A randomised comparison of the novel nucleoside analogue sapacitabine with low-dose cytarabine in older patients with acute myeloid leukaemia.

The development of new treatments for older patients with acute myeloid leukaemia (AML) is an active area, but has met with limited success. Sapacitabine is a novel orally administered nucleoside analogue that has shown encouraging activity in unrandomised early-stage trials. We randomised 143 untreated patients with AML or with high-risk myelodysplastic syndrome (>10% marrow blasts) between sapacitibine and low-dose ara-C (LDAC) in our 'Pick a Winner' trial design. At the planned interim analysis there was no difference between LDAC and sapacitibine in terms of remission rate (CR/CRi, 27% vs 16% hazard ratio (HR) 1.98(0.90-4.39) P=0.09), relapse-free survival (10% vs 14% at 2 years, HR 0.73(0.33-1.61) P=0.4) or overall survival (OS; 12% vs 11% at 2 years, HR 1.24(0.86-1.78) P=0.2). Sapacitibine was well tolerated, apart from more grade 3/4 diarrhoea. On the basis of these findings sapacitibine did not show sufficient evidence of benefit over LDAC for the trial to be continued.

Chemotherapy drug scheduling for the induction treatment of patients with acute myeloid leukemia.

Leukemia is an immediately life-threatening cancer wherein immature blood cells are overproduced, accumulate in the bone marrow (BM) and blood and causes immune and blood system failure. Treatment with chemotherapy can be intensive or nonintensive and can also be life-threatening since only relatively few patient-specific and leukemia-specific factors are considered in current protocols. We have already presented a mathematical model for one intensive chemotherapy cycle with intravenous (i.v.) daunorubicin (DNR), and cytarabine (Ara-C). This model is now extended to nonintensive subcutaneous (SC) Ara-C and for a standard intensive chemotherapy course (four cycles), consistent with clinical practice. Model parameters mainly consist of physiological patient data, indicators of tumor burden and characteristics of cell cycle kinetics. A sensitivity analysis problem is solved and cell cycle parameters are identified to control treatment outcome. Simulation results using published cell cycle data from two acute myeloid leukemia patients are presented for a course of standard treatment using intensive and nonintensive protocols. The aim of remission-induction therapy is to debulk the tumor and achieve normal BM function; by treatment completion, the total leukemic population should be reduced to at most 10(9) cells, at which point BM hypoplasia is achieved. The normal cell number should be higher than that of the leukemic, and a 3-log reduction is the maximum permissible level of population reduction. This optimization problem is formulated and solved for the two patient case studies. The results clearly present the benefits from the use of optimization as an advisory tool for treatment design.

A real-time multi-channel monitoring system for stem cell culture process.

A novel, up to 128 channels, multi-parametric physiological measurement system suitable for monitoring hematopoietic stem cell culture processes and cell cultures in general is presented in this paper. The system aims to measure in real-time the most important physical and chemical culture parameters of hematopoietic stem cells, including physicochemical parameters, nutrients, and metabolites, in a long-term culture process. The overarching scope of this research effort is to control and optimize the whole bioprocess by means of the acquisition of real-time quantitative physiological information from the culture. The system is designed in a modular manner. Each hardware module can operate as an independent gain programmable, level shift adjustable, 16 channel data acquisition system specific to a sensor type. Up to eight such data acquisition modules can be combined and connected to the host PC to realize the whole system hardware. The control of data acquisition and the subsequent management of data is performed by the system's software which is coded in LabVIEW. Preliminary experimental results presented here show that the system not only has the ability to interface to various types of sensors allowing the monitoring of different types of culture parameters. Moreover, it can capture dynamic variations of culture parameters by means of real-time multi-channel measurements thus providing additional information on both temporal and spatial profiles of these parameters within a bioreactor. The system is by no means constrained in the hematopoietic stem cell culture field only. It is suitable for cell growth monitoring applications in general.

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 microl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-gamma. Absolute numbers of CD8(+) and CD8(-) (putative CD4(+)) T-cells, NK cells (CD8(+)/CD3(-)) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8(-) and CD8(+) T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-gamma. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8(lo)/CD3(lo-int) lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.

Response of human CD34+ cells to CXC, CC, and CX3C chemokines: implications for cell migration and activation.

Ultrastructural studies of marrow and examination of the in vivo processes of stem cell homing and mobilization show that multipotential hematopoietic progenitors are able to traverse endothelial cells. The regulation of this process by various classes of chemokines was studied in this report, using an in vitro model of transendothelial migration. Human umbilical vein endothelial cells (HUVECs) or bone marrow-derived endothelial cells (BMECs) were grown to confluence on 3-microm microporous membrane inserts and placed in 24-well culture plates. CD34(+) cells isolated from normal volunteer donor marrow by immunoadsorption or magnetic bead selection techniques were added to the inserts and various individual chemokines were added to the lower chamber of the culture plates in serum-free conditions. After 24 h, the percentage of transmigrated cells was determined. A mean of 8.5% of unfractionated marrow CD34(+) populations migrated, and all chemokines tested, with the exception of macrophage inflammatory protein-1alpha (MIP-1alpha), had some positive effect on this migration. The greatest effects were seen with stroma-derived factor-1alpha (SDF-1alpha) and stroma-derived factor-1beta (SDF-1beta), with lesser effects noted for other chemokines and cytokines. When the CD34(+) population was subselected for expression of CD38, a greater fraction of the CD38(-) cells migrated as compared to the CD38(+) fraction. CD34(+) cells isolated from mobilized peripheral blood and cord blood also migrated in response to chemokines. Chemokines of the CC, CXC, and CX(3)C classes as well as other hematopoietic cytokines may modulate the process of stem cell transmigration of endothelial cells. Further understanding of this process may help elucidate the mechanism of stem cell mobilization and homing.

Localization of androgen receptor expression in human bone marrow.

Androgens have been shown to modulate the haematopoietic and immune systems and have been used clinically for stimulating haematopoiesis in bone marrow failure conditions. To identify the bone marrow cell types as potential targets of androgens, an androgen receptor (AR)-specific antibody was used to localize the AR in normal human bone marrow biopsies. The results show that AR was ubiquitously expressed in the bone marrow of both males and females. Furthermore, the AR expression pattern did not change with age. Stromal cells, macrophages, endothelial cells, myeloblasts, myelocytes, neutrophils, and megakaryocytes expressed AR. In contrast, AR was not detected in the lymphoid and erythroid cells, or in eosinophils. These results indicate that androgens may exert direct modulating effects on a wide spectrum of bone marrow cell types via AR-mediated responses.

Human immunodeficiency virus and the hematopoietic repertoire: implications for gene therapy.

The human immunodeficiency virus (HIV) infects primarily the hematopoietic and immune systems. At the onset of infection, an initial activation of the immune system occurs, with a subsequent suppression thereafter due to direct viral infection of cells, inhibitory effects of HIV proteins, an altered microenvironment with cytokine imbalance, and increased apoptosis of both infected and non-infected cells. The CD34+ hematopoietic stem cell, however, remains free of infection. Novel methods in gene therapy utilize viral vectors that can introduce genes with good efficiency into the non-dividing stem cell. Therefore, HIV-resistance genes can be introduced into stem cells using these vectors. This confers resistance to infection to their respective progeny, and concurrently allows for repopulation of the immuno-hematopoietic repertoire. Applications of this technology to the patient infected with HIV are discussed in the context of myeloablative therapy and stem cell rescue.