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Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate–dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.

TO THE EDITOR: The discovery of prognostically informative mutations in patients with primary myelofibrosis (PMF)[1][1] prompted the development of mutation-enhanced risk scores.[2][2][⇓][3][⇓][4][⇓][5]-[6][6] Among these mutations, those in ASXL1 are invariably associated with adverse

Background: Until recently, ruxolitinib (RUX) was the only drug approved for treatment of intermediate- or high-risk myelofibrosis (MF). Many patients discontinue RUX due to lack of response, loss of efficacy, or intolerance. Fedratinib (INREBIC) is an oral, selective kinase inhibitor with activity against mutant and wild-type JAK2 and FLT3. Fedratinib is approved in the United States, Canada, European Union, United Kingdom, and elsewhere as front-line therapy for treatment of patients with JAK-inhibitor-naïve MF and those previously treated with RUX. In the single-arm, phase 2 JAKARTA2 trial of fedratinib 400 mg/day (starting dose) in patients with MF relapsed, refractory, or intolerant to prior RUX, 31% of pts achieved a spleen volume response and 27% achieved a symptom response with fedratinib (Harrison, Am J Hematol 2020). Gastrointestinal (GI) events were among the most common adverse events (AEs) reported during fedratinib treatment in JAKARTA2. A clinical hold was placed on fedratinib in November 2013 due to suspected cases of Wernicke encephalopathy (WE); the hold was later lifted but long-term data, including survival outcomes, are limited for fedratinib. Two phase 3 trials, FREEDOM (NCT03755518) and FREEDOM2 (NCT03952039) are ongoing to assess the safety and efficacy of fedratinib in patients with MF previously treated with RUX. Unlike earlier studies of fedratinib, these two trials prospectively include GI-directed mitigation strategies and thiamine monitoring and supplementation. Preliminary data from the single-arm FREEDOM trial show reduced incidence of GI AEs with these mitigation strategies (Gupta, CLML 2020). Here we describe the study design and methods for the FREEDOM2 trial, the first trial to compare fedratinib vs. active MF therapies. Study Design and Methods: The ongoing multicenter, open-label, randomized, phase 3 FREEDOM2 trial is enrolling patients with MF in the European Union, China, Russia, South Korea, and Australia. Key eligibility criteria include age ≥ 18 years; primary, post-polycythemia vera (post-PV), or post-essential thrombocythemia (post-ET) MF; DIPSS Intermediate-2 or High-risk disease; splenomegaly, defined as spleen volume ≥ 450 cm 3 by MRI/CT and palpable spleen measuring ≥ 5 cm below the left costal margin; ECOG PS score ≤ 2; and platelet count ≥ 50 ×10 9/L (Figure). Patients must have been relapsed or refractory to prior RUX treatment for ≥ 3 months, or intolerant to RUX after ≥ 28 days of treatment (i.e., developed RBC transfusion dependence [≥ 2 units/month for 2 months] or grade ≥ 3 thrombocytopenia, anemia, or hematoma/hemorrhage). Eligible patients are randomized 2:1 to receive fedratinib 400 mg/day or investigator-selected best available therapy (BAT), including RUX or other approved JAK inhibitors, in repeated 28-day treatment cycles (Figure). Spleen volume is assessed by MRI/CT scan at screening, end of cycles (EOC) 3, 6, 12, 18, 24, and at the end of treatment. The primary endpoint is spleen volume response rate (SVRR), the proportion of patients in each arm achieving a ≥ 35% reduction in spleen volume from baseline at EOC6, and the key secondary endpoint is MF symptom response rate, the proportion of patients achieving a ≥ 50% reduction from baseline in total symptom score (TSS) on the Myelofibrosis Symptom Assessment Form (MFSAF) at EOC6. Other secondary endpoints include durations of spleen volume and symptom responses, reductions in spleen size by palpation, overall and progression-free survival, effectiveness of the risk mitigation strategies for GI AEs and WE, patient-reported health-related quality of life (HRQoL) measures, and safety. Exploratory endpoints include relationships between fedratinib efficacy and prognostic markers (eg, mutations) and pharmacodynamic activity (eg, circulating cytokines, hematopoietic cell profiling). Patients in the BAT arm can cross-over to receive fedratinib after EOC6, or before EOC6 if the patient experiences confirmed progression of splenomegaly. As the first trial to compare fedratinib against other active therapies for MF, results from FREEDOM2 are eagerly anticipated. As of June 2021, 135 patients had been randomized, with enrollment expected to be completed by the end of 2021. Figure 1 Figure 1. Disclosures Benevolo: BMS: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Amgen: Speakers Bureau. Vannucchi: 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; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Harrison: CTI BioPharma: 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; Abbvie: 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; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Incyte Corporation: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Shire: 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; Galacteo: 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; 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. Loschi: CELGENE/BMS: Honoraria; AbbVie: Ended employment in the past 24 months, Honoraria; Gilead: Ended employment in the past 24 months, Honoraria; Novartis: Ended employment in the past 24 months, Honoraria; Servier: Ended employment in the past 24 months, Honoraria; MSD: Honoraria. Al-Ali: Incyte: Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Pfizer: Consultancy; AbbVie: Consultancy, Honoraria. Bonifacio: Amgen: 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; Pfizer: Membership on an entity's Board of Directors or advisory committees. Cervantes: Bristol Myers Squibb: Consultancy, Honoraria. Wrobel: Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BeiGene: Honoraria, Speakers Bureau. Kiladjian: AbbVie: Consultancy; AOP Orphan: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Verstovsek: PharmaEssentia: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Ital Pharma: Research Funding; Incyte Corporation: Consultancy, Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Roche: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. Mesa: Promedior: Research Funding; Pharma: Consultancy; Gilead: Research Funding; CTI: Research Funding; CTI: Research Funding; Novartis: Consultancy; Sierra Oncology: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; Celgene: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; AOP: Consultancy; Abbvie: Research Funding; La Jolla Pharma: Consultancy; Samus: Research Funding; Genentech: Research Funding. Rose: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Gharpure: Bristol Myers Squibb: Current Employment. Hernandez: Bristol Myers Squibb: Current Employment. Zhang: Bristol Myers Squibb: Current Employment. Passamonti: BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

INTRODUCTION: Ruxolitinib (RUX) has demonstrated efficacy in patients (pts) with MF, but many pts discontinue (D/C) RUX due to loss of response or treatment (Tx)-related cytopenias. Fedratinib (FEDR) is an oral, selective JAK2 inhibitor approved for Tx of pts with MF, including those previously treated with RUX. In the single-arm, phase 2 JAKARTA2 trial of FEDR 400 mg/day (d) in pts with MF resistant/intolerant to prior RUX, 31% of pts achieved a spleen volume response and 27% achieved a symptom response with FEDR. The most common adverse events (AEs) in JAKARTA2 were gastrointestinal (GI) events, including diarrhea in 62% of pts, nausea in 56%, and vomiting in 41%. A temporary clinical hold was placed on FEDR in 2013 due to suspected cases of Wernicke's encephalopathy (WE). The ongoing, single-arm, phase 3b FREEDOM trial (NCT03755518) further evaluates the safety and efficacy of FEDR in pts with MF previously treated with RUX. Unlike JAKARTA2 and other early FEDR trials, FREEDOM includes prospective strategies for preventing or mitigating GI AEs, thiamine level decreases, and potential WE. We investigated the safety of FEDR 400 mg/d in FREEDOM, including the effectiveness of these strategies. METHODS: Eligible pts are aged ≥ 18 years with DIPSS-defined intermediate- or high-risk, primary or post-PV/ET MF, ECOG PS ≤ 2, platelet count ≥ 50 ×10 9/L, and spleen volume ≥ 450 cm 3 by MRI/CT or palpable spleen ≥ 5 cm below the left costal margin (LCM). Pts must have previously received RUX for ≥ 3 months (mo), or for ≥ 28d with development of RBC transfusion requirement (≥ 2 units/mo for 2 mo) or grade ≥ 3 thrombocytopenia, anemia, hematoma, or hemorrhage. All pts received FEDR 400 mg QD in continuous 28d cycles. AE mitigation strategies include prophylactic and symptomatic use of anti-nausea/vomiting and anti-diarrheal Tx, thiamine supplementation, FEDR dosing modifications, and administration of FEDR with food. All pts who received ≥ 1 FEDR dose were evaluated for safety. AEs were coded using MedDRA v24.0 and graded (G) by CTCAE v5.0. RESULTS: At data cutoff (April 9, 2021), 34 pts had been enrolled and 16 pts continued to receive FEDR. Reasons for FEDR Tx D/C in > 1 pt were lack of efficacy (n = 5) AEs (n=4; 1 was Tx-related [G3 thrombocytopenia]), disease progression (n = 2), pt decision (n = 2), and to undergo transplant (n = 2). Median FEDR Tx duration was 28.3 weeks (range 1.6-101.3); 20 pts (59%) had received > 6 Tx cycles and 14 pts (41%) completed > 12 cycles. Median average FEDR dose was 400 mg/d (range 298-400).At BL, median (range) age was 68.5 years (49-82), time from MF diagnosis was 3.4 years (0.1-17.4), and spleen size was 15 cm (3-31). Most pts (62%) had primary MF, and all pts had received ≥ 3 mo of prior RUX Tx; the most common reason for prior RUX D/C was loss of response/Tx failure (41%). During FEDR Tx, 22 pts (65%) received ondansetron and 11 (32%) received loperamide. GI AEs reported in > 10% of pts were constipation (47%), diarrhea (35%), nausea (26%), abdominal pain (24%), and vomiting (18%). The frequency of nausea, vomiting, and diarrhea decreased as Tx continued (Figure). Most GI AEs were G1/2 (including all events of nausea, vomiting and diarrhea) and there were no Tx-related G3/4 GI AEs. No pt required FEDR dose-reduction, interruption, or D/C due to a Tx-related GI AE Overall, Tx-related G3/4 AEs were reported in 11 pts (32%), including anemia in 7 pts (21%), and neutropenia, thrombocytopenia, and hyperkalemia in 2 pts each (6%). The cases of hyperkalemia occurred in the setting of acute renal failure or of relevant concomitant conditions. At BL, 1 pt had a thiamine level below the lower limit of normal (LLN; 70 nmol/L); thiamine level was normalized before the pt received FEDR Tx. Thiamine levels dropped below the LLN during FEDR Tx for 4 pts between cycles 2-3 (and for 1 pt at end of Tx); levels returned to normal for these pts with thiamine supplementation and did not require FEDR interruption or reduction. Five other pts received prophylactic thiamine supplementation. There were no reported cases of WE. CONCLUSIONS: FEDR was generally well tolerated. These data suggest the frequency and severity of GI AEs may be reduced via early implementation of GI prophylaxis. Use of GI-directed therapies may have increased the incidence of low-grade constipation. Monitoring thiamine levels before FEDR initiation and periodically during FEDR therapy is recommended. Figure 1 Figure 1. Disclosures Gupta: Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; Incyte: Honoraria, Research Funding; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Yacoub: Agios: Membership on an entity's Board of Directors or advisory committees; Acceleron Pharma: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau. Verstovsek: Gilead: Research Funding; Genentech: Research Funding; Ital Pharma: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; PharmaEssentia: Research Funding; Protagonist Therapeutics: Research Funding; Incyte Corporation: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; Roche: Research Funding; Sierra Oncology: Consultancy, Research Funding; AstraZeneca: Research Funding; Novartis: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. Mesa: Novartis: Consultancy; CTI: Research Funding; Promedior: Research Funding; AOP: Consultancy; Sierra Oncology: Consultancy, Research Funding; La Jolla Pharma: Consultancy; Genentech: Research Funding; CTI: Research Funding; Celgene: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Pharma: Consultancy; Incyte Corporation: Consultancy, Research Funding; Samus: Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Harrison: BMS: 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; Roche: 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; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Sierra Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kiladjian: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; Taiho Oncology, Inc.: Research Funding; Incyte Corporation: 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. Fazal: Agios: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Sanofi Genzyme: Consultancy, Honoraria, Speakers Bureau; Stemline: Consultancy, Honoraria, Speakers Bureau; Taiho: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Tolero: Consultancy. Foltz: Incyte: Research Funding; CTI Biopharma: Research Funding; Constellation: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra: Research Funding; Amgen: Other: Spouse employment and equity ownership.. Miller: CTI: Consultancy, Research Funding; Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria; Takeda: Honoraria, Speakers Bureau; Celgene: Speakers Bureau. Gharpure: Bristol Myers Squibb: Current Employment. Hernandez: Bristol Myers Squibb: Current Employment. Wang: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; VIR Biotechnology: Current equity holder in publicly-traded company. Talpaz: Imago: Consultancy; Celgene: Consultancy; Constellation: 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: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Other: Grant/research support .

Introduction: MF is a clonal stem cell disorder characterized by progressive bone marrow failure, extramedullary hematopoiesis, and debilitating constitutional symptoms. Approximately 60% of pts develop anemia within a year of diagnosis; pts who are transfusion dependent have worse survival and quality-of-life outcomes (Tefferi A, et al. Mayo Clin Proc 2012;87:25-33). Treatment for MF-associated anemia remains critically unaddressed, with no approved therapies available. Luspatercept is a first-in-class erythroid maturation agent that has been shown to increase hemoglobin and reduce transfusion burden in pts with myelodysplastic syndromes (Fenaux P, et al. N Engl J Med 2020;382:140-51) and β-thalassemia (Cappellini MD, et al. N Engl J Med 2020;382:1219-31). Pts have also achieved multiple response episodes with luspatercept (Fenaux P, et al. Blood 2019;134:841). We previously reported findings from the ongoing open-label, phase 2 ACE-536-MF-001 trial evaluating luspatercept in pts with MF-associated anemia (Gerds AT, et al. Blood 2019;134:557). Here we report updated study results, focusing on response in pts requiring RBC transfusions. Methods: 79 pts with MF and anemia were enrolled (Figure). Of these, 43 were requiring RBC transfusions, defined as 6-12 U/84 d prior to treatment, or 4-12 U/84 d for the 3 expansion cohort pts. Pts in Cohort 3B received a stable ruxolitinib (RUX) dose for ≥ 16 wks (40 wks for expansion cohort pts), whereas Cohort 2 pts did not (Figure). Pts in Cohort 3B received a median RUX dose of 20 mg/d (range 5-40) for a median of 73 wks (range 29-359) prior to treatment. Pts received luspatercept every 21 d at a starting dose of 1.0 mg/kg (1.33 mg/kg for 3 expansion cohort pts in Cohort 3B), with doses escalating up to 1.75 mg/kg; 81% and 50% of pts in Cohorts 2 and 3B escalated to 1.75 mg/kg. Primary endpoint for pts receiving transfusions was RBC transfusion-independence (RBC-TI) for ≥ 12 consecutive wks within the first 24 wks on study. However, as responses were observed that did not fall within the 24-wk response window, additional assessments were conducted for the entire treatment duration for RBC-TI and ≥ 50% decrease in RBC transfusions (minimum 4 RBC U decrease) from baseline. Intent-to-treat data were analyzed as of March 29, 2020. Results: Pts in Cohorts 2 and 3B received a median of 8 cycles of luspatercept (range 1-39), for a median (mean) duration of 24 (31) and 23 (39) wks, respectively. Pts in Cohorts 2 and 3B received a median of 2.7 RBC U/28 d (range 1-5) and 2.7 RBC U/28 d (range 2-4), respectively. For the primary endpoint, 2/21 (10%) and 6/22 (27%) pts receiving transfusions in Cohorts 2 and 3B achieved RBC-TI over any consecutive 12 wks during the first 24 wks. Median time to start of first RBC-TI response period in Cohorts 2 and 3B was 1.5 days and 37 days; median duration of response was 49 wks (range 16-82) and 42 wks (range 12-111), respectively. When assessing the entire treatment period, 4/21 (19%) and 8/22 (36%) pts in Cohorts 2 and 3B achieved RBC-TI ≥ 12 wks, respectively. 25% of the RBC-TI responders in both cohorts experienced 2 separate episodes of RBC-TI ≥ 12 wks. Median cumulative duration of all individual ≥ 12-wk response episodes of RBC-TI was 59 wks (range 24-82) in Cohort 2 and 55 wks (range 12-116) in Cohort 3B. 8/21 (38%) and 10/22 (46%) pts in Cohorts 2 and 3B achieved a ≥ 50% reduction in RBC transfusion burden (minimum ≥ 4 U reduction) over 12 wks; of these, 38% and 20% of responders in Cohorts 2 and 3B experienced 2 separate ≥ 12-wk response episodes, and 1 pt (13%) in Cohort 2 experienced 3 separate responses. 4/15 (27%) and 8/14 (57%) pts in Cohorts 2 and 3B who reached 24 wks of treatment achieved clinical benefit and therefore continued luspatercept on an ongoing basis. Safety is reported for all 79 pts on study. 5 (6%) pts had grade 3-4 treatment-related adverse events (AEs); these AEs were diarrhea (n = 2), dehydration (n = 1), and hypertension (n = 3). There were no treatment-related deaths. Treatment-related AEs (any grade) occurring in ≥ 5% of pts were hypertension (13%), bone pain (9%), and diarrhea (5%). 8 (10%) pts had ≥ 1 AE leading to discontinuation. 16 (20%) pts remain on treatment. Conclusions: These longer-term findings suggest durable activity of luspatercept in pts with MF-associated anemia. In addition, a quarter of pts receiving transfusions achieved more than one ≥ 12-wk episode of RBC-TI response with luspatercept. The incidence of grade 3-4 AEs with luspatercept was low. Disclosures Gerds: Roche/Genentech: Research Funding; Celgene: Consultancy, Research Funding; Sierra Oncology: Research Funding; AstraZeneca/MedImmune: Consultancy; Incyte Corporation: Consultancy, Research Funding; Pfizer: Research Funding; Apexx Oncology: Consultancy; CTI Biopharma: Consultancy, Research Funding; Gilead Sciences: Research Funding; Imago Biosciences: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Passamonti:Novartis: Speakers Bureau; BMS: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support. Kremyanskaya:Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Bristol Myers Squibb: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Astex Pharmaceuticals: Research Funding. Gotlib:CTI Biopharma: Research Funding; Kartos: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; BMS: Research Funding. McCaul:BMS: Speakers Bureau; Seattle Genetics: Speakers Bureau; Karyopharm: Speakers Bureau; Incyte: Speakers Bureau. Ribrag:argenX: Research Funding; pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; servier: Consultancy; nanostring: Honoraria, Membership on an entity's Board of Directors or advisory committees; gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AZD: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other; Infinity: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Membership on an entity's Board of Directors or advisory committees; gustave roussy comprehensive cancer center: Current Employment; EPZ: Honoraria, Membership on an entity's Board of Directors or advisory committees; epizyme (EPZ): Research Funding. Mead:Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy; CTI: Consultancy; Gilead: Consultancy. Harrison:Roche: Honoraria; Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau; Janssen: Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Sierra Oncology: Honoraria; Celgene: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria. Mesa:Sierra Oncology: Consultancy; LaJolla Pharmaceutical Company: Consultancy; Novartis: Consultancy; Incyte: Research Funding; CTI BioPharma: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Promedior: Research Funding; Bristol Myers Squibb: Research Funding; Samus Therapeutics: Research Funding. 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; 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. Barosi:Bristol Myers Squibb: Consultancy. Gerike:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Shetty:BMS: Current Employment, Current equity holder in publicly-traded company. Pariseau:Bristol Myers Squibb: Current Employment. Miranda:Bristol Myers Squibb: Current Employment. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Giuseppi:BMS: Current Employment, Current equity holder in publicly-traded company. Zhang:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Backstrom:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company; BMS: Current equity holder in publicly-traded company. Verstovsek:AstraZeneca: Research Funding; Roche: Research Funding; CTI Biopharma Corp: Research Funding; Genentech: Research Funding; Incyte Corporation: Consultancy, Research Funding; NS Pharma: Research Funding; Novartis: Consultancy, Research Funding; PharmaEssentia: Research Funding; ItalPharma: Research Funding; Sierra Oncology: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding.

Background: Plasma soluble interleukin-2 receptor alpha (sIL-2Rα), that is released from the cell surface of activated T and B lymphocytes, is one of the best markers of immune activation. High levels of sIL-2Rα have been correlated with poor prognosis in different types of cancers. In patients with primary myelofibrosis (PMF) elevated plasma levels of the receptor are associated with risk of blast transformation and death. In this study we aimed to analyze how increased levels of sIL-2Rα mark the disease progression to improve our knowledge on the immune regulation of the disease. Methods: sIL-2Rα plasma concentration values were obtained by a commercial immunoassay (R&D Systems), according to the Manufacturer's Instructions, and expressed in pg/ml. Health care data of persons with PMF were obtained from the data-base of the Center for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. The final study cohort consisted of 300 subjects. Thirty-four healthy normal subjects matched for age and sex represented the control group. The study was approved by the local Ethic Committee, and was conducted complying with the principles of the Declaration of Helsinki. Results: In patients with PMF, plasma sIL-2Rα values ranged from 275 to 13,860 pg/ml, with a mean value of 1693 ± 1529 pg/mL standard deviation (SD). sIL2Rα metrics in healthy controls were: mean, 740 ± 346 pg/mL (SD), range, 207 to 1718 pg/mL. Difference in plasma sIL2Rα levels between subjects with PMF and normals was significant (P 10 cm from the left costal margin), 26 (24.1%) leukocytosis (WBC >12 x109/L), and 22 (20.4%) an alarming number of HSP-CD34+ cells in PB (>100 x 106/L). Moreover, 10 (9.2%) received an allogeneic hematopoietic stem cell transplantation (HSCT), 15 (13.8%) incurred in blast transformation (BT), and 8 (7.4%) died. In patients with JAK2V617F mutation, having elevated level of sIL-2Rα gave a higher risk of incurring in severe anemia (hazard ratio (HR), 3.70, 95% CI, 1.31-10; P=0.009), large splenomegaly (HR, 8.33; 95% CI, 2.12-33.3; P=0.002) and alarming HSP-CD34+ cells (HR, 4.14; 95% CI, 1.02-20; P=0.050). Moreover, JAK2V617F mutants with elevated levels of sIL-2Rα had higher risk of death than those with non-elevated sIL-2Rα (HR, 9.09; 95% CI, 1.75-50; P=0.008). On the contrary, in CALR mutants no interaction with the levels of sIL-2Rα and risk of disease progression was evidenced. By using a composite endpoint that considered death for any cause, BT or HSCT, PMF patients with JAK2V617F mutation carrying an elevated sIL-2Rα presented a shorter time to event as compared to those with non-elevated sIL-2Rα (P=0.011), while in CALR mutants this difference disappeared (P=0.80) (Figure). Conclusion: The results of this study give a strong proof of concept that the immune landscape of PMF is associated with the presence/absence of JAK2V617F or CALR mutation. Acquiring direct evidence that elevated levels of sIL-2Rα represents an immune- mediated modality of disease progression in JAK2V617F but not in CALR mutated patients opens new areas of investigation towards an individualized therapeutic approach of PMF. Figure Disclosures No relevant conflicts of interest to declare.

Background: A myeloproliferative neoplasm (MPN) is frequent underlying cause of splanchnic vein thrombosis (SVT). We reported that ruxolitinib, a JAK1/2 inhibitor, was safe in patients (pts) with MPN-associated SVT and effective in reducing spleen size at the planned primary analysis at 24 weeks (w) in an investigator-initiated phase II clinical trial (Pieri L, AJH 2016). Herein we present final long term follow up (FU) data. Methods. A total of 21 MPN-SVT entered the trial in 2012. Pts who completed the 24w core study and well tolerated the drug and had evidence of clinically-significant improvement were allowed to enter an extension phase up to w72, then entered a long term FU. The drug was provided by Novartis, that had no role in trial design nor in data analysis. Safety data were reported as cumulative incidence of adverse events (CTCAE v4.03). Clinical responses were defined according to ELN and IWG-MRT criteria. Results. Patients disposition. Eighteen patients were alive at last follow-up at a median of 5.5y, range 3.7-6.5y. Two pts died before w72 (hepatocarcinoma; unknown cause), 1 pt died at w252 (sepsis). Diagnosis of the 18 pts was: PMF 8 (44%), PV 5 (28%), ET 4 (22%), PET-MF 1 (6%). The DIPSS risk score of the 8 pts with PMF was low in 4 and intermediate-1 in 4. At last FU, 14 patients are still on ruxolitinib (78%), 10 pts still on clinical trial, 4 pts shifted to commercial drug. Four pts (22%) withdrawn treatment after w72 because of inefficacy, withdrawal of consent, unknown reasons, shift to observation only (1 each). Safety. Thirteen pts (72.2%) had adverse events; the median number of events per patient was 5 (range, 0-37). Five pts had G2 thrombocytopenia plus 1 pt G3 (28%), 9 had G2 anemia (50%), 1 had G2 neutropenia (6%). There were 4 non-hematologic G3 adverse events (22%), one drug-related (CPK increase) that resolved with dose reduction. Six pts developed G1-2 infections (33%), 4 had Herpes Zoster reactivation (22%), 1 developed second cancer (pheochromocytoma at w96; 6%). Efficacy. Spleen response (defined as percentage reduction of spleen length from left costal margin (LCM) from baseline value as by IWG-MRT criteria) was available in 16 of 18 pts on treatment. Of 8 pts who obtained spleen response at w72, 4 pts had a complete response, while the remaining 4 pts had a spleen reduction >50%. At last FU, 6 of 8 pts (75%) adjudicated as spleen responders at w72 maintained the response. None of the 8 pts who had not obtained a spleen response at w72 acquired it at the last FU. According to ELN/IWG-MRT criteria of disease response, 7 pts had partial response, 11 had stable disease. At abdominal vessel eco scan, thrombosis improved in 3 of 12 evaluated pts (25%) and remained stable in the remaining 9. Of the 16 pts with endoscopic evaluation at last FU, complete resolution of oesophageal varices was documented in 3/12 with baseline varices (25%), worsening in 4 (33%), stable in 5 (42%), while no de-novo formation of F1 varices was documented in the remaining 4 (33%). No pt had gastrointestinal bleeding episodes. Exploratory endpoints. Included changes in the level of JAK2V617F variant allele burden (VAF) at last FU, available in 13 of 15 JAK2V617F mutated pts. VAF remained stable in 2 pts (13%), increased by 34% (range 25-40) in 3 pts, while 8 pts (62%) had a reduction >10% (range 18-96), of whom 6 (46%) had a reduction >50% (median 82%, range 70-96). By correlative analysis of JAK2 VAF changes and spleen response, 3 of 6 pts with VAF reduction of >50% had complete spleen response, as compared to none of the 3 pts who had an increase of VAF. Conclusions. After a median follow-up of 5.5 years, ruxolitinib continues to be safe in pts with MPN-associated SVT and maintains efficacy against splenomegaly in 33% of the pts. Of note, the large majority of pts (67%) showed stabilization or improvement of oesophageal varices, supporting the hypothesis that sustained reduction of enlarged spleen might contribute to decrease the upstream venous system pressure. Significant reduction of JAK2V617F VAF>50% was documented in 40% of the pts, although it was not clearly correlated with clinical improvement. Disclosures Rumi: novartis: Honoraria, Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ruxolitinib use in essential thrombocythemia

Primary myelofibrosis (PMF) is the most aggressive Philadelphia-negative myeloproliferative neoplasm. Heterogeneity in disease phenotype and outcome led to investigate the influence of germline host genetic variations on disease onset and evolution. We recently found that patients with secondary myelofibrosis are enriched in the polymorphic allele variant of the -2518 A/G SNP of the Monocyte Chemoattractant Protein-1 (MCP-1), and that this SNP correlates with disease severity (Masselli E. et al, Leukemia 2018). Here, we examined the interactive effects of MCP-1 allele variants with the baseline phenotype in PMF, and how these variants impact the disease progression. MCP-1 -2518 SNP genotyping was performed by TaqMan Predesigned SNP Genotyping Assays on DNA extracted from peripheral blood granulocytes of 201 PMF patients and 249 matched local control subjects (CTRL). One-hundred and nineteen out of 201 PMF were males (59.2%). Median age was 52y (18-80y). N.103 patients (51.2%) were pre-fibrotic PMF. Driver mutations occurred as follows: 123 (61.2%) harbored a JAK2V617F mutation, 47 (23.4%) had CALR mutations, 20 (9.9%) were triple negative and n.8 (4%) had MPL mutations. Three patients had both JAK2V617F and CALR mutation. After a median follow-up of 84.7 months, 37 patients (18.4%) died, 38 (18%) experienced blast transformation (BT) and 23 patients (11.4%) received allogeneic hematopoietic stem cell transplantation (ASCT). Median age of CTRL was 64y (28-85y), and 132/249 (53%) were males. Consistently with our previous report, PMF displayed similar genotypic and allelic frequencies of the local control population (O.R., 1.04). The G allele distribution was also similar in JAK2V617Fpos vs. JAK2V617Fneg PMF and did not correlate with the allele burden (O.R., 1.29 and 1.51, respectively). After verifying that PMF with only 1 copy of the risk allele (A/G), had a similar risk of the reference group with 0 copies (A/A), we used a recessive genetic model to compare PMF with 2 copies of the risk variant (G/G, hereby referred as the MCP-1 high-risk group) vs. A/A+A/G PMF (the MCP-1 low-risk group). Genotype-phenotype correlations with disease parameters at the time of diagnosis were performed by χ2/Fisher exact test. We found that the MCP-1 high risk group displayed a significantly higher frequency, at the time of diagnosis, of severe anemia, massive splenomegaly, elevated LDH and higher levels of hs-CRP (high-sensitivity C-reactive protein) (see Table). These results indicate that MCP-1 high-risk variant has a role in determining the disease phenotype and the pro-inflammatory microenvironment of PMF. Additionally, we assessed whether the MCP-1 high-risk variant could predispose the acquisition of risk factors in PMF. On the basis of the recessive genetic model, we found that the MCP-1 high-risk group was about four times as likely to progress to leukocytosis as was the reference group (HR, 4.16; 95% CI; 1.37 to 12.5; P=0.011). To weight the clinical relevance of our findings, we tested whether the MCP-1 high-risk variant might affect the disease progression. ASCT occurrence resulted significantly higher in the high-risk group as compared to the low-risk (HR = 3.22; 95% CI, 1.06 to 10; P=0.038). In the composite endpoint model that considered death for any cause, BT or ASCT, PMF carrying the high-risk genotype presented a shorter time to event as compared to the low-risk group (HR, 2.56; 95% CI, 1.19 to 5.55; P=0.016). In conclusion, the MCP-1 SNP appears to identify PMF patients at higher chance to present with adverse hematological features and more likely to progress rapidly to either death, BT or ASCT. Table. Disclosures No relevant conflicts of interest to declare.

Several evidences indicate that abnormal megakaryocyte (MK) maturation drives the development of Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative neoplasms: a) presence in bone marrow (BM) of numerous clusters of MK with ultrastructural abnormalities indicating retarded maturation was the first cell hallmark reported for MF (Zucker-Franklin, Dahlem Workshop on Myelofibrosis-Osteosclerosis Syndrome Germany-West: Pergammon Press; 1975); b) studies in mouse models with MK-restricted JAK2V617F expression demonstrated that abnormal MK are necessary and sufficient to induce MF (Zhan, Leukemia 2016;30:23324) and c) both in patients and mouse models, the driver mutations induce a RSP14 ribosomopathy that reduces expression of GATA1 in MK, leading to retarded maturation (Vannucchi, AmJPathol 2005;167:849; Gilles, JCI 2017;127:1316). Due to fibrosis and hematopoietic failure in BM, MF patients develop hematopoiesis in spleen which also is rich of MK. Whether in MF MK in BM and spleen express similar ultrastructural abnormalities has not been investigated as yet. By transmission electron microscopy, we compared the ultrastructural features of MK from BM of 5 MF patients and spleen of 11 MF patients (9 JAK2V617Fhomo, 1 JAK2V617Fhomo/ASXl1R1068X, 2 JAK2V617Fhetero, 1 JAK2V617F hetero/ASXl1R693X, 1 CARLtyp1/TET2R1214W and 2 triple negative; 12 primary and 4 post-PV, IPSS score: low-intermedium 2, DIPSS score: intermedium 1-2). MF underwent splenectomy to reduce discomfort of large spleen size (>20 cm from the left costal margin). BM from 2 healthy controls (HC) and post-trauma spleen from 1 HC were analyzed as control. MK were numerous both in BM (43±5 vs 30±2/area in MF vs HC, p=0.027, Anova) and spleen (47±18 in MF vs below detection in HC). The majority (98%) of MK from MF BM had immature ultrastructural morphology while 59% of MK from HC BM were mature (p=0.00003). To a surprise, the majority (69%) of MK from MF spleen had also mature morphology with developed platelet territories. However, by contrast with the heavy electron dense granules found in the cytoplasm of HC MK, the cytoplasm of MK from both BM (56%) and spleen (32%) contained granules with light electron density, an indication of reduced protein content. MK from MF BM and spleen consistently expressed distinctive ultrastructural abnormalities: 100% of BM MK presented dilated nuclear membranes with abnormally large nuclear pores which in 33% of the cases were associated with thickening of the plasma membrane with extrusion of pseudopods and presence of glycogen droplets. By contrast, these abnormalities were not presented by spleen MK, 49% of which presented instead mitochondria with reduced crests. These results suggest that MK from BM and spleen express distinctive metabolic abnormalities: those from BM express insufficient lipid metabolism impairing membrane biosynthesis while those from spleen have impaired aerobic metabolism. As predicted by the impaired RSP14 signature reported in MF (Gilles, JCI 2017;127:1316), the cytoplasm of MK from MF BM contained reduced number of ribosomes (455±125 vs 1059±41 in MF vs HC, p=0.004) but the percent of ribosomes engaging with the rough endoplasmic reticulum (RER) was the same in both cases (41±2 vs 46±10%, p=0.80). The number of ribosomes was lower also in MK from spleen (157±85, p=0.0003 ) but in addition, MK from the spleen were poorly engaged with the RER (33±22%, p=0.008). These results indicate that in addition to low number of ribosomes resulting in defective mRNA translation, MK from spleen express ribosomes that engage poorly with RER leading to defective folding check and post-translational modification of nascent proteins. These results predict that partially different mechanisms drive the alterations of platelet territories observed in MK from BM and spleen in MF: defective membrane biosynthesis drives the paucity of the granules and the retarded maturation of the platelet territories observed in MK from BM. Insufficient proteins synthesis drives the poor protein content of granules found in MK both from BM and spleen but in addition the function of the proteins in the granules of MK from spleen may be impaired by improper protein folding check/post-translational modification. These results suggests lipid metabolism and RER functions as two potential novel therapeutic targets to restore MK function in MF. Disclosures Vannucchi: Incyte: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.

Introduction: Approximately two-thirds of patients with primary or post-essential thrombocythemia/polycythemia vera myelofibrosis (MF) have anemia, many of whom require red blood cell (RBC) transfusions. In this heavily transfused population there are severely limited treatment options; effective treatment for anemia in MF is a critically unmet medical need. Luspatercept is a first-in-class erythroid maturation agent which binds to select TGF-β superfamily ligands to reduce aberrant Smad2/3 signaling and enhance late-stage erythropoiesis. Here we report the interim results of the ongoing open-label, phase 2 trial evaluating luspatercept in patients with MF and anemia. Methods: Seventy-four patients with MF and anemia were enrolled, including 41 not receiving concomitant ruxolitinib at study entry who received either no RBC transfusions (n = 20; Cohort 1) or 2-4 RBC U/28 d in the 12 wks prior to treatment (n = 21; Cohort 2). Thirty-three enrolled patients were receiving a stable dose of ruxolitinib for at least 16 wks, of which 14 were not receiving RBC transfusions (Cohort 3A) and 19 were (Cohort 3B). Patients in Cohorts 3A and 3B received a median dose of ruxolitinib of 20 mg/d (range, 5-50 mg/d) for a median of 41 and 43 wks, respectively. Patients received luspatercept every 21 d at a starting dose of 1.0 mg/kg in doses escalating up to 1.75 mg/kg. The primary endpoint in patients not receiving transfusions was a hemoglobin (Hb) increase ≥ 1.5 g/L at every assessment from baseline for ≥ 12 consecutive wks, within the first 24 wks on study. In patients receiving RBC transfusions, the primary endpoint was RBC transfusion-independence (RBC-TI) for ≥ 12 consecutive wks, within the first 24 wks on study. Additional endpoints included proportion of patients achieving mean Hb increase ≥ 1.5 g/dL in Cohorts 1 and 3A, and ≥ 50% decrease in RBC transfusions (minimum 4 RBC U decrease from baseline) in Cohorts 2 and 3B, with both responses lasting ≥ 12 consecutive wks, within 24 wks of study entry. Intent-to-treat (ITT) data were analyzed as of May 10, 2019. Results: Median age was 71 y (range, 50-88 y) and 42 (57%) were male. Median interval from MF diagnosis to study entry was 3.3 y (range, 19 d-13.5 y). Seven patients were Dynamic International Prognostic Scoring System (DIPSS) intermediate-1, 58 were intermediate-2, and 8 were high risk. Median Hb concentration at study entry was 8.8 g/dL (range, 6.7-9.8 g/dL) for Cohort 1 and 8.6 g/dL (range, 6.7-9.1 g/dL) for Cohort 3A. Patients in Cohort 2 received a median of 2.7 RBC U/28 d (range, 1-5 U/28 d) and patients in Cohort 3B received a median of 2.3 RBC U/28 d (range, 2-4 U/28 d). Patients received a median of 8 cycles of luspatercept (range, 1-24 cycles). Efficacy data are displayed in the Table, per ITT data. Two of twenty (10%) and 3/14 (21%) patients in Cohorts 1 and 3A, respectively, achieved an absolute Hb increase ≥ 1.5 g/dL at every measurement from baseline over any consecutive 12 wks. Two of twenty-one (10%) and 6/19 (32%) patients in Cohorts 2 and 3B, respectively, achieved RBC-TI over any consecutive 12 wks. Median duration of Hb response was 20 wks (range, 12-27 wks) in Cohort 1 and 12 wks (range, 12-13 wks) in Cohort 3A. Median duration of RBC-TI was 23 wks (range, 16-31 wks) in Cohort 2 and 32 wks (range, 12-65 wks) in Cohort 3B. Three (15%) and 8 (57%) patients in Cohorts 1 and 3A, respectively, achieved a mean Hb increase of ≥ 1.5 g/dL. Eight (38%) and 10 (53%) patients in Cohorts 2 and 3B, respectively, achieved a ≥ 50% reduction in RBC transfusion burden from baseline. Ruxolitinib exposure remained stable throughout the 24-wk treatment period in both Cohorts 3A and 3B. There was no difference in spleen size between responders and non-responders. Four (5%) patients had grade 3-4 treatment-related adverse events (AEs). There were no treatment-related deaths. Treatment-related AEs occurring in ≥ 3% of patients were hypertension (11%), bone pain (8%), and diarrhea (4%). Seven (9%) patients had ≥ 1 AE resulting in treatment discontinuation; 23 (31%) remain on study. Conclusions: The initial results from this ongoing study suggest clinically significant activity of luspatercept in patients with MF-associated anemia, including those receiving concomitant ruxolitinib. A minority of AEs were grade 3-4 in severity, consistent with previous studies in MDS and beta-thalassemia. Disclosures Gerds: CTI Biopharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Pfizer: Consultancy; Imago Biosciences: Research Funding; Roche: Research Funding; Celgene Corporation: Consultancy, Research Funding; Sierra Oncology: Research Funding. Vannucchi:Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; CTI: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: 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. Passamonti:Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau. Kremyanskaya:La Jolla: Consultancy; Incyte, Celgene, Constellation, Protagonist.: Research Funding. Gotlib:Celgene Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Promedior: Consultancy, Research Funding; Kartos: Consultancy, Honoraria, Research Funding; CTI Biopharma: Research Funding. Ribrag:BMS: Membership on an entity's Board of Directors or advisory committees; AZ: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; argenX: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Epizyme: Honoraria, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees. Mead:Celgene: Consultancy, Research Funding; CTI: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; Bristol Myers-Squibb: Consultancy; Pfizer: Consultancy. Harrison:Sierra Oncology: Honoraria; Gilead: Speakers Bureau; CTI: Speakers Bureau; Roche: Honoraria; Promedior: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Incyte: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; AOP: Honoraria; Shire: Speakers Bureau. Mesa:Shire: Honoraria; Promedior: Research Funding; Genotech: Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses; LaJolla: Consultancy; AbbVie: Research Funding; NS Pharma: Research Funding; CTI: Research Funding; Gilead Sciences: Research Funding; Samus: Research Funding; Baxalta: Consultancy; Galena Biopharma: Consultancy; Celgene Corporation: Research Funding; Sierra Oncology: Consultancy; PharmaEssentia: Research Funding; Genentech: Consultancy; Pfizer: Research Funding; AOP Orphan Pharmaceuticals: Honoraria, Other: travel, accommodations, expenses; Incyte: Other: travel, accommodations, expenses, Research Funding. Kiladjian:AOP Orphan: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Consultancy. Gale:Celgene Corporation: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Pariseau:Celgene Corporation: Employment. Gerike:Celgene Corporation: Employment. Zhang:Celgene Corporation: Employment, Equity Ownership. Linde:Abbott Laboratories, Inc.: Equity Ownership; Acceleron Pharma: Employment, Equity Ownership; Fibrogen, Inc.: Equity Ownership. Reynolds:Acceleron Pharma: Employment, Equity Ownership. Verstovsek:Gilead: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Roche: Research Funding; Incyte: Research Funding. OffLabel Disclosure: Luspatercept is an investigational therapy that is not approved for any use in any country. Luspatercept is currently being evaluated for potential use in patients with anemia due to myelodysplastic syndromes, beta-thalassemia, or myelofibrosis.

The current document is a revision of the International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria for treatment response in myelofibrosis (MF) and represents a collaborative effort by the IWG-MRT and the European LeukemiaNet to objectively assess the value of new drugs in inducing morphologic remission or improvement in MF-associated symptomatic burden (MF-SB). Some of the changes in the current revision include stricter definitions of red cell transfusion dependency and independency and consideration of the Myeloproliferative Neoplasm Symptom Assessment Form as a tool to quantify meaningful changes in disease-related symptoms. Six response categories are listed: complete remission (CR) and partial remission signify treatment effects that are consistent with disease modification, whereas drug-induced improvements in MF-SB were annotated as clinical improvement, anemia response, spleen response, or symptoms response. Additional criteria are provided for progressive disease, stable disease, and relapse. The document also includes recommendations for assessing cytogenetic and molecular remissions, without mandating their inclusion for CR assignment.

Standardized response criteria to interpret and compare clinical trials are needed for approval of new therapeutic agents by regulatory agencies. The European LeukemiaNet (ELN) response criteria for essential thrombocythemia (ET) and polycythemia vera (PV) issued in 2009 have been widely adopted as end points in a number of recent clinical trials. However, evidence exists that they do not predict response or provide clinically relevant measures of benefit for the patients. This article presents revised recommendations for assessing response in ET and PV provided by a working group established by ELN and International Working Group-Myeloproliferative Neoplasms Research and Treatment. New definitions of complete and partial remission incorporate clinical, hematological, and histological response assessments that include a standardized symptom assessment form and consider absence of disease progression and vascular events. We anticipate that these criteria will be adopted widely to facilitate the development of new and more effective therapies for ET and PV.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

To the editor: Although primary myelofibrosis (PMF) is generally regarded as arising from a mutated stem or progenitor hematopoietic cell, immune dysregulation is common. For example, there are increased plasma levels of inflammatory cytokines and clinical and laboratory manifestations of

The frequency of A3669G single nucleotide polymorphism (SNP) of human glucocorticoid receptor has been reported increased in polycythemia vera. We investigated the frequency of A3669G SNP and its impact on disease phenotype and progression in 499 patients with primary myelofibrosis (PMF). The distribution of the A3669G allele differed between PMF patients and 2 healthy control populations (odds ratio, 1.6 and 1.8). The variant allele at the homozygous state (G/G) was associated with higher white blood cell count, larger spleen index, and higher frequency of circulating CD34+ cells at diagnosis. The latter association remained significant after correction for the JAK2V617F genotype. In patients JAK2V617F mutated, the G/G genotype was associated with shorter overall survival (77.6 months vs 298 months, P = .049) and blast transformation (BT)–free survival (76.7 months vs 261 months; P = .018). The latter association remained significant after correction for the known BT risk factors, such as age, sex, white blood cell count, percentage of blasts, IPSS prognostic score, and homozygosity for JAK2V617F (hazard ratio = 3.3; P = .006). In conclusion, the glucocorticoid receptor A3669G is a susceptibility allele for PMF: it contributes to confer the phenotype of excess myeloproliferation, and it cooperates with the JAK2V617F mutation in determining BT.

We genotyped 370 subjects with primary myelofibrosis (PMF) and 148 with postpolycythemia vera/postessential thrombocythemia (PPV/PET) MF for mutations of EZH2. Mutational status at diagnosis was correlated with hematologic parameters, clinical manifestations, and outcome. A total of 25 different EZH2 mutations were detected in 5.9% of PMF, 1.2% of PPV-MF, and 9.4% of PET-MF patients; most were exonic heterozygous missense changes. EZH2 mutation coexisted with JAK2V617F or ASXL1 mutation in 12 of 29 (41.4%) and 6 of 27 (22.2%) evaluated patients; TET2 and CBL mutations were found in 2 and 1 patients, respectively. EZH2-mutated PMF patients had significantly higher leukocyte counts, blast-cell counts, and larger spleens at diagnosis, and most of them (52.6%) were in the high-risk International Prognostic Score System (IPSS) category. After a median follow-up of 39 months, 128 patients (25.9%) died, 81 (63.3%) because of leukemia. Leukemia-free survival (LFS) and overall survival (OS) were significantly reduced in EZH2-mutated PMF patients (P = .028 and P < .001, respectively); no such impact was seen for PPV/PET-MF patients, possibly due to the low number of mutated cases. In multivariate analysis, survival of PMF patients was predicted by IPSS high-risk category, a < 25% JAK2V617F allele burden, and EZH2 mutation status. We conclude that EZH2 mutations are independently associated with shorter survival in patients with PMF.

Symptomatic burden in myeloproliferative neoplasms is present in most patients and compromises quality of life. We sought to validate a broadly applicable 18-item instrument (Myeloproliferative Neoplasm Symptom Assessment Form [MPN-SAF], coadministered with the Brief Fatigue Inventory) to assess symptoms of myelofibrosis, essential thrombocythemia, and polycythemia vera among prospective cohorts in the United States, Sweden, and Italy. A total of 402 MPN-SAF surveys were administered (English [25%], Italian [46%], and Swedish [28%]) in 161 patients with essential thrombocythemia, 145 patients with polycythemia vera, and 96 patients with myelofibrosis. Responses among the 3 administered languages showed great consistency after controlling for MPN subtype. Strong correlations existed between individual items and key symptomatic elements represented on both the MPN-SAF and the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-C30. Enrolling physicians' blinded opinion of patient symptoms (6 symptoms assessed) were highly correlated with corresponding patients' responses. Serial administration of the English MPN-SAF among 53 patients showed that most MPN-SAF items are well correlated (r > 0.5, P < .001) and highly reproducible (intraclass correlation coefficient > 0.7). The MPN-SAF is a comprehensive and reliable instrument that is available in multiple languages to evaluate symptoms associated with all types of MPNs in clinical trials globally.

Background. Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder characterized by extramedullary haematopoiesis and abnormal neoangiogenesis in both the bone marrow (BM) and the spleen. We previously provided evidence that endothelial cells (ECs) from either the spleen or the splenic vein of PMF patients frequently share the JAK2V617F mutation with the hematopoietic malignant cells. More recently, we confirmed this observation also in BM-derived ECs of PMF patients. The mechanism underlying this phenomenon remains, however, not yet clarified. RUNX1 is a critical regulator of hematopoiesis, required for hematopoietic stem cell (HSC) generation and function. In human embryo, it is expressed in all emerging HSCs and progenitors and it is a necessary transcription factor for endothelial to hematopoietic transition. In the adult humans it is expressed in all blood cells, in decreasing intensity according to the maturation status, except erythrocytes. In angiogenesis, it induces endothelial differentiation and maturation as well as vascular network formation by promoting expression of VE-cadherin. Finally, it is involved in retinal aberrant neoangiogenesis. Aim. To assess if neoangiogenetic activity observed in spleen and BM of PMF patients is associated with RUNX1 expression in ECs. Patients and Methods. Paraffin-embedded BM sections from patients with MF (n=3), and from patients with lymphomas (named as CTRLs), who underwent BM biopsy for disease staging (n=2), were used for immunostaining analysis. Spleens samples were collected from 3 patients with PMF, who received splenectomy for clinical reasons, and from 2 healthy subjects (HS), who underwent surgery after traumatic damage. Fresh spleen samples were embedded in OCT, and then snap-frozen and stored in liquid nitrogen. Endothelial colony forming cells (ECFCs) were obtained from peripheral blood (n=1 PMF, n=1 HS), PMF spleen tissue (n=1) and cord blood (n=1), according to Ingram et al (Blood 2004;104:2752) and cytospun on slides at confluence. ECFCs, BM and spleen sections were stained with antibodies directed against RUNX1 and VE-cadherin. The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGEJ software. We evaluated 10 fields for each BM and spleen section and measured the number of RUNX1-positive vessels/total vessels. We considered positive a vessel that contained at least one RUNX1-positive cell. The results are shown as mean ± SD. Results. Immunofluorescence staining of spleen and BM sections confirmed the presence of increased neoangiogenetic processes in samples from PMF patients with respect to CTRLs. The staining of BM and spleen sections obtained from PMF patients with antibodies anti-RUNX1 and anti-VE-cadherin (endothelial marker) allowed the detection of RUNX1+VE-cadherin- cells in the parenchima of BM and spleen (Figure 1A, 1C), occasionally in perivascular position (Figure 1A, arrowhead). Interestingly, in all patients analyzed, we could detect RUNX1+VE-cadherin+ cells in both the parenchima and in the vessels both in the BM and the spleen (Figure 1A, 1C, arrow). In BM, 22 ± 12% of the vessels had at least one double positive cell in the microvessel wall; in spleen tissue the percentage of RUNX1+ ECs increased to 60 ± 13%. We detected RUNX1+VE-cadherin- cells only in the BM parenchima of CTRLs but not in the spleen of HS, whereas RUNX1+VE-cadherin+ cells were never observed in the vessels of neither the spleen nor the BM (Figure 1B, 1D) of HS and CTRLs, respectively. The circulating ECFCs obtained from both PMF and HS were, as expected, VE-cadherin+ but did not express RUNX1; however, 30% of spleen-derived-ECFCs of the PMF patient were RUNX1+. In cord blood-derived ECFCs a small percentage (3%) of RUNX1+ cells was observed. Conclusions. Our data show a selective expression of RUNX1 in splenic- and BM-ECs of PMF patients, suggesting that activation of RUNX1 expression could be associated with the neoangiogenetic processes that characterize the disease. The expression of RUNX1 in ECFCs from spleen but not in their circulating counterpart suggests a role for the splenic microenvironment in determining RUNX1 expression in ECs. Further studies are ongoing to assess the genotype of RUNX1+ ECs of PMF patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Fibronectin (FN) is a glycoprotein of approximately 220 kDa, whose mRNA has three alternative splicing sites (termed EDA, EDB and IIICS) that allow 20 different isoforms of FN mRNA. Circulating plasma FN lacks both EDA and EDB segments and is a soluble form secreted by the hepatocytes, while cellular FN contains variable proportions of EDA and EDB segments and is organized as fibrils in the tissue matrix. FN containing EDA segment (EDA FN) presents unique biochemical properties as compared to the isoform lacking this domain, specifically: 1) pro-fibrotic, as the EDA-containing isoform becomes predominantly expressed during wound healing to sustain myofibroblast differentiation; 2) pro-inflammatory, as inclusion of EDA domain activates Toll Like Receptor 4 (TLR4), involved in the defense of the innate immune response following recognition of pathogens associated molecular patterns resulting in NF-κβ-dependent cytokine release; 3) pro-thrombotic, as EDA FN was shown to promote agonist-induced platelet aggregation and thrombus formation in vivo through TLR4 dependent mechanisms. EDA FN is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We identified the up-regulation, at both molecular and protein levels, of EDA FN in an experimental mouse model of BM fibrosis treated with supra-pharmacological doses of the thrombopoietin (TPO) mimetic Romiplostim (TPOhigh). Higher expression levels of EDA FN were detected in endothelial and stromal cells after inducing experimental fibrosis in wild type (WT) mice as compared to untreated controls. To unravel the role of EDA segment on the progression of BM fibrosis in vivo, we exploited two unique transgenic mouse models that present an aberrant splicing of the EDA exon. Mice with constitutive inclusion of EDA exon (EIIIA+/+), in tissues, were more prone to BM fibrosis development as compared to knockout mice (EIIIA-/-). Upon fibrosis induction with TPO mimetic treatment, EIIIA+/+ mice presented extensive reticulin deposition, thrombocytosis, splenomegaly and increased extra-medullary hematopoiesis. Applying numerous in vitro methods, we demonstrated that EDA FN as opposed to plasma FN lacking the EDA domain, induces megakaryocyte (Mk) proliferation in a TPO-independent fashion through engagement of TLR4 and activation of STAT5 and ERK 1/2 signalling pathways. Additionally, in vitro activation of EDA FN/TLR4 axis on Mks resulted in lipopolysaccharide-like responses, such as NF-kb activation, the release of pro-fibrotic Interleukin-6 (IL-6), and no anti-fibrotic TNF-a. Pharmacological inhibition of TLR4 in TPOhigh and EIIIA+/+/TPOhigh mice or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis and splenomegaly. Finally, developing a novel ELISA assay, we analysed samples from patients affected by primary myelofibrosis (PMF). PMF is one of the Philadelphia-negative Myeloproliferative Neoplasms (MPNs), a group of heterogeneous clonal disorders affecting the hematopoietic stem cell. MPNs are characterised by neoplastic proliferation of the myeloid lineage leading to an abnormal increase of platelet count in essential thrombocythemia (ET), red blood cells in polycythemia vera (PV), or Mks with BM fibrosis in primary myelofibrosis (PMF). To date, three major driver mutations have been identified affecting, respectively, the TPO receptor gene (MPL), the intracellular Janus Kinase 2 gene (JAK2) and the latest discovered mutations affecting the endoplasmic reticulum (ER) molecular chaperone calreticulin gene (CALR). All of these mutations induce a permanent activation of the JAK/STAT signalling pathways. We found that the EDA FN is increased in plasma and BM biopsies of PMF patients, as compared to healthy controls and ET patients, correlating with the fibrotic phase. In conclusion, we identified EDA FN/TLR4 as a new pathological axis that sustains Mk expansion and inflammation during BM fibrosis progression. EDA FN and TLR4 targeting has very promising potential for new therapeutic approaches in PMF patients with high phase BM fibrosis. Disclosures No relevant conflicts of interest to declare.

Despite new targeted therapies, patients with high risk myeloproliferative neoplasms (MPNs) have an increased chance of transforming into acute myeloid leukemia (AML) and can only be cured with allogeneic hematopoietic stem cell transplant (HSCT). JAK2V617F mutation is common in MPNs and is associated with genomic instability, with baseline DNA double strand breaks and homologous recombination (HR) activity as a result of stalled replication forks. Poly(ADP-ribose) polymerase-1 (PARP-1) detects disrupted replication forks and recruits HR repair enzymes to restart DNA replication. Because busulfan, which is used in preparative regimens for HSCT, also leads to stalled replication forks through DNA strand crosslinking, we hypothesized that the PARP-1 inhibitor veliparib and busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated two JAK2V617F positive MPN cell lines with increasing doses (from 0.1 to 100μM) of veliparib in liquid cultures and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3μM and 74.2μM respectively). We next treated the cell lines with increasing doses of busulfan in combination with a fixed subtherapeutic dose of veliparib (4μM) for 48 hours. With combination treatment, the busulfan IC50 decreased from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. The combination was synergistic with a mean combination index (CI) of 0.55 for SET2 and 0.40 for HEL cells. Combination treatment resulted in a higher fraction of cells in G2M arrest than with veliparib alone (p=0.03) or busulfan alone (p=0.04). Similarly, when the combination treatment was tested on CD34+ cells obtained from 5 patients with JAK2V617F mutated and 2 CALR mutated PMF in a standard clonogenic (CFU) assay, it caused a greater inhibition of colony formation than busulfan or veliparib alone (p=0.001). Veliparib alone did not show any effect on colony formation from healthy control CD34+ cells. Finally we utilized a xenograft model with NOD/SCID/IL-2Rγnull (NSG) mice to test the in-vivo effect of combined low doses of busulfan and veliparib in JAK2V617F MPNs. In order to establish disease, mice were injected with 5x106 SET2 cells via tail vein 12 hours after sub-lethal irradiation. Then, 14 days after SET-2 injection 4 groups of mice (n=5 each) were treated for up to 3 weeks with intra-peritoneal vehicle (control); or a subtherapeutic dose of veliparib (3mg/kg daily) for 5 days a week; or busulfan (25mg/kg weekly); or a combination of both drugs. Death from leukemia was documented by marrow and spleen cell immunophenotype using non-cross reactive anti-human CD33 and CD34 antibodies. Veliparib alone did not improve survival vs control. On the contrary, survival was increased by the combination treatment vs busulfan alone (p=0.02). Here we show that treatment with the PARP-1 inhibitor veliparib and busulfan elicits a synergistic cytotoxicity in MPN cells both in-vitro and in-vivo. Our data provide the rationale for testing novel pre-transplant conditioning regimens by combining veliparib with alkylating agents, such as busulfan or melphalan, in HSCT for high risk MPNs or MPN-AML. Disclosures Patel: Janssen: Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

The BCR-ABL-negative Myeloproliferative Neoplasms (MPNs), are a group of clonal hematopoietic malignancies, that consist of three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Approximately one quarter of patients with ET or PMF carry a somatic mutation of CALR, the gene encoding for the endoplasmic reticulum (ER) chaperone calreticulin. A 52-bp deletion (Type I) and a 5-bp insertion (Type II mutation) are the most frequent lesions. Both generate a new carboxy-terminus of the mutant proteins, in which the ER retention signal KDEL is lost, causing abnormal interaction of the mutants with the thrombopoietin (TPO) receptor (MPL), thus constitutively activating its downstream signaling in megakaryocytes. CALR Type I mutation causes important changes in the charge of the carboxyl-terminal tail, which is responsible for calcium (Ca2+) binding activity. As a consequence, megakaryocytes from CALR Type I patients present enhanced activation of Store-Operated Ca2+ Entry (SOCE), the mechanisms that trigger extracellular Ca2+ inflow upon agonist-induced intracellular Ca2+ mobilization from the ER. A fine control of SOCE and overall Ca2+ homeostasis is fundamental to ensure proper megakaryocyte function. Despite this knowledge, the role of a crosstalk among MPL, CALR and SOCE in regulating megakaryopoiesis has never been hypothesised. Our research analysed the activation of MPL downstream pathways and Ca2+ signalling in human cord blood and peripheral blood-cultured megakaryocytes, upon stimulation with recombinant human TPO (rhTPO). Additionally, we cultured megakaryocytes from the peripheral blood of patients carrying the CALR Type I mutation to investigate whether CALR and SOCE alterations determine abnormal activation of the MPL downstream pathway in MPNs. We demonstrated that binding of rhTPO to MPL induces a series of downstream signaling events that lead to the activation of STAT5, AKT and ERK1/2 in human megakaryocytes. This activation relies on significant accumulation of inositol triphosphate (IP3), and consequent intracellular Ca2+ release from the ER followed by extracellular Ca2+ entry, indicative of activated SOCE. Pharmacologic inhibition of the IP3 receptor or SOCE, with 2-APB or BTP-2 respectively, resulted in the complete absence of rhTPO-induced MPL signaling activation. SOCE activation is thus dependent on the dissociation of CALR and STIM1, a protein of the SOCE machinery, which normally forms a complex in un-stimulated megakaryocytes. Importantly, we verified that in human megakaryocytes in rhTPO-starved conditions, CALR forms a molecular complex with its co-chaperone, ERp57, and STIM1, while they dissociate upon rhTPO stimulation. Conversely, we observed that CALR, ERp57 and STIM1 are constitutively dissociated in megakaryocytes from CALR Type I patients, even in complete absence of rhTPO. This dissociation resulted in markedly increased TPO-induced cytosolic Ca2+ flows with a consequent abnormal megakaryocyte proliferation that could be counteracted by pharmacological inhibition of SOCE with BTP-2. Our findings provide the first evidence that CARL Type I, in addition to MPL activation, concurs to the alteration of SOCE, which sustains the phosphorylation of STAT5, AKT and ERK1/2, and induces megakaryocyte proliferation. Further investigation is needed to understand whether this altered megakaryocyte function is determined by the decrease in CALR levels or by the presence of CALR mutants in the ER. Nevertheless, the abnormal regulation of Ca2+ flows may represent a potentially actionable target. Disclosures No relevant conflicts of interest to declare.

Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.

Myelofibrosis with myeloid metaplasia (MMM) is a clinicopathologic entity characterized by stem cell-derived clonal myeloproliferation, ineffective erythropoiesis, extramedullary hematopoiesis, and bone marrow fibrosis and osteosclerosis. Patients with MMM have shortened survival and their quality of life is compromised by progressive anemia, marked hepatosplenomegaly, and severe constitutional symptoms including cachexia. After decades of frustration with ineffective therapy, patients are now being served by promising treatment approaches that include allogeneic hematopoietic stem cell transplantation and immunomodulatory drugs. Recent information regarding disease pathogenesis, including a contribution to the myeloproliferative disorder phenotype by a gain-of-function JAK2 mutation (JAK2(V617F)), has revived the prospect of targeted therapeutics as well as molecular monitoring of treatment response. Such progress calls for standardization of response criteria to accurately assess the value of new treatment modalities, to allow accurate comparison between studies, and to ensure that the definition of response reflects meaningful health outcome. Accordingly, an international panel of experts recently convened and delineated 3 response categories: complete remission (CR), partial remission (PR), and clinical improvement (CI). Bone marrow histologic and hematologic remissions characterize CR and CR/PR, respectively. The panel agreed that the CI response category is applicable only to patients with moderate to severe cytopenia or splenomegaly.

Idiopathic myelofibrosis (IM) is characterized by the constitutive mobilization of CD34+ cells. IM peripheral blood (PB) CD34+ cells had a reduced cloning efficiency and a lower frequency of cobblestone areas compared with normal granulocyte colony-stimulating factor (G-CSF)-mobilized PB CD34+ cells. IM CD34+ cells engrafted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, demonstrating that they contain bone marrow (BM)-repopulating cells. G-CSF-mobilized CD34+ cells produced multiple hematopoietic lineages within the NOD/SCID mice with a predominance of CD19+ cells. By contrast, IM CD34+ cells produced predominantly CD33+ cells, increased numbers of CD41+ cells, but fewer CD19+ cells. Transcriptional clonality assays of the engrafted human IM cells demonstrated their clonal origin. CD34+ cells from one patient isolated prior to leukemic transformation were capable of generating acute leukemia in NOD/SCID mice. The engrafted human cells exhibited the same abnormal karyotype as primary cells in a portion of the population. These findings demonstrate that BM-repopulating cells and more differentiated progenitor cells are constitutively mobilized into the PB in IM, and that their differentiation program is abnormal. In addition, the NOD/SCID model may be useful in gaining an understanding of the events occurring during the transition of IM to acute leukemia. (Blood. 2005;105:1699-1705)

From 2007 to 2011, 66 patients with primary myelofibrosis or myelofibrosis (MF) preceded by essential thrombocythemia or polycythemia vera were enrolled into a prospective phase 2 clinical trial of reduced-intensity allogeneic hematopoietic stem cell transplantation (AHSCT), Myeloproliferative Disorder Research Consortium 101 trial. The study included patients with sibling donors (n = 32) receiving fludarabine/melphalan (FluMel) as a preparative regimen and patients with unrelated donors (n = 34) receiving conditioning with FluMel plus anti-thymocyte globulin (ATG). Patient characteristics in the 2 cohorts were similar. Engraftment occurred in 97% of siblings and 76% of unrelated transplants, whereas secondary graft failure occurred in 3% and 12%, respectively. With a median follow-up of 25 months for patients alive, the overall survival (OS) was 75% in the sibling group (median not reached) and 32% in the unrelated group (median OS: 6 months, 95% confidence interval [CI]: 3, 25) (hazard ratio 3.9, 95% CI: 1.8,8.9) (P < .001). Nonrelapse mortality was 22% in sibling and 59% in unrelated AHSCT. Survival correlated with type of donor, but not with the degree of histocompatibility match, age, or JAK2V617F status. In patients with MF with sibling donors, AHSCT is an effective therapy, whereas AHSCT from unrelated donors with FluMel/ATG conditioning led to a high rate of graft failure and limited survival. This trial was registered at www.clinicaltrials.gov as #NCT00572897.

The revised 2016 World Health Organization (WHO) classification of myeloid neoplasms dictated distinct criteria for prefibrotic (prePMF) and overt primary myelofibrosis (PMF), based on bone marrow (BM) morphology, including fibrosis grade (G) AIM: to describe the characteristics and outcome of patients (pts) with a diagnosis of prePMF versus PMF according to the 2016 WHO criteria. METHODS. We used a database of ca 800 pts collected in 5 Italian tertiary centers of the AGIMM project. Pts annotated with diagnosis of pre/early PMF and PMF were identified, and BM biopsies were re-classified according to current criteria. A total of 639 pts with full information were retrieved; all were annotated for both driver mutations (JAK2V617F, MPLW515x, CALR) and High Molecular Risk mutations (HMR; Vannucchi et al, Leukemia 2013;1861), including ASXL1, EZH2, SRSF2, IDH1/2. RESULTS. Of the 639 pts, 274 (42.8%) were re-classified as prePMF and 365 (57.2%) as PMF. After a median follow-up (FU) of 3.6y, 212 pts (33.2%) had died, 23% prePMF vs 40.8% PMF (P65y old individuals (36.1% vs 46.8%, P= .004), had higher Hb (12.7 vs 10.7g/dL; P1% were found in 12.0% vs 26.3% (P25x109/L were similar. Abnormal karyotype in 18.5% prePMF vs 38.6% PMF (n=317; P10cm in 10.5% vs 24.1% (P The proportion of patients with JAK2V617F (and their median allele burden), MPLW515x and CALR (type I and type II) mutations was similar in prePMF and PMF; triple-negative (TN) pts were slightly more frequent in PMF (14%) than prePMF (9.9%; P=.041). Conversely, 24.8% of prePMF pts vs 41.1% PMF were HMR (P2 HMR mutations, that are prognostically negative (Guglielmelli et al, Leukemia 2014; 28), were found in 11.8% of PMF pts vs 4.7% prePMF (P Median survival (OS) was 17.6y in prePMF vs 7.2y in PMF (P2 HMR mutations (HR 9.3, 4.5-19.0 and 3.4,2.1-5.3, respectively; P CONCLUSIONS. This analysis of pts with contemporary diagnosis of prePMF and PMF disclosed important clinical, hematologic and molecular differences between the two, and indirectly suggested that they might represent a phenotypic continuum where increased grade of fibrosis associates with worsening of disease manifestations and outcome. Disclosures Vannucchi: Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

BACKGROUND: Symptom burden in essential thrombocythemia (ET) and polycythemia vera (PV) is severe even among individuals with low risk disease (Blood 2012. 12;123(24):3803-10). New therapies exist which alleviate the severe symptom burden and reduce splenomegaly in ET and PV patients (N Engl J Med 2015; 372:426-435). This analysis is the first to date to evaluate thresholds at which symptom-based treatment can be considered for ET and PV patients who are intolerant or resistant to hydroxyurea (HU). METHODS: Patient demographics, symptom burden, and disease traits were collected from ET and PV patients at a single time point during therapy. The MPN-10 total symptom score (TSS, JCO 2012;30(33)4098-103) was utilized to assess symptom burden. Symptom criteria models were determined as previously described among a population of MF patients (Scherber et. al. EHA 2016: a2250). Cutoffs were then evaluated in a cohort of ET and PV patients to assess for utility as a symptom model among this population. RESULTS: Demographics and symptom burden: 838 PV and 867 ET patients with previous hydroxyurea therapy were included in this analysis. Patients were of mean age (54.9 years ET, 64.0 years PV) and gender (69.2% female ET, 55.7% female). Mean disease duration was 6.0 years for ET and 7.3 years for PV.Among ET and PV patients, 15.0% and 24.2% had prior thrombosis respectively. In evaluating prognostic risk, ET patients tended to be low (45.5%) or intermediate risk (42.9%) with only a minority of patient meeting criteria forhigh risk disease (11.6%). Laboratory findings: ET patients had a mean platelet value of 598.7x 109/L(SD=283.4). Among PV patients, mean hematocrit was 45.8% (SD=8.1) and 42.6% of patients had a hematocrit of greater than 45%. White blood cell count was normal between the two groups (ET mean 8.3 x 109/L, PV mean 9.0 x 109/L). Symptoms: Mean worst symptom severity was 6.4 out of 10 (SD=2.7). Among ET patients, worst symptom was most frequently fatigue (32.7%, mean 5.0/10, SD=3.1, overall prevalence 88%) followed by night sweats (13.6%, mean 2.0/10, SD=3.0, overall prevalence 53%) and concentration difficulties (8.6%, mean 3.1/10, SD=3.0, overall prevalence 68%). For PV, worst individual symptom items were most frequently fatigue (29.2%, mean 5.2/10, SD=3.0, overall prevalence 91%), pruritus (14.1%, mean 3.2/10, SD=3.2, overall prevalence 69%), and night sweats (12.8%, mean 2.5/10, SD=3.0, overall prevalence 57%). Cutoff Scoring: 47.0% of ET patients fit criteria for TSSgreater than to equal to 20; 59.0%% had a single itemgreater than 5; and 45.7% had both a TSS greater than or equal to 20 and a single item greater than 5. Among PV participants, 54.5% had aTSS greater than to equal to 20; 66.1% had a single itemgreater than 5; and 51.5% had both a TSS greater than or equal to 20 and a single item greater than 5. Each scoring method was significantly associated with individual item scores (Table 1). Prognostic scoring was not significantly associated with any of the symptom cutoffs evaluated. Correlations: Among ET patients, a prior history of thrombosis was significantly associated with having a worst symptom item greater than 5 (p=0.043). ET patients with lower hemoglobin were significantly more likely to meet criteria for a MPN-10 score greater than or equal to 20 or to meet combined criteria for a MPN-10 greater than or equal to 20 and single worst item greater than 5 (for both p=0.01 or less). For PV, lower hematocrit levels were significantly associated with having an individual worst symptom score of greater than 5 (44.9% versus 46.7%, p=0.0376). CONCLUSION: Assessment of ET and PV symptoms, now measurable through standardized and practical instruments such as the MPN-10, is an integral part of determining therapeutic impact of newer therapies in both clinical practice and trial settings. In our modeling, patients with severe symptom burden profiles are well represented by utilizing cutoff criteria including aworst individual symptom item of greater than 5 out of 10, an MPN-10 score of greater than or equal to 20, or combined criteria of both cutoffs. These cutoffs can be considered when determiningwhich HU intolerant or resistant patients would most benefit fromsymptom orientedtreatment. Disclosures Kiladjian: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding. Schouten:Novartis: Consultancy; Sanofi: Consultancy. Etienne:BMS: Speakers Bureau; ARIAD: Speakers Bureau; Pfizer: Speakers Bureau; novartis: Consultancy, Speakers Bureau. Harrison:Incyte Corporation: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Radia:Pfizer: Honoraria; Novartis: Honoraria. Cervantes:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mesa:Promedior: Research Funding; Celgene: Research Funding; CTI: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy.

BACKGROUND: The presence of constitutional symptoms has been associated with increased mortality risk in myelofibrosis (MF) (Blood 2010;115(9):1703-8). New therapies exist which alleviate the severe symptom burden profile observed in MF patients but are only approved for use in those with intermediate-2 or high risk disease (N Engl J Med 2012;366:787-798). However, it has been proposed that there are patients who may benefit from symptom based treatment regardless of prognostic score (Am Soc Hematol Educ Program 2014;2014:277-286). We have recently characterized symptom score cutoffs at which patients would statistically benefit from treatment based on symptom scores alone (Scherber et. al. EHA 2016: a2250). These treatment thresholds included aMyeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS or MPN-10) total score of greater than or equal to 20, a worst individual item score of greater than 5, or a combined criteria of those with both an MPN-10 total score of greater than or equal to 20 and a worst individual item score greater than 5. This abstract represents an additional analysis of our MF cohort to better characterize the profile of patients who meet criteria for symptom-based therapy. METHODS: Patient demographics, symptom burden via the MPN-10 score (JCO 2012;30(33)4098-103), and disease traits were collected from MF patients and their physicians at a single time point during therapy. Previously we identified MPN-10 cutoffs via AkaikeÕs Information Criterion (AIC) analysis (Ecology 2014;95: 631-6), which represented the optimal model among all models specified for the data at hand to determine which patients would most benefit from symptom-directed therapy. RESULTS: Demographics. 695 MF patients without previousruxolitinib therapy were included in this analysis. Overall, of 455 patients (65.4%) fit a cutoff of having a single worst symptom item of greater than 5/10. 401 patients (57.7%) had a MPN-10 score of equal to or greater than 20. A total of 381 (54.8%) patients fit both of these criteria. A distribution of worse MPN-10 individual scores is shown in Table 1. Mean TSS score was 26.4 (SD=17.7). Symptom Criteria Associations. Demographics and disease traits: Neither mean age or age greater than 60 was significantly associated with meeting any of the symptom score cutoff criteria. Females were significantly more likely to meet any of the symptom score cutoffs (for all criteria, p=0.0003 or less). Patients with splenomegaly, particularly spleen size of greater than 15cm below the LCM, were significantly more likely than those with a normal sized spleen to meet any of the three criteria (spleen enlargement of any size p=0.014 or less; spleen greater than 15cm p=0.0114 or less). Patients who met any of the three symptom criteria tended to have a longer MPN duration, although this trend did not meet significance. A prior history of thrombosis was not associated with achieving any cutoff criterions. Symptom burden: Individuals who met the any symptom criteria were significantly more likely to have higher DIPSS prognostic risk score (for all p=0.0002 or less). Laboratory values: For those meeting criteria for a worst symptom greater than 5, mean WBC was 11.7 vs 9.1 x 109/L (p=0.025) and platelet count was 238.7 versus 329.1 (p=0.023). For those meeting criteria for a TSS greater than or equal to 20, mean WBC was 11.8 vs 9.5 x 109/L (p=0.04). For individuals meeting both criteria, mean WBC was 11.9 vs 9.5 x 109/L (p=0.034). The presence of peripheral blasts were significantly more common in patients with an individual worst symptom score greater than 5 (p=0.0364). Hemoglobin level was not significantly associated with symptom criteria for any cutoffs. CONCLUSION: Our analysis indicates that patients who would be treated based on symptom criteria are similar to patients who would be treated based on high risk features such as high DIPSS prognostic score, concerning blood count abnormalities (i.e., leukocytosis, thrombocytopenia, presence of peripheral blasts), and splenomegaly (particularly massive splenomegaly). Thrombosis history and age were not associated with criterion cutoff assignment, and it is notable that elderly age nor history of thrombosis alone would likely alter treatment choice other than anticoagulation. This data supports that JAK2 inhibitor treatment be strongly considered in patients meeting symptom based criteria. Disclosures Dueck: Bayer: Honoraria. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Zweegman:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Schouten:Sanofi: Consultancy; Novartis: Consultancy. Etienne:ARIAD: Speakers Bureau; Pfizer: Speakers Bureau; novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau. Harrison:Shire: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Incyte Corporation: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Radia:Novartis: Honoraria; Pfizer: Honoraria. Cervantes:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mesa:Promedior: Research Funding; Celgene: Research Funding; CTI: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy.

Primary Myelofibrosis (PMF) belongs to the Philadelphia negative Myeloproliferative Neoplasms (MPNs) and is characterized by hematopoietic stem-cell derived clonal myeloproliferation, involving especially megakaryocyte (MK) lineage, bone marrow fibrosis and extramedullary hematopoiesis. Recent studies have suggested that alterations in miRNAs expression could play a critical role in MPN's pathogenesis. In order to shed some light on this issue, we have previously performed the integrative analysis (IA) of gene and miRNA expression profiles of PMF CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patients compared with 31 healthy donors (R. Norfo et al., Blood, 2014). IA identified miR-494-3p as one of the most upregulated miRNAs in PMF CD34+ cells associated to the highest number of downregulated predicted targets (eighty-six, Fig. 1). In order to study the role of miR-494-3p in hematopoietic commitment and differentiation, and to elucidate its possible involvement in PMF pathogenesis, we performed miRNA overexpression experiments in cord blood (CB) CD34+ cells through miRNA mimic electroporation. The data showed that miR-494-3p promotes MK differentiation of HSPCs. Indeed, the fraction of cells expressing MK surface antigen CD41 was steadily increased in miR-494-3p overexpressing samples compared with controls (75.4±0.3% vs 53.2±3.5% at day 8, 82.6±1.3% vs 60.4±4.3% at day 10 of culture, p Taken together, our results showed that miR-494-3p overexpression promotes megakaryocytopoiesis in HSPCs. Moreover, we demonstrated for the first time that SOCS6 is a direct target of miR-494-3p. Since SOCS6 downregulation promotes MK differentiation of HSPCs, SOCS6 could be considered, at least in part, responsible for the biological effects observed after miR-494-3p overexpression. As miR-494-3p and SOCS6 showed the same expression trend in PMF CD34+ cells, our results could suggest that miR-494-3p/SOCS6 axis is involved in the induction of MK hyperplasia typically observed in PMF patients. Figure Figure. Disclosures Vannucchi: Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

A number of evidences indicate that inflammation and deranged innate immunity intervene in the pathobiology of PMF. In this study we aimed to analyze the inflammatory components of PMF by measuring hs-CRP in a vast population of patients. hs-CRP can detect minimal variations of serum CRP levels even in the range of normal limits, and thus is a sensitive marker of low grade systemic inflammation. We included PMF patients from the data base of the Center for the Study of Myelofibrosis in Pavia with available archived plasma. We excluded patients who had been treated with disease-modifying agents at any time before or on the date of base-cohort entry (hydroxyurea, interferon, ruxolitinib, corticosteroids) and patients with acute inflammatory or autoimmune disease, malignancy, severe liver or renal dysfunction. In this study we report the results on the determinants of levels of circulating hs-CRP in PMF in an analysis in which we used parameters that could be hypothesized with a biological plausibility to influence the level of hs-CRP. Selected predictors were: age, sex, the five different genotypes reflecting the presence or absence of the driver mutations (JAK2V617F with low allele burden (1 mg/L). The mean age of the patients was 54.4 years. Increasing age was correlated with increasing hs-CRP (R=0.45;P Figure Figure. Disclosures No relevant conflicts of interest to declare.

Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F+ ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

Background: The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) all have a time dependent risk of progression to either an advanced myelofibrotic state (post ET/PV MF) and/or to acute myeloid leukemia. The impact of disease duration upon the MPN symptom burden is not well understood, nor are the precise mechanisms of disease progression. We sought to better understand the impact of disease duration on MPN symptom burden. Methods: Symptom burden data was collected utilizing the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) amongst MPN patients, collected at the time of an office visit in an international cohort of MPN patients as previously described (Scherber et. al.). Symptom burden assessment was a previously validated 27-item symptom burden questionnaire scored on a 0-10 scale (0= as good as it can be, 10 = as bad as it could be). The patient or provider was asked to report the time since MPN diagnosis. MPN duration was determined to be early if the diagnosis was established between 0 to 5 years ago, intermediate if the diagnosis was established between 6 to 10 years ago, and late if the diagnosis was established 11 years ago or more. Anemia was defined as a red blood cell count less than 10 g/dL, leukopenia was defined as a white blood cell count was below 4 x 109, and thrombocytopenia if the platelet count was below 150 x109. Statistical significance was calculated using ANOVA f-test and chi squared. Results: Patient demographics and disease burden: A total of 1443 patients responded to the survey, including 592 (41%) ET, 549 (38%) PV, and 302 (21%) MF patients, including 181 (60%) primary MF, 67 (22%) post-ET MF, and 54 (18%) post-PV MF. Among MF patients, mean duration of MPN diagnosis was 9 years, and mean duration MF diagnosis was 4.7 years. Among respondents, 757 fit criteria for early disease duration, 353 fit criteria for intermediate disease duration, and 333 fit criteria for late disease duration. Respondent mean age was 62 years and approximately half of respondents were female (55%). Patients with longer diagnosis duration tended to be older (p=0.009) and were most likely to have anemia (0.02), leukopenia (p=0.01), or thrombocytopenia (p=0.03). These individuals were also most likely to have a history of hemorrhage (p=0.007) or require red blood cell transfusions (p Combined cohort symptom burden: On average among the combined cohort of ET, PV and MF patients, symptoms tended to worsen with time with this effect being significant for symptom items of fatigue (BFI, p Symptom burden in MPN subtypes. When evaluating specific MPN types, patients with essential thrombocythemia experienced significantly greater sexual difficulties over time (p=0.03). The severity (p=0.01) and incidence (p=0.03) of pruritus and incidence of night sweats (p Discussion Overall, significant worsening in symptom burden can be recognized over time for individuals diagnosed with MPNs. Diagnosis may not necessarily correlate with disease duration as the timing of diagnosis may be delayed from onset of disease. Given the intent of this abstract to evaluate changes with disease duration, we did not investigate correlations between symptom burden and cytopenias. We do know that risk factors for survival in the MPNs include older age and thrombosis, however, disease duration should be investigated as an alternative marker of burden in future survival studies. Disclosures Harrison: CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Shire: Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Kiladjian:Incyte Corporation: Consultancy; Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot); Novartis: Consultancy. Zweegman:Celgene: Research Funding; Janssen: Research Funding; Takeda: Research Funding. Barbui:Novartis: Speakers Bureau. Etienne:Novartis: Consultancy, Honoraria, Other: Congress Travel/Accomodations, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria, Speakers Bureau. te Boekhorst:CTI Biopharma: Consultancy; Novartis: Consultancy. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cells, characterized by an increased number of circulating CD34+ cells. Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. A subset of patients display immune-related abnormalities that suggest an (auto)immune pathogenesis. In addition, it has been reported a significant association between personal history of a broad span of autoimmune diseases and subsequent risk of myeloproliferative neoplasm (Kristinsson SY Haematologica 2010; 95:1216-1220). CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN) (Hasselbalch HC et al. Leuk Res 2013;37:214-20), suggesting that T cells play an important role in their pathogenesis. A recent paper by Keohane C et al (BJH 2015; doi 10.1111/bjh 13519) shows that peripheral blood (PB) T regulatory cells (Tregs) are reduced in MPN patients compared to healthy subjects (CTRLs) and that this decrease is even more pronounced following JAKi therapy (Massa M et al Leukemia 2014;28:449-51). Unpublished data from our group further indicate a significant (p Study design. To investigate the possible factors favouring a decrease of Tregs, we examined in patients with PMF and Results. 1) The percentages of PB CD4+, CD4+CD25+, or CD4+CD25++CD127low/- cells expressing membrane Annexin V were comparable in patients with PMF and CTRLs (data not shown). 2) The phosphorilation of Stat1, Stat3, Stat5, Stat6, and p38-MAPK (expressed as mean fluorescence intensity: MFI) on CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells before and after stimulation with IFNα, IL6, IL4, or PMA was comparable in patients and CTRLs. The Erk1,2 phosphorilation was comparable in patients and CTRLs at baseline condition, while in patients with PMF Erk1,2 phosphorilation following stimulation with PMA was significantly lower in both CD4+CD25+ cells (p=0.039; median MFI 5.4, range 0.3-40) and CD4+CD25++FOXP3+ cells (p=0.01; median MFI 4.3, range 1.4-9.3) than in CTRLs (CD4+CD25+ cells median MFI 8.1, range 4.9-20; CD4+CD25++FOXP3+ cells median MFI 7.3, 3.7-10.8). 3) sIL2rα plasma levels were higher (p=0.01) in patients with PMF (1658 pg/ml, range 476-8968) than in CTRLs (1040 pg/ml, 482-1719); the sIL2rα plasma levels were significantly (R=-0.35, p=0.039) inversely correlated with the percentages of circulating Tregs. No significant correlation was found with the presence (n=13)/absence (n=14) of JAK2V617F mutation. Conclusions. These results indicate that in patients with PMF Tregs present an untuned Erk signaling; moreover, sIL2rα plasma levels are higher in patients than in CTRLs, and significantly inversely correlated with Treg percentages. These findings may partly explain the reduced frequency of Tregs in PMF since Erk signaling is pivotal in T cell differentiation, regulation of T cell homeostasis, and defects in Erk expression have been associated with autoimmunity (Chiung-Fang Chang J Immunol 2012; 189:721-731). Moreover, increased levels of sIL2rα may impact on IL-2-mediated functions and alterate the optimal activation of Erk1/2 that is IL-2 dependent (Lequn Li, Blood. 2005;106:3068-3073), Disclosures No relevant conflicts of interest to declare.

Background: Thrombotic and hemorrhagic complications are commonly encountered in uncontrolled essential thrombocythemia (ET). Both anagrelide and hydroxyurea (HU) have proven efficacious in cytoreduction as well as reducing these events and remain first line therapy for most high-risk ET patients. Independent of their role in risk-reduction, little is known about how these therapies impact patient symptomatology or quality of life. In this study, we compared the clinical and symptomatic profiles of ET patients receiving HU or anagrelide against patients with no previous experience with these agents. Methods: Data was assessed from a prospectively collected international database of ET patients in which demographics, disease features, and ET symptoms utilizing the myeloproliferative neoplasm symptom assessment form (MPN-SAF; Scherber et al, 2011). The MPN-SAF includes the patient's perceptions of common MPN-related symptoms and overall quality of life (QOL) on a 0 (absent) to 10 (worst imaginable) scale. Total symptom score (TSS) was computed based on symptom items using the published scoring algorithm on a 0 (all reported symptoms absent) to 100 (all reported symptoms worst imaginable) scale. ET risk scores were calculated using the IPSET scoring algorithm (Passamonti 2012). Thrombocytopenia was defined as a platelet count Results Hydroxyurea vs. HU Naive A total of 402 ET patients with active HU use were compared to 392 ET patients with no history of HU use. Patients using HU were older (63.5 years vs. 52.3 years, p Anagrelide vs. Anagrelide Naive A total of 49 ET patients with active anagrelide use were compared to 794 ET patients with no history of anagrelide use. Patients using anagrelide had a longer mean disease duration (8.1 years vs. 5.8 years) and were more anemic (9.1% vs. 1.2%, p HU vs. Anagrelide A total of 402 patients currently using HU were compared to 39 patients currently using anagrelide. Overall, HU users were slightly older (63.5 years vs. 55.1 years, p Discussion In this retrospective analysis, it does not appear cytoreduction with either HU and/or anagrelide has a significant impact on ET symptom burden despite reducing vascular events. Importantly, the higher risk scores in HU patients did not translate directly into greater patient symptomatology supporting previous studies demonstrating a poor association between these two items. Prospective trials measuring ET symptom change, in the setting of randomized trials will better quantify impact of cytoreduction on symptom burden as well as quantify impact of newer agents such as interferon or jak inhibition. Figure 1. Symptom Comparisons Between HU, Anagrelide and Other Therapy Figure 1. Symptom Comparisons Between HU, Anagrelide and Other Therapy Disclosures Kiladjian: Incyte Corporation: Consultancy; Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot); Novartis: Consultancy. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison:Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau; Gilead: Honoraria; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cervantes:Sanofi-Aventis: Consultancy; Novartis: Consultancy, Speakers Bureau; CTI-Baxter: Consultancy, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Etienne:ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Congress Travel/Accomodations, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Roy:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding. te Boekhorst:Novartis: Consultancy; CTI Biopharma: Consultancy. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Background. The prognostic significance of bone marrow (BM) fibrosis grade in pts with primary myelofibrosis (PMF) is debated. A fibrosis grade greater than 1 was associated with a 2-fold higher risk of death compared with pts with early/prefibrotic MF (grade 0) [Thiele J, Ann Hematol 2006]. Recent data suggest that more accurate prediction of survival is achieved when fibrosis grade is added to IPSS [Verner C, Blood 2008; Giannelli U, Mod Pathol 2012]. Aim. To analyze the prognostic impact of fibrosis in diagnostic BM samples of 540 WHO-2008 diagnosed PMF pts with extensive clinical and molecular information collected in 6 Italian centers belonging to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative). Methods. The clinical variables assessed were those previously identified as prognostically relevant in the IPSS score. Published methods were used to screen mutations of JAK2, MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. European consensus scoring system was used to grade fibrosis (on a scale of MF-0 to MF-3). The prognostic value of fibrosis with regard to overall survival (OS) was estimated by Kaplan-Meier method and Cox regression. Results. Pts' median age was 61y; median follow-up 3.7y; median OS 10.5y; 184 pts (34.1%) died. IPSS risk category: low 33.7%, Int-1 27.7%, Int-2 19.1%, High-risk 19.5%. Mutational rate: JAK2 V617F 62.6%, CALR 20.7% (type-1/1-like 77.7%, type2/2-like-2 21.4%), MPL W515 5.9%; 62 (11.5%) were triple negative (TN). 171 pts (31.7%) were High-Molecular Risk (HMR) category (Vannucchi AM, Leukemia 2013); mutation rate: EZH2 7.2%, ASXL1 22.2%, IDH1-2 2.4%, SRSF2 8.3%. According to fibrosis grading, 50 pts were MF-0 (9.3%), 180 MF-1 (33.3%), 196 MF-2 (36.3%), 114 MF-3 (21.1%). Compared with both MF-0 and MF-1, MF-2 and MF-3 pts presented more frequently constitutional symptoms (P There was no correlation between fibrosis grade and phenotypic driver mutations; in particular, TN pts were equally distributed among MF fibrosis grades (10%, 10.6%, 14.3% and 8.8% from MF-0 to -3, respectively). Conversely, the frequency of HMR pts increased progressively according to fibrosis grade: 8 pts MF-0 (16%), 46 MF-1 (25.6%), 66 MF-2 (33.7%) and 51 MF-3 (44.7%) (P Median OS was significantly shorter in pts with MF-2 (OS 6.7y, HR 7.3, IC95% 2.7-20.0; P Conclusions. Overall, these results indicate that higher grades (MF-2 and MF-3) of fibrosis correlate with defined clinical and molecular variables and independently negatively impact on OS in PMF, suggesting the opportunity to explore its value in the setting of clinical and molecular prognostic scores for PMF. Table. Multivariate Analysis Variables HR 95% CI P value HMR status 2.4 1.5-3.7 Figure 1. Figure 1. Disclosures Passamonti: Novartis: Consultancy, Honoraria, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Vannucchi:Shire: Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Baxalta: 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.

Background Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) associated with a high degree of symptomatology, progressive cytopenias and potential to transform into acute myelogenous leukemia (AML). Thrombocytopenia amongst MF patients is a proven negative prognostic indicator and predictor of transformation to AML. Ruxolitinib is an effective JAK inhibitor for MF symptoms and splenomegaly, but is not indicated in patients with severe thrombocytopenia. Phase III trials of pacritinib have shown alleviation of the MF symptom burden amongst patients with thrombocytopenia (ASCO 2015 Mesa et. al.). In this study, we assessed the symptom burden of MF patients with significant thrombocytopenia who were naïve to pacritinib. Methods Data was assessed from a prospectively collected international database of MF patients in which demographics, disease features, and MF symptoms utilizing the myeloproliferative neoplasm symptom assessment form (MPN-SAF; Scherber et al, 2011). The MPN-SAF includes the patient's perceptions of common MPN-related symptoms and overall quality of life (QOL) on a 0 (absent) to 10 (worst imaginable) scale. Total symptom score (TSS) was computed based on symptom items using the published scoring algorithm on a 0 (all reported symptoms absent) to 100 (all reported symptoms worst imaginable) scale. MF risk scores were calculated using the DIPSS criteria (Gangat, 2011). Thrombocytopenia was defined as a platelet count Results Demographic and Disease Features: A total of 418 patients with (n=89) and without (n=329) thrombocytopenia completed the MPN-SAF. Patients with thrombocytopenia were slightly younger (57.4 vs. 61.0, p=0.01) with longer disease durations (11.9 years vs. 8.8 years, p=0.0489) and had a higher prevalence of primary myelofibrosis (PMF; 82% vs. 66%, p=0.01). The presence of thrombocytopenia was associated with other laboratory abnormalities including anemia (60.7% vs. 25.3%, p Symptoms Scores for individual MPN-SAF items were assessed for each subgroup. Patients with thrombocytopenia had markedly higher total symptom scores than patients without thrombocytopenia (32.8 vs. 24.1, p Discussion MF patients with thrombocytopenia have distinctive clinical characteristics and face a significantly more severe symptom burden. Importantly, despite thrombocytopenia being a recognized risk factor for disease advancement, no correlations are noted between patient symptomatology and risk category. In addition, patients with severe thrombocytopenia do not differ symptomatically from patients with moderate thrombocytopenia despite having more severe anemia, leukopenia and transfusion requirements. This implies that the degree of symptomatology expressed by thrombocytopenic MF patients occurs independent from the exact platelet value. Conclusion The results of this study suggest that patients with thrombocytopenia will benefit from aggressive symptomatic control, potentially from targeted agents. Figure 1. MF Symptoms in Patients With and Without Thrombocytopenia Figure 1. MF Symptoms in Patients With and Without Thrombocytopenia Disclosures Kiladjian: Novartis: Consultancy; Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot); Incyte Corporation: Consultancy. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison:Shire: Speakers Bureau; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau. Cervantes:Novartis: Consultancy, Speakers Bureau; Sanofi-Aventis: Consultancy; CTI-Baxter: Consultancy, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Etienne:ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Congress Travel/Accomodations, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Roy:BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Vannucchi:Shire: Speakers Bureau; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Background: Philadelphia-negative Myeloproliferative Neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis, both Primary (PMF) and secondary to PV or ET (PPV-MF and PET-MF). A MPN is frequently the underlying cause of splanchnic vein thrombosis (SVT). Ruxolitinib, a JAK1/2 inhibitor, efficiently reduced spleen volume and improved symptoms in patients (pts) with MF and PV in the COMFORT-I/II and RESPONSE phase III trials, and in ET pts in a phase II study. We reported (Blood 2014 124:3192) that ruxolitinib was safe in pts with MPN associated to SVT and effective in reducing spleen size at the planned primary endpoint analysis at 24 weeks (w) in a phase II clinical trial. Herein we present follow up data with cut off at 1 year after core period (a total of 72 w of treatment). Methods: Main enrolment criteria included diagnosis of PV, ET, PMF or PPV-/PET-MF associated with SVT, splenomegaly >5 cm below costal margin (bcm), active anticoagulant or antiaggregant thrombosis prophylaxis, platelet count (plt) >100 x109/L, neutrophils count >1x109/L, normal hepatic and renal function, absence of esophageal varices >grade 2. Pts who completed the 24 w of study treatment and tolerated well the drug and had evidence of clinically-significant improvement were allowed to enter an extension phase aimed at collecting and reviewing safety and efficacy data. The drug was provided free of charge by Novartis, that had no role in trial design nor in data analysis. Results: Diagnosis of MPN were: PMF 8 (38.1%), PV 5 (23.8%), ET 4 (19.1%), PPV-MF 3 (14.3%), PET-MF 1 (4.8%). Nineteen pts had spleno-porto-mesenteric thrombosis and 3 Budd-Chiari syndrome (BCS); one pt had both sites involved. Initial dose of ruxolitinib was 10 mg BID for PV, 25 mg BID for ET, 15 mg BID for MF pts with baseline (bl) platelet count of 100 to 200x109/L and 20 mg BID for platelet count >200x109/L. Currently 17/21 pts are on active treatment, 14 completed w72; final data for all 17 pts will be available at meeting. One pt with MF discontinued from the study being shifted to commercial ruxolitinib at w60, one ET and one MF pt discontinued for inefficacy at w24 and one MF pt for an unrelated adverse event after w72. Efficacy: 13/21 (61.9%) pts obtained a ≥50% spleen length (sl) reduction by palpation at w24, that was maintained at w72 in 8/14 pts (57.1%). Median sl reduction at w72 was 63% (range 0-100). No significant differences in resistive or pulsatility index of splanchnic artery were noted, nor in esophageal varices status evaluated at w72. 10/11 evaluated pts with echocardiography at w72 showed a median reduction of the cardiac output of 20.1% (range 2.3-42.2) mainly due to a reduction of heart rate and of cardiac index (-21.9%, range 8.8-44.3) due to increase in body surface area. The first effect could be attributed to decrease of proinflammatory cytokines, the second to weight gain associated with ruxolitinib. Symptomatology was evaluated by MPN-SAF up to w24, showing a median total symptom score reduction from 65 to 42. Safety: regardless of drug relationship, the most common adverse events (AE) (% any grade, % grade ≥3) were thrombocythopenia (57.1%; 14.3%) and anemia (33.3%, 19%) that were the main reasons for dose adjustments. Other AE included AST or ALT increase (42.9%, 0%), diarrhea (28.6%, 0%), abdominal pain (23.8%, 0%), ascites (19%, 0%), fever (23.8%, 0%), neutropenia, (9.5%, 9.5%), upper airways infection (19%, 0%), weight gain (14.3%, 4.8%), muscle cramps (14.3%, 0%). Three serious AE occurred: one case of hepatocarcinoma in a pts with BCS, one grade 2 pneumonia and one grade 2 haematemesis not related to esophageal varices. Median ruxolitinib total daily dose at w72, after dose adjustments, was 19.1 mg for MF, 16 mg for PV and 28.3 mg for ET. Median hemoglobin reduced from 12.9 gr/dL (range 9.4-16.7) at bl to 10.7 (8.4-14.4) at w16 and recovered at w72 (12.1, range 10.8-14.7). No pts received transfusions. Median platelet count was 212 x109/L (100-389) at bl, reached to the lowest level at w4 (139, range 48-252) and improved to 160 (69-285) at w72. Median leukocyte count decreased from 7.3 x109/L (1.8-16.4) at bl to 4.08 (1.2-21.7) at w 24, and remained substantially stable through w 72 (4.96; range 2.45-17.3). Median reduction of JAK2 allele burden at w72 was 9% (range 0-38). Conclusions: At w 72 follow up, ruxolitinib continues to be safe in pts with MPN associated to SVT and maintains efficacy against splenomegaly in 57% of the pts. Disclosures De Stefano: Roche: Research Funding; GlaxoSmithKline: Speakers Bureau; Bruno Farmaceutici: Research Funding; Novartis: Research Funding, Speakers Bureau; Janssen Cilag: Research Funding; Celgene: Speakers Bureau; Shire: Speakers Bureau; Amgen: Speakers Bureau. Barbui:Novartis: Speakers Bureau. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: 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.

Clinical data suggest that in 8-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (S-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 8-thalasemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with B-thalassemia) and 6 presented lower HbF levels (p+ -thalassemia). The two groups of patients did not differ in age (1 5.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (S-Epo) was correlated inversely with Hb (r = - 0.47; P < .Ol), and directly with HbF (r = .55; P < .OOl). Multivariate regression analysis showed that Hb and HbF were independently correlated ETA-THALASSEMIA intermedia is a genetically heterogeneous disorder defined according to degree of anemia that allows patients to survive without blood transfusions and grow without severe bone deformities or impairment of sexual development.’,* Nevertheless, the phenotype of this disorder varies with respect to age of first symptoms, splenomegaly, jaundice, cardiomegaly, and the incidence of extramedullary erythropoiesis.“’ The expansion of erythropoiesis, resulting from an erythropoietin drive secondary to chronic anemia, is recognized as the most important determinant of the phenotypic expression of the disease; however, it has also been claimed that increased amounts of circulating fetal hemoglobin (HbF) may exert an independent effect on erythroid expansion.’-5 HbF, in fact, shifts the oxygen dissociation curve of the blood towards higher affinit~,~.~ thus potentially producing a functional anemia and an adjunctive stimulus to erythropoiesis. Even though patients with high HbF have been reported to have earlier disease presentation3 and higher incidences of extramedullary erythropoiesis4 and splenomegaly3 than those with low HbF at comparable levels of anemia, the influence of HbF on the regulation of erythropoiesis in @-thalassemia intermedia has never been quantitatively investigated. This is of overwhelming importance in view of the recent introduction of therapies aimed at increasing the production of HbF to ameliorate the anemia and the symptoms of &globin In this study we assessed the influence of high HbF levels on the regulation of erythropoiesis in p-thalassemia intermedia. We evaluated serum erythropoietin (S-Epo) in relation to the degree of anemia in 30 patients with different HbF concentrations. Moreover, because measurement of serum transfemn receptor (S-TfR) has recently been proposed as a convenient method for monitoring erythropoiesis,” we determined S-TfR levels and correlated them with that of s-Epo. The results of this investigation show that, in &thalassemia intermedia patients, HbF values greater than 40% are responsible for higher S-Epo activity than expected for the degree of anemia. This produces an oversetting of erythropoiesis to a degree that may be clinically significant.

A definition of response by cytoreductive therapy in essential thrombocythemia was recently provided by the European LeukemiaNet (ELN). Complete, partial, or no clinicohematologic responses were defined on the bases of platelet count, disease-related symptoms, spleen size, and white blood cell count. To provide estimates and clinical correlation of responses according to these criteria, we retrospectively examined 416 essential thrombocythemia patients treated with hydroxyurea for at least 12 months. Complete response, partial response, and no response were 25%, 58%, and 17%, respectively. Age more than 60 years and JAK2V617F mutation were significant predictors of response. After a median follow-up of 3.9 years, we registered 23 deaths, 16 hematologic transformations, and 27 thrombotic events (rate, 1.66% patients/year). Age, previous thrombosis, leukocytosis (white blood cell count > 10 × 109/L), but not ELN responses, were independently associated with higher risk of thrombosis. The actuarial probability of thrombosis was significantly influenced by leukocytosis (P = .017) and not by platelet count, indicating that platelet number does not seem of prime relevance in the definition of ELN response.

We have used recombinant human erythropoietin (rHuEPO) in a phase I/II clinical trial to evaluate its ability to reverse refractory anemia in hematologic disorders. rHuEPO was administered subcutaneously 5 days per week at escalating doses (50 to 150 U/kg per day). The aim of treatment was a hemoglobin (Hb) level greater than or equal to 10 g/dL without blood transfusion. Of 25 patients treated, 17 were evaluable, most of them with a regular need for transfusion. Eight of these had lymphoproliferative disorders (three cases of malignant lymphoma and five of monoclonal gammopathy) and were exposed to cytotoxic therapy. The other nine patients had hematopoietic stem cell disorders (four cases of myelodysplastic syndrome, three of idiopathic myelofibrosis, and two of chronic myelogenous leukemia). All patients with lymphoproliferative disorder had serum EPO levels inappropriately low for the degree of anemia, while patients with stem cell disorder showed variable values. Erythroid marrow activity was inadequate in all cases. Seven of eight patients with lymphoproliferative disorder responded to treatment maintaining Hb above 10 g/dL without transfusion. The median dose of rHuEPO required for correction of anemia was 75 U/kg. In four cases response was maintained with 50 U/kg, three times per week. There was no complete response among patients with hematopoietic stem cell disorder, although transfusion requirement was eliminated or reduced in four cases. Four patients developed functional iron deficiency during rHuEPO treatment and required iron supplementation to obtain response. Aggravation of splenomegaly was observed in two cases of myeloproliferative disorder. We conclude that: (1) subcutaneous administration of rHuEPO can be effective and safe in patients with lymphoproliferative disorder exposed to chemotherapy and showing inappropriate EPO response to anemia; (2) this is less likely in hematopoietic stem cell disorders, although favorable responses may be observed in occasional patients; and (3) functional iron deficiency as a cause of nonresponse to rHuEPO is frequent also in nonrenal anemia.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Among 994 patients with essential thrombocythemia (ET) who were genotyped for the MPLW515L/K mutation, 30 patients carrying the mutation were identified (3.0%), 8 of whom also displayed the JAK2V671F mutation. MPLW515L/K patients presented lower hemoglobin levels and higher platelet counts than did wild type (wt) MPL; these differences were highly significant compared with MPLwt/JAK2V617F–positive patients. Reduced hemoglobin and increased platelet levels were preferentially associated with the W515L and W515K alleles, respectively. MPL mutation was a significant risk factor for microvessel disturbances, suggesting platelet hyperreactivity associated with constitutively active MPL; arterial thromboses were increased only in comparison to MPLwt/JAK2wt patients. MPLW515L/K patients presented reduced total and erythroid bone marrow cellularity, whereas the numbers of megakaryocytes, megakaryocytic clusters, and small-sized megakaryocytes were all significantly increased. These data indicate that MPLW515L/K mutations do not define a distinct phenotype in ET, although some differences depended on the JAK2V617F mutational status of the counterpart.

To the editor: The Budd-Chiari syndrome (BCS) and extrahepatic portal vein obstruction (EHPVO) are splanchnic vein thromboses (SVT) that may occur as presenting complications of undiagnosed chronic myeloproliferative disorders (CMPD). Diagnosis of the underlying CMPD may be difficult because

The Janus kinase 2 mutation, JAK2617V>F, is myeloid neoplasm-specific; its presence excludes secondary polycythemia, thrombocytosis, or bone marrow fibrosis from other causes. Furthermore, JAK2617V>F or a JAK2 exon 12 mutation is present in virtually all patients with polycythemia vera (PV), whereas JAK2617V>F also occurs in approximately half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Therefore, JAK2 mutation screening holds the promise of a decisive diagnostic test in PV while being complementary to histology for the diagnosis of ET and PMF; the combination of molecular testing and histologic review should also facilitate diagnosis of ET associated with borderline thrombocytosis. Accordingly, revision of the current World Health Organization (WHO) diagnostic criteria for PV, ET, and PMF is warranted; JAK2 mutation analysis should be listed as a major criterion for PV diagnosis, and the platelet count threshold for ET diagnosis can be lowered from 600 to 450 × 109/L. The current document was prepared by an international expert panel of pathologists and clinical investigators in myeloproliferative disorders; it was subsequently presented to members of the Clinical Advisory Committee for the revision of the WHO Classification of Myeloid Neoplasms, who endorsed the document and recommended its adoption by the WHO.

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF), peripheral blood CD34+ cells isolated from patients with IMF, polycythemia vera (PV), and G-CSF–mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is, therefore, likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-β (TGF-β) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9, therefore, likely contribute to the development of many pathological epiphenomena associated with IMF.

BACKGROUND. In primary myelofibrosis (PMF), survival from time of diagnosis is predicted by the international prognostic scoring system (IPSS), and from time of referral, by the dynamic IPSS (DIPSS) or DIPSS-plus. JAK2/CALR/MPL mutational status and presence and number of other prognostically-relevant mutations (ASXL1, SRSF2, EZH2, IDH1/2) provide IPSS/DIPSS-plus independent prognostic information. The objective in the current study was to revise IPSS, by including mutation-relevant prognostic information. METHODS. The study included 986 PMF patients divided into learning (n=588) and validation (n=398) cohorts. Previously published methods were used to screen for mutations involving JAK2,MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. The clinical variables assessed were those previously identified as being prognostically-relevant by IPSS or DIPSS-plus. The prognostic model (MIPSS) was developed through a stepwise selection process, based on a z-test of the regression coefficients, and its relative quality was measured by means of the Akaike information criterion. RESULTS. In the Italian learning cohort (n=588),median follow-up was 3.6years (95% CI, 0.03-30.8) for alive patients and 196 (33.3%) deaths and 67 leukemic transformations were documented. IPSS risk distribution was low in 26%, intermediate-1 in 30%, intermediate-2 in 24% and high in 20%. Mutational frequencies were approximately 63% for JAK2, 20% CALR, 6% MPL, 7% EZH2, 22% ASXL1, 2.5% IDH1/2 and 9% SRSF2. Constitutional symptoms were recorded in 28.6% of patients, splenomegaly 75% (18% >10cm from LCM) and grade 3 fibrosis 21%. Overall 252 patients were evaluable for karyotype and abnormalities were detected in 35%, including 9.5% unfavorable. Univariate analysis identified the following risk factors for inferior survival: age >60 years, constitutional symptoms, hemoglobin 25x109/L, platelets 10cm, grade 3 fibrosis, unfavorable karyotype, triple-negativity for JAK2, MPL and CALR (TN), JAK2 or MPL mutation, and mutations in ASXL1, SRSF2, EZH2 or IDH1/2. In multivariable analysis, age >60 years, constitutional symptoms, hemoglobin In the Mayo Clinic validation cohort (n=398), median age 63 years; males 64%,median follow-up for alive patients was 7.5 years and 271 (68%) deaths and 51 (13%) leukemic transformations were documented. DIPSS-plus risk distribution was low in 13%, intermediate-1 18%, intermediate-2 37% and high 32%. Mutational frequencies were approximately 51% for JAK2, 30% CALR, 7.5% MPL, 5% EZH2, 36% ASXL1, 5.6% IDH1/2 and 13% SRSF2. MIPSS performed equally well in this cohort, as illustrated in figure 1B, which includes number of patients in each risk category with their median survival and HR and 95% CI values. Leukemia-free survival in the Mayo cohort was also predicted by high (p CONCLUSIONS. MIPSS addresses the mutation-relevant prognostic information gap in IPSS and performs equally well in the setting of patients seen at time of diagnosis or referral. Karyotype carries MIPSS-independent prognostic value that is recognized in an alternative GIPSS prognostic model (see accompanying ASH 2014 abstract), which complements MIPSS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Splenic extramedullary hematopoiesis is a major clinico-pathological feature of patients with myelofibrosis. As in the bone marrow (BM), hematopoiesis in the spleen occurs thank to the interplay of hematopoietic progenitor cells with the microenvironment, which provides the regulatory mechanism for their differentiation, proliferation and trafficking. Among other components, such as vessels and extra-cellular matrix proteins, this microenvironment encompasses different types of accessory cells, including mesenchymal stromal cells (MSCs). We have recently reported that MSCs from the BM of patients with myelofibrosis harbor genetics abnormalities and display an altered functional activity, suggesting that a primary MSC defect may either lead to or favor the pathogenesis of the disease. Here, we describe the phenotypical, functional, and genetic profile of MSCs isolated from the spleen of 23 patients with myelofibrosis, who underwent splenectomy for anemia and/or for excessive size of the spleen, and compare them to splenic mesenchymal stromal cells (s-MSCs) from 7 healthy subjects (HSs) who were splenectomized following traumatic lesion. The study was approved by the institutional review board of IRCSS Policlinico San Matteo Foundation; patients and HSs gave written informed consent for participating to the study. Mononuclear cells (MNCs) were obtained by dissociation of small spleen fragments by means of the GentleMacs Dissociator device (Miltenyi Biotech, Germany), and s-MSCs were isolated and expanded according to the standard procedures used for BM-MSCs. S-MSCs were obtained in 9/23 patients and in 3/7 HSs and displayed no significant differences for morphology and differentiation ability into adipocytic and osteoblastic lineages. However, the clonogenic efficiency of s-MSCs from patients with myelofibrosis was statistically higher than that of HSs (0.07 colonies/106 MNCs, range 0.03-0.01, vs 0.03/106 MNCs, range 0.03-0.04, respectively; p=0.048), whereas doubling time and time to senescence were not statistically different. Flow cytometric assessment of standard surface antigens (CD13, CD14, CD34, CD45, CD73, CD90, CD105) confirmed the mesenchymal nature of the cells grown in the cultures, and was similar between patients’ and HSs’ s-MSCs. When nestin expression was determined, no significant differences in the frequency of MSCs expressing this antigen was observed; however, nestin Mean Fluorescence Intensity (MFI) of patients’ s-MSCs was significantly lower than that of s-MSCs from HSs (22, range 6-45, vs 97, range 65-100, respectively; p=0.035). Patients’ s-MSCs also displayed a reduced capacity to sustain long term hematopoiesis in vitro in a classical Long Term Culture-Initiating Cell assay. However, when normal cord blood-derived CD34+ cells were co-cultured onto patients’ s-MSCs in a transwell system for 13 days, the output of CD41+ megakaryocytic cells increased with respect to culture where CD34+ cells were plated onto HSs' s-MSCs [21,5% vs 14,2% w/o recombinant human thrombopoietin (rhTPO), respectively, p=0,043; 60,2% vs 33,6% with rhTPO, respectively, p=0,01] at detriment of CD33+ cells (41,5% vs 48,6% w/o rhTPO, respectively, p=0,049; 10,4% vs 29,4% with rhTPO, respectively, p=0,012]. Finally, an abnormal karyotype [46XXt(5;17)(4-12)] was detected in 1 out of 18 metaphases of 1 out of 3 patient s-MSCs, while a normal karyotype was always observed in 2 out of 2 HSs’ s-MSC. This extensive characterization of s-MSCs shows that s-MSCs of patients with myelofibrosis display functional and genetic abnormalities compared to those isolated from HSs. The low level of nestin expression suggests that the hematopoietic niche of the spleen of patients with myelofibrosis can be defective and responsible for the increased trafficking of CD34+ cells that is observed in these patients, whereas the increased differentiation into the megakaryocytic lineage indicates a role of the splenic niche in leading hematopoiesis toward a pathological profile. All together, our data suggest that s-MSCs play a role in the pathogenesis of myelofibrosis and could be, therefore, a potential target for the treatment of the disease. Disclosures No relevant conflicts of interest to declare.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare clonal disorder of hematopoietic stem cells causing complement-mediated intravascular haemolysis, bone marrow aplasia and thromboembolic events. Allogeneic stem cell transplantation (SCT) has the potential to cure the disease but it is burdened by transplant-related mortality (TRM), which is lower in younger patients and with reduced-intensity conditioning regimens. Eculizumab is a monoclonal antibody suited to stop the abnormal complement cascade and capable of reducing thromboembolic events and related fatalities. Which of the two therapeutic options achieves the best health-care benefit to patients is uncertain as well as the economic burden of the two options onto health-care budget. Methods: We built a decision tree comparing upfront allogeneic SCT with lifelong eculizumab. Only patients with no prior thrombosis were analysed. A Markov model tracked PNH patients lifelong and accounted for persisting health-care modifications (states, such as transfusion-dependence, long-term disability related to severe thromboembolic events or GVHD, as well as for transitory modifications (events), such as acute phase of thromboembolic events and the first 3 months after SCT. TRM for sibling and unrelated fully matched donors was considered to be 15%, as reported by EBMT for patients transplanted for aplastic anemia after the year 2002 and by other institutions after reduced-intensity conditioning (de Latour 2012; Pantin 2014). However, patients 30-40 years of age were assigned a TRM of 20% and those older than 40 a TRM of 30%. The probability of thrombotic events was estimated based onthe reported cumulative rate of 40% in 15 years (de Latour 2008), but the risk was 4 times as higher for recurrent events. The fatality rate of thrombotic events was assumed to be 10%. The probability of death not related to thrombotic events was calculated based on the rate (8% in 10 years) reported by the French retrospective study (de Latour 2008) for individuals diagnosed after 1996. Patients being treated with eculizumab, however, were assigned thrice as higher the mortality probability of age-matched general population according to the 2008 Italian life tables (Hillmen 2011). Quality of life for PNH patients was calculated from EORTC scores reported by the International PNH Registry (conversion algorithm reported by Kontodimopoulos 2009): it was 0.81 in non-transfused patients, but decreased to 0.71 after thrombotic events, and to 0.51 in transfusion-dependent patients and in those with severe GVHD. Allogeneic SCT was assigned a cost of €80,000 upfront and €100 per month subsequently. Eculizumab was assigned a monthly cost of €20,000 assuming standard scheduling (600 mg weekly for 4 weeks followed by 900 mg biweekly). Future life years and costs were discounted by 3.5% per year, according to international guidelines. The model was built and analyzed with TreeAgePro2014. Results: At baseline analysis, patients aged 35 years would expect 41.32 life years after allogeneic SCT versus 35.0 on lifelong eculizumab. At two-way sensitivity analysis, we analyzed the role of TRM and relative survival of patients treated with eculizumab, as compared with healthy individuals. Allogeneic SCT is expected to provide better life expectancy so far as mortality in patients treated with eculizumab is >38% higher than general population. At TRM rates of 30%, allogeneic SCT keeps more effective than eculizumab only provided that PNH-related mortality is > 100% higher than general population. Similar results were obtained for discounted quality-adjusted life expectancy, which was 37.1 QALYs after allogeneic SCT versus 26.8 QALYs with lifelong eculizumab. In patients aged more than 52 years, eculizumab provided a better life expectancy than SCT. Lifelong discounted costs were estimated to be €113,341 after allogeneic SCT versus €8,362,079 with eculizumab. Conclusions: In an era of improved transplant yield and of budget constraints, allogeneic SCT should still be considered a therapeutic option for younger PNH patients with an aplastic phenotype. Disclosures No relevant conflicts of interest to declare.