Etude des altérations génomiques des leucémies aiguës myéloïdes de l’adulte : vers une approche thérapeutique personnalisée ?

Acute myeloid leukemia (AML) represents a heterogenous disease with dismal outcome. The current European LeukemiaNet (ELN) 2017 AML risk stratification integrates limited cytogenetic and molecular alterations. However, over the past years, outstanding advances in molecular technologies have pointed out new alterations of potential clinical significance and refined the genomic profile of AML in younger adults. Growing evidence that integrating both clinical and biological parameters may help to refine AML prognostic led to multi-stage models development.This research project focused on younger AML patients included in the ALFA-0702 trial. In the randomized cohort of this study, SNP-array analysis revealed a new subset of patients with adverse prognostic, from at least 4 and more alterations. Interestingly, such patients benefited from alternative consolidation regimen such as the clofarabine.Secondly, we confirmed that applying the knowledge bank (KB) algorithm developed by Gerstung et al in the 656 patients from the ALFA-0702 trial provided more accurate outcome predictions than the current ELN 2017 risk stratification. Notably, we have shown that the decision to perform hematopoietic stem cell transplantation in first complete remission could be personalized by integrating ELN 2017 risk stratification, NPM1 minimal residual disease and KB simulations.Interestingly, cytogenetic and molecular alterations stand for new theranostic biomarkers in the era of new drugs for AML treatment. These biomarkers offer new hope to improve AML management.This research project offered the opportunity to identify new prognostic biomarkers, and to personalize and improve patient management through the integration of numerous pre- and post-treatment biomarkers in the treatment decision-making.Les leucémies aiguës myéloïdes (LAM) de l’adulte jeune représentent des entités hétérogènes avec un pronostic contrasté. La classification pronostique de l’European LeukemiaNet (ELN) révisée en 2017 intègre un nombre restreint d’altérations cytogénétiques et moléculaires. Néanmoins, le développement notable des techniques de biologie moléculaire au cours des dernières années a permis l’identification de nouveaux marqueurs d’intérêt pronostique potentiel. La prise en compte d’un nombre plus exhaustif de paramètres cliniques et biologiques pourrait aider à mieux évaluer le pronostic des LAM et a conduit au développement d’approches prédictives personnalisées multi-étapes.Ce projet de recherche était centré sur les adultes jeunes diagnostiqués pour une LAM inclus dans le protocole ALFA-0702. Au sein de la cohorte randomisée de l’étude, l’analyse en SNP-array a permis la caractérisation d’un nouveau groupe de patients avec un pronostic défavorable, caractérisé par la présence d’au moins 4 anomalies. Nous avons montré que ces patients bénéficiaient d’un régime alternatif de consolidation par clofarabine.Par ailleurs, à partir de l’analyse par séquençage haut débit de 656 patients inclus dans le protocole ALFA-0702, nous avons confirmé que l’approche « banque de connaissance » développée par Gerstung et al. était plus performante pour prédire le pronostic des patients que la classification pronostique de l’ELN 2017. Nous avons montré que l’intégration de la classification ELN 2017 et le suivi de la maladie résiduelle sur le marqueur NPM1 à l’approche « banque de connaissance » permettait de prédire de façon personnalisée le sous-groupe de patients susceptible de bénéficier de l’allogreffe de cellules souches hématopoïétiques en première rémission complète.Enfin, les altérations cytogénétiques et moléculaires constituent de nouveaux marqueurs théranostiques d’intérêt à l’heure du développement des thérapies ciblées dans les LAM.Ainsi, ce projet offre de nouvelles perspectives dans la prise en charge des LAM, selon une approche plus personnalisée, par l’identification et l’intégration de multiples marqueurs pré- et post-thérapeutiques

Genomic alterations in younger adults with acute myeloid leukemia : toward a personalized molecular-based management ?

Les leucémies aiguës myéloïdes (LAM) de l’adulte jeune représentent des entités hétérogènes avec un pronostic contrasté. La classification pronostique de l’European LeukemiaNet (ELN) révisée en 2017 intègre un nombre restreint d’altérations cytogénétiques et moléculaires. Néanmoins, le développement notable des techniques de biologie moléculaire au cours des dernières années a permis l’identification de nouveaux marqueurs d’intérêt pronostique potentiel. La prise en compte d’un nombre plus exhaustif de paramètres cliniques et biologiques pourrait aider à mieux évaluer le pronostic des LAM et a conduit au développement d’approches prédictives personnalisées multi-étapes.Ce projet de recherche était centré sur les adultes jeunes diagnostiqués pour une LAM inclus dans le protocole ALFA-0702. Au sein de la cohorte randomisée de l’étude, l’analyse en SNP-array a permis la caractérisation d’un nouveau groupe de patients avec un pronostic défavorable, caractérisé par la présence d’au moins 4 anomalies. Nous avons montré que ces patients bénéficiaient d’un régime alternatif de consolidation par clofarabine.Par ailleurs, à partir de l’analyse par séquençage haut débit de 656 patients inclus dans le protocole ALFA-0702, nous avons confirmé que l’approche « banque de connaissance » développée par Gerstung et al. était plus performante pour prédire le pronostic des patients que la classification pronostique de l’ELN 2017. Nous avons montré que l’intégration de la classification ELN 2017 et le suivi de la maladie résiduelle sur le marqueur NPM1 à l’approche « banque de connaissance » permettait de prédire de façon personnalisée le sous-groupe de patients susceptible de bénéficier de l’allogreffe de cellules souches hématopoïétiques en première rémission complète.Enfin, les altérations cytogénétiques et moléculaires constituent de nouveaux marqueurs théranostiques d’intérêt à l’heure du développement des thérapies ciblées dans les LAM.Ainsi, ce projet offre de nouvelles perspectives dans la prise en charge des LAM, selon une approche plus personnalisée, par l’identification et l’intégration de multiples marqueurs pré- et post-thérapeutiques.Acute myeloid leukemia (AML) represents a heterogenous disease with dismal outcome. The current European LeukemiaNet (ELN) 2017 AML risk stratification integrates limited cytogenetic and molecular alterations. However, over the past years, outstanding advances in molecular technologies have pointed out new alterations of potential clinical significance and refined the genomic profile of AML in younger adults. Growing evidence that integrating both clinical and biological parameters may help to refine AML prognostic led to multi-stage models development.This research project focused on younger AML patients included in the ALFA-0702 trial. In the randomized cohort of this study, SNP-array analysis revealed a new subset of patients with adverse prognostic, from at least 4 and more alterations. Interestingly, such patients benefited from alternative consolidation regimen such as the clofarabine.Secondly, we confirmed that applying the knowledge bank (KB) algorithm developed by Gerstung et al in the 656 patients from the ALFA-0702 trial provided more accurate outcome predictions than the current ELN 2017 risk stratification. Notably, we have shown that the decision to perform hematopoietic stem cell transplantation in first complete remission could be personalized by integrating ELN 2017 risk stratification, NPM1 minimal residual disease and KB simulations.Interestingly, cytogenetic and molecular alterations stand for new theranostic biomarkers in the era of new drugs for AML treatment. These biomarkers offer new hope to improve AML management.This research project offered the opportunity to identify new prognostic biomarkers, and to personalize and improve patient management through the integration of numerous pre- and post-treatment biomarkers in the treatment decision-making

Targeting RUNX1 in acute myeloid leukemia : preclinical innovations and therapeutic implications

Introduction: RUNX1 is an essential transcription factor for normal and malignant hematopoiesis. RUNX1 forms a heterodimeric complex with CBFB. Germline mutations and somatic alterations (i.e. translocations, mutations and abnormal expression) are frequently associated with acute myeloid leukemia (AML) with RUNX1 mutations conferring unfavorable prognosis. Therefore, RUNX1 constitutes a potential innovative and interesting therapeutic target. In this review, we discuss recent therapeutic advances of RUNX1 targeting in AML.Areas covered: Firstly, we cover the clinical basis for RUNX1 targeting. We have subdivided recent therapeutic approaches either by common biochemical pathways or by similar pharmacological targets. Genome editing of RUNX1 induces anti-leukemic effects; however, off-target events prohibit clinical use. Several molecules inhibit the interaction between RUNX1/CBFB and control AML development and progression. BET protein antagonists target RUNX1 (i.e. specific BET inhibitors, BRD4 shRNRA, proteolysis targeting chimeras (PROTAC) or expression-mimickers). All these molecules improve survival in mutant RUNX1 AML preclinical models.Expert opinion: Some of these novel molecules have shown encouraging anti-leukemic potency at the preclinical stage. A better understanding of RUNX1 function in AML development and progression and its key downstream pathways, may result in more precise and more efficient RUNX1 targeting therapies

Diagnostic challenge in a case of ambiguous lineage acute leukemia with PICALM::MLLT10 rearrangement

International audienc

Germline pathogenic variants in transcription factors predisposing to pediatric acute myeloid leukemia: results from the French ELAM02 trial

International audienc

Frugal alignment-free identification of FLT3-internal tandem duplications with FiLT3r

International audienceAbstract Background Internal tandem duplications in the FLT3 gene, termed FLT3 -ITDs, are useful molecular markers in acute myeloid leukemia (AML) for patient risk stratification and follow-up. FLT3 -ITDs are increasingly screened through high-throughput sequencing (HTS) raising the need for robust and efficient algorithms. We developed a new algorithm, which performs no alignment and uses little resources, to identify and quantify FLT3 -ITDs in HTS data. Results Our algorithm (FiLT3r) focuses on the k -mers from reads covering FLT3 exons 14 and 15. We show that those k -mers bring enough information to accurately detect, determine the length and quantify FLT3 -ITD duplications. We compare the performances of FiLT3r to state-of-the-art alternatives and to fragment analysis, the gold standard method, on a cohort of 185 AML patients sequenced with capture-based HTS. On this dataset FiLT3r is more precise (no false positive nor false negative) than the other software evaluated. We also assess the software on public RNA-Seq data, which confirms the previous results and shows that FiLT3r requires little resources compared to other software. Conclusion FiLT3r is a free software available at https://gitlab.univ-lille.fr/filt3r/filt3r . The repository also contains a Snakefile to reproduce our experiments. We show that FiLT3r detects FLT3-ITDs better than other software while using less memory and time

Clonal Dynamics of FLT3 -ITD Positive Acute Myeloid Leukemia Patients with Relapsed/Refractory Disease Following Intensive Chemotherapy +/- Midostaurin

International audienceIntroduction: Despite the wider use of midostaurin (MIDO) in combination with intensive chemotherapy (ICT) as the 1st-line treatment for FLT3-mutated acute myeloid leukemia (AML), complete remission (CR) rates are close to 60%, and relapses occur in over 40% of cases, demonstrating the ability of leukemic cells to resist and evade therapy ( Stone et al., NEJM 2017). Conventional fragment-length analyses of paired diagnosis/relapse samples have shown that FLT3-internal tandem duplications (ITDs) are retained in 80% and 50% of cases following ICT alone and MIDO+ICT respectively ( Schmalbrock et al., Blood 2021). Only limited data are available on the dynamics of FLT3-ITDs or other co-mutations in refractory patients (pts). Here, we conducted a retrospective study involving 115 pts with relapsed/refractory AML harboring FLT3-ITD at diagnosis. Materials and methods: Clonal evolution was examined in paired diagnosis/progression blood or bone marrow samples from 115 pts with FLT3-ITD+ AML treated with MIDO+ICT (n=33) or ICT alone (n=82). Among them, 21 pts had primary refractory disease (MIDO+ICT, n=8; ICT, n=13) and 94 pts relapsed after achieving CR (MIDO+ICT, n=25; ICT, n=69). FLT3-ITDs and co-mutations were screened on genomic DNA by high-throughput sequencing at both timepoints using a custom-designed panel. For accurate annotation and quantification of FLT3-ITDs from sequencing data, we used the recentlypublished FiLT3r algorithm ( Boudry et al., BMC Bioinformatics 2022). For each ITD detected, FiLT3r allelic ratio (AR) was assessed by the ratio between the mutated allele and the wild-type allele. Results: A total of 226 FLT3-ITDs were detected in 115 pts at AML diagnosis, among which 120 (53%) ITDs were found with an AR below 0.05 ( Figure 1). Among pts who achieved CR and experienced relapse (n=94), 48 had multiple FLT3-ITDs at diagnosis and 46 had a single FLT3-ITD at diagnosis. Overall, we observed a simplification of the FLT3-ITD repertoire upon relapse with the persistence of at least one initial clone in 8/12 [67%] and 24/36 [67%] pts with multiple ITDs receiving MIDO+ICT and ICT alone respectively. In relapsed pts who initially had a single FLT3-ITD clone at diagnosis, the addition of MIDO to ICT was associated with a higher rate of FLT3-ITD negativity compared to pts receiving ICT alone (6/13 [46%] vs 5/33 [15%]; P = 0.05) ( Figure 2). Interestingly, among 21 pts with primary refractory AML, we observed that FLT3-ITD mutation status became negative in 5/8 pts (62%) and 2/13 pts (15%) after induction with MIDO+ICT and ICT alone respectively. We then compared the initial characteristics between retained and lost FLT3-ITDs at AML relapse. Lost FLT3-ITDs had significantly lower AR than retained clones in both treatment groups. In order to limit the impact of sample dilution on the allele burden, we defined adjusted variant allele frequencies (VAFs) as the VAFs of FLT3-ITDs normalized to the VAFs of NPM1 mutations, whenever applicable. In so doing, we observed that adjusted VAFs of retained FLT3-ITDs increased at relapse, regardless of the treatment group (adjusted VAF, diagnosis vs relapse: 0.28 vs 0.86 and 0.88 vs 1.6 in the MIDO+ICT group and ICT alone group; P = 2.3e-03 and P = 2.4e-04). Importantly, an adjusted VAF higher than 1 was strongly suggestive of a homozygous state of FLT3-ITD. Such situation was found to be more prevalent at relapse in both treatment groups. Besides the selection of a dominant FLT3-ITD clone, other relapse-related changes including the acquisition of additional mutations will be presented. Conclusion: Our study of the clonal dynamics of AML with FLT3-ITD mutations provides insights into the mechanisms underlying therapy escape. Our data suggest that clonal interference characterized by multiple FLT3-ITD clones is associated with a greater ability to select a FLT3-ITD-positive clone at relapse in pts receiving MIDO+ICT. Although the addition of MIDO to ICT increases the probability of eradicating a single FLT3-ITD clone, FLT3-ITD+ relapses remain common following this combination, often with the selection of homozygous FLT3-ITD clones and/or the emergence of new mutations. Finally, our data in refractory situation emphasize the need to reassess mutational status at each stage of progression before implementing targeted therapy