G. B. Tennant

Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia.

Myelodysplastic syndromes (MDS) are characterized by a clonal disorder of haemopoiesis with defective growth in vitro. The long‐term culture system was used to examine aspects of stromal function in MDS patients. Primary long‐term cultures of MDS bone marrow showed poor myelopoiesis with progenitors being detected for a median 3·5 weeks (n = 12) compared with 18 weeks in cultures of normal marrow (n = 10; P < 0·0001). The haemopoietic function of adherent layers was assessed in secondary co‐cultures seeded with 5 × 106 cord blood mononuclear cells on irradiated normal (n = 27; aged 38–82 years) or MDS (n = 32; aged 41–86 years) adherent layers (> 60% confluent). The median myeloid progenitor number/cord blood co‐culture was 135 in 5‐week‐old cultures with normal adherent layers and 22 in those with MDS layers (P < 0·0001). Myeloid colonies were detectable for a median 11 weeks with normal adherent layers and 6 weeks with MDS adherent layers (P < 0·0001); erythroid colonies were detectable for 7 weeks (normal) compared with 5 weeks (MDS) (P < 0·01). The differences in granulocyte‐macrophage colony forming unit (CFU‐GM) generation were not related to patient age. Cells from adherent layers of at least half of the primary normal (n = 48) and MDS (n = 26) long‐term cultures expressed cytokines [interleukin (IL)‐3, IL‐1β, thrombopoietin (Tpo) and erythropoietin (Epo)] and receptors for retinoic acid (RARα) [IL‐2, IL‐3, macrophage colony stimulating factor (M‐CSF) (Fms) and Tpo (Mpl)]. Only IL‐1β expression was reduced in week‐5 MDS cultures compared with those from normal marrows (P < 0·05). There was also a highly significant decline in IL‐1β expression in normal (but not MDS) adherent layers between week 5 and week 10. Thus, the adherent layers in cultures grown from MDS patients were haemopoietically defective and showed abnormal IL‐1β expression.

Colony-cluster ratio and cluster number in cultures of circulating myeloid progenitors as indicators of high-risk myelodysplasia.

Circulating myeloid progenitors were assayed in 172 normal subjects and 147 patients with myelodysplastic syndrome (MDS). Patients whose cultures had colony/cluster ratios (CCR) < 0·3 had significantly shorter survival periods than comparable patients with CCR > 0·3. A second prognostic indicator, which complemented CCR, was identified in patients with < 5% blasts. Median survival was significantly reduced in patients with > 15 clusters/ml blood despite colony and cluster numbers being predominantly within the normal range. Characteristic differences were found in three FAB groups large enough to allow statistical analysis. Survival amongst patients with refractory anaemia with excess of blasts (RAEB) was related to CCR and was independent of cluster number. Amongst sideroblastic patients (SA) survival related only to cluster number. Refractory anaemia (RA) patients included individuals in both high‐risk groups with only three patients out of 64 showing both features. Amongst all the MDS patients, those with CCR > 0·3 and < 15 clusters/ml blood formed a low‐risk group (n= 60) with a relatively good prognosis of whom 85% survived the study period (median duration 938 d) including 94% of those in this group with < 5% marrow blast cells.

Prognosis of myelodysplasic patients: non-parametric multiple regression analysis of populations stratified by mean corpuscular volume and marrow myeloblast number.

Summary.  Myelodysplastic (MDS) patients at diagnosis (n = 162) were analysed by the International Prognostic Scoring System (IPSS). The two intermediate groups were not significantly different. The IPSS was of limited value in predicting survival of MDS patients after preliminary separation into subgroups with < 5%, or ≥ 5%, myeloblasts. Coxs proportional hazards analysis of these subgroups enabled discrimination of highly significant prognostic groups. In both subgroups, longer survival was associated with macrocytosis. Mean corpuscular volume (MCV) and marrow myeloblast number were used to define four groups with prognostic significance similar to the IPSS. A low‐risk group was described by macrocytosis associated with < 5% myeloblasts and high risk was related to blast counts ≥ 5% and MCV < 100 fl. Further analysis defined factors identifying very high‐risk patients and those with benign disease, together with many intermediate survival patterns. Results were consistent in two time‐separated patient groups.

Effect of 5637-conditioned medium on peripheral blood granulocyte-macrophage progenitors in normal subjects and patients with the myelodysplastic syndrome

There was no overall increase in PB-CFU-GM from normal subjects, or MDS patients, when exogenous CSA (5637-CM) was added to the culture medium. However, there was a sub-group of MDS patients (seven of 35) whose PB-CFU-GM numbers were significantly stimulated by 5637-CM. In addition, there were 11 (out of 48) MDS patients with undetectable PB-CFU-GM in assays without exogenous CSA but only two when 5637-CM was added (p less than 0.01). This sub-group is of particular interest as it is known that those without detectable PB-CFU-GM tend to have significantly shorter survival times than others. The mechanism of the functional abnormality is yet to be determined.

Undetectable peripheral blood CFU-GM as a prognostic indicator in myelodysplastic syndrome

The survival of MDS patients without detectable circulating CFU-GM (median = 188 days) was significantly lower than those in whose peripheral blood CFU-GM were detected (median greater than 1000 days) (p less than 0.01). About half of those with detectable PB-CFU-GM died within 2 yr whilst the remainder survived more than ca 3 yr. There was no significant difference in the distribution of patients having 0-5% and greater than 5% marrow blast cells within the three groups.

Long-term survival of myelodysplastic patients with macrocytosis

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Control of pH in human long-term bone marrow cultures with low-glucose medium containing zwitterion buffer lengthens the period of haemopoietic activity

Primary long‐term bone marrow cultures grown in 40 mm HEPES‐buffered McCoys 5A medium produced granulocyte–macrophage colony‐forming units (CFU‐GM) for a median of 9 weeks compared with 7 weeks with CO2/bicarbonate‐buffered cultures. Reducing the medium glucose concentration (from 12·5 to 2·75 mm) extended the culture longevity to 17 weeks. The median period of erythroid colony detection increased from 6 to 8 weeks. Secondary cultures (5 × 106 cord blood mononuclear cells seeded on irradiated stroma) showed statistically similar myeloid and erythroid longevity to primary cultures. Improved control of medium pH significantly improved the capacity of long‐term stromal layers to maintain stem cells in vitro.

Growth response to cytokines of circulating myeloid progenitors from myelodysplastic patients at diagnosis and more than 600 days after diagnosis

Abstract: Myeloid colony growth from the peripheral blood of myelodysplastic (MDS) patients was assessed for abnormal in vitro response to haemopoietic growth factors (granulocyte colony‐stimulating factor (G‐CSF), macrophage colony‐stimulating factor (M‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐1 (IL‐l), interleukin‐3 (IL‐3)). Abnormal colony growth, increased or reduced, was observed with each of the factors. No specific growth pattern was related to any of the French‐American‐British classification (FAB) types of disease. MDS patients who had survived >600 days after diagnosis (n = 34) showed significantly fewer abnormalities than patients assayed at the time of diagnosis (n = 37), the major difference being less frequent stimulation of colony growth. These findings indicate that the time of sampling relative to diagnosis needs to be considered when interpreting the in vitro response to growth factors of myeloid colonies from MDS patients.

A novel CSF-1 binding factor in a patient in complete remission following cytotoxic therapy for lymphoma.

Summary. A novel colony stimulating factor‐1 (CSF‐1) binding factor present in the serum from a patient in remission from lymphoma is described. Radioimmunoassay (RIA) repeatedly failed to detect circulating levels of CSF‐1 in the peripheral blood system of this patient. Molecular analysis showed a normal CSF‐1 gene structure by Southern blot analysis and a 46, XX karyotype by cytogenetic analysis. CSF‐1 mRNA expression in peripheral blood leucocytes was confirmed using reverse transcriptase polymerase chain reaction analysis. Morphological analysis of bone marrow cells was normal and peripheral blood progenitor cell colony assays showed a pattern of growth within the normal range in response to CSF‐1 alone and in combination with other cytokines. Analysis of the patients plasma and conditioned media prepared from peripheral blood mononuclear and granulocytic cell fractions for their ability to bind 125Iodine‐labelled CSF‐1 revealed the presence of a plasma CSF‐1 binding factor. This binding factor was not present in the patients urine, because CSF‐1 was detected by RIA and production of the binding factor by the patients peripheral blood white cells could not be demonstrated in vitro. To our knowledge, this is the first reported case of a soluble CSF‐1 binding factor.

Normal haemopoietic progenitors grow better on stromal layers from normal, rather than myelodysplastic, marrow [Abstract]

1 NORMAL HAEMOPOIETIC PROGENITORS GROW BETTER 3 THE MDR1 ,GENE AND INTRACELLULAR GLUTATHIONE IN ON STROMAL LAYERS FROM NORMAL, RATHER THAN DRUG RESISTANCE IN HUMAN LEUKAEMIA IMPLICATIONS MYELODYSPUSTIC, MARROW. FOR OVERCOMING MDR WITH PSC 833 Jiane, Kelsey SM, G.B. Tennant, L.N. Truran, A.K. Bumett, Department of Haematology, Devadasan C, Gutteridge CN & Newland AC. Department of Haematology, University of Wales College of Medicine, Cardiff, UK The London Hospital Medical College, London El 2AD