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We evaluated the use of the turbidimetric HemosIL von Willebrand Factor (VWF) Activity assay (VWF:Act) on the STA-R automated coagulometer (Stago, Asnières, France) for the diagnosis of von Willebrand disease (VWD). For this, we prospectively screened 268 patients. As a second part, we retrospectively assayed 111 patients with well-defined VWD subtype. In the first prospective study, we demonstrate that in most cases of VWD, VWF ristocetin cofactor activity (VWF:RCo) and VWF:Act are highly correlated but that they both cannot be considered a good screening assay when used alone, since they could miss about 25% of VWF abnormalities. However, the association of VWF:Act analysis and the Platelet Function Analyzer-100 (PFA-100) test constitutes an excellent screening strategy. In our second retrospective study concerning VWD subtypes, VWF:RCo and VWF:Act were well correlated but could be very discrepant, especially for some cases of type 2M VWD. We consider that VWF:RCo remains the “reference assay” for VWD subtype classification.

Les leucemies aigues myeloides (LAM) constituent un groupe de cancers tres heterogene sur les plans genotypiques et phenotypiques. Cette heterogeneite se manifeste notamment par la disparite des taux et des durees de remission obtenus apres chimiotherapie. Le recours a l’allogreffe de cellules souches hematopoietiques est un reel benefice pour certains patients en termes de survie. A l’heure actuelle, la cytogenetique reste un element pronostique indispensable. Ces dernieres annees, les progres realises dans le domaine de la biologie moleculaire ont permis des avancees considerables sur la comprehension des processus de leucemogenese. Cette revue est une synthese de ces dernieres avancees qui permettent a a la fois d’affiner le pronostic des patients atteints de LAM, et d’assurer un suivi de plus en plus specifique et sensible de la maladie residuelle et d’identifier de nouvelles cibles therapeutiques. Nous proposons, en outre, un schema de depistage et du suivi des anomalies moleculaires qui peuvent influer sur la prise en charge therapeutique des patients.

We recently identified that the MEK/ERK1/2 pathway synergized with retinoic acid (RA) to restore both transcriptional activity and RA-induced differentiation in RA-resistant acute promyelocytic leukemia (APL) cells. To target the MEK/ERK pathway, we identified glycogen synthase kinase-3β (GSK-3β) inhibitors including lithium chloride (LiCl) as activators of this pathway in APL cells. Using NB4 (RA-sensitive) and UF-1 (RA-resistant) APL cell lines, we observed that LiCl as well as synthetic GSK-3β inhibitors decreased proliferation, induced apoptosis and restored, in RA-resistant cells, the expression of RA target genes and the RA-induced differentiation. Inhibition of the MEK/ERK1/2 pathway abolished these effects. These results were corroborated in primary APL patient cells and translated in vivo using an APL preclinical mouse model in which LiCl given alone was as efficient as RA in increasing survival of leukemic mice compared with untreated mice. When LiCl was combined with RA, we observed a significant survival advantage compared with mice treated by RA alone. In this work, we demonstrate that LiCl, a well-tolerated agent in humans, has antileukemic activity in APL and that it has the potential to restore RA-induced transcriptional activation and differentiation in RA-resistant APL cells in an MEK/ERK-dependent manner.

BACKGROUND: Wilms tumor 1 (WT1) is a transcription factor that is overexpressed in most acute myeloid leukemias (AMLs). Recently, 2 groups reported that WT1 mutations occur in approximately 10% of normal karyotype AMLs and are an independent predictor of poor outcome in this subgroup of patients with AML. METHODS: The authors studied a cohort of 268 young adults (ages 15-50 years) with AML who were treated on the Acute Leukemia French Association 9802 trial. WT1 exon 7 and 9 mutations were screened retrospectively by polymerase chain reaction and direct sequencing. The patients also were assessed for the presence of the fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD), FLT3-D835/I836, nucleophosmin 1 (NPM1), and CCAAT/enhancer binding protein α (CEBPA) mutations. RESULTS: WT1 mutations were identified in 14 patients (5%) and were associated with a younger age (P = .02) and an FLT3-ITD (P = .03). No mutation was detected in patients who had favorable cytogenetics. Patients who had WT1 mutations had a shorter overall survival at 4 years (22% vs 56%; P = .01) and a higher risk of recurrence at 4 years (82% vs 46%; P = .0008) compared with patients who had wild-type WT1. Within the subgroup of patients who had normal karyotype AML (n = 106), WT1 mutation was identified as an independent adverse prognostic factor for the risk of recurrence. CONCLUSIONS: The current results indicted that WT1 mutations represent an adverse prognostic factor in young adults with AML. Prospective trials should confirm the clinical relevance of WT1 mutations in relation to other prognostic factors in patients with AML. Cancer 2009. © 2009 American Cancer Society.

Introduction: The impact of gene mutations, i.e. poor-prognosis FLT3 internal tandem duplications (ITDs) and good-prognosis NPM1 or CEBPA mutations, has been welldocumented in several recent reports dealing with younger patients with acute myeloid leukemia (AML). As these mutations were associated with cytogenetically normal (CN) AML, most of these reports focused on CN-AML patients. Both FLT3-ITD and NPM1 mutations were also associated with higher WBC. The objective of the present study was to evaluate the incidence, correlations, and prognostic value of these mutations in older patients with the disease. Methods: The French ALFA group has screened a total of 583 patients, including 333 younger patients (15–50 years) treated in the ALFA-9802 trial and 250 older patients (50–70 years) treated in the ALFA-9801 trial. The older ALFA-9801 trial included 468 patients with previously untreated de novo AML and studied the role of idarubicin (IDA) as compared to high-dose daunorubicin (DNR) as well as interleukine-2 as a maintenance therapy (C. Pautas et al. ASH 2007, abstract #162). Comparison between the 250 patients tested for mutations in that trial and the 218 patients not tested showed no differences in age, sex ratio, FAB classification, bone marrow blasts percentage, randomization arm, and performance status at entry in the study. There was, however, a higher rate of patients with intermediate cytogenetics (p=.01) or increased WBC (p=.01) in the former subgroup. Results: Median age of the 250 patients tested was 60 years. Cytogenetics was studied in 232 patients (12 favorable, 174 intermediate, 46 unfavorable). One hundred twenty-two patients (49%) had CN-AML. CR rate was 67.5% and estimated 4-year OS was 26% (95% CI, 20–33). Incidences of FLT3-ITD, NPM1, and CEBPA mutations were 37/250 (15%), 64/249 (26%), and 20/249 (8%), respectively. These incidences were very similar than in the younger ALFA-9802 population [50/329 (15%), 76/321 (24%), and 24/316 (8%), respectively]. In these older AML patients, the correlation between increased WBC and FLT3-ITD (p Conclusion: This study conducted in a large cohort of patients aged 50 to 70 years and prospectively treated in the same trial showed that gene mutational status still affect the outcome of older patients with AML. Mutation incidences are in the same range than in younger patients. Nevertheless, their impact on OS appeared to be less marked than in younger patients, probably due to the worse general outcome observed in these older patients.

Abstract 2614 We previously demonstrated that although retinoic acid (RA) has targeted efficacy in Acute Promyelocytic Leukemia (APL), heterogeneity exists leading to the appearance of un-targeted clones at the time of relapse. Characterization of these clones is not yet fully unraveled though we and others have previously highlighted the roles of RARα mutations, pharmacogenomics or APL miRNome. We recently identified that the ERK1/2 pathway synergized with RA to restore the transcriptional activity of RA in resistant APL cells, thus restoring RA induced differentiation (Cassinat et al. Mol Cell Biol 2011). These results suggest that targeting interconnected signaling pathways could optimize differentiation therapy efficacy. To this effect, we studied known signaling pathway activators or inhibitors that could potentiate with RA and identified Lithium chloride (LiCl). Treatment of the ATRA sensitive-APL NB4 cell line with LiCl (25mM) decreases proliferation and increases apoptosis (25% and 40% of Annexin V-positive cells at day 1 and 2 respectively) with evidence of caspase 3 cleavage at day 2. Because NB4 cells fully differentiated with RA alone we were unable to observe any synergy when combined with LiCl. Treatment of the RA-resistant APL UF-1 cell line with RA or LiCl alone does not induce differentiation. Combination of RA+LiCl restores differentiation after 3 days of culture (65% CD11b positive and 55% NBT test positive cells). Similar results were obtained with different GSK3 inhibitors, suggesting that the LiCL effects were in part linked to its well characterized GSK3 inhibitory activity. Interestingly, we noted that LiCl treatment induces rapid phosphorylation of ERK1/2 and pretreatment with the MEK/ERK1/2 inhibitor UO126 fully abolished the differentiation induced by the RA+LiCl combination. The combination restores in UF-1 the expression of RA target genes (such as RARα2) to the same levels obtained in NB4 cells treated by RA alone. The level of luciferase activity of an RA responsive element reporter gene was increased with the RA+LiCl combination compared to RA alone. Both target gene expression and luciferase activiy were abolished after inhibition of the MEK/ERK1/2 pathway. Thus, increase in differentiation of UF-1 cells by RA+LiCl is linked to increased RA transcriptional activation. Similar studies in fresh APL patient cells confirmed both the increase in differentiation and level of RA target gene expression and their inhibition by UO126. Finally, to translate these findings in vivo, we used the APL-transplantable mouse model. Plasma lithium levels in treated mice were measured between 0.6 and 1.05 mmol/l, levels reached in humans. When LiCl was combined with RA we repeatedly observed a pronounced survival advantage compared to mice treated by RA alone as evaluated by Kaplan Meier analysis. In this work we demonstrate that LiCl, a well tolerated agent in humans, has the potential, when combined with RA, to restore RA induced transcriptional activation and differentiation in RA resistant APL cells. Furthermore, this combination also increases RA efficacy in an in vivo APL mouse model. Disclosures: Off Label Use: Lithium is a mood modulator administered for bipolar disorders.