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The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

During SARS-CoV-2 infection, the innate immune response can be inhibited or delayed, and the subsequent persistent viral replication can induce emergency signals that may culminate in a cytokine storm contributing to the severe evolution of COVID-19. Cytokines are key regulators of the immune response and virus clearance, and, as such, are linked to the—possibly altered—response to the SARS-CoV-2. They act via a family of more than 40 transmembrane receptors that are coupled to one or several of the 4 Janus kinases (JAKs) coded by the human genome, namely JAK1, JAK2, JAK3, and TYK2. Once activated, JAKs act on pathways for either survival, proliferation, differentiation, immune regulation or, in the case of type I interferons, antiviral and antiproliferative effects. Studies of graft-versus-host and systemic rheumatic diseases indicated that JAK inhibitors (JAKi) exert immunosuppressive effects that are non-redundant with those of corticotherapy. Therefore, they hold the potential to cut-off pathological reactions in COVID-19. Significant clinical experience already exists with several JAKi in COVID-19, such as baricitinib, ruxolitinib, tofacitinib, and nezulcitinib, which were suggested by a meta-analysis (Patoulias et al.) to exert a benefit in terms of risk reduction concerning major outcomes when added to standard of care in patients with COVID-19. Yet, only baricitinib is recommended in first line for severe COVID-19 treatment by the WHO, as it is the only JAKi that has proven efficient to reduce mortality in individual randomized clinical trials (RCT), especially the Adaptive COVID-19 Treatment Trial (ACTT-2) and COV-BARRIER phase 3 trials. As for secondary effects of JAKi treatment, the main caution with baricitinib consists in the induced immunosuppression as long-term side effects should not be an issue in patients treated for COVID-19.We discuss whether a class effect of JAKi may be emerging in COVID-19 treatment, although at the moment the convincing data are for baricitinib only. Given the key role of JAK1 in both type I IFN action and signaling by cytokines involved in pathogenic effects, establishing the precise timing of treatment will be very important in future trials, along with the control of viral replication by associating antiviral molecules.

Systemic mastocytosis (SM) encompasses a heterogeneous group of clonal disorders characterized by abnormal expansion of mast cells (MCs). Beyond KIT and other genes recurrently mutated in myeloid neoplasms, several genetic variants have been described as predisposing to the development of the disease and influencing its clinical phenotype. Increased copy number variants of the TPSAB1 gene were identified as a cause of nonclonal elevated tryptasemia and defined as hereditary α-tryptasemia (HαT). Moreover, HαT is enriched in patients with SM, where it can affect the incidence of mediator-related symptoms.In a multicenter data set of 444 patients with MC disorders, we aimed to investigate the clinical correlates of germline TPSAB1 copy number gains.Droplet digital PCR was performed in all cases to ascertain the presence of HαT. Clinical history along with blood values and bone marrow examination were analyzed.We confirmed a higher incidence of HαTThese findings on a large patient series support and extend previous data, and suggest that knowledge of HαT status may be useful for personalized management of patients with SM.

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

Introduction. MPN-COVID is a European LeukemiaNet cohort study, launched in March 2020 in patients with myeloproliferative neoplasms (MPN) with COVID-19. The first cohort of 175 cases was analyzed at the end of first wave (July 2020) and results provided estimates and risk factors of overall mortality (Barbui T. Leukemia, 2021), thrombosis incidence (Barbui T. Blood Cancer J, 2021), and post-COVID outcomes (Barbui T. Blood Cancer J, 2021). In the second wave of pandemic (June 2020 to June 2021), case-fatality risk in the general population has been found variable across different countries, and no information is available in MPN patients with COVID-19 diagnosed during the second wave in comparison with those of the first wave. Methods. In an electronic case report form, we registered a total of 479 cases of ET (n=161, 34%), PV (n=135, 28%), pre-PMF (n=49, 10%) and overt MF (n=134, 28%), from 39 European hematology units (Italy, Spain, Germany, France, UK, Poland, Croatia). Of these, 304 were diagnosed COVID-19 during the second wave. Results. Patients in the second wave were significantly different from those in the first wave, including parameters such as age (median: 63 vs. 71 years, p Conclusions. This is the largest series of MPN patients who incurred COVID-19 from June 2020 onward, namely during the "second COVID-19 wave". Compared to the first wave, the second one recorded a lower overall COVID-19 severity, but Ruxolitinib discontinuation still remained a risk factor for a dismal outcome. Greater vulnerability of ET than PV in developing venous thrombosis was confirmed also during the second wave. This finding suggests that ET warrants a specific antithrombotic prophylaxis in addition to heparin. Figure 1 Figure 1. Disclosures Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Iurlo: Novartis: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Sobas: Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Fox: Novartis: Honoraria; Sierra: Honoraria. Palandri: AOP: Membership on an entity's Board of Directors or advisory committees; CTI: Consultancy; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Benevolo: Novartis: 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; Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau. Harrison: 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; Sierra Oncology: Honoraria; Galacteo: 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; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; BMS: 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; Janssen: 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; Geron: 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; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bonifacio: Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Kiladjian: Taiho Oncology, Inc.: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; 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; PharmaEssentia: Other: Personal fees; 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. Patriarca: Incyte: Honoraria; Takeda: Honoraria; Argenix: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria. Griesshammer: Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria; CTI: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria. Garcia Gutierrez: Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Osorio: Janssen, Abbvie, Roche: Consultancy. Koschmieder: CTI: Membership on an entity's Board of Directors or advisory committees, Other; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Abbvie: Other: Travel support; Alexion: Other: Travel support; Karthos: Other: Travel support; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Image Biosciences: Other: Travel support; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Roche: 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, Other: Travel support, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other. 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.

Background. The tendency towards thrombosis in myeloproliferative neoplasms (MPNs) is linked to the JAK2-mutant clone which leads to hypercellularity and functional interplay between abnormal erythrocytes, platelets, leukocytes and dysfunctional endothelium. The resulting cell activation that also involves stromal cells in their microenvironment, has been shown associated with chronic, systemic, subclinical pro-inflammatory state, and has been implicated in the pathogenesis of thrombosis in MPN. Neutrophil to lymphocyte ratio (NLR) is a novel inflammatory marker found to be associated with the severity and prognosis of cardiovascular diseases but there is little evidence for its prognostic significance in MPN. We investigated whether NLR could predict the onset of arterial and venous thrombosis in polycythemia vera (PV). Methods. Subjects of this study were 1508 patients included in the ECLAP trail with NLR evaluation available. In addition to standard statistical methods, we used proportional hazards additive models (GAM) as they can provide an excellent fit in the presence of nonlinear relationships, for the prediction of total, arterial and venous thrombosis as smooth functions with cubic splines of different blood parameters. Results. After a median follow-up of 2.76 years, 169 thrombotic events (10.3%) were objectively diagnosed and analyzed: 87 arterial (MI, Stroke, TIA, PAT) and 88 venous thrombosis (DVT±PE, superficial thrombophlebitis). In univariate analysis, arterial thrombosis was associated with age (p=0.003) and previous thrombosis, especially if arterial (p Conclusions. The NLR is an inexpensive and easily accessible test compared to other inflammatory markers. We found that NLR-alone is an independent predictor of venous thrombosis and could be included in a new scoring system. In addition to guiding the stratification of thrombotic risk, these data confirm that inflammation is a relevant target of antithrombotic therapy in PV. Figure 1 Figure 1. Disclosures Vannucchi: 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; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction. Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells that include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. 10-20% of MPN pts transform to secondary acute myeloid leukemia (sAML), unresponsive to conventional therapy and associated with dismal outcome (Dunbar A, 2020). In addition to somatic driver mutations affecting JAK2, CALR or MPL, several additional variants are harbored by MPN pts inb chronic phase, and a restricted set of them were associated with risk of leukemic evolution (Vannucchi AM, Leukemia 2013; Tefferi A, Blood ASdv 2016) However, the molecular mechanisms underlying leukemic transformation remain largely unknown. Although bulk next generation sequencing (NGS) highlights the overall mutation landscape, it cannot distinguish which mutations occur in the same clone(s), nor elucidate the order of mutations or resolve clonal complexity. Conversely, single-cell sequencing (SCS) might allow to resolve clonal heterogeneity and reconstruct clonal phylogenies at each disease phase (Parenti, NPJ Prec Onc 2021)). Aim: To delineate the clonal landscape of sAML, we performed single-cell mutational profiling in 10 pts with MPNs who progressed to sAML. Methods. There were 2 set of samples/approaches: (i) 15 paired samples (chronic (CP)/blast phase (BP)) from 7 pts were analyzed using the Mission-Bio Tapestri platform and the Myeloid panel in order to target SNVs and indels across 45 myeloid genes with 312 amplicons. In one pt we also analyzed an intermediate phase corresponding to progression from PV to PPV-MF before BP development. (ii) 7 further paired CD34+ samples from 3 pts were analyzed using a 239-amplicon custom panel including 29 genes frequently mutated in myeloid neoplasms. SCS libraries were sequenced on Illumina Novaseq. Data were processed by using Mission Bio's Tapestri Pipeline and analyzed with Mission Bio's Tapestri Insights software package and R software. CNV analysis was performed by using an integrated pipeline for multiomics analysis (Mosaic, Mission Bio) Results. (i) A total of 78,354 single cells were sequenced (average 5,223) using Tapestri Myeloid panel, with an average of 28,303 reads per cell and coverage of 97X. SCS was able to identify 17 low-frequency variants not detected in bulk analysis; however, it failed to discriminate homopolymeric regions including the ASXL1 G646Wfs*12. (ii) A total of 25,417 single cells were sequenced (average 3636) using a custom panel, with a coverage of 186X and an average Allele Dropout Rate of 8.6%. This panel was able to identify ASXL1 G646Wfs*12 variant. Overall, we found a significant correlation of variant allele frequency (VAF) measured by bulk and SCs approach (R =0.84, p20 copies in 2 pts), NRAS (8 copies in 2pts) and BRAF (8 copies in 2pts). Other subclonal ploidy abnormalities were also observed in RUNX1, EZH2, U2AF1 and ZRSR2 (5-18 copies). Conclusions. Together, these data suggest that MPN present a complex clonal combination evolving over time. SCS allows to resolve this milieu, that is largely missed by conventional bulk NGS, in particularly SCS identifies rare leukemia-driving clones that were already present in chronic phase and describes their dynamics during leukemic progression. Leukemic transformation after MPN is a highly heterogeneous process with mutations and CNVs acquired in different genes and different clones. Overall, our findings provide further insights into the pathogenesis of AML transformation of MPN. Supported by AIRC, Mynerva project no.21267 Figure 1 Figure 1. Disclosures Vannucchi: BMS: 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; AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background The detrimental effect of leukocytosis on survival in myeloproliferative neoplasms (MPN) has been well established for primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET ) (JCO. 2018;36:310; BJH. 2020;189:291) Previous studies have also implicated leukocytosis as a risk factor for leukemic transformation (Mayo Clin Proc. 2017;92:1118) and thrombosis in MPN (Blood Adv. 2019;3:1729). However, it is currently not clear as to which component(s) of white blood cells is responsible for these observations. In the current study, we sought to examine the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts, on overall (OS), leukemia-free (LFS), and myelofibrosis-free (MFFS) survival and in ET. Methods Study patients (n=349) were retrospectively recruited from the Mayo Clinic MPN database of 1,249 WHO-defined ET patients, evaluated over five decades (1967-2021), based on availability of information on ANC, ALC and AMC. Conventional criteria were used for diagnosis and definitions of major complications, including leukemic or fibrotic transformation (Blood 2016;127:2391). Conventional statistical methods were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for survival. Results 349 patients (median age 57 years, range 18-89; females 61%) with ET were included in the study: 46% JAK2, 34% CALR, 16% triple-negative and 4% MPL mutated; IPSET risk category high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1-16.4) for hemoglobin, 8.2 x 10(9)/L (3.2-52) for leukocyte count, and 859 x 10(9)/L (451-3460) for platelet count; palpable splenomegaly was present in 48 (14%); median followup was 10 years (range 0-47), during which time 118 deaths, 52 fibrotic progressions, and 14 leukemic transformations were documented. Multivariable analysis identified older age (p60 years, 3.1 (2.1-4.6; p60 years), 2 for increased ANC (≥8 x 10(9)/L) and 1 for ALC ( An external validation cohort from the University of Florence (n=485) confirmed the independent survival risk contribution from age >60 years (p Conclusions: The current study identifies increased ANC and decreased ALC as age-independent risk factors for survival in ET, thus allowing the development of a globally applicable simple to use "Triple A" risk model that is based on Age, ANC and ALC. Decreased ALC also predicted fibrotic and leukemic progression. Our observations suggest potential value for immune profiling as an additional prognostic tool in MPN. Figure 1 Figure 1. Disclosures Szuber: Novartis: Honoraria. Vannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Introduction: Myelofibrosis (MF), whether primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocytemia, is characterized by a complex and partially undeciphered molecular architecture. Besides mutations in driver genes (JAK2, CALR, MPL), somatic mutations in selected myeloid-associated genes have been shown to impact prognosis of MF patients (pts). Among these, ASXL1 mutations (ASXL1MTs) are associated with poor outcomes in myeloid malignancies including PMF, where they are included in the category of "high molecular risk" (HMR) mutations along with EZH2MTs, IDH1/2MTs, and SRSF2MTs (Vannucchi AM, Leukemia 2013). However, a recent study (Luque Paz D, Blood Adv 2021) questioned the value of ASXL1MTs in MF. The current study aimed at further characterizing the prognostic role of ASXL1MTs in MF. Methods: After IRB approval, pts with WHO-defined MF were included in the study. Mutational analysis by targeted NGS was performed as previously described (Guglielmelli P, JCO 2017). All deposited variants were manually curated to assess pathogenicity. In this study, we also used the molecular model proposed by Luque Paz et al. that identifies 4 genetic groups: TP53MT; High-risk (≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1/2); ASXL1MT-only; and "Others". Results: A total of 525 pts were included in the study, including 331 (63%) PMF and 194 (37%) SMF. Median age at diagnosis was 89 (18-90) years, 314 (60%) were male. The median follow-up time was 80 (98% CI, 68-90) months. Overall, 324 (62%) pts were JAK2MT, 126 (24%) CALRMT, 24 (5%) MPLMT, 40 (8%) triple negative (TN), and 11 (2%) double mutated. Among non-driver genes, ASXL1MTs were found in 158 (30%) pts, EZH2MTs in 45 (9%), SRSF2MTs in 37 (7%), NRASMTs in 30 (6%) U2AF1MTs in 27 (5%), TP53MTs and CBLMTs in 25 (5%) each, IDH1/2 MTs in 18 (3%), and KRAS MTs in 15 (3%). Pts in the HMR category were 125 (38%) in PMF and 63 (32%) in SMF. According to the above model, distribution of pts was as follows: TP53MT n=25 (5%), High-risk n=137 (26%), ASXL1MT-only n=64 (12%), and Others n=299 (57%). Pts in the TP53MT and ASXL1MT-only groups were more likely to be diagnosed with SMF compared to pts in the High-risk and Others groups (44% and 48% vs 28% and 38%, respectively). In addition, the High-risk group was enriched in TN pts (16%), while CALRMTs were more common in the ASXL1MT-only and Others compared to the TP53MT and High-risk groups (25% and 27% vs 12% and 18%, respectively). In univariate analysis, the TP53MT and High-risk groups were associated with the worst overall survival (OS), with median values of 38 (14-110) and 55 (45-85) months (P=.0039), respectively (Fig 1A). Albeit remarkably better, the OS of pts in the ASXL1MT-only group was inferior compared to pts in the Others group (median 124 [91-156] vs 193 [142-NR] months; P=.0118) (Fig 1A). We then analyzed separately PMF and SMF cohorts. In the former, the TP53MT and High-risk groups remained associated with the worst OS (median 58 [20-126] vs 55 [36-85] months), although with no significant difference, likely due to the low frequency (4%) of TP53MTs mutations in PMF (Fig 1B). Concurrently, the negative prognostic impact of the ASXL1MT-only group was confirmed in comparison to the Others group (median 103 [78-NR] vs 320 [178-NR] months; P=.0170). In pts with SMF, while the TP53MT group (6%) had by far the worst OS (median 13 [6-NR] months), the OS of the ASXL1MT-only group (median 141 [56-171] months) was comparable to that of the Others group (median 131 [106-NR] months; P=.5188) and not different from the High-risk group (median 58 [45-174] months; P=.3606) (Fig 1C). In a further analysis including only pts in the High-risk group, ASXL1MTs were found in 62% and 63% of patients with PMF and SMF, respectively. In survival analysis, the presence of ASXL1MTs was associated with an increased risk of death only in PMF (median OS 47 [31-73] vs 102 [34-317] months; P=.0240), unlike in SMF (median OS 90 [47-174] vs 25 [16-338] months; P=.3296) (Fig 1D-E). Conclusion: In the current study, we critically re-addressed the prognostic impact of ASXL1MTs by applying a genetic model recently developed by Luque Paz et al. to our cohort of molecularly annotated, WHO-defined MF pts. Overall, our results confirm that ASXL1MTs -even in the absence of other co-occurring high-risk mutations- harbor a negative prognostic impact mainly in PMF. These findings also reinforce the idea that PMF and SMF represent two different biological entities. Figure 1 Figure 1. Disclosures 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.

Background In addition to its influence on survival, leukocytosis in myeloproliferative neoplasms (MPN) has also been implicated as a risk factor for thrombosis, including venous thrombosis in PV (Blood Cancer J, 2017;7:662) and arterial thrombosis in ET (Blood. 2011;117:5857). In the current study, we sought to clarify the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts, for arterial and venous thrombotic events in essential thrombocythemia (ET) and polycythemia vera (PV). Methods The current study included 487 patients with ET (n=349) or PV (n=138), recruited from the Mayo Clinic MPN database, based on availability of information on ANC, ALC and AMC. Conventional criteria were used for diagnosis (Blood 2016;127:2391) and definitions of major vascular events (Blood Cancer J. 2018;8:25). Conventional statistical models were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for arterial or venous thrombosis. Results Essential thrombocythemia patients: 349 patients (median age 57 years, range 18-89; females 61%) with ET were included in the study: 46% JAK2, 34% CALR, 16% triple-negative and 4% MPL mutated; IPSET risk category high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1-16.4) for hemoglobin, 8.2 x 10(9)/L (3.2-52) for leukocyte count, and 859 x 10(9)/L (451-3460) for platelet count; palpable splenomegaly was present in 48 (14%) patients and cardiovascular risk factors in 56%; median follow-up was 10 years (range 0-47). There were 38 (11%) documented venous events at diagnosis and 31 (9%) after diagnosis and 42 (12%) arterial events at diagnosis and 64 (18%) after diagnosis. Polycythemia vera patients: 138 patients (median age 62 years, range 20-94; females 50%) with PV were included in the study; presenting median (range) values were 17.9 g/dL (16.1-24) for hemoglobin, 11.8 x 10(9)/L (2.7-65.8) for leukocyte count, and 434 x 10(9)/L (44-1679) for platelet count; 57% of patients presented with leukocytosis >11 x 10(9)/L; palpable splenomegaly was present in 37 (27%) patients; abnormal karyotype was documented in 17% of patients. Median follow-up was 11 years (range 0.03-36.7). There were 22 (16%) documented venous events at diagnosis and 21 (16%) after diagnosis and 28 (20%) arterial events at diagnosis and 15 (11%) after diagnosis. ANC/ALC/AMC associations with thrombosis in ET and PV: In both ET and PV, ANC/ALC/AMC did not correlate with arterial thrombosis at/prior to diagnosis (p>0.1 in all instances). By contrast, in both ET and PV, higher ANC (p=0.05 and 0.04, respectively) and higher AMC (p=0.005 and 0.07), but not ALC (p=0.6 and 0.7), were correlated with venous thrombosis at/prior to diagnosis. Arterial thrombosis-free survival was not affected by ANC/ALC/AMC in either ET or PV (p>0.1 in all instances). By contrast, venous thrombosis-free survival in both ET and PV was compromised by higher ANC (p=0.05 and 0.003, respectively), but not ALC or AMC. The significant association between higher ANC and inferior venous thrombosis-free survival in both ET (p=0.01) and PV (p=0.007) was sustained during multivariable analysis that included history of venous thrombosis, age and sex; the only other variable of significance was older age in ET (p Conclusions: The current study identifies ANC as having a direct correlation with venous thrombosis in both ET and PV, independent of currently known risk factors. An additional risk contribution from AMC was apparent for events occuring at or prior to but not after diagnosis. Taken together, these observations flag both neutrophils and monocytes as potential contributors to venous but not arterial thrombosis in MPN, at least not by themselves. Additional collaborative studies are ongoing in order to integrate and reconcile our observations in the context of neutrophil/lymphocyte ratio (Carobbio et al) and JAK2V617F allele burden (Loscocco et al), on venous thrombosis-free survival in PV, reported in concomitantly submitted ASH 2021 abstracts, especially in light of the relatively small number of events in the current study. Disclosures Barbui: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Vannucchi: AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: 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.

Introduction Hereditary alpha-Tryptasemia (HαT) is a group of genetically defined traits that share increased copy number of TPSAB1 gene encoding for both the α- and β-alleles (Lyons et al 2018). Increased copy number (CN) of the α-tryptase coding sequence in TPSAB1 on one or both alleles represents the genetic base of HαT (Lyons et al 2016). HαT is characterized by mild elevation in serum tryptase levels and a variety of mast cell (MC) activation symptoms, including recurrent anaphylaxis. Prevalence in the general population is up to 5%, that increases up to 20% in systemic mastocytosis (SM); it has been suggested that HαT might be a germline variant predisposing to SM development. In SM, HαT correlated with higher incidence of mediator-related symptoms (Greiner G et al, Blood 2021). Due to its complexity, the assay for TPSAB1 CN is performed in few centers and the actual prevalence of HαT in selected subsets is still to be elucidated. To this end, we screened for HαT 2 groups of subjects, the first with MC activation symptoms and no evidence of SM, the second with diagnosis of SM according to WHO-2016. Methods Droplet digital PCR (ddPCR) was used to measure CN variation (CNV) in TPSAB1 by adapting standard CNV ddPCR protocol to genotype for both TPSAB1 and TPSB2 (Fig.1A). The high homology between α and β encoding isoforms and the presence of paralogous genes in a single locus makes TPSAB1 CNV detection very challenging. ddPCR was performed on genomic DNA with/without BamHI, using the PrimePCR ddPCR Copy Number reference AP3B1 (BioRad). Accuracy and precision of the ddPCR protocol was assessed by analyzing 10 samples in triplicate in 3 separate experiments. Data robustness and repeatability can be appreciated in Fig 1B. Results We studied 41 subjects with mediator-related symptoms and augmented basal serum tryptase (BST) (cohort 1) and 150 patients with ascertained diagnosis of mastocytosis (cohort 2). The BST threshold established for cohort 1 was equal or higher than 11 mcg/L. Median age was 64.7 yr, males 54%; median BST levels was 15.3 (range 12.3-21 mcg/L); 29% of the pts had history of anaphylaxis. In cohort 2, 134/150 (89.3%) pts had a diagnosis of SM, whereas 13/150 (8,6%) were Cutaneous Mastocytosis (CM). Among SM patients, 113(84.3%) presented with non-advanced SM variants. Advanced forms including aggressive SM (ASM) and SM with an associated hematological neoplasm (SM-AHN) were diagnosed in 6 (4.5%) and 8 (6%) respectively. In 3 pts, SM subtype was not available. Median age was 49 yr, males 55%; 41.7% of the pts had history of anaphylaxis. HαT was documented in 27 (65.9%) subjects in cohort 1, and 14 (9.3%) in cohort 2. In cohort 1, 3 α-tryptase (3α) copy number was observed more frequently (59.2% of HαT+ pts); conversely 3α and 2α-tryptase copy number were observed at a similar rate (42,8%) in cohort 2. HαT+ pts in cohort 1 presented significantly higher BST (17.1 vs 12.05 mgc/L, P In cohort 2, BST levels were similar in HαT+ and HαT wt pts (24.6 and 24.3 mcg/L), as were anaphylaxis episodes (50% and 41%, respectively). A trend for lower MC burden in HαT+ as assessed by flow cytometry was demonstrated (% of bone marrow MC: 0.01% in HαT+ vs 0.07%) whereas no meaningful differences emerged regarding the symptom burden. In addition, a lower prevalence of KIT 816V mutation was observed in HαT+ (71.4% vs 89.5%; p=0.073). Conclusions In our study HαT+ was observed in around 10% of patients with SM, a prevalence lower than previously reported (Greiner et al, Blood 2021) and remarkably lower than in a selected cohort of subjects with raised BTL and history of mediator-released symptoms (66%). ddPCR represents a suitable method to investigate the presence of CNV in the α-tryptase coding sequence. Genetic testing for HαT+ should be considered in the diagnostic workout of patients presenting with anaphylaxis or MC mediator-related symptoms and no suspicion/evidence of SM. The clinical correlates of HαT in SM remain to be fully ascertained. Supported by IMH no.GR-2016-02362631 and AIRC, Mynerva project no21267 Figure 1 Figure 1. Disclosures Elena: CELGENE: Other: funding for meeting participation; PFIZER: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees. Vannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; 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.

Introduction: Myelofibrosis -either primary (PMF) or post-polycythemia vera/essential thrombocythemia (collectively, secondary MF [SMF])- is characterized by biologic and clinical heterogeneity, with some patients (pts) presenting with features of myeloproliferation (MyP) and others exhibiting a myelodepletive (MyD) phenotype. Although not well defined, the latter is marked by cytopenias involving one or more hematopoietic lineages, in some respects mimicking a myelodysplastic or bone marrow (BM) failure syndrome. MyD features have been associated with poor prognosis in patients with MF, but cytopenias have been usually considered individually. In the current study, we aimed at investigating the phenotypic and prognostic correlates of a MyD phenotype in a large cohort of MF patients. Methods: After IRB approval, 704 consecutive patients with WHO-defined MF referring to CRIMM (Florence), were included in the study. There were 442 (63%) PMF and 262 (37%) SMF. Cytopenias at diagnosis were defined as follows: white blood cell (WBC) 15×10 9/L, Hb >16.5 g/dL for male and >16 g/dL for female, Plt >450×10 9/L). Pts not included in the MyD group were considered as having a MyP phenotype. Mutational analysis by targeted NGS was available for 594/704 patients (PMF 368/442; SMF 226/262). Results: Overall, 166 pts were diagnosed as having a MyD MF, including 108 (24%) PMF and 58 (22%) SMF. Among PMF patients, leukopenia, sex-adjusted anemia and thrombocytopenia were present in 38%, 84% and 35%, respectively; in SMF, corresponding figures were 24%, 91% and 24%. Two or more cytopenias were found in 11% and 6% of PMF and SMF patients, respectively. In PMF, a MyD phenotype was associated with male gender (P=.0468), older age (P=.0002), lower peripheral blast count (P=.0006), higher prevalence of splenomegaly (P=0.0142), constitutional symptoms (P Phenotypic differences between MyD and MyP patients were less evident in SMF, a part for older age of the former (P=.0207). The molecular background was likewise different: a MyD feature was enriched in mutations in TP53 (P=.0024), U2AF1 (P Conclusion: The current study provides a comprehensive analysis of the MyD phenotype in pts with PMF and SMF, partly recapitulating findings of previous studies (Marcellino BK et al. Clin Lymphoma Myeloma Leuk 2020). We showed that the MyD phenotype is associated with high-risk clinical and molecular features, particularly in PMF, and correlates with inferior OS. Of interest, U2AF1 mutations emerged as a distinct molecular marker of MyD in both PMF and SMF, while mutations in TP53 appear prevalent in MyD SMF. Figure 1 Figure 1. Disclosures 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.

BACKGROUND The rate of major arterial and venous thrombosis in primary myelofibrosis (PMF) and post-ET (PET) and post-PV (PPV) secondary myelofibrosis has been evaluated in a limited number of studies. In the present paper we describe the clinical epidemiology of thrombosis in a large series of patients with overt PMF and PPV/PET MF looking at the rate and risk factors. Moreover, we report findings on thrombosis rate in two cohorts of patients treated with Hydroxyurea (HU) or Ruxolitinib (Ruxo). METHODS Patients were registered in the European Registry for Myeloproliferative Neoplasms (ERNEST). This project, promoted by the European LeukemiaNet, is coordinated by FROM - Foundation for Research, Papa Giovanni XXIII Hospital, Bergamo (Italy) and supported by Novartis through a research collaboration . Patients were diagnosed in 6 Centers from Italy, Spain and Sweden, between Jan, 2001 and Dec, 2012, with the required follow-up information. Patients (n= 1010) with PMF (n=584, 59%), PET-MF (n=207, 20%) and PPV-MF (n=219, 21%) were evaluated for incident thrombosis as primary endpoint. Considering death as a competitive event, uni-and multivariate analyses were performed by applying Fine & Gray competing-risk regression models. RESULTS After a median follow-up of 3.8 years (IQR: 1.8-7.1) from diagnosis, 108 thromboses (10.7%) occurred, for an overall incidence rate of 2.0% pts-yr (95% CI: 1.7-2.5). Arterial thromboses were found in 50 patients (46.3%) including cerebral (n=21, 19.4%), myocardial infarction (n=13, 12.0%) and peripheral events (n=9, 8.3%). Venous thromboses were 58 (53.7%), of which 25 (23.0%) were DVT ± PE and 11 (10.2%) were splanchnic. Thrombosis rate was 1.91, 1.60 and 2.79% pts-yr in PMF, PET-MF and PPV-MF, respectively. In univariate analysis, factors significantly associated with an increased thrombotic risk in PMF were age (p=0.013) and the presence of the JAK2 mutation (p=0.003); in addition, a significant higher proportion of PMF patients at low and intermediate-1 vs intermediate-2 or high risk IPSS score, had thrombosis during the follow-up (p=0.008). In multivariate analysis, only JAK2 mutation retained statistical significance (SHR=3.12, 95% CI: 1.40-6.94, p=0.005). Conversely, neither in univariate nor in multivariable analysis, significant risk factors were not found.To investigate the possible interaction of IPSS score and JAK2 mutation we created a model whose results are presented in Fig. 1A: the cumulative incidence function (CIF) of thrombosis was significantly lower in patients with JAK2 wild-type and intermediate-2 or high IPSS score (CIF: 4% projected at 10 years; SHR=1 [reference category]), while patients at the highest risk for thrombosis harbored JAK2 mutation and were categorized at low or intermediate-1 by IPSS score (CIF: 20% projected at 10 years, SHR=7.13, p=0.008). Of note, thrombosis had a significant impact on mortality. After adjusting for sex, age, year of diagnosis, type of MF and IPPS, HR was 1.51, (95% CI. 1.15-1.98, p=0.003).The influence of drug exposure to incident thrombosis was investigated in two cohorts of 559 consecutive patients exposed to HU (n=470) or to Ruxo (n=89), median treatment 2.6 and 3.0 years, respectively. HU- compared to Ruxo-treated patients were older (median age 67 vs. 63 years, p=0.001), more frequently triple negatives (12% vs. 2%, p=0.036), less splenomegalic (spleen length >10 cm: 30% vs. 88%, p CONCLUSION IPSS score, in addition to the survival risk assessment, may be useful, if associated with the JAK2 mutation, to recognize patients at vascular risk and to suggest appropriate anti-thrombotic prophylaxis. The trend towards a benefit of Ruxo, compared to HU, warrants a study in larger case series. Figure 1 Figure 1. Disclosures Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Passamonti: Janssen: 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; Celgene: 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, Research Funding, Speakers Bureau; AbbVie: Speakers Bureau. 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, Other: Lectures; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Lectures.

Background: Thrombosis is the main cause of morbidity and mortality in pts with Polycythemia Vera (PV). Current risk stratification is based on 2 variables: age >60y and history of thrombosis. Additional thrombotic risk factors in PV are generic cardiovascular risk factors and leukocytosis. JAK2V617F (JAK2VF) variant allele frequency (VAF) at diagnosis is highly heterogeneous. A VAF>75% was associated with higher rate of all thrombosis after diagnosis (Vannucchi AM et al, Leukemia 2007), and a VAF ≥ 60% correlated with increased rate of venous thrombosis (VT) in high-risk pts (Guglielmelli P et al, ASH 2018); however, predictive role of JAK2VF VAF is still debated. Aim: To evaluate the impact of JAK2VF VAF on rate of arterial and venous thrombosis in PV pts. Patients and methods: A cohort of 576 strictly 2016 WHO-defined PV pts followed at Univ. of Florence (1981-2020) were included. All pts were annotated for JAK2VF VAF, determined Results: Median age was 61.4 y (range, 16.2-91.8), 58.2% were male; 62% were high-risk based on current classification. Median JAK2VF VAF was 41.5% (range, 0.3-100). A total of 76 (13.2%) pts had an AT event before/at PV diagnosis and 49 (8.5%) pts had an AT during FU. As regards VT, 64 (11.1%) and 39 (6.8%) pts had a VT before/at or after PV diagnosis, respectively. We found that JAK2 VAF as a continue variable was correlated with the risk of VT in FU (p=0.003) but not with AT (p=0.8). ROC analysis to determine the best cut-off level for JAK2 VAF predicting VT had an AUC of 0.72 and a best cut-off value of VAF=50%. VT at FU were significantly enriched in pts with VAF >50%: 14.5% versus 2.4%, p=50% (HR 4, CI 1.9-8.6, p50%, univariate analysis for VT-FS identified history of VT (HR 2.9; CI 1.4-6.1, p=0.006), leukocytosis ≥11x10 9/L (HR 1.9; CI 1.1-3.4, p=0.02) and palpable splenomegaly (HR 1.9, CI 1-3.6; p=0.04) as risk factors. Multivariable analysis confirmed VAF>50% (HR 3.8, CI 1.8-8.1, p=0.0006) and previous VT (HR 2.4, CI 1.1-5.1; p=0.02) as independent risk factors for future VT. In contrast, univariate analysis for AT-free survival (AT-FS) identified history of AT (HR 2.5; CI 1.3-4.9, p=0.007), diabetes (HR 3.3; CI 1.6-6.5, p=0.0007), hyperlipidemia (HR 3.1; CI 1.7-5.6, p=0.0003) and hypertension (HR 2, CI 1.1-3.8; p=0.03) as predictors of future AT; age >60y showed only a trend (p=0.08). Multivariable analysis for AT-FS identified diabetes (HR 2.4, CI 1.2-5; p=0.02), hyperlipidemia (HR 2.3; CI 1.2-4.3, p=0.01) and previous AT (HR 2.1, CI 1-4.2; p=0.04) as independent predictors of future AT. Validation: Our findings were validated in an independent cohort of 315 2016-WHO defined PV pts from Policlinico Gemelli, Catholic Univ., Rome. After exclusion of 26 pts with SVT, analysis was conducted on 289 pts, 38 of them with thrombosis as heralding event (21 AT and 17 VT). Multivariable analysis confirmed JAK2VF VAF >50% (HR 2.3, CI 1.03-5.0, p=0.04) and previous VT (HR 4.5, CI 2.0-10.1; p=0.0003) as independent risk factors for future VT. In pts with VAF >50%, the rate of VT at FU was 19.9% vs 7.7%, P=0.005. KM curve showed that VT-FS was significantly shorter in pts with a JAK2VF VAF >50% (HR 2.2, CI 1.2-4.2; p=0.01) (Figure 1B). Of note, impact of JAK2 VAF>50% on VT at FU was statistically significant particularly in conventionally low-risk pts, accounting for an HR of 9.4 (CI 1.2-72) and HR 3.6 (CI 1.3-10) in Florence and Rome cohorts, respectively. Conclusions: These data support JAK2VF VAF as a strong independent predictor for future venous thrombosis in PV, in association with history of prior venous events, reinforcing that AT and VT are associated with unique risk factors in pts with PV. Supported by AIRC, Project Mynerva n.21267 Figure 1 Figure 1. Disclosures Vannucchi: BMS: 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; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

// Niccolo Bartalucci 1, 2, 3 , Laura Calabresi 1, 2, 3 , Manjola Balliu 1, 2, 3 , Serena Martinelli 1, 2, 3 , Maria Caterina Rossi 2, 3 , Jean Luc Villeval 4 , Francesco Annunziato 2, 3 , Paola Guglielmelli 1, 2, 3 and Alessandro M. Vannucchi 1, 2, 3 1 CRIMM, Centro di Ricerca e Innovazione per le Malattie Mieloproliferative, Azienda Ospedaliera Universitaria Careggi, Florence, Italy 2 Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 3 DENOTHE Excellence Center, Florence, Italy 4 INSERM, Unite Mixte de Recherche (UMR) 1170, Institut Gustave Roussy, Villejuif, France Correspondence to: Alessandro M. Vannucchi, email: amvannucchi@unifi.it Keywords: STAT5, phosphorylation, MPN, ruxolitinib, PI3K/mTOR inhibitors Received: February 28, 2017 Accepted: May 10, 2017 Published: May 22, 2017 ABSTRACT Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in JAK2V617F mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in JAK2V617F mutated cell lines, primary patients’ cells, and JAK2V617F knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials.

Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. Results PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. Conclusions These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

BACKGROUND RUX, a JAK 1/2 inhibitor, is a second-line treatment for symptom management and control of trilineage expansion in patients (pts) with PV who are resistant or intolerant to hydroxyurea (HU). In the RESPONSE study, RUX showed a superior and durable response in controlling hematocrit (HCT) and improving splenomegaly in PV pts resistant or intolerant to HU. RESPONSE-2 (NCT02038036) is a multicenter, open-label, Phase 3 trial comparing RUX with best available therapy (BAT) in HU-resistant/intolerant PV pts without splenomegaly. The primary analysis at Week (Wk) 28 and follow-ups at Wks 80 and 156 proved superiority of RUX over BAT. Here we present the final study results of the 5-year follow-up (Wk 260). METHODS Pts were randomized (1:1) to RUX 10 mg twice daily or BAT; crossover from BAT to RUX was allowed from Wk 28. The final analysis at Wk 260 evaluated durability of HCT control, complete hematologic response (CHR), and safety. Durability of HCT control was defined as absence of phlebotomy eligibility from Wk 8 to Wk 260 with ≤1 phlebotomy eligibility post-randomization to Wk 8. CHR was defined as HCT control with a white blood cell count RESULTS Pts were randomized to RUX (n=74) or BAT (n=75). A total of 59/74 RUX pts and 61/75 BAT pts completed the treatment duration. Among the 75 BAT pts, 58 pts crossed over to RUX, with 38 of them completing the study, allowing for long-term follow-up of 97 pts treated with RUX. Median (range) exposure was 260.0 (0.1 - 273.1) wks in RUX, 28.4 (6.7 - 83.0) wks in BAT, and 224.7 (2.7 - 236.1) wks in crossover arms. At Wk 260, 21.6% of RUX pts (16/74) achieved durable HCT control. The Kaplan-Meier (KM) -estimated median duration of HCT control was not reached (Fig. A). Durable CHR was achieved in 24.3% of RUX pts (18/74; estimated median duration, 34.0 wks). Crossover pts derived RUX benefit by achieving HCT control with HCT levels decreasing over time (median HCT was 46.2% at the crossover baseline and 38.9% at the end of treatment). JAK2 V617F allele burden reduced over time for pts treated with RUX from baseline with a median decrease of 14.8% in RUX (59 evaluable pts) and 13.5% in crossover arms (42 evaluable pts). RUX showed durable improvement in PV-associated symptoms; about 45% of RUX pts continued to achieve ≥50% reduction in MPN-SAF TSS at Wk 260. RUX pts also continued to experience improvements in all 5 dimensions of the EQ-5D-5L assessment. Total number of phlebotomies was 60 in the RUX arm (at Wk 260) vs 106 for BAT (Wk 80). At Wk 260, RUX- (vs BAT-) treated pts required >0 − ≤2 phlebotomies in 16.2% (vs 38.7%), >2 − ≤4 in 9.5% (vs 21.3%), >4 − ≤6 in 5.4% vs (2.7%), and >6 − ≤8 in none (vs 1.3%). The most common AEs (>5%, exposure-adjusted rate) were anemia, arthralgia, and increased weight in the RUX arm and anemia, and hypertension in pts after crossover. Of note, exposure-adjusted rates of herpes zoster were 3.9 in both RUX (Grade 3/4, 0.9) and crossover pts (no Grade 3/4) and none in the BAT arm. The exposure-adjusted rate of thromboembolic events was higher with BAT than RUX (3.7 vs 1.5). As expected given prior HU exposure, non-melanoma skin cancer was the most common secondary malignancy in RUX (exposure-adjusted rates: randomized, 2.7; crossover, 2.9) and BAT-treated pts (1.9). No RUX pts developed AML, whereas myelofibrosis was reported in 2 pts (exposure-adjusted rate, 0.6%). On-treatment death was reported in 5 pts: 1 each in the RUX arm (metastatic melanoma) and BAT arm (septic shock), and 3 in crossover (1 pt each - cardiac disorder, general health deterioration, and PV progression). The KM-estimated median overall survival was not reached in both RUX (95.8%; 95% CI: 87.4, 98.6) and BAT arms (90.7%; 95% CI: 80.3, 95.7; Fig. B) at ≥260 wks. Transformation-free survival (KM-estimated, 95% CI) in the RUX arm was 94.18% (85.23, 97.78) at ≥260 wks. CONCLUSIONS In line with the RESPONSE study outcomes, final analysis from the 5-yr follow-up of RESPONSE 2 proved superiority of RUX over BAT with a safety profile that remained consistent with previous reports. RUX provided durable HCT control with a decrease in the need for phlebotomies, CHR, improved PV-associated symptoms and PRO scores, with fewer thromboembolic events in PV pts without splenomegaly, who are HU resistant or intolerant. Disclosures Palandri: Novartis: Consultancy, Honoraria. Callum:Canadian Blood Services: Research Funding; Octapharma Canada: Research Funding. Devos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Vannucchi: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; Blueprint: Membership on an entity's Board of Directors or advisory committees; 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. Zor:Novartis: Current Employment. Gilotti:Novartis: Current Employment. Zhang:Novartis: Current Employment. Griesshammer:Celgene: Honoraria, Speakers Bureau; AOP Orphan: Honoraria, Speakers Bureau; CTI: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

BACKGROUND RUX, a potent oral JAK1/JAK2 inhibitor, has shown superiority over standard therapies in MF. RUX was approved for the treatment of MF on the basis of the phase 3 COMFORT trials, in which RUX led to sustained improvements in splenomegaly and symptom burden as well as longer survival compared with placebo (COMFORT-I) and best available therapy (COMFORT-II). The starting dose (prescribing information) is based on PLT count: > 200 × 109/L, 20 mg bid; 100-200 × 109/L, 15 mg bid. Due to limited data available at the time, the recommended starting dose in pts with PLT counts of 50 to < 100 × 109/L was determined to be 5 mg bid. However, findings from COMFORT-I and Study 258 (phase 2 study of RUX in pts with MF with low PLT count) demonstrated that final titrated doses of ≥ 10 mg bid were safe and resulted in larger improvements in spleen volume and MF-related symptoms compared with titrated doses of ≤ 5 mg bid. The EXPAND study established a maximum safe starting dose (MSSD) in pts with low baseline PLT count (50 to < 100 × 109/L) and evaluated the safety and tolerability of RUX in this population. The 24-wk (Vannucchi AM, et al. ASH 2015 #2817) and 48-wk (Vannucchi AM, et al. Haematologica. 2019) analyses determined the MSSD to be 10 mg bid for pts with baseline PLT count of 50 to < 100 × 109/L. We present the final results from the study, confirming 10 mg bid as a safe starting dose for pts with low PLT count. METHODS EXPAND is a phase 1b, dose-finding study (NCT01317875) in pts with MF and baseline PLT count of 50 to < 100 × 109/L (Stratum [S] 1, 75 to < 100 × 109/L; S2, 50 to < 75 × 109/L). The study had a core period (up to wk 24) consisting of 2 phases (dose escalation and safety expansion) followed by an extension period (up to 3 years total). The primary objective was determination of the MSSD for both strata; safety and efficacy were secondary objectives. RESULTS Of 69 enrolled pts, 38 received RUX at the MSSD of 10 mg bid (S1, n = 20; S2, n = 18) and are the focus of this analysis. Baseline characteristics were indicative of advanced disease in both strata. Overall, 50% of pts in S1 and 83% of pts in S2 discontinued study treatment. Primary reasons for discontinuation were progressive disease (15%), adverse events (AEs; 10%), and other (10%) in S1 and AEs (33%), death (17%), and physician decision (17%) in S2. Median (range) duration of exposure was 155 (4-210) wk in S1 and 83 (4-161) wk in S2; 17/20 pts (85%) in S1 and 11/18 (61%) in S2 received RUX for ≥ 48 wk. AEs were consistent with the known safety profile of RUX (Table). The most common AEs were thrombocytopenia (S1, 50%; S2, 78%) and anemia (S1, 55%; S2, 44%); other common (> 30% all grade) AEs included diarrhea, ecchymosis, PLT decrease, and pyrexia (each 30%) in S1, and cough (33%) in S2. A total of 85% of pts in S1 and 89% in S2 had grade ≥ 3 AEs; thrombocytopenia (S1, 40%; S2, 78%) and anemia (25%; 17%) were most common. Overall, 20% and 50% of pts in S1 and S2 had AEs leading to study drug discontinuation, most commonly thrombocytopenia (S1, 5%; S2, 20%); other AEs led to discontinuation in 1 pt each. On-treatment deaths included 1 cardiac arrest (S1), 1 AML (S1), 1 multiorgan failure (S2), and 1 sepsis (S2). The death due to cardiac arrest was assessed as related to study drug; the others were unrelated. Overall, 40% (6/15) and 38% (3/8) of evaluable pts in S1 and S2 achieved a spleen response (≥ 50% reduction in spleen length from baseline) at wk 24; 33% (5/15) and 30% (3/10) of evaluable pts in S1 and S2 achieved a spleen response at wk 48; 55% (11/20) and 67% (12/18) of pts achieved a response at any time. A symptom response (≥ 50% reduction in MF-SAF TSS from baseline to wk 24) was achieved by 31% (4/13) of evaluable pts in S1 and 40% (4/10) in S2; at wk 24, there was a mean (SD) decrease (improvement) in total daily score from baseline by 7.7 (9.7) in S1 and 3.9 (11.4) in S2. There was no significant difference in the PK of RUX among pts with low PLT counts in this study compared with that in pts with PLT ≥ 100 × 109/L. CONCLUSIONS A starting dose of RUX 10 mg bid was manageable in this previously unstudied MF pt population with low PLT counts (50 to < 75 × 109/L and 75 to < 100 × 109/L). AEs were consistent with the known safety profile of RUX, with no new or unexpected adverse findings. This was supported by the PK/PD results as well. RUX treatment at a starting dose of 10 mg bid provided the benefit of spleen length reductions and clinical symptom improvements. EXPAND results confirm that a starting dose of RUX 10 mg bid is suitable for pts with MF and low PLT counts. Disclosures 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. Vannucchi:Incyte: 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; Novartis: 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; Blueprint: Membership on an entity's Board of Directors or advisory committees. He:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; LongBio Pharma: Consultancy, Research Funding. Verstovsek:ItalPharma: Research Funding; Protagonist Therapeutics: Research Funding; AstraZeneca: Research Funding; PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; CTI Biopharma Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Incyte Corporation: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Roche: Research Funding; NS Pharma: Research Funding. Boyer-Perrard:Novartis (RUMYCUP study only): Consultancy. Niederwieser:Novartis: Speakers Bureau; Amgen: Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi: Research Funding. Liberati:Janssen: Honoraria, Research Funding; Onconova: Research Funding; Verastem: Research Funding; Oncopeptides: Research Funding; Incyte: Honoraria; GSK: Research Funding; Novartis: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Research Funding. Harrison:AOP Orphan Pharmaceuticals: Honoraria; Roche: Honoraria; Promedior: Honoraria; Incyte Corporation: Speakers Bureau; Janssen: Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; Sierra Oncology: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau. Roussou:Novartis: Current Employment, Other: I have Novartis shares. Wroclawska:Novartis Pharma AG: Current Employment. Majidi:Novartis: Current Employment. Gisslinger:Janssen-Cilag: Honoraria; Roche: Honoraria; Celgene: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MyeloPro Diagnostics and Research: Honoraria; PharmaEssentia: Honoraria. OffLabel Disclosure: Label dose in this patient population is 5 mg bid starting dose. EXPAND presents data for a 10 mg bid starting dose.

Patients with Philadelphia-negative myeloproliferative neoplasm (MPN) are prone to the development of second cancers, but the factors associated with these events have been poorly explored. In an international nested case-control study, we recruited 647 patients with carcinoma, nonmelanoma skin cancer, hematological second cancer, and melanoma diagnosed concurrently or after MPN diagnosis. Up to 3 control patients without a history of cancer and matched with each case for center, sex, age at MPN diagnosis, date of diagnosis, and MPN disease duration were included (n = 1234). Cases were comparable to controls for MPN type, driver mutations and cardiovascular risk factors. The frequency of thrombosis preceding MPN was similar for cases and controls (P = .462). Thrombotic events after MPN and before second cancer were higher in cases than in controls (11.6% vs 8.1%; P = .013), because of a higher proportion of arterial thromboses (6.2% vs 3.7%; P = .015). After adjustment for confounders, the occurrence of arterial thrombosis remained independently associated with the risk of carcinoma (odds ratio, 1.97; 95% confidence interval, 1.14-3.41), suggesting that MPN patients experiencing arterial events after MPN diagnosis deserve careful clinical surveillance for early detection of carcinoma. This study was registered at www.clinicaltrials.gov as NCT03745378.

Introduction Malignancy can be heralded by unprovoked venous thromboembolism (VTE) but also by arterial thrombosis. To date it is unknown whether this association is present also in myeloproliferative neoplasms (MPN), in which arterial thrombosis is more frequent that venous thrombosis and solid tumors are reported with an increased frequency. The MPN-K nested case-control study addressed the impact of cytoreductive drugs on the risk of developing second cancer in MPN patients (Barbui T et al, Leukemia 2019); here we re-examined the study database to evaluate the frequency and type of vascular complications in MPN patients with second cancer excluding leukemia and to establish whether arterial and venous thrombosis during follow-up after diagnosis of MPN could predict the occurrence of a second cancer. Patients and methods Cases were patients with second cancer diagnosed concurrently or subsequent to the diagnosis of MPN. Controls were MPN patients without second cancer. For each case with second cancer, up to 3 cancer-free controls were matched by each center for sex, age at MPN diagnosis, date of MPN diagnosis, and MPN disease duration. Each set consisting of one case and their matched-controls had a similar observational period (from MPN diagnosis until the index date of diagnosis for the second cancer). The study included 647 cases with second cancer (carcinoma, non-melanoma-skin cancers, hematological secondary cancer and melanoma). The most frequent category was carcinoma (n=426, 65.8%). Cases were comparable with the 1,234 matched controls for demographics, type of MPN, and exposure to potential confounders such as mutational profile, abnormal karyotype and cardiovascular risk factors. The thrombotic events of interest were ischemic stroke, transient ischemic attacks, acute coronary syndromes, peripheral arterial thrombosis, deep venous thrombosis (including thrombosis of cerebral and splanchnic veins) and pulmonary embolism. Thrombosis had to be concurrent with or in the 2 years before MPN diagnosis or occurring after MPN diagnosis. The cumulative incidence of either arterial or venous thrombosis from MPN diagnosis was estimated by the Kaplan-Meier method and was compared between cases and controls using the log-rank test. A conditional logistic regression model estimated the Odds Ratio (OR) with 95% Confidence Interval (CI) of second cancer associated with the occurrence of thrombosis before/at diagnosis of MPN and during follow-up. Other covariates were patient age, cardiovascular risk factors, the JAK2V617F mutation, and treatment during follow-up. Results Approximately 20% of either MPN cases or controls had thrombosis before MPN or at diagnosis (19.8% vs. 21.1%, respectively, p=0.462). After a median observation time from diagnosis of MPN to an index date of 4.5 years (interquartile range 1.5-8.2) in cases and 3.7 years (interquartile range 1.5-7.5) in controls, cases showed a percentage of thrombosis higher than in controls (75/647, 11.6% vs. 100/1234, 8.1%, respectively, p=0.013). Approximately one-third of thrombosis preceding cancer occurred in the 12 months before the diagnosis of second cancer (22/75, 29.3%). The excess of thrombosis in cases was due to a higher frequency of arterial thrombosis (6.2% vs. 3.7%, p=0.015), whereas no significant difference was found for venous thrombosis (5.4% vs. 4.3%, p=0.277). While the cumulative incidence of venous thrombosis over time was similar among cases and controls (p=0.864), the cumulative incidence of arterial thrombosis was higher in cases with second cancer (p=0.006) (Figure 1). The excess of arterial thrombosis after MPN diagnosis was limited to cases with carcinoma (6.8% vs 3.9%, p=0.027). In a multivariable model, arterial thrombosis during the follow-up was confirmed to be an independent predictor factor for carcinoma, with an odds ratio of 1.97 (95%CI 1.14-3.41, p=0.015). Conclusions. These findings reveal an association of arterial thrombosis with subsequent second cancer (namely carcinoma) in MPN patients. A possible biological plausibility for this link may be related to an underlying common pathogenic mechanism such as an aberrant inflammatory response consistently found in MPN. This observation may have practical implications and suggests careful clinical surveillance for early diagnosis of second cancer in MPN patients with arterial thrombosis during the follow-up. Disclosures Palandri: Novartis: Consultancy, Honoraria. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Bonifacio:Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Rumi:novartis: Honoraria, Research Funding. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Benevolo:Novartis Pharmaceuticals: Consultancy. McMullin:Italopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Daiko Sanyo: Membership on an entity's Board of Directors or advisory committees. Griesshammer:Novartis: Consultancy, Honoraria, Speakers Bureau. Vannucchi:CTI: 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; Italfarmaco: 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 Corporation: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Skoda et al. provide new insights into the collaboration between epigenetic regulator Ezh2 and a key hematopoietic tyrosine kinase in disease initiation and progression., Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2. In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2−/− mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2+/− mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F–expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

In this issue of Blood , Newberry et al provide evidence that clonal evolution in patients with myelofibrosis (MF) who are receiving therapy with ruxolitinib correlates with discontinuation of treatment, mainly because of loss of response or progression, and also predicts shorter survival thereafter. 1

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.

Annals of hematology 96(10), 1653-1665 (2017). doi:10.1007/s00277-017-3082-y, Published by Springer, Berlin ; Heidelberg ; New York

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR-494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494-3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients.

Introduction: Polycythemia vera (PV) is a myeloproliferative neoplasm for which no curative treatment currently exists. However, the discovery of abnormally activated JAK/STAT signaling pushed the clinical development of JAK1/JAK2 inhibitors and other target drugs, including inhibitors of histone deacetylases (HDACi). Areas covered: We reviewed preclinical studies and clinical trials with givinostat, a Class I and II HDACi, through PubMed research and communications to the meeting of the most important hematology associations. Expert opinion: Proof of evidence has been obtained with givinostat earlier in JAK2V617F mutated cells and then in two clinical trials in patients with PV. Treatment resulted effective in inducing hematological response, symptomatic improvement and reduction of enlarged spleen, with good tolerability. Further clinical development is required to more clearly define the role of Givinostat in the therapeutic armamentarium of PV and exploit the full activity of the drug against the myel...

CALR (calreticulin) trails JAK2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type-1, 52-bp deletion (p.L367fs*46), and type-2, 5-bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test (n = 402) and validation (n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK2, 11 (3%) Myeloproliferative leukemia virus oncogene (MPL), and 114 (28%) CALR mutations; 12% were wild-type for all three mutations (i.e., triple-negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type-1 and 44 (39%) type-2 variants; compared to mutant JAK2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type-1 (P = 0.005) and younger age with type-2 (P = 0.001) variants. Notably, platelet count was significantly higher in type-2 vs. type-1 CALR-mutated patients (P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR-mutated ET patients (P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild-type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis.

Background: Patients (pts) with myelofibrosis (MF) suffer from a wide range of possibly debilitating disease manifestations (e.g. splenomegaly, weight loss, fatigue, night sweats, pain etc) resulting in severe symptom burden and quality of life decrement. Compared to INT-2/HIGH IPSS risk categories, lower risk pts display a less severe disease burden at diagnosis; nonetheless, with no active therapy, disease severity increases over time in all risk categories (Mesa et al, Leuk Res 2013). ROMEI (CINC424AIT04-Ruxolitinib Observational study in Myelofibrosis treated patiEnts in Italy) is a prospective observational study focused on real-life management of MF pts treated with ruxolitinib (RUX). The prospective assessment of the Myeloproliferative Neoplasm 10 (MPN 10) total score over time and the EQ-5D-5L and its related general health EQ-VAS values over time were included as primary endpoints and changes in spleen length were instead included as secondary endpoint. Aims: To analyze the extent lower-risk MF pts treated with RUX may differentiate from higher-risk pts treated with RUX in terms of disease characteristics at treatment start, toxicity and response. A sub-analysis of the ROMEI trial was performed to evaluate differences in disease burden (i.e symptoms according to MPN-10, general health status according to EQ-VAS and spleen length) at RUX start and its modification after 24 weeks (wks) of treatment between pts with different BL IPSS category. Methods: Out of 215 enrolled pts, 183 (96 male) were evaluable for this sub-analysis: 34 INT-1, 89 INT-2 and 60 HIGH risk pts based on IPSS. BL (BL) pts characteristics are shown in table 1. Pts started RUX according to Summary of Product Characteristics; MPN-10 scores and EQ-VAS values were collected at BL and at wks 4, 8, 12 and 24 after treatment start; spleen length by manual palpation was assessed at the same time points. Differences in IPSS subgroups were compared by means of analysis of variance (ANOVA) applied on rank transformed data in case of non-normal distribution. The BL value was included in the models on scoring changes. Results: MPN-10. In the overall cohort, we observed an improvement of MPN-10 score upon RUX start from 34.86±17.90 (range 0-88) at BL to 21.55±15.25 (range 0-70) at wk 24. No statistically differences were observed on MPN-10 total score between IPSS risk categories both at BL (P=0.83) and at wk 24 (P=0.53). Furthermore, the improvement in pts' symptoms evaluated by means of change of MPN-10 total score between wk 24 and BL resulted similar between the different IPSS risk categories (P=0.87). EQ-VAS. In the overall group, EQ VAS values increased in mean from 59.88±20.23 (min; max: 0; 100) at BL to 67.36±18.57 (min; max: 10; 100) at wk 24. No differences between IPSS risk category were observed at BL (P=0.23, table 1).After 24 wks, EQ VAS was, in mean, 73.70±16.74 in INT-1 risk, 67.78±18.59 in INT-2 risk and 62.45±18.93 in HIGH risk with statistically differences between IPSS risk category (P=0.02) and, in particular, between HIGH and INT-1 risk (P=0.02). Difference between IPSS risk category was observed also when considering the change of EQ VAS between wk 24 and BL (P=0.02): the improvement in pts' health status resulted statistically lower in pts with HIGH compared to INT-1 (P=0.01) while no difference between INT-2 and INT-1 risk was observed (P=0.07). Spleen length. Among evaluable pts, spleen length at BL seemed to increase as the IPSS risk category increased: 7.84±5.64 cm in INT-1 risk, 8.40±5.08 cm in INT-2 risk and 9.17±6.24 cm in HIGH, but no statistical difference was found between the groups (P=0.57). At week 24 mean spleen length decreased in all subgroups: 4,53±5.99 cm in INT-1 risk, 3.90±4.29 cm in INT-2 risk and 5.39±6.52 cm in HIGH risk but again with no statistical difference between groups (P= 0.77). Furthermore, the change of palpable spleen length between wk 24 and BL resulted similar between IPSS risk categories (P=0.55). Conclusion: Our sub analysis suggests that disease burden can be very disabling even in INT-1 pts and comparably as in higher-risk categories. We also demonstrate that the EQ-VAS questionnaire may detect and quantify a wide range of symptoms not adequately included in IPSS risk stratification. Despite improvements in symptoms and splenomegaly were comparable across age groups, general health status improvement was significantly better in INT-1 pts. Further analysis will be available at the meeting. Disclosures Palandri: Novartis: Consultancy, Honoraria. Breccia:Novartis: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Celgene: Honoraria; Incyte: Honoraria. Palumbo:Teva: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Hospira: Honoraria. Abruzzese:BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Liberati:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Roche: Other: Clinical trial support; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Celgene: Honoraria, Other: Clinical trial support; Bristol-Myers Squibb: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Clinical trial support; Janssen: Honoraria. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Tiribelli:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Foà:Celltrion: 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; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Caocci:Novartis: Honoraria; Celgene: Honoraria. Morelli:Novartis: Employment. Passamonti:Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau.

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

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms that can progress to post-PV (PPV) myelofibrosis (MF) and post-ET (PET) MF, also known as secondary myelofibrosis (SMF). The 2009 International Working Group for Myelofibrosis Research and Treatment group (IWG-MRT) diagnostic criteria for SMF require the presence of grade 2 or 3 bone marrow fibrosis (BMF) according to the European classification. The prognostic value of BMF in primary MF has been recently explored. Aim of this study is to describe the relevance of BMF grading in terms of presentation and outcome in SMF. Methods: The MYSEC (Myelofibrosis Secondary to PV and ET Collaboration) project is an international retrospective collaboration that collected 805 SMF cases. BMF grade at time of SMF evolution was defined as grade 2 (BMF2) in case of a diffuse increase in reticulin with extensive intersections, occasional bundles of collagen and/or osteosclerosis, or as grade 3 (BMF3) if presenting with coarse bundles of collagen, often associated with osteosclerosis. Chi-square or Fisher exact test for categorical variables, Wilcoxon rank-sum test for continuous ones and multiple logistic regression analysis were applied in order to explore pattern of association between BMF grading and patients' characteristics. A Poisson regression model was applied to estimate incidence rate of events, together with 95% Confidence Interval (CI). Survival was estimated using the Kaplan-Meier method. Differences between BMF groups were compared by a Log-rank test. The MYSEC study was approved by the Review Board of each Institution and conducted in accordance with the Declaration of Helsinki. Results: Detailed information about BMF grade was available in 675 patients: BMF2 was reported in 443 (65.6%) and BMF3 in 232 (34.4%). We did not find any correlation between BMF grade and advanced age, marrow blasts percentage, spleen and liver size, presence of constitutional symptoms, abnormal karyotype at the time of SMF diagnosis. No imbalance was evident for gender and for time to progression from PV/ET to SMF. Looking at genotype, BMF grading was not differently distributed among the 3 phenotypic driver mutations as well as the "triple negative" cases. On the contrary, in univariate analysis we found a significant association between BMF3 and previous diagnosis of ET vs. PV, decreased hemoglobin levels, reduced platelet and leukocyte counts, higher percentage of circulating blast cells and higher LDH value (Table 1). In a multivariate analysis that took into consideration SMF subtypes, complete blood count and circulating blast cells in 437 SMF patients, PET-MF, lower hemoglobin levels and reduced platelet count maintained their association with BMF3 (Table 2). The cumulative incidence of thrombosis after SMF was 3.2% (CI 95%: 2.4-4.2) person-year of follow up (PYFU) in BMF2, equivalent to BMF3 (2.7% PYFU, CI 95%: 1.2-5.7) (P = 0.44). Besides, BMF grade did not impact on the cumulative incidence of post-SMF blast phase, which resulted 2.4% (CI 95%: 1.8-3.3) PYFU in case of BMF2 and 3% (CI 95%: 1.3-6.9) PYFU in the BMF3 cohort (P = 0.68). Overall, MYSEC-PM (MYSEC-Prognostic Model) risk categories were differently distributed between BMF grades: patients with BMF2 were more frequently included in lower risk groups, while those with BMF3 were enriched in the higher categories (Table 1). Finally, we found a significantly higher mortality in patients with BMF3 vs. BMF2, mainly due to SMF progression (Table 1). Figure 1 shows the Kaplan-Meier estimate of survival in the 2 populations, resulting significantly different. Nevertheless, when considering the MYSEC-PM score in a multivariate analysis, BMF grade lost its prognostic impact on survival. Conclusions: This study provides evidence that BMF grade does correlate with specific clinical phenotype in SMF. BMF3 was associated with a more "cytopenic" phenotype (lower hemoglobin levels and reduced platelet count) and clustered with higher MYSEC-PM scores. Survival is significantly reduced in patients with BMF3. In conclusion, irrespectively of the PV/ET duration, progressive anemia and thrombocytopenia imply more frequently BMF3, finally resulting in a worse survival. This highlights the need of performing bone marrow biopsy earlier in disease evolution. Disclosures Rumi: novartis: Honoraria, Research Funding. Rambaldi:Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau. Komrokji:Incyte: Consultancy; pfizer: Consultancy; JAZZ: Speakers Bureau; celgene: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; Agios: Consultancy; DSI: Consultancy. Gotlib:Deceiphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Promedior: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Celgene: Consultancy. Cervantes:Novartis: Honoraria, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Palandri:Novartis: Consultancy, Honoraria. Silver:PharmEssentia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Benevolo:Novartis Pharmaceuticals: Consultancy. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. 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. Passamonti:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Background: Non-adherence to medications is recognized as one of the most important and costly worldwide healthcare problems in the 21st century; according to an EU report, non-adherence to therapies is responsible for 194,500 deaths and costs €125 billion annually. Taking into account those data, the use of adherence measurements in clinical trials could be extremely useful, in order to better understand patients (pts) behaviours and outcomes. The 8-item Morisky Medication Adherence Scale (MMAS-8, Morisky DE et al, J Clin Hypertens,2008 - Krousel-Wood MA et al, Am J Manag Care 2009- Morisky DE et al, J Clin Epidemiol. 2011) is a widely used questionnaire to asses indirectly pts adherence to medications. The first seven items are yes/no questions, and the last item is a five point Likert-scale. Ruxolitinib (RUX) is an oral JAK1/2 inhibitor approved for the treatment of symptoms and/or splenomegaly in myelofibrosis (MF) pts. ROMEI (CINC424AIT04-Ruxolitinib Observational study in Myelofibrosis treated patients in Italy) is a prospective observational study focused on real life management of MF pts with RUX; the trial enrolled 215 pts and included a prospective evaluation of MMAS-8 as a secondary endpoint. To date, no data regarding MMAS-8 and RUX are available and here we show a descriptive analysis of MMAS-8 after 24 weeks (wks) of RUX treatment. Methods: After informed consent signature, pts started RUX according to Summary of Product Characteristics; MMAS-8 questionnaire was administered at wks 4, 8, 12 and 24 after RUX initiation. MMAS-8 were analysed only if pts provided all responses to the questionnaire; a score ˂ 6 indicates low adherence, a score between 6 and 8 indicates medium adherence, and a score = 8 suggests high adherence. As per protocol, only adherence classes (low-medium-high) assessment was scheduled and in case of low or medium adherence (ie suboptimal adherence), no action in term of target education or psychological interventions was planned. Results: Out of 215 enrolled pts, 188 were evaluable for MMAS-8. The MMAS-8 was completed by 163 pts (87%) at week (wk) 4, 156 pts (83%) at wk 8, 146 pts (78%) at wk 12 and 134 pts (71%) at wk 24. A total of 101 out of 188 eligible pts (54%) completed all 4 scheduled questionnaires. MMAS-8 total score seems to remain stable over the time. Mean MMAS-8 total score was 7.54±0.77 (min; max: 4.75; 8.00) at wk 4 and 7.67±0.70 (min; max: 4.25; 8.00) at wk 24, resulting in a mean change from wk 4 of 0.14±0.79 (min; max: -2.25; 2.25). Regarding adherence classes, the distribution between low, medium and high adherence over time is shown in table 1. Likewise, if we consider pts who completed all scheduled questionnaires, MMSA-8 total score remains stable over the wks, with total score of 7.53±0.79 (min; max: 4.75; 8.00) at wk 4, 7.60±0.71 (min; max: 4.75; 8.00) at wk 8, 7.69±0.69 (min; max 4.50; 8.00) at wk 12 and 7.67±0.73 (min; max: 4.25; 8.00) at wk 24, as well as adherence classes distribution (Fig 1). Regarding adherence classes in this subgroup of pts, seems that pts with high adherence at wk 4 are more prone to maintain the same class during time. Finally, we tried to perform an exploratory analysis relating spleen response according to IWG-MRT criteria with adherence classes at each time points: considering the small number of evaluable pts with complete information regarding both available parameters, no correlation between spleen responses and adherence classes was demonstrated (data available at the meeting). Conclusion: This is the first time of MMAS-8 applied in MF pts as well as those treated with RUX. Our analysis showed a not irrelevant percentage of pts (ranging from 25% to 40%) belonging to a low-intermediate adherence classes during treatment. Surprisingly overall approximately one third of patients could be exposed to a suboptimal treatment over time and probably clinicians underestimate the number of those pts in clinical practice. Although we did not find in our cohort a correlation between adherence classes and spleen responses, probably due to low number of evaluable pts for both variables, the knowledge regarding pts attitude to therapy, especially for long-term and chronic treatments, remains crucial from clinical and pharmacoeconomic perspectives. Standardized methods to assess pts adherence such as MMAS-8 should be implemented in prospective well-designed clinical trials and could be a precious tool guiding clinicians in the everyday practice. Disclosures Palandri: Novartis: Consultancy, Honoraria. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Palumbo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morelli:Novartis: Employment. Passamonti:Celgene Corporation: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Breccia:Bristol-Myers Squibb, Celgene, Incyte, Novartis, Pfizer: Honoraria.

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.

Background. MPN are clonal disorders of hematopoietic stem cell (HSC) that include polycythemia vera (PV), essential thrombocytemia (ET) and myelofibrosis (MF), either secondary to ET-PV, or primary (PMF), including pre-fibrotic (pre-PMF) and overt. In MF, multi-lineage dysplasia (MLD) of varying degree can be detected by morphologic analysis of bone marrow (BM), but appraisal of MLD is intrinsically operator-dependent and lacks quantitative information. Multi-parameter flow cytometry (MFC) is used to study dysplasia by investigating aberrant antigen expression profiles during hematopoietic cells' differentiation and maturation, allowing: i) analysis of a much greater cell number; ii) standardization of data by referring to controls; iii) definition of scores to estimate MLD extent. Aim. The aim of the study was to assess MLD in MPN by MFC and correlate with disease' variant, clinical and biological features. Methods. Patients: pts were diagnosed according to 2016 WHO criteria. Driver and high molecular risk mutations (HMR) were determined. MFC: Different BM cell compartments were analyzed with a panel of 28 monoclonal antibodies (MoAb) by 2 acquisition steps: Step 1) HSC and progenitor (GMP and MEP) cells up to >5 x 106 cells/sample were studied by Lin/CD34/CD38/CD90/CD45/CD49f/CD45RA/CD135 MoAb. Step 2) Precursor and mature compartments (B-, neutrophilic, monocytic, erythroid, plasmacytoid dendritic cells, basophils) were identified within CD34+ and/or CD117+ subset. Uncommitted CD38+ precursors were obtained by subtraction. CD117-neg, maturing cells were identified by specific features, ie CD34, CD45 and scatter. MLD was assessed by 2 phenotypic scores: i) one validated by ELN in MDS (Ogata score) includes 4 parameters; ii) the second one, adapted from Matarraz et al (2010; Salamanca score), 63 parameters. Normal phenotypic profile was defined as the interval between mean value (M) ± 2SD per parameter in healthy donors' BM samples. Each parameter scored 0.5, 1 or 2 for values between M ± 2 SD and M ± 3 SD, M ± 3 SD and M ± 4 SD, and over M ± 4 SD of the normal profile, respectively. Results. 51 consecutive MPN pts at diagnosis were enrolled, including 30 MF (7 pre-PMF, 14 overt PMF, 9 post-PV/ET MF); 13 ET, 5 PV, 1 MPN-unclassifiable and 2 MDS/MPN. Median age was 59y (range 24-83). 19 (63.3%) MF patients had JAK2V617F, 9 (30.0%) CALR, 2 (6.7%) MPLW515L mutation. 11 pts (36.7%) harbored at least 1 HMR mutation. According to MIPSS, 6 (20.0%), 11 (36.7%) and 10 (33.3%) were low, intermediate and high risk. By comparing MF pts to controls and ET-PV, altered proportion of immature cell subsets was observed. Specifically, HSC and uncommitted progenitors in MF were increased of 3.8 and 1.5-fold, respectively whereas GMP and downstream precursors were significantly reduced 2 to >6-fold. No significant differences emerged comparing mature compartments, except for an increase of basophils and a decrease of pDC in MF subset (Table 1). When considering MLD, MF patients ranked higher scores for both the Ogata and Salamanca score. Furthermore, Salamanca score values had a trend to correlate with MIPSS risk categories, with median value of 7.75, 13.25 and 15.5 in low, intermediate and high category (KW test P=0.06). MF cases were separated according to Salamanca score ≥ or < to the median value of 13.5: pts with higher score were featured by significant differences in hemoglobin (11.9 vs 13.7 g/dL; P=.015), platelet count (330 vs 558 x109/L; P=.004), LDH (380 vs 275 U/L; P=.034), circulating CD34+ cells (60.4 vs 5.3/mL; P=.007). We also compared prePMF and ET pts, two categories with challenging differential diagnosis: prePMF displayed expansion of HSC (0.015) compared to ET (0.0056; P=0.028) and reduced proportions of downstream precursors, similar to what observed in MF vs non-MF comparison. Conversely, MLD scores were similar: Ogata 1.0 vs 1.0, Salamanca 9.0 vs 7.5 for ET and prePMF, respectively. Conclusions. The assessment of MLD by MFC correlated with MPN variants according to WHO; MF was associated with higher deviation from normal phenotypic profile. MLD scores delineated clinical phenotype and were consistent with MIPSS risk stratification. With the limits of number of pts analyzed, these preliminary data also support a biologic continuum of pre-PMF and MF, the first being characterized by abnormal expansion of immature cell compartments compared to ET but minimal signs of dysplastic maturation unlike overt MF. Disclosures Vannucchi: Novartis: 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; 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.