ALBERT

Description: The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.

Description: Introduction: Next generation sequencing (NGS) studies identified additional somatic mutations impacting disease evolution and prognosis in MPN. SF3B1, a component of the U2 small nuclear ribonucleoprotein splicing complex, is frequently mutated in myelodysplastic syndromes where it has been proposed to define a new entity (Malcovati et. al. Blood 2020). In MPN, SF3B1 is mutated in approximately 10% of patients with primary myelofibrosis (PMF) and 3-5% with polycytemia vera or essential thrombocytemia (ET). Recent reports suggested that spliceosome mutations may adversely affect myelofibrosis free survival (MFS) in ET (Tefferi et. al. Br J Haematol. 2020). The main objective of this study was to evaluate the impact of the concomitant presence of driver (JAK2, MPLor CALR) and SF3B1 clonal or sub-clonal mutations on MPN phenotype and evolution in a large single center cohort of MPN patients. Methods: A total of 1243 consecutive patients were diagnosed with MPN according to WHO criteria between January 2011 and May 2020 in our center, of whom 707 had molecular analysis by NGS targeting a panel of 36 myeloid genes performed at diagnosis and/or during follow-up. Significant variants were retained with a sensitivity of 0.5%. Patients were grouped according to variant allele frequencies (VAF) determined by NGS as "driver" SF3B1mutated patients when driver and SF3B1 mutations VAF were similar (double mutated clone), and as "non-driver" SF3B1 mutants when SF3B1VAF was lower than that of the driver mutation, suggestive of a sub-clone. 4 patients with SF3B1 mutations but no driver mutation were excluded. We then compared the characteristics and outcomes of 3 groups of patients according to their SF3B1 mutational status: wild type (WT), driver and non-driver SF3B1 mutations. Results: A total of 39/703 (5.6%) patients had SF3B1 mutations, of whom 11/39 (28.2%) and 28/39 (71.8%) harbored driver and non-driver SF3B1mutations, respectively. Driver SF3B1 mutations were associated with PMF (OR 6.1, 95%CI [1.1; 33.6], p= 0.039) and MPN unclassified (OR 15.6,95%CI [1.1; 116.5], p= 0.007) subtypes, presence of immature myeloid cells ≥ 2% (OR 9.3, CI [2.6; 32.8], p= 0.001) and peripheral blasts ≥ 1% (OR 5.0,95%CI [1.0; 24.7], p= 0.047) at diagnosis (Figure A). Other variables were not significantly different between patients with driver, non-driver and WT SF3B1, including age, driver mutation type, MPN-related symptoms, cytogenetics and high molecular risk mutations (ASXL1, EZH2, SRSF2, IDH1/2or U2AF1). There was no significant difference in the response to therapy: complete hematological response was seen in 5/11 (45.5%), 10/28 (35.7%) and 327/664 (49.3%) of patients with driver, non-driver and WT SF3B1 respectively. After a median follow-up of 103.7 months IQR [47.2; 175.6], evolution to myelofibrosis occurred in 5/7 (71.4%), 6/20 (30.0%) and 99/564 (17.6%) of patients with driver, non-driver and WT SF3B1 respectively. Interestingly, driver SF3B1 but not non-driver SF3B1 mutational status adversely impacted MFS (OR 7.56,95%CI[2.95; 19.38], p

Description: Background: Although patients with MPN may achieve complete hematological remission (CHR) with cytoreductive therapy (CRT), lifelong treatment represents a major burden for these chronically ill patients. IFN has emerged as a major MPN therapy thought to positively alter the natural history of MPN and appears to be the only drug that can provide long-term CHR after discontinuation in some patients (Hasselbalch HC et al., Seminars in Immunopathology, 2019). In this study, we aimed to identify clinical and molecular factors associated with long-term CHR after IFN treatment discontinuation and to compare clinical outcome of patients who discontinued therapy, to patients who continued IFN treatment despite achieving a CHR. Methods: All MPN patients followed between January 2000 and May 2020 in our institution who received at least 3 months of IFN were included. Logistic regression analysis was performed to identify variables associated with long-term CHR after discontinuation. Cumulative incidence of relapse was calculated from time of IFN discontinuation, considering death without relapse as a competitive event. Overall survival (OS) and thrombotic/hemorrhagic events free survival (EFS) curves were estimated using the Kaplan-Meier method and compared by cause-specific hazard Cox models. Results: 381 patients treated with IFN were included in the study. Median age at MPN diagnosis was 44 years [interquartile range: 33-54]. 171 had polycythemia vera (PV), 169 essential thrombocythemia (ET) and 34 primary myelofibrosis (PMF). JAK2V617F was the most frequent driver mutation (78.8% of patients), while CALR and MPL were mutated in 15.5%, and 2.9% of patients respectively. Median driver variant allele frequency (VAF) at time of IFN therapy initiation was 34% [12; 51]. Reasons to start IFN included age younger than 50 years in 44.9%, intolerance/resistance to previous therapies in 17.8% or pregnancy in 1.8% patients. CHR was achieved with IFN in 77.2% of patients. After a median follow-up of 72.4 months [28.4; 119.7] from IFN initiation, 131 patients were still on IFN treatment, while 250 patients had discontinued therapy. No significant difference was observed between continued and discontinued IFN patients in terms of MPN subtype, initial clinical, biological or molecular characteristics. Reasons for discontinuation were toxicity in 128 (50.4%), prolonged hematological CHR in 76 (29.9%), failure in 16 (6.3%) and other in 30 (11.8%) patients. At time of IFN discontinuation, 170 (66.9%) patients were in CHR and the median driver mutation VAF was 12% [3; 35]. Of note, IFN was re-introduced in 61 patients who lost CHR with a second CHR rate of 83.6%, arguing for the absence of development of IFN resistance in post-discontinuation relapses. In multivariate logistic regression analysis, stopping for prolonged CHR (OR 3.52, 95%CI [1.18; 10.56], p=0.024) was associated with a higher CHR without CRT rate, while VAF ≥ 10% at time of IFN discontinuation (OR 0.26, 95%CI [0.1; 0.65], p=0.004) was associated with lower long-term CHR without CRT. Accordingly, VAF ≥ 10% at time of IFN discontinuation (HR 3.06 [1.59; 5.90], p=0.001) was independently associated with a higher cumulative incidence of relapse after IFN discontinuation (Figure A). Driver VAF at IFN discontinuation time was available for 117 patients. 53 patients (45.3%) had a driver VAF 〈 10% at IFN discontinuation. Finally, we compared discontinued IFN patients who stopped IFN for CHR (n=76) to patients who obtained CHR and are still on IFN therapy (n=116). Hematological transformation could not be evaluated due to very low incidence of events: 2 patients from each group progressed to myelofibrosis while leukemic progression was only observed in 1 patient. Importantly, OS (HR 0.23, 95%CI [0.5; 1.14], p=0.07) and EFS (HR 0.53, 95%CI [0.19; 1.45], p=0.217) were not significantly different between patients who discontinued and those who continued IFN. Conclusion: Overall, our study demonstrates that IFN discontinuation represents a safe strategy in MPN patients who achieved CHR, and particularly in patients with a driver VAF lower than 10% at time of discontinuation. Importantly, relapsed patients did not develop IFN resistance. Our data contributes to set the frame for future clinical trials evaluating the possibility of IFN treatment holidays in good responding MPN patients. Disclosures Benajiba: Gilead Foundation: Research Funding. Kiladjian:Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Clonal hematopoiesis (CH) is an age-related condition in which somatic mutations can be detected in the blood of healthy individuals. In the non-transplant setting, CH is associated with an elevated risk of developing hematologic malignancy and an increased risk of non-hematologic outcomes due to altered inflammatory signaling. During hematopoietic cell transplantation (HCT), CH in older donors can engraft in recipients and could therefore influence outcomes through effects on graft immunologic function or by causing donor cell leukemia. A definitive link between donor CH and recipient outcomes has not been established. We therefore evaluated the impact of CH in donors aged 40 years or older on recipient clinical outcomes in 1727 donor-recipient pairs. Methods: We performed targeted, error-suppressed sequencing of 46 genes on 1727 samples from donors age 40 and older. We defined CH as pathogenic mutations at variant allele fraction (VAF) 0.005 or greater. Median donor age was 51 (range 40-80) and median recipient age was 55 (range 1-78). There were 889 matched related donors (51.5%), 454 haploidentical donors (26.3%), 273 matched unrelated donors (15.8%), 71 mismatched unrelated donors (4.1%), and 38 mismatched related donors (2.2%). 929 recipients (53.8%) had myeloid malignancies, 718 (41.6%) had lymphoid malignancies, and 80 (4.6%) had non-malignant diseases. 1022 (59.2%) recipients received peripheral blood stem cell products and 703 (40.7%) received bone marrow. 672 recipients (38.9%) received post-transplant cyclophosphamide (PTCy) for graft versus host disease (GVHD) prophylaxis. Median follow-up for survivors was 6.0 years. Results: We identified CH in 388 of 1727 (22.5%) donor samples. Mutations in DNMT3A were most common (302 mutations in 253 donors), followed by TET2 (96 mutations in 89 donors), ASXL1 (n=22) and PPM1D (n=14). No other genes were mutated in more than 10 donors (0.5%). The presence of donor CH was independently associated with older donor age, but not with donor sex, donor-recipient relatedness, graft source, or recipient disease. The presence of donor CH at VAF 0.01 or greater was associated with improved progression-free survival (PFS) in a multivariable model that included donor and recipient age, HCT-CI, disease category, Disease Risk Index score, conditioning intensity, and donor type (HR for death or relapse 0.81, 95% CI 0.68-0.97, P=0.019). This effect was driven by DNMT3A mutations, which were independently associated with improved overall survival (HR for death 0.75; 0.59-0.95, P=0.016) and reduced risk of relapse (sHR 0.76; 0.59-0.97, P=0.029) in the same model. CH involving other gene mutations, including TET2 and genes other than DNMT3A/TET2, was not significantly associated with any recipient outcome. Smaller clones (VAF 0.005-0.01) had no effect on any outcome. The association between donor DNMT3A-CH and recipient outcomes was limited to those who did not receive PTCy for GVHD prophylaxis (Figure 1A-C). In the model described above, donor DNMT3A-CH in the absence of PTCy was independently associated with improved PFS (HR 0.61; 0.45-0.84, P=0.002), reduced risk of relapse (sHR 0.59; 0.39-0.9, P=0.014) and an elevated risk of chronic GVHD (sHR 1.36; 1.01-1.83, P=0.042) compared to those without DNMT3A-CH. In recipients who received PTCy, there was no significant effect of donor DNMT3A-CH on PFS, relapse, or cGVHD (1D). Eight recipients developed donor cell leukemia (DCL), for a cumulative incidence of 0.7% at 10 years. In seven of these cases, we identified gene mutations in the corresponding donor products that matched the mutations found in the subsequent DCL, including 2 with TP53 mutations, 3 with splicing factor mutations, and 2 with germline DDX41 mutations that were present in both donor and recipient. No recipients who received products with sole DNMT3A-CH developed DCL. Conclusions: In HCT donors age 40 or older, the presence of DNMT3A clonal hematopoiesis at VAF 〉/= 0.01 is independently associated with prolonged overall and progression-free survival in transplant recipients. This effect is driven by reduced risk of disease relapse and confined to recipients who do not receive PTCy, suggesting that it is mediated at least in part by effects on donor T cells. The risk of direct evolution of DNMT3A-CH to donor cell leukemia is low, and most DCLs were traced to atypical donor CH involving MDS-associated genes or germline risk alleles. Disclosures Nikiforow: Novartis: Honoraria; Nkarta Therapeutics: Honoraria; Kite/Gilead: Honoraria. DeZern:MEI: Consultancy; Abbvie: Consultancy; Astex: Research Funding; Celgene: Consultancy, Honoraria. Ritz:Rheos Medicines: Consultancy; Infinity Pharmaceuticals: Consultancy; Amgen: Research Funding; Equillium: Research Funding; Kite Pharma: Research Funding; Avrobio: Consultancy; Falcon Therapeutics: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; LifeVault Bio: Consultancy. Soiffer:VOR Biopharma: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Juno: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; alexion: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy. Lindsley:Jazz Pharmaceuticals: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy; MedImmune: Research Funding; Bluebird Bio: Consultancy.

Description: Introduction: Approximately 20% of Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML) are associated with SIAD, but pathophysiological mechanisms underlying this association remain largely unknown and data on somatic mutations are very scarce. Autoreactive T lymphocytes play a major role in SIAD development. The main objective of this study was to investigate the mutational landscape of MDS/CMML associated with SIAD, and its impact on the immunological phenotype of T lymphocytes. Patients and Methods: This retrospective study was conducted in the hematology department of Hôpital Saint-Louis, Paris, in all MDS/CMML patients diagnosed between 2012 and 2017, and with a molecular analysis by NGS targeting a panel of 80 genes. MDS/CMML patients with associated SIAD (n= 85, whose diagnosis was based on usual international criteria) were compared with MDS/CMML patients without SIAD (n=319) who constituted the control cohort. Flow cytometry was performed on peripheral blood samples from 28 MDS/CMML patients (of whom 12/28 had SIAD) and 18 healthy controls, on a BD Fortessa X20 to study CD8+T lymphocytes subsets and the expression of immune checkpoints. Analysis was performed with FlowAI R v1.14.0 and Cytobank website for T cells clustering with unsupervised Citrus algorithm. Results: We included 404 MDS/CMML patients (323 MDS, 81 CMML), of whom 85 (21%) had SIAD diagnoses including 35 (34%) inflammatory arthritis, 20 (19%) systemic vasculitis, 16 (15%) autoimmune cytopenias, 15 (14%) connective tissue diseases, 9 (9%) neutrophilic dermatosis, 6 (6%) inflammatory bowel disease, and 3 (3%) unclassified SIAD. Both SIAD and control cohorts were similar in terms of age, levels of cytopenias, MDS/CMML subtypes and cytogenetic features. There was a preponderance of low risk MDS/CMML (85.9% and 82.8% in SIAD and control cohort), and the number of patients with CMML did not differ between the 2 groups (p=0.36). Median follow-up was 32.2 months in the whole cohort. Median overall survival was 95.7 months [60.7 - not estimable] and 101.0 months [70.8 - not estimable] in the control and SIAD cohort respectively (p=0.91). Figure A represents the mutational landscape of MDS/CMML patients for the recurrent mutated genes. Median number of mutations was 2 [1-4] in both cohorts (ns). TET2mutations were found in 39/85 (46%) and 108/319 (34%) patients in the SIAD and control cohorts respectively (p=0.04), and IDH1/2 mutationsin 12/85 (14%) patients in the SIAD cohort vs 14/319 (4%) in the control group (p

Description: Introduction: Ruxolitinib (RUX) is a JAK 1/2 inhibitor approved for the treatment of intermediate and high-risk primary or secondary MF. In addition to the intrinsic immune system modulation in MF patients, JAK inhibition exerts an immunosuppressive effect that may increase the risk of infections (Heine A et al., Blood, 2013). We aimed to investigate the association between treatment with RUX and infectious complications in a large monocentric cohort of MF patients. Methods: We performed a retrospective study including all patients diagnosed with primary, post-polycythemia vera or post-essential thrombocythemia MF in our MPN clinic between January 2011 and April 2020. Patients were considered RUX treated when exposed to treatment for at least two consecutive months. Clinical and biological characteristics at time of MF diagnosis and follow-up were collected from medical charts and electronic medical records. We used logistic regression models to identify specific infection subtypes associated with RUX treatment. Univariate and multivariate analysis assessing the impact of categorical and continuous variables on infectious events cumulative incidence were performed using Cox models. Results: 213 MF patients were included in the study. 114 (51.1%) patients were treated with RUX while 99 didn't receive RUX. Overall 102 patients exhibited a total of 231 infectious episodes. To search for factors associated with occurrence of infectious events, we evaluated the characteristics of MF, potential clinical and biological immune suppression factors (immunosuppressive treatment, prior non-MPN cancer, diabetes, hepatic/renal failure, neutropenia, lymphopenia, hypo-gammaglobulinemia, complement deficiency, prior history of infection), use of infection prophylaxis and RUX treatment using Cox models. In this cohort of MF patients, we found that RUX treatment (HR 5.91, 95%CI[3.48; 10.04], p 2 (HR 4.91, 95%CI[2.62; 9.21], p

Description: Introduction: MPN are a heterogeneous group of chronic hematological malignancies often resulting from a combination of a driver gene mutation (JAK2, MPL or CALR) and a variety of somatic mutations harboring diverse prognosis values. A subset of MPN patients carry somatic mutations in the hematopoietic transcription factor NFE2 (nuclear factor erythroid 2) resulting in a functionally enhanced truncated form of NFE2 (Jutzi JS et al., JEM, 2013). Moreover, epigenetically induced overexpression of NFE2 has recently been reported in the majority of MPN patients (Peeken JC et al., Blood, 2018). In transgenic murine models, NFE2 overexpression results in an MPN phenotype (thrombocytosis, leukocytosis, EPO-independent colony formation, characteristic bone marrow histology and expansion of stem and progenitor compartments) and has recently been shown to predispose to the acquisition of additional genetic abnormalities and subsequent leukemic transformation (Kaufmann KB et al., JEM, 2012) (Jutzi JS et al., Blood, 2019). However, clinical impact of NFE2 mutations in MPN patients remains unknown. The aim of this study was to evaluate the phenotypic characteristics and prognostic impact of NFE2 somatic mutations in a large mono-centric cohort of MPN patients. Methods: A total of 1243 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and May 2020. This study included 707 of them in whom a next-generation sequencing (NGS) molecular analysis targeting 36 myeloid genes was performed at diagnosis and/or during follow-up. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Statistical analyses were performed using the STATA software (STATA 15.0 Corporation, College Station, TX). Results: In our cohort, 411 patients presented with polycythemia vera (PV), 577 with essential thrombocythemia (ET), 184 with primary or pre-fibrotic myelofibrosis (PMF), 59 with unclassified MPN and 12 with MDS/MPN. Median age at diagnosis was 51 years [40-63]. 73.1% patients had a JAK2V617F mutation, 14.1% a CALR mutation and 3.3% a MPL mutation. Overall, 64 (9.05%) patients harbored a NFE2 mutation with a variant allelic frequency (VAF) ≥ 0.5% and 36 had a VAF ≥ 5%, the latest were considered as NFE2 mutated for the rest of the study as VAF