching for these antigens can be an effective donor-selection strategy to increase the number of donors for alloimmunized recipients.     

2-chlorodeoxyadenosine induces durable remissions and prolonged suppression of CD4+ lymphocyte counts in patients with hairy cell leukemia

A number of effective treatments are available for patients with hairy cell leukemia (HCL). 2-Chlorodeoxyadenosine (2-CdA) induces more than 80% complete responses, but is associated with profound suppression of CD4+ lymphocyte counts. However, the duration of each is uncertain. We have analyzed a previously reported cohort of 40 patients who had responded to 2-CdA. Eight patients (20%) have relapsed at a median of 16 months (range, 3 to 23 months). The remaining 32 patients were observed for a median of 30 months (range, 7 to 43 months). No patients have died. At 3 years, the actuarial disease-free survival rate is 77% (95% confidence interval, 70% to 84%). The median CD4+ lymphocyte count before therapy was 743/microL (range, 58 to 2,201/microL). The median CD4+ nadir after treatment was 139/microL (range, 25 to 580/microL). There was a single opportunistic infection and no second malignancies observed. Although there was evidence of some improvement in CD4+ lymphocyte counts on sequential testing, CD4+ counts remained significantly lower than baseline (P < .0001) at a median of 23 months after therapy (median, 237/microL; range, 25 to 514/microL), and were also lower than baseline (P < .002) in those patients with more than 1 year of follow-up (median, 27 months; range, 13 to 42 months). The median time to reach an absolute CD4+ lymphocyte count of 365/microL, the lower limit of the normal range, was 40 months. Although responses to 2-CdA are durable in the majority of patients with HCL, the uncertain long-term consequences of the observed CD4+ lymphocytopenia suggest caution in the broad application of this therapy.     

Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3-4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3-4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3-4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3-3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3-3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3-3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3-4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3-3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3-4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3-3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.     

Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony- forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony- forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.     

The effect of lymphocytotoxic antibody reactivity on the results of single antigen mismatched platelet transfusions to alloimmunized patients

Despite selection strategies that attempt to maximize the platelet donor pool, significant numbers of alloimmunized patients have few if any available donors. Although the number of potential donors increases when one antigen mismatched platelet transfusions (OAMPT) are considered, transfusions from such donors are often cited to fail to produce satisfactory platelet count increments. The presence of lymphocytotoxic antibody (LCTAB) correlates well with responsiveness to random donor platelet transfusions and serves as a good serologic screen for the diagnosis of alloimmunization. We therefore reviewed the results of OAMPT to alloimmunized patients and assessed the relationship between LCTAB levels in the recipient and posttransfusion platelet count increments. We noted an unexpectedly high percentage of good responses in our patient population: 73% of all OAMPT to recipients with LCTAB < 60% reactive, resulted in successful increments. In recipients with LCTAB > or = 60%, 58% of all transfusions were still successful. Despite a statistically significant inverse relationship between the level of LCTAB and the response of OAMPT to alloimmunized patients, 58% to 73% of recipients will have a satisfactory platelet recovery posttransfusion. These data support extending donor searches for alloimmunized patients to include any single mismatch particularly if a recipient's LCTAB has lower reactivity.     

Treatment of patients over 50 years of age with acute myelogenous leukemia with a combination of rubidazone and cytosine arabinoside, vincristine, and prednisone (ROAP)

We administered a combination of rubidazone, cytosine arabinoside, vincristine, and prednisone (ROAP) to 91 patients with acute myelogenous leukemia who were 50 yr of age or older. These patients had been identified in previous studies to be a group with a relatively poor prognosis. One-third of the patients had an antecedent hematologic disorder prior to treatment. Forty patients (48%) obtained a complete hematologic and clinical remission. A history of an antecedent hematologic disorder, male sex, and absence of Auer rods were adverse factors for achieving remission in this older population. More than half of the patients achieved remission in one course. The major cause of failure to obtain a remission was death due to infection, 40% of which were caused by fungi. Resistance to chemotherapy, although uncommon, was noted more frequently in patients with an antecedent hematologic disorder. Univariate and multivariate prognostic factor analysis was used to compare these patients with a historical control group treated with a program in which adriamycin was used instead of rubidazone (AdOAP). No significant difference in remission rate was detected. Cyclocytidine was used as a maintenance agent in this study, and while the median remission duration was only 37 wk, 30% of patients are expected to be in remission for 2 yr. Chemotherapy programs combining an anthracycline with cytosine arabinoside, given to older patients in similar fasion to younger patients will achieve remissions in one-half of a group of older patients. These remissions are of comparable quality to those of younger patients. Mathematical models derived from analysis of prognostic factors are of use in identifying patients likely to fail these programs who are in need of innovative approaches to treatment.