Dual regulation of SPI1/PU.1 transcription factor by heat shock factor 1 (HSF1) during macrophage differentiation of monocytes.

In addition to their cytoprotective role in stressful conditions, heat shock proteins (HSPs) are involved in specific differentiation pathways, for example, we have identified a role for HSP90 in macrophage differentiation of human peripheral blood monocytes that are exposed to macrophage colony-stimulating factor (M-CSF). Here, we show that deletion of the main transcription factor involved in heat shock gene regulation, heat shock factor 1 (HSF1), affects M-CSF-driven differentiation of mouse bone marrow cells. HSF1 transiently accumulates in the nucleus of human monocytes undergoing macrophage differentiation, including M-CSF-treated peripheral blood monocytes and phorbol ester-treated THP1 cells. We demonstrate that HSF1 has a dual effect on SPI1/PU.1, a transcription factor essential for macrophage differentiation and whose deregulation can lead to the development of leukemias and lymphomas. Firstly, HSF1 regulates SPI1/PU.1 gene expression through its binding to a heat shock element within the intron 2 of this gene. Furthermore, downregulation or inhibition of HSF1 impaired both SPI1/PU.1-targeted gene transcription and macrophage differentiation. Secondly, HSF1 induces the expression of HSP70 that interacts with SPI1/PU.1 to protect the transcription factor from proteasomal degradation. Taken together, HSF1 appears as a fine-tuning regulator of SPI1/PU.1 expression at the transcriptional and post-translational levels during macrophage differentiation of monocytes.

Age, JAK2(V617F) and SF3B1 mutations are the main predicting factors for survival in refractory anaemia with ring sideroblasts and marked thrombocytosis.

Refractory anaemia with ring sideroblasts (RARS) and marked thrombocytosis (RARS-T) is a provisional entity in the World Health Organisation 2008 classification and has previously been shown to have a high proportion of JAK2(V617F) (Janus Kinase 2) and SF3B1 (Splicing Factor 3B subunit 1) mutations. The purpose of the present study was to analyse the frequency of SF3B1 mutations in a large cohort of 111 patients with RARS-T and 33 patients with RARS and to explore the prognostic impact of SF3B1 mutational status on RARS-T. The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) and RARS (28/33, 84.8%) was similar. In RARS-T, median survival was better in SF3B1-mutated patients than in SF3B1-non-mutated patients (6.9 and 3.3 years, respectively, P=0.003). RARS can be differentiated from RARS-T by the frequency of JAK2(V617F) (0% vs 48.6%). In RARS-T patients, SF3B1 (P=0.021) and JAK2 mutations (P=0.016) were independent factors for a better prognosis. Altogether, our results confirm that RARS-T is an independent entity that should be recognised by the next World Health Organisation classification. The assessment of SF3B1 mutations is of prognostic interest in RARS-T patients. Younger age, JAK2(V617F) and SF3B1 mutations are the main predicting factors for survival in RARS-T.

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Mutations with epigenetic effects in myeloproliferative neoplasms and recent progress in treatment: Proceedings from the 5th International Post-ASH Symposium.

Immediately following the 2010 annual American Society of Hematology (ASH) meeting, the 5th International Post-ASH Symposium on Chronic Myelogenous Leukemia and BCR-ABL1-Negative Myeloproliferative Neoplasms (MPNs) took place on 7-8 December 2010 in Orlando, Florida, USA. During this meeting, the most recent advances in laboratory research and clinical practice, including those that were presented at the 2010 ASH meeting, were discussed among recognized authorities in the field. The current paper summarizes the proceedings of this meeting in BCR-ABL1-negative MPN. We provide a detailed overview of new mutations with putative epigenetic effects (TET oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), isocitrate dehydrogenase (IDH) and enhancer of zeste homolog 2 (EZH2)) and an update on treatment with Janus kinase (JAK) inhibitors, pomalidomide, everolimus, interferon-α, midostaurin and cladribine. In addition, the new 'Dynamic International Prognostic Scoring System (DIPSS)-plus' prognostic model for primary myelofibrosis (PMF) and the clinical relevance of distinguishing essential thrombocythemia from prefibrotic PMF are discussed.

Minor dysplastic changes are frequently observed in the bone marrow aspirate in elderly patients without haematological disease.

Bone marrow aspirates were obtained by sternal puncture prior to sternotomy in 54 volunteers (40 males and 14 females) aged 60 years or more. All underwent surgery for cardiological diseases and had normal blood counts, without any haematological abnormalities. Quantitative examination of these bone marrow aspirates yielded reference ranges for each cell type similar to those obtained in younger adults. However, qualitative analysis revealed certain discrepancies: dysplastic changes were observed frequently, mainly in megakaryocytes and erythroblasts, with a normal growth pattern of haematopoietic progenitor cells. A low proportion of macrophages and mast cells were noted in 30% of the bone marrow aspirates. Lymphoid aggregates, seen in 13% of these samples, were generally of moderate size, contained few mast cells and were non-clonal on immunophenotypic analysis.

Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.

Acute myeloid leukemia with hypergranular cytoplasm: a differential diagnosis of acute promyelocytic leukemia.

We report here the case of a woman with acute myeloid leukemia with some blast cells exhibiting acute promyelocytic leukemia (APL)-like hypergranular cytoplasm. The cytologic and cytochemical aspects as well as the mature myeloid phenotype and hemostasis disorders were consistent with the diagnosis of APL. However, no t(15;17), or RARalpha gene, MLL gene or PML gene rearrangement was observed, or any other cytogenetic clonal abnormality. Coexpression on blast cells of CD33 and CD56 without CD34, CD16 or HLA-DR, suggested a myeloid/natural killer cell acute leukemia.

Increase therapy-related leukemia secondary to breast cancer.

Therapy-related leukemia associated with chemotherapy, particularly alkylating agents and topoisomerase II inhibitors, are being reported with increasing frequency in the literature mainly after breast cancer. We also observed an increasing number of such leukemias in the data base of the specialized registry of hematological malignancies of the Côte d'Or department. Between 1980 and 1998, 156 AML and RAEB-t were registered in women in Côte d'Or. Among them, 12 occurred in women with breast cancer history (7.7%). Analysis by period of time shows a significant increase in the proportion of therapy-related leukemia secondary to breast cancer (P < 0.02). Chemotherapy including topoisomerase II inhibitors was used in 10 cases in which mitoxantrone was used in eight cases. In these eight cases, leukemia had clinical and biological characteristics usually described with topoisomerase II inhibitors but 44% were promyelocytic sub-type with the t(15;17) specific karyotypic abnormality. These data on a well-defined population demonstrate the increased proportion of therapy-related leukemia secondary to breast cancer, probably due to the use of mitoxantrone.

Immunophenotype of a transient myeloproliferative disorder in a newborn with trisomy 21.

Cytologic, immunologic, and cytogenetic studies were performed on the blast cells of a newborn with Down syndrome and transient myeloproliferative disease. This hematologic disorder is uncommon, and occurs primarily in infants with Down syndrome. This boy presented with a high white blood cell count and a high percentage of blast cells, without anemia or thrombocytopenia. Chromosome analysis showed a constitutional trisomy 21 without any other clonal abnormality. A three-color flow cytometric analysis was performed and revealed two different CD45 dim, CD34(+), CD117(+), CD56(+) immature subpopulations: the normal immature myeloid precursor and an immature blast cell population that expressed CD41, CD42, CD61, CD36, CD13, CD1a, and CD2. We postulate that this population could be the leukemic precursor involved in the acute megakaryoblastic leukemia frequently observed in children with Down syndrome.

Effect of a two-year supplementation with low doses of antioxidant vitamins and/or minerals in elderly subjects on levels of nutrients and antioxidant defense parameters.

BACKGROUND: Eighty-one elderly hospitalized subjects (> 65 years) were recruited for a double-blind placebo-controlled study to examine low dose supplementation of antioxidant vitamins and minerals on biological and functional parameters of free radical metabolism. Subjects were randomly assigned to one of the four treatment groups, daily receiving for 2 years: placebo group; mineral group: 20 mg zinc, 100 micrograms selenium; vitamin group: 120 mg vitamin C (Vit C), 6 mg beta-carotene (beta CA), 15 mg vitamin E (Vit E); mineral and vitamin group: Zn 20 mg, Se 100 micrograms, Vit C 120 mg, beta CA 6 mg, Vit E 15 mg. RESULTS: Fifty-seven subjects completed the study. A large frequency of Vit C, Zn and Se deficiencies were observed at baseline. As early as 6 months of treatment, a significant increase in vitamin and mineral serum levels was observed in the corresponding groups. The increases ranged from 1.1-4.0 fold depending on the nutrient. Antioxidant defense, studied in vitro with a test using red blood cells in presence of 2,2'-azo-bis (2-amidinopropane) by hydrochloride, showed an increase of cell resistance in patients receiving vitamins (p = 0.002); it was positively correlated with serum Vit C (p < 0.0001), alpha-tocopherol/cholesterol (p = 0.06), beta CA (p = 0.0014), serum Cu and Se (p < 0.05). Moreover, red blood cell antioxidant defense was reduced in elderly compared with young control subjects (50% hemolysis time: 69 +/- 14 mn and 109 +/- 12 mn, respectively). Erythrocyte glutathione peroxidase activity was enhanced in groups receiving minerals, whereas no significant change was observed for other indicators of oxidative stress (erythrocyte superoxide dismutase activity, thiobarbituric acid-reactive substances, total glutathione, reduced and oxidized forms). DISCUSSION: Our results provide experimental evidence that a low dose supplementation with vitamins and minerals was able to normalize biological nutrient status as early as 6 months of treatment. In addition, our data indicate that antioxidant defense in elderly subjects was improved with low doses of vit C, vit E and beta CA as studied by means of a functional test utilizing red blood cells challenged in vitro with free radicals.