Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial.

This study evaluated azacitidine as treatment of minimal residual disease (MRD) determined by a sensitive donor chimerism analysis of CD34(+) blood cells to pre-empt relapse in patients with CD34(+) myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). At a median of 169 days after HSCT, 20/59 prospectively screened patients experienced a decrease of CD34(+) donor chimerism to <80% and received four azacitidine cycles (75 mg/m(2)/day for 7 days) while in complete hematologic remission. A total of 16 patients (80%) responded with either increasing CD34(+) donor chimerism to ≥80% (n=10; 50%) or stabilization (n=6; 30%) in the absence of relapse. Stabilized patients and those with a later drop of CD34(+) donor chimerism to <80% after initial response were eligible for subsequent azacitidine cycles. A total of 11 patients (55%) received a median of 4 (range, 1-11) additional cycles. Eventually, hematologic relapse occurred in 13 patients (65%), but was delayed until a median of 231 days (range, 56-558) after initial decrease of CD34(+) donor chimerism to <80%. In conclusion, pre-emptive azacitidine treatment has an acceptable safety profile and can substantially prevent or delay hematologic relapse in patients with MDS or AML and MRD after allogeneic HSCT.

CD49d blockade by natalizumab in patients with multiple sclerosis affects steady-state hematopoiesis and mobilizes progenitors with a distinct phenotype and function.

Therapeutic application of natalizumab, an anti-CD49d Ab, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. We analyzed the frequency, phenotype and functional activity of CD34+ HSC in blood and BM of patients with MS who were treated with natalizumab. Compared with healthy controls and untreated MS patients, natalizumab treatment increased CD34+ cells in the peripheral blood 7-fold and in BM 10-fold. CD34+ cells derived from blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules than G-CSF-mobilized CD34+ cells. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared with that of CD34+ cells mobilized by G-CSF (median 43.9 vs 15.1%). This was associated with a more than doubled migration capacity toward a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more mRNA for p21 and less for matrix metallopeptidase 9 compared with G-CSF-mobilized hematopoietic stem cell (HSC). Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.

Kidney transplantation substantially improves endothelial progenitor cell dysfunction in patients with end-stage renal disease.

Endothelial progenitor cells (EPC) are involved in endothelial repair and maintenance. Dysfunction of EPC may contribute to accelerated arteriosclerosis in chronic kidney disease. Kidney transplantation (KTx) improves both survival and endothelial function of dialysis patients. In a prospective study, we tested to which extent KTx changes EPC biology. We studied number and function (migratory activity, adhesion to extracellular matrix proteins and to mature endothelial cells [EC]) of EPC in 20 patients during dialysis and 3, 6, 9 and 12 months after KTx. Twenty-two healthy volunteers served as matched controls. Circulating precursor populations were measured by flow cytometric analysis. Cytokines relevant for EPC mobilization were monitored. Compared to the dialysis state, KTx increased the migration of EPC to approximately 2-fold. Adhesion to fibronectin and to collagen type IV was significantly increased after KTx. An improved adhesion rate of EPC to mature EC was observed. The number of EPC decreased. The amount of precursor populations showed no difference compared to the pretransplant state. Our study shows an improved function of EPC after KTx. This finding indicates an improved potential for endothelial repair which in turn may contribute to enhanced endothelial function and reduced cardiovascular morbidity after KTx.

Endothelial dysfunction in patients with rheumatoid arthritis is associated with a reduced number and impaired function of endothelial progenitor cells.

BACKGROUND: Rheumatoid arthritis (RA) is associated with increased morbidity and mortality attributable to accelerated atherosclerosis and cardiovascular events. OBJECTIVE: To determine the role played by endothelial progenitor cells (EPC) in the defence system against arteriosclerosis. METHODS: The number and function of EPC in 13 young patients with RA with low disease activity (DAS28 3.5 (0.3)) and 13 healthy control subjects was studied. Endothelial function was investigated by agonist-induced, endothelium dependent vasodilatation measured by the forearm blood flow technique. Migratory activity and adhesion of EPC to tumour necrosis factor alpha (TNFalpha) activated mature endothelial cells and components of the extracellular matrix were tested in vitro. Putative precursor populations (CD34(+), CD34(+)/CD133(+), and CD34(+)/KDR(+) haematopoietic stem cells) were measured by flow cytometric analysis. RESULTS: Acetylcholine-induced, endothelium dependent vasodilatation was reduced by about 50% in patients with RA, indicating endothelial dysfunction, whereas endothelium-independent vasodilatation in response to glyceryl trinitrate was at control level. Significantly reduced numbers of EPC were found in the patients compared with controls. Migratory activity of EPC was decreased in patients with RA. Adhesion to mature endothelial cells after activation with TNFalpha was enhanced only in controls. The adhesion to matrix proteins and the number of putative precursor cell lineages was comparable in both groups. CONCLUSION: Endothelial dysfunction in patients with RA with low grade inflammation is associated with a reduced number and partial dysfunction of EPC. Further studies are needed to explore whether interventions that potentially ameliorate the number and function of EPC also improve endothelial function in these patients.

HPC enumeration with the Sysmex XE-2100 can guide further flow cytometric CD34(+) measurements and timing of leukaphereses.

BACKGROUND: The aim of this study was to evaluate whether HPC counts measured with the hematology analyzer can predict CD34+ levels in peripheral blood and in the apheresis product, as detected by standard flow cytometry. The main focus was the evaluation of HPC counts in poor mobilizers. METHODS: Progenitor cell quantification was performed measuring HPC counts provided by the Sysmex XE-2100 hematology analyzer and CD34+ counts obtained in parallel by flow cytometry. Peripheral blood of patients who had received chemotherapy and G-CSF (142 measurements) and healthy donors mobilized with G-CSF alone (106 measurements) was investigated HPC counts in peripheral blood were also correlated with apheresis yield. RESULTS: HPC counts were significantly higher than CD34+ counts (3.5 fold inpatients and 1.7 fold in healthy donors, p= 0.0015). Our data indicate that HPC counts < or = 10/microL in pretreated patients predict a low probability of adequate CD34+ counts in peripheral blood and yields < 2 x 10(6)/kg in subsequent aphereses. Furthermore, repetitive low HPC enumerations in an individual were followed by insufficientCD34+ counts in peripheral blood or aphereses in 81% of investigations. In healthy donors low HPC counts (< or = 10/microL; 12/106 measurements) did not exclusively predict low CD34+ counts (median 23/microL). DISCUSSION: HPC counts can be used to schedule the start of CD34+ measurements(threshold > 10 HPC/microL) in patients mobilized after chemotherapy for autologous donation. Thus, expensive and time-consuming CD34+ enumerations can perhaps be minimized HPC measurements cannot completely replace flow cytometric CD34+ enumeration. In particular healthy stem-cell donors should be monitored with both methods to exclude false negative HPC measurements.

A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes and granulocytes.

PNH is a disorder of the pluripotent stem cells resulting in a deficient expression of membrane-bound GPI-anchored proteins in different cell types. Several flow cytometric approaches are designed to detect this antigen deficiency. But they all require drawing and testing of normal samples as control. Therefore, in the present study two flow cytometric assays for the detection of CD55 and CD59 deficiency in erythrocytes (REDQUANT CD55/CD59) and granulocytes (CELLQUANT CD55/CD59) are proposed. Precalibrated beads are used to define the cut off between normal and deficient cell populations. The specificity of the tests has been evaluated in healthy blood donors (n=52) resulting in a clear and reproducible cut off (3%) for the normal percentage of GPI-deficient cells. This cut off has been confirmed in leukaemia and lymphoma patients not suspected for developing PNH. The sensitivity has been tested in patients suffering from known PNH (n=23). Both tests performed in combination allowed a reliable detection of PNH in all patients showing antigen deficiencies in both cell types in most patients (20/23). In contrast, the PNH clones in the investigated patients with MDS (4/19) or AA (4/22) were present in granulocytes or erythrocytes, only. This underlines the necessity of analysing erythrocytes as well as granulocytes. Preliminary data regarding a possible correlation between disease activity and percentage of antigen-deficient cells lead to the assumption that haemolytic crises can only be determined on granulocytes whereas deficient erythrocytes disappeared due to complement-mediated lysis of the PNH clone. In conclusion, the combination of the test kits enables the differential diagnosis of PNH clones in a standardized, simple and rapid approach which may have therapeutic consequences.

Flow cytometric DNA-quantification of three-color immunophenotyped cells for subpopulation specific determination of aneuploidy and proliferation.

A method is described for three-color immunophenotyping and simultaneous DNA-quantification using a flow cytometer equipped with a 488-nm argon laser and a mercury lamp (UV). The approach includes reproducible immunophenotyping comparing antigen expression before and after cell manipulation for DNA-measurement. The coefficients of variation after DNA-staining (CV=3.13 for T-cells in peripheral blood and CV=3.38 for T-cells in bone marrow) were adequate for exact DNA-analysis. For aneuploidy detection, a true internal standard was established measuring, for example, the DNA-content of T-cells in B-cell disease simultaneously with the DNA-content of the malignant cells. Using this method, aneuploidies could be unequivocally detected in 17 out of 24 patients with multiple myeloma. Furthermore, intratumor heterogeneities in DNA-content and antigen expression could be recognized, allowing an exact separation of tumor cells and normal hematopoiesis. The study also demonstrated the importance of exact immunophenotypic characterization of lymphocyte subpopulations and the determination of their specific proliferation, for example after proliferation induction in cell cultures. Future studies should address the applicability of this rather simple multiparameter approach for simultaneous immunophenotyping and DNA-measurement especially in the detection of minimal amounts of aneuploid cells after chemotherapy.

Sensitivity of combined DNA/immunophenotype flow cytometry for the detection of low levels of aneuploid lymphoblastic leukemia cells in bone marrow.

By simultaneous DNA staining and immunophenotyping, the aneuploid blasts of acute lymphoblastic leukemia (ALL) can be quantitated by flow cytometry. The present study evaluates the application of this method for the detection of minimal residual disease (MRD). For this purpose leukemia cells with known aneuploidy were serially diluted with diploid bone marrow cells obtained after chemotherapy. These mixed samples were immunophenotyped using antibodies CD34, CD10, or CD19 and processed for DNA staining with propidium iodide. Fifty thousand cells were measured for each experiment and the results were compared with the analysis of control sample without added aneuploid cells. Aneuploid cells diluted to 1% were not detectable in 6/15 cases when DNA content analysis was used alone, and in dilutions of < 1%, these cells were not detectable. When DNA content and immunophenotype were simultaneously measured, aneuploidy was detectable in 15/15 cases at 1.00%, 6/6 at 0.50%, 16/16 at 0.10%, and 16/17 at 0.05%. Therefore this method may be useful for the detection of minimal residual disease in cases of aneuploid lymphoblastic leukemia.