Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes.

In this paper, we report analysis of differentiation in human hemopoietic colonies derived from a single cell. Cord blood mononulear cells and panned My-10 antigen-positive bone marrow and cord blood cells were plated in methylcellulose medium containing erythropoietin and conditioned medium. Initially, we performed mapping studies to identify candidate colony-forming cells. Subsequently, using a micromanipulator, we transferred single cells individually to 35-mm dishes for analysis of colony formation. Cellular composition of the colony was determined by identifying all of the cells in the May-Grunwald-Giemsa stained preparation. Of 150 single candidate cells replated, 63 produced colonies. The incidences of single lineage colonies included 19 erythroid, 17 monocyte-macrophage, and 9 eosinophil colonies. There were 18 mixed hemopoietic colonies consisting of cells in two, three, four, and five lineages in varying combinations. In some instances, we noted the predominance of one lineage and the presence of very small populations of cells in a second or third lineage. These results provide evidence for the single-cell origin of human multilineage hemopoietic colonies, and are consistent with the stochastic model of stem cell differentiation in man. They also indicate that restriction of the proliferative potential of committed progenitors is a stochastic process.

Elimination of clonogenic malignant human T cells using monoclonal antibodies in combination with 2'-deoxycoformycin.

2'Deoxycoformycin (dCF) specifically inhibits adenosine deaminase (ADA) and causes selective cytotoxicity of normal and malignant T cells. In clinical trials, dCF caused rapid lysis of malignant T lymphoblasts. Although dCF has been associated with dose-limiting nonhematopoietic toxicities, myelosuppression has not been observed. Since dCF is relatively nontoxic to hematopoietic stem cells, we tested dCF for utility in the ex vivo purging of malignant T lymphoblasts from remission leukemic bone marrow for autologous bone marrow transplantation. We found that T lymphoblast cell lines were sensitive to dCF (plus deoxyadenosine [dAdo]) under conditions that did not ablate human hematopoietic colony-forming cells. Moreover, combined pharmacologic (dCF plus dAdo) and immunologic (anti-T cell monoclonal antibodies [McAb] plus complement) purging resulted in additive reduction in clonogenic T lymphoblasts. These results provide the basis for a clinical trial of bone marrow transplantation using combined pharmacologic/immunologic purging of T lymphoblasts from patients' harvested autologous marrow.

Invasive fungal disease in pediatric acute leukemia patients with fever and neutropenia during induction chemotherapy: a multivariate analysis of risk factors.

We evaluated the courses of 115 consecutive cases of pediatric acute leukemia treated with induction chemotherapy. Seventy-two patients developed fever associated with neutropenia; 15 developed systemic fungal infections. We reviewed multiple demographic and treatment characteristics of these patients in an attempt to identify potential risk factors for the development of invasive fungal disease (IFD). Risk factors identified in a univariate analysis included duration of neutropenia after first fever (P less than .0001), diagnosis of acute nonlymphocytic leukemia (ANLL) (P = .003), onset of fever and neutropenia within 5 days of starting induction chemotherapy (P = .009), and multiple (greater than one) surveillance culture sites positive for fungal organisms (P = .02). In a multiple logistic regression analysis, duration of neutropenia (P less than .001) remained a significant risk factor. The study group of patients had a significantly higher risk of fungal infections than a matched group of leukemia patients developing fever with neutropenia due to postremission consolidation chemotherapy (P = .003). In the first 48 patients, 14 (29%) developed IFD. In the subsequent patients (n = 24), intravenous miconazole (5 mg/kg every 8 hours) was begun at the time of the first fever. One of the 24 patients (4%) given miconazole developed IFD. The use of miconazole was a negative risk factor for the development of IFD in univariate (P = .01) and multivariate (P = .05) analysis. We conclude that pediatric leukemia patients who develop fever associated with neutropenia during induction chemotherapy are at high risk for developing IFD. The role of intravenous miconazole at the time of the first fever in this group deserves further study.

Treatment of recurrent and refractory pediatric solid tumors with high-dose busulfan and cyclophosphamide followed by autologous bone marrow rescue.

PURPOSE: The purpose of this study was to determine the toxicities of and responses to high-dose busulfan and cyclophosphamide with autologous bone marrow transplant (ABMT) in patients with recurrent or refractory pediatric solid tumors. PATIENTS AND METHODS: We treated 18 patients (ages, 2 to 38 years; median, 14) who had tumors that were resistant to conventional chemotherapy and radiotherapy with busulfan 16 mg/kg and cyclophosphamide 200 mg/kg. Seventeen patients received bone marrow purged with 4-hydroperoxycyclophosphamide; one received unpurged marrow. RESULTS: Despite extensive prior treatment, including radiotherapy in 16 patients, toxicity generally was acceptable. For seven patients with measurable disease, there were three partial responses of 2, 10, and 20 months' duration, three patients with stable disease (SD), and one early, toxic death. Of the 11 patients with no measurable disease at the time of transplantation, one patient with osteosarcoma continues in remission at 57+ months and one third of the patients survived for at least 16 months. Mucositis was the predominant nonhematopoietic toxicity. CONCLUSION: Although the high-dose busulfan and cyclophosphamide combination showed modest activity, changes in the preparative regimen should be considered to improve the response rate in refractory tumors.

Initiation in DNA synthesis by colony-stimulating factors in subsets of human CD34+ marrow cells.

Initiation of DNA synthesis by recombinant colony-stimulating factors (CSFs) was assessed in normal human marrow blast cells isolated by expression of CD34 antigen (tritiated thymidine incorporation). Continuous exposure to CSF was required. A mild increase in DNA synthesis was initiated by granulocyte CSF (G-CSF; greater than or equal to 1 ng/ml), to approximately 1.5 times control levels. A greater increase was initiated by granulocyte-macrophage CSF (GM-CSF), with a threshold of approximately 0.1 ng/ml and a plateau increment 2.5 times control levels. CD34+ cells were stimulated by interleukin 3 (IL-3) over a wide concentration range: two times control at 0.1/ml, three times control at 1 ng/ml, and four times control at 10 ng/ml. Overlap between responding populations was analyzed. G-CSF plus GM-CSF induced DNA synthesis greater than GM-CSF alone and supported the growth of much larger granulocyte-monocyte colonies. At saturating IL-3 concentrations, neither G-CSF nor GM-CSF induced additional DNA synthesis; at lower concentrations of IL-3, however, GM-CSF recruited additional cells into DNA synthesis. Using CD10 and CD19 antibodies to separate B-lineage cells, the CD34+ cells responding to CSF were observed to be in the non-B-lineage subset. Therefore 1) the response of CD34+ cell subsets CSFs is IL-3 greater than GM-CSF greater than G-CSF, and the IL-3-responsive population is heterogeneous for dose requirement; 2) a CD34+ subpopulation responding to concurrent G-CSF and GM-CSF includes increased proliferative potential cells; 3) IL-3-responsive cells include GM-CSF- and G-CSF-responsive cells, but cells responding to lower IL-3 concentration do not respond to GM-CSF; and 4) B-cell precursors do not respond to GM-CSF or IL-3 in this assay.

Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10-positive progenitor cells in normal human bone marrow.

We have previously shown [1] that the anti-My-10 murine monoclonal antibody detected an epitope of a 115-kDa glycoprotein expressed specifically on KG-1a leukemia cells and a small subset of normal human bone marrow cells. This My-10+ marrow cell subset was shown to contain a highly enriched population of morphologic blast cells and hematopoietic colony-forming cells [1]. In this report, My-10+ cells were characterized, by flow cytometry, as an approximately 1% subpopulation of normal human bone marrow cells. My-10+ cells were slightly larger than lymphocytes and agranular, as determined by their fluorescence-activating cell sorting (-er) (FACS) light-scattering properties. In two-color immunofluorescence experiments, My-10+ cells coexpressed the HLA-DR antigen. However, there was no detectable cellular coexpression of My-10 with either the Leu 1-5, 7, 9, 11, 15, M3, or My-18 antigens. There was an average of approximately 50,000 My-10 molecules per My-10+ marrow cell. This provides further evidence that the My-10 molecule is expressed, at relatively low levels, selectively on early human marrow cells but not on mature lymphohematopoietic cells.

Antigenic analysis of hematopoiesis. IV. The My-11 hematopoietic cell surface antigen is expressed by myelomonocytic and lymphoid, but not erythroid, progenitor cells.

The anti-My-11 murine monoclonal antibody reported on in this work identifies a KG-1a cell surface glycoprotein with apparent molecular mass of 210,000 daltons. Peripheral blood B-lymphocytes, and a novel subset of T-lymphocytes (not coinciding with helper or cytotoxic subsets) express My-11 antigen; granulocytes, red cells, and platelets are antigen negative. In normal bone marrow, lymphoid progenitors (TdT positive) and most granulocyte-monocyte progenitors express My-11, but erythroid and multilineage progenitors are My-11 negative. Approximately half of acute leukemia blast cell specimens are My-11 positive. The My-11 antigen distinguishes between lymphohematopoietic cells on the basis of lineage, and assists in the purification of hematopoietic progenitor cells and the subclassification of leukemias and normal lymphocytes.

Antigenic analysis of hematopoiesis. V. Characterization of My-10 antigen expression by normal lymphohematopoietic progenitor cells.

The My-10 glycoprotein is an hematopoietic cell surface antigen expressed specifically by undifferentiated (blast) cells, constituting 1%-4% of normal adult bone marrow leukocytes. We used several immunological and in vitro culture methods to analyze the expression of this unique antigen on a variety of lymphohematopoietic progenitor cells. Colony-forming cells (CFC) for granulocyte-monocyte colonies (CFC-GM) and erythroid colonies (BFU-E) were predominantly My-10 positive. CFC with higher proliferative potential were more strongly My-10 positive than CFC with lower proliferative potential, and those for mixed-lineage and blast cell colonies were even more uniformly My-10 positive. Cells maintaining CFC-GM number in short-term marrow culture (pre-CFC) were found to be My-10 positive, as were lymphoid precursors defined by their content of intranuclear terminal deoxynucleotidyl transferase. More mature erythroid precursors (CFU-E) were heterogeneous for antigen expression and lost My-10 antigen progressively, in parallel with advancing maturational stage. The My-10 antigen permits rapid identification and purification of hematopoietic progenitor cells for further study or potential clinical application. The disappearance of the My-10 antigen, moreover, may be a probe for differentiation-linked cellular events.

Two primary brain tumors in one child.

We studied a 4-year-old boy with symptoms and signs of a posterior fossa tumor. CT showed two separate intracranial tumors: a fourth ventricle choroid plexus papilloma and a frontal subependymal giant-cell astrocytoma. This case emphasizes that, even in the absence of special genetic predisposition to CNS tumors, two separate intracranial masses may not represent CSF metastasis of a single primary tumor.

Development of a simplified counterflow centrifugation elutriation procedure for depletion of lymphocytes from human bone marrow.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic bone marrow (BM) transplantation. Techniques that effectively purge BM of mature T lymphocytes should reduce the incidence of GVHD and improve survival. We have developed a simplified, two-flow rate, fixed rotor speed counterflow centrifugation-elutriation (CCE) procedure that reproducibly depletes 99% of lymphocytes from Ficoll-Hypaque(F/H)-separated BM or BM buffy-coat. Two predetermined flow rates (24 and 28 ml/min) were used to purge small and intermediate-to-large lymphocytes, respectively, whereas faster sedimenting cells were recovered at the termination of the run. Lymphocyte depletion was substantiated by pan-T monoclonal antibody analysis as well as by complete loss of responsiveness to alloantigens and mitogens. Despite the lack of mature T cells, the depleted marrow fraction retained lymphoid colony-forming ability. Lymphocyte-purged marrow was obtained in high yield (72%), and retained high viability (greater than 97%) and hematopoietic colony-forming ability (greater than 99%). The ratio of total myeloid/erythroid colony-forming cells to T lymphocytes was 73-fold higher in the lymphocyte-depleted fraction than in unseparated BM. We concluded that a two-step CCE procedure can be used to rapidly deplete lymphocytes from both F/H-separated and buffy-coat BM inocula without altering hematopoietic capacity as measured by the in vitro clonogenic assays. It may be possible to adapt this procedure to the separation of the large number of marrow cells required for human BM transplantation.

A simple method of estimating cerebrospinal fluid pressure during lumbar puncture.

It is often difficult to measure cerebrospinal fluid (CSF) pressure in children. CSF flow through a spinal needle is described by the equation: Flow = pressure/(needle constant x relative viscosity). Thus, CSF flow rate during lumbar puncture can be used to estimate CSF pressure. Because the viscosity of CSF is approximately the same as that of normal saline, 0.9% NaCl was used to model CSF flow in vitro. Flow of saline through various spinal needles was measured as pressure and temperature were varied to determine needle constants and variation in viscosity with temperature. Counting periods for which the number of drops counted equals the pressure (in centimeters of H2O) then were determined for each needle size. At patient temperatures less than 40 degrees C, counting periods were calculated at 21, 39, and 12 seconds, for 22-gauge 1.5-inch, 22-gauge 3.5-inch, and 20-gauge 3.5-inch spinal needles, respectively. Viscosity decreased slightly above 40 degrees C, and counting periods became 20, 37, and 11 seconds. Finally, the method was tested prospectively in 12 patients by comparing drop count (over the calculated counting period) to manometric pressure measurement. Drop counts were within 15% of manometric pressure in all patients. This method allows simple and rapid estimation of CSF pressure during lumbar puncture.

Acquired chromosome rearrangements, including fine interstitial deletions, in a patient with Down syndrome and monoblastic leukemia.

A female child with Down syndrome who developed acute monoblastic leukemia is reported. Anemia associated with milk leukopenia was first recognized when the patient was 14 months old. Acute monoblastic leukemia was diagnosed 1 year later; cytogenetic studies were performed on circulating leukemic cells at this time. Analysis of elongated, finely banded chromosomes revealed three structural rearrangements, including two rather subtle interstitial deletions, in addition to trisomy 21 which was representative of the patient's constitutional karyotype. The karyotype of the leukemic cells was 47,XX,+21,t(3;18)(p23;q11.2), del(7)(q31.1q31.3), del(9)(p22p24 or p21p23). The patient received no cytostatic chemotherapy and died 4 months after the diagnosis of acute leukemia was made.

Lymphohematopoiesis: role of growth factors in leukemogenesis and therapy.

The interrelationship between proliferation, differentiation, and activation responses of hematopoietic progenitor cells and mature blood cells is complex. Therefore, we are only now learning what role colony-stimulating factors play in the regulation of normal hematopoiesis in vivo and in the dysregulation of hematopoiesis in leukemia. Recent advantages in molecular hematology have opened the door to the therapeutic administration of recombinant growth factors. Through continued preclinical trials in animals and by clinical trials in humans, a better understanding of the precise target cells and mechanisms of action of hematopoietic growth factors will improve the therapeutic index of administering colony-stimulating factors. Better understanding of hematopoietic growth factors will, in turn, suggest novel approaches to therapy of acute lymphoblastic leukemia.

Two-phase [11C]L-methionine PET in childhood brain tumors.

Thirteen children (1.8-15.8 years of age) with brain tumors were studied with [11C]L-methionine positron emission tomography (METPET). Patients were injected intravenously with tracer before a baseline PET scan was obtained. To assess the sensitivity of [11C]L-methionine uptake to competitive inhibition, 10 patients received oral L-phenylalanine (100 mg/kg); 1 hour later, a second METPET was obtained. Subjective assessment of [11C]L-methionine uptake closely paralleled results of quantitative examination (r = 0.81). [11C]L-methionine uptake in tumor-containing brain was increased in 11 patients (mean ratio of [11C] radioactivity in tumor to normal brain: 1.5 +/- 0.57; range: 1.13-2.98). Increased tracer uptake occurred in ependymomas (3), medulloblastoma (1), and astrocytomas (5), but was less intense in low-grade tumors. L-phenylalanine reduced L-methionine uptake (25-69%) in 70% of studies. L-methionine uptake was not sensitive to competitive inhibition in brain radiation injury. Two-phase METPET is of potential value in difficult clinical situations evident in children with brain tumors, including the differential diagnosis of tumor recurrence and cerebral radiation injury.

Treatment of intraocular retinoblastoma with carboplatin and etoposide chemotherapy.

PURPOSE: Management of intraocular retinoblastoma was initiated with 2-drug chemotherapy in an effort to improve the rate of vision preservation and ocular salvage and to avoid or delay the use of external beam radiation treatment. METHODS: Six patients with intraocular retinoblastoma (five bilateral; one unilateral, 1 month old) received 6 to 7 monthly cycles of intravenously administered carboplatin and etoposide (VP-16) as primary treatment. No eyes were enucleated primarily. Twelve of the 33 discrete tumors in the 11 study eyes received prophylactic supplemental treatment with cryotherapy or laser hyperthermia. Response was documented with frequent eye examinations with the patient under general anesthesia and with repeated fundus photography. RESULTS: All eight larger tumors (> 10-mm diameter) underwent dramatic regression after treatment with chemotherapy alone, and six of these tumors ultimately became fully calcific. One larger tumor and two smaller tumors showed post-treatment growth, each within 2 months after completion of chemotherapy. Six larger tumors were observed without growth or further treatment for 7 to 21 months after completion of chemotherapy. Subretinal fluid resorbed completely in four of four eyes with extensive retinal detachment, and vitreous seeding diminished considerably in four of four eyes. In five eyes, intraocular disease recurrence at a distance from any initially observed tumor eventually required treatment with external beam radiation (three eyes) or enucleation (three eyes). Eight of 11 involved eyes were salvaged, including 5 of 8 with larger tumors and 4 of 4 with vitreous seeding; 4 retained eyes received no radiation exposure, including 3 with larger tumors and 1 with vitreous seeding. Good vision was preserved in six eyes, two of which were markedly improved after occlusion therapy for amblyopia. There was no extraocular disease recurrence and no serious harm from treatment during observation ranging from 12 to 40 months after diagnosis. CONCLUSION: Chemotherapy with carboplatin and etoposide shows promise as initial treatment for intraocular retinoblastoma. Further study is indicated to define its proper role in the management of this disease.

Localization of hematopoietic progenitor cells in tissue with the anti-My-10 monoclonal antibody.

Putative hematopoietic progenitor cells were localized in human lymphoid and nonlymphoid tissues with the use of the anti-My-10 antibody and avidin-biotin immunoperoxidase. Groups of My-10+ round mononuclear cells were evident in the splenic marginal zone near the follicular mantle. My-10+ cells were also evident in the hepatic portal triads and rare cells were seen near Peyer's patches. Except for My-10+ endothelial cells, there was no staining of the thymus, lymph node, skin, or kidney specimens.

The role of hematopoietic growth factors and oncogenes in leukemogenesis.

Understanding of the roles and molecular mechanisms of hematopoietic growth factors has increased greatly in recent years. This past decade has also brought us tantalizingly close to linking a group of genes normally involved in the regulation of growth and differentiation--the cellular proto-oncogenes--to the process of malignant transformation. In this article, we review the known actions of hematopoietic growth factors in normal hematopoiesis and in hematologic malignancies. We then discuss current concepts of the roles of proto-oncogenes in normal cells and their potential involvement in leukemogenic events. A merging of these concepts with classical hypotheses of multistage carcinogenesis is emphasized.

Antigenic analysis of hematopoiesis. I. Expression of the My-1 granulocyte surface antigen on human marrow cells and leukemic cell lines.

Five monoclonal antibodies that identify the My-1 human granulocyte surface antigen were not reactive with other peripheral blood cells. These antibodies effected complement-dependent cytolysis of a large fraction of normal human marrow leukocytes. This My-1-positive marrow cell population consisted of morphologically identifiable granulocytic precursor cells. Colony-forming cells of the granulocyte-monocyte lineage (CFC-GM) did not express My-1, suggesting that the My-1 antigen is expressed later in normal granulocytic maturation. However, these antibodies did react with myeloid leukemic cell lines. The significance and potential utility of these probes for the understanding of granulopoietic differentiation is discussed.

Secondary leukemia following successful treatment of Wilms' tumor.

Leukemia accounts for 15-20% of the secondary malignancies among survivors of Wilms' tumor. We report three patients who developed leukemia after the successful treatment of Wilms' tumor, each of whom demonstrates the importance of close long-term medical surveillance. The first patient developed Philadelphia chromosome positive chronic myelogenous leukemia (CML) 6 years after the diagnosis of Wilms' tumor. This is the second report of CML occurring after Wilms' tumor. The other two patients developed acute nonlymphocytic leukemia (ANLL) 3 and 18 years after successful treatment of Wilms' tumor. In one patient, the clinical manifestations were subtle, and in the other the latency period was the longest reported for secondary leukemia following Wilms' tumor. We conclude that survivors of childhood cancer require frequent medical surveillance even in their adult years.