Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches

Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects

Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2 D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2 D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2 D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies

Thiopurine Enhanced ALL Maintenance (TEAM) : study protocol for a randomized study to evaluate the improvement in disease-free survival by adding very low dose 6-thioguanine to 6-mercaptopurine/methotrexate-based maintenance therapy in pediatric and adult patients (0-45 years) with newly diagnosed B-cell precursor or T-cell acute lymphoblastic leukemia treated according to the intermediate risk-high group of the ALLTogether1 protocol

Background: A critical challenge in current acute lymphoblastic leukemia (ALL) therapy is treatment intensification in order to reduce the relapse rate in the subset of patients at the highest risk of relapse. The year-long maintenance phase is essential in relapse prevention. The Thiopurine Enhanced ALL Maintenance (TEAM) trial investigates a novel strategy for ALL maintenance. Methods: TEAM is a randomized phase 3 sub-protocol to the ALLTogether1 trial, which includes patients 0-45 years of age with newly diagnosed B-cell precursor or T-cell ALL, and stratified to the intermediate risk-high (IR-high) group, in 13 European countries. In the TEAM trial, the traditional methotrexate (MTX)/6-mercaptopurine (6MP) maintenance backbone (control arm) is supplemented with low dose (2.5-12.5 mg/m(2)/day) oral 6-thioguanine (6TG) (experimental arm), while the starting dose of 6MP is reduced from 75 to 50 mg/m(2)/day. A total of 778 patients will be included in TEAM during similar to 5 years. The study will close when the last included patient has been followed for 5 years from the end of induction therapy. The primary objective of the study is to significantly improve the disease-free survival (DFS) of IR-high ALL patients by adding 6TG to 6MP/MTX-based maintenance therapy. TEAM has 80% power to detect a 7% increase in 5-year DFS through a 50% reduction in relapse rate. DFS will be evaluated by intention-to-treat analysis. In addition to reducing relapse,TEAM may also reduce hepatotoxicity and hypoglycemia caused by high levels of methylated 6MP metabolites. Methotrexate/6MP metabolites will be monitored and low levels will be reported back to clinicians to identify potentially non-adherent patients. Discussion: TEAM provides a novel strategy for maintenance therapy in ALL with the potential of improving DFS through reducing relapse rate. Potential risk factors that have been considered include hepatic sinusoidal obstruction syndrome/nodular regenerative hyperplasia, second cancer, infection, and osteonecrosis. Metabolite monitoring can potentially increase treatment adherence in both treatment arms.Peer reviewe

A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95%CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatmentrelated mortality (TRM) of 9.0% (95%CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95%CI: 65.7-82.4) and 66.4% (95%CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT >6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with three or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced yet still substantial relapse incidence. By integrating genetic information with clinical and hematological features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit novel therapeutic agents and post-transplant strategies

Germline bi-allelic <i>SH2B3/LNK</i> alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of 2 unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, 8 from 5 families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematological JMML features with neonatal onset and marked thrombocytopenia. They were characterized by absence of additional genetic alterations and a hypomethylated DNA profile with fetal characteristics. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that the JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options

Juvenile myelomonocytic leukaemia, a disease of the hematopoietic development ?

La LMMJ est un syndrome myéloprolifératif et myélodysplasique rare du jeune enfant, secondaire à une hyperactivation de la voie RAS/MAPK suite à la mutation de RAS (NRAS, KRAS) ou de ses régulateurs (PTPN11, NF1 ou CBL). La LMMJ est une hémopathie sévère dont le seul traitement est l’allogreffe de moelle osseuse. Cependant, sa présentation et son évolution sont particulièrement hétérogènes puisqu’un tiers des patients présentent une transformation en leucémie aiguë quand d’autres présentent des formes plus indolentes, voire des rémissions spontanées. Les LMMJ ont par ailleurs la particularité de se développer chez les très jeunes enfants et de présenter des caractéristiques que l’on rencontre au cours du développement hématopoïétique physiologique ou dans certains cancers de l’enfant avec initiation anténatale. Malgré les progrès réalisés dans la compréhension des bases génétiques de la LMMJ ces dernières années, ceux-ci n’expliquent qu’en partie l’hétérogénéité phénotypique des patients. Le stade auquel apparait la cellule initiatrice de LMMJ au sein de la pyramide hématopoïétique mais aussi au cours du développement ontogénique pourrait apporter des réponses à ces questions et faire émerger des nouvelles pistes thérapeutiques. Une collaboration avec l’équipe de D. Bonnet (Crick Institute) nous a permis d’établir un modèle murin de xénotransplantation dans des souris immunodéficientes de type NSG ou NSG-S et de montrer que la capacité de propagation de la leucémie est bien portée par la fraction souche, mais s’étend aussi chez certains patients à des fractions plus différenciées. L’architecture clonale des LMMJ est dans la majorité des cas compatible avec une acquisition linéaire des altérations, mais une architecture complexe est parfois observée, avec coexistence de clones distincts, dont les plus faiblement représentés sont susceptibles de devenir dominants lors de la rechute. Le séquençage de sous-populations isolées et des colonies obtenues par culture des progéniteurs en méthylcellulose a montré que l’ensemble des mutations (initiatrice et additionnelles) est présent dès les fractions les plus immatures et qu’elles coexistent dans les mêmes cellules, sans qu’il soit possible de hiérarchiser leur ordre de survenue, témoignant d’un avantage sélectif majeur de leur association dès la cellule souche. Nous avons poursuivi l’analyse phénotypique et transcriptionnelle (RNAseq) des sous fractions hématopoïétiques triées de LMMJ et les avons comparées à leurs contreparties saines à différents stades de l'ontogénie (foie fœtal, moelle osseuse fœtale, et moelle osseuse d'enfants appariés en âge). Le profil d'expression des progéniteurs hématopoïétiques divise les LMMJ en deux groupes dont l’un présente des similitudes transcriptionnelles avec les échantillons embryo-fœtaux en lien avec l’expression accrue de gènes spécifiques des cellules monocytaires et dendritiques et des composants de l'inflammasome. L’autre groupe de LMMJ présente une signature transcriptionnelle distincte des prélèvements sains mais surexprime 2 facteurs de transcription onco-fœtaux: Lin28b et Wt1. Au total, nos résultats montrent que si la cellule initiatrice de la LMMJ est la CSH quel que soit le groupe génétique, les capacités de propagation de la maladie sont également présentes dans les progéniteurs plus engagés. Ceux-ci mettent en place des processus oncogéniques hétérogènes qui s’appuient cependant tous sur des mécanismes utilisés au cours du développement anté-natal.JMML is a rare myeloproliferative and myelodysplastic syndrome of early childhood, consecutive to the hyperactivation of the RAS/MAPK pathway following the mutation of RAS (NRAS, KRAS) or its regulators (PTPN11, NF1 or CBL). JMML is a severe malignancy for which the only treatment is bone marrow transplant. However, its presentation and evolution are particularly heterogeneous since a third of patients present a transformation into acute leukaemia while others harbour more indolent forms, and even spontaneous remissions. Moreover, JMML have the particularity to arise in very young children and to present characteristics that are encountered during physiological haematopoietic development or in certain childhood cancers with antenatal initiation. Despite the progress made in understanding the genetic basis of JMML in recent years, this only partly explains the phenotypic heterogeneity of patients. The stage at which the JMML initiating cell appears within the haematopoietic pyramid but also during ontogenic development could provide answers to these questions and lead to the emergence of new therapeutic approaches. A collaboration with D. Bonnet's team (Crick Institute) has enabled us to establish a mouse model of xenotransplantation in immunodeficient mice of the NSG or NSG-S type and to show that the capacity for spreading leukaemia is well carried by the stem cell fraction, but also extends in certain patients to more differentiated hematopoietic fractions. The clonal architecture of JMML is in the majority of cases compatible with a linear acquisition of alterations, but a complex architecture is sometimes observed, with the coexistence of distinct clones, the weakest of which are likely to become dominant during relapse. Sequencing of isolated sub-populations and colonies obtained by culture of methylcellulose progenitors has shown that all the mutations (initiating and additional) are present from the most immature fractions and that they coexist in the same cells, without it being possible to prioritise their order of occurrence, demonstrating a major selective advantage of their association from the stem cell onwards. We continued the phenotypic and transcriptional analysis (RNAseq) of the sorted hematopoietic sub-fractions of JMML and compared them to their healthy counterparts at different stages of ontogeny (fetal liver, fetal bone marrow, and bone marrow of age-matched children). The expression profile of hematopoietic progenitors divided the JMML into two groups, one of which had transcriptional similarities with embryo-fetal samples in relation to increased expression of monocyte and dendritic cell-specific genes and inflammasome components. The other group of JMML has a transcriptional signature distinct from healthy samples but overexpresses 2 onco-fetal transcription factors: Lin28b and Wt1. In all, our results show that if the initiating cell of JMML is HSC regardless of the genetic group, the capacity to spread the disease is also present in the more committed progenitors. These set up heterogeneous oncogenic processes that, however, all rely on mechanisms used during antenatal development

La leucémie myélomonocytaire juvénile, une pathologie du développement hématopoïétique ?

JMML is a rare myeloproliferative and myelodysplastic syndrome of early childhood, consecutive to the hyperactivation of the RAS/MAPK pathway following the mutation of RAS (NRAS, KRAS) or its regulators (PTPN11, NF1 or CBL). JMML is a severe malignancy for which the only treatment is bone marrow transplant. However, its presentation and evolution are particularly heterogeneous since a third of patients present a transformation into acute leukaemia while others harbour more indolent forms, and even spontaneous remissions. Moreover, JMML have the particularity to arise in very young children and to present characteristics that are encountered during physiological haematopoietic development or in certain childhood cancers with antenatal initiation. Despite the progress made in understanding the genetic basis of JMML in recent years, this only partly explains the phenotypic heterogeneity of patients. The stage at which the JMML initiating cell appears within the haematopoietic pyramid but also during ontogenic development could provide answers to these questions and lead to the emergence of new therapeutic approaches. A collaboration with D. Bonnet's team (Crick Institute) has enabled us to establish a mouse model of xenotransplantation in immunodeficient mice of the NSG or NSG-S type and to show that the capacity for spreading leukaemia is well carried by the stem cell fraction, but also extends in certain patients to more differentiated hematopoietic fractions. The clonal architecture of JMML is in the majority of cases compatible with a linear acquisition of alterations, but a complex architecture is sometimes observed, with the coexistence of distinct clones, the weakest of which are likely to become dominant during relapse. Sequencing of isolated sub-populations and colonies obtained by culture of methylcellulose progenitors has shown that all the mutations (initiating and additional) are present from the most immature fractions and that they coexist in the same cells, without it being possible to prioritise their order of occurrence, demonstrating a major selective advantage of their association from the stem cell onwards. We continued the phenotypic and transcriptional analysis (RNAseq) of the sorted hematopoietic sub-fractions of JMML and compared them to their healthy counterparts at different stages of ontogeny (fetal liver, fetal bone marrow, and bone marrow of age-matched children). The expression profile of hematopoietic progenitors divided the JMML into two groups, one of which had transcriptional similarities with embryo-fetal samples in relation to increased expression of monocyte and dendritic cell-specific genes and inflammasome components. The other group of JMML has a transcriptional signature distinct from healthy samples but overexpresses 2 onco-fetal transcription factors: Lin28b and Wt1. In all, our results show that if the initiating cell of JMML is HSC regardless of the genetic group, the capacity to spread the disease is also present in the more committed progenitors. These set up heterogeneous oncogenic processes that, however, all rely on mechanisms used during antenatal development.La LMMJ est un syndrome myéloprolifératif et myélodysplasique rare du jeune enfant, secondaire à une hyperactivation de la voie RAS/MAPK suite à la mutation de RAS (NRAS, KRAS) ou de ses régulateurs (PTPN11, NF1 ou CBL). La LMMJ est une hémopathie sévère dont le seul traitement est l’allogreffe de moelle osseuse. Cependant, sa présentation et son évolution sont particulièrement hétérogènes puisqu’un tiers des patients présentent une transformation en leucémie aiguë quand d’autres présentent des formes plus indolentes, voire des rémissions spontanées. Les LMMJ ont par ailleurs la particularité de se développer chez les très jeunes enfants et de présenter des caractéristiques que l’on rencontre au cours du développement hématopoïétique physiologique ou dans certains cancers de l’enfant avec initiation anténatale. Malgré les progrès réalisés dans la compréhension des bases génétiques de la LMMJ ces dernières années, ceux-ci n’expliquent qu’en partie l’hétérogénéité phénotypique des patients. Le stade auquel apparait la cellule initiatrice de LMMJ au sein de la pyramide hématopoïétique mais aussi au cours du développement ontogénique pourrait apporter des réponses à ces questions et faire émerger des nouvelles pistes thérapeutiques. Une collaboration avec l’équipe de D. Bonnet (Crick Institute) nous a permis d’établir un modèle murin de xénotransplantation dans des souris immunodéficientes de type NSG ou NSG-S et de montrer que la capacité de propagation de la leucémie est bien portée par la fraction souche, mais s’étend aussi chez certains patients à des fractions plus différenciées. L’architecture clonale des LMMJ est dans la majorité des cas compatible avec une acquisition linéaire des altérations, mais une architecture complexe est parfois observée, avec coexistence de clones distincts, dont les plus faiblement représentés sont susceptibles de devenir dominants lors de la rechute. Le séquençage de sous-populations isolées et des colonies obtenues par culture des progéniteurs en méthylcellulose a montré que l’ensemble des mutations (initiatrice et additionnelles) est présent dès les fractions les plus immatures et qu’elles coexistent dans les mêmes cellules, sans qu’il soit possible de hiérarchiser leur ordre de survenue, témoignant d’un avantage sélectif majeur de leur association dès la cellule souche. Nous avons poursuivi l’analyse phénotypique et transcriptionnelle (RNAseq) des sous fractions hématopoïétiques triées de LMMJ et les avons comparées à leurs contreparties saines à différents stades de l'ontogénie (foie fœtal, moelle osseuse fœtale, et moelle osseuse d'enfants appariés en âge). Le profil d'expression des progéniteurs hématopoïétiques divise les LMMJ en deux groupes dont l’un présente des similitudes transcriptionnelles avec les échantillons embryo-fœtaux en lien avec l’expression accrue de gènes spécifiques des cellules monocytaires et dendritiques et des composants de l'inflammasome. L’autre groupe de LMMJ présente une signature transcriptionnelle distincte des prélèvements sains mais surexprime 2 facteurs de transcription onco-fœtaux: Lin28b et Wt1. Au total, nos résultats montrent que si la cellule initiatrice de la LMMJ est la CSH quel que soit le groupe génétique, les capacités de propagation de la maladie sont également présentes dans les progéniteurs plus engagés. Ceux-ci mettent en place des processus oncogéniques hétérogènes qui s’appuient cependant tous sur des mécanismes utilisés au cours du développement anté-natal

Immunisation à la L-asparaginase dérivée de l escherichia coli au cours du traitement de la leucémie aiguë lymphoblastique de novo chez l'enfant dans le cadre du protocole Fralle 2000

Objectif : La L-asparaginase est une molécule de chimiothérapie utilisée dans le traitement des LAL de l'enfant. Elle induit des manifestations allergiques responsables d'une baisse de son activité enzymatique. Les conséquences de l'allergie silencieuse, c'est-à-dire sans hypersensibilité clinique, sont mal connues. Le but de cette étude est de déterminer l'incidence, les facteurs favorisants l'apparition de ces anticorps et leurs conséquences pharmacodynamiques. Patients et méthodes : 33 enfants traités pour une LAL de novo dans le protocole FRALLE 2000 ont été inclus prospectivement dans l étude de 2002 à 2005. Pour chaque enfant une mesure d anticorps, d'activité asparaginase, et d'asparagine était réalisée avant chaque injection de L-asparaginase de l induction à l'intensification n2. Résultats : 18% des patients ont présenté une allergie silencieuse. Tous sauf un étaient traités dans les bras B-T du protocole FRALLE. Parmi les enfants ayant une allergie clinique, 87% appartenaient au bras A (p=0,002). Il n était pas retrouvé de différence significative entre l activité L-asparaginase et la déplétion en asparagine entre les patients non immunisés, présentant une allergie clinique et présentant une allergie silencieuse. Conclusion : La corticothérapie et la chimiothérapie concomitantes au traitement par L-asparaginase diminuent la production d'anticorps et favorisent leur maintien à l'état silencieux.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Life in the woods: Taphonomic evolution of a diverse saproxylic community within fossil woods from Upper Cretaceous submarine mass flow deposits (Mzamba Formation, southeast Africa)

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