The effect of stem-cell factor,interleukin-3 and erythropoietin on in vitro erythropoiesis in myelodysplastic syndromes

An inherent defect of erythroid differentiation at the colony-forming unit blast (CFU-blast) compartment and (or) an impaired response of early erythroid progenitors (BFU-E) to growth stimulation are both considered to contribute to anemia in myelodysplastic syndromes (MDS). With the intention of improving survival and growth of early erythroid progenitors we investigated the effect of stem-cell factor (SCF) and interleukin-3 (IL-3) alone and in combination with erythropoietin, on the in vitro erythropoiesis of 13 patients with MDS and of three normal controls. SCF and IL-3 alone did not promote erythroid colony growth in MDS, while 3 cases responded to erythropoietin alone. In each of these, BFU-E colony growth could be increased by SCF, which was also found in all normal bone marrows. Altogether 6 cases showed a significant enhancement of BFU-E colony numbers by the combination of SCF and erythropoietin as compared to erythropoietin alone (P=0.036). Out of the 6 responding cases, 5 belonged to the FAB-classified subgroups refractory anemia (RA) and refractory anemia with ringed sideroblasts (RA/RS) (5/5), while 1 patient was classified as having refractory anemia with excess of blasts (RAEB) (1/4). No patient with refractory anemia with excess of blasts in transformation (RAEB-T) (0/4) responded. In spite of these positive effects, the absolute number of BFU-E colonies remained reduced in all MDS cases when compared to normal controls. IL-3 proved ineffective in increasing the response to erythropoietin in MDS although it increased erythropoietin-induced BFU-E formation in normal controls significantly. We conclude that the striking synergistic effect of SCF and erythropoietin on erythroid colony formation seen with normal bone marrow is conserved in most cases with RA and RA/RS. In RAEB and RAEB-T the intrinsic defect of the erythroid differentiation pathway cannot be overcome by SCF.Abbreviation MDS myelodysplastic syndrome - SCF stem-cell factor - II-3 interleukin-3 - RA refractory anemia - RA/RS RA with ringed sideroblasts - RAEB RA with excess of blasts - RAEB-T RA with excess of blasts in transformation - Epo erythropoietin     

In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.     

Evidence for nonclonal hematopoietic progenitor cell populations in bone marrow of patients with myelodysplastic syndromes

Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS) was analyzed by X-chromosome inactivation pattern using polymerase chain reaction (PCR). Five female patients were included in this study; two with refractory anemia (RA) and three with RA with excess blasts (RAEB). They were heterozygous for BstXI restriction fragment length polymorphisms (RFLP) of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each patient, erythroid and nonerythroid colonies, grown in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibited no remarkable difference in clonal constitution. Two patients showed only one methylation pattern, suggesting the monoclonal origin of hematopoietic progenitor cells. Colonies of two other patients exhibited predominant and minor methylation patterns in PGK gene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony- stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells can persist in a substantial number of MDS patients.     

High doses of intravenous recombinant erythropoietin for the treatment of anaemia in myelodysplastic syndrome

Summary. We studied the efficacy of high doses (100 000 IU intravenously (IV)/twice a week) of human recombinant erythropoietin (rHuEpo) in patients with transfusion dependent myelodysplastic syndromes (MDS). Rationale for such dose of IV Epo was the poor in vitro response of MDS erythroid progenitors (CFU-E) to physiological concentrations of Epo, and the usual high endogenous serum Epo levels of MDS patients. Seventeen patients (nine males, eight females) were included, five refractory anaemia (RA), six RA with blasts excess (RAEB), five RA with ringed sideroblasts (RARS).
Tolerance was good, except in three patients who experienced severe flu-like syndrome after Epo injection. None of the patients showed hypertension or developed anti rHuEpo antibodies. Three patients (17.6%) with RAEB had 35–60% reduction of transfusion requirements. No progression of disease occurred. Percentage of erythroblasts, endogenous baseline Epo level and in vitro cultures of erythroid progenitors did not correlate with response to Epo treatment. This study shows that very high IV doses induce only seldom and partial improvement in the status of transfusion dependent MDS. This rate of response, not higher than described with lower dosage, probably represents the maximum expectable response to rHuEpo in this category of patients.     

Recombinant human granulocyte-macrophage colony-stimulating factor plus recombinant human erythropoietin may improve anemia in selected patients with myelodysplastic syndromes

The purpose of this study was to improve erythropoiesis in patients with anemia due to myelodysplastic syndromes (MDS). We treated 13 patients first with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 6 weeks, then with recombinant human erythropoietin (rhEpo) and rhGM-CSF for the next 12 weeks. Five patients had refractory anemia (RA), 3 refractory anemia with ringed sideroblasts (RAS), and 5 refractory anemia with excess of blasts (RAEB). Ten patients were transfusion-dependent at the time of inclusion. Eleven patients completed this phase II study. Five responded with an increase in hemoglobin level (3 patients) or a reduction in transfusion requirement (2 patients). We registered no response in the remaining 6 patients during treatment. Patients responding to combined treatment had relatively low concentrations of plasma Epo and plasma ferritin before treatment with rhEpo and a normal karyotype throughout the study. Long-term bone marrow cultures did not predict the response. Still, responders seemed to have a higher number of colony-forming progenitors than non-responders. In conclusion, combined therapy with rhGM-CSF and rhEpo may stimulate hematopoiesis and correct or improve anemia in some patients with MDS. © 1993 Wiley-Liss, Inc.     

Regulatory abnormalities in the marrow of patients with myelodysplastic syndromes

Regulation of haemopoiesis in the marrow of patients with myelodysplastic syndromes (MDS) was evaluated by assaying (1) the production of haemopoietic regulators acting upon multipotent and committed progenitors by MDS marrow cells, and (2) the responsiveness of MDS marrow progenitors to stimulation with granulocyte colony-stimulating factor (G-CSF). The levels of multipotent progenitor cell colony-stimulating activity (CFU-GEMMCSA) in 7 d bone marrow-conditioned medium (BMCM) from MDS patients were markedly reduced as compared to controls. MDS BMCM also exhibited reduced levels of burst-promoting activity (BPA) for primitive erythroid (BFU-E) progenitors. Both CFU-GEMMCSA and BPA detected in BMCM were completely neutralized by antibodies directed against interleukin-3. MDS BMCM exhibited markedly reduced levels of murine-active CSA. This activity was partially neutralized by anti-CSF-1 antibodies. Levels of regulators in BMCM of refractory anaemia (RA), sideroblastic anaemia. RA with excess blasts, and chronic myelomonocyte leukaemia were virtually the same. CFU-GEMM and BFU-E growth in MDS marrow (n = 9) was markedly reduced. A 5-fold saturating dose of G-CSF induced an approximately 2-fold increase in CFU-GEMM in four of eight MDS and a 1.5-fold increase of BFU-E in five of nine MDS, but not in control (n = 5) marrow cell cultures. Impaired haemopoiesis in MDS marrow may be related to abnormalities both in regulator production by marrow accessory cells and in regulator responsiveness of multipotent and committed progenitors.     

Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors

Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders.     

Erythroid colony studies on sickle cell anemia in hypoproliferative crisis

Bone marrow cells, peripheral blood lymphocytes, and sera from patients with sickle-cell anemia in hypoproliferative crisis were studied in the plasma clot culture system in the presence or absence of erythropoietin (Epo). Bone marrow cells from five patients demonstrated a marked ability to form erythroid colonies in the presence of Epo. These studies also suggested that bone marrow cells from some patients may have an increased sensitivity to Epo. The most outstanding observation in the present study was the marked erythroid colony inhibition by serum taken from one patient during crisis. Serum taken from the same patient two months after hypoproliferative crisis had no suppressive effect on erythroid colony formation. Lymphocytes taken from three patients in crisis had a stimulatory effect on erythroid colony formation when included in culture. The conclusion is that the defect of erythropoiesis in sickle-cell anemia during hypoproliferative crisis is not due to the absence of erythroid precursor cells or to the presence of suppressor lymphocytes, but may in some cases be associated with a circulating inhibitor of erythroid maturation.     

Circulating myeloid colony-forming cells predict survival in myelodysplastic syndromes

The growth characteristics and the prognostic value of cytokine-stimulated myeloid colony formation from peripheral blood mononuclear cells (PBMC) of patients with myelodysplastic syndromes (MDS) are largely unknown. In this study we have determined the number of myeloid colony-forming units (mCFUs) in semisolid medium from 112 MDS patients and correlated them with French-American-British (FAB) type, the international prognostic scoring system (IPSS), karyotype, peripheral blood (PB) and bone marrow (BM) blast cells, cytopenias, lactate dehydrogenase (LDH), and survival data. Concerning the FAB classification, lower median mCFUs were found in patients with refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) compared to refractory anemia with excess of blast cells (RAEB) and refractory anemia with excess of blasts cells in transformation (RAEB-T). In vitro growth in MDS clearly correlated with the cytogenetic risk groups defined by the IPSS (30.5/10(5) PBMCs with favorable karyotypes, 191 in the intermediate prognostic group, 677 with unfavorable cytogenetics, p=0.015 favorable vs unfavorable). BM blast cells >5% (60.5 vs 255 colonies, p=0.032) as well as LDH levels above the normal limit (64.5 vs 425 colonies, p=0.045) were also associated with higher colony formation. Patients were stratified according to the number of circulating mCFUs into a low growth, intermediate growth and high growth group. Median survival was 343 days in the high growth, 1119 days in the low growth, and 2341 days in the intermediate growth group ( p=0.0002). Multivariate analyses revealed colony growth ( p=0.0056), PB blast cells ( p=0.0069), cytogenetic risk group ( p=0.024), and platelet count ( p=0.018) to predict survival in our patients. After inclusion of the IPSS risk categories, mCFU levels remained a highly predictive parameter for survival ( p=0.0056) and acute myeloblastic leukemia (AML) transformation ( p=0.0003).     

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.     

骨髓增生异常综合征免疫表型分析

目的：探讨免疫表型测定在骨髓增生异常综合征（MDS）诊断及分型中的价值。方法：采用一组系列相关单克隆抗体和流式细胞术对19例MDS患者免疫表型进行检测，并对其中的10例进行了细胞遗传学检查。结果：MDS患者骨髓单个核细胞（MNC）CD13，CD33抗原表达率平均分别为36．69％和41．86％，而T淋巴系抗原CD3的表达平均仅为14．49％，且随着低危的难治性贫血（RA）向高危的难治笥贫血伴原始细胞增多（RAEB）或难治性贫血伴原始细胞增多－转变型（RAEB-t)的进展，较早期的髓系抗原CD13，CD33及干（祖）细胞抗原CD34的表达升高，并伴有T淋巴系抗原CD3的表达降低。10例进行了细胞遗传学检查的患者中，5例有染色体核型异常，染色体核型异常的患者与染色体核型正常的患者在抗原表达上存在区别。结论：对MDS患者进行免疫表型检查有助于MDS的诊断分型研究。     

Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)-induced BFU-E and CFU-E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo-R in MDS marrow, we focused on Epo-induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro-apoptotic signals rather than the defect of anti-apoptotic pathways resulting from Epo-stimulated Jak2-STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.     

Inhibition of erythroid colony formation by autologous bone marrow adherent cells from patients with the anemia of chronic disease

To determine the role marrow-adherent cells may play in the anemia of chronic diseases, marrow samples were collected from ten patients with the anemia of chronic disease, seven control patients with cancer but without the anemia of chronic disease, and five normal volunteers. Marrow was either cultured directly or first depleted of adherent cells and then cultured. Plasma clots containing 6 X 10(4) nonadherent marrow cells were cocultured with marrow-adherent cells prepared by incubating 6 X 10(4)-6 X 10(2) unfractionated marrow cells in microtiter plates and removing the nonadherent cells. Adherent cell depletion of marrow from patients with anemia of chronic disease significantly increased erythroid colony formation. Coculture of adherent cells from anemic patients with autologous nonadherent marrow cells inhibited erythroid colony-forming unit (CFU-E) proliferation in patients with the anemia of chronic disease. In contrast, adherent cells from control patients did not affect autologous erythroid colony formation, and adherent cells from normal volunteers stimulated autologous erythroid colony formation. Coculture of adherent cells from anemia patients with nonadherent marrow from control patients failed to inhibit allogeneic erythroid colony formation. Media conditioned by adherent cells from patients with the anemia of chronic disease failed to suppress consistently the formation of allogeneic erythroid colonies. These data suggest that marrow-adherent cells normally stimulate erythropoiesis, but suppress erythroid progenitors, in patients with the anemia of chronic disease and may in part be responsible for their anemia.     

Kit ligand in synergy with interleukin-3 amplifies the erythropoietin- independent, globin-synthesizing progeny of normal human burst-forming units-erythroid in suspension cultures: physiologic implications

Although the proliferative effects of hematopoietic cytokines on erythroid progenitors are well known, parameters that influence the initiation of expression of specialized or lineage-restricted genes are not clear. We have studied the acquisition of erythroid-differentiative features from enriched populations of human early erythroid progenitors (burst-forming unit-erythroid [BFUe]) in suspension culture and the influence of several cytokines on this process. In suspension cultures containing no erythropoietin (Epo), we have found that kit ligand (KL) in synergy with interleukin-3 not only increases the proliferation of cells and of progenitors but also consistently amplifies a population of cells that contain globin within 1 week. Our experiments suggest that neither extraneously provided nor endogenously produced Epo is critical for the generation of globin-synthesizing cells. Globin- producing cells generated mostly from late BFUe or pre-CFUe with a CD34- /EP-1+ phenotype in this system do not all express a well-coordinated erythroid program accompanied by heme or glycophorin A expression and most die maintaining an immature state. Therefore, conditions that are responsible for initiation of globin expression in these cells are not sufficient to carry them to terminal maturation. The data point to an expanded target cell population for KL, as they suggest an influence of KL on survival and/or amplification of late erythroid cells previously thought to be influenced only by Epo. Our results in aggregate are of relevance to the physiology of normal erythropoiesis and the role of Epo and KL in the initial stages of lineage-restricted gene expression. In addition, they provide insight into the understanding of anemia in W and Steel mutants in which expansion of the late erythroid progenitor pool, normally dependent on the synergistic action of KL and Epo, is curtailed.     

In polycythemia vera human interleukin 3 and granulocyte-macrophage colony-stimulating factor enhance erythroid colony growth in the absence of erythropoietin

To further define the growth factors required for the in vitro proliferation of erythroid progenitors in polycythemia vera (PV), we have compared the ability of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to support the growth of erythropoietin (Epo)-dependent and -independent erythroid colony formation. By using nonadherent mononuclear cells from peripheral blood, Epo-dependent colony formation was enhanced by IL-3 and GM-CSF in PV patients. Comparable results were obtained with normal erythroid progenitors. Augmenting effects of IL-3 and GM-CSF were observed on spontaneous erythroid colony formation, i.e., erythroid colony formation in the absence of exogenous supplied Epo. This was not due to a small amount of Epo in the culture media because an anti-Epo antibody did not prevent endogenous colony formation, nor did it prevent the enhancing effects of IL-3. Finally it was observed that in contrast to IL-3, monocyte depletion was required for the enhancing effects of GM-CSF on erythroid colony formation. These results provide evidence that endogenous colony formation in PV is independent of Epo but can be augmented by IL-3 or GM-CSF.     

Comparison of hemin enhancement of burst-forming units-erythroid clonal efficiency by progenitor cells from normal and HIV-infected patients.

The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.     

Prognostic value of clonal chromosomal abnormalities in patients with primary myelodysplastic syndromes

Chromosome analyses were carried out on bone marrow cells from 43 consecutive patients with primary myelodysplastic syndromes (MDS), classified according to the French-American-British (FAB) cooperative group criteria. The objective was to evaluate the prognostic value of clonal chromosomal abnormalities and of an excess of blasts for early death from acute nonlymphocytic leukemia (ANLL) and/or bone marrow failure (BMF). Patients were subdivided into two main groups: (1) refractory anemia without an excess of blasts (RAWEB), grouping patients with refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS), and (2) refractory anemia with an excess of blasts (RAEB), grouping patients with refractory anemia with an excess of blasts (RAEB) and refractory anemia with an excess of blasts in transformation (RAEBt). There were 29 patients with RAWEB and 14 with RAEB. The median time of observation was 26 months for RAWEB and 12 months for RAEB. Ten RAWEB patients (34%) and 11 RAEB patients (78%) had clonal chromosomal abnormalities. Among the ten RAWEB patients with clonal abnormalities, one (10%) died from ANLL, while of 19 RAWEB patients with a normal karyotype, two (10%) died from ANLL or BMF. The median survival for patients with RAWEB and an abnormal karyotype was not reached. In contrast, eight of the 11 RAEB patients with clonal chromosomal abnormalities (74%) died from ANLL or BMF. The median survival in this sub-group was 7 months. By using a Cox proportional hazard regression analysis, it was determined that a karyotype abnormality was not a significant predictory of survival once the contribution of the RAWEB/RAEB variable was taken into account. Being in the RAEB group was associated with a relative risk of 10.6 of dying from ANLL or BMF (beta = 2.36, standard error (SE) = 0.68, P = .0001). We conclude that classifying patients according to an excess of blasts will lead to a better prediction of survival than determining karyotype abnormality.     

Erythropoietin-independent erythroid colony formation in patients with erythroleukaemia (M6) and related disorders

In vitro erythropoietin (Ep) responsiveness of human bone marrow mononuclear cells was determined in 12 normal human volunteers and four patients with erythroleukemia (EL), two patients with refractory anaemia with excess blasts (RAEB), and one patient with de novo acute myelogenous leukaemia (AML). The bone marrow cells were cultured in a microtitre methylcellulose system containing 30% human AB serum and human urinary Ep in concentrations ranging from 0 to 2 units/ml. Erythroid colony growth from normal marrow cultures was Ep-dependent. It was augmented by added Ep and inhibited by Ep antiserum. Marrow cells from one patient with EL and one patient with RAEB after transformation to AML had no erythroid colony formation with or without added Ep. All of the remaining patients formed 'spontaneous' or endogenous erythroid colonies (EEC) without the addition of Ep. In three of these (two with EL and one with de novo AML), the erythroid colony formation was augmented by added Ep. In three other patients (one with EL and two with RAEB), erythroid colony growth was unaffected by added Ep or Ep antiserum, and thus appeared to be Ep-independent.     

Primary refractory anemia: clinical and laboratory study of erythropoiesis in 16 patients

Erythropoiesis was studied in vitro in 16 selected patients with primary refractory anemia without excess of blasts who have been followed for an average of 4.8 years. The number of erythroid colonies and bursts grown in vitro from the patients' marrows did not correlate with any parameter of their disease or their prognosis. The response of marrow erythroid precursor cells to erythropoietin was found to be normal. In no case was a serum or IgG inhibitor of erythropoiesis detected either by quantitation of heme synthesized by marrow cells or by the erythroblast cytotoxicity assay. A clinically significant response of the anemia to corticosteroids was noted in three out of 14 patients. Ten patients died during the followup period, eight of them as a consequence of their hematologic disorder. Bone marrow aplasia with pancytopenia developed in six cases, increased number of marrow blasts in two cases, myelofibrosis with myeloid metaplasia in one case and a spontaneous remission in another case. Refractory anemia without excess of blasts is a heterogeneous disorder with variable natural history including evolution into marrow aplasia.