Leucémie lymphoïde chronique et stroma médullaire (étude préliminaire des interactions entre les cellules mésenchymateuses et le clone malin)

La leucémie lymphoïde chronique (LLC) est la plus fréquente des lymphopathies de l adulte et reste une maladie incurable. L étude des cellules de LLC est difficile puisqu elles entrent rapidement en apoptose in vitro. La localisation médullaire initiale de la maladie, ainsi que la constatation que cette apoptose puisse être retardée si les lymphocytes malins sont cultivés en présence de cellules adhérentes, suggèrent une importante interaction entre le clone lymphocytaire et le microenvironnement, dont les cellules mésenchymateuses (CM) sont un élément essentiel. Cette étude préliminaire s est intéressée à l influence des facteurs solubles présents dans le sérum des malades sur les CM médullaires, et l influence des CM normales sur la survie du clone B in vitro. L étude comparative du sérum de veau fœtal (SVF), d un sérum humain normal (sAB) et du sérum de LLC (sLLC) a démontré un effet stimulant particulier du sLLC sur la prolifération des CM humaines normales. La co-culture de lymphocytes de LLC et de CM en présence des 3 sérums met en évidence un effet positif du sLLC sur la survie du clone B et confirme celui des CM. Dans ces conditions, 40% des lymphocytes survivent à J4. Une série limitée de moelles osseuses de malades (n=8) met en évidence la difficulté de détection des CFU-F, dont la fréquence est faible (50% d échec) et la prolifération limitée en présence de SVF. Leur effet sur la survie des lymphocytes est observé en présence de sLLC mais pas de sAB. Par ailleurs, nous avons constaté, à J4, la persistance de polynucléaires basophiles et l apparition de cellules de type macrophagique (et/ou dentritique) qui pourraient jouer un rôle dans la survie du clone. Ce travail préliminaire suggère que la survie du clone malin dépend de facteurs solubles sériques et de contacts cellulaires. Les CM des malades ne sont pas équivalentes aux cellules témoins et pourraient procurer un environnement adapté aux lymphocytes de LLC.CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Cryopreservation of mononuclear cells before extracorporeal photochemotherapy does not impair their anti-proliferative capabilities

International audienceBackground aims. The clinical benefi ts of extracorporeal photochemotherapy (ECP) are well recognized, but its clinical use is limited by logistical diffi culties, especially because of the need to perform repeated aphereses. The cryopreservation of mononuclear cells could allow maintenance of the ECP schedule while reducing the number of aphereses. The aim of this work was to assess whether previous cryopreservation impairs the immunomodulatory function of ECP-treated peripheral blood mononuclear cells (PBMC). Methods. Fresh or previously cryopreserved PBMC were exposed to ECP and added on day 0 into a mixed leukocyte reaction. Proliferation of alloreactive lymphocytes was measured by carboxyfl uorescein succinimidyl ester (CFSE) dye dilution. Apoptosis was quantifi ed by annexin-7AAD staining. Results. ECP-induced apop-tosis was slightly increased in cryopreserved cells but the kinetics of apoptosis were similar to fresh cells. Lymphocytes stimulated in the presence of ECP-treated PBMC displayed a signifi cant decrease in proliferation. The suppression was enforced when ECP-treated cells had been activated previously by allogeneic stimulation. Cryopreservation before ECP exposure did not impact apoptosis triggering or anti-proliferative properties of ECP-treated cells. Conclusions. Cryopreser-vation before ECP does not impair the immunomodulatory effects of treated cells. These data warrant investigation of the clinical use of cryopreserved PBMC for ECP

Double L611S/V617F JAK2 mutation in a child with erythrocytosis

International audienc

Outcome and impact of post-remission strategy after MIDAM regimen in patients with relapsing or refractory acute myeloid leukemia

International audienc

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes. A diagnostic accuracy study.

International audienceSuspicion of myelodysplastic syndromes is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood cytopenia of unclear etiology. A peripheral blood assay that accurately rules out myelodysplastic syndromes would have major benefits. The diagnostic accuracy of the intraindividual robust coefficient of variation for neutrophil myeloperoxidase expression measured by flow cytometric analysis in peripheral blood was evaluated in a retrospective derivation study (44 myelodysplastic syndrome cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected myelodysplastic syndromes). Compared with controls, myelodysplastic syndrome cases had higher median robust coefficient of variation values for neutrophil myeloperoxidase expression (40.2% versus 30.9%, P<.001). The area under the receiver operating characteristic curve estimates were 0.94 (95% confidence interval [CI], 0.86-0.97) and 0.87 (95% CI, 0.76-0.94) in the derivation and validation studies, respectively. A robust coefficient of variation lower than 30% ruled out myelodysplastic syndromes with 100% sensitivity (95% CI, 78-100%) and 100% negative predictive value (95% CI, 83%-100%) in the prospective validation study. Neutrophil myeloperoxidase expression measured by flow cytometric analysis in peripheral blood might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected myelodysplastic syndromes

Strong correlation between VEGF and MCL-1 mRNA expression levels in B-cell chronic lymphocytic leukemia.

International audienceExpression of the anti-apoptotic myeloid cell leukemia-1 (MCL-1) gene is a novel prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL). Vascular and endothelial growth factor (VEGF) and interleukin-6 (IL-6) are able to upregulate MCL-1 via autocrine signaling loops. In 88 B-CLL patients, we found a strong correlation of MCL-1 gene expression with VEGF (P<10(-7)) but not with IL-6 mRNA levels. VEGF but not IL-6 expression influenced patient prognosis. VEGF may be a positive autocrine in vivo regulator of MCL-1 in B-CLL. Inhibition of VEGF and its signaling may prove to be useful in the treatment of B-CLL patients

Hematopoietic progenitor cell mobilization and harvesting in children with malignancies: do the advantages of pegfilgrastim really translate into clinical benefit?

International audienceOur purpose was to assess success rates in children of achieving optimal hematopoietic progenitor cells (HPCs) harvest after mobilization with 300 microg/kg pegfilgrastim. Between January 2005 and January 2007, 26 children with solid malignancies who were referred for HPC collection were consecutively included. Hematopoietic progenitor cell mobilization consisted of one s.c. injection of 300 microg/kg body weight (BW) of pegfilgrastim. The success criterion was defined as at least 5 x 10(6) CD34+ cells/kg during the first standard apheresis (less than 3 blood volumes processed (BVP)). After 26 inclusions, the Bayesian analysis gave a mean estimated success rate of 60.7% (95% credibility interval: 42.0-78.0%). The first apheresis allowed the collection of 8.3 x 10(6) CD34+ cells/kg BW (range 0.6-37.8), with a median of 2.8 BVP (range 1.4-3.0). Overall, the median of CD34+ cells collected was 12.4 x 10(6)/kg (range 2.7-37.8). The cumulative dose of anthracyclin was the only variable associated with the total number of CD34+ collected cells (P or =3 occurred. We conclude that a single injection of 300 microg/kg pegfilgrastim in the hematological steady state is an efficient and well-tolerated method of HPC mobilization in children with solid malignancies

Linking the KIR phenotype with STAT3 and TET2 mutations to identify chronic lymphoproliferative disorders of NK cells

International audienceDistinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell expansion is challenging. We assessed the value of killer immunoglobulin-like receptor(KIR) phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (n = 46) and a reactive NK group (n = 68). We then developed an NK-cell clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the patients with CLPD-NK, constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK preferentially exhibited a CD16low phenotype, more frequently displayed a lower platelet count, and was associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed whole-exome sequencing of sorted, myeloid, T, and NK cells and found that TET2 mutations were shared by myeloid and NK cells in 3 of 4 cases. Thus, we hypothesized that TET2 alterations occur in early hematopoietic progenitors which could explain a potential link between CLPD-NK and myeloid malignancies. Finally, we analyzed the transcriptome by RNA sequencing of 7 CLPD-NK and evidenced 2 groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including 2 TET2-mutated cases, significantly underexpressed genes known to be downregulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK-cell proliferative disorders and, potentially, new therapeutic opportunities