Facteurs de réponse dans les leucémies aiguës myéloblastiques traitées par azacytidine

Contexte : Les leucémies aiguës myéloblastiques (LAM) ont un pronostic sombre, nécessitant de nouveaux traitements. Des facteurs pronostiques ont été validés dans le cadre de traitements conventionnels afin d adapter le traitement aux caractéristiques de chaque patient. L azacytidine est un agent déméthylant ayant démontré son efficacité dans les syndromes myélodysplasiques de haut risque dont l efficacité et les facteurs pronostiques de réponse nécessitent d être explorés dans les LAM. Méthodes : De manière rétrospective, les patients du Nord-Pas-de-Calais et d Amiens traités par azacytidine pour une LAM entre 2007 et 2011 ont été recensés. Une recherche de mutations de 12 gènes sur ADN génomique par biologie moléculaire classique et séquençage ultra haut débit a été réalisée afin d en évaluer leur impact pronostique. Résultats : Dans cette cohorte de 102 patients, l âge médian était de 68 ans [20-82]. Les LAM secondaires représentaient 51% des cas. 57% étaient traités en 1ere ligne, 20% en 2e ligne et 23% en 3e ligne et plus. 51% des patients présentaient un risque cytogénétique défavorable. La blastose médullaire médiane était de 35% [9 ; 93]. Le taux de réponse globale était de 19% avec une durée médiane de 9,5 mois [2,5-38,7]. 9 patients étaient allogreffés au terme du traitement par azacytidine. 39% des patients n atteignaient pas l administration des 4 cures dont 20% en raison d une progression de la maladie. La ligne thérapeutique apparaît comme pronostique de la réponse à l azacytidine avec 26% de réponse globale en première ligne contre 9% en deuxième ligne et plus (p=0,03). IDH2 muté apparaît comme un facteur pronostic péjoratif de réponse (40% vs 0%) parmi la recherche de mutations réalisées. La survie médiane de la cohorte était de 7,1 mois [IC95 : 5,3-8,8]. La ligne thérapeutique avait un impact péjoratif avec une survie médiane diminué à 5,3 mois en cas de 3e ligne et plus (P=0,04) et à 2,5 mois en cas d antécédent d allogreffe (p=0.02). A l inverse, la réalisation d une allogreffe à l issu de traitement par azacytidine permet un allongement de la survie médiane à 12,1 mois (p=0,01). Conclusion : L azacytidine est un traitement qui est à envisager chez le patient âgé en première ligne pour une LAM à caryotype complexe permettant une prise en charge ambulatoire. L allogreffe à conditionnement atténué devra être envisagée chaque fois que possible chez ces patients. IDH2 muté apparaît dans une première analyse avoir un impact péjoratif sur l obtention de la réponse.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

International audienceBACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also usefulmarkers of pathologies. In leukemia, they are used to quantify the minimal residual disease duringpatient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamedV(D)J junctions and gather them into clones for quantification. This analysis is based on a seedheuristic and is fast and scalable because in the first phase, no alignment is performed with germlinedatabase sequences. The algorithms were applied to TR HTS data from a patient with acutelymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified themain clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-troughput sequencing data forleukemia, and also to the quantitative assessment of any immunological profile. The methodsdescribed here are implemented in a C++ open-source program called Vidjil

Multi-loci diagnosis of acute lymphoblastic leukaemia with high-throughput sequencing and bioinformatics analysis

International audienceHigh-throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow-up. As part of an immunoglobulin/T cell receptor-based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients

BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

International audienceBACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also usefulmarkers of pathologies. In leukemia, they are used to quantify the minimal residual disease duringpatient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamedV(D)J junctions and gather them into clones for quantification. This analysis is based on a seedheuristic and is fast and scalable because in the first phase, no alignment is performed with germlinedatabase sequences. The algorithms were applied to TR HTS data from a patient with acutelymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified themain clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-troughput sequencing data forleukemia, and also to the quantitative assessment of any immunological profile. The methodsdescribed here are implemented in a C++ open-source program called Vidjil

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow.

Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr-/- cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34+ cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Abstract: Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Abstract: Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options