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Background: The prognosis of central nervous system (CNS) involvement in adult patients with acute lymphoblastic leukemia (ALL) has been historically associated with a dismal outcome. Whereas the prognosis of adult patients with ALL has greatly improved since the advent of pediatrics-inspired regimens, the prognostic impact of CNS involvement has not been formerly reevaluated. We report herein the impact of CNS involvement in patients included in the pediatric-inspired prospective GRAALL-2005 study. Methods: All patients received a 5-drug induction therapy with native E. Coli-ASP intravenous injections. Patients in complete remission (CR) received two consolidation courses with alternating cycles including high dose cytarabine (2g/m2/12h on days 1 and 2), high dose methotrexate (3 g/m2 on day 1), and cyclophosphamide. All patients in persistent CR and with no indication for allogeneic stem cell transplantation (SCT) received late intensification, followed by one last consolidation course. Patients with initial CNS involvement, clinically and/or cytologically (cerebrospinal fluid), were recommended to receive an increased number of triple intrathecal therapy, CNS irradiation, and were eligible for allogeneic SCT in first CR. They received less Asp injections during induction therapy to avoid CNS adverse events. CNS irradiation included two lateral fields encompassing the skull, facial, the base of the skull, and the first two cervical vertebrae at a dose of 24 grays for those not receiving allogeneic SCT and 15 grays for those receiving allogeneic SCT. Results: Between 2006 and 2014, 784 adult patients with newly diagnosed Philadelphia-negative ALL were included with 55 (7%) having initial CNS involvement. These patients were more likely to be of T-phenotype (51 versus 32%, p=.004) and had more white blood cells at diagnosis (median 23 G/l versus 11 G/l, p=.02). Most patients (36 pts, 66%) were classified as CNS-3 (> 5 white blood cells/µl and a positive cytospin and/or clinical signs) whereas 5 patients (9%) were CNS-2 (< 5 white blood cells/µl and a positive cytospin), and 14 (25%) have data pending. Among patients with details regarding CNS involvement, 25/41 (61%) had clinical signs including trigeminal anesthesia (9 pts, 36%), facial paralysis (4 pts, 16%), extremities paresthesia (4 pts, 16%), visual signs (2 pts, 8%), meningeal syndrome (2 pts, 8%), and motor deficit (2 pts, 8%), and 4/18 (22%) had radiological signs. Induction death, CR1 rate, and negative minimal residual disease after induction were similar whether patients had CNS involvement or not (6 vs 6%, 89 vs 89%, 73 vs 62%, 26 vs 22%, respectively). Patients with CNS involvement had a worse outcome than those without with a median event-free survival (EFS) of 391 days (versus not reached for patients without CNS involvement, HR: 1.7, 95% CI: 1.2 - 2.5, p=.002) and a median overall survival (OS) of 608 days (versus not reached for patients without CNS involvement, HR: 1.8, 95% CI: 1.3 - 2.6, p=.001) (figure). Similar results were observed when patients who received allogeneic SCT in CR1 were censored at the time of graft. As recommended, patients with CNS involvement were more likely to receive allogeneic SCT than those without (53 versus 34%, p=.01), with a median time of 169 days. A 150-day landmark analysis, excluding 12 patients with an EFS event before 150 days, was performed to study the impact of allogeneic SCT on the outcome of patients with CNS involvement. Allogeneic SCT had no impact on either EFS (HR: .5, 95% CI: .2 - 1.2, p=.15) or OS (HR: .8, 95% CI: .3 - 1.8, p=.53). Conclusion: Despite improved outcome in young adult ALL patients with pediatrics-inspired protocols, CNS involvement remains a poor-risk feature. The historical use of allogeneic SCT does not improve outcome. Specific regimens should be developed for adult ALL patients with CNS involvement. Figure 1 Figure 1. Disclosures Huguet: Amgen: Other: Advisor; BMS: Other: Advisor; Celgene: Other: Advisor; Jazz Pharmaceuticals: Other: Advisor; Novartis: Other: Advisor; Pfizer: Other: Advisor. Barbieux: ASTRA-ZENECCA: Consultancy. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. 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. Boissel: Bristol-Myers Squibb: Honoraria, Research Funding; Servier: Consultancy, Honoraria; Incyte: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; JAZZ Pharma: Honoraria, Research Funding; CELGENE: Honoraria; SANOFI: Honoraria; PFIZER: Consultancy, Honoraria. Mathilde: ABBVIE: Consultancy; SERVIER: Consultancy.

On behalf of the GRAALL group. Introduction. Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of patients diagnosed with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). In a previous randomized trial (GRAAPH-2005; Chalandon Y., Blood 2015), our group demonstrated that anthracycline and cyclophosphamide could be safely omitted during the first Hyper-CVAD chemotherapy cycle when combined to imatinib. In the present GRAAPH-2014 trial, we investigated whether the use of nilotinib, a 2 nd generation TKI, would allow further decreasing chemotherapy intensity through the omission of high-dose cytarabine (HD-AraC) during consolidation cycles 2 and 4. Methods. From March 2016, patients aged 18-60 years old were randomized frontline to receive 4 cycles (1=3, 2=4) of chemotherapy combined with continuous nilotinib 400 mg bid. Cycle 1 and 3 were similar in both arms, identical to the less-intensive first cycle of the previous GRAAPH-2005 trial with weekly vincristine and dexamethasone and nilotinib instead of imatinib. Cycle 2 and 4 differed between control arm A (MTX 1 g/sqm over 24h, HD-AraC 3 g/sqm/12h for 2 days) and experimental arm B (MTX 1 g/sqm over 24h only). Marrow minimal residual disease (MRD) was assessed by both BCR-ABL1 and IG-TCR qPCR after each cycle (MRD1-4). Patients in complete remission (CR) after cycle 4 were eligible to receive allogeneic stem cell transplant (alloSCT). Non allografted patients had the option to receive autologous SCT (autoSCT) if a major molecular response (MMolR) (MRD4 BCR-ABL1 40y) and BCR-ABL1 breakpoint region (M/m-bcr). MMolR rate at MRD4 was the primary endpoint of this non-inferiority study, with a margin set at 0.15. In February 2019, the study DSMB decided to stop the randomizations after an unplanned analysis demonstrating a significant excess of relapse in arm B (without HD-AraC). An intention-to-treat analysis is provided. All patients gave informed consent. The study is registered at EudraCT under the number: 2014-002146-44. Results. A total of 156 patients were randomized (77 in arm A, and 79 in arm B). Median age was 47.1 years old (range 18.1-59.9). One-hundred and ten patients had the m-bcr (70.5%) and 46 (29.5%) the M-bcr. An IKZF1 intragenic deletion was found in 87/153 (56.9%). Median follow-up was 2.8 years (95%CI 2.6-3.1). All evaluable patients reached CR after a maximum of two cycles. Three patients died during cycle 1 (2 in arm A, 1 in arm B), and 2 during cycle 2 (1 per arm). Most patients received the 4 scheduled cycles (n=143, 91,7%). AlloSCT was performed in 91 patients (58.3%), whereas 41 patients received an autoSCT (26.3%) with no difference in allo/autoSCT rates between arms. A non-inferiority in late MRD response (MRD4) between the two arms was observed (primary endpoint). MMolR was reached in 55/75 (73.3%) and 61/78 (78.2%) of CR patients in arm A and B, respectively, with an estimated 95% CI of difference of -11% to +16% (-9.2% to +20.8% in the ITT analysis treating missing data as failures). At 3 years, overall survival (OS) was 86.0% in arm A versus 74.2% in arm B (p=.08, Figure A). Progression-free survival (PFS) was 79.6% in arm A versus 57.2% in arm B (p=.008, Figure B). Thirty-one patients experienced hematological relapse (8 in arm A and 23 in arm B). Twenty-height out of 31 relapsed patients were tested for the acquisition of BCR-ABL1 mutations, 20/28 (71%) had at least one mutation and 10/28 (36%) had a T315I mutation. At 3 years, the cumulative incidence of relapse (CIR) was 21.3%, significantly higher in arm B than in arm A (30.8% vs 10.6%, p=.007). When analyzing the CIR considering alloSCT as a competing event for relapse, a significant higher CIR was still observed in arm B as compared to arm A (Figure C). Conclusion. Four cycles of the combined administration of nilotinib and chemotherapy is very efficient for bridging younger adults with Ph-positive ALL to SCT. However, omitting HD-AraC is associated with a higher relapse incidence despite non-inferior levels of BCR-ABL1 MRD4. Figure 1 Figure 1. Disclosures Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Chalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board. Huguet: Novartis: Other: Advisor; Jazz Pharmaceuticals: Other: Advisor; Celgene: Other: Advisor; BMS: Other: Advisor; Amgen: Other: Advisor; Pfizer: Other: Advisor. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Raffoux: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ASTELLAS: Consultancy; ABBVIE: Consultancy. Boissel: Amgen: Consultancy, Honoraria, Research Funding; CELGENE: Honoraria; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte: Honoraria; SANOFI: Honoraria; PFIZER: Consultancy, Honoraria; JAZZ Pharma: Honoraria, 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. OffLabel Disclosure: Nilotinib as a therapy for PH-positive acute lymphoblastic leukemia

In myeloid malignancies, presence of 'multi-hit' TP53 mutations is associated with lack of response to conventional therapy and dismal outcomes, particularly when found in combination with a complex karyotype. Therefore, it is crucial to understand the biological basis of TP53-mutant driven clonal evolution, suppression of antecedent clones and eventual disease transformation to inform the development of more effective therapies. Myeloproliferative neoplasms (MPN) represent an ideal tractable disease model to study this process, as progression to secondary acute myeloid leukemia (sAML) frequently occurs through the acquisition of TP53 missense mutations. To characterize tumor phylogenies, cellular hierarchies and molecular features of TP53-driven transformation, we performed single-cell multi-omic TARGET-seq analysis (PMID: 33377019 & 30765193) of 22116 hematopoietic stem and progenitor cells (HSPCs) from 35 donors and 40 timepoints (controls, MPN in chronic phase, pre-AML and TP53-mutated sAML; Figure1a). TARGET-seq uniquely enables single-cell mutation analysis with allelic resolution with parallel transcriptomic and cell-surface proteomic readouts. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type alleles upon transformation, including parallel evolution of separate TP53 "multi-hit" subclones in the same patient (n=4/14) and JAK2-negative progression (n=2/14). Complex clonal evolution driven by chromosomal abnormalities (CAs) was present in all patients and TP53 multi-hit HSPCs without CAs were rarely observed. Subclones with recurrent CA such as monosomy 7 showed upregulation of RAS-associated transcription and preferentially expanded in xenograft models. Together, these data indicate that TP53 missense mutation, loss of TP53 wild-type allele and cytogenetic evolution are collectively required for leukemic stem cell (LSC) expansion. Integrated transcriptomic analysis of sAML samples (Figure1b) revealed three major populations: (1) a TP53-mutant cluster (Figure1c) characterized by an erythroid signature (e.g. KLF1, GATA1, GYPA; an unexpected finding as no cases showed diagnostic features of erythroid leukemia), (2) an LSC TP53-mutant cluster (Figure1d) and (3) a TP53-WT preleukemic cluster (Figure1e). The LSC cluster showed dysregulation of key stem cell regulators, from which we derived a novel 48-gene LSC score with prognostic impact in an independent AML cohort (HR=3.13; Figure1f). Importantly, this score was predictive of outcome irrespective of TP53 status for both de novo and sAML, demonstrating its broader potential clinical utility. TARGET-seq analysis uniquely allowed us to characterize rare TP53-WT preleukemic cells (preLSCs), which were almost exclusively confined to the immunophenotypic lineage-CD34+CD38-CD90+CD45RA- HSC compartment. PreLSC from sAML samples presented increased stemness, increased quiescence, aberrant inflammatory signaling and differentiation defects (Figure1g) as compared to HSCs from normal or MPN donors, both at the transcriptional and functional levels through in vitro long-term and short-term cultures. This indicates cell-extrinsic suppression of residual TP53-WT hematopoiesis. Longitudinal analysis of TP53-heterozygous mutant HSPCs at different stages of disease evolution (Figure1a) revealed that aberrant inflammatory signalling (e.g. BST2, IFITM1, IFITM3) in the genetic ancestors of TP53 "multi-hit" LSCs, but not the presence of TP53-mutations alone, was predictive of subsequent transformation. In a mouse model system, TP53-mutant cells challenged with sustained inflammatory stimuli acquired a mean 3-fold competitive advantage in WT: TP53 R172H/+chimeras. This indicates that pro-inflammatory cues from the tumour microenvironment promote fitness advantage of TP53-mutant cells whilst supressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis of the genetic, cellular and molecular landscape of TP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia (Figure1h). Since TP53 is the most commonly mutated gene in human cancer, we anticipate these findings will be of broader relevance to many other cancer types. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment. Drummond: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mead: Abbvie: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau.

Introduction Clonal selection is one of the mechanisms leading to therapy-related myeloid neoplasms (TRMN). A preexisting somatic mutation in hematopoietic stem cell (defined as clonal hematopoiesis [CH]) emerges under pressure of chemotherapy or radiotherapy, leading to TRMN development. Most of these mutations belong to the DNA damage response (DDR) pathway as TP53 or PPM1D mutations and are known to confer a dismal prognosis. Recently authorized for the treatment of ovarian cancers (OC), the poly (ADP-ribose) polymerase inhibitors (PARPi) represent a promising targeted therapy. However, by inducing DNA damage and altering DNA repair process, PARP inhibition could represent a challenge for the genetic stability of the healthy tissues. Thus, we assessed the effect of PARP inhibition on the development of CH and TRMN after PARPi treatment for OC (TRMN-PARPi) in combination with chemotherapies. Methods Firstly, we performed a targeted 77 genes mutational analysis using Next Generation Sequencing (NGS) in 13 patients exposed to PARPi without TRMN. Secondly, we retrospectively identified, with the help of the UNIHEM group of Unicancer, 17 patients who experienced TRMN-PARPi. Clinical, biological and survival data were collected and compared to 23 OC patients with TRMN not treated with PARPi (Gustave Roussy institutional database). Lastly, NGS was performed for 3 patients with TRMN-PARPi with sequential sampling. Patient's samples were obtained with informed consent. Results Thirteen OC patients during maintenance treatment with PARPi without TRMN were explored by NGS. Median age at NGS was 64.5 years old (yo) (40.5-75.3). 4/13 (31%) patients harbored BRCA1/2 germline mutation. Time between OC diagnosis and NGS was 4.3 y (1-11.6). The median number of chemotherapy line at PARPi initiation was 2 (1-3). 7 received Olaparib, 5 Niraparib and 1 Rucaparib. The median duration of PARPi treatment before NGS was 4.7 months (1.1-25.1). 12/13 patients experienced hematological toxicities during the PARPi treatment. CH was found in 10/13 (77%) patients (Figure 1a), including mutations of DDR genes in 8/10 (80%). 6/8 (75%) patients had 2 or more gene mutations. Next we identified 17 cases of TRMN occurring during or after PARPi administration for OC (6/17 [35%] t-AML and 11/17 [65%] t-MDS). 12/17 (71%) patients had BRCA germline mutations (7 BRCA1 and 5 BRCA2). All received Olaparib with a median dose of 600mg/d (400-1200). Median duration of Olaparib treatment was 1.7 years (0.2-4.6). TRMN-PARPi were described 1.4 months (0-10.9) after the end of PARPi administration. We compared these patients to a cohort of TRMN post OC not treated by PARPi. Number of therapy lines for OC, time between TRMN and OC diagnosis, median age at TRMN, were, for TRMN-PARPi, 2 (1-8), 5.9 y (0.9-20.8), 64.4 yo (46-74); respectively, compared to 3 (1-8), 4.9 y (1.7-36.9), 59.3 yo (35.7-85.7); (p=ns). TRMN-PARPi cytogenetic was unfavorable for 16/17 (94%) (including 11/17 [65%] complex karyotype) compared to 16/23 (70%) (11/23 [48%] complex karyotype). C Median survival was significantly lower in the TRMN-PARPi group 3.9 months 95%CI [2.0-9.7] and 6.1 months 95%CI [4.1-15.8] respectively, p=0.046, Fig 1b). However, median survival from OC diagnosis was not different between the two groups 6.2 y 95%CI [5.6-NA] for TRMN-PARPi vs 5.6 y 95%CI [5.0-11.6]. NGS was available for 8/17 TRMN-PARPi and revealed mutations in DDR genes in 7/8 patients (6 patients with TP53 mutation, 2 with PPM1D mutation). For 3 patients, we had samples from OC stage, before PARPi administration. We found that mutations from TRMN stage were present at lower frequency, confirming clonal selection by PARPi treatment (Figure 1c). Conclusions Here we described, for the first time, a cohort of TRMN patients previously treated with PARPi for an OC. Intriguingly, most of these TRMN occurred with a short latency at the end of PARPi treatment, with unfavorable cytogenetic and very short OS. Moreover, we found a very high percentage of CH involved in the DDR pathway (62%) in patients under PARPi treatment without TRMN suggesting a potential clonal selection which could lead ultimately to TRMN. PARPi are now indicated in 1rst line high grade OC regardless of BRCA status, which should expand indications. Benefit for OC patients is not questionable; however, caution will be warranted for patients with CH before PARPi treatment, especially implicating DDR mutations. Disclosures Etienne: Incyte: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses.

Introduction Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). These are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. MPN are caused by three main recurrent mutations: JAK2V617F, mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL) genes. Interferon alpha (IFNα) treatment induces not only a hematological response in around 70% of ET, PV and early myelofibrosis, but also a significant molecular response on both JAK2V617F- and CALR-mutated cells. However, a complete molecular response is only achieved in around 20% of patients. Our aim is to predict the long-term efficacy of IFNα in JAK2V617F- and CALR-mutated patients by monitoring the fate of the disease-initiating mutated HSC in order to better stratify the molecular responders. Methods A longitudinal observational study (3-5 years) was performed in 46 IFNα-treated patients. The MPN disease distribution was 42% ET, 47% PV and 11% PMF. We detected 33 patients with JAK2V617F mutation, 11 with CALR mutations (7 type 1/type 1-like and 4 type 2/type 2-like), 1 with both JAK2V617F and CALR mutation and 1 with JAK2V617F, CALR mutation and MPLS505N. At 4-month intervals, the JAK2V617For CALR mutation variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was determined by studying the presence of the mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38-HSC-enriched, CD90-CD34+CD38- immature and CD34+CD38+committed progenitors). We used a combination of mathematical modeling (Michor et al., Nature, 2005) and Bayesian analysis to infer the long-term behavior of mutated HSC. Results After a median follow-up of 40 months, IFNα targeted more efficiently and more rapidly the HSPC, particularly the HSC-enriched progenitors, than the mature blood cells in JAK2V617Fpatients (p Since it is very difficult to purify true HSC from patients, we used a combination of mathematical and statistical modeling to infer the behavior and the kinetics of IFNα-targeted mutated HSC. The model gave a good fit to the data and indicated that mutated HSC are exhausted slowly (> 1 year) with concomitant increase in mutated HSPC and granulocytes in well-responding patients. We calculated the rate of HSC decrease for each patient. Rates of decrease are very low for heterozygous JAK2V617F and CALR-mutated HSC and greater for homozygous JAK2V617FHSC, but all increase with high IFNα dose (>100 µg/week). Moreover, very low proportion of heterozygous mutated HSC compared to high proportion can be targeted more easily in patients. The associated mutations at diagnosis and at the last timepoint were also investigated using an NGS-targeted myeloid panel. Results indicate that IFNα does not induce any further mutations on additional genes and the mathematical approach predicts that associated mutations have no major impact on the ratio of HSC decrease. Conclusion Altogether, using a rigorous method of statistical inference, our results show that IFNα exhaust the human mutated HSC by differentiation in HSPC and mature cells. This is likely due to IFNα inducing a stronger proliferation of mutated compared to wild-type HSC, as previously shown in a mouse model (Mullally et al., Blood, 2013). Our study predicts that IFNα can slowly eradicate the mutated HSC, but this beneficial effect would be more efficient: i) in patients with homozygous JAK2V617F versus those with heterozygous JAK2V617F or CALR-mutated, ii) with high IFNα dose, iii) in patients with very low proportion of heterozygous JAK2V617F and CALR-mutated HSC. Thus, this study will help to stratify patients for IFNα treatment.These results might also explain the different outcomes in current IFNα clinical trials. Disclosures Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AlsaTech: Other: Co-Founde; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics; Wiley & Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine. Kiladjian:AOP Orphan: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Consultancy.

In 2015, a germline copy number variation of chromosome 14 (CNVdup14) including ATG2B and GSKIP genes was described as a predisposition genetic factor responsible of familial myeloproliferative neoplasms from French West Indies (Saliba et al, Nat Gen 2015), frequently progressing to AML. In this study, we looked at the presence of this CNVdup14 in a cohort of Caribbean islands patients (pts) with non-secondary aggressive hematological malignancies (HM). We also studied the expression of ATG2B and GSKIP genes in a cohort of acquired AML pts. This is a retrospective multicenter study of adults Caribbean islands pts treated at Gustave Roussy Cancer Center (Villejuif, France) and at the French West Indian hospitals (Martinique and Guadeloupe) between May 2000 and May 2018. We included pts with AML, acute undifferentiated leukemia (AUL), acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Pts with personal history of myeloproliferative or myelodysplastic syndromes before the onset of aggressive HM were not included in this study. The presence of the CNVdup14 was carried out by PCR analyses in all the pts. For the second part of the study, expression of ATG2B and GSKIP genes were assessed in newly diagnosed de novo AML pts with normal karyotype or trisomy 8 (samples from the GOELAMSTHEQUE) by quantitative RT-PCR and expressed as relative expression PPIA/HPRT/H2A.Z. One hundred pts were analyzed. Median age was 52 years (IQ 40-62) with male predominance (61%). Fifty eight pts came from Martinique, 42 pts from the rest of the Caribbean islands (including 28, 4, 3, 2 pts from Guadeloupe, Haiti, Saint Martin, Dominican Republic, respectively). Seventy eight pts had AML. Among them, according to revised MRC cytogenetic classification, 11 (14%) were favorable, 47 (60%) intermediate and 20 (26%) adverse. Seventeen pts had ALL, 3 LL, and 2 AUL. On the entire cohort, all except nine pts were treated with intensive chemotherapy, 80 reached complete remission, 29 relapsed, 46 pts died. Thirty two pts received hematopoietic stem cell transplantation (HSCT). Six pts were positive for the CNVdup14 by PCR (confirmed by SNP array in the 5 pts with leftover DNA available). All had an AML (no pts with favorable AML) and were originated from Martinique. These pts represented 14% of the 43 AML from Martinique in our cohort (17% if we excluded favorable AML). One was known to be part of an ATG2B/GSKIP family, 2 pts had no familial history of myeloid malignancies and 2 new families were discovered. Median white blood cell, hemoglobin and platelets counts were 17.7 G/l (IQ 6.7-48), 8.15 g/dl (6.9-9.7) and 58 G/l (20-132), respectively. Median age at AML diagnosis was 49 years (34-55), 3/6 (50%) had extramedullary localization compared to 11/78 (14%) for others AML pts. Karyotype was normal for 4, or showed a monosomy 7 for 2 pts. NGS panel showed distinct abnormalities compared to the entire cohort (Fig A). None had JAK2, MPL, CALR, P53, RUNX1, DNMT3A, FLT3-ITD mutations. All harbored an epigenetic and/or spliceosome mutation (IDH n=3, TET2 n=3, ASLX1 n=3, SRSF2 n=3). Five out of the six pts received intensive treatment and 4 achieved complete remission. Two received HSCT, 2 relapsed and 4 died. Median overall survival (OS) of the entire cohort was 35.7 months (22.5-89.5) and progression free survival (PFS) 27.6 months (15.6 -56.1). As CNVdup14 pts had AML only, we next evaluated survival according to the predisposition status in the AML cohort. Pts with CNVdup14 had a median OS and PFS of 19 (6.5-29) and 11.4 (6.5-29) months, respectively, compared with 52.6 (22.9-100.2) and 30 months (15.6-60) in CNV wild-type counterparts (PFS Fig B). We next evaluated ATG2B and GSKIP expression in a cohort of 46 random de novo AML pts (GOELAMS-LAM-IR-2006 multicenter trial). Median expression of ATG2B and GSKIP were 4.8 (2.6-12.9) and 5.3 (0.3-5.1) respectively. No CNVdup14 was detected. Interestingly we found a correlation between the two genes expression (Pearson Correlation Coefficients 0.55 and linear regression p< 0.001, Fig C). Expression of ATG2B and GSKIP was also correlated with leukocytosis (p=0.003 and p=0.07) (Fig D). We found no impact on OS and PFS. For the first time, we described a high percentage of the germline CNVdup14 in de novo AML pts from Martinique (14%). Moreover evaluation of ATG2B and GSKIP expression suggested that the role of theses 2 genes in leukemogenesis is not limited to pts with the CNVdup14. Figure. Figure. Disclosures de Botton: Agios: Research Funding; Celgene: Honoraria, Research Funding. Benabelali:CERBA laboratory: Employment.

Primary Familial and Congenital Polycythemia (PFCP) is a non-malignant pathology of the erythroid lineage, characterized by an isolated increase of the red cell mass without evolution into myelofibrosis or acutisation. Around twenty constitutive non-sense and missense mutations located in the exon 8 of the erythropoietin receptor (EPOR) gene have been described so far. They all lead to the truncation of the C-terminal part of the protein and the loss of several cytoplasmic tyrosines. The erythropoietin (EPO) hypersensitivity of the PFCP erythroid progenitors is usually explained by the disappearance of these negative signaling regulation and internalization domains (Figure 1a). Nonetheless, relatively few functional studies have been carried out. We therefore investigated the mechanism of EPOR mutations in PFCP. We identified and extensively studied a new constitutive heterozygous frameshift EPOR mutation, p.Gln434Profs*11, which generates a new 11 amino acid (AA) C-terminal tail and a STOP codon at position 444, leading to the truncation of 63 AA of the wild-type receptor (Figure 1c). The primary progenitor cells displayed a major hypersensitivity to EPO, similar to Polycythemia Vera (PV) patients, as well as a spontaneous and persistent JAK2 and STAT5 phosphorylation, compared to the control cells. To study the mechanism of this new EPOR mutant, Ba/F3 cells were transduced with different retroviruses encoding either the HA-tagged wild-type EPOR (EPOR WT)or a truncated receptor at position 444, p.Gln444* (EPOR STOP) or the frameshift EPOR p.Gln434Profs*11mutation (EPOR FS), identical to the patient's mutation (Figure 2). As observed in primary cells, EPOR FS conferred a spontaneous STAT5 phosphorylation and a 4- to 5-fold EPO hypersensitivity to Ba/F3 cells (IC50 of 0.003 U/mL vs 0.01 U/mL) compared to both EPOR WT and EPOR STOP. As expected, the loss of negative regulatory domains in the C-terminal part of the receptor induced a persistent STAT5 activation in EPOR FS and EPOR STOP Ba/F3 cells. Moreover, EPOR FS was more stable (half-life of 120 minutes vs 60 minutes) and displayed a higher level of localization at the cell surface (more than 2-fold), compared to EPOR WT and EPOR STOP. However, no modification of the EPOR FS internalization pattern was observed during 125I-EPO labeling experiments and cytometry analysis. Furthermore, a dileucine motif, known to be a potential clathrin-dependent endocytosis site, is lost in the new C-terminal tail of EPOR FS mutant, yet its abrogation in EPOR WT and EPOR STOP did not modify the phenotype of Ba/F3 cells. Therefore, unlike previous reports, the major EPO hypersensitivity induced by EPOR p.Gln434Profs*11 cannot be explained by the receptor truncation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling. We wondered if this model could be extended to other EPOR mutations already described in PFCP (Figures 1a-b and 2). We therefore measured the impact on Ba/F3 cells proliferation of the frameshift EPOR p.Pro438Metfs*6 and its non-sense mutant counterpart, p.Pro443*, which retains the tyrosine at position 426, a binding site for the negative signaling regulators SOCS3 and CIS. EPO-hypersensitivity (4- to 5-fold) was only induced by EPOR p.Pro438Metfs*6, suggesting a common mechanism for the frameshift EPOR mutations. Interestingly, two proximal non-sense mutations, EPOR p.Glu399* and p.Glu425*, lacking 7 of the 8 cytoplasmic tyrosines that compose EPOR negative regulatory and internalization domains, were also able to confer a high EPO hypersensitivity to Ba/F3 cells. To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We highlighted that this pathology is much more complex than expected, since different mechanisms are involved in the EPO hypersensitivity phenotype, according to the type of EPOR mutation. Indeed, extensive truncations are sufficient by themselves to confer the EPO hypersensitivity phenotype due to the loss of all negative regulatory and internalization domains, whereas more distal truncations induced by frameshift mutants confer EPO hypersensitivity because of the appearance of a new C-terminal tail. The latter, by increasing EPOR stability at the cell surface, may cause pre-activation of both receptor and JAK2, constitutive signaling and hypersensitivity to EPO close to that of JAK2V617F-positive PVs. Disclosures No relevant conflicts of interest to declare.

Recent advances in myeloproliferative neoplasms (MPN) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. CALR mutations were reported in 25% of ET and in 35% of MF patients, which were non-mutated for JAK2 and MPL. CALR mutations lead to a frame-shift generating a common 36 amino acids C-terminal end and loss of the KDEL motif. Two variants account for 85% of the CALR mutations in ET and PMF: type 1, a 52-bp deletion and type2, a 5-bp insertion. 572 MPN patients negative for JAK2 and MPL mutations were collected from several French and Belgian hospitals. In our series, 396 patients were diagnosed as ET, 108 as MF and 68 as mixed MDS/MPN. We identified mutations of CALR in 368 patients (63.3%). The remaining 204 patients were designated as triple negative. In MF there was an over representation of type 1 mutation (70%) and an under representation of type 2 mutation (13%) as compared to patients with ET. This bias was associated with a higher allelic burden of CALR mutation in MF. MF patients represent a quite homogeneous group, mostly composed of men diagnosed at a median age of 62.5 with a low hemoglobin concentration (10.1 g/dl) and a low platelet count (median at 237 x 109/l). In ET patients the clinical presentation was more heterogeneous. They were mostly women (more than 61%) at a median age of diagnosis of 57 with a median platelet count of 724 x 109/l. In CALR mutated patients there were no sex prevalence and a more important thrombocytosis (785 x 109/l). The type of CALR mutation impacted also age and platelet count. We report the caracterisation of triple negative patients. In ETs they were mostly women (76.9%), particularly for ET patients under 50 years old that were almost exclusively women (27/28). In MF, triple negative patients presented a low hemoglobin concentration (8.85 g/dl) and a low leukocyte count (1.995 x 109/l). A striking characteristic is their platelet count, which was significantly lower than their group mates either in ET or in MF. This lower platelet count may suggest that in the general population, putative asymptomatic triple negative ET male patients could be retrieved, which would only be diagnosed at more advanced age with a symptomatic MF. Taken together, our results underline the differences between the two most frequent types of CALR mutation and show that CALR mutated patients should not be considered as a single entity. Disclosures No relevant conflicts of interest to declare.

Abstract 436 Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patients. IFNα was shown to induce complete long-term hematologic and molecular remissions in JAK2V617F-positive MPD patients, suggesting a possible curative effect of IFNα. In order to better understand mechanisms of action of this drug, experiments were perfomred on cell lines, patient cells and mice cells harboring a JAK2V617F mutation. We hypothesized that IFNα may target directly MPD cells through binding to its specific receptor, in addition to the potential immunological effect of this molecule and could induce cell cycle entry of the pathological quiescent HSCs. Human cell lines harboring JAK2 mutation or BCR-ABL oncogene were treated with increasing doses of IFNα. A significant anti-proliferative effect at low concentrations (100 IU/ml) on the JAK2V617F-positive HEL cell line was observed. On the contrary, at this low dose IFNα did not influence growth of the BCR-ABL-positive K562 and the non-mutated UT-7 cell lines. This result supported a direct effect of IFNα in JAK2V617F cells. Suppressor of cytokine signaling (SOCS) are potent inhibitors of the JAK-STAT pathway by proceeding to a classic negative regulation loop proteins. Following IFNα stimulation of HEL cells, SOCS1 and SOCS3 mRNAs expression were induced (p=0.00036 and p=0.0012, respectively) and efficient knock-down of either SOCS1 or SOCS3 by shRNAs expression in HEL cells was able to counteract the anti-proliferative effect of IFNα (p=0.028 and p=0.031, respectively). We concluded that SOCS1 and SOCS3 are involved, in IFNα proliferative inhibition activity of HEL cells. To determine whether JAK2V617F confer hypersensitivity to IFNα inhibitory effect, proliferation and genotyping of CD34+ progenitors isolated from the bone marrow of JAK2V617F–positive MPD patients (n=5) and control subjects (n=4) were plated at one cell per well in 96-well plates and counted and genotyped at Day 10-12. A significant decrease of the patients progenitors clonogenicity was observed when exposed to IFNα (10 000 IU/ml, p Lastly, in order to explain the long term molecular responses observed in PV patients treated by IFNα, we investigated the effect of IFNα on the cell cycle in more immature cells. BrdU assay on JAK2V617F Knock-in (KI) mice was performed. Five-months old JAK2V617F KI and wild-type mice received or not 10,000 UI of murine IFNα during 3 days. Sixteen hours before analysis, BrdU was injected i.p. in the animals. Bone marrow Lin-Sca+cKit+cells (LSK) were stained with CD150 and CD48 antibodies before BrdU labelling was analyzed by flow cytometry. Analysis of BrdU postive cells confirmed that i-JAK2V617F induces LSK CD150+CD48- to enter cell cycle ( 8.55+/-3.12% for WT cells versus 19.92+/-3,19% for JAK2V617F KI cells respectively (p = 0.04)) and ii- That IFNα induces CD150+CD48- LSK to enter cell cycle whatever the JAK2 WT (8.55+/-3.12% for non treated animals versus 15.43+/-3.19% for IFNα receiving mice (p=0.02)) or mutated status but this induction was more statistically significant in JAK2V617F mice (19.92+/-1.82% versus 25.23+/-0.57% respectively( p=0.02)). In conclusion, we gave rise to a double effect of IFNα in MPD cells: A direct preferential anti-proliferative effect of IFNα on JAK2V617F–positive MPD progenitor cells, possibly through the induction of SOCS over-expressions and a direct cell cycling effect on JAK2V617F stem cells suggesting that IFNa containing treatments could be of interest for JAK2V617F patients cure. Disclosures: No relevant conflicts of interest to declare.

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in approximately 50% of adult acute myeloid leukemia (AML) with normal karyotype. More than 40 mutant variants have been identified. Most of these mutations consist of a 4-bp insertion, which can be used as a target for minimal residual disease (MRD) monitoring. We previously checked the stability of NPM1 mutations at relapse in 21 NPM1-mutated patients at initial diagnosis. In this prospective study, we evaluated MRD by real-time quantitative PCR (RQ-PCR) in 36 NPM1-mutated AML patients corresponding to 33 adult and 3 pediatric cases, treated according to the French ALFA9801 or ALFA9802 and ELAM02 protocols, respectively. Out of these patients, 31/34 (91%) had normal karyotype, 13/33 (39%) had a high initial white blood cell count, and 10/36 (28%) were FLT3-Intern Tandem Duplication (FLT3-ITD) positive. 28 (78%) patients carry NPM mutation A, 3 (8%) mutation B and 5 (14%) other rare variants. RQ-PCR assays using a mutation-specific primer were performed on cDNA for mutation A and B and on genomic DNA for other NPM1 mutants. In our experiments, the maximal reproductible sensitivity of NPM1-based MRD detection is about 10−4 on genomic DNA and 10−5 to 10−6 on cDNA. The median follow-up was 260 days [40–791]. 2 to 9 follow-up samples from bone marrow and/or peripheral blood were analysed per patient. No correlation was found between leukocytosis at diagnosis and initial expression ratio of NPM1 mutation. The study of MRD log reduction after induction therapy shows that molecular responses are very heterogeneous (from 4.10−2 to more than 1.10−5), but 50% of patients reach at least a 4 log reduction in NPM1 levels. Patients with FLT3-ITD tend to have lower log reduction after induction than patients without FLT3-ITD, although not statistically significant (P=0.07). The analysis of NPM1-MRD in bone marrow and in peripheral blood at the same follow-up time-points shows that NPM1 levels can be until 1 log higher in bone marrow. This indicates that the evaluation of NPM1-MRD in bone marrow is more informative than in peripheral blood. We found all relapses had NPM1-MRD levels comparable to those observed at diagnosis. Among the 5 patients who relapsed so far, 2 were predictable by increasing MRD levels 1 to 4 months before hematological relapse. In 29 out of 36 cases, we could monitor MRD by both NPM1 mutation and WT1 gene expression. The comparison of the MRD profiles obtained by these two approaches reveals some discordant results, which can be, at least in part, explained by difference in the sensitivity of the RQ-PCR techniques, since the sensitivity of WT1 expression as MRD target is generally not higher than 10−3. In conclusion, NPM1 mutations are very specific and sensitive markers for MRD monitoring in AML. Further studies are required to determine if NPM1-MRD provides an independent prognostic factor and may be useful for therapeutic stratification in AML patients with intermediate risk cytogenetics.