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Background A better understanding of how the care of acute leukemia patients is managed in the last days of life would help clinicians and health policy makers improve the quality of end-of-life care. This study aimed: (i) to describe the intensity of end-of-life care among patients with acute leukemia who died in the hospital (2017–2018) and (ii) to identify the factors associated with the intensity of end-of-life care. Methods This was a retrospective cohort study of decedents based on data from the French national hospital database. The population included patients with acute leukemia who died during a hospital stay between 2017 and 2018, in a palliative care situation (code palliative care Z515 and-or being in a inpatient palliative care support bed during the 3 months preceding death). Intensity end-of-life care was assessed using two endpoints: High intensive end-of-life (HI-EOL: intensive care unit admission, emergency department admission, acute care hospitalization, intravenous chemotherapy) care and most invasive end-of-life (MI-EOL: orotracheal intubation, mechanical ventilation, artificial feeding, cardiopulmonary resuscitation, gastrostomy, or hemodialysis) care. Results A total of 3658 patients were included. In the last 30 days of life, 63 and 13% of the patients received HI-EOL care and MI-EOL care, respectively. Being younger, having comorbidities, being care managed in a specialized hospital, and a lower time in a palliative care structure were the main factors associated with HI-EOL. Conclusions A large majority of French young adults and adults with acute leukemia who died at the hospital experienced high intensity end-of-life care. Identification of factors associated with high-intensity end-of-life care, such as the access to palliative care and specialized cancer center care management, may help to improve end-of-life care quality.

Purpose: data on the interest of hypomethylating agents in pediatric hematologic malignancies are missing. Administration of Azacitidine (Aza), regarding indication, efficacy and toxicity, in a cohort of children and young adults, has been studied. Methods: we retrospectively collected data of patients aged under 25 years old, treated with Aza in 3 university hospitals, between 2009 and 2019. Results: 32 patients (median age = 11.7 y), treated with 35 courses of Aza, have been included. Diagnosis were: 16 acute myeloid leukemias (AML), 6 juvenile myelomonocytic leukemias (JMML), 7 myelodysplastic syndromes (MDS), 2 mixed-type acute leukemias (AL), and 1 sarcoma. 137 cycles have been administrated and were well tolerated. At the end of course 23/35 treatments with Aza had failed. 24/32 patients died, and overall survival at 5 years was 23%, IC95% [11 ;48]. No difference on overall survival according to diagnosis has been observed (p=0,85). Four patients with refractory AML achieved remission prior to bone marrow transplant and were alive in complete remission (median follow-up = 29.5 months). 3/6 AL treated with Sorafenib and Aza, and 2/3 JMML with PTPN11 mutation have presented a response. 1 MDS presented an extended stable disease after palliative treatment with Aza. Conclusion: Aza was well tolerated, but overall outcome was poor, probably due to the fact that most of the patients have been heavily pretreated. Further international prospective investigations are needed in order to confirm some of the results, and better define pediatric patients who might benefit from hypomethylating agents, in particular in combination with kinase inhibitors or proapoptotic agents.; Objectif : il existe peu de données sur l’intérêt des agents hypométhylants dans les hémopathies malignes de l’enfant. Nous avons étudié l’indication, l’efficacité et la tolérance de l’Azacitidine (Aza) dans une cohorte d’enfants et jeunes adultes. Méthodes : les données de patients (âge = 25 ans) traités par Aza dans 3 centres d’hématologie, entre 2009 et 2019, ont été recueillies rétrospectivement. Résultats : 32 patients (âge médian = 11,7 ans), ayant reçu 35 cycles d’Aza, ont été inclus. Les pathologies comprenaient : 16 leucémies aiguës myéloïdes (LAM), 6 leucémies myélomonocytaires juvéniles (LMMJ), 7 syndromes myélodysplasiques (SMD), 2 leucémies aiguës biphénotypiques et 1 sarcome. Les 137 cures d’Aza administrées ont été bien tolérées. 23/35 traitements se sont soldés par un échec. 24/32 patients sont décédés et la survie globale à 5 ans était de 23% IC95% [11 ; 48], sans différence selon la pathologie (p = 0,85). 4 patients avec une LAM réfractaire d’emblée ont été greffés et étaient vivants en rémission à 29,5 mois de recul médian. 3/6 LA traitées par Sorafénib et Aza, et 2/3 LMMJ avec mutation de PTPN11 ont présenté une réponse. 1 SMD en traitement palliatif a présenté une maladie stable prolongée. Conclusion : la tolérance de l’Aza a été globalement bonne mais son efficacité limitée, probablement en partie à cause du profil des patients déjà lourdement traités. Des études prospectives internationales sont nécessaires pour confirmer certains résultats et identifier les patients pédiatriques qui pourraient bénéficier des agents hypométhylants, en particulier en combinaison avec d’autres agents comme les inhibiteurs de kinase ou les agents proapoptotiques.

International audience; Transcriptional deregulation caused by epigenetic or genetic alterations is a major cause of leukemic transformation. The Spi1/PU.1 transcription factor is a key regulator of many steps of hematopoiesis, and limits self-renewal of hematopoietic stem cells. The deregulation of its expression or activity contributes to leukemia, in which Spi1 can be either an oncogene or a tumor suppressor. Herein we explored whether cellular senescence, an anti-tumoral pathway that restrains cell proliferation, is a mechanism by which Spi1 limits hematopoietic cell expansion, and thus prevents the development of leukemia. We show that Spi1 overexpression triggers cellular senescence both in primary fibroblasts and hematopoietic cells. Erythroid and myeloid lineages are both prone to Spi1-induced senescence. In hematopoietic cells, Spi1-induced senescence requires its DNA-binding activity and a functional p38MAPK14 pathway but is independent of a DNA-damage response. In contrast, in fibroblasts, Spi1-induced senescence is triggered by a DNA-damage response. Importantly, using our well-established Spi1 transgenic leukemia mouse model, we demonstrate that Spi1 overexpression also induces senescence in erythroid progenitors of the bone marrow in vivo before the onset of the pre-leukemic phase of erythroleukemia. Remarkably, the senescence response is lost during the progression of the disease and erythroid blasts do not display a higher expression of Dec1 and CDKN1A, two of the induced senescence markers in young animals. These results bring indirect evidence that leukemia develops from cells which have bypassed Spi1-induced senescence. Overall, our results reveal senescence as a Spi1-induced anti-proliferative mechanism that may be a safeguard against the development of acute myeloid leukemia.

HIV (human immunodeficiency virus) infected patients can develop tumor or infectious complications. Data on HIV patients (HIVP) with acute leukemia (AL, acute myeloid leukemia (AML) or non Burkitt acute lymphoid leukemia (ALL)) are very poor especially on their outcome. Our aim was to precise the epidemiology, the best therapeutic strategies as well as patient’s prognostics, and to compare their outcome to those of seronegative patients with AL. Our retrospective, national, multicentric study shows that AL incidence is not different than in general population but AL occurred earlier (49.29 years [44.21 ; 57.47]) and secondary AL are more frequent (42.55%). With a global and multidisciplinary approach these patients can be treated with intensive chemotherapy resulting on good efficiency (complete remission (CR)=84.38%)) and tolerance. Based on a multivariable model, only absence of CR was associated with hazard of death (p=0.01). HIVP’s 2 years overall survival (OS) was 29% CI95% [15 ; 54] for AML and 40% CI95% [14 ; 100] for ALL. There was no difference in OS between our HIVP and seronegative controls with AL after propensity score matching (HR=1.347 [0.6486-2.796]; p=0,42). Patients were completely matched except for HIV status so HIV status has no prognostic impact on AL patient’s outcome. HIVP with acute leukemia should thus be included in clinical trials to improve and standardize their therapeutic management.; Les patients infectés par le virus d’immunodéficience humaine (VIH) peuvent développer des complications infectieuses ou tumorales. Peu de données existent sur les patients VIH+ (PVVIH) ayant une leucémie aiguë (LA, LA myéloïde (LAM) ou LA lymphoïde (LAL) non Burkitt) et notamment sur leur devenir. Notre objectif était de préciser l’épidémiologie, la prise en charge thérapeutique optimale ainsi que le pronostic de ces patients et de comparer leur devenir à celui de patients séronégatifs ayant une LA. Notre étude rétrospective, nationale, multicentrique montre que l’incidence des LA n’est pas différente de celle de la population générale mais que les LA surviennent plus précocement (49,29 ans [44,21 ; 57,47]) et qu’il y a plus de LA secondaires (42,55%). Pris en charge de façon globale et pluridisciplinaire ces patients peuvent être traités par chimiothérapie intensive efficacement (rémission complète (RC)=84,38%) et avec une bonne tolérance. Le seul facteur influençant le risque de décès après analyse multivariée était l’absence de RC (p=0,01). La survie globale à 2 ans des PVVIH ayant une LAM était de 29% IC95% [15 ; 54] et de 40% IC95% [14 ; 100] pour les LAL. Il n’a pas été retrouvé de différence de survie globale (HR=1,347 [0,6486- 2,796]; p=0,42) entre nos PPVIH et leurs témoins séronégatifs ayant une LA appariés via un score de propension excepté pour le statut VIH. Le statut VIH n’a donc pas d’impact pronostique sur ledevenir des patients ayant une LA. Les PVVIH atteints de LA devraient ainsi, comme les patients séronégatifs, être inclus dans les essais cliniques afin d’améliorer et d’harmoniser leur prise en charge.

International audience; Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3β (GSK3β) by serine 9-dephosphorylation. Strikingly, GSK3β-mediated survival was restricted to leukemic progenitors from female patients. GSK3β inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)β(1)-integrin engagement, was specifically upregulated and controlled GSK3β activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3β-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3β as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

International audience; OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.

MHC-matched hemopoietic stem cell transplantation is commonly used for the treatment of some forms of leukemia. Conditioning regimens before transplant act to reduce the burden of leukemic cells and the graft-vs-leukemia (GvL) effect can eliminate residual disease. The GvL effect results largely from the recognition of minor histocompatibility Ags by donor T cells on recipient tissues. These Ags are generally widely expressed and also provoke graft-vs-host (GvH) disease. Manipulation of immunity to promote GvL while curtailing GvH would greatly improve clinical outcome. To develop strategies that may achieve this, the parameters which control immunity to minor histocompatibility Ags need to be defined. In this study, we have analyzed responses to the mouse HY minor histocompatibility Ag using hemopoietic cell and skin grafts as surrogate GvL and GvH targets, respectively. We show that natural regulation of CD8 T cell responses to HY operates at multiple levels. First, CD4 T cell help is required for primary CD8 responses directed at hemopoietic cells. However, although CD4 T cells of H2k mouse strains recognize HY, they provide ineffective help associated with a proportion of recipients developing tolerance. This was further investigated using TCR-transgenic mice which revealed H2k-restricted HY-specific CD4 T cells are highly susceptible to regulation by CD25+ regulatory T cells which expand in tolerant recipients. A second level of regulation, operating in the context of skin grafts, involves direct inhibition of CD8 T cell responses by CD94/NKG2 engagement of the nonclassical MHC class I molecule Qa1.

BCR ABL; International audience; BCR-ABL induces an intrinsic genetic instability in chronic myeloid leukemia (CML). The protein breast cancer 1, early onset (BRCA1)-associated protein 1 (BAP1) is a deubiquitinase interacting with the DNA repair regulator BRCA1 and is frequently inactivated in many cancers. Here, we report that BAP1 mRNA and protein levels are downregulated in a BCR-ABL1-expressing hematopoietic cell line (UT-7/11). A decrease of BAP1 transcripts is also observed in newly diagnosed CML patients. Moreover, BAP1 protein levels are low or undetectable in CD34(+) cells from CML patients at diagnosis as compared with CD34(+) cells from normal donors. In addition, BRCA1 protein level is reduced in BCR-ABL1-expressing UT-7/11 cells. Finally, the enforced expression of BAP1 is associated with BRCA1 protein deubiquitination and restoration. These results demonstrate BAP1 as a major link with the BCR-ABL-induced downregulation of BRCA1 in CML.

// Stephane Vispe 1 , Arthur Deroide 1 , Emeline Davoine 1 , Cecile Desjobert 1 , Fabrice Lestienne 2 , Lucie Fournier 1 , Natacha Novosad 1 , Sophie Breand 3 , Jerome Besse 3 , Florence Busato 4 , Jorg Tost 4 , Luc De Vries 2 , Didier Cussac 2 , Joelle Riond 1 , Paola B. Arimondo 1 1 Unite de Service et de Recherche n°3388 CNRS-Pierre Fabre, ETaC Epigenetic Targeting of Cancer, CRDPF, Toulouse, France 2 Molecular and Cellular Biology Department, Centre de Recherche Pierre Fabre, Castres, France 3 Informatique de Recherche (Bioinformatics and Statistics), Centre de Recherche Pierre Fabre, Castres, France 4 Laboratory for Epigenetics and Environment, Centre National de Genotypage, CEA-Institut de Genomique, Evry, France Correspondence to: Stephane Vispe, e-mail: stephane.vispe@pierre-fabre.com Paola B. Arimondo, e-mail: paola.arimondo@etac.cnrs.fr Keywords: leukemia, DNA methylation, DNMT, 5-aza-2’-deoxycytidine, DNA damage Received: December 28, 2014 Accepted: February 08, 2015 Published: March 20, 2015 ABSTRACT 5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.

International audience; Human leukocyte antigen G (HLA-G) molecules exhibit immunomodulatory properties corresponding to nonclassic class I genes of the major histocompatibility complex. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membrane-bound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-gamma in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.

International audience; OBJECTIVES: To investigate the relation between childhood acute leukaemia and household exposure to pesticides. METHODS: The study included 280 incident cases of acute leukaemia and 288 controls frequency matched on gender, age, hospital, and ethnic origin. The data were obtained from standardised face to face interviews of the mothers with detailed questions on parental occupational history, home and garden insecticide use, and insecticidal treatment of pediculosis. Odds ratios were estimated using unconditional regression models including the stratification variables parental socioeconomic status and housing characteristics. RESULTS: Acute leukaemia was observed to be significantly associated with maternal home insecticide use during pregnancy (OR = 1.8, 95% CI 1.2 to 2.8) and during childhood (OR = 1.7, 95% CI 1.1 to 2.4), with garden insecticide use (OR = 2.4, 95% CI 1.3 to 4.3), and fungicide use (OR = 2.5, 95% CI 1.0 to 6.2) during childhood. Insecticidal shampoo treatment of pediculosis was also associated with childhood acute leukaemia (OR = 1.9, 95% CI 1.2 to 3.3). CONCLUSION: The results reported herein support the hypothesis that various types of insecticide exposure may be a risk factor for childhood acute leukaemia. The observed association with insecticidal shampoo treatment of pediculosis, which has never been investigated before, requires further study.

International audience; INTRODUCTION: We investigated the role of maternal alcohol and coffee drinking and parental smoking on the risk of childhood acute leukemia in a multicenter case-control study. METHODS: The study included 280 incident cases and 288 hospitalized controls, frequency matched with the cases by age, gender and center. Data collection was completed by face-to-face standardized interviews of the case and control mothers. RESULTS: An association with maternal alcohol consumption during pregnancy was observed with acute lymphoid leukemia (ALL) (OR=2.0 [1.4-3.0]) and acute non-lymphoid leukemia (ANLL) (OR=2.6 [1.2-5.8]). Maternal coffee consumption during pregnancy was associated with childhood acute leukemia, ORs increasing in ALL with coffee consumption (OR=1.1 [0.7-1.8], OR=2.4 [1.3-4.7] and OR=3.1 [1.0-9.5], respectively, for < or =3, 4-8 and >8 cups/day). No association with maternal smoking during pregnancy or parental smoking before or after the index child's birth was observed. DISCUSSION: Our results suggest an association with maternal alcohol and coffee drinking during pregnancy and call for further investigations. Besides, the present study does not support the hypothesis of an increase in the risk of childhood leukemia related to parental smoking.

Motivation: The most used criterion in microarray data analysis is nowadays the false discovery rate (FDR). In the framework of estimating procedures based on the marginal distribution of the P-values without any assumption on gene expression changes, estimators of the FDR are necessarily conservatively biased. Indeed, only an upper bound estimate can be obtained for the key quantity π0, which is the probability for a gene to be unmodified. In this paper, we propose a novel family of estimators for π0 that allows the calculation of FDR. Results: The very simple method for estimating π0 called LBE (Location Based Estimator) is presented together with results on its variability. Simulation results indicate that the proposed estimator performs well in finite sample and has the best mean square error in most of the cases as compared with the procedures QVALUE, BUM and SPLOSH. The different procedures are then applied to real datasets. Availability: The R function LBE is available at http://ifr69.vjf.inserm.fr/lbe Contact: broet@vjf.inserm.fr

International audience; Plasmacytoid dendritic cell (PDC) leukemia/lymphoma is a rare neoplasm presenting cutaneous lesions at the time of diagnosis, followed by dissemination to bone marrow, lymph nodes, and other lymphoid and nonlymphoid organs. Since these leukemic counterparts of human PDC are similar to normal PDC, we studied their chemokine receptor equipment and their migratory capacities. We found both in skin lesions and in invaded lymph nodes an expression by tumor cells of CXCR3, CXCR4, and CCR7, and the concomitant expression by cells in the microenvironment of their respective ligands CXCL9, CXCL12, and CCL19. Moreover, flow cytometry phenotype of leukemic PDC (LPDC) revealed an unexpected expression of CCR6. We show that fresh tumor cells are able to migrate in response to CXCR4, CCR2, CCR5, CCR6, and CCR7 ligands, and the ability of CXCR3 ligands to increase the responsiveness to CXCL12. IL-3- or virus-induced activation of LPDC leads to downregulation of CXCR3 and CXCR4, and upregulation of CCR7, associated with the loss of response to CXCL12, and the acquisition of sensitivity to CCL19. Altogether, these results suggest that the preferential accumulation of LPDC in the skin or lymph nodes could be orchestrated by CXCR3, CXCR4, CCR6, and CCR7 ligands, found in nontumoral structures of invaded organs.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

International audience; Diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) or plasmacytoid dendritic cell leukemia (pDCL) is mainly based on immunophenotypical characterization of leukemic cells in blood or bone marrow samples. We tested by flow cytometry intracellular expression of the proto-oncogene T-cell leukemia 1 (TCL1), as well as membrane and intracellular expression of immunoglobulin-like transcript 7 (ILT7) in 21 pDCL samples and 61 non-pDC acute leukemia samples [i.e., 14 B-acute lymphoblastic leukemia (B-ALL), 9 T-ALL and 38 acute myeloid leukemia (AML)]. TCL1 is highly expressed in all pDCL samples while at a statistically lower level in all B-ALL and 34% of AML. Statistical analysis shows that intensity of TCL1 expression is a good marker for differential diagnosis of pDCL versus other acute leukemia (area under the receiver-operating characteristic curve, [AUC]: 0.96). By contrast, ILT7 positivity is limited to few pDCL samples and cannot be useful for diagnosis purpose. In conclusion, high intracellular intensity of TCL1 expression is currently the best marker for pDC lineage assignment by flow cytometry, which is particularly useful to distinguish pDCL from CD4(+) CD56(+/-) undifferentiated or monoblastic acute leukemia. Thus, intracellular TCL1 detection should be included in acute leukemia diagnosis panels used in hematology laboratories. © 2012 International Society for Advancement of Cytometry.

International audience; Here we describe a protocol established in our laboratory to quantify early common progenitors/stem cells grown as spheres in non-adherent culture conditions. This protocol is based on the combination of two functional tests: the mammosphere assay to maintain and enrich early mammary progenitors/stem cells, and the epithelial colony-forming cells (E-CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH-3T3 fibroblasts for further processing, as in the E-CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named ECP-DC, standing for Early Common Progenitor-Derived Colonies assay.

The identification of the causative organism in invasive pulmonary aspergillosis (IPA) is recommended. We investigated whether a mycologic diagnostic strategy could be optimized based on patient characteristics. Fifty-five patients were enrolled in a prospective study. The presence of Aspergillus in respiratory samples occurred more frequently in non-acute leukemia (AL) patients than in AL patients (P = .0003), and in patients with leukocyte counts more than 100/mm3 (P = .002). In a logistic regression model, these 2 factors appeared to be independent, with an adjusted odds ratio of 7.14 (95% confidence interval, 1.40-36.5) for non-AL patients and an adjusted odds ratio of 6.97 (95% confidence interval, 1.33-36.5) for patients with leukocyte counts more than 100/mm3. A positive mycologic result was also more frequent among patients with lung CT scan signs of airway-invasive disease than among other patients (P = .043). Airway-invasive signs were more frequent among non-AL patients (P = .049), whereas angioinvasive disease was more frequent among both AL patients (P = .01) and patients with leukocyte counts less than 100/mm3 (P = .001). A concomitant pulmonary infection was identified more frequently among non-AL patients (P = .005 vs allogeneic hematopoietic stem cell transplant and P = .048 vs others). Our results suggest that different strategies for diagnosing IPA should be considered based on the underlying condition.

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10). Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The 1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3- one 4 (EC50 vs EZH2 = 74.9 μM), tested in U937 cells at 50 μM, induced massive cell death and 28% of granulocytic differentiation, highlighting the potential use of EZH2 inhibitors in cancer. © 2012 Elsevier Masson SAS. All rights reserved.

International audience; Diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) or plasmacytoid dendritic cell leukemia (pDCL) is mainly based on immunophenotypical characterization of leukemic cells in blood or bone marrow samples. We tested by flow cytometry intracellular expression of the proto-oncogene T-cell leukemia 1 (TCL1), as well as membrane and intracellular expression of immunoglobulin-like transcript 7 (ILT7) in 21 pDCL samples and 61 non-pDC acute leukemia samples [i.e., 14 B-acute lymphoblastic leukemia (B-ALL), 9 T-ALL and 38 acute myeloid leukemia (AML)]. TCL1 is highly expressed in all pDCL samples while at a statistically lower level in all B-ALL and 34% of AML. Statistical analysis shows that intensity of TCL1 expression is a good marker for differential diagnosis of pDCL versus other acute leukemia (area under the receiver-operating characteristic curve, [AUC]: 0.96). By contrast, ILT7 positivity is limited to few pDCL samples and cannot be useful for diagnosis purpose. In conclusion, high intracellular intensity of TCL1 expression is currently the best marker for pDC lineage assignment by flow cytometry, which is particularly useful to distinguish pDCL from CD4(+) CD56(+/-) undifferentiated or monoblastic acute leukemia. Thus, intracellular TCL1 detection should be included in acute leukemia diagnosis panels used in hematology laboratories. © 2012 International Society for Advancement of Cytometry.

There is a paucity of information regarding the factors that affect nonrelapse mortality (NRM) and overall survival among children that develop chronic graft-versus-host disease (cGVHD). We performed multivariate analyses using data from the Center for International Blood and Marrow Transplant Research to identify risk factors for NRM and survival in 1117 pediatric subjects with leukemia or myelodysplastic syndrome, transplanted from related donors, unrelated donors (URD), or unrelated cord blood between 1995 and 2004. We identified 4 variables associated with higher NRM: HLA partially matched or mismatched URD, peripheral blood cell graft, Karnofsky/Lansky score < 80 at cGVHD diagnosis, and platelets < 100 × 109/L at cGVHD diagnosis. Factors associated with significantly worse survival were: age > 10 years, transplantation from HLA partially matched or mismatched URD, advanced disease at transplantation, Karnofsky/Lansky < 80; and platelets < 100 × 109/L. Cumulative incidence of discontinuation of systemic immune suppression at 1, 3, and 5 years after diagnosis of cGVHD were 22% (20%-25%), 34% (31%-37%), and 37% (34%-40%), respectively. This is the largest study elucidating variables affecting outcome after diagnosis of cGVHD in pediatric allograft recipients. These variables may be useful for risk stratification, development of future clinical trials, and family counseling in children with cGVHD.

International audience; Allogeneic haematopoietic stem-cell transplantation (HSCT) is the only curative treatment for myelofibrosis. We retrospectively analyzed the outcome of patients who underwent allogeneic HSCT, 1994-2008, and the potential risk factors affecting non-relapse mortality (NRM), OS and relapse-free survival (RFS). A total of 39 patients, 15-65 (median 49) years old, diagnosed with primary (n=27) or secondary (n=12) myelofibrosis underwent HSCT (25 related and 14 unrelated). In ten patients, disease had transformed into acute leukaemia. Lille prognosis score was low for 9, intermediate for 16 and high for 14 patients. The conditioning regimen was myeloablative (MAC) for 15 and reduced-intensity (RIC) fludarabine-based for 24, with successful engraftment in 38 patients. A total of 31 patients developed grade I-IV GvHD; 19 developed chronic GvHD. The 3-year OS, RFS and NRM rates (95% confidence interval) were 60% (42-74), 54% (37-59) and 30% (30-45), respectively.

International audience; BACKGROUND: Acute myeloblastic leukaemia (AML) patients are at high risk of suffering from invasive fungal infections (IFI). Posaconazole demonstrated higher efficacy than standard azole agents (SAA) in the prophylaxis of IFI in this population. The authors estimated the cost effectiveness of posaconazole versus SAA in France. METHODS: A decision-tree model was developed to compare posaconazole with SAA with the results of a published clinical trial. Clinical events were modelled with chance nodes reflecting probabilities of IFI, IFI-related death, and death from other causes. Medical resource consumption and costs were obtained from results of the clinical trial and from a dedicated survey on the costs of treating IFI using a retrospective chart review design. RESULTS: IFI treatment costs were estimated using medical files from 50 AML patients from six French centres, with a proven and probable IFI, who had been followed-up for 298 days on average. Direct costs directly related to IFI were estimated at €51,033, including extra costs of index hospitalisation, costs of antifungal therapy and additional hospitalisations related to IFI treatment. The model indicated that the healthcare costs for the posaconazole strategy were €5,223 (€2,697 for prophylaxis and €2,526 for IFI management), which was €859 less than the €6,083 in costs with SAA (€469 for prophylaxis and €5614 for IFI management). A sensitivity analysis indicated that there was an 80% probability that prophylaxis using the posaconazole strategy would be superior. CONCLUSION: The findings from this analysis suggest that posaconazole use is a clinically and economically dominant strategy in the prophylaxis of IFI in AML patients, given the usual limits of economic models and the uncertainty of costs estimates.

International audience; BACKGROUND: There is limited information regarding the efficacy and long term safety of intrathecal injection of liposomal cytarabine in leukemic or lymphomatous meningitis. DESIGN AND METHODS: We studied 20 consecutive HIV-negative patients with leukemic or lymphomatous meningitis who were treated with intrathecal liposomal cytarabine between 2004 and 2007. We focused on efficacy and on any late effects of the drug. RESULTS: Twenty patients who received intrathecal liposomal cytarabine injection as part of their treatment; of these, 9 were alive and in complete remission at the end of the study. Median survival from the time of the first injection was 22.7 months (range, 0.5 to 64 months). Short-term toxicity related to intrathecal of liposomal cytarabine was observed in 2 cases; headache in 1 case and regressive facial palsy and diplopy in 1 case. Long-term toxicity was seen in 2 cases; clinical symptoms were urinary and fecal dysfunction with confusion in 1 case, and urinary dysfunction in 1 case. Both patients had been heavily pre-treated with neurotoxic drugs and neuraxis irradiation. CONCLUSION: In our experience, intrathecal liposomal cytarabine injections were convenient in the management of leukemic and lymphomatous meningitis, and can lead to long-term survival. Although neurotoxicity was rare, clinicians should exercise caution when retreatment is required in relapsing patients.

To the editor: Lymphocytes from granulocyte colony-stimulating factor (G-CSF)–mobilized donors display epigenetic and genetic alterations similar to those observed in leukemia patients.[1][1] To further evaluate the scope and duration of G-CSF–induced genetic alterations, 24 healthy donors were

International audience; INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

International audience; The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4(+) CD56(+) lineage(neg) CD45RA(+)/RO(neg) CD11c(neg) CD116(low) CD123(+) CD34(neg) CD36(+) HLA-DR(+). Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC - lymphoid or myeloid - acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4(+) CD56(+/-) MPO(neg) cCD3(neg) cCD79a(neg) CD11c(neg) profile and then the CD123(high), BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL.

Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA and PLZF/RARA fusion proteins through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is still poorly understood. Here we show that, like PML/RARA, PLZF/RARA expression leads to recruitment of the Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements. Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. We further show that Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1 in PLZF/RARA-mediated myeloid leukemic development.

International audience; Bis(tbutyl-S-acyl-2-thioethyl)-cytidine monophosophate is a new cytotoxic mononucleotide prodrug which have been developed to reverse the cellular resistance to nucleoside analogues. Unfortunately, its in vivo utilisation was hampered by its poor water solubility, raising the need of a molecular vector capable to mask its physicochemical characteristics although without affecting its cytotoxic activity. Hydroxypropyl-beta-cyclodextrin was used to prepare the prodrug inclusion complexes, allowing it to be solubilized in water and hence to be used for in vitro and in vivo experiments. A molar ratio of the cyclodextrin: prodrug of 3 was sufficient to obtain complete solubilization of the prodrug. The inclusion complex was characterized by differential scanning calorimetry, which revealed the disappearance of the melting peak of the prodrug suggesting the formation of inclusion complex. Proton Nuclear Magnetic Resonance spectroscopy provided a definitive proof of the inclusion complex formation, which was evidenced by the large chemical shift displacements observed for protons located in the interior of the hydrophobic cyclodextrin cavity. The complex retained its cytotoxic activity as shown by in vitro cell survival assays on murine leukemia cells. These results provided a basis for potential therapeutic applications of co-formulation of this new nucleotide analogue with hydroxypropyl-beta-CD in cancer therapy.

International audience; BACKGROUND: Extranodal natural killer (NK)/T-cell lymphoma, nasal type, and aggressive NK-cell leukemia are highly aggressive diseases with a poor outcome. PATIENTS AND METHODS: We report a multicentric French retrospective study of 15 patients with relapsed, refractory, or disseminated disease, treated with L-asparaginase-containing regimens in seven French centers. Thirteen patients were in relapse and/or refractory and 10 patients were at stage IV. RESULTS: All but two of the patients had an objective response to L-asparaginase-based treatment. Seven patients reached complete remission and only two relapsed. CONCLUSION: These data, although retrospective, confirm the excellent activity of L-asparaginase-containing regimens in refractory extranodal NK/T-cell lymphoma and aggressive NK-cell leukemia. Therefore, L-asparaginase-based regimen should be considered as a salvage treatment, especially for patients with disseminated disease. First-line L-asparaginase combination therapy for extranodal NK/T-cell lymphoma and aggressive NK-cell leukemia should be tested in prospective trials.

Purpose Acute graft-versus-host disease (GVHD) causes substantial morbidity and mortality after human leukocyte antigen (HLA)-identical sibling transplants. No large registry studies of acute GVHD risk factors have been reported in two decades. Risk factors may have changed in this interval as transplant-related techniques have evolved. Patients and Methods Acute GVHD risk factors were analyzed in 1,960 adults after HLA-identical sibling myeloablative transplant for acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), or chronic myeloid leukemia (CML) reported by 226 centers worldwide to the Center for International Blood and Marrow Transplant Research from 1995 to 2002. Outcome was measured as time from transplant to onset of grade 2 to 4 acute GVHD, with death without acute GVHD as a competing risk. Results Cumulative incidence of grade 2 to 4 acute GVHD was 35% (95% CI, 33% to 37%). In multivariable analyses, factors significantly associated with grade 2 to 4 acute GVHD were cyclophosphamide + total-body irradiation versus busulfan + cyclophosphamide (relative risk [RR] = 1.4; P < .0001), blood cell versus bone marrow grafts in patients age 18 to 39 years (RR = 1.43; P = .0023), recipient age 40 and older versus age 18 to 39 years receiving bone marrow grafts (RR = 1.44; P = .0005), CML versus AML/ALL (RR = 1.35; P = .0003), white/Black versus Asian/Hispanic race (RR = 1.54; P = .0003), Karnofsky performance score less than 90 versus 90 to 100 (RR = 1.27; P = .014), and recipient/donor cytomegalovirus-seronegative versus either positive (RR = 1.20; P = .04). Stratification by disease showed the same significant predictors of grade 2 to 4 acute GVHD for CML; however, KPS and cytomegalovirus serostatus were not significant predictors for AML/ALL. Conclusion This analysis confirmed several previously reported risk factors for grade 2 to 4 acute GVHD. However, several new factors were identified whereas others are no longer significant. These new data may facilitate individualized risk estimates and raise several interesting biologic questions.

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)–like receptors (KIR) ligand-mismatched donors. We showed that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype, as well as through phenotypic and functional analyses of NK cells, both at the polyclonal and clonal level. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether, these results may have important clinical implications for the selection of optimal donors for haplo-HSCT.

Background Prior to the era of immunomodulating or immunosuppressive (IS) treatments Multiple Sclerosis (MS) was linked to reduced rates of cancer. Method A descriptive study of MS patients with a documented oncological event was performed. From 1 January 1995 to 30 June 2006, we collected and studied the profile of 7418 MS patients gathered from nine French MS centers. We evaluated the incidence of cancer in a Cancer Risk In MS Cohort. Results Thirty one patients (1.75%) with confirmed MS had a history of cancer: mean age at MS diagnosis of 37.9 years and a mean age at cancer diagnosis of 46.4 years. The most frequent cancers were breast (34.5%), gynecological (12.5%), skin (10.2%), acute leukemia and lymphoma (5.9%), digestive (8.8%), kidney and bladder (5.1%), lung (3.4%) and central nervous system (3%). Calculated standardized incidence rates were 0.29 (0.17—0.45) for men and 0.53 (0.42—0.66) for women. The incidence of cancer in this MS population was lower than that expected for the general population. Matched to age, gender and histology, cancers in MS were associated with a young age and exposure to IS treatments. When considering all patients, treated patients had a 3-fold higher risk of developing cancer, if they had a history of IS ( P = 0.0035). For treated patients, the cancer sites were more likely the breast, the urinary tract, the digestive system and the skin. Conclusion Our data suggest that MS patients do not have an increased risk of cancer. Rather for several types of cancer a significantly reduced risk was observed, except for breast cancer in women treated with IS. The relative increased risk of breast cancer in MS women under IS treatment warrants further attention. Multiple Sclerosis 2008; 14: 399—405. http://msj.sagepub.com

International audience; BACKGROUND: Chronic disseminated candidiasis (CDC) is typically observed during neutrophil recovery in patients with acute leukemia and requires protracted antifungal therapy. OBJECTIVE: Our objective was to document the efficacy and tolerance of corticosteroid therapy (CST) in patients with symptomatic CDC, including those who experienced fever and abdominal pain despite ongoing antifungal therapy. METHODS: We performed a retrospective, multicenter study involving 10 pediatric and adult patients who experienced ongoing symptomatic CDC despite receipt of appropriate antifungal therapy for whom adjuvant oral CST was initiated. RESULTS: All cases of CDC were proven or probable, as determined on the basis of the European Organization for Research and Treatment of Cancer-Mycosis Study Group definition criteria, and occurred in patients with leukemia. CDC-attributable clinical symptoms resolved with CST, which was started a mean of 33.8 days after antifungal therapy had been initiated. Fever and abdominal pain disappeared a median of 4-5 days, and serum fibrinogen and C-reactive protein levels returned to normal values within 14-30 days. The median duration of hospitalization after CST initiation was 8.8 days. Hepatosplenic microabscesses decreased or disappeared within a mean period of 107 days (range, 30-210 days). No relapses of CDC were observed during a median duration of follow-up of 6.5 years (range, 4-9 years). CONCLUSIONS: In children and adults who experience persistently symptomatic CDC despite ongoing receipt of antifungal therapy, CST involving a prednisone equivalent at a dosage of > or =0.5 mg/kg per day for at least 3 weeks is associated with a prompt resolution of symptoms and of inflammatory response. These findings support the pathophysiological hypothesis that CDC belongs to the spectrum of fungus-related immune reconstitution inflammatory syndrome.

International audience; A case-control study was conducted to investigate the role of a familial history of cancer in the etiology of childhood acute leukemia. The history of cancer in the relatives of 472 cases was compared with that of 567 population-based controls. Recruitment was frequency matched on age, sex and region. The familial history of cancer in each child's relatives was reported by the mother in response to a standardized self-administered questionnaire. A familial history of solid tumor in first or second-degree relatives was associated with an increased risk of acute lymphoblastic leukemia (odds ratio (OR)=1.6 [95% confidence interval, 1.2-2.1]), while a familial history of hematopoietic malignancies in first or second-degree relatives was associated with an increased risk of acute myeloid leukemia (OR=4.3 [1.4-13]). The ORs for the histories of cancer increased with the number of relatives with cancer (OR=1.5 [1.1-2.0] for one relative and OR=2.3 [1.3-3.8] for two relatives or more; Ptrend

International audience; For several decades, organometallic and organometalloid compounds have played an important part in anticancer chemotherapies, in particular those derived from platinum. Trivalent arsenic, in the form of arsenic trioxide (As2O3; Trisenox(R)) is currently used in the treatment of refractory leukemias, but at the cost of major adverse effects. Moreover, recent studies showed that the trypanocide melarsoprol, could be more effective than arsenic trioxide on myelogenous leukaemias. We have synthesized a series of derivatives from 2-phenyl-[1,3,2]dithiarsolan-4-yl)-methanol (AsIII). Our work shows that the substitution of the aromatic ring by an iodine atom in the ortho position or by an amino-dimethoxytriazin group in the para position increases very significantly the antileukemic activity and improves the therapeutic index (IT=LD50/IC50) of these melarsoprol-derivatives molecules, as compared to arsenic trioxide. However, one of the most promising compounds seems to be arsthinol, an old drug used in the past as an amebicide. Nanoparticle carriers of melarsoprol were also prepared for the purpose of modifying its tissue distribution reduce its brain toxicity.

Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP / follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFΒ) superfamily such as activin A and myostatin. We have previously shown that TGFβ and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFΑ) could regulate FLRG expression since TNFΑ plays a critical role in hematopoietic malignancies. We demonstrate that TNFΑ activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not very common among non-coding sequences. Each DNA repeat contains one TNFΑ responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-ΚB) transcription factors. We also show that TGFΒ, through the Smad proteins, potentates the effect of TNFΑ on FLRG expression. This cooperation is unexpected since TGFΒ and TNFΑ usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis.

Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP / follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFΒ) superfamily such as activin A and myostatin. We have previously shown that TGFβ and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFΑ) could regulate FLRG expression since TNFΑ plays a critical role in hematopoietic malignancies. We demonstrate that TNFΑ activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not very common among non-coding sequences. Each DNA repeat contains one TNFΑ responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-ΚB) transcription factors. We also show that TGFΒ, through the Smad proteins, potentates the effect of TNFΑ on FLRG expression. This cooperation is unexpected since TGFΒ and TNFΑ usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis.

International audience; Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP/follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFbeta) superfamily such as activin A and myostatin. We have previously shown that TGFbeta and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFalpha) could regulate FLRG expression since TNFalpha plays a critical role in hematopoietic malignancies. We demonstrate that TNFalpha activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats, which are evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not common among non-coding sequences. Each DNA repeat contains one TNFalpha responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-kappaB) transcription factors. We also show that TGFbeta, through the Smad proteins, potentates the effect of TNFalpha on FLRG expression. This cooperation is unexpected since TGFbeta and TNFalpha usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis.

BACKGROUND: In 2002, a poster alerted the French health authorities to the possibility that the risk of childhood leukaemia might be increased by hepatitis B vaccination. Elucidating the role of vaccination in the aetiology of childhood acute leukaemia (AL) was therefore included in the objectives of an ongoing national study. METHODS: The ESCALE study was a French national population-based case-control study conducted in France in 2003 and 2004 in order to investigate the role of infectious, environmental and genetic factors in four childhood neoplastic diseases (leukaemia, lymphoma, neuroblastoma and brain tumour). The controls were randomly selected from the French population and age and gender frequency matched with the cases. A total of 776 cases of AL (91% of the eligible cases) and 1681 controls (71% of the eligible controls) were included. In a specific standardized telephone interview, which was the same for both the cases and controls, each mother was asked to read out her child's complete vaccination record. RESULTS: No association between vaccination and the risk of childhood AL: acute lymphoblastic leukaemia or acute myeloblastic leukaemia was observed. No relationship between the risk of leukaemia and the type of vaccine, number of doses of each vaccine, total number of injections, total number of vaccine doses or number of early vaccinations was evidenced. No confounding factor was observed. CONCLUSION: The study did not show any evidence of a role of vaccination in the aetiology of childhood leukaemia.

The aim of this study was to determine the therapy-related risk factors for the occurrence of leukaemia after childhood solid cancer. Among 4204 3-year survivors of a childhood cancer treated in eight French and British centres before 1986, 11 patients developed leukaemia as a second malignant neoplasm (SMN). Compared with the leukaemia incidence in the general French and British populations, the standardised incidence ratio (SIR) of leukaemia was 7.8 (95% CI 4.0-13.4). It decreased from 20.3 (95% CI 8.3-41.2) during the first years of follow-up, to 2.2 (95% CI 0.1-9.7) between 10 and 20 years, but rose again to 14.8 (95% CI 3.7-38.3) 20 or more years after the first cancer. Radiotherapy appeared to increase the risk of leukaemia at moderate weighted doses to active bone marrow; the relative risk (RR) was 4.2 (95% CI 0.8-20.7) for doses ranging from 3 to 6.6 Gy. A greater RR was observed for epipodophyllotoxins and for vinca alkaloids. No specific type of first malignant neoplasm (FMN) was found to lead to a higher risk of secondary leukaemia. Epipodophyllotoxins and vinca alkaloids at high doses and moderate weighted radiation doses to active bone marrow may contribute independently to an increased risk of leukaemia for patients treated for childhood cancer. Our results suggest that the long-term risk of secondary leukaemia could be higher than previously reported.

This study aimed to investigate the spatial and space-time distributions of cases of childhood acute leukaemia (CL) during 1990-2000 over the whole French territory. A global spatial heterogeneity and a spatial autocorrelation were first considered using the methods proposed by Potthoff and Whittinghill, Moran and Rogerson methods. The presence of space-time interaction between the places of residence and the dates of diagnosis was investigated with the Knox's test. Finally, the Kulldorff's statistic permitted to scan the whole territory in search for localised clusters. Two time periods were considered (1990-1994, 1995-2000). Overall, a statistically significant spatial heterogeneity of a very small magnitude was observed in the incidence of CL over 1990-1994, but neither over 1995-2000 nor over the whole time period. Moreover, a significant overdispersion of 5.5% was evidenced for 0-4 year children living in isolated areas with more than 50 inhabitants per km(2). Cases older than 10 years living in the same area at diagnosis also tended to cluster within 6 months.

International audience; Pifithrin alpha (PFTalpha) is a chemical compound that inhibits p53-mediated gene activation and apoptosis. It has also been recently shown to alter metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs). This has led us to examine the effect of PFTalpha on the activity of cytochrome P-450 (CYP) 1 isoforms, known to metabolize PAHs, such as benzo(a)pyrene (BP), into mutagenic metabolites. We report that PFTalpha caused a potent inhibition of CYP1-related activity as measured by ethoxyresorufin O-deethylase activity in CYP1-containing MCF-7 cells and liver microsomes. It also directly affected the catalytic activity of human recombinant CYP1A1, CYP1A2 and CYP1B1 isoforms, with a potent inhibitory effect towards CYP1B1. The nature of this CYP1B1 inhibition by PFTalpha was mixed-type with an apparent K(i) of 4.38 nM. Blockage of CYP1 activity by PFTalpha was associated with a decreased metabolism of BP, a reduced formation of BP-derived adducts and a diminished BP-induced apoptosis in human cultured cells targets for PAHs like primary human macrophages and p53-negative KG1a leukaemia cells. These data further substantiate an unexpected and p53-independent action of PFTalpha for preventing toxicity of chemical carcinogens such as PAHs, through inhibition of CYP1 enzyme activities, especially that of CYP1B1.

International audience; INTRODUCTION: Mitoxantrone (Mx) is used as a second-line treatment in multiple sclerosis. Since 1998, eight cases of acute leukemia (AL) have been described. We report two new cases of myeloid AL that occurred during treatment with Mx. OBSERVATIONS: The first case concerned a women who was treated with Mx for 3 months. In spite of a very low total dose (58.32 mg), she developed promyelocytic AL. The second patient died of myeloid AL, 27 months after the last injection of Mx. DISCUSSION: All the reported cases of AL occurring after Mx respond to the criteria of leukemia induced by anti-topoisomerases II. Epidemiological data and those from animal experiments suggest that Mx has direct role in the occurrence of leukemia. CONCLUSION: It must be remembered that even if the risk of Mx-induced leukemia is low, blood cell counts must be closely monitored for at least five years after the last injection of this treatment.

The association between the risk of childhood leukaemia before age 7 years and population-mixing at the place of residence at birth was investigated by retrospectively considering all the children born in mainland French communes between 1st January 1990 and 31st December 1998. An increased risk of acute lymphoblastic leukaemia was found with higher levels of migration for children residing at birth in isolated communes with a population density > or =50 people per km2 (SIRR = 2.59, 95% CI: 1.48-4.49). No association was observed with lower population densities. For children residing in non-isolated communes at birth, the results were similar but less marked. The risk tended to increase only for population densities > or =5000 people per km2 (SIRR = 1.57, 95% CI: 0.99-2.52). The findings are consistent with epidemic models and support the hypothesis of an infectious aetiology relating to population-mixing. Population density may be seen as an indicator of the opportunity of contacts between inhabitants and should therefore be taken into account when investigating an infectious hypothesis. This is the first systematic study of population-mixing at the place of residence at the time of birth to be conducted on a national scale.

International audience; As a single signal, retinoids induce terminal differentiation. This implies that they activate differentiation and apoptosis in a temporally defined order to allow expression of the differentiated phenotype well before death. We report that two apparently contradictory retinoid-induced programs have the capacity to define cellular life span. Anti-apoptotic factors are activated concomitantly with differentiation, while retinoids induce at the same time also pro-apoptotic signaling. We have assessed the roles of two key factors, Bcl2A1 and TRAIL, in the temporal programming of cell death and differentiation. We demonstrate that PLB985 are type II cells in which TRAIL induces apoptosis through the extrinsic and--via Bid activation--also the intrinsic death pathways. Bcl2A1, ectopically over-expressed, or endogenously induced by retinoic acid receptor agonists, protected cells from apoptosis triggered by TRAIL, whose induction required the activation of both the retinoic acid and retinoid X receptors. Bcl2A1 prevented loss of mitochondrial membrane potential and caspase-9, but not caspase-8, activation. The expression of anti-sense Bcl2A1 sensitized PLB985 cells to TRAIL. Co-culture experiments revealed protection from fraternicide if sister cells were pre-exposed to retinoic acid. Collectively, our data support a model in which retinoids orchestrate a life span-regulatory program comprising Bcl2A1 induction to temporally protect against concomitantly induced TRAIL death signaling. Termination of this life span in presence of Bcl2A1 is most likely a consequence of the Bid-independent TRAIL action. Thus, depending on the retinoic acid and retinoid X receptor activation potential of a ligand and the relative efficacies of the intrinsic and extrinsic death pathways in a given cell, a single retinoid triggers the life span of a differentiated phenotype.

Recently, regression analysis of the cumulative incidence function has gained interest in competing risks data analysis, through the model proposed by Fine and Gray (JASA 1999; 94: 496-509). In this note, we point out that inclusion of time-dependent covariates in this model can lead to serious bias. We illustrate the problems arising in such a context, using bone marrow transplant data as a working example and numerical simulations. Practical advices are given, preventing the misuse of this model.

International audience; Metabolic polymorphisms may influence the risk of childhood leukaemia related to maternal tobacco, coffee or alcohol consumption. The data were extracted from a case-control study including 280 cases of acute leukaemia and 288 controls. Blood sampling was obtained for a representative subset of 219 cases and 105 controls. Gene-environment interactions were estimated using both case-control and case-only analyses. The polymorphisms of CYP1A1, GSTM1, GSTP1, GSTT1 and NQO1 were not associated with the risk of leukaemia. The slow EPHX1 allele was negatively associated with childhood leukaemia while an inverse non-significant association was observed with the fast EPHX1 allele. Maternal smoking during pregnancy was not related to leukaemia, but an interaction was observed in the case-only analysis with CYP1A1*2A variant allele (odds ratio (OR) 2.2 [1.0-4.9]) and with GSTM1 deletion (OR 2.3 [1.2-4.4]). Conversely, coffee drinking interacted negatively with NQO1 polymorphism in the case-only analysis (OR 0.6 [0.3-1.2] and 0.4 [0.1-1.0] for light and heavy coffee consumptions, respectively). This study suggests that maternal smoking may be a risk factor for leukaemia in children who carry CYP1A1 or GSTM1 genotypes, which might increase reactive metabolites of polycyclic aromatic hydrocarbons.