Erythropoietin-independent erythroid colony formation by bone marrow progenitors exposed to interleukin-11 and interleukin-8.

OBJECTIVE: Endogenous erythroid colonies (EECs), formed in vitro without erythropoietin (EPo) or other exogenous cytokines, are characteristic of Polycythemia vera (PV). Our aim was to identify specific conditions of culture of bone marrow (BM) progenitors allowing formation of erythroid colonies without EPo. METHODS: BM mononuclear cells (BMMCs), purified CD34+ cells, and purified CD36+ erythroid progenitors were cultured in serum-free media without and with cytokines: EPo, stem cell factor (SCF), and interleukin (IL)-11 and IL-8, produced by BM stromal cells and found elevated in PV. RESULTS: EECs were formed in PV cultures of either BMMCs or CD34+ cells, which include cytokine-secreting cells, but not in cultures of purified CD36+ erythroid progenitors (EP). Despite expression of V617F JAK-2, no constitutive activation of JAK-2, Stat-5, or Erk-1/2 was detected in erythroblasts issued from PV CD36+ progenitors. However, when SCF was provided, PV CD36+ progenitors formed erythroid colonies without EPo. The ability to form erythroid colonies with SCF alone was conferred to BM progenitors of healthy donors and secondary erythrocytosis by exposure to IL-11 and IL-8. Both IL-11 and IL-8 enhanced formation of erythroid colonies in response to EPo and interfered with the activation of Erk-1/2 and Stat-5 induced, respectively, by SCF and EPo in erythroblasts. Anti-IL-11 antibody inhibited formation of erythroid colonies by PV BMMCs and CD34+ cells. CONCLUSION: The data indicate that PV erythroid progenitors remain cytokine-dependent and that normal BM progenitors exposed to IL-11 and IL-8 can acquire the ability to form erythroid colonies without EPo.

Patients with CD45 negative multiple myeloma receiving high-dose therapy have a shorter survival than those with CD45 positive multiple myeloma.

BACKGROUND AND OBJECTIVES: CD45 is a critical regulator of signaling threshold in immune cells. There are clinical and animal studies suggesting that the CD45-negative phenotype is the phenotype of progressive multiple myeloma (MM). The aims of this study were to confirm this hypothesis and to test the prognostic value of CD45 expression in newly diagnosed MM patients. DESIGN AND METHODS: In a retrospective study of 95 newly diagnosed MM patients treated with high dose therapy we used 4-color flow cytometry to determine CD45 expression and correlated the immunophenotipic data with clinical data. RESULTS: Thirty of 95 patients (31.5%) lacked CD45 expression at diagnosis. The CD45 phenotype significantly affected the overall survival (OS) of the patients, like the most common presenting prognostic parameters analyzed including b-2-microglobulin, age and 14q32 translocations. CD45 negative MM patients had a significantly worse OS than did CD45 positive cases of MM: 28.7% cumulative survival at 4 years, median 42 months vs not reached; p = 0.004. Furthermore, CD45 remained the only parameter adversely affecting OS in multivariate analysis. INTERPRETATION AND CONCLUSIONS: The CD45 negative phenotype could reflect the phenotype of progressive disease in relation to the intrinsic malignancy of the MM clone. Indeed, CD45 negative myeloma cells appear to have a greater capacity to circulate, disseminate and clone as well as being less sensitive to apoptosis.

A standardized endogenous megakaryocytic erythroid colony assay for the diagnosis of essential thrombocythemia.

BACKGROUND AND OBJECTIVES: The reliability of assays of endogenous megakaryocytic colony (EMC) and endogenous erythroid colony (EEC) formation for the diagnosis of thrombocytoses remains controversial. We tested the suitability of a recently developed collagen-based assay of EMC formation for the diagnosis of essential thrombocythemia (ET). DESIGN AND METHODS: This was a multicenter (8 laboratories) study including 121 patients: 82 with ET and 39 with reactive thrombocytoses (RT). EMC and EEC were assessed in each laboratory in serum-free, cytokine-free, standardized collagen gel assays; bone marrow (BM) and peripheral blood (PB) were tested in parallel. RESULTS: In PB cultures, only EEC were specific for ET. In BM cultures, both EMC and EEC were specific for ET and present in assays of 77.8% (EMC) and 33.3% (EEC) of ET patients. Altogether, 80.2% of ET patients had BM EMC and/or EEC, whereas none of the patients with RT did. INTERPRETATION AND CONCLUSIONS: When performed with BM progenitors for the diagnosis of thrombocytoses, positivity of the standardized EMC/EEC assay in collagen is specific (100%) and detects 80% of ET.

Standardization and comparison of endogenous erythroid colony assays performed with bone marrow or blood progenitors for the diagnosis of polycythemia vera.

The reliability of the assay of endogenous erythroid colony (EEC) formation in serum-free, cytokine-free collagen-based media was investigated in a multicentric study including 140 patients with polyglobuly (80 polycythemia vera (PV), 54 secondary erythrocytosis (SE), six idiopathic erythrocytosis (IE)) and 10 healthy donors. In each center, EEC assays were performed in parallel with progenitor cells from bone marrow (BM) and peripheral blood (PB); two commercialized media and 'low' and 'high' cell plating densities were tested. Negativity of EEC assays was considered certain only when sufficient BFU-E growth was obtained in control cultures with cytokines. In the two media, EEC formation was specific - never observed in cultures of healthy donors or SE patients - and comparable. BM EEC assays were positive (presence of eythroid colonies) for 75% ('low' plating) to 100% ('high' plating) of PV patients; PB EEC assays were positive for 83.3% ('low' plating) to 93.7% ('high' plating) of PV patients (differences not significant). Depending on the medium, 86.2-93.7% of patients with a positive BM EEC assay had a positive PB EEC assay. Hence, a standardized collagen-based EEC assay can be performed with either BM or PB progenitors; the EEC assay described here is positive for at least 75% of PV patients when a single EEC assay is performed, and for at least 94% of PV patients when both BM and PB EEC assays are performed.

Abnormal production of interleukin (IL)-11 and IL-8 in polycythaemia vera.

We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242pg/ml, vs 4.4pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508pg/ml) were significantly higher than those of IE patients (120pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (x6.4) and IL-8 (x8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC.

Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

CD20 is associated with a small mature plasma cell morphology and t(11;14) in multiple myeloma.

CD20 has been reinvestigated in 66 patients with multiple myeloma (MM). Twelve of the patients (18%) expressed CD20, including 5 of 50 patients at diagnosis presenting 100% CD20+ cells. Seven (58%) of 12 CD20+ patients with MM had a small mature plasma cell morphology as opposed to 4 (7%) of 54 with CD20- MM (P =.0001). Of note, 10 (83%) of 12 patients with CD20+ MM had t(11;14) as opposed to 5 of 54 (9%) CD20- patients (P <.001). All the patients with 100% CD20+ cells presented with t(11;14) and 4 of 5 with a small mature plasma cell morphology. Thus, 66% of the patients with t(11;14) expressed CD20, whereas only 4% of the 51 patients lacking such translocation expressed CD20 (P <.0001). In conclusion, CD20 expression is associated with small mature plasma cell morphology and with t(11;14) in patients with MM.