Soluble angiogenic factors in MDS BM microenvironment.

<p>The box plot compares median levels of sENG, sFLT-1 and sVEGF in BM supernatant fluid of various types of MDS. To measure the levels of angiogenic factors present in the BM supernatant fluid in the different MDS groups, ELISA assays were carried out in the BM supernatant fluid from MDS patients and controls. Whiskers represent the range. Mann-Whitney test showed that sENG concentrations in BM supernatants was higher in RCMD with respect to the healthy cases (<i>p</i><0.005), the remaining low-risk MDS (<i>p</i><0.05) and high-risk patients (<i>p</i> = 0.05) (A). RCDM displayed higher levels of sFLT-1 with respect to the controls (<i>p</i> = 0.001), the remaining low-risk MDS (<i>p</i><0.005) and the high-risk MDS patients (<i>p</i><0.005) (B). No significant differences in sVEGF concentration of MDS groups were found (C). MDS: myelodysplastic syndrome; BM: bone marrow; ENG: endoglin; sFLT-1: fms-like tyrosine kinase 1; VEGF: vascular endothelial grow factor; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 24; Low-Risk MDS excluding RCMD n = 15; RCMD n = 15; High-Risk MDS n = 6).</p

<i>ENG</i> and <i>VEGF</i> RNA expression in mononuclear BM cells of MDS subtypes.

<p>The box plot compares median of <i>ENG</i> and <i>VEGF</i> expression levels in BM mononuclear cells between the different MDS groups and controls. The gene expression levels were analyzed by RT-PCR. Each sample was performed in triplicate. Value of each patient is the mean of these three experiments. Mann-Whitney test was used to analyze the results. The box plot compares the RNA expression in BM mononuclear cells of subtypes of MDS. Whiskers represent the range. A down-regulation of <i>ENG</i> was showed in RCMD cases (<i>p</i><0.05). By contrast, <i>ENG</i> expression in high-risk MDS patients was higher than in controls or in the other MDS (<i>p</i><0.05). No significant differences in low-risk MDS excluding RCMD patients in <i>ENG</i> expression with respect to the healthy controls were found (A). The low-risk MDS groups showed over-expression of <i>VEGF</i> with respect to the control group (<i>p</i><0.05). Moreover, patients with RCMD showed the highest values in the expression of this gene with respect to the other low-risk MDS. No significant differences in high-risk MDS patients in <i>VEGF</i> expression with respect to the healthy controls were found (B). ENG: endoglin; VEGF: vascular endothelial grow factor; BM: bone marrow; MDS: myelodysplastic syndrome; RCMD: refractory cytopenia with multilineage dysplasia; RAEB: refractory anaemia with excess of blasts. (Controls n = 13; Low-Risk MDS excluding RCMD n = 22; RCMD n = 12; High-Risk MDS n = 16).</p

Effect of the MDS BM microenvironment on BMEC-1 and HMVEC-L tube formation.

<p>BMEC-1 (A) and HMVEC-L (B) were seeded at a concentration of 8,000 cells per well of 96-well plate and incubated for 7 h at 37°C in 5% CO₂. The endothelial tube formation was photographed at 5 h using a phase contrast inverted microscope. Each experiment was performed in duplicate. The pictures show the appearance of endothelial cell tubes on Matrigel® precoated plates in culture medium (i) and BM supernatant fluid from healthy control (ii), RA (iii), RARS (iv), 5q syndrome (v), RAEB (high-risk MDS) (vi) and RCMD (vii-viii) patients at 1∶10 dilution in culture medium. As the arrows show in the figure, the tube morphology was strikingly influenced by BM supernatant fluid from MDS (iii-viii) with respect to the controls (ii). The tubes originated after the incubation of BMEC-1 or HMVEC-L with the BM supernatant fluid from RCMD patients (vii-viii) were almost completely disrupted and formed closed capillary networks. MDS: myelodysplastic syndrome; BM: bone marrow; BMEC-1: bone marrow endothelial cells; HMVEC-L: lung-derived normal human microvascular endothelial cells; RA: refractory anemia; RARS: refractory anemia with ring sideroblast; RAEB refractory anemia with excess of blasts; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 13; RA n = 5; RARS n = 6; 5q syndrome n = 2; RAEB n = 4; RCMD n = 7).</p

Effect of MDS BM microenvironment on BMEC-1 proliferation.

<p>(A) BMEC-1 proliferation curve. To analyze the effect of the BM supernatant fluid from MDS patients and controls on BMEC-1 proliferation, the cell line was incubated with BM supernatant fluid. The cell number was estimated by MTT at two, four or six days. The measurement of absorbance is indicative of the rate of cell proliferation and each value of each patient is the mean of four independent experiments. Each point is the mean of these values ± SEM. The graphics show the increase of proliferation in MDS patients. ANOVA test was used to analyze the overall MDS results at sixth day. The proliferation was 2.4 times higher in MDS than controls (<i>p</i><0.005). (B) The box plot compares median levels of BMEC-1 proliferation at sixth day in the different subtypes of MDS. Whiskers represent the range. Significant differences between RCMD and the control group (<i>p</i><0.01), the other low-risk MDS and the control group (<i>p</i><0.05) and high-risk MDS patients and the controls (<i>p</i><0.05) were observed by Mann-Whitney test. MDS: myelodysplastic syndrome; BM: bone marrow; BMEC-1: bone marrow endothelial cells; MTT: Thiazolyl Blue Tetrazolium Bromide; SEM: standard error of the mean; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 8; MDS n = 14; Low-Risk MDS excluding RCMD n = 6; RCMD n = 4; High-Risk MDS n = 4).</p

Identification of two novel mutations in <i>RASGRP2</i> affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

<p>The <i>RASGRP2</i> gene encodes the Ca²⁺ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new <i>RASGRP2</i> variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in <i>RASGRP2</i>: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn^2<b>+</b>-induced fibrinogen binding. In summary, we have identified two new <i>RASGRP2</i> mutations that can be added to this rapidly growing form of inherited platelet function disorder.</p

(A) Time to first therapy (TFT) and (B) overall survival (OS) of 197 patients with 11q deletion CLL and <40% or ≥40% FISH losses.

<p>(A) Time to first therapy (TFT) and (B) overall survival (OS) of 197 patients with 11q deletion CLL and <40% or ≥40% FISH losses.</p

Multivariate Cox regression analysis of overall survival in 11q- CLL patients with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).^*

<p>*The following covariates were included in the final model: age, sex, Binet stage, splenomegaly, extended lymphadenopathies, LDH, β₂ microglobulin, CD38, ZAP70, <i>IGHV</i> mutation status and percentage 11q deleted nuclei.</p><p>Multivariate Cox regression analysis of overall survival in 11q- CLL patients with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143073#t002fn001" target="_blank">*</a>}</p

Characteristics of 197 patients with 11q deletion with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).

<p>*Number of cases.</p><p>Characteristics of 197 patients with 11q deletion with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).</p

Multivariate Cox regression analysis of time to first therapy in 11q- CLL patients with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).^*

<p>*The following covariates were included in the final model: age, sex, Binet stage, splenomegaly, extended lymphadenopathies, LDH, β₂ microglobulin, CD38, ZAP70, <i>IGHV</i> mutation status and percentage 11q deleted nuclei.</p><p>Multivariate Cox regression analysis of time to first therapy in 11q- CLL patients with respect to the number of losses detected by FISH: <40% (n = 51) or ≥40% (n = 146).^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143073#t002fn001" target="_blank">*</a>}</p

Distribution of mutations of <i>ATM</i>, <i>SF3B1</i>, <i>NOTCH1</i>, <i>TP53</i>, <i>XPO1</i> and <i>BIRC3</i> among 11q- CLL patients with respect to the percentage of 11q- cells.

<p>In the heat maps, rows correspond to identical genes, and columns represent individual patients color-coded on the basis of gene status (white: wild type; grey: mutated gene).</p