CD34-positive cell selection by immunomagnetic beads and chymopapain.

BACKGROUND. Pluripotent hemopoietic stem cells, progenitors of all hemolymphopoietic lineages, and clonogenic cells from many patients with acute nonlymphocytic leukemia (ANLL) and chronic myeloid leukemia (CML) express the CD34 antigen on their surface. Isolation of these cell populations is of primary experimental and clinical importance. METHODS. Six bone marrow (BM) and 10 peripheral blood (PB) samples were obtained from 2 normal individuals, 3 patients with CML and 9 with ANLL. The CD34+ cell fraction was isolated using MY10 antibody, sheep anti-mouse immunomagnetic beads and the enzyme chymopapain. Indirect immunofluorescence and semisolid culture were employed to evaluate the percentage of CD34+ cells and that of clonogenic cells in each cell fraction. RESULTS. The frequency of CD34+ cells in the original unseparated populations was (mean +/- SE) 24.3 +/- 7.3%, and reached 85.0 +/- 2.7% in the isolated CD34-positive fractions; in the negative fractions it was only 2.7 +/- 1.7%. According to these results, the great majority of clonogenic cells was separated in the CD34-positive fractions and depleted in those CD34-negative. Moreover, chymopapain was shown to be non-toxic to the clonogenic cells. CONCLUSIONS. Positive immunoselection using My10 Ab, immunomagnetic beads and chymopapain is a method for isolating almost pure progenitors from the BM and PB of normal individuals and patients with myeloid leukemias.     

Secretion of tumor necrosis factor-alpha by fresh human acute nonlymphoblastic leukemic cells: role in the disappearance of normal CFU-GM progenitors.

The disappearance of normal hematopoiesis during acute nonlymphoblastic leukemia (ANLL) is poorly understood. Several reports indicate that conditioned medium obtained from leukemic cells might inhibit the formation of normal hematopoietic progenitors. However, these blast-conditioned medium (BCM) inhibitory activities are not well characterized. In order to evaluate whether BCM might contain an activity inhibiting the growth of normal marrow progenitors, BCM from 13 consecutive patients with ANLL were tested on normal bone marrow in methylcellulose assays. In all the cases, a significant inhibition of the growth of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors was observed, whereas erythroid burst-forming unit (BFU-E) progenitors were not affected. Further characterization of the BCM inhibitory activity showed using both a biological assay and RIA, the presence of tumor necrosis factor-alpha (TNF-alpha) in 10 out of 13 BCM. Northern blot analysis performed in six patients showed a correlation between the expression of TNF-alpha mRNA by leukemic cells and the presence of TNF-alpha in BCM. Moreover, the BCM inhibitory activity could be neutralized with an anti-TNF-alpha antiserum. These data indicate that leukemic cells express and release frequently TNF-alpha, which may therefore play an important role in the inhibition of granulopoiesis during leukemia.     

Influence of recombinant alpha and gamma interferons on the in vitro proliferation of myeloid and leukemic progenitors

Abstract: We have compared the effect of alpha 2-C and gamma recombinant interferons (rIFNs) on normal myeloid progenitors (N-CFU-GM), chronic myeloid leukemia (CML) progenitors (CML-CFU-GM) and leukemic progenitors (L-CFU) of acute non-lymphoblastic leukemia (ANLL) patients. Within 14 days of continuous exposure in culture, a dose-dependent inhibition of CFU-GM was seen for most normal subjects. Resistance to rIFNs was frequent in leukemic patients and even more in acute leukemia than in CML. Stimulation of clonogenic cell growth was seen for a minority of leukemic patients. When only the sensitive cases were considered, no difference in sensitivity was noticed between normal, CML and ANLL patients. A good correlation was observed between the activity or the lack of activity of alpha and gamma rIFNs.     

Natural killer cell-mediated inhibition of growth of myeloid and lymphoid clonogenic leukemias

We have shown that peripheral blood (PB) lymphocytes of normal donors inhibit colony formation by myeloid (K-562) and lymphoid (Molt-4) leukemia cell lines in a clonogenic assay in vitro. The inhibitor cells were identified as natural killer (NK) cells based on their large granular lymphocyte (LGL) morphology and CD16+CD5- cell surface phenotype. The levels of inhibition were dependent both on the leukemia: effector (L:E) cell ratio, as well as on the time of preincubation of the leukemic and effector cells; maximum inhibition was observed at a 1:20 L:E cell ratio, and required 6-16 h preincubation of the leukemia and effector cells. Colony formation of both K-562 and Molt-4 was also inhibited by a soluble factor derived from coculture of K-562 and PB lymphocytes. The finding that the growth of clonogenic Molt-4 cells was almost completely abolished following treatment with interferon (IFN)-alpha rA in the dose of 10(3) U/ml, whereas growth of K-562 cells was only slightly affected by similar treatment, suggested that NK cells may mediate inhibition of clonogenic leukemias through various mechanisms.     

Effect of pharmacologic manipulation of protein kinase C by phorbol dibutyrate and bryostatin 1 on the clonogenic response of human granulocyte-macrophage progenitors to recombinant GM-CSF

The effect of pharmacologic manipulation of protein kinase C (PK-C) activity on the response of committed human myeloid progenitor cells (CFU-GM) to recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF) was assessed. Coadministration of the PK-C activating agents, phorbol dibutyrate (PDBu) or bryostatin 1, with rGM-CSF resulted in a dose-dependent and, under some conditions, highly synergistic increase in the number of CFU-GM. With optimal combinations, colony formation far exceeded that which could be obtained with high concentrations of rGM-CSF alone. High concentrations of PDBu (e.g. greater than or equal to 50 nM), but not bryostatin 1, completely inhibited the CFU-GM response. These inhibitory effects could be reversed by bryostatin 1, but not by high concentrations of rGM-CSF. Bryostatin 1 also potentiated colony formation in response to rGM-CSF, and blocked the inhibitory effects of high concentrations of PDBu in bone marrow cells highly enriched for progenitors bearing the MY-10 antigen. The increase in CFU-GM induced by PDBu or bryostatin 1 was associated with little change in the morphologic type of colony observed. Continuous exposure of cells to the calcium ionophore, ionomycin (500 nM), reduced the number of granulocyte-macrophage colonies, but produced little change in the concentration-response of rGM-CSF and PK-C activating agents. Finally, the PK-C inhibitors H-7 and tamoxifen, when administered at concentrations exhibiting minimal inhibitory effects in the presence of rGM-CSF alone, led to no change or small increases in the numbers of colonies formed in response to rGM-CSF and bryostatin-1, and a substantial increase in the number of colonies formed in the presence of rGM-CSF and PDBu. These results suggest that PK-C activation may play a complex role in regulating the response of normal myeloid progenitors to growth factors such as rGM-CSF. They also raise the possibility that under some circumstances the phorbol ester PDBu may trigger events that inhibit the growth of myeloid progenitors, and that this process may be blocked by bryostatin 1.     

Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias

Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in "serum-free" cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM- CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two "de novo" leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.     

Significance of in vitro cultures in myelodysplastic syndromes

Bone marrow samples from 20 controls, 41 patients suffering from various types of myelodysplasia and 19 suffering from ANLL were investigated by in vitro cultures. The cultures were stimulated by various concentrations of leucocyte conditioned medium (LCM) and PHA-stimulated conditioned medium (PCM) and were examined after 7 and 14 d. We found that, in clinically stable MDS, growth patterns and dose-response to CM's were mostly within the normal range. With progressive blastic transformation, these features became abnormal with an increase in cluster growth. Clusters responding to a high dose of LCM, persisting after 14 d and enhanced by PCM may represent 'early' clonogenic cells. These clusters were found in progressive MDS with increased numbers of blast cells. Clusters formed by 'late' clonogenic cells were found in normal bone marrow and stable MDS. In ANLL the disturbance of proliferation and maturation seems to be much more pronounced than in progressive MDS with blastic transformation. We conclude that the interpretation of in vitro bone marrow culture data in terms of a disorderly arrangement of clonogenic cells in MDS and ANLL is facilitated by comparing different conditioned medium stimulations and by scoring after different time intervals.     

A micromethod for determination of terminal deoxynucleotidyl transferase (TdT) in the diagnostic evaluation of acute leukemias

Summary A micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 x 10⁶ leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 x g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinicalmorphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.Abbreviations ALL acute lymphoblastic leukemia - ANLL acute non-lymphoblastic leukemia - ATP adenosine triphosphate - AUL acute undifferentiated leukemia - B cell bone marrow related lymphocyte - CFU-c Colomy-forming unit in soft agar culture (myeloid committed stem cell) - CM chronic myelogenous leukeima - E-rosette sheep erythrocyte rosette - (³H) dGTP tritium-labeled deoxyguanosine triphosphate, incorporated into nucleic acid as monophosphate: (³H) dGMP - HTLA human T-lymphoeyte antigen - MDS myelodysplastic syndrome - MPS myeloproliferative syndrome - MPS-AP in acute or blastic phase - Oligo d(pA)_12-18 polymer of deoxyadenylic acid, chain length 12–18 residues - PB-T peripheral blood T cell - slg surface immunoglobulins - T cell thymus-related lymphocyte - TdT terminal deoxynucleotidyl transferase - TdT specific TdT activity in bone marrow mononuclear cells <0.10 units/10⁸ cells - TdT+ TdT activity >0.10 units/10⁸ cells - thy-T thymocyte This research was supported in part by the following grants: ACS PDT-95, NCI-CA-17404, NCI-CA-19267, NCI Program Project Grant 3 PO1 CA-20194, NCI Grant CA-08748, the Gar Reichman Foundation and the Zelda R. Weintraub Cancer Fund, and a research career development award no. 1 KO4-CA-00545 from the National Cancer Institute to MJM     

AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1

AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.     

Comparison of in vitro growth characteristics of blast cell progenitors (CFU-L) in patients with myelodysplastic syndromes and acute myeloid leukemia.

Current knowledge is inadequate to explain the different patterns of blast cell accumulation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We compared the growth patterns of blast cell progenitors (CFU-L) in 23 patients with advanced MDS and 32 patients with de novo AML. Circulating blast progenitors were identified in 74% of MDS and 81% of AML samples. Primary plating efficiencies (PE1) were similar in both disorders, despite marked differences in peripheral blast cell concentrations. By cytological and cytochemical examination, colonies from MDS patients were indistinguishable from those obtained in AML. Cell cycle status was assessed by loss of colony formation following short-term exposure to cytosine arabinoside. CFU-L suicide rates (median, range) were 40% (12% to 77%) in MDS and 60.5% (27% to 98%) in AML. Actively proliferating blast cell progenitors are thus not confined to AML, but are also present in the majority of MDS patients. An important difference between MDS and AML was found when self-renewal capacity of CFU-L was examined by means of secondary plating efficiencies (PE2). Colonies could be successfully replated in 74% of AML cases. PE2 showed marked heterogeneity (2 to 730 colonies/10(5) mononuclear cells), with some values indicating excessive self-renewal capacity of CFU-L. In contrast, 62% of the MDS specimens failed to produce any secondary colony growth, and PE2 in the remaining cases was low (5 to 99/10(5) MNC). We conclude that a different balance between self-renewal and determination could be responsible for a slower pace of clonal expansion in MDS, even if the proliferative activity of clonogenic cells is similar to that in AML.     

Bryostatin 1 induces differentiation of B-chronic lymphocytic leukemia cells

Peripheral blood cells from nine patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate). Like the phorbol ester 12-0-tetradecanoyl-phorbol 13-acetate (TPA), bryostatin 1 activates protein kinase C (PKC), which plays a central role in the phosphatidylinositol signal transduction pathway. The effects of bryostatin 1 alone and in combination with TPA or with the calcium mobilizing ionophore A23187 were assessed by morphological appearance, cell adherence and aggregation, RNA and DNA synthesis, and immunoglobulin (Ig) production. While eight of nine B-CLL cultures remained proliferatively inert, bryostatin 1 could effectively trigger activation and differentiation of B-CLL cells in all cases as inferred by the induction of morphological changes, RNA synthesis, and monotypic Ig production. Addition of calcium ionophore A23187 to bryostatin 1-exposed cells resulted in significantly increased values for RNA synthesis and Ig production and in the acquisition of plasmacytoid morphology. Bryostatin 1 and the dual signal of bryostatin 1 plus A23187 mimicked the stimulatory action of TPA and the combination of TPA plus A23187, respectively. Overall, bryostatin 1 was less active than equivalent concentrations of TPA. This lesser efficacy may, however, reflect a quantitative rather than qualitative difference. Bryostatin 1 partially antagonized TPA-mediated effects on B-CLL cells suggesting different modes of action by the two activators. These studies indicate that bryostatin 1 has effective differentiation-inducing properties on B-CLL cells that can differentiation-inducing properties on B-CLL cells that can be accentuated by a calcium ionophore.     

Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta

CD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105(+) cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105(intermediate) population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105(high) population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process.     

IGF signaling contributes to malignant transformation of hematopoietic progenitors by the MLL-AF9 oncoprotein

Malignant transformation of normal hematopoietic progenitors is a multistep process that likely requires interaction between collaborating oncogenic signals at critical junctures. For instance, the MLL-AF9 fusion oncogene is thought to contribute to myeloid leukemogenesis by driving a hematopoietic stem cell-like "self-renewal" gene expression signature in committed myeloid progenitors. In addition, insulin-like growth factor (IGF) signaling has been implicated in self-renewal/pluripotency in hematopoietic and embryonic stem cell contexts and supports cell growth/survival by activation of downstream pathways, including phosphatidylinositol 3-kinase/Akt and Ras/Raf/extracellular signal-regulated kinase. We hypothesized that IGF signaling could be an important contributor in the process of cellular transformation and/or clonal propagation. Utilizing an MLL-AF9 mouse bone marrow transplantation model of acute myelogenous leukemia, we discovered that committed myeloid progenitor cells with genetically reduced levels of IGF1R were less susceptible to leukemogenic transformation due, at least in part, to a cell-autonomous defect in clonogenic activity. Rather unexpectedly, genetic deletion of IGF1R by inducible Cre recombinase had no effect on growth/survival of established leukemia cells. These findings suggest that IGF1R signaling contributes to transformation of normal myeloid progenitor cells, but is not required for propagation of the leukemic clone once it has become established. We also show that treatment of mouse MLL-AF9 acute myelogenous leukemia cells with BMS-536924, an IGF1R/insulin receptor-selective tyrosine kinase inhibitor, blocked cell growth, suggesting its efficacy in this model may be due to inhibition of insulin receptor and/or related tyrosine kinases, and raising the possibility that similar IGF1R inhibitors in clinical development may be acting through alternate/related pathways.     

Effect of stem cell factor (c-kit ligand) on clonogenic leukemic precursor cells: Synergy with other hematopoietic growth factors

Using clonogenic assay we investigated the effect of stem cell factor (SCF) on the in vitro growth of clonogenic precursor cells from acute myeloid leukemia (AML) and myelodys-plastic syndromes (MDS) in the presence or absence of recombinant human erythropoietin (rhEpo) or recombinant human granulocyte colony-stimulating factor (rhG-CSF). SCF as a single factor did not induce significant colony formation, and even in the presence of rhEpo or rhG-CSF it very weakly stimulated erythroid colony formation and was rarely capable of inducing myeloid colony formation by clonogenic leukemic cells. In culture dishes supplemented with SCF, both myeloid and erythroid colony formations were dramatically enhanced in MDS, regarding both colony number and size. Cotony-formation abilities by MDS progenitors were improved following costimulation with SCF and rhEpo. These results suggest that SCF may have a therapeutic role in restoring hematopoiesis in patients with MDS. © 1994 Wiley-Liss, Inc.     

The action of bryostatin on normal human hematopoietic progenitors is mediated by accessory cell release of growth factors

Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34-positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone-marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.     

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied. MGF as a single factor did not induce significant colony formation by clonogenic leukemic precursor cells. In the presence of IL-3 and/or GM-CSF, MGF weakly stimulated the colony formation by clonogenic precursor cells from patients with AML. In contrast, in the presence of IL-3 and/or GM-CSF, MGF strongly induced both size and number of leukemic colonies from patients with CML in chronic phase. Furthermore, in the presence of EPO, MGF strongly stimulated erythroid colony formation by CML precursor cells. Cytogenetic analysis of the colonies showed that all metaphases after 1 week of culture were derived from the leukemic clone. In patients with MDS, MGF strongly stimulated myeloid colony formation in the presence of IL-3 and/or GM-CSF (up to fourfold), and erythroid colony formation in the presence of EPO (up to eightfold). Not only the number, but also the size of the colonies increased. In the presence of MGF, the percentage of normal metaphases increased in three patients tested after 1 week of culture compared with the initial suspension, suggesting that the normal HPC were preferentially stimulated compared with the preleukemic precursor cells. In the absence of exogenous EPO and in the presence of 10% human AB serum, MGF in the presence of IL-3 and/or GM-CSF induced erythroid colony formation from normal bone marrow and patients with MDS or CML, illustrating that MGF greatly diminished the EPO requirement for erythroid differentiation. These results indicate that MGF may be a candidate as a hematopoietic growth factor to stimulate normal hematopoiesis in patients with acute myeloid leukemia, or with myelodysplastic syndromes.     

Phase I-II study of continuous-infusion high-dose human lymphoblastoid interferon and the in vitro sensitivity of leukemic progenitors in nonlymphocytic leukemia

Twelve pediatric patients with nonlymphocytic leukemia were treated for 10 days with high-dose (15, 20, or 30 million U/m2/day) human lymphoblastoid interferon (Wellferon) administered by continuous iv infusion. Nine children had acute nonlymphocytic leukemia (ANLL) in relapse, two had Philadelphia chromosome-positive chronic myelocytic leukemia in myeloblastic crisis, and one had juvenile chronic myelocytic leukemia. Blast cell counts in the peripheral blood decreased in five patients with ANLL treated with the higher interferon doses; however, there was no evidence of an antileukemic effect in the marrow. Dose-limiting toxicity, which included malaise, hepatotoxicity, and coagulation abnormalities, was observed in patients given 20 or 30 million U/m2/day. Studies of the growth of leukemic progenitor cells in vitro in the presence of interferon disclosed a concentration-related inhibition of colony formation. Patients who had a decrease in peripheral blast cell counts demonstrated greater in vitro inhibition of clonogenic leukemic progenitors than patients whose blast cell counts did not decrease. However, the serum interferon concentrations in patients given clinically tolerable doses were lower than those concentrations which inhibited leukemic cell growth in vitro by a median of 42% (1000 U/ml). This study failed to demonstrate clinically significant antileukemic activity against nonlymphocytic leukemia in patients given high-dose constant-infusion interferon, and the toxicity of this approach was prohibitive.     

Comparison of in vitro inhibition of etoposide (VP16) on leukemic and normal myeloid, erythroid clonogenic cells

We have compared in various clonogenic assays the in vitro sensitivity to etoposide (VP16) of 1) human leukemic precursors (leukemia colony-forming units; L-CFU), 2) normal erythroid progenitors (erythroid burst-forming units; BFU-E, and 3) normal committed myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM and more primitive hemopoietic precursors (PPC) that adhere to cultured marrow stromal cells. Bone marrow samples were obtained from 15 normal subjects and 16 leukemic patients: 9 in the acute phase of acute nonlymphoblastic leukemia (ANLL) and 7 in complete remission. VP16 was tested at concentrations ranging from 10(-8) to 10(-3) M. The median recoveries at 10(-3) M VP16 were respectively 0%, 0.5%, 0%, and 0% for leukemic progenitors, CFU-GM from leukemic patients in complete remission, normal CFU-GM, and BFU-E, and 23% for PPC. This indicates that CFU-GM, BFU-E, and L-CFU are highly sensitive to VP16, whereas PPC, more primitive myeloid precursors, are spared. These results suggest that VP16 may be used as an "ex vivo" purging agent for autologous bone marrow.