Acute promyelocytic leukemia: All-trans retinoic acid (ATRA) along with chemotherapy is superior to ATRA alone

This study was conducted to compare the results of treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid alone (ATRA) or a combination therapy of ATRA followed by chemotherapy. Forty-three patients treated between February 1992 and February 1996 were included in this study. Eighteen patients were treated with ATRA alone and 25 patients were treated with ATRA followed by chemotherapy. The cytogenetic analysis was done in 41 patients at presentation, following treatment, and at follow-up. A complete response (CR) was achieved in 13 (72%) patients on ATRA and 19 (76%) on ATRA followed by chemotherapy. Eleven of 13 patients with response to ATRA alone relapsed with median survival of eight months (range, 1 to 28). One patient died of hepatitis in CR and one patient is alive 2 years after diagnosis. In the combination therapy arm, 10 patients are in CR with a median follow-up of 22 months (range, 6 to 56 months). After achieving a CR, four patients died due to infections during chemotherapy therapy, and only 5 of 19 patients have relapsed. Major cytogenetic response was seen in 8 of the 10 patients in whom cytogenetic data was available after treatment with ATRA at the time of remission. Similarly, 13 of 15 for whom data was available showed a major cytogenetic response after treatment with ATRA plus chemotherapy. Prior to relapse, 80% of the patients had an increase in the percentage of t(15;17) cells in the marrow. Patients with a complete hematological response but no cytogenetic response relapsed within six months. Ten patients died prior to response evaluation. Two patients who received ATRA died of retinoic acid syndrome, one of pneumonia, and one of intracranial hemorrhage. Of the six patients on ATRA and chemotherapy, four died of retinoic acid syndrome (RAS), one of intracranial hemorrhage, and one of left ventricular failure. Only one patient is alive at 24 months following treatment with ATRA alone. The relapse-free survival is 42% at four years for patients treated with ATRA followed by chemotherapy. This trial is a historical comparison of ATRA alone and ATRA with subsequent combination chemotherapy. Nonetheless, the trial shows a significant improvement in the event free survival of patients receiving chemotherapy as consolidation following ATRA. Am. J. Hematol. 60:87–93, 1999. © 1999 Wiley-Liss, Inc.     

All-trans retinoic acid delays the differentiation of primitive hematopoietic precursors (lin-c-kit+Sca-1(+)) while enhancing the terminal maturation of committed granulocyte/monocyte progenitors.

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin-c-kit+Sca-1(+)) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin- cells: 31.5% at day 7 and 4% at day 14; Sca-1(+): 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin-c-kit+Sca-1(+) hematopoietic precursors, ATRA did not exert the same effects on the more committed lin-c-kit+Sca-1(-) progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.     

All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells

The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 micromol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin- c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre-CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin- c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre-CFU-S during the 14 days of culture. In marked contrast, lin- c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre-CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation. (Blood. 2000;95:470-477)     

SDF-1 enhances the expansion and maintenance of highly purified human hematopoietic progenitors

The stromal-derived factor-1 (SDF-1) chemokine and its putative receptor, CXCR4, have been implicated in hematopoiesis. Here we aim to characterize the effects of cytokine-induced CXCR4 expression and SDF-1 treatment on primitive human umbilical cord blood (CB) cells in vitro. Highly purified CD34+CD38-Lin-CXCR4- blood cells were capable of forming CD34+CXCR4+ cells during short-term liquid culture, but maintained distinct erythroid and myeloid progenitor composition, similar to the parent population prior to culture. In vitro, SDF-1 enhanced the expansion and differentiation of primitive CB cells in a manner that was dependent upon both the concentration of SDF-1 and the presence of specific cytokines. In the absence of cytokine addition, cultures seeded with CD34+CD38-Lin- cells demonstrated substantial cell death; however, the addition of SDF-1 alone preferentially increased progenitor cell frequency. Our study demonstrates that induction of CXCR4 expression does not alter the differentiative potential of human blood progenitors and suggests a role for SDF-1 as a growth factor required for human hematopoiesis.     

E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors

OBJECTIVE: FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. MATERIALS AND METHODS: FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. RESULTS: Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. CONCLUSIONS: The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.     

Effects of erythroid differentiation factor (EDF) on proliferation and differentiation of human hematopoietic progenitors.

The effects of erythroid differentiation factor (EDF) on normal human hematopoietic progenitor cells were examined by bone marrow colony assay. Addition of EDF to the erythroid colony assay system enhanced erythroid burst-forming unit (BFU-E)-derived colony formation, and this effect disappeared on removal of adherent cells. Conditioned medium of EDF-treated monocytes also enhanced BFU-E colony formation, whereas conditioned medium of EDF-treated T cells did not. In contrast, EDF inhibited erythroid colony-forming unit (CFU-E) colony formation dose-dependently, although it had no effect on colony formation by myeloid cells. These data show that EDF has a specific effect on human hematopoietic progenitors of the erythroid lineage. The results also indicate that EDF enhanced BFU-E colony formation by stimulating adherent cells to produce factors with burst-promoting activity (BPA), but suppressed CFU-E colony formation by promoting differentiation of these cells.     

Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.     

A unique case of acute promyelocytic leukemia showing monocytic differentiation after ATRA (all-trans retinoic acid) therapy

Acute promyelocytic leukemia (PML) is characterized by a reciprocal translocation between chromosomes 15 and 17 resulting in a chimeric PML and retinoic acid receptor alpha (RARA) oncogene. The resultant fusion protein (PML/RARA) is thought to block differentiation of bone marrow cells arrested at the promyelocytic stage. In vitro and in vivo studies have shown that the large majority of APL cells undergo granulocytic maturation after ATRA therapy. We report a unique case of a PML/RARA positive APL patient exhibiting extensive monocytic differentiation after ATRA therapy as documented by morphology, flow cytometry, and FISH studies. We discuss potential dual capability for granulocytic/monocytic differentiation of PML/RARA positive APL cells and implications of monocytic differentiation in the management of APL patients treated with ATRA.     

All-trans retinoic acid directly inhibits granulocyte colony- stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte- macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)- induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.     

Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture

Cellular and molecular analysis of megakaryocytopoiesis has been hampered thus far by the lack of pure and abundant megakaryocyte (MK) cell populations. In this study, hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood, were induced to megakaryocytic differentiation/maturation in serum-free liquid suspension culture treated with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal maturation; (2) further addition of mpIL increased the MK purity level to 80% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell number (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphological evaluation indicated the presence of putative mononuclear MK precursors and then of mature polynucleated platelet- forming MKs, peaking at days 5 and 12, respectively. Membrane phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before mature MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature MKs. Furthermore, MKs synthesize and secrete low but significant amounts of both IL-6 and granulocyte- macrophage colony-stimulating factor. Comparative culture studies were performed on purified bone marrow CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no proliferation. In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of highly purified or "pure" megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis.     

Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer ("stroma-free") or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane ("stroma-noncontact"). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a "stroma-free" culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in "stroma-noncontact" cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in "stroma-noncontact" cultures supplemented with or without IL-3 than in "stroma-free" cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.     

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.