Summary.We investigated the role of the drug resistancerelated proteins LRP, MRP and Pgp and the apoptotic suppressor, bcl-2, in relation to other clinical characteristics, with respect to response and survival in 91 patients with newly diagnosed AML, treated with standard chemotherapy. Multivariate analysis showed that poor response to chemotherapy was associated with increasing age (P ¼ 0·0004), LRP expression (P ¼ 0·0001) and Pgp function (P ¼ 0·015). The significant predictors of both leukaemia-free survival (LFS) and overall survival (OS) were LRP (LFS, P ¼ 0·01; OS, P ¼ 0·0001), Pgp function (LFS, P ¼ 0·0001; OS, P ¼ 0·0003) and cytogenetic abnormalities (LFS, P ¼ 0·0001; OS, P ¼ 0·0005). Patients with the lowest expression of LRP and Pgp function and favourable karyotype (group I) had an LFS of 30·2 months compared to 8·5 months in the group with the highest expression of LRP and Pgp and poor prognosis karyotype (group III, P ¼ 0·002). OS decreased from 75·4 months in group I to 7·9 months in group III patients (P < 0·0001). Neither MRP nor bcl-2 were significantly associated with chemotherapy response and survival. Correlations were found between increasing expression of LRP and older age (P ¼ 0·05) and an unfavourable karyotype (P ¼ 0·005), but these variables were independent of each other in analysis of treatment response and patient survival. Our findings suggest that both LRP and Pgp are clinically relevant drug-resistance proteins and it may be necessary to modulate both LRP and Pgp functions in order to reverse the multidrug resistance phenotype in AML.

P-glycoprotein (Pgp) expression, which is associated with the multi-drug resistance (MDR) phenotype, has been reported to be a useful predictor of treatment outcome in acute leukaemia. We have examined the expression of Pgp on acute myeloid leukaemia (AML) cells in 54 newly diagnosed patients, using a novel streptavidin-biotin complex (ABC) technique. 55% of patients at diagnosis were positive for Pgp with JSB-1, a monoclonal antibody that binds to an internal epitope of Pgp. All patients received intensive induction chemotherapy. Post-remission treatment consisted of further chemotherapy +/- bone marrow transplantation. Complete remission (CR) rates were significantly lower in the Pgp positive group than in the Pgp negative group (60% v 92%; P = 0.02). The overall survival for Pgp-positive patients was significantly shorter (329 v 534d, P = 0.004), disease-free survival was also reduced but the difference was not statistically significant (median 277 v 522d, P = 0.16). In this study CD34 expression was not predictive of response to chemotherapy nor was it associated with Pgp expression. Our results confirm the prognostic value of Pgp expression in AML at diagnosis and we suggest that Pgp could be a useful therapeutic target for reversing multi-drug resistance. Furthermore, our simple and sensitive method of detecting Pgp should enable widespread testing to be performed.

This study has provided information about survival rates and risk factors for survival in horses undergoing relaparotomy that can assist clinicians and owners when determining whether to perform relaparotomy and in predicting the likely surgical outcome.

Summary.The results for colony forming unit granulocytemacrophage (CFU-GM) assays vary substantially between centres. It is possible that colony counting is largely responsible for this discrepancy. In order to examine this exclusively from the many factors that make up the CFU-GM assay, we performed a colony counting exercise involving 11 laboratories. Two-way analysis of variance showed a highly significant difference (P ¼ 0 : 0001) in the counts obtained from the centres. One centre was found to score consistently high and two others scored consistently low numbers of colonies. This suggests that identification of colonies is a major source of variation between centres.

Summary. Megakaryocytes (Mks) are found in the lungs and the blood stream as well as in the bone marrow. We modified a whole blood filtration method for Mks by immunostaining for CD61 using biotin streptavidin, and used this technique to study Mks and their morphology in the central venous and arterial circulations before, during and after cardiopulmonary bypass (CPB) in haematologically normal patients undergoing routine cardiac surgery.Blood samples were taken immediately after the insertion of central venous (V) and arterial (A) catheters and after thoracotomy, immediately before bypass. Further samples were taken after 60-90 min on-CPB and 180-240 min postbypass. In comparison with the steady state before bypass, circulating Mk levels in blood on bypass increased dramatically, from (V) 10 . 93 Ϯ 3 . 94/ml (mean Ϯ SD) to 36 . 48 Ϯ 11 . 52/ml and from (A) 8 . 37 Ϯ 4 . 39/ml to 38 . 65 Ϯ 20 . 68/ml. This effect was still present, to a lesser extent, 180-240 min post-bypass. Circulating levels of Mks were consistently lower in the arterial circulation than in the venous circulation off bypass, but levels in the two circulations were comparable during CPB, confirming previous suggestions that the lungs are net removers of Mks from the circulation. Type 4 Mks, the largest and most normal morphologically, were rarely seen in arterial blood, but increased significantly during CPB, indicating that the lungs selectively remove large Mks.The lungs appear to play an active role in the regulation of Mk levels. This is lost during CPB and despite the extracorporeal 40 mm arterial line filter, large Mks enter the systemic circulation. More effective extracorporeal filtration of large Mks might reduce the neurological impairment seen in some patients who have undergone CPB.

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia Summary. Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo. We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61 þ cells in 7 d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61 þ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61 þ cells were increased 8-10-fold by the

Summary. The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP). An inverse and signi®cant association was found between IDA and Pgp-related ef¯ux activity (r À0´31, P 0´01) and also MRP (r À0´25, P 0´04) but not with LRP (r À0´13, P 0´28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was signi®cantly higher than that of blasts carrying two MDR proteins (0´466 vs. 0´296, P 0´046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of ef¯ux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no signi®cance was found on multifactorial analysis. There was a non-signi®cant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16´3 months compared with 21´1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined ef¯ux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.

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