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Purpose: Acute myeloid leukemias (AML) are clonal diseases that develop from leukemic stem cells (LSC) that carry an independent prognostic impact on the initial response to induction chemotherapy, demonstrating the clinical relevance of LSC abundance in AML. In 2018, the European LeukemiaNet published recommendations for the detection of measurable residual disease (Bulk MRD) and suggested the exploration of LSC MRD and the use of multiparametric displays. Experimental Design: We evaluated the performance of unsupervised clustering for the post-induction assessment of bulk and LSC MRD in 155 patients with AML who received intensive conventional chemotherapy treatment. Results: The median overall survival (OS) for Bulk+ MRD patients was 16.7 months and was not reached for negative patients (HR, 3.82; P < 0.0001). The median OS of LSC+ MRD patients was 25.0 months and not reached for negative patients (HR, 2.84; P = 0.001). Interestingly, 1-year (y) and 3-y OS were 60% and 39% in Bulk+, 91% and 52% in Bulk-LSC+ and 92% and 88% in Bulk-LSC−. Conclusions: In this study, we confirm the prognostic impact of post-induction multiparametric flow cytometry Bulk MRD in patients with AML. Focusing on LSCs, we identified a group of patients with negative Bulk MRD but positive LSC MRD (25.8% of our cohort) with an intermediate prognosis, demonstrating the interest of MRD analysis focusing on leukemic chemoresistant subpopulations.

Supplemental figure 1. Flow chart for sample availability of patients included in our study and their outcome. Supplemental figure 2. Supplemental figure 3. Size of LSC nodes in CD34+CD38- subpopulation in AML-CD34+ and AMLCD34- populations. Mann-Whitney test. Supplemental figure 4. LSC phenotypic profiles according to MRD status. Normalized MFI of CD34, CD38, CD45RA, CD135 and CD133 in LSC nodes used to measure LSC MRD. Mann-Whitney tests. Supplemental figure 5. Landmark analysis at 6 months of the impact of HSCT according to MRD status. Supplemental figure 6. Evaluation of immunophenotypic markers in chemoresistant and chemosensitive AML patients at diagnosis. Supplemental figure 7. Evaluation of LSC phenotypic markers in clinically relevant AML patients in relation to Figure 4.

Purpose:Acute myeloid leukemias (AML) are clonal diseases that develop from leukemic stem cells (LSC) that carry an independent prognostic impact on the initial response to induction chemotherapy, demonstrating the clinical relevance of LSC abundance in AML. In 2018, the European LeukemiaNet published recommendations for the detection of measurable residual disease (Bulk MRD) and suggested the exploration of LSC MRD and the use of multiparametric displays.Experimental Design:We evaluated the performance of unsupervised clustering for the post-induction assessment of bulk and LSC MRD in 155 patients with AML who received intensive conventional chemotherapy treatment.Results:The median overall survival (OS) for Bulk+ MRD patients was 16.7 months and was not reached for negative patients (HR, 3.82; P < 0.0001). The median OS of LSC+ MRD patients was 25.0 months and not reached for negative patients (HR, 2.84; P = 0.001). Interestingly, 1-year (y) and 3-y OS were 60% and 39% in Bulk+, 91% and 52% in Bulk-LSC+ and 92% and 88% in Bulk-LSC−.Conclusions:In this study, we confirm the prognostic impact of post-induction multiparametric flow cytometry Bulk MRD in patients with AML. Focusing on LSCs, we identified a group of patients with negative Bulk MRD but positive LSC MRD (25.8% of our cohort) with an intermediate prognosis, demonstrating the interest of MRD analysis focusing on leukemic chemoresistant subpopulations.

Supplemental Table 1. Characteristics of AML patients. Supplemental Table 2. Flow cytometry antibodies. Supplemental Table 3. Multivariate Cox regression analysis of factors contributing to overall survival of AML patients. Supplemental Table 4. Multivariate Cox regression analysis of factors contributing to leukemia free survival of AML patients. Supplemental Table 5. Multivariate Cox regression analysis of factors contributing to overall survival of AML patients including the association of bulk and LSC MRD parameters. Supplemental Table 6. Multivariate Cox regression analysis of factors contributing to leukemia free survival of AML patients including the association of bulk and LSC MRD parameters.

We analyzed 526 consecutive acute myeloid leukemia patients refractory to or relapsing after chemotherapy. 270 patients received intensive salvage chemotherapy (IC), 97 azacitidine (AZA) and 159 best supportive care (BSC). Complete response was obtained in 37/19/0% (

Key Points Typical features of GATA2 deficiency were associated with a de novo tandem duplication of GATA2 and increased expression of GATA2-AS1.The duplication contained a maternally inherited deletion copy number polymorphism esv2725896/nsv513733., A 3-year-old girl of nonconsanguineous healthy parents presented with cervical and mediastinal lymphadenopathy due to Mycobacterium fortuitum infection. Routine blood analysis showed normal hemoglobin, neutrophils, and platelets but profound mononuclear cell deficiency (monocytes < 0.1 × 109/L; B cells 78/μL; NK cells 48/μL). A 548 902-bp region containing GATA2 was sequenced by targeted capture and deep sequencing. This revealed a de novo 187-kb duplication of the entire GATA2 locus, containing a maternally inherited copy number variation deletion of 25 kb (GRCh37: esv2725896 and nsv513733). Many GATA2-associated phenotypes have been attributed to amino acid substitution, frameshift/deletion, loss of intronic enhancer function, or aberrant splicing. Gene deletion has been described, but other structural variation has not been reported in the germline configuration. In this case, duplication of the GATA2 locus was paradoxically associated with skewed diminished expression of GATA2 messenger RNA and loss of GATA2 protein. Chimeric RNA fusion transcripts were not detected. A possible mechanism involves increased transcription of the anti-sense long noncoding RNA GATA2-AS1 (RP11-472.220), which was increased several fold. This case further highlights that evaluation of the allele count is essential in any case of suspected GATA2-related syndrome.

Objectives SSc is an autoimmune disease characterized by fibrosis, microangiopathy and immune dysfunctions including dysregulation of proinflammatory cytokines. Clonal haematopoiesis of indeterminate potential (CHIP) is defined by the acquisition of somatic mutations in haematopoietic stem cells leading to detectable clones in the blood. Recent data have shown a higher risk of cardiovascular disease in patients with CHIP resulting from increased production of proinflammatory cytokines and accelerated atherosclerosis. Eventual links between CHIP and autoimmune diseases are undetermined. The aim of our study was to evaluate the prevalence of CHIP in SSc patients and its association with clinical phenotype. Methods Forty-one genes frequently mutated in myeloid malignancies were sequenced in peripheral blood mononuclear cells from 90 SSc patients and 44 healthy donors. Results A total of 15 somatic variants were detected in 13/90 SSc patients (14%) and four somatic variants in 4/44 (9%) healthy donors (HD) (P = 0.58). The prevalence of CHIP was significantly higher in younger SSc patients than in HD: 25% (6/24) vs 4% (1/26) (P = 0.045) under 50 years and 17% (7/42) vs 3% (1/38) (P = 0.065) under 60 years. The prevalence of CHIP in patients over 70 years was similar in SSc patients and healthy donors. The most common mutations occurred in DNMT3A (seven variants). No major clinical differences were observed between SSc patients with or without CHIP. Conclusion Whether CHIP increases the risk to develop SSc or is a consequence of a SSc-derived modified bone marrow micro-environment remains to be explored.

Acute myeloid leukemia (AML) relapse is defined as the presence of bone marrow blasts equal or superior to 5% or reappearance of blasts in the blood or development of extramedullary disease after h...

Key Points We detail at clinical, cytological, cytogenetic, and molecular levels 113 cases of MDS and MDS/MPN with del(11q), a rare recurrent event.CADM1, a tumor suppressor gene identified initially in solid tumors, ATM, CBL, and KMT2A are deleted and/or mutated in del(11q)., Visual Abstract, Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic, and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count, and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A, and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as Tumor Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid-to-myeloid ratio in bone marrow, although not altering their multilineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM, and CBL and mutations of ASXL1, SF3B1, and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.

Somatic mutations of isocitrate dehydrogenase 1 (IDH1R132) genes are found in 6–10% of AML [1]. IDH1R132 mutations are most frequent in cytogenetically normal AML and significantly associated with ...

International audience; No abstract available

International audience; Dendrogenin A (DDA) is a mammalian cholesterol metabolite that displays potent antitumor properties on acute myeloid leukemia (AML). DDA triggers lethal autophagy in cancer cells through a biased activation of the oxysterol receptor LXRβ, and the inhibition of a sterol isomerase. We hypothesize that DDA could potentiate the activity of an anticancer drug acting through a different molecular mechanism, and conducted in vitro and in vivo combination tests on AML cell lines and patient primary tumors. We report here results from tests combining DDA with antimetabolite cytarabine (Ara-C), one of the main drugs used for AML treatment worldwide. We demonstrated that DDA potentiated and sensitized AML cells, including primary patient samples, to Ara-C in vitro and in vivo. Mechanistic studies revealed that this sensitization was LXRβ-dependent and was due to the activation of lethal autophagy. This study demonstrates a positive in vitro and in vivo interaction between DDA and Ara-C, and supports the clinical evaluation of DDA in combination with Ara-C for the treatment of AML.

Two recent phase 3 trials showed that outcomes for relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) patients may be improved by a single-agent tyrosine kinase inhibitor (TKI) (i.e., quizartinib or gilteritinib). In the current study, we retrospectively investigated the characteristics and real-world outcomes of R/R FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) patients in the Toulouse-Bordeaux DATAML registry. In the study, we included 316 patients with FLT3-ITD AML that received intensive chemotherapy as a first-line treatment. The rate of complete remission (CR) or CR without hematological recovery (CRi) was 75.2%, and 160 patients were R/R after a first-line TKI-free treatment (n = 294). Within the subgroup of R/R patients that fulfilled the main criteria of the QUANTUM-R study, 48.9% received an intensive salvage regimen, none received hypomethylating agents or low-dose cytarabine. Among the R/R FLT3-ITD AML patients with CR1 durations &lt, 6 months who received intensive TKI-free treatment, the rate of CR or CRi after salvage chemotherapy was 52.8%, and these results allowed a bridge to be transplanted in 39.6% of cases. Finally, in this QUANTUM-R standard arm-matched cohort, the median overall survival (OS) was 7.0 months and 1-, 3- and 5-year OS were 30.2%, 23.7% and 21.4%, respectively. To conclude, these real-world data show that the intensity of the second-line treatment likely affects response and transplantation rates. Furthermore, the results indicate that including patients with low-intensity regimens, such as low-dose cytarabine or hypomethylating agents, in the control arm of a phase 3 trial may be counterproductive and could compromise the results of the study.

The prognostic impact of immunophenotypic CD34+CD38-CD123+ leukemic stem cell (iLSC) frequency at diagnosis has been demonstrated in younger patients treated by intensive chemotherapy, however, this is less clear in older patients. Furthermore, the impact of iLSC in patients treated by hypomethylating agents is unknown. In this single-center study, we prospectively assessed the CD34+CD38-CD123+ iLSC frequency at diagnosis in acute myeloid leukemia (AML) patients aged 60 years or older. In a cohort of 444 patients, the median percentage of iLSC at diagnosis was 4.3%. Significant differences were found between treatment groups with a lower median in the intensive chemotherapy group (0.6%) compared to hypomethylating agents (8.0%) or supportive care (11.1%) (p &lt, 0.0001). In the intensive chemotherapy group, the median overall survival was 34.5 months in patients with iLSC &le, 0.10% and 14.6 months in patients with &gt, 0.10% (p = 0.031). In the multivariate analyses of this group, iLSC frequency was significantly and independently associated with the incidence of relapse, event-free, relapse-free, and overall survival. However, iLSC frequency had no prognostic impact on patients treated by hypomethylating agents. Thus, the iLSC frequency at diagnosis is an independent prognostic factor in older acute myeloid patients treated by intensive chemotherapy but not hypomethylating agents.

Objectives Complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug. Here, we report a single-center experience of compassionate use of dactinomycin in untreated or relapsed/ refractory NPM1-mutated AML. Methods From September 2015 to February 2019, 26 adult patients with NPM1-mutated AML received dactinomycin in different situations: front-line treatment in 4 unfit patients (16%); morphologic (n = 16, 62%), molecular relapses (n = 4, 16%), refractory disease (n = 1, 13%), or postremission therapy in second complete response (n = 1, 13%). Results Median age was 62.5 years. Median number of dactinomycin cycle was 1 (1-8), and 7 patients (27%) received more than 3 cycles. Three out of 17 patients (18%) in morphologic relapse or refractory to chemotherapy achieved complete remission after the first cycle of dactinomycin. None of the 4 patients unfit for intensive chemotherapy responded to dactinomycin as front-line therapy. Grade 3-4 adverse events were thrombocytopenia (n = 11, 42%), neutropenia (n = 11, 42%), GI toxicity (n = 6, 23%), mucositis (n = 5, 19%), lung infection (n = 5, 19%), and skin rash (n = 2, 7.6%). Conclusions Dactinomycin is an inexpensive and easily available drug that may induce significant responses in few AML patients with NPM1 mutations with an acceptable safety profile.

The prognostic impact of immunophenotypic CD34+CD38−CD123+ leukemic stem cell (iLSC) frequency at diagnosis has been demonstrated in younger patients treated by intensive chemotherapy, however, this is less clear in older patients. Furthermore, the impact of iLSC in patients treated by hypomethylating agents is unknown. In this single-center study, we prospectively assessed the CD34+CD38−CD123+ iLSC frequency at diagnosis in acute myeloid leukemia (AML) patients aged 60 years or older. In a cohort of 444 patients, the median percentage of iLSC at diagnosis was 4.3%. Significant differences were found between treatment groups with a lower median in the intensive chemotherapy group (0.6%) compared to hypomethylating agents (8.0%) or supportive care (11.1%) (p 0.10% (p = 0.031). In the multivariate analyses of this group, iLSC frequency was significantly and independently associated with the incidence of relapse, event-free, relapse-free, and overall survival. However, iLSC frequency had no prognostic impact on patients treated by hypomethylating agents. Thus, the iLSC frequency at diagnosis is an independent prognostic factor in older acute myeloid patients treated by intensive chemotherapy but not hypomethylating agents.

A recent phase 3 trial showed that the outcome of patients with relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) improved with gilteritinib, a single-agent second-generation FLT3 tyrosine kinase inhibitor (TKI), compared with standard of care. In this trial, the response rate with standard therapy was particularly low. We retrospectively assessed the characteristics and outcome of patients with R/R FLT3-mutated AML included in the Toulouse&ndash, Bordeaux DATAML registry. Among 347 patients who received FLT3 TKI-free intensive chemotherapy as first-line treatment, 174 patients were refractory (n = 48, 27.6%) or relapsed (n = 126, 72.4%). Salvage treatments consisted of intensive chemotherapy (n = 99, 56.9%), azacitidine or low-dose cytarabine (n = 9, 5.1%), other low-intensity treatments (n = 17, 9.8%), immediate allogeneic stem cell transplantation (n = 4, 2.3%) or best supportive care only (n = 45, 25.9%). Among the 114 patients who previously received FLT3 TKI-free intensive chemotherapy as first-line treatment (refractory, n = 32, 28.1%, relapsed, n = 82, 71.9%), the rate of CR (complete remission) or CRi (complete remission with incomplete hematologic recovery) after high- or low-intensity salvage treatment was 50.0%, with a bridge to transplant in 34.2% (n = 39) of cases. The median overall survival (OS) was 8.2 months (interquartile range, 3.0&ndash, 32), 1-, 3- and 5-year OS rates were 36.0% (95%CI: 27&ndash, 45), 24.7% (95%CI: 1&ndash, 33) and 19.7% (95%CI: 1&ndash, 28), respectively. In this real-word study, although response rate appeared higher than the controlled arm of the ADMIRAL trial, the outcome of patients with R/R FLT3-mutated AML remains very poor with standard salvage therapy.

A recent phase 3 trial showed that the outcome of patients with relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) improved with gilteritinib, a single-agent second-generation FLT3 tyrosine kinase inhibitor (TKI), compared with standard of care. In this trial, the response rate with standard therapy was particularly low. We retrospectively assessed the characteristics and outcome of patients with R/R FLT3-mutated AML included in the Toulouse–Bordeaux DATAML registry. Among 347 patients who received FLT3 TKI-free intensive chemotherapy as first-line treatment, 174 patients were refractory (n = 48, 27.6%) or relapsed (n = 126, 72.4%). Salvage treatments consisted of intensive chemotherapy (n = 99, 56.9%), azacitidine or low-dose cytarabine (n = 9, 5.1%), other low-intensity treatments (n = 17, 9.8%), immediate allogeneic stem cell transplantation (n = 4, 2.3%) or best supportive care only (n = 45, 25.9%). Among the 114 patients who previously received FLT3 TKI-free intensive chemotherapy as first-line treatment (refractory, n = 32, 28.1%; relapsed, n = 82, 71.9%), the rate of CR (complete remission) or CRi (complete remission with incomplete hematologic recovery) after high- or low-intensity salvage treatment was 50.0%, with a bridge to transplant in 34.2% (n = 39) of cases. The median overall survival (OS) was 8.2 months (interquartile range, 3.0–32); 1-, 3- and 5-year OS rates were 36.0% (95%CI: 27–45), 24.7% (95%CI: 1–33) and 19.7% (95%CI: 1–28), respectively. In this real-word study, although response rate appeared higher than the controlled arm of the ADMIRAL trial, the outcome of patients with R/R FLT3-mutated AML remains very poor with standard salvage therapy.

Hypomethylating agents are a classical frontline low-intensity therapy for older patients with acute myeloid leukemia. Recently, TP53 gene mutations have been described as a potential predictive biomarker of better outcome in patients treated with a ten-day decitabine regimen., However, functional characteristics of TP53 mutant are heterogeneous, as reflected in multiple functional TP53 classifications and their impact in patients treated with azacitidine is less clear. We analyzed the therapeutic course and outcome of 279 patients treated with azacitidine between 2007 and 2016, prospectively enrolled in our regional healthcare network. By screening 224 of them, we detected TP53 mutations in 55 patients (24.6%), including 53 patients (96.4%) harboring high-risk cytogenetics. The identification of any TP53 mutation was associated with worse overall survival but not with response to azacitidine in the whole cohort and in the subgroup of patients with adverse karyotype. Stratification of patients according to three recent validated functional classifications did not allow the identification of TP53 mutated patients who could benefit from azacitidine. Systematic TP53 mutant classification will deserve further exploration in the setting of patients treated with conventional therapy and in the emerging field of therapies targeting TP53 pathway.

Germline GATA2 mutations are identified in a complex disorder termed GATA2 deficiency syndrome. Clinical heterogeneous manifestations are associated with a wide diversity of molecular alterations (missense, frameshift, nonsense, intronic or splicing mutations). Truncating mutations decrease GATA2 expression suggesting a haploinsufficiency mechanism while molecular consequences of missense mutations are ill-known. We focus our research on the most recurrent GATA2 mutation, GATA2 R396Q. Our in vitro results indicate that the ectopic expression of GATA2 R396Q has a strong impact on the clonogenicity associated with an excessive granulocyte differentiation. This data motivates the elaboration of a more physiological approach through the development of a new knock-in mouse model with endogenous expression of Gata2 R396Q mutation. This model allowed us to address the question of the impact of this missense mutation on hematopoiesis at steady state and in challenging conditions. The Gata2R396Q/+ mice showed an abnormal distribution of the immature progenitors Lin - Sca-1 + Kit + (LSK) subpopulations, mainly defines by an increase of the number of aberrant Long-Term Hematopoietic Stem cells (LT-HSC) contrasting with the decrease of LT-HSC population in Gata2+/- mouse model. Functional assays on Gata2R396Q/+ LSK cells revealed also qualitative defects. Indeed, their reconstitution capacity and their response to inflammatory or chemotherapy stimuli seemed to be largely affected. To address at the molecular level the impact of the missense mutation in these progenitors, we combined gene expression analysis with chromatin gene accessibility. These analyses revealed a strong disruption of the cells' ability to respond to stimuli, such as IFN or TNFα signaling, confirming what we observed at the cellular level in functional assays. Furthermore, specific RNA sequencing of the LT-HSC, ST-HSC and MPP3-4 subpopulations showed that the majority of these molecular perturbations are detected in every subpopulation of the LSK cells. In addition, we are able to establish a precise genetic signature in LT-HSC that may be related to their major functional defects. Combination of in vitro and in vivo approaches leads to improve our understanding of GATA2 deficiency syndrome. Modelization of the most recurrent germline Gata2 mutation revealed a different phenotype than the haploinsufficient mouse model. Furthermore, expression of Gata2 R396Q mutation qualitatively impacts the LSK compartment mimicking functional and molecular defects observed in the GATA2 deficiency patients. Disclosures Delabesse: Novartis: Consultancy; Astellas: Consultancy. Pasquet: Novartis: Consultancy.

Background: The WHO 2016 classification identifies myeloid malignancies with germline predisposition as a distinct subgroup (Arber DA et al., Blood 2016). We and other reported mutations of the DEAD-box RNA helicase 41 gene (g DDX41m) as the most common predisposition to MDS/AML (Sébert et al., Blood 2019). Relatively good outcomes have been suggested in small cohorts receiving heterogeneous treatment, but the prognostic significance of g DDX41m in AML patients (pts) treated with intensive chemotherapy (IC) has never been reported. Here, we analyzed the prognostic impact of g DDX41m in a large cohort of newly diagnosed AML pts treated with IC in 5 prospective ALFA (Acute Leukemia French Association) and FILO (French Innovative Leukemia Organization) trials. Methods: We retrospectively screened 1690 AML pts (aged 18-85y) treated in ALFA0701 (EudraCT 2007-002933-36), ALFA0702 (NCT00932412), ALFA1200 (NCT01966497), ALFA1401 (NCT02473146) and LAM-SA (NCT00590837) clinical trials for DDX41 mutations using High Throuput Sequencing. DDX41 variants with a variant allele frequency (VAF) >40% were interpreted as causal if they were pathogenic or likely pathogenic by the American College of Medical Genetics and Genomics (ACMG) guidelines. The concurrence of a somatic DDX41 mutation was also considered as a strong evidence for the causality. Correlation between g DDX41m and covariates was realized using point biserial correlation and Fisher test for continuous and dichotomic variables, respectively. Allogeneic hematopoietic stem cell transplantation (HSCT) was considered as a time-depending variable, and all outcome analyses were stratified on the clinical trial. Results: We identified 86 unrelated pts with DDX41-related AML representing 5% of the whole cohort; 66 (77%) of them had additional somatic DDX41 mutations. Most common germline variants were p.D140fs (21%), p.M1? (8%), p.L283fs (7%) and p.K331del (5%); p.R525H and p.G530D/C/S accounted for 80% and 10% of all somatic mutations respectively. Compared to wild-type pts, DDX41-related AML were significantly older (65.5 vs 64y, p=0.036), with male predominance (74 vs 54%, p=0.002), had higher rates of normal karyotypes (77 vs 57%, p=0.006), lower WBC (2.0 vs 7.9 G/L, p After one induction course, CR/CRp was achieved in 81 (94%) DDX41-related AML compared to 1164 (73%) in DDX41-wt pts. In a multivariate analysis including WBC, ELN-2017 classification and clinical trial, presence of a g DDX41m was associated with significant higher CR/CRp achievement (OR, 5.39 [95% CI, 2.33-15.67]; p=0.0004) (Figure 1B). After a median follow-up of 47.8 months, DDX41-related AML had a median OS of 39.7 (IQR, 19.4-66.4) months compared to 29.1 (IQR,10-not reached [NR]) months in DDX41wt (p=0.045). However, the prognostic impact on OS of g DDX41m was not independent of WBC and ELN-2017 classification (p=0.5). Relapse rates in DDX41-related pts were lower at 1-year (11 vs 30%), but then increased to join the relapse rates of DDX41-wt pts at 3 years (50 vs 50%, Figure 1C). Finally, 35 DDX41-related and 288 non-favorable DDX41-wt pts received an HSCT in first CR. HSCT was associated with a prolonged OS in the non-favorable DDX41-wt cohort (HR, 0.61 [95% CI, 0.49-0.76]; p Conclusion: This is the first study evaluating the prognostic impact of g DDX41m in a large cohort of AML pts prospectively treated with IC. DDX41-related AMLs represented a rare specific entity and were associated with a higher response rate, prolonged time to relapse without independent OS advantage compared to DDX41-wt AML. These results suggest that consolidation/maintenance strategy might be adapted in these pts. Figure 1 Figure 1. Disclosures Lambert: ASTELLAS: Consultancy; CELGENE/BMS: Consultancy. de Botton: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics: Honoraria, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Syros: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pierre Fabre: Other; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Recher: Jazz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MaatPharma: Research Funding; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pigneux: Amgen: Consultancy; Sunesis: Consultancy, Research Funding; BMS Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Dombret: Amgen: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Honoraria, Research Funding; Servier: Research Funding; Abbvie: Honoraria; BMS-Celgene: Honoraria; Daiichi Sankyo: Honoraria. Delabesse: Astellas: Consultancy; Novartis: Consultancy. Sebert: Abbvie: Consultancy; BMS: Consultancy.

INTRODUCTION Cytokines are involved in many processes, including hematopoiesis and inflammation. Aging is associated with the onset of clonal hematopoiesis (CH) of indeterminate potential, putatively associated with a higher risk of progression to hematological malignancies such as myelodysplastic syndromes or acute myeloid leukemia. Moreover, CH may participate to create a pro-inflammatory environment contributing to the pathogenesis of age-related diseases, such as cardiovascular diseases. This is likely to be driven by or translated in changes in bone marrow (BM) and/or peripheral blood (PB) soluble factors for which reference values still remain unclear, because BM cytokines levels have never been determined in strictly selected healthy people. Indeed, control BM samples classically used in studies are from subjects undergoing surgeries for non-hematologic causes, such as total hip replacement or cardiac surgery, patients suffering from immune thrombocytopenic purpura, brain death patients or allogeneic BM donors. In this study, the BM and PB plasma concentrations of 49 hematopoietic and inflammatory cytokines were measured in a representative panel of 94 healthy adult volunteers and the results were analyzed considering their age and presence of CH. METHOD Ninety-four healthy donors aged from 18.6 to 80.1 years old (yo), including 58 women were recruited for this study (HEALTHOX protocol, CPP Tours, AFSSAPS identifier ID-RCB: 2016-A00571-50 and ClinicalTrials.gov # NCT02789839). The presence or absence of CH (>1% of variant allele frequency) in this cohort is already known (Guermouche H, Ravalet N et al, Blood Adv 2020;4(15):3550-3557). BM samples were obtained through sternal aspiration using a classical procedure in France, and PB sampling was performed at the same time by venipuncture. Samples were collected on sodium heparin or EDTA, centrifuged twice (1200 g, 10 min, 20 C°), aliquoted and stored at -80°C. A 48-plex human cytokine panel assay was used to quantify 48 human cytokines in PB and BM plasmas [beta-NGF, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL27, CLEC11A, CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, FGF-2, G-CSF, GM-CSF, HGF, IFN-alpha-2, IFN-gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-2-RA, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-12B, IL-13, IL-15, IL-16, IL-17A, IL-18, KITLG, LIF, LT-alpha, M-CSF, MIF, PDGF subunit B, TNF, TNFSF10, VEGF-A]. MIF and FLT3L quantification were performed by ELISA. Regarding CH, the controls were subjects older than 50 yo without CH. Statistical analyses were performed with the R (3.6.3) and Rstudio version 1.2.5042 (www.rstudio.org) software. Comparisons were computed by Wilcoxon and Kruskal-Wallis tests. All pairwise multiple comparisons were performed using Dunn's-test for multiple comparisons of independent samples (PMCMR package). Correlations between cytokine levels in PB and BM were tested with Pearson and Spearman methods. Correlation matrices were plotted using the "corrplot" package. RESULTS CH was detected in 16 volunteers, mostly in individuals over 50 yo. BM and PB plasma samples were studied in 3 age-groups: 18-40, 40-60 and 60-80 yo. With aging, variations were observed for 18 BM cytokine levels, with 7 increasing (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreasing (G-CSF, TNF, IL-2, IL-15, IL-17a, IL-4, LT-alpha, IL-1 alpha). In PB, 10 cytokines significantly increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with significantly higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF (Table). CONCLUSION In this study and for the first time, we concomitantly analyzed BM and PB concentrations of a panel of hematopoietic and inflammatory cytokines in a cohort of strictly selected healthy volunteers. In addition to the establishment of reference values, useful for various biological studies, and correlations between blood and BM levels, we identify variations in the BM of key cytokines according to age and CH. The differences in these cytokine concentrations, either as causes or as consequences, may shape both the BM microenvironment and hematopoietic processes, eventually leading to the beginning of age-related myeloid malignancies or inflammatory conditions. Figure 1 Figure 1. Disclosures Hirsch: Daiichi Sankyo Oncology: Consultancy; Novartis Pharma: Consultancy. Suner: Sanofi - Genzyme: Consultancy. Delhommeau: Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy.