BCL-2 Inhibitor ABT-737 effectively targets leukemia-initiating cells with differential regulation of relevant genes leading to extended survival in a NRAS/BCL-2 mouse model of high risk-myelodysplastic syndrome

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment

Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients

Resistant acute myeloid leukemia (AML) exhibits mitochondrial energy metabolism changes compared to newly diagnosed AML. This phenotype is often observed by evaluating the mitochondrial oxygen consumption of blasts, but most of the oximetry protocols were established from leukemia cell lines without validation on primary leukemia cells. Moreover, the cultures and storage conditions of blasts freshly extracted from patient blood or bone marrow cause stress, which must be evaluated before determining oxidative phosphorylation (OXPHOS). Herein, we evaluated different conditions to measure the oxygen consumption of blasts using extracellular flow analyzers. We first determined the minimum number of blasts required to measure OXPHOS. Next, we compared the OXPHOS of blasts cultured for 3 h and 18 h after collection and found that to maintain metabolic organization for 18 h, cytokine supplementation is necessary. Cytokines are also needed when measuring OXPHOS in cryopreserved, thawed and recultured blasts. Next, the concentrations of respiratory chain inhibitors and uncoupler FCCP were established. We found that the FCCP concentration required to reach the maximal respiration of blasts varied depending on the patient sample analyzed. These protocols provided can be used in future clinical studies to evaluate OXPHOS as a biomarker and assess the efficacy of treatments targeting mitochondria

Étude du chimérisme après allogreffe de cellules hématopoïétiques : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC)

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Impact of DNMT3a Status on Post Induction NPM1 MRD Predictive Value on Survival in Elderly AML Patients Treated Intensively

International audienceMinimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached ≥ 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse

The Impact of DNMT3A Status on NPM1 MRD Predictive Value and Survival in Elderly AML Patients Treated Intensively

International audienceSimple Summary DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of acute myeloid leukemia (AML) patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. A 4log reduction of NPM1 MRD was associated with a better outcome. DNMT3A negative patients who achieved a 4log reduction had a superior outcome to those who did not. However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identify a subgroup of patients at high risk of relapse. Minimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a >= 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached >= 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse

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

Genetic identification of patients with AML older than 60 years achieving long-term survival with intensive chemotherapy

International audienceTo design a simple and reproducible classifier predicting the overall survival (OS) of patients with acute myeloid leukemia (AML) >= 60 years of age treated with 7+3, we sequenced 37 genes in 471patients fromtheALFA1200 (Acute Leukemia FrenchAssociation) study (median age, 68 years). Mutation patterns and OS differed between the 84 patients with poor-risk cytogenetics and the 387 patientswith good (n = 13), intermediate (n = 339), or unmeasured (n = 35) cytogenetic risk. TP53 (hazards ratio [HR], 2.49; P = .0003) and KRAS (HR, 3.60; P = .001) mutations independently worsened the OS of patients with poor-risk cytogenetics. In those without poor-risk cytogenetics, NPM1 (HR, 0.57; P = .0004), FLT3 internal tandem duplications with low (HR, 1.85; P = .0005) or high (HR, 3.51; P = 60 years of age treated with intensive chemotherapy. This model can instruct the design of trials comparing the 7+3 standard of care with less intensive regimens