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Allogeneic bone marrow transplantation (BMT) can induce long-term complete remission (CR) in patients with multiple myeloma but it is not yet clear whether the disease can be eradicated. We have used immunoglobulin gene fingerprinting, a PCR-based technique, to evaluate minimal residual disease in 5 patients in unmaintained CR 9-60 months after allogeneic BMT. All 5 patients were PCR-positive within the first year after BMT, suggesting that early PCR positivity is common and not predictive of relapse. Three patients were studied at > 1 year post-transplant; one had become PCR-negative at 1 year, a second at 2 years and the third at 4.5 years post-BMT. The ability of the technique to detect clonal evolution was demonstrated by serial studies in a further patient who relapsed post-BMT. The absence of any detectable disease at the molecular level in 3 patients in long-term CR post-transplant suggests that cure of multiple myeloma may be a realistic goal.

The CD34-positive population of bone marrow cells contain not only committed myeloid, erythroid, megakaryocytic and lymphoid progenitor cells but also more primitive stem cells capable of initiating haemopoiesis in long-term marrow culture that are termed long-term culture initiating cells (LTC-IC). Recently, different subpopulations of BM CD34+ cells have been defined. Thus the CD34+/CD45RO+ fraction contains both BFU-E and LTC-IC whereas CFU-GM are mainly CD34+/CD45RA+ and CD34+/CD33+. As human umbilical cord blood (UBC) also contains CD34+ cells and has been used as a source of material for clinical transplantation, we have undertaken a comparative study of the expression of CD34RO, CD45RA, CD33 and CD38 on CD34+ cells from UCB and BM using directly conjugated antibodies and flow cytometry. The frequency of CD34 cells in UCB (mean 1.0% +/- 0.3%) was similar to that in harvested pelvic BM (0.8% +/- 0.4%). The frequencies of CD34+ cells co-expressing CD33 (UBC: 54.1% +/- 28.7%; BM: 52.9% +/- 18.5%) and CD45RA (UBC: 24.8% +/- 16.6%; BM: 21.1% +/- 19.7%) were not significantly different between UCB and BM. In contrast, a significantly higher percentage of UCB-CD34+ cells co-expressed CD45RO+ (19.7% +/- 10.3%) compared with BM-CD34+ cells (7.2% +/- 5.3%; p < 0.001). The absolute number of CD34+ cells in UCB correlated with the number of committed myeloid (CFU-GM) and erythroid (BFUe) progenitors; a similar relationship was found for the absolute number of CD34+/CD33+ cells. However, the numbers of CD34+/CD45RO+ cells showed no correlation with numbers of committed progenitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Although the combination of cyclosporin A (CYA) and methotrexate has been reported to reduce the incidence of acute GVHD in patients undergoing allogeneic BMT for leukaemia, it has been associated with a higher risk of leukaemic relapse. Since 1987 we have used the combination of CYA and methotrexate for GVHD prophylaxis in 24 patients undergoing allogeneic BMT for leukaemia or myelodysplasia. Over the first 50 days post-transplantation, CYA dosage was adjusted to keep within a therapeutic range of 95-205 ng/ml. This resulted in a 60% reduction in CYA dosage by day 50 post-transplant compared to the original Seattle protocol. Despite the low dosage of CYA administered, the incidence of acute GVHD was only 25% with no patient having greater than grade I GVHD. There have been no leukaemic relapses in low risk patients. The results indicate that decreasing CYA dosage does not increase the incidence of GVHD but may reduce the risk of leukaemic relapse following allogeneic BMT.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is active in enhancing the production of mature myeloid cells in vitro and several phase I/II clinical trials have suggested that its administration may accelerate neutrophil recovery after autologous bone marrow transplantation (ABMT). We have conducted a multicentre randomized double-blind placebo controlled trial in patients with poor prognosis malignant lymphoma receiving an identical high-dose combination chemotherapy regimen with ABMT. 61 patients were entered and 29 in each arm of the trial were evaluated. Treatment with GM-CSF did not affect the period of severe neutropenia (absolute neutrophil count (ANC) of < 0.1 x 10(9)/l) but accelerated recovery to an ANC of 0.5 x 10(9)/l (median 14 d v 20 d in controls, P = 0.001). There was no significant difference in platelet recovery between the groups (GM-CSF group platelet dependent for 25 d v control 19 d, P = NS). The number of positive blood cultures was similar in both groups (GM-CSF 14 v placebo 13) and there were no differences in days of fever > 37.5 degrees C (median 8 v 6) or days on parenteral antibiotics (11 v 10). Patients receiving GM-CSF had a median period of hospitalization following BMT of 24 d (control 25). No significant major toxicity attributable to GM-CSF administration was detected. We have confirmed in a randomized trial that GM-CSF accelerates neutrophil but not platelet recovery following ABMT. We were unable to demonstrate any accompanying changes in clinical outcome and believe that further trials are necessary to assess the clinical value of GM-CSF in BMT.

Studies on the structure of haemopoiesis in acute myeloblastic leukaemia (AML) has shown the presence of a small population of malignant cells with extensive proliferative and self-renewal properties which are features of stem cells. The requirements of these cells for proliferation have been studied both in clonogenic assays in semi-solid media and in liquid suspension culture. These have demonstrated that AML clonogenic cells from the majority of patients, can be stimulated to proliferate by colony-stimulating factors (GM-CSF, G-CSF and IL-3) as well as other cytokines including interleukin-1 and interleukin-6, all of which are known to stimulate normal haemopoietic progenitors. Unlike normal haemopoietic cells, leukaemic blasts from many patients with AML express transcripts for haemopoietic growth factors including GM-CSF, G-CSF and IL-1 but not IL-3, and secrete growth factor protein. When leukaemic cells are cultured at sufficiently high density to permit cell-cell interactions, autonomous growth of clonogenic cells can be seen. Autonomous growth is related to the autocrine secretion of haemopoietic growth factors including GM-CSF, G-CSF and IL-6. The degree of autonomous colony growth is variable but approximately 70% of AML samples exhibit either partial or totally autonomous growth; the remaining cells being absolutely dependent on exogenous CSF or fail to grow in the culture systems employed. Similar patterns of growth have been found in murine haemopoietic cells lines which have been transformed as the result of the retroviral insertion of genes for GM-CSF or IL-3.(ABSTRACT TRUNCATED AT 250 WORDS)

The blast cells from some patients with acute myeloblastic leukemia (AML) proliferate autonomously in vitro. We have previously identified four groups of AML blasts based upon their growth characteristics in vitro, in particular the degree of autonomous growth. We have now measured the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-1 beta (IL-1 beta) by AML cells with different growth characteristics, using two sensitive enzyme-linked immunosorbent assays. Our results show a correlation between the capacity of AML blasts to produce GM-CSF and IL-1 beta and the ability to grow autonomously in vitro. Blasts from cells with no autonomous growth (n = 5) secreted low or undetectable amounts of GM-CSF and IL-1 beta. Blasts with totally autonomous growth (n = 10) secreted the highest levels of GM-CSF (mean 2469 pg/10(3) cells) and IL-1 beta (mean 3156 pg/10(6) cells). Whereas blasts with partially autonomous growth (n = 9) secreted intermediate levels of GM-CSF (mean 270 pg/10(6) cells) and IL-1 beta (mean 931 pg/10(6) cells). In order to determine whether GM-CSF production was autocrine or the consequence of paracrine secretion by differentiated leukemic cells, we studied the degree of autonomous growth and production of GM-CSF by CD34-positive blasts from eight patients whose unfractionated cells produced GM-CSF. We found that CD34-positive blasts from six of these cases grew autonomously to a degree comparable to that of the unfractionated cells, and that CD34-positive blasts produced GM-CSF either autonomously or in response to recombinant IL-1 beta. Our data suggests that in the majority of cases, CD34-positive blasts are capable of autonomous growth and autocrine GM-CSF production, however this is variably regulated by the paracrine production of IL-1 beta by CD34-negative cells.

A 21-year-old patient developed interstitial pneumonitis nine months post bone marrow transplant for acute myeloblastic leukaemia. Immunofluorescence of broncheoalveolar lavage fluid revealed the presence of respiratory syncytial virus (RSV). Aerosolized ribavarin therapy resulted in rapid resolution of the pneumonitis with full recovery without any side effects. Ribavarin therapy should be considered early in the management of BMT patients who develop RSV pneumonitis.

Aluminium-transferrin (Al-Tf) and gallium-transferrin caused a dose-dependent decrease in proliferation of human peripheral blood lymphocytes cultured for 3 days with phytohaemagglutinin (PHA). Addition of apotransferrin reduced the inhibitory effect. Al added as AlCl3 or aluminium citrate had no effect, and there was no significant difference in the response of cells from renal failure patients with or without high serum Al levels or controls. Lymphocytes cultured in the presence of Al-Tf showed a dose-dependent uptake of Al, whereas uptake from aluminium citrate was low and not dose-dependent. Uptake from AlCl3 was very high but probably involved a nonspecific uptake mechanism. Levels of Al in freshly isolated lymphocytes were approximately 1.6 ng/10(6) cells, there being no difference between cells from patients and controls. It is concluded that Al, when bound to transferrin, may have a detrimental effect on lymphocyte function and might contribute to the decreased immune responsiveness of renal failure patients on haemodialysis. However, lymphocyte Al levels are probably not useful as a marker of Al overload in such patients.

The clinical effects of cyclosporin were evaluated during cytotoxic treatment in a 61 year old man with acute myeloid leukaemia. He had required a renal transplant 18 months before presenting with acute myeloid leukaemia (FAB subtype M4). He had received cyclosporin 3.5-4.0 mg/kg daily to maintain a plasma cyclosporin concentration of 75-150 ng/ml. Cyclosporin was continued during induction chemotherapy with daunorubicin, cytarabine, and 6-thioguanine (DAT). He had fever and oropharyngeal candidiasis that was unresponsive to anti-bacterial drugs but responsive to systemic amphotericin. Bone marrow examination 14 days after chemotherapy showed complete haematological remission. Subsequently he tolerated consolidation treatment with DAT with no serious complications. Unfortunately he developed fatal septicaemia following a second consolidation with mitozantrone and cytarabine. Inhibition of P-glycoprotein activity by cyclosporin may not significantly increase the toxicity of aggressive chemotherapeutic regimens, and as benefit may be achieved by this approach further clinical evaluation is justified.

The role of interleukin-1 (IL-1) as a regulator of the autonomous growth of the blast cells of acute myeloid leukaemia (AML) has been studied on samples isolated from 15 patients. In nine out of 10 patients with evidence of partial autonomous blast cell growth. IL-1 further stimulated cell growth in both suspension culture and in a clongenic assay. IL-1 also stimulated cell growth in two out of three cases with no evidence of autonomous growth whereas no additional stimulation was observed in two cases with totally autonomous growth. In blast cells stimulated by IL-1, synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) was increased and the proliferative response to IL-1 was inhibited by an antibody to GM-CSF. In all samples with evidence of autonomous growth, blast cell conditioned medium (BCCM) contained IL-1 activity (range 0.7-74.2 units ml) and a polyclonal antibody to IL-1 markedly inhibited autonomous growth in four samples. BCCM from three of these four samples contained GM-CSF, the synthesis of which was suppressed when BCCM was prepared in the presence of anti-IL-1. Our data suggests that endogenous IL-1 is an important factor in the regulation of the production of GM-CSF and hence of autonomous growth of AML blasts, but that other mechanisms regulating GM-CSF production may exist.

In long-term culture (LTBMC) of marrow from patients with myelodysplastic syndromes (MDS) abnormal patterns of growth, dysplastic cell morphology and cytogenetic changes present at initiation of culture persisted, providing a model in which to investigate the effects of hemopoeitic growth factors. In this model, rGM-CSF added weekly (100 µ/ml) failed to increase the number of non-adherent cells, adherent cells or CFU-GM, but markedly increased the number or blast cells (from 11 ± 5%) to 44 ± 6% after 3.5 weeks), with a commensurate fall in the numbers of mature myeloid cells. In contrast, in LTBMC of normal marrow, rGM-CSF increased the non-adherent cell count by almost 5-fold. The increase was almost entirely due to enhanced production of myeloid precursors, predominantly mature myeloid cells, with a small increment in CFU-GM (2 fold: p < 0.05). These results suggest that in MDS GM-CSF enhances the "differentiation block" and may accelerate leukemic transformation.

Hairy cell leukemia was diagnosed in a 74-year-old man. He was followed for 5 years when he developed confusion and focal neurological signs. Despite investigation and treatment he died. Postmortem study revealed isolated primary necrotizing vasculitis affecting the cerebral arteries. No leukemic infiltration of the central nervous system was found. There is a recognized association between hairy cell leukemia and generalized necrotizing vasculitis of polyarteritis type, however, this is the first case report of isolated cerebral vasculitis associated with this condition. The importance of excluding infective (mycotic) arteries in this type of case is emphasized.

The effects of the nucleoside deoxyadenosine (AdR) towards 2'deoxycoformycin (dCF) treated peripheral blood T-lymphocyte and bone marrow myeloid progenitor cells has been studied. Mononuclear cell preparations were exposed to dCF and AdR in suspension culture prior to washing and culture in a medium free of exogenous nucleosides. The combination of dCF and AdR was found to be highly toxic to peripheral blood T lymphocyte colony forming cells (CFU-TL) following prolonged (18 h) incubation. CFU-GM were markedly less sensitive to the combination of dCF and AdR, concentrations of dCF (10(-5) M) and AdR (10(-5) M) which produced an almost total inhibition of CFU-TL only inhibited CFU-GM by a mean of 10%.

Plasma fibrinolytic activity was measured in 34 patients with systemic lupus erythematosus (SLE) and 12 normal subjects. Patients with SLE showed a significantly reduced resting level of plasma tissue plasminogen activator (t-PA) compared to normal. The reduction in t-PA was demonstrated both by a functional assay (fibrin-plate lysis, FP) and an immunochemical assay (ELISA). Measurement of the fibrinolytic response following venous occlusion allowed division of the patients into two groups. In the first (24 patients), there was a normal increase in t-PA response, demonstrated both by the functional and immunochemical assays. In the second (10 patients), there was a significantly reduced plasma t-PA response measured by FP. Seven of the patients in this second group, which included four patients with histological evidence of vasculitis, showed a similar failure of t-PA response (ELISA) after venous occlusion. These results suggest that their impaired fibrinolytic response may be related to defective t-PA release secondary to endothelial cell damage. The remaining three patients in the latter group had a normal t-PA (ELISA) response despite a reduced FP response, suggesting the presence of an inhibitor.

The toxicity of the purine nucleoside, deoxyadenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin and of deoxyguanosine in the presence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine was measured against two Thy-leukemic cell lines. Toxicity was assessed by the survival of clonogenic cells in a colony assay. The kill of clonogenic Thy-leukemic cells was 99.99% with both nucleoside enzyme inhibitor combinations following 4-h incubations when 50 microM concentration of nucleoside were used. With these nucleoside concentrations some reduction in toxicity was apparent when drug treated cells were cultured in the presence of deoxycytidine (50 microM), however, this reduction in toxicity was not apparent when higher nucleoside concentrations were used (100 microM). Survival of bone marrow myeloid progenitor cells (CFU.GM) was only slightly reduced by these nucleoside concentrations following 4 hour incubations. The presence of a twenty-fold excess of normal bone marrow cells reduced the cytotoxic effect but clonogenic cell incubation still ranged from 99.98 to 99.99% for deoxyguanosine and deoxyadenosine respectively. These combinations of nucleosides and enzyme inhibitors may have a therapeutic role in the elimination of malignant Thy cells from human bone marrow.

The occurrence of aplastic anaemia in pregnancy has been long recognized but its rarity has made it difficult to establish the relationship between the two conditions and the optimal management. We now report five cases of aplastic anaemia in pregnancy and offer some recommendations for treatment. In two patients the pregnancy was allowed to continue to term and the disease persisted post-partum leading to death in one case. The other three patients had their pregnancies terminated; one subsequently deteriorated and died, two had spontaneous remissions of their aplasia. We suggest that patients presenting with severe aplastic anaemia in early pregnancy should be offered termination because this may be followed by haematological improvement. If haematological improvement does not occur allogeneic bone marrow transplantation (BMT) may be considered. Aplastic anaemia presenting in late pregnancy should be treated with supportive care until delivery. On the basis of our experience, antilymphocyte globulin may safely be given during pregnancy.

A child presented with raised intracranial pressure for which no cause was established. Five months later a further cerebrospinal fluid examination showed lymphoblasts expressing the common acute lymphoblastic leukaemia phenotype. Bone marrow infiltration was not present. This case illustrates the difficulties that may be encountered in establishing the diagnosis of central nervous system leukaemia.

Platelet aggregation and the platelet prostaglandin pathway have been investigated in two patients with preleukaemic states who had a haemorrhagic tendency but a normal platelet count. In both patients platelet aggregation induced by collagen adenosine diphosphate (ADP) and arachidonic acid (AA) were abnormal. Malonyldiadehyde (MDA) production from exogenous AA was normal in both patients thus excluding cyclo-oxygenase deficiency. The platelet aggregating and rabbit aorta contracting activities of thromboxane A2 (TxA2) were very low in both patients. Production of thromboxane B2 (TxB2) assessed by thin layer chromatographic separation of the metabolites of [1(-14)C]AA and by radioimmunoassay, was normal. These abnormalities of platelet function appear to be due to the production of TxA2 with a low biological activity.

In four patients with Thy-acute lymphoblastic leukaemia changes in blast cell deoxynucleoside triphosphate concentrations and, in three, changes in blast cell S-adenosyl homocysteine hydrolase activity were measured during treatment with 2' deoxycoformycin, a potent inhibitor of adenosine deaminase. These studies were aimed at identifying the molecular basis of cell killing by this drug. In three patients an increase in blast deoxyadenosine triphosphate (dATP) concentration occurred which was found to be temporally related to cell killing and was accompanied by decreased concentrations of the other three deoxyribonucleoside triphosphates. In the one patient with Thy-ALL who responded poorly to treatment, the increase in dATP concentration was delayed and was not accompanied by a fall in the concentrations of the other deoxyribonucleoside triphosphates. Progressive inactivation of blast cell S-adenosyl homocysteine hydrolase was found to occur in the three patients tested but was maximal only after a substantial reduction of peripheral blast cell count. These results show that 2' deoxycoformycin has a potent cytoreductive effect in Thy-ALL and suggest that the molecular basis of this toxicity is related both to the intracellular accumulation of dATP with inhibition of ribonucleotide reductase. Inactivation of S-adenosyl homocysteine hydrolase may be of importance as an additional mechanism.

Seventeen patients with acute leukaemia refractory to conventional chemotherapeutic agents were treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). Of the twelve patients with acute lymphoblastic leukaemia of the thymic phenotype (Thy-ALL), seven went into complete remission after one or two courses of therapy. Two other (Thy-ALL) patients showed good partial response, and three were resistant to dCF. The five patients with acute leukaemia of other phenotypes had progression of disease despite treatment with dCF. Response to dCF can be predicted from the pattern of change in cellular nucleotide levels in blood and/or bone marrow blasts which have been treated in vitro with dCF and deoxyadenosine. The main adverse effects of dCF therapy were renal and liver dysfunction, conjunctivitis, and haemolysis.

The ability of blast cells to grow autonomously and to produce autostimulatory growth factors has been investigated in 25 consecutive patients with AML. An autostimulatory index (ASI) was calculated (no. of colonies without CSF divided by no. of colonies with CSF) and patients classified into four groups: Group 1 (n = 3): non-growers; Group 2 (n = 4): CSF-dependent (ASI less than 0.1); Group 3 (n = 11): partially autonomous (ASI 0.1-0.8); and Group 4 (n = 7): fully autonomous/CSF-unresponsive (ASI greater than 0.8). In Group 3 patients colony formation and DNA synthesis were significantly (P less than 0.01) augmented by CSFs but at high cell concentrations became CSF-independent. Blast cell-conditioned medium (BCCM) from these patients exhibited potent autostimulatory activity, increasing DNA synthesis by less than or equal to 5-fold, and also stimulated CSF-dependent homologous blasts by less than or equal to 20-fold. In 5/5 this activity was neutralized by anti-GM-CSF, which also inhibited autonomous proliferation of their blast cells. Group 4 blasts also secreted GM-CSF but their BCCM possessed no autostimulatory activity, and anti GM-CSF failed to inhibit their autonomous growth. No membrane-associated CSF activity was found, however purified cytosolic fractions stimulated proliferation of CSF-dependent homologous blasts, consistent with production and secretion of CSF which is present in active form in the cytosol but does not autostimulate via membrane receptors. These results suggest that autocrine mechanisms are important in regulating blast cell proliferation, but that the mechanisms are heterogeneous.

The role of cellular interactions in stimulating leukemic cell proliferation was studied in AML. When peripheral blood blasts were cultured in suspension in flat-bottomed vessels at low cell numbers (15.6-500 x 10(3)/ml), the rate of DNA synthesis [( 3H]thymidine incorporation) was proportional to cell density. When cells were cultured under otherwise identical conditions in round-bottomed vessels, to induce cell crowding, the rate of DNA synthesis was independent of cell numbers and was significantly augmented (p less than or equal to 0.01). Physical separation of cells in round-bottomed (crowded) cultures by latex beads reduced DNA synthesis to the same rate as that in flat-bottomed (non-crowded) cultures. In contrast, addition of mitomycin-treated autologous blasts had little effect in crowded cultures but increased DNA synthesis in non-crowded cultures. DNA synthesis was also enhanced: by reducing culture volume to concentrate any diffusible factors produced by the cells or by blast cell conditioned medium from autologous blasts grown under crowded conditions. Cells cultured in suspension for 72 hr under crowded/non-crowded conditions both formed blast cell colonies in methyl cellulose; however, the number of colonies derived from crowded cultures was greater. These results indicate that cell proximity promotes proliferation of blasts and blast cell progenitors; both close cell contact and autostimulatory soluble factors are important in regulating growth of AML blasts.

Intracellular deoxynucleoside triphosphate pools of normal bone marrow, thymocytes and cells from patients with ALL and AML were measured after 2 h incubation with deoxycoformycin (dCF) 10(-5) M and deoxyadenosine (AdR) 10(-4) M in vitro and after another 30 min incubation in the absence of dCF and AdR ('chase' experiment). Incubation with dCF and AdR resulted in a significant rise of dATP concentrations in all groups (the highest rises occurring in the leukaemic groups particularly in AML and Thy-ALL). The concentrations of the other three deoxyribonucleoside triphosphates fell in all groups. The dATP level fell during the 'chase' period but in Thy-ALL and thymocytes this fall was insignificant and slower than in the other groups. This suggests that not only intracellular build-up of dATP but also the capacity of the cell to degrade dATP is important for in vivo cytotoxicity of dCF treatment. These results help to explain the differences in response to dCF of the different leukaemias.

Expression of the transferrin receptor (TfR) was studied on peripheral blood blast cells from 11 patients with acute myeloid leukaemia (AML). Using a monoclonal anti-TfR antibody (OKT9) and a polyclonal antibody against surface membrane-bound transferrin, a proportion of blasts from all the patients was found to express receptors for transferrin. Further analysis of OKT9 expression using a fluorescent activated cell sorter (FACS) showed that the TfR was heterogeneously distributed in the blast cell population. In five out of six samples studied, stimulation of DNA synthesis following short-term culture induced a several-fold increase in TfR display as analysed by flow cytometry using OKT9 or FITC-conjugated transferrin. Blasts from seven patients stained with OKT9 were separated on the FACS into positive and negative or weakly positive fractions. Culture of the TfR negative population in a blast cell colony assay produced no colonies in either of two patients. In a further five patients the colony forming cells were predominantly associated with the TfR strongly positive fraction (52 +/- 25 colonies/10(4) cells) rather than the TfR weakly positive fraction (12 +/- 11 colonies/10(4) cells). Analysis of colony size showed that clones derived from the weakly positive fraction were smaller than clones from the TfR strongly positive fraction. These results suggest that TfR display by AML blasts is related to their proliferative capacity and is expressed by the leukaemic stem cell fraction.