Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

Stromal-derived factor 1 and matrix metalloproteinase 9 levels in bone marrow and peripheral blood of patients mobilized by granulocyte colony-stimulating factor and chemotherapy. Relationship with mobilizing capacity of haematopoietic progenitor cells.

The roles of the chemokine stromal-derived factor 1 (SDF-1) and the matrix metalloproteinase 9 (MMP-9) in haematopoietic progenitor cell (HPC) mobilization are still unclear, particularly when patients are mobilized by granulocyte colony-stimulating factor (G-CSF) plus chemotherapy. We determined bone marrow (BM) and peripheral blood (PB) plasma levels of SDF-1, together with CXC-chemokine receptor 4 (CXCR-4) expression on CD34+ cells, and interleukin 8 (IL-8) and MMP-9 in 55 patients mobilized for autologous PB transplantation compared with 10 normal BM and PB samples. Plasma samples were tested at steady state (SS-) and after mobilization by cyclophosphamide and G-CSF administration (M-). SDF-1, CXCR-4, IL-8 and MMP-9 levels were significantly lower in SS- and M-PB than in SS-BM. Differences in SDF-1 levels between SS-PB and SS-BM were also observed after mobilization. We showed for the first time a clear relationship between the levels of circulating HPC, both at steady state and after mobilization, and those of secreted MMP-9 but not of SDF-1 or IL-8. However, a negative correlation was observed between mobilizing capacity and CXCR-4 expression on CD34+ cells. These findings suggest that G-CSF-induced mobilization of HPC from BM involves MMP-9, without reversing the positive gradient of SDF-1 between BM and PB.

Long-term marrow reconstitutive ability of autologous grafts in lymphoma patients using peripheral blood mobilized with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor compared to bone marrow.

OBJECTIVES: The aim of this study was designed to compare the in vivo long-term hematopoietic potential of bone marrow and peripheral blood grafts. MATERIALS AND METHODS: Marrow progenitor cell recovery was assessed for up to 4 years in 227 patients. One hundred patients were treated for malignant lymphomas by autologous bone marrow transplantation (BMT) and 127 by peripheral blood progenitor cell transplantation (PBPCT). RESULTS: Marrow progenitor cell counts were decreased for several years with both bone marrow and peripheral blood grafts. They were not different according to the origin of the graft, despite the reduced duration of peripheral blood cell recovery observed after PBPCT. Granulocyte colony-stimulating factor (G-CSF) used for PB graft mobilization and after transplantation resulted in faster neutrophil recovery compared to granulocyte-macrophage colony-stimulating factor (GM-CSF) with no evidence of decreased marrow progenitor cell recoveries. On the other hand, postgraft administration of GM-CSF enhanced long-term colony-forming unit granulocyte-macrophage reconstitution only after BMT. Factors that influenced marrow progenitor cell reconstitution have been identified by univariate and multivariate analysis: age, gender, type of lymphoma, and postgraft administration of hematopoietic growth factors (HGF) for the whole patient group; gender, graft progenitor cell yields, and type of HGF (G-CSF vs GM-CSF) for the PBPCT group; and only type of HGF for the BMT group. Despite faster peripheral blood cell recovery, persistent deficiency of marrow progenitor cells was found several years after PBPCT, as observed after BMT. G-CSF-mobilized PBPCT resulted in faster neutrophil recovery compared to GM-CSF mobilization, with no difference in long-term hematopoietic reconstitution.

Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

Frequency and differentiation capacity of circulating LTC-IC mobilized by G-CSF or GM-CSF following chemotherapy: a comparison with steady-state bone marrow and peripheral blood.

OBJECTIVE: The present study was designed to compare directly the frequency of circulating LTC-IC and E-LTC-IC mobilized in peripheral blood (PB) after chemotherapy supported by either G-CSF (PB-G) or GM-CSF (PB-GM) in comparison to steady-state bone marrow (BM) and PB (PB-ST) values in the same patients. MATERIALS AND METHODS: Long-term cultures (LTC) were performed from 20 patients with malignant lymphoma at saturating cell concentrations to assess bulk progenitor cell production and by limiting dilution assay (LDA) to measure both frequency of LTC-IC and their proliferative and differentiation capacities. RESULTS: While CFC production in bulk LTC was higher at weeks 3-5 with PB-G than with PB-GM samples, week-5 LTC-IC and week-10 LTC-IC (E-LTC-IC) frequencies were not different using a LDA. However, the number of CFC derived from a single LTC-IC was higher in PB-G patients than in PB-GM patients (p = 0.01). Interestingly, the frequency of LTC-IC per 1 x 10(5) MNC in mobilized PB positively correlated with one-year marrow progenitor cell recovery, in contrast to the number of autografted CD34(+) cells and CFU-GM per kg. CONCLUSION: Both G-CSF and GM-CSF resulted in similar increases in LTC-IC and E-LTC-IC in PB at comparable levels to those present in BM. However, the differentiation capacity of LTC-IC was higher after mobilization with G-CSF than with GM-CSF, suggesting qualitative differences in LTC-IC mobilized with these growth factors.