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The mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients’ cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.

Supplementary Table 6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 2 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 3 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Figure 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 5 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Methods, Table Legends 1-6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 4 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

There are few data regarding the association of autoimmune and inflammatory diseases (AID) with Philadelphia negative myeloproliferative neoplasms (MPN). In this retrospective study, we describe the prevalence, clinical and biological features and outcome of AID association in MPN. A total of 1541 MPN patients were included, encompassing 95 (6%) patients with AID. Female patients were predominant within the AID group (65% versus 54%, p=0.03). A total of 103 AID diagnoses were reported in 95 patients, including 48 organ-specific AID, 13 inflammatory arthritis, 9 connective tissue diseases, 9 dermatosis, 6 systemic vasculitis and 18 unclassified AID. The prevalence of TET2 mutations was higher in the AID cohort (32% versus 22%), although not statistically significant (p=0.08). In subgroup analysis of patients with myelofibrosis, TET2 mutations were more prevalent in AID group (p=0.025). The prevalence of driver and other additional mutations did not differ between the 2 groups. The association with AID did not impact overall survival (p=0.67), transformation-free survival (p=0.37) or secondary myelofibrosis-free survival (p=0.91). Our data suggest that the prevalence of AID is similar in MPN patients to that of the general population. TET2 mutations are highly prevalent in MPN patients with AID potentially suggesting a shared physiopathology.

Superior rates of deep molecular response (DMR) have been reported with the combination of tyrosine kinase inhibitors and pegylated-interferon-alpha (Peg-IFN) in patients with newly diagnosed chronic phase-chronic myeloid leukaemia (CP-CML). In this setting, this study investigated the efficacy and safety of dasatinib combined to Peg-IFN-α2b (Dasa-PegIFN, NCT01872442). A total of 79 patients (age ≤65 years) started dasatinib; 61 were eligible for Peg-IFNα-2b add-on therapy at month 3 for a maximum 21-months duration. Dasatinib was continued thereafter. The primary endpoint was the cumulative rate of molecular response 4.5 log (MR

Musculoskeletal (MSK) pains have been reported during TKI treatment or after its discontinuation in patients with chronic myeloid leukemia (CML). We hypothesized that MSK pains originate from calcific tendinopathy according to preliminary clinical observations. We conducted a retrospective study including CML patients divided into three groups: patients with MSK pain during TKI treatment; asymptomatic patients during TKI treatment; patients with MSK pain after TKI discontinuation. Patients with MSK pain were clinically evaluated, and the presence of calcific deposits was assessed in X-rays of both shoulders and pelvis. Forty-five patients were included; 14 described MSK pain during TKI treatment and 12 after TKI discontinuation. A diagnosis of rotator cuff tendinopathy was retained for 57.7% of patients and of gluteus tendinopathy in 19.2%. The prevalence of calcifications in shoulders and/or hips was 64.3% in symptomatic patients receiving TKIs, 63.2% in asymptomatic patients and 75.0% in patients with MSK pain after TKI treatment.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Myeloproliferative neoplasms (MPNs) are uncommon in children/young adults. Here, we present data on unselected patients diagnosed before 25 years of age included from 38 centers in 15 countries. Sequential patients were included. We identified 444 patients, with median follow-up 9.7 years (0-47.8). Forty-nine (11.1%) had a history of thrombosis at diagnosis, 49 new thrombotic events were recorded (1.16% patient per year [pt/y]), perihepatic vein thromboses were most frequent (47.6% venous events), and logistic regression identified JAK2V617F mutation (P = .016) and hyperviscosity symptoms (visual disturbances, dizziness, vertigo, headache) as risk factors (P = .040). New hemorrhagic events occurred in 44 patients (9.9%, 1.04% pt/y). Disease transformation occurred in 48 patients (10.9%, 1.13% pt/y), usually to myelofibrosis (7.5%) with splenomegaly as a novel risk factor for transformation in essential thrombocythemia (ET) (P= .000) in logistical regression. Eight deaths (1.8%) were recorded, 3 after allogeneic stem cell transplantation. Concerning conventional risk scores: International Prognostic Score for Essential Thrombocythemia-Thrombosis and new International Prognostic Score for Essential Thrombocythemia-Thrombosis differentiated ET patients in terms of thrombotic risk. Both scores identified high-risk patients with the same median thrombosis-free survival of 28.5 years. No contemporary scores were able to predict survival for young ET or polycythemia vera patients. Our data represents the largest real-world study of MPN patients age < 25 years at diagnosis. Rates of thrombotic events and transformation were higher than expected compared with the previous literature. Our study provides new and reliable information as a basis for prospective studies, trials, and development of harmonized international guidelines for the specific management of young patients with MPN. ispartof: BLOOD ADVANCES vol:6 issue:17 pages:5171-5183 ispartof: location:United States status: published

Summary We have reconciled steady-state and stress hematopoiesis in a single mathematical model based on murine in vivo experiments and with a focus on hematopoietic stem and progenitor cells. A phenylhydrazine stress was first applied to mice. A reduced cell number in each progenitor compartment was evidenced during the next 7 days through a drastic level of differentiation without proliferation, followed by a huge proliferative response in all compartments including long-term hematopoietic stem cells, before a return to normal levels. Data analysis led to the addition to the 6-compartment model, of time-dependent regulation that depended indirectly on the compartment sizes. The resulting model was finely calibrated using a stochastic optimization algorithm and could reproduce biological data in silico when applied to different stress conditions (bleeding, chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependent model of immature hematopoiesis provides new avenues to a better understanding of both normal and pathological hematopoiesis., Graphical abstract, Highlights • We describe a new 6-compartment time-dependent regulated model of hematopoiesis • Biological data under steady state and stress and cell dynamics were used • Modeling is able to recapitulate effects from chemotherapy, bleeding, or HSC depletion, Cell biology; Stem cells research; In silico biology

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM=125%), 92·1% and 90% respectively. A very-low erythropoietin level (=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Myelodysplastic syndromes and acute myeloid leukemia with TP53 mutations are characterized by frequent relapses, poor or short responses, and poor survival with the currently available therapies including chemotherapy and 5-azacitidine (AZA). PRIMA-1Met(APR-246,APR) is a methylated derivative of PRIMA-1, which induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function of mutant p53. Here we show that low doses of APR on its own or in combination with AZA reactivate the p53 pathway and induce an apoptosis program. Functionally, we demonstrate that APR exerts these activities on its own and that it synergizes with AZA in TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cell lines and in TP53-mutated primary cells from MDS/AML patients. Low doses of APR on its own or in combination with AZA also show significant efficacy in vivo Lastly, using transcriptomic analysis, we found that the APR + AZA synergy was mediated by downregulation of the FLT3 pathway in drug-treated cells. Activation of the FLT3 pathway by FLT3 ligand reversed the inhibition of cell proliferation by APR + AZA. These data suggest that TP53-mutated MDS/AML may be better targeted by the addition of APR-246 to conventional treatments.

The effectiveness of tyrosine kinase inhibitors (TKIs) has made it possible to consider treatment discontinuation in chronic myeloid leukaemia (CML) patients that achieve an excellent response. However, a few of the patients included in the Europe Stop Tyrosine Kinase Inhibitors (EURO-SKI) trial reported musculoskeletal pain shortly after stopping TKIs, considered as a withdrawal syndrome (WS). To identify factors that may predispose to TKI WS, we analysed the pharmacovigilance declarations for the 6 months after stopping TKIs in a large cohort of CML (n = 427) that combined the French patients included in the STop IMatinib 2 (STIM2; n = 224) and EURO-SKI (n = 203) trials. Among these patients, 23% (99/427) developed TKI WS after stopping imatinib (77/373; 20·4%), nilotinib (12/29; 41·4%) or dasatinib (10/25; 40%). WS concerned mainly the upper body joints, and required multiple symptomatic treatments in 30% of patients. Univariate and multivariate analyses identified two risk factors: duration of TKI treatment [risk ratio (RR) = 1·68 (1·02-2·74)] with a 93-month cut-off time, and history of osteoarticular symptoms [RR = 1·84 (1·04-3·28)]. These findings confirm that WS is a TKI class effect. CML patients should be carefully screened before treatment initiation to identify pre-existent osteoarticular symptoms. Moreover, before TKI discontinuation, patients should be informed of the possibility of WS, particularly after a long treatment period.

Introduction Les maladies auto-immunes et inflammatoires (MAI) sont associees a un sur-risque de survenue de neoplasies myeloproliferatives (NMP) [1] . Chez les patients presentant un syndrome myelodysplasique (SMD) ou une leucemie myelomonocytaire chronique (LMMC), les mutations des regulateurs epigenetiques TET2 et IDH1/2 etaient significativement plus frequentes chez les patients porteurs de MAI comparativement a une cohorte temoin de patients SMD/LMMC sans MAI [2] . Dans le contexte des NMP, le phenotype des MAI associees et leur statut mutationnel ont ete peu decrits. Les objectifs de notre etude etaient de decrire les caracteristiques cliniques et genetiques des patients presentant une NMP avec une MAI et d’evaluer leur impact pronostique. Patients et Methodes Dans notre centre, 1541 patients ont ete diagnostiques d’une NMP selon les criteres OMS, entre janvier 2011 et janvier 2021. Des analyses moleculaires par sequencage haut debit (NGS) ciblant un panel de 36 genes mutes dans les NMP ont ete realisees pour 998 d’entre eux, au diagnostic ou durant le suivi. Tous les cas de MAI ont ete revus par un interniste et les diagnostics evalues selon les criteres internationaux correspondants. Les diagnostics de MAI post-interferon ont ete exclues. Resultats Notre cohorte comprenait 522 (34 %) polyglobulie de Vaquez (PV), 709 (46 %) thrombocytemie essentielle (TE) et 229 (15 %) myelofibrose primitive (MFP). Cent (6.6 %) patients presentaient une MAI associee et les 1441 patients restants ont constitue notre population temoin. L’âge median de nos patients dans la cohorte globale etait de 54 ans [IQR 40.4–63.2] et il y avait une predominance de femmes dans le groupe avec MAI associee (66 (66 %) versus 769 (53 %), p = 0.019). Il n’y avait pas de difference significative entre les 2 groupes concernant les sous-types de NMP, les mutations « driver » (JAK2, MPL et CALR) ou encore l’hemogramme au diagnostic des NMP. L’incidence d’episodes thrombotiques ou hemorragiques etait aussi similaire dans les 2 groupes (44 (44 %) versus 564 (39 %), p = 0.356). Le diagnostic de MAI etait anterieur a celui de NMP dans 34 % des cas, concomitant dans 12 % et ulterieur dans 31 %. Sur les 100 MAI, 45 etait des maladies auto-immunes specifiques d’organe, principalement des thyroidites auto-immunes, 13 rhumatismes inflammatoires chroniques, 9 connectivites, 8 dermatoses inflammatoires, 7 vascularites et 18 inclasses. Au total, 70 % des maladies auto-immunes remplissaient leurs criteres de classification. Parmi les patients ayant beneficie d’une analyse moleculaire, 62 % avaient une mutation additionnelle a la mutation « driver ». Les mutations de TET2 etaient plus frequemment retrouvees chez les patients avec une MAI associee (24 (34 %) versus 205 (22 %), OR = 1.84 95 %CI [1.08–3.07], p = 0.028), mais elle n’etait pas associee a un sous-type de MAI particulier. Il existait une tendance a une surrepresentation des mutations IDH1/2 dans le groupe MAI (4 (6 %) versus 27 (3 %), OR = 2.02 95 %CI[0.74–5.51], p = 0.27), bien que non statistiquement significatif. Nous n’avons pas retrouve d’autres mutations de facteurs epigenetiques, de transcription, d’epissage ou de mutation a haut risque moleculaire, associees preferentiellement au groupe NMP avec MAI associee. Apres un suivi median de 8.3 ans dans la cohorte globale, la presence d’une MAI n’avait pas d’impact defavorable sur la survie globale (p = 0.82), ni sur la survenue de myelofibrose secondaire (p = 0.98), ou la survenue de transformation en SMD/LAM (p = 0.53). Conclusion Au sein de cette large cohorte retrospective de NMP decrite sur le plan clinique et moleculaire, la prevalence de MAI est de 6.6 %, similaire a celle de la population generale. Une sur-prevalence de la mutation de TET2 a ete mise en evidence dans le groupe de patients presentant une MAI associee. Nos resultats suggerent une susceptibilite genetique commune, secondaire a la mutation de regulateur epigenetique comme TET2, potentiellement responsable des phenotypes inflammatoire et hematologique.

We aimed to study the prognostic impact of the mutational landscape in primary and secondary myelofibrosis. The study included 479 patients with myelofibrosis recruited from 24 French Intergroup of Myeloproliferative Neoplasms (FIM) centers. The molecular landscape was studied by high-throughput sequencing of 77 genes. A Bayesian network allowed the identification of genomic groups whose prognostic impact was studied in a multistate model considering transitions from the 3 conditions: myelofibrosis, acute leukemia, and death. Results were validated using an independent, previously published cohort (n = 276). Four genomic groups were identified: patients with TP53 mutation; patients with ≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS, or KRAS (high-risk group); patients with ASXL1-only mutation (ie, no associated mutation in TP53 or high-risk genes); and other patients. A multistate model found that both TP53 and high-risk groups were associated with leukemic transformation (hazard ratios [HRs] [95% confidence interval], 8.68 [3.32-22.73] and 3.24 [1.58-6.64], respectively) and death from myelofibrosis (HRs, 3.03 [1.66-5.56] and 1.77 [1.18-2.67], respectively). ASXL1-only mutations had no prognostic value that was confirmed in the validation cohort. However, ASXL1 mutations conferred a worse prognosis when associated with a mutation in TP53 or high-risk genes. This study provides a new definition of adverse mutations in myelofibrosis with the addition of TP53, CBL, NRAS, KRAS, and U2AF1 to previously described genes. Furthermore, our results argue that ASXL1 mutations alone cannot be considered detrimental.

International audience; No abstract available

BCR-ABL-negative myeloproliferative neoplasms (MPNs) in transformation have a dismal prognosis, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the sole curative therapeutic option. We retrospectively analyzed 53 molecularly annotated patients treated at Saint Louis Hospital, Paris, diagnosed with MPN in transformation between 2008 and 2018. The median patient age was 65 years, and the median interval between MPN diagnosis and MPN transformation was 46 months. The median overall survival (OS) of the entire cohort after transformation was 7.1 months. OS was better for patients treated with hypomethylating agents (HMAs) or with chemotherapy compared than for those treated by best supportive care or single-agent targeted therapy (median, 9.1 months versus 1.5 months; P.001). Patients treated with chemotherapy more often achieved complete remission compared with those treated with HMAs (68% versus 29%; P = .02), and were more often candidates for transplantation (59% versus 14%; P = .02), but the median OS was similar in the 2 groups. We then compared the outcomes in transplant recipients and nonrecipients using the Mantel-Byar methodology and found that allo-HSCT did not improve survival. In multivariate analysis, independent factors in prognosis were performance status at transformation (P.01), initial treatment with HMAs or chemotherapy (P = .02), and the ability to achieve complete remission during follow-up (P.01). Our data demonstrate that the indication for allo-HSCT for high-risk MPN should be discussed before transformation, because transplantation rescues few patients after transformation.

Myeloproliferative neoplasms (MPNs) are the most frequent underlying causes of splanchnic vein thromboses (SVTs). MPN patients with SVTs (MPN-SVT) often have a unique presentation including younger age, female predominance, and low Janus kinase 2 (JAK2) mutation allele burden. This study aimed at identifying risk factors for adverse hematologic outcomes in MPN-SVT patients. We performed a retrospective study of a fully characterized cohort of MPN-SVT patients. The primary outcome was the incidence of evolution to myelofibrosis, acute leukemia, or death. Eighty patients were included in the testing cohort. Median follow-up was 11 years. Most of the patients were women with a mean age of 42 years and a diagnosis of polycythemia vera. The primary outcome was met in 13% of the patients and was associated with a JAK2V617F allele burden ≥50% (odds ratio [OR], 14.7) and presence of additional mutations in genes affecting chromatin/spliceosome (OR, 9). We identified high-risk patients (29% of the cohort) as those harboring at least 1 molecular risk factor: JAK2-mutant allele burden ≥50%, presence of chromatin/spliceosome/TP53 mutation. High-risk patients had worse event-free survival (81% vs 100%; P = .001) and overall survival at 10 years (89% vs 100%; P = .01) than low-risk patients. These results were confirmed in an independent validation cohort of 30 MPN-SVT patients. In conclusion, molecular profiling identified MPN-SVT patients with dismal outcome. In this high-risk population, a disease-modifying therapy should be taken into consideration to minimize the probability of transformation.

Introduction The pathogenesis of myeloproliferative neoplasms (MPNs) is closely related to the acquisition of specific molecular alterations in JAK2, MPL, or CALR genes, the presence of which represent major diagnostic criteria in the WHO classification. The CALR exon 9 insertions and deletions are very heterogeneous, and their detection mainly relies on polymerase chain reaction (PCR) fragment length analysis. Methods We report on the rare nonclassical profiles that we observed among the 1382 patients analyzed by PCR fragment length analysis. In difficult cases, we tested germline DNA and performed NGS analysis. Results We faced some troubling results because of the presence of several unexpected peaks. Our investigations showed that they resulted from a mix of isolated or double somatic mutations combined with germline alterations which could be misleading for a correct diagnosis. The precise interpretation of such difficult cases is mandatory as a misinterpretation could lead to the prescription of cytoreductive drugs to nondiseased persons or to an absence of treatment in true MPN patients. Conclusion Our observations showed that every mutation should be verified by direct Sanger sequencing, and we show that sometimes it may be necessary to study germline DNA and to complement with NGS analysis to precisely interpret the molecular alterations.

Polycythemia vera is characterized by the acquisition of the JAK2V617F mutation. Recommended treatments include hydroxyurea and interferon-alpha. Several groups have reported a reduction in the JAK2 mutant allele burden in interferon-treated patients, but significance of this observation is questioned. We characterized the activity of ropeginterferon alpha-2b, a novel form of interferon-alpha recently shown to be safe and efficacious in polycythemia vera. Ropeginterferon was able to inhibit the proliferation of the HEL, UKE-1, and UT-7 JAK2-mutant cell lines while sparing JAK2-wild-type UT-7 and normal CD34+ cells growth. In vitro treatment of erythroid progenitors derived from PV patients showed that ropeginterferon could considerably inhibit the growth of endogenous erythroid colonies, a hallmark of polycythemia vera. Finally, we could study in sequential samples the clonal architecture of erythroid progenitors derived from patients included in a randomized study comparing hydroxyurea to ropeginterferon. After 1 year of treatment with ropeginterferon, the ratio of JAK2-mutated to wild-type colonies grown from bone marrow progenitors was reduced by 64%, compared to 25% in patients receiving hydroxyurea. This study shows that ropeginterferon has a potent targeted activity against JAK2-mutant cells and is able to drastically reduce the proportion of malignant progenitors in patients treated with this drug.

5,10-Methylene-tetrahydrofolate reductase (MTHFR) deficiency is a genetic disorder that can occur at any age and can be easily detected by increased homocysteinemia. In adolescence/adult onset forms, the clinical picture is often complex with association of various neurological features and thrombosis. Here we report the cases of two adult siblings who experienced focal epilepsy at 18 years old as a first disease manifestation, without other symptom during several years. Upon diagnosis, both patients received metabolic treatment comprising B9, B12 and betaine which has stopped the occurrence of seizures, allowing discontinuation of anti-epileptic drugs. Among 24 reviewed adolescent/adult onset patients with MTHFR deficiency in the literature, clinical manifestations included gait disorder (96%, from motor central or peripheral origin), cognitive decline (74%), epileptic syndromes (50%), encephalopathy (30%), psychotic symptoms (17%), and thrombotic events (21%). A total of 41% presented a single neurological manifestation that could stay isolated during at least 3 years, delaying achievement of the diagnosis. Brain MRI showed a mostly periventricular white matter changes in 71% of cases. All patients stabilized or improved following metabolic treatment. Despite being rare, adolescence/adult onset MTHFR deficiency can nevertheless be successfully treated. Therefore, homocysteinemia should be tested in various unexplained neuro-psychiatric syndromes like epilepsy or spastic paraparesis, even if isolated, since waiting for completion of the clinical picture is likely to increase the risk of irreversible neurological damage. Electronic supplementary material The online version of this article (10.1186/s13023-018-0767-9) contains supplementary material, which is available to authorized users.

Introduction: Auto-immune and Inflammatory Diseases (AID) have been associated with myeloproliferative neoplasms (MPN) in a large population-based study (Kristinsson et al. Haematologica 2010). In myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), epigenetic regulators TET2 and IDH1/2 were more frequently mutated in patients with AID, suggesting potentially common pathogenesis pathways (Zhao et al. Leukemia 2021). However, in the context of MPN, AID specific features remain poorly characterized, and no study has reported to date the mutational landscape of MPN patients with AID. The objectives of our study were to describe the clinical and molecular characteristics of MPN patients with associated AID and evaluate its impact on patient's outcome. Methods: A total of 1541 patients were diagnosed with MPN according to WHO criteria between January 2011 and January 2021 in our center, of whom 998 had a molecular analysis by next generation sequencing (NGS) targeting a panel of 36 genes involved in MPN, performed at diagnosis and/or during follow-up. AID diagnosis was based on international criteria, and all cases have been reviewed by internal medicine experts. Patients with AID induced by interferon-alpha treatment were not included. Results: The median age of our whole cohort was 51.3 years IQR[40.4-63.2]. Our cohort included 522 (34%), 709 (46%) and 229 (15%) diagnosis of polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (MF) respectively. A total of 100 patients (6.6%) had AID and were compared to the remaining 1441 MPN patients without AID. There were more females (66 (66%) versus 769 (53%), p=0.019) within the AID group compared to non-AID patients. MPN subtype, driver mutation, complete blood counts at diagnosis did not differ between the two groups. Occurrence of thrombosis and hemorrhage episodes did not vary either (44 (44%) versus 564 (39%), p=0.356). AID diagnosis was prior to MPN in 34% of cases, concomitant in 12% and posterior in 31% of cases. AID diagnosis included 45 (45%) organ-specific AID (mainly autoimmune thyroiditis, n=34), 13 (13%) inflammatory arthritis, 9 (9%) connective tissue diseases, 8 (8%) inflammatory dermatosis, 7 (7%) systemic vasculitis and 18 (18%) unclassified AID (Figure 1A). The AID fulfilled the required classification criteria in 70 (70%) cases, while complete criteria were not reached in 30 (30%) cases. The median interval of time between MPN diagnosis and NGS was 7.2 years IQR[2.1-13.3] in the whole cohort. Among patients with available molecular analysis, 44 (62%) and 571 (62%) patients had at least one additional non-driver mutated gene in the AID and control groups respectively. Interestingly, TET2 mutations were more frequent in MPN patients with AID (24 (34%) versus 205 (22%), OR=1.84 95%CI[1.08-3.07], p=0.028, Fig 1B). The prevalence of TET2 mutations did not significantly differ between the AID categories. When focusing on IDH1/2 mutations, as they act on the same biological epigenetic pathway as TET2, IDH1/2 mutations were more frequent in the AID cohort although not statistically significant (4 (6%) versus 27 (3%), OR=2.02 95%CI[0.74-5.51], p=0.27). No other mutations including other epigenetic factors, splicing regulators, transcription factors or high molecular risk mutations, were significantly associated with AID. After a median follow up of 8.3 years IQR[3.7-14.3] in the whole cohort, 10 (10%) and 122 (8%) patients died in the AID and control groups respectively. The presence of AID did not impact overall survival (p=0.82), secondary myelofibrosis free (p=0.98) or MDS/AML transformation free (p=0.53) survivals. Conclusion Our study reports on a large retrospective clinically and molecularly annotated cohort the prevalence of AID in MPN patients (6.6%). This prevalence did not differ from that of the general population. Interestingly, our data emphasize a high prevalence of TET2 mutations in patients with both AID and MPN, compared to MPN patients without AID. Although other studies are warranted to better define the causal relationship between MPN and AID, our results may suggest a common pathophysiology as it has been proposed in MDS patients, based on shared genetic susceptibilities with mutations in TET2 that could occur within early hematopoietic progenitors and give rise to both the inflammatory phenotype and myeloid malignancy. DE and LPZ contributed equally to this work. Figure 1 Figure 1. Disclosures Fenaux: Celgene/BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Syros Pharmaceuticals: Honoraria. Ades: Novartis: Honoraria; Takeda: Honoraria; Abbvie: Honoraria; JAZZ: Honoraria; Celgene: Honoraria, Research Funding. Raffoux: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ABBVIE: Consultancy; ASTELLAS: Consultancy. Kiladjian: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; PharmaEssentia: Other: Personal fees. Benajiba: Pfizer: Research Funding; Gilead: Research Funding.

The purpose of this study was to identify the incidence, causes and impact of non-adherence to oral and subcutaneous chronic treatments for patients with polycythemia vera or essential thrombocythemia. Patients receiving cytoreductive drugs for polycythemia vera or essential thrombocythemia were recruited at our institution (Observatoire Brestois des Néoplasies Myéloprolifératives registry). They completed a one-shot questionnaire designed by investigators (Etude de l’Observance Thérapeutique et des Effets Secondaires des Traitements study). Data about complications (thrombosis, transformation and death) at any time in the patient’s life (before diagnosis, up until consultation and after the completion of the questionnaire) were collected. Sixty-five (22.7%) of 286 patients reported poor adherence (

BACKGROUND Several studies have demonstrated that approximately one-half of patients with chronic myeloid leukemia (CML) who receive treatment with tyrosine kinase inhibitors (TKIs) and achieve and maintain a deep molecular response (DMR) are able to successfully discontinue therapy. In patients who have a molecular relapse, a DMR is rapidly regained upon treatment re-initiation. METHODS The authors report the results from RE-STIM, a French observational, multicenter study that evaluated treatment-free remission (TFR) in 70 patients who re-attempted TKI discontinuation after a first unsuccessful attempt. After the second TKI discontinuation attempt, the trigger for treatment re-introduction was the loss of a major molecular response in all patients. RESULTS The median follow-up was 38.3 months (range, 4.7-117 months), and 45 patients (64.3%) lost a major molecular response after a median time off therapy of 5.3 months (range, 2-42 months). TFR rates at 12, 24, and 36 months were 48% (95% confidence interval [CI], 37.6%-61.5%), 42% (95% CI, 31.5%-55.4%), and 35% (95% CI, 24.4%-49.4%), respectively. No progression toward advanced-phase CML occurred, and no efficacy issue was observed upon TKI re-introduction. In univariate analysis, the speed of molecular relapse after the first TKI discontinuation attempt was the only factor significantly associated with outcome. The TFR rate at 24 months was 72% (95% CI, 48.8%-100%) in patients who remained in DMR within the first 3 months after the first TKI discontinuation and 36% (95% CI, 25.8%-51.3%) for others. CONCLUSIONS This study is the first to demonstrate that a second TKI discontinuation attempt is safe and that a first failed attempt at discontinuing TKI does not preclude a second successful attempt. Cancer 2017;123:4403-10. © 2017 American Cancer Society.

Myeloproliferative neoplasms are characterized by transduction pathway recognized as mutually exclusive molecular abnormalities such as BCR-ABL translocation, JAK2V617F or JAK2 exon 12 mutations, MPL w515, and CALR mutations. However, in some rare cases, associations of such mutations are found in 1 patient. This can be related to 2 pathologies (at least 2 different clones harboring 2 mutations) or associated mutations in 1 clone. We describe here such an association of CALR and MPL mutations in a patient harboring the second mutation in a subclone during the phenotypic evolution of the myeloproliferative neoplasms.

TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients. Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more). Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations. Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment. Disclosures Leblond: Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.

The transcription factor p53 is overexpressed in the lung of patients with emphysema, but it remains unclear if it has a deleterious or protective effect in disease progression. We investigated the role of p53 in the elastase-induced emphysema model and the molecular underlining mechanisms. Wild-type (WT) and p53(-/-) mice were instilled with pancreatic porcine elastase. We quantified emphysema (morphometric analysis), chemokine (C-C motif) ligand 2 (CCL2), and TNF-α in bronchoalveolar lavage (BAL) (ELISA), oxidative stress markers [heme oxygenase 1 (HO1), NAD(P)H dehydrogenase quinone 1 (NQO1), and quantitative RT-PCR], matrix metalloproteinase 12 (MMP12) expression, and macrophage apoptosis (cleaved caspase-3, immunofluorescence). p53 gene expression was up-regulated in the lung of elastase-instilled mice. p53 deletion aggravated elastase-induced emphysema severity, pulmonary inflammation (macrophage and neutrophil numbers and CCL2 and TNF-α levels in BAL), and lung oxidative stress. These findings, except for the increase in CCL2, were reproduced in WT mice transplanted with p53(-/-) bone marrow cells. The increased number of macrophages in p53(-/-) mice was not a consequence of reduced apoptosis or an excess of chemotaxis toward CCL2. Macrophage expression of MMP12 was higher in p53(-/-) mice compared with WT mice after elastase instillation. These findings provide evidence that p53(-/-) mice and WT mice grafted with p53(-/-) bone marrow cells are more prone to developing elastase-induced emphysema, supporting a protective role of p53, and more precisely p53 expressed in macrophages, against emphysema development. The pivotal role played by macrophages in this phenomenon may involve the MMP12-TNF-α pathway.

Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia.

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 41 (22.2%) and 16 (34.0%) infections were of grade 3-4, 61 (33.1%) and 19 (40.4%) required hospitalization, while 8 (4.3%) and 2 (4.2%) resulted in patients' death, in RUX treated and RUX naïve patients, respectively (all non-significantly different). Bacterial infections represented 48.0%, viral infections 16.0% and fungal infections 4.8% of the total infectious events. The most frequently infected sites were lungs/respiratory tract (28.1%) and skin/soft tissue (24.2%). RUX treatment was significantly associated with recurrence of HSV-1/2 or VZV (OR 7.57, 95%CI[1.01-57.30], p=0.050), and skin/soft tissue infections including the above herpetic recurrences (OR 3.33, 95%CI[1.13-9.78], p=0.029). 17 (14.9%) patients presented with HSV/VZV recurrence in the RUX treated group compared to 1 (1.0%) in non-treated patients (Figure B). We then thought to evaluate the impact of potential infections risk factors in RUX treated patients at time of treatment initiation in order to inform clinicians' therapeutic decision and follow-up guidance. For this analysis, we evaluated the characteristics of MF, potential clinical and biological immune suppression factors, treatment lines prior to RUX, dose and cumulative exposition to RUX. No clinical or biological factor was significantly associated with the overall risk of infectious events. However, hypo-gammaglobulinemia at time of RUX initiation was independently associated with HSV and/or VZV recurrence (HR 4.57, 95%CI[1.29-16.16], p=0.018). The higher rate of infections in RUX treated patients was associated with a not statistically significant trend towards shorter OS (HR 1.97, 95%CI[0.98; 3.94], p=0.055). Conclusion: This retrospective study shows an association between the occurrence of infectious complications and Ruxolitinib treatment in MF patients, and in particular, a higher incidence of HSV/VZV recurrence and skin/soft tissue infection. Patients with hypo-gammaglobulinemia at time of RUX initiation were at higher risk of HSV/VZV recurrence, suggesting that protein electrophoresis should be performed prior to treatment initiation and primary antiviral prophylaxis proposed to patients with hypo-gammaglobulinemia. Disclosures Rea: BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kiladjian:AbbVie: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

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 Conclusion: In this retrospective study we show that presence of NFE2 mutations with a VAF ≥5% is independently associated with an increased risk of leukemic transformation and shorter overall survival. These findings are in line with recently reported animal models and suggest that NFE2 mutations screening should be routinely performed in MPN patients. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

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 Conclusion: This study in a large cohort of MPN patients highlights for the first time to our knowledge the adverse impact on MFS of SF3B1 and MPN-driver co-mutated clones. In contrast, presence of an SF3B1 mutation at sub-clonal level didn't increase the risk of MF development. In line with our findings, a recent study reported an association between rapid progression to myelofibrosis and SF3B1 mutations in patients with age-related clonal hematopoiesis (Bartels et al. Leukemia 2020). Further studies are warranted to confirm our results on independent cohorts and to investigate the mechanisms of bone marrow fibrosis development in patients with SF3B1 and MPN-drivermutations. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

DLP, OM and JR contributed equally to this work.; International audience

The JAK2V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R2 = 0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution.

Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.