Oncogenic pathways underlying in the development of myelodysplastic syndromes (MDS) remain poorly characterized, but mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed. In the present work, we evaluated the prognostic impact of TET2 mutations in MDS. Frameshift, nonsense, missense mutations, or defects in gene structure were identified in 22 (22.9%) of 96 patients (95% confidence interval [CI], 14.5-31.3 patients). Mutated and unmutated patients did not significantly differ in initial clinical or hematologic parameters. The 5-year OS was 76.9% (95% CI, 49.2%-91.3%) in mutated versus 18.3% (95% CI, 4.2%-41.1%) in unmutated patients (P = .005). The 3-year leukemia-free survival was 89.3% (95% CI, 63.1%-97.0%) in mutated versus 63.7% (95% CI, 48.2%-75.4%) in unmutated patients (P = .035). In univariate analysis (Cox proportional hazard model), the absence of TET2 mutation was associated with a 4.1-fold (95% CI, 1.4-12.0-fold) increased risk of death (P = .009). In multivariate analysis adjusted for age, International Prognostic Scoring System, and transfusion requirement, the presence of TET2 mutation remained an independent factor of favorable prognosis (hazard ratio, 5.2; 95% CI, 1.6-16.3; P = .005). These results indicate that TET2 mutations observed in approximately 20% of patients, irrespective of the World Health Organization or French-American-British subtype, represent a molecular marker for good prognosis in MDS.

Simultaneously to phospholipid flip-flop that supports the procoagulant activity of activated platelets, blebs, supported by actin reorganization, are formed at the plasma membrane and generate microvesicles. The molecular mechanism of microvesicle shedding from activated platelets implicates Ca2+ influx and Ca2+-dependent protease, calpain. We previously demonstrated that the formation of lamellipodias and filopodias associated with platelet shape change involved the reorganization of actin filaments through a Cdc42/Rac1/p21-activated kinase (PAK)-dependent pathway. Here, we investigated whether platelet blebbing also depends on the Cdc42/Rac1/PAK pathway. Exposure of platelets in vitro to either a mixture of thrombin receptor-activating peptide (TRAP) and collagen or the Ca2+ ionophore A23187 in the presence of Ca2+ generates microvesicles that can be identified by flow cytometry. The calpain inhibitor, calpeptin, diminished microvesicle formation induced by the Ca2+ ionophore A23187, confirming the role of calpain in this process. PAK1/2 is cleaved in a calpain-dependent manner, and calpeptin prevents this cleavage and allows a transient activation of the kinase. Inhibition of Cdc42 and Rac1 by toxin B from Clostridium difficile, that suppresses PAK1/2 activation induced by TRAP and collagen or by A23187 in the presence of calpeptin, decreases polymerization of actin, lamellipodia and filopodia formation and interferes with the shedding of microvesicles. We conclude that the Rac1/Cdc42/PAK pathway controls actin reorganization that is necessary for microvesicle shedding.

We analyzed prognostic factors of response, response duration, and possible impact on survival of epoetin alpha, epoetin beta, or darbepoetin alpha (DAR) with or without granulocyte colony-stimulating factor in 403 myelodysplastic syndrome (MDS) patients. Sixty-two percent (40% major and 22% minor) and 50% erythroid responses were seen, and median response duration was 20 and 24 months according to IWG 2000 and 2006 criteria, respectively. Significantly higher response rates were observed with less than 10% blasts, low and int-1 International Prognostic Scoring System (IPSS), red blood cell transfusion independence, serum EPO level less than 200 IU/L, and, with IWG 2006 criteria only, shorter interval between diagnosis and treatment. Significantly longer response duration was associated with major response (IWG 2000 criteria), IPSS low to INT-1, blasts less than 5%, and absence of multilineage dysplasia. Minor responses according to IWG 2000 were reclassified as "nonresponders" or "responders" according to IWG 2006 criteria. However, among those IWG 2000 minor responders, response duration did not differ between IWG 2006 responders and nonresponders. Multivariate adjusted comparisons of survival between our cohort and the untreated MDS cohort used to design IPSS showed similar rate of progression to acute myeloid leukemia in both cohorts, but significantly better overall survival in our cohort, suggesting that epoetin or DAR treatment may have a favorable survival impact in MDS.

Diagnosis of leukemic B-cell chronic lymphoproliferative disorders (B-CLPD) is a frequent challenge in hematology. In this multicentric study, we prospectively studied 165 new consecutive leukemic patients with B-CLPD selected on the basis of Royal Marsden Hospital scoring system ≤3. The primary aim of the study was to try to decipher the atypical cases and identify homogenous subgroups. Overall, morphological examination contributed to diagnosis in only 20% cases, all of them CD5 negative. Thirty additional cases were CD5 negative suggestive of leukemic marginal zone lymphoma in most cases. The significantly poorer survival of the 26 cyclin D1 positive cases justifies recommending its systematic determination among atypical B-CLPD. CD20 expression segregated clearly two subgroups among CD5 positive cyclin D1 negative B-CLPD. The 17 patients with the CD20 dim profile represent a homogeneous subgroup very close to typical B-cell chronic lymphocytic leukemia (B-CLL) on morphological, phenotypical and cytogenetical criteria. In contrast, the subgroup of 51 patients with a CD20 bright profile is heterogeneous. Their significantly lower p27 expression level suggest the presence of a proliferative component, underlying a more aggressive disease. Further genomic studies are warranted to establish their precise nature. These cases should not be included in the same therapeutic trials as B-CLL.

Background: MDS with del 5q are characterized by profound anemia, which until the recent introduction of lenalidomide (N Engl J Med 2005; 352: 549–57, J Clin Oncol 2005; 16S:5), was considered generally unresponsive to available treatments. In order to reevaluate the outcome of those patients in the pre-lenalidomide era, we analyzed response of anemia in MDS with del 5q treated with EPO ± G-CSF and thalidomide in previous GFM trials.

Given the high level of background flora in sludge, methods for detecting Listeria monocytogenes are not well established. In this study, two critical parameters for the detection of L. monocytogenes were evaluated: the concentration of Listeria sp. in a modified Fraser broth (first stage of the method) and the proportion of L. monocytogenes on Palcam agar (second stage of the method). Concentrations of Listeria sp. estimated in 118 modified Fraser enrichment broths inoculated with four types of sludge, reached 104 bacteria per mL for 83% of the positive enrichment broths. Proportion of L. monocytogenes on Palcam agar, which was estimated by transferring all characteristic colonies of Listeria sp. onto Rapid’L Mono agar, was highly variable regardless of the type of sludge. According to these results, we proposed a protocol that consisted of an enrichment in modified Fraser broth for 48h at 37°C, followed by plating 0.1mL of appropriate dilutions of broth onto Palcam agar. After an incubation of 48h at 37°C, a systematic identification of characteristic colonies of Listeria sp. on Rapid’L Mono agar allowed to enhance the detection of Listeria monocytogenes.

Les syndromes myelodysplasiques sont des pathologies clonales acquises de la cellule souche hematopoietique, se caracterisant par une moelle riche contrastant avec des cytopenies peripheriques. Leur physiopathologie est complexe. Une de leurs caracteristiques est un defaut de croissance des progeniteurs immatures en particulier erythroides de type BFU-E et CFU-E. Ce defaut de croissance des progeniteurs hematopoietiques a ete lie a une insensibilite aux cytokines hematopoietiques, alors que l'expression et la fonctionnalite du recepteur a l'erythropoietine semblent conserves, et plus recemment a une augmentation d'apoptose des cellules medullaires. Cette apoptose implique tous les compartiments cellulaires, est massive aux stades precoces de myelodysplasies et diminue avec l'evolution blastique de la maladie. L'implication du compartiment de cellules souches CD34+ reste a preciser. Une activation proteolytique des caspases est observee et un role essentiel du systeme Fas/Fas-l deregule est fortement suggeree dans le mecanisme de cette apoptose accrue. Le micro-environnement medullaire anormal au cours des SMD produit des quantites accrues de cytokines inhibitrices de l'hematopoiese telles que le TNF-alpha et l'IFN-gamma, qui pourraient contribuer au phenomene apoptotique. Des therapeutiques a visee anti-apoptotique (facteurs de croissance, thalidomide, anticorps anti-TNF-alpha) ameliorent les cytopenies des patients, laissant suggerer qu'une meilleure comprehension des mecanismes biologiques conduisant a cette apoptose massive, pourrait deboucher sur des avancees therapeutiques.

Myelodysplastic syndromes (MDSs) are heterogeneous diseases of bone marrow (BM) cell precursors for which immunophenotypic characterization is still considered irrelevant despite the accuracy and sensitivity of flow cytometry techniques. The aim of this study was to determine whether immunophenotypic abnormalities could be defined in MDSs and could correlate with the French-American-British classification and cytogenetics. Analysis was performed on 275 BM samples (207 MDS patients, 68 controls) and 25 control blood samples. Immunophenotyping was based on a primary gating of blast cells, monocytes, and granulocytes according to CD45 antigen expression and side scatter light diffraction. Immunophenotypic hierarchical clustering was performed to analyze the results. The data obtained show that (1) immunophenotypic clustering partly discriminates patients with refractory anemia with excess blasts/refractory anemia with excess blasts in transformation (RAEB/RAEB-T), chronic myelomonocytic leukemia (CMML), and refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) for CD45(lo) blast cells and patients with RA/CMML, RARS, and RAEB/RAEB-T for CD45(hi)/side scatter(hi) (SS(hi)) granulocytes; (2) the most discriminating markers were CD16, CD34, CD36, CD38, CD71, and HLA-DR for blast cells and CD11b, CD13, CD33, CD36, CD38, CD71, and HLA-DR for CD45(hi)/SS(hi) granulocytes; (3) clusters related to CD34 expression were associated with high levels of blast cells on BM smear; (4) clusters related to high levels of CD36 expression on CD45(lo) blast cells and CD45(hi)/SS(hi) granulocytes were associated with a poor International Prognosis Scoring System score; and (5) high levels of CD71 expression on CD45(hi)/SS(hi) granulocytes were associated with the RARS category. These results show a close relationship between immunophenotypic abnormalities and BM dysplasia and suggest that flow cytometry could be a future tool for the characterization of MDSs.

The new WHO classification abolishes the frontier between RAEB-t with 20% of blasts and leukemia with 30% of blasts. We review the definitions of erythroleukemia and discuss the relationship between FAB AML6, RAEB-t and AML6 variant. We ask whether secondary erythroleukemias are the same entity as RAEB-t on survival, karyotype and cytologic characteristics. We suggest that ‘AML6 variant’ with pure erythroid lineage proliferation would be the real de novo erythroleukemia. Current FAB AML6 entity will probably be classified in either subgroup (1) multilineage dysplasia; (2) therapy-related leukemia; or (3) acute erythroid leukemia subdivided into erythroleukemia (erythroid/myeloid) and pure erythroid leukemia, in the WHO classification – a classification which highlights the importance of clinical and cytogenetic prognostic factors.

Platelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and microparticles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow, cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p < 0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients With proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.

We report here a case of acute polymyositis associated with a Ki-1 non-Hodgkin's lymphoma (NHL). This anaplastic large cell malignant lymphoma was a primary T-cell NHL lymphoma of the bone marrow. The malignant cells expressed the CD30 (Ki-1), CD3, and CD4 antigens, the beta chain of the interleukin 2 receptor (CD25), and the betaF1 antigen (alpha/beta T-cell receptor). Chemotherapy and high dose methylprednisolone pulse therapy were initiated. However, no clinical improvement was noticed, because the patient rapidly died of an acute respiratory distress syndrome. To our best knowledge, this represents the first case of Ki-1 lymphoma associated with Polymyositis.

Ribrag, V.; Salmon, D.*; Picard, F.; Guesnu, M.; Sicard, D.*; Dreyfus, F.; Kostolansky, F.*; Li, I. I.; Friedman-Kien, A. E. Author Information

Oxygenated derivatives of cholesterol, such as 25-OH-cholesterol, are potent inhibitors of cholesterol synthesis in various cell types. Although the main site of 25-OH-cholesterol action appears to be mediated by the inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, one of the key steps in the pathway of cholesterol formation, there is no indication on the molecular mechanism leading to enzyme inhibition. We have therefore compared in isolated mouse thymocytes the mode of action of 25-OH-cholesterol to that of dexamethasone which has also been demonstrated to block cholesterol synthesis. 25-OH-cholesterol induced a rapid, dose dependent, inhibition of [3H]acetate incorporation into the sterol fraction. This effect was specific for the cholesterol biosynthetic pathway and, in contrast to dexamethasone, did not affect either lipid synthesis or RNA formation. Unlike glucocorticoid action, the effect of 25-OH-cholesterol was not suppressed in the presence of actinomycin D and did not appear to be mediated via an interaction with specific receptors. It thus appears that the mechanism of action of 25-OH-cholesterol on cholesterol biosynthesis is not comparable to that of steroid hormones.

Glucosteroids, as well as oxygenated derivatives of cholesterol, have been demonstrated to inhibit cholesterol biosynthesis in stimulated lymphocytes and Hela cells. We have therefore measured the effect of various steroids on the incorporation of [3H]-acetate into digitonin-precipitable material in mouse thymocytes. Dexamethasone, induces a marked inhibition of cholesterol synthesis (40–50% inhibition after 6 h incubation in the presence of 10−6 M dexamethasone). This inhibitory effect appears specific of glucocorticoids as the other steroids tested (testosterone, estradiol, progesterone) are minimally effective. Moreover, the decrease in cholesterol synthesis produced by dexamethasone is abolished in the presence of Actinomycin D suggesting that the effects of steroids on cholesterol synthesis are mediated via glucocorticoid receptor occupancy and macromolecular synthesis. In contrast, human circulating lymphocytes and corticoresistant thymocytes do not show a significant inhibition of cholesterol synthesis in the presence of dexamethasone. As the rate of cholesterol synthesis is considered to represent a major factor controlling cell proliferation, our results suggest that part of the effects of glucosteroids on cell growth could result from an inhibition of cholesterol synthesis.