Replacement of serum by hemolysate as growth promoter for murine leukemic and normal hemopoietic progenitor cells in culture.

The present study was undertaken to test whether lysates prepared from rat erythrocytes can replace serum as in vitro growth promoters for murine leukemic and normal hemopoietic progenitor cells (CFU-C). Normal bone marrow and three leukemic cell lines (P-1081, L-1210 and P-815) were used in all the experiments. The soft agar technique for cloning hemopoietic cells was used to quantitate cell proliferation. Addition of hemolysate to the agar medium at a final concentration of 4% promoted the growth of a maximal number of leukemic colonies, similar to the number of colonies obtained when 20% serum was added to the soft agar. As for normal CFU-C, addition of 10% hemolysate or 40% horse serum promoted the growth of comparable numbers of colonies. Rat hemolysate can therefore replace horse serum as a growth promoter of leukemic and normal CFU-C present in culture. It was also shown that rat hemolysate cannot substitute for the colony stimulating factor (CSF) needed for the cloning of CFU-C. However, addition of hemolysate, CSF and serum to the soft agar cultures promoted a potentiated rather than an additive growth of CFU-C.     

Chronic neutrophilic leukemia and chronic myelomonocytic leukemia: WHO defined

The World Health Organization (WHO) classification of myeloid disorders has provided updated parameters for the consistent diagnosis of two previously less than optimally defined chronic myeloid disorders, CNL and CMML. The classification of these disorders, which had been controversial, is now better defined and provides more clinically and biologically relevant disease definitions to enable uniform diagnosis and a framework to evaluate natural history and therapeutic interventions. CNL is now recognized as a distinct entity among the chronic myeloproliferative disorders and CMML is included within the new category of 'myelodysplastic/myeloproliferative diseases' (MDS/MPD). Predominant neutrophilia defines CNL whereas CMML is defined by predominant and monocytosis. In each case these defining features must be distinguished from reactive causes for the same in the absence of clear evidence of myeloid clonality (CNL and CMML) or dysplasia (CMML). The exclusion of underlying bcr/abl-driven oncogenesis is an essential component in the diagnosis of these chronic leukemic processes. The optimal therapy for both CNL and CMML remains uncertain. Current management decisions are based on small studies or extrapolated from therapeutic strategies that are effective in similar chronic, clonal myeloid disorders. Given the potential for evolution to acute leukemia or progressive refractory leucocytosis or cytopenias, allogeneic stem cell transplantation might be appropriate for younger patients. Continued reporting and investigation of specific therapeutic strategies and responses must be encouraged.     

The effects of PGF2 alpha, PGI2, and TxB2 on human CFU-C in healthy and leukemic patients

This study was undertaken to test the effects of certain arachidonate derivatives, PGF2 alpha, PGI2 and TxB2 on in vitro bone marrow granulocyte colony growth (CFU-C) in leukemia patients receiving maintenance chemotherapy and in normal controls. The addition of PGF2 alpha did not result in increased numbers of colonies, but it did cause a shift in the size of the colonies so that there was a significant increase in larger colonies (P less than 0.001) and significantly fewer small colonies (P less than 0.05) as compared to untreated samples. Of the prostenoids tested in a Tris-buffered system, PGI2 affected the greatest increase in CFU-C (P less than 0.01) followed by PGF2 alpha (P less than 0.05) whereas 6-keto-PGF1 alpha (the stable hydrate of PGI2) did not affect colony growth. Time-response curves revealed a linear growth pattern for PGF2 alpha with a peak at 10 days, whereas there was a 6-day growth lag with PGI2 followed by linear growth with a peak at 13 days. TxB2 added to cultures significantly reduced the number of bone marrow CFU-C at all doses tested. The prostanoid effects on CFU-C derived from leukemic patients on maintenance chemotherapy and from normal individuals were identical in every respect.     

Leukemic cluster growth in culture is an independent risk factor for acute myeloid leukemia and short survival in patients with myelodysplastic syndrome

In patients with myelodysplastic syndrome (MDS) precursor cell cultures (colony-forming unit cells, CFU-C) can provide an insight into the growth potential of malignant myeloid cells. In a retrospective single-center study of 73 untreated MDS patients we assessed whether CFU-C growth patterns were of prognostic value in addition to established criteria. Abnormalities were classified as qualitative (i.e. leukemic cluster growth) or quantitative (i.e. strongly reduced/absent growth). Thirty-nine patients (53%) showed leukemic growth, 26 patients (36%) had strongly reduced/absent colony growth, and 12 patients showed both. In a univariate analysis the presence of leukemic growth was associated with strongly reduced survival (at 10 years 4 vs. 34%, p = 0.004), and a high incidence of transformation to AML (76 vs. 32%, p = 0.01). Multivariate analysis identified leukemic growth as a strong and independent predictor of early death (relative risk 2.12, p = 0.03) and transformation to AML (relative risk 2.63, p = 0.04). Quantitative abnormalities had no significant impact on the disease course. CFU-C assays have a significant predictive value in addition to established prognostic factors in MDS. Leukemic growth identifies a subpopulation of MDS patients with poor prognosis.     

Preferential growth inhibition of human leukemic versus normal myeloid colony-forming cells by 12-O-tetradecanoylphorbol-13-acetate

Myeloid leukemic cells can be induced to differentiate by various compounds, suggesting the possibility of controlling leukemia through induced differentiation. For this to be feasible, the growth of leukemic progenitor cells should be inhibited by these compounds, with the inhibition preferentially affecting leukemic over normal hemopoietic progenitor cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was chosen as a differentiation inducer and was studied for its effect on the growth of leukemic colony-forming cells (L-CFU) in ten patients with acute nonlymphocytic leukemia in comparison with normal myeloid colony-forming cells (CFU-C). Growth inhibition of both L-CFU and CFU-C was observed with TPA concentrations as low as 10(-10) M. With increasing concentrations of TPA, survival of L-CFU tended to decline more precipitously than that of CFU-C. In eight of ten patients, inhibition of L-CFU was significantly greater (P less than 0.01) than CFU-C with TPA concentrations of 10(-9) M or higher. This study indicates that a compound capable of inducing differentiation of leukemic cells can inhibit growth of leukemic progenitor cells and that this growth inhibition applies preferentially to leukemic cells as compared with normal hemopoietic cells.     

Myelodysplastic syndromes: analysis of 131 cases according to the FAB classification

The clinical and haematological findings in 131 patients with myelodysplastic syndromes (MDS), none of which had previously received chemotherapy or radiotherapy, classified according to the FAB criteria, were analysed. The distribution among the 5 subgroups was: RA 31 patients, RAS 19, RAEB 23, CMML 29 and RAEBT 29 patients. There were difficulties in the classification of 24 patients. These included, first, 8 cases with myeloid hyperplasia of the bone marrow (BM) but without monocytosis or excess of blasts of the BM. They were classified as RA. Second, 8 cases with sideroblastosis but with monocytosis or excess of blasts of the BM were classified 3 as RAEB, 2 as CMML and 3 as RAEBT. Finally, 8 cases with absolute monocytosis and BM blasts 15-30% were classified as CMML. 37 of 82 dead patients (45.1%) had transformed to acute non-lymphoblastic leukaemia (ANLL). The incidence of evolution to ANLL was low for RA and RAS (6.30% and 12.5% respectively), while it was 37.5% for RAEB, 57.1% for CMML and 77.2% for RAEBT. The median survival for each subgroup was: RA 18 months; RAS 25; RAEB 13; CMML 14 and RAEBT 10 months. It is concluded that the FAB classification with some modifications recognises group of MDS with different prognosis.     

Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.     

Leukemic pneumonitis as a poor prognostic factor in chronic myelomonocytic leukemia.

We report the case of a 69-year-old man with chronic myelomonocytic leukemia (CMML) that was complicated by fatal respiratory failure. Bilateral pulmonary infiltrates were demonstrated by chest roentgenograms and worsened with an increase in the leukocyte count. Postmortem examination confirmed the presence of leukemic monocyte infiltration of the pulmonary interstitial spaces. In CMML with marked monocytosis, respiratory failure may be one of the important causes of death in addition to infection and bleeding.     

Juvenile chronic myelogenous leukemia: characterization of the disease using cell cultures

To characterize juvenile chronic myelogenous leukemia (JCML), the proliferative properties of bone marrow (BM) and peripheral blood (PB) cells from nine patients were studied using assays for CFU-C and CFU- GEMM and liquid cultures. All specimens showed two reproducible abnormalities: impaired growth of normal hematopoietic progenitors and excessive proliferation of monocyte-macrophage colonies in the absence of exogenous colony-stimulating activity (CSA). Cytogenetic studies in one patient indicated that the CFU-C were malignant because BM cells at diagnosis and monocyte-macrophage colonies showed an abnormal karyotype, whereas PB lymphocytes did not. In contrast to JCML, PB from six adults with Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia (Ph1 + CML) yielded CSA-dependent CFU-C colonies which were composed of granulocytes, macrophages, or both, as well as exuberant growth of BFU-E colonies. Co-cultures of JCML BM adherent or nonadherent cells with normal BM resulted in suppression of normal hematopoietic colony formation. Supernatant from JCML adherent cells in liquid culture or plasma from newly diagnosed untreated JCML patients also suppressed control BM colony growth in a dose-dependent manner. These findings confirm that JCML is a malignant disorder of monocytic lineage and suggest that the mechanism of hematopoietic failure in JCML is mediated by an inhibitory monokine secreted by malignant JCML cells.     

Reduction of granulocyte-macrophage progenitor cells (CFU-C) and fibroblastoid colony-forming units (CFU-F) by leukemic cells in human and murine leukemia

The number of bone marrow granulocyte-macrophage progenitor cells (CFU-C) and fibroblastoid colony-forming units (CFU-F) was significantly smaller in acute leukemia patients. To analyze the mechanism by which CFU-C and CFU-F were reduced, the study on murine myeloid leukemia was carried out. In murine leukemia, bone marrow CFU-C and CFU-F decreased in number in correlation with the proliferation of leukemic cells. Murine leukemic cells suppressed the growth of CFU-C and CFU-F in the mixed culture of leukemic cells and normal murine marrow cells. Furthermore, leukemic cell-conditioned medium inhibited the growth of CFU-F. The results show that murine leukemic cells themselves and/or humoral factors produced by them give inhibitory effects on the growth of not only CFU-C but also CFU-F.     

A clinical study on myelodysplastic syndrome. Report of 64 cases

Systematic clinical and laboratory observations of 64 cases of myelodysplastic syndrome (MDS) were made. The results showed that malignant clone already exists in the bone marrow of patients with MDS. Eighteen cases of them have transformed into AML. The rates of transforming into AML in RAEB, RAEB-T and CMML were markedly higher than that in RA. The courses of MDS which transformed into AML from the various types were different. The transformation in RA was obviously longer than those in RAEB and RAEB-T. Two cases transformed into myelofibrosis. Diagnosis of CMML according to the FAB classification criteria revealed that it has two forms. One shows only increase of mature monocytes and it has no relation with the types of acute leukemia to be transformed. It is considered as a reactive monocytosis. The other form, in addition to increase of mature monocytes, also has increase of a few monoblasts and promonocytes. These are considered as true CMML and usually quickly developed into M4 or M5 which are related with monocytes.     

Pathological splenic rupture: a rare complication of chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is an uncommon disorder characterized by monocytosis of the peripheral blood, absence of the Philadelphia chromosome, fewer than 20% blasts, and one or more lineages showing dysplastic features. Splenomegaly is frequently seen and may be massive. A 56-year-old man with stable CMML and moderate splenomegaly presented to the emergency department with generalized abdominal pain and abrupt drop in hemoglobin. Abdominal imaging revealed splenic rupture and emergency splenectomy was undertaken, with complete recovery. Atraumatic rupture of the spleen has rarely been reported as a complication of CMML or other myelodysplastic disorders. This report should alert physicians to consider this diagnosis in patients with CMML and acute abdominal pain.     

Chronic lymphocytic leukemia coexisting with chronic myelomonocytic leukemia.

A case with coexisting chronic lymphocytic leukemia (CLL) and chronic myelomonocytic leukemia (CMML) is described. A 74-year-old man with a typical B-CLL also showed sustained peripheral blood and bone marrow monocytosis. Typical myelodysplastic changes and monosomy 7 were also found. Cytographic and immunophenotypic analysis confirmed the presence of two distinct cell populations, i.e., lymphoid and monocytoid. Both malignancies presented an extraordinarily benign prognosis. It remains uncertain whether monocytosis was either the expression of a distinct myelomonocytic clone or the progeny of a B/monocytic bipotential precursor able to feed both leukemic phenotypes.     

慢性粒—单核细胞白血病10例临床分析

本文报告了10例慢粒单白血病,并提出CMML可分为不典型CML伴单核细胞增多与RAEB或RA伴单核细胞增多两种类型。     

Constitutional Aplastic Anaemia: Defective Haematopoietic Stem Cell Growth in Vitro

S ummary . . Seven children with constitutional aplastic anaemia (one dyskeratosis congenita, one constitutional type without anomalies, five classical Fanconi's anaemia) were studied to define the pathogenesis of the marrow failure. Haematopoiesis was assessed by assay of granulocytic (CFU-C) and erythrocytic (CFU-E) colonies in vitro .
Numbers of CFU-C were profoundly reduced or absent in marrow cultures from all patients (range 0-5 CFU-C/10⁵, control range 15-106/10⁵). Numbers of CFU-E were also strikingly reduced or absent (range 0-2 CFU-E/10⁵, control range 31-317/10⁵) with one exception. No CFU-C could be grown from peripheral blood. Incubation of the patients' sera with control marrow failed to demonstrate inhibition of CFU-C, colony stimulating activity (CSA), CFU-E or erythropoietin (EPO). The peripheral blood lymphocytes from these patients in co-culture experiments failed to inhibit CFU-C or CFU-E growth from control marrow. Aplastic marrow co-cultured with control marrow also failed to demonstrate cellular inhibition. CSA production from patients'peripheral blood leucocytes averaged 93% of the activity of a standard control CSA, suggesting normal granulopoietic stimulation.
The data indicate a problem early in haematopoiesis. Although a defect in the stromal microenvironment cannot be excluded, it is possible the defect is intrinsic to the haematopoietic stem cells, either an absence or marked reduction in stem cells, or a failure of stem cells to grow colonies in Vitro .     

Chronic myelomonocytic leukemia requires granulocyte-macrophage colony-stimulating factor for growth in vitro and in vivo

OBJECTIVE: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease with no effective treatments or cure. Several factors have been implicated in its pathogenesis. In the current study, we studied the dependence of CMML on granulocyte-macrophage colony-stimulating factor (GM-CSF). MATERIALS AND METHODS: We used in vitro colony assays in methylcellulose where CMML cells were tested in the presence or absence of the specific GM-CSF antagonist E21R. We also developed an in vivo model in which CMML cells were tested for their ability to engraft into immunodeficient mice transgenic for human GM-CSF. RESULTS: Bone marrow cells from seven of seven patients with CMML formed spontaneous colonies that were sensitive to E21R treatment, with reduction in colony growth by up to 92%. E21R also inhibited colony formation by CMML patient cells stimulated by exogenously added GM-CSF but not interleukin-3. In in vivo experiments we observed engraftment of CMML cells (but not normal cells) in immunodeficient mice transgenic for human GM-CSF. None engrafted in nontransgenic mice. Cell dose escalation showed that the optimal number was 0.5 to 1 x 10(8) peripheral blood mononuclear cells per mouse, which is equivalent to an infusion of 0.2 to 3.6 x 10(6) CD34(+) cells. Time course experiments showed that maximal engraftment occurred 6 weeks after injection. CONCLUSIONS: These results demonstrate that in some CMML patients, GM-CSF produced by either autocrine or paracrine mechanisms is a major growth determinant. The results suggest that therapies directed at blocking this cytokine could control the growth of some CMML patients in vivo.     

Bone Marrow Culture Studies in Refractory Cytopenia and ''Smouldering Leukaemia''

As an adjunct to conventional haematological and cytogenetic data, 22 cases of refractory cytopenia, and five with chronic myelomonocytic leukaemia, (CMML) were studied by bone marrow culture. Cultures from II such patients without an excess of marrow myeloblasts usually showed low, or undetectable, numbers of cells capable of giving rise to colonies of granulocytes and/or macrophages (CFUc) but near-normal numbers of cluster-forming cells and cells capable of forming erythroid colonies (CFUE). Those with similar blood pictures, but in whom the marrow contained a slight excess of myeloblasts (II cases), showed a more profound defect in growth patterns: low or undetectable numbers of CFUC, clusters and CFUE, results similar to those found in acute myeloblastic leukaemia, into which three of this group evolved. The patients with CMML gave comparatively normal CFUC, cluster and CFUE growth patterns.     

Increased Levels of Myeloid Progenitor Cells in the Blood of Patients with Metastatic Invasion of the Marrow

Granulocyte-macrophage clusters and colony-forming cells (CFU-C) in the peripheral blood have been studied in 26 cancer patients with neoplastic bone marrow involvement. The concentration of CFU-C in the blood of normal individuals and of cancer patients with no bone marrow invasion, ranged from 0 to 99 ml. In contrast, out of 27 cancer patients with marrow invasion, 9 (35%) showed a significant increase of blood CFU-C (100 to 21000/ml) and of those 5 (19%) showed an increase of blood colonies (41 to 9000/ml). There was a strong correlation between increased CFU-C or colony concentration and the presence of myeloid or/and erythroid immature cells in the peripheral blood. On the other hand, there was no apparent correlation between an increased CFU-C level and anaemia or abnormal blood leucocyte count or marrow fibrosis. These observations suggest that bone marrow involvement by neoplastic cells may cause spatial redistribution of the grnulocyte macrophage progenitor cells.     

Chronic and subacute myelomonocytic leukaemia in the adult: a report of 60 cases with special reference to prognostic factors

We report 60 cases of chronic and subacute myelomonocytic leukaemias (CMML and SMML) in the adult, using the FAB group criteria. The M/F sex ratio was 3.3 and the mean age 67.5 years. Splenomegaly was found in 32% of cases, hyperleucocytosis in 52% of cases and mean blood monocytosis was 4.3 X 10(9)/l. Marrow smears showed an excess of blasts in 57% of patients, a moderate increase in monocytes in most cases and frequent myelodysplastic features. An increase in serum lysozyme and polyclonal hypergammaglobulinaemia were usual and clonal cytogenetic anomalies found in about half of the patients tested. Treatment was usually palliative and the median survival was 28 months, a blastic transformation being responsible for a third of the deaths. Prognostic factors at diagnosis were analysed retrospectively in the 46 patients who had sufficient follow up. Percentage of marrow blasts haemoglobin level and blood monocytosis at diagnosis, were subject to multivariate analysis, resulting in a discriminant 'score'. This allowed assignment of each patient into one of two prognostic subgroups (10.9% probability of error): a poor prognosis one, with a life expectancy of less than 1 year and a high risk of acute transformation (subgroup termed SMML) and a better prognosis subgroup (termed CMML), with some CMML patients surviving over 5 years.