High incidence of translocations t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with plasma cell malignancies.

Abnormalities involving the 14q32 region are recurrent chromosomal changes in plasma cell malignancies. Recent preliminary molecular analyses found IGH rearrangements in almost 100% of human myeloma cell lines and in 75% of patients. However, no systematic study analyzing the nature of the partner chromosomal regions have been reported thus far. To define the exact incidence of illegitimate IGH rearrangements and the respective incidence of partner genes cloned to date, we analyzed 141 patients with either multiple myeloma (MM, n = 127) or primary plasma cell leukemia (PCL, n = 14) using fluorescence in situ hybridization. The overall incidence of illegitimate recombinations was 57% (80 of 141 patients). Analysis of this incidence according to Durie and Salmon stage, patients' status, i.e., MM versus primary PCL and diagnosis versus relapse, immunoglobulin type and subtype, and beta2-microglobulin value, did not show any correlation. To analyze the nature of the partner chromosomal region, we selected probes specific for the following genes: FGFR3 (4p16), MYC (8q24), CCND1 (11q13), MAF (16q23), and BCL2 (18q21). These probes, combined with differentially labeled 14q32 probes, were used for dual-color fluorescence in situ hybridization on interphase plasma cells. Among the 80 patients with illegitimate IGH rearrangement, we identified 23 IGH-CCND1 fusion cases [i.e., t(11;14)], 17 IGH-FGFR3 fusion cases [i.e., t(4;14)], 3 IGH-MYC fusion cases [i.e., t(8;14)], and only one IGH-MAF fusion case. No IGH-BCL2 fusion case was detected. In 37 of 80 patients, none of these partner genes was involved. Analysis of cases with specific translocations according to their bioclinical features at diagnosis did not show any correlation. This study demonstrated that CCND1 and FGFR3 genes are involved together in about 50% of MM and primary PCL patients with illegitimate IGH rearrangements.

PAF and haematopoiesis: III. Presence and metabolism of platelet-activating factor in human bone marrow.

Platelet-activating factor (PAF) is a phospholipid compound with major immunoregulatory activities. The present study shows that human bone marrow contains 576 +/- 39 pg PAF/ml (n = 35). Bone marrow-derived PAF exhibits the same biophysical and biological properties that synthetic PAF. PAF concentrations in bone marrow are correlated with the granulocyte (r = 0.4, P = 0.02) but not with the lymphocyte (r = 0.24, P = 0.17) and the monocyte (r = 0.12, P = 0.48) counts. In bone marrow PAF is inactivated by a plasma PAF acetylhydrolase activity (48.0 +/- 2.3 nmol/min per ml, n = 34). Experiments with [3H]PAF indicate that human bone marrow cells actively metabolize this potent molecule by the deacetylation-transacylation pathway. Results of this investigation indicate the permanent presence of significant amounts of PAF in bone marrow suggesting its putative involvement in the processes of bone marrow cell proliferation and maturation.

Arachidonic acid and human bone marrow stromal cells.

Human bone marrow stromal cells regulate the growth of marrow hematopoietic progenitors by secreting cytokines. Arachidonic acid (AA) is the fatty acid precursor of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that modulate the growth of human bone marrow progenitors. We have investigated the incorporation of AA in human bone marrow stromal cell cultures, their production of PGE2 and LTB4 and the effect of AA on their growth. Gas chromatography analysis reveals the presence of AA in the human bone marrow plasma and in bone marrow stromal cell cultures. In stromal cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids. Prelabeling-chase experiments indicate a preferential incorporation of AA into phosphatidylethanolamine and no trafficking of labeled AA between phospholipid species. Bone marrow stromal cells release PGE2 and LTB4 in response to phorbol myristic acetate (PMA) (1 microM) and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml). Exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow stromal cells capt exogenous AA and, thus, may participate to the control of marrow AA concentrations. They may also regulate human marrow hematopoiesis by secreting AA metabolites such as PGE2 and LTB4.

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

Interleukin-4 (IL-4), but not IL-10, regulates the synthesis of IL-6, IL-8 and leukemia inhibitory factor by human bone marrow stromal cells.

Leukemia inhibitory factor (LIF), interleukin 6 (IL-6) and IL-8 are important regulators of inflammation and hematopoiesis. Human bone marrow stromal cells regulate marrow hematopoiesis by secreting cytokines. By using reverse-transcriptase polymerase chain reaction (RT-PCR), we demonstrate that human bone marrow stromal cells constitutively express LIF, IL-6 and IL-8 transcripts. By using specific ELISAs, we found that their spontaneous productions of LIF, IL-6 and IL-8 are elevated in response to serum and after stimulation with the pro-inflammatory cytokines IL-1alpha and TNF-alpha. The anti-inflammatory cytokine IL-4 reduces their serum- and cytokine-induced LIF secretion. By contrast, IL-4 stimulates their serum- and IL-1alpha-induced IL-6 synthesis. IL-4 has no effect on the serum-induced IL-8 synthesis by marrow stromal cells, but stimulates their cytokine-induced IL-8 production. The anti-inflammatory cytokine IL-10 has no effect on the serum- and cytokine-induced LIF, IL-6 and IL-8 synthesis by bone marrow stromal cells. RT-PCR experiments reveal the presence of IL-4 receptor alpha-chain mRNA and IL-10 receptor mRNA in cultured bone marrow stromal cells. The differential regulation by IL-4 of two related cytokines, such as LIF and IL-6, and the enhanced effect of this 'anti-inflammatory' cytokine on IL-6 and IL-8 synthesis highlight the tightly controlled regulation and the complexity of the cytokine production within the human bone marrow.

IgM peak independently predicts treatment-free survival in chronic lymphocytic leukemia and correlates with accumulation of adverse oncogenetic events.

We examined the significance of IgM peaks in chronic lymphocytic leukemia (CLL), including its association with newly reported MYD88, BIRC3, NOTCH1 and SF3B1 mutations. A total of 27, 25, 41 and 57 patients with monoclonal IgM or IgG peaks (IgM and IgG groups), hypogammaglobulinemia (Hypo-γ group) and normal immunoglobulin serum levels (normal-γ group) were, respectively, included. IgM peaks were mainly associated with Binet stage C and the del(17p). Biased usage of IGHV3-48 was shared by both IgM and IgG groups. IGHV3-74 and IGHV4-39 gene rearrangements were specific for IgM and IgG peaks, respectively. SF3B1, NOTCH1, MYD88 and BIRC3 mutation frequencies were 12%, 4%, 2% and 2%, respectively, being over-represented in IgM, IgG and Hypo-γ groups for SF3B1, and being equal between normal-γ and IgM groups for MYD88. Overall, 76%, 87%, 49% and 42% of cases from IgM, IgG, Hypo-γ and normal-γ groups had at least one intermediate or poor prognosis genetic marker, respectively. By multivariate analysis, IgM peaks were associated with shorter treatment-free survival independently from any other univariate poor prognosis biological parameters, including IgG peaks, Hypo-γ, IGHV status, SF3B1 mutations, cytogenetics and lymphocytosis. Therefore, as with IgG peaks, IgM peaks aggravated the natural course of CLL, with increased accumulation of adverse genetic events.

Thrombocytopenia in the sepsis syndrome: role of hemophagocytosis and macrophage colony-stimulating factor.

BACKGROUND: Thrombocytopenia is frequently encountered in critically ill patients with the sepsis syndrome, but its mechanisms frequently remain undetermined. Hemophagocytosis has been reported as a cause of thrombocytopenia in various diseases. This prospective study was designed to assess: (1) the incidence of hemophagocytosis in patients suffering from both the sepsis syndrome and unexplained thrombocytopenia, and (2) the circulating level of the macrophage-colony-stimulating factor (M-CSF) according to the presence or absence of hemophagocytosis. METHODS: Fifty consecutive patients diagnosed with both the sepsis syndrome and thrombocytopenia of undetermined origin were studied. Hemophagocytosis was diagnosed based on microscopical examination of sternal bone marrow aspiration by two independent observers. Serum M-CSF concentrations were measured in each patient and compared with levels of a normal population (n = 59). Causes and severity of sepsis syndromes as well as serum M-CSF levels were compared between patients with and without hemophagocytosis. RESULTS: Hemophagocytosis was diagnosed in 32 patients (64%). Mean serum M-CSF levels were increased in patients when compared with normal subjects (539 +/- 141 versus 208 +/- 82 IU/mL: P < 0.001), and higher in patients with than without hemophagocytosis (580 +/- 145 versus 457 +/- 89 IU/mL: P = 0.01). Multiorgan dysfunction and infection were independent risk factors of hemophagocytosis (odds ratio = 31.3 and 6.8, 95% confidence interval (CI) = 5.4 to 177.6 and 1.0 to 47.4, P <0.0001 and P = 0.03, respectively). CONCLUSIONS: Hemophagocytosis is a frequent cause of unexplained thrombocytopenia in patients with severe sepsis syndrome. Our results suggest that M-CSF is overproduced in the sepsis syndrome, particularly when hemophagocytosis is present. The role of M-CSF in the initiation and development of hemophagocytosis remains to be determined.

Deletion of chromosome 20q associated with hypereosinophilic syndrome. A report of two cases.

Deletion of the long arm of chromosome 20--del(20q)--is commonly associated with myeloid malignancies, in particular with myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), and acute myelogenous leukemia (AML). Its association with the hypereosinophilic syndrome (HES) had never been reported. In the present study we describe two patients with long-standing hypereosinophilia and features of atypical MPD or MDS carrying a del(20q) as a constant and isolated chromosomal abnormality. One patient, with an aggressive clinical course, died within 2 years, despite several lines of treatment. The other patient had a more indolent course consistent with that of an atypical MDS with eosinophilia.

Detection of functional platelet-activating factor receptors on leukemic B cells of chronic lymphocytic leukemic patients.

Although platelet-activating factor receptors (PAF-R) are reported on normal B cells, few results are available concerning leukemic ones. We demonstrated functional PAF-R on cell and nuclear surfaces of leukemic B cells of chronic lymphocytic leukemic (CLL) patients. Analysis of 102 patients revealed dramatic differences for their membrane PAF-R expression, a result that might be related to their plasma IL-4 levels. In the light of the potent immunoregulatory role of PAF on B cell physiology, it is suggested that the presence or absence of PAF-R on leukemic B cells may profoundly affect their in vivo behavior.

Leukemia inhibitory factor concentrations in the bone marrow plasma of healthy subjects and patients with hematologic malignancies.

Several cytokines may play a role in the pathogenesis of various hematopoietic malignancies. As cytokines work locally, we have investigated leukemia inhibitory factor (LIF) concentrations in the bone marrow plasma of healthy subjects and patients with hematopoietic malignancies by using a specific enzyme-linked immunosorbent assay. LIF levels in patient's samples were significantly higher in bone marrow plasmas (330.3 +/- 43.6 pg/ml; n = 29) than LIF levels in blood plasmas (178.9 +/- 16.8 pg/ml; n = 43) (p = 0.0006, Mann-Whitney U-test). Marrow stromal cells which constitutively produce LIF and enhance their synthesis in response to LPS and PMA might be the cell source of the bone marrow-derived LIF. No statistical difference was documented between marrow plasma LIF concentrations of 24 patients with hematological malignancies and healthy controls. At the present time, the role of LIF in the human marrow cytokine network requires further evaluation.

[Reactive hemophagocytic syndrome: a probably underestimated cause of thrombocytopenia in intensive acre patients]. / Syndrome hémophagocytaire réactionnel: une cause probablement sous-estimée de thrombopénie en réanimation.

Thrombocytopenia is a common feature in ICU patients which occurs usually in case of infection or septic shock. Its mechanisms, which are often unclear, include the haemophagocytic syndrome initially linked with histiocytic proliferation but probably also associated with infectious diseases. This syndrome is characterized by a phagocytosis of medullar blood cells. Reactive haemophagocytic syndrome can probably lead to thrombocytopenia in ICU patients as in this case report of a E. Coli infection.

[Pancytopenia and pulmonary tuberculosis. Significance of a hemophagocytic syndrome]. / Pancytopénie et tuberculose pulmonaire. Implication d'un syndrome hémophagocytaire.

Haemophagocytic syndromes or syndromes involving macrophage activation are rare complications of tuberculosis, whether they be pulmonary or polyvisceral. They are characterised by an anomalous increase in the phagocytic power of macrophages with phagocytosis of the formed elements of blood. The clinical biological picture associates a change in the general physical state accompanied by organomegaly, hyperferritinaemia and pancytopenia. Their occurrence is a poor prognostic factor and few treatment seem to check this mechanism. The authors report a rare case of marked macrophage activation syndrome complicating pulmonary tuberculosis in a patient who was HIV negative without an underlying blood disturbance and a favourable outcome.

[Hemophagocytosis during multiple organ failure: M-CSF overproduction or viral reactivation?]. / Hémophagocytose au cours des défaillances polyviscérales: implication du M-CSF ou réactivation virale?

OBJECTIVE: This study was aimed to assess the potential role of M-CSF and viral reactivation in the genesis of haemophagocytosis during the multiple organ failure (MOF) syndrome. METHODS: Twenty-five patients (mean age: 60 +/- 16 years; Apache II: 23 +/- 5) sustaining MOF with an unexplained thrombocytopenia were studied. In each patient, a bone marrow aspirate, serum M-CSF concentration, and a virological examination (Herpes viruses) were obtained on admission. In addition, 20 patients (mean age: 57 +/- 15 years; Apache II: 24 +/- 7) with at least two organ failures but no thrombocytopenia constituted the control group. Circulating M-CSF levels and the frequency of virus reactivation were compared between groups. RESULTS: Haemophagocytosis was diagnosed in 11/25 patients (44%). No viral reactivation was found. Serum M-CSF concentrations were higher in the presence of haemophagocytosis (699 +/- 242 vs 438 +/- 157 IU.mL-1; p < 0.05). Ferritin levels were also increased in the presence of a macrophage activation (3,258 +/- 2,807 vs. 520 +/- 280 mg.L-1; p < 0.0001). In contrast, both circulating M-CSF and ferritin levels were similar between thrombocytopenic patients with no hemophagocytosis and controls. CONCLUSIONS: This study confirmed the high incidence of haemophagocytosis in critically ill patients sustaining MOF. In this setting, circulating M-CSF levels were markedly elevated, whereas no Herpes viruses reactivation was found.

Effects of lipid mediators on the synthesis of leukaemia inhibitory factor and interleukin 6 by human bone marrow stromal cells.

Human bone marrow stromal cells regulate haematopoiesis by releasing cytokines such as leukaemia inhibitory factor (LIF) and interleukin 6 (IL-6). We have investigated the effects of 5 lipid mediators (i.e. 12-HETE, 15-HETE, platelet-activating factor (PAF), LTB4 and prostaglandin E2 (PGE2)) on their LIF and IL-6 synthesis. 12-HETE, 15-HETE and LTB4 (both at 1 microM) stimulate the LIF production by human bone marrow stromal cells grown in 10% fetal calf serum (FCS). In contrast, PAF and PGE2 have no effect. 12-HETE (1 microM) enhances LIF synthesis in serum free medium 7.7-fold and stimulates IL-1 induced LIF production. 12-HETE, 15-HETE, PAF and LTB4 have no effect on the spontaneous, serum- and cytokine-induced IL-6 synthesis by bone marrow stromal cells. In contrast PGE2 significantly stimulates serum-induced IL-6 synthesis. This study reports for the first time that lipid mediators may act on human haematopoiesis by modulating LIF and IL-6 synthesis by bone marrow stromal cells. Particularly, 12-HETE enhances LIF but not IL-6 synthesis. The different regulation of IL-6 and LIF synthesis in response to lipid mediators highlights the complexity of the cytokine regulation into the human bone marrow.

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells.

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.

Platelet-activating factor acetylhydrolase and haemophagocytosis in the sepsis syndrome.

Sepsis syndrome (SS) is associated with depressed PAF acetylhydrolase, the enzyme responsible for the degradation of platelet activating factor. PAF acetylhydrolase is in a large part produced by macrophages, whose inadequate activation with haemophagocytosis is frequent in patients with SS. The aim of this study was to test the hypothesis that PAF acetylhydrolase levels could be affected in these critically ill patients, because of the large amounts produced by activated macrophages in vitro and in vivo in animal models. The levels of serum PAF acetylhydrolase were assessed in 90 SS patients, who were divided into three groups: patients with (n = 34) or without haemophagocytosis (n = 31), and patients without thrombocytopenia (n = 25) who were used as a control group. The number of organ dysfunctions was matched between patients with haemophagocytosis and controls. Normal reference values were obtained in 59 randomly selected blood donors. Circulating levels of PAF acetylhydrolase were significantly (p = 0.0001) decreased in patients with SS (57+/-3 nmol/ml/min, n = 90) when compared with healthy subjects (69+/-3 nmol/ml/min, n = 59). PAF acetylhydrolase levels were greater in the presence of a haemophagocytosis but without statistical significance (64.2+/-6.5 vs. 50.1+/-2.8:p = 0.25). Despite the fact that macrophagic activation stimulates the in vitro release of PAF acetylhydrolase, no difference was found between patients with or without haemophagocytosis. The mechanism and the role of the PAF acetylhydrolase reduction in SS patients remain to be determined.