Myeloproliferative Neoplasms Pts Research Articles

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.

Anxieties, age and motivation influence physical activity in patients with myeloproliferative neoplasms - a multicenter survey from the East German study group for hematology and oncology (OSHO #97).

Physical activity (PA) is a non-pharmacological approach to alleviate symptom burden and improve health-related quality of life (HrQoL) in cancer patients (pts). Whether pts with myeloproliferative neoplasms (MPN) PA behavior changes due to symptom burden and/or knowledge of the putative beneficial effects of PA has not yet been investigated. We performed a large questionnaire study in MPN pts. Self-reported PA behavior and potential influencing factors of 634 MPN pts were analyzed. Questionnaires were used to assess demographics, anxiety, severity of symptoms, HrQoL, current level of everyday and sports activities, and the level of information regarding the importance/possibilities of PA. According to their PA, the pts were assigned to the three groups: "inactive", "non-targeted active", and "sporty active" and compared with each other. Key findings are that in 73% of the pts, the disease had an impact on PA, with 30% of pts reducing their PA. The prevalence of anxieties (e.g., occurrence of thrombosis and bleeding) regarding PA was 45%. Sporty active pts had a lower symptom burden and better HrQoL (p ≤ 0.001) compared to the other groups. Inactive pts were significantly older and had a higher body mass index than sporty active pts. Inactive and non-targeted active pts felt less informed about the importance/possibilities of PA (p = 0.002). Our results suggest that especially older and non-sporty MPN pts could benefit from motivational as well as disease-specific PA information. This study was registered at the German Registry of Clinical Trials, DRKS00023698.

Protection Against Breakthrough Delta/Omicron Variants in Vaccinated Patients with Myeloproliferative Neoplasms (MPN)

Background Information on breakthrough SARS-CoV-2 infections in MPN vaccinated patients (pts) is very limited. In these diseases, effectiveness of coronavirus disease 2019 (Covid-19) vaccines may be influenced not only by the MPN phenotype and the intrinsic reduced immunological competence, but also by the prior Covid-19 infection that can elicit a natural immunity against reinfections. Patients In the European LeukemiaNet (ELN) observational registry, 863 MPN pts with Covid-19 have been enrolled since February 2020 and in 649 of them, information on the vaccination status is now available. To ensure that well characterized reinfections were considered, diagnosis of breakthrough Covid-19 included a positive naso-pharyngeal swab and symptoms highly suggestive for SARS CoV-2 infection. ResultsBreakthrough infections in vaccinated patients with previous Covid-19 infection The effectiveness of previous Covid-19 infection in preventing reinfections was tested in a group of 131 and 287 unvaccinated and vaccinated MPN pts, respectively. Prior Covid-19 occurred during the first and second wave in 74% and 98% pts, respectively and the interval between Covid-19 and the breakthrough infection in unvaccinated and in vaccinated was similar (8.5 months). Administered vaccine doses (Pfizer in 71%) were 1-2 and 3-4 in 77% and 23% of vaccinated cases, respectively. The proportion of PV, ET, pre-PMF was similar in the two groups while the number of MF pts was lower (19%) than in the unvaccinated group (28%) (p=0.024). Reinfections occurred during delta (n=4) or omicron variants period (n=14) in 8 (6.1%) and 10 (3.5%) unvaccinated and vaccinated pts (p=ns) and hospitalization was required in 0 and 2 pts, respectively. Severity of the acute infection was variable and 1 death was registered.Breakthrough infections in vaccinated patients without previous Covid-19 infection Breakthrough infections were reported in 231 vaccinated cases (ET=89, PV=75, MF=54 and pre-PMF=13) without prior Covid-19. In 51% of pts the administered vaccine doses were 1-2 and 3-4 in 49%. No relevant vaccine related side effects were registered. Time interval from vaccination to Covid-19 was 8.1 months. Twenty-six pts (11%) required hospitalization and 205 (89%) were managed at home. Hospitalized pts were older (median age 76), males (69%) with MF (39%), and prior exposure to ruxolitinib (42%, 7 MF and 4 PV). Of note, values of C-reactive protein (CRP) and neutrophil/lymphocyte ratio (NLR) were significantly more elevated in hospitalized cases (CRP=33.1 vs. 2.0 and NLR=5.9 vs. 3.3, p<0.001). Although some infections occurred in the second wave, corresponding to alpha/beta/gamma coronavirus infection (6%), the majority of breakthrough episodes occurred in delta and omicron variant periods (41% and 53%). Three deaths were registered in hospitalized pts. We explored the risk of hospitalization and evaluated the marginal effect of gender, age and ruxolitinib exposure in a logistic model fitted to predict this event. We found that the risk of hospitalization was substantially higher only in males on ruxolitinib and increased with age (Figure). We also found that the inflammatory status measured with NLR could explain these results (data not shown). Conclusion The effectiveness of previous Covid-19 in preventing reinfection occurring during the delta and omicron variants of SARS-CoV-2 was robust (94% and 97% in unvaccinated and vaccinated pts, respectively). This is good news for MPN patients who recovered from Covid-19 and the hope is that this effectiveness can work against any future SARS-CoV-2 variant. In the cohort of vaccinated pts without prior Covid-19, we identified a subgroup of patients with more severe delta and omicron disease, at high risk of hospitalization, consisting of males who were on ruxolitinib and had elevated inflammatory biomarkers. Subsequent studies are needed to interpret these latest results. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1066: OUTCOMES OF COVID-19 IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS - AN OBSERVATIONAL COHORT STUDY FROM A LARGE ACADEMIC CANCER CENTER IN THE UNITED STATES

Background: Infections are one of the main causes of morbidity and mortality in patients with Myeloproliferative Neoplasms (MPN). Several studies from all over the world has reported on outcomes of the SARS-CoV-2 infection (COVID-19) pandemic in MPN pts but there is no robust data on characterizing the clinical outcomes of patients with MPN and COVID-19 in the United States (US). Aims: Therefore, we aimed to investigate the effect of COVID-19 on MPN patients’ clinical outcomes including the effect of MPN-subtypes, and SARS-CoV-2 vaccination on COVID-19 infection. Methods: We conducted a single center, retrospective, observational cohort study. Between April 2020 – December 2021, data was obtained from 388 patients flagged in the electronic health records system with MPN and COVID-19, of which 53 MPN patients with positive SARS-CoV-2 test were identified. COVID-19 infection was ascertained by a positive real-time reverse transcriptase polymerase chain reaction from nasal swab. Presenting clinical information was abstracted from review of unstructured notes as well as structured data from the foundry. Demographic and baseline parameters were summarized. The Kaplan-Meier method was utilized to compare the median time from date of diagnosis to date of last follow up or death. Results: Among 53 patients with MPN and COVID-19 infection, the median age was 58 yrs (range,21-83) with equal distribution between men and women of which 46% had a body mass index ≥30. Most common comorbidities included hypertension (60%), and diabetes (19%). 17 (33%) patients required hospitalization of which all (33%) of them required oxygen supplementation. 12 pts (23%) had radiological evidence of pneumonia of which 4 pts (8%) required high flow oxygen and 3 pts (6%) required mechanical ventilation. 2 pts had new thrombosis of portal vein and coronary artery each. 10 pts (19%) received remdesivir and 4 (8%) received tocilizumab, and 9 pts (17%) received steroids. At a median follow up of 11.8 months, 7 pts died at a median of 24 days after COVID-19 diagnosis and the case fatality rate was 13%. Except for 1 pt,all the other 6 pts who died had BMI≥30. In the US, COVID-19 predominantly occurred in pts with myelofibrosis (56%), polycythemia vera (29%), and essential thrombocythemia (15%). 54% received MPN directed therapy of which 17 (33%) pts received ruxolitinib, and 11 pts (21%) received hydroxyurea. At the time of infection, median WBC was 6.2 x 109/µL (range,0.3-16.3),Hb was 11.6 gm/dL (range,6.8-15.8),platelet count was 178 x 109/µL (range,2-766), and the median neutrophil to lymphocyte ratio was 4.1. In this cohort, 22 pts (42%) were infected prior to the availability of vaccination. 42% of the entire cohort has received vaccination of which 45% pts were infected after first dose of vaccination. Among the 7 deaths, 6 patients had underlying myelofibrosis, and one pt had polycythemia vera, and all deaths occurred in unvaccinated population except for one death that occurred post vaccination. Hospitalization rate was higher in the unvaccinated (33%) pts than those who were vaccinated (22%). Summary/Conclusion: To date this is the largest cohort of MPN patients with COVID-19 infection in the US accounting for 14% of the MPN patients with COVID-19 in our center. BMI≥30 appear to be an important risk factor for COVID-19 infection and mortality in MPN pts. SARSCOV2-vaccination appear to decrease the risk of mortality and hospitalization. Patterns of inflammatory markers, and long term follow up of recovered MPN pts with COVID-19 will be presented in the meeting.

Spleen and Liver Fibrosis Is Associated to Treatment Response and Prognosis in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Spleen and Liver Fibrosis Is Associated to Treatment Response and Prognosis in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Excess Mortality in Young Patients with Myeloproliferative Neoplasms

▪Introduction: Myeloproliferative neoplasms (MPNs) - essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) - are associated with significant morbidity and shortened survival, irrespective of age (Srour et al. Br J Hematol. 2016). Despite shortened survival, young MPN patients (pts) are often classified as “low-risk” and managed with aspirin alone, phlebotomy-only (PHL), or observation instead of potentially life-prolonging cytoreductive and/or disease-modifying treatment. Age-biased risk assessment and treatment practices deprive young pts from early intervention and access to clinical trials. Our goal is to highlight the unmet need for identifying and implementing life-prolonging treatment in young MPN pts. Here, we determine and compare the excess mortality from MPNs in young (<60 years) to older pts (≥60 years) in the United States (US).Methods: In this population-based study, demographic (age, sex, race), survival, and cause-of-death data were collected for MPN pts from the US Survey, Epidemiology, and End Results (SEER) registry between 2001-2017; and for the general US population from the Centers for Disease Control and Prevention Wide-ranging OnLine Data for Epidemiologic Research (CDC WONDER) database. SEER reports cancers classified by International Classification of Diseases for Oncology, version 3 (ICD-O-3), which evolved with the WHO classification of myeloid neoplasms in 2001 for ET, PV and PMF. Cause-of-death is reported by ICD-10 codes, which we categorized into cardiovascular (CVE), second cancers (CA), acute leukemia (AL), and others. Excess, or relative mortality was determined by the ratio of observed mortality in MPN pts (Kaplan-Meier estimate) over the expected mortality of the US population matched by age, sex, race, and birth-year (actuarial estimate). A one-sided log-rank test was used. Excess mortality of young compared to older pts was determined from a random-effects model using restricted maximum-likelihood estimator method. All analyses were performed using RStudio software v 1.4.1106.Results: The SEER query identified 40,333 MPN pts (ET: 17,420 (43%), PV: 18,027 (45%), PMF: 4,886 (12%)) diagnosed at a median age of 66 years (ET: 67, PV: 65, PMF: 69) of whom 20,470 (51%) were females (ET: 10,709 (61%), PV: 7,768 (43%), PMF: 1,993 (41%)). The 10-year mortality for ET, PV, and PMF pts <60 was 13%, 18%, and 49% respectively (versus 6% for controls); and ≥60 was 59%, 59%, and 87% respectively (versus 35% for controls) (Figure 1A). Excess all-cause mortality was higher in pts <60 compared to ≥60 with ET (2.75 versus 1.82, p<0.001), PV (3.16 versus 1.92, p<0.001), and PMF (10.6 versus 5.73, p<0.001) (Figure 1B). Excess CVE mortality was higher in PV<60 compared to PV≥60 (3.36 versus 2.55, p=0.036) but not in ET or PMF. Excess CA mortality was higher in PV<60 than PV≥60 (2.36 versus 1.53, p=0.002) but not in ET or PMF. Excess AL mortality was high for all pts, particularly PMF, and was not significantly different in pts <60 compared to ≥60Discussion: Excess or relative mortality emphasizes the burden of MPN-related death with current treatment. In this population-based study, we found excess mortality in young MPN pts (<60), greater than in older pts (≥60). This difference may result from age-biased risk assessment and treatment recommendations that recommend lower intensity therapy for young pts. It may be time to revisit these recommendations since accruing evidence demonstrates long-term safety, efficacy and potential survival benefit of available cytoreductive agents. Certain treatments such as interferon-alpha (IFN) may improve survival in PV (Abu-Zeinah et al. Leukemia. 2021), and a recent randomized clinical trial established superiority of IFN over standard of care PHL in young PV pts (Barbui et al. Lancet Hematol. 2021). These data advocate for early intervention with potentially disease-modifying treatment for all pts regardless of age. These data also call into question age-biased exclusions of many young pts from participation in clinical trials of established or promising drugs.Conclusion: Excess mortality of young MPN pts (<60) is unacceptably high and greater than that of older pts (≥60). Clinical trials testing early intervention with potentially life-prolonging treatments are required. [Display omitted] DisclosuresSilver: Abbvie: Consultancy; PharamEssentia: Consultancy, Speakers Bureau. Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Abu-Zeinah: PharmaEssentia: Consultancy.

Hematopoietic Stem and Progenitor Cell Fitness As a Novel Prognostic and Monitoring Biomarker for JAK2 V617F Myeloproliferative Neoplasms (MPNs)

Hematopoietic Stem and Progenitor Cell Fitness As a Novel Prognostic and Monitoring Biomarker for JAK2 V617F Myeloproliferative Neoplasms (MPNs)

Single Cell Mutation Analysis Delineates Clonal Architecture in Leukemic Transformation of Myeloproliferative Neoplasms

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, p<.0001). Epigenetic variants (i.e. ASXL1, TET2, EZH2) account for around half of the mutations and affect a large fraction of CP cells (3 representative samples in Fig.1). In 8/10 pts, leukemic clone emerged from a driver mutation-positive cells (JAK2V617F n=4; CALR Type1 n=4). In all pts we are able to identify at least 3 mutated clones and in 7 pts the dynamics of the clones allowed to identify the one(s) responsible for evolution to sAML. In 7/10 pts, the leukemic clones were already detectable at low frequency (<2%) at CP and became dominant in BP; these low-frequency clones were missed by bulk sequencing. Furthermore, SCS revealed acquisition of 3 mutually exclusive mutations in RAS pathway in one pt: two NRAS mutations and a KRAS mutation. Copy number variation (CNV) could be assessed in 4 pts. Of note, during progression to sAML, we found single cells with amplification of ETV6 (>20 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 [Display omitted] DisclosuresVannucchi: 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.

The Impact of Somatic Mutations upon the Response to Combination Therapy with Ruxolitinib and Interferon in MPN Patients

Introduction: The Philadelphia-negative myeloproliferative neoplasms (MPN) are associated with driver mutations in JAK2, CALR, and MPL genes. Non-driver mutations affect disease progression and treatment response. Combination therapy with pegylated interferon-alpha2 (IFN) and ruxolitinib has recently been shown to induce hematologic and molecular responses in patients (pts) with MPN. We studied 25 pts by targeted next generation sequencing (NGS) of 42 genes and investigated the impact of somatic mutations (mut) on treatment response.Methods: Twenty-five MPN pts with polycythemia vera (PV) (n=16, all JAK2V617F) and myelofibrosis (MF) (n=9, JAK2V617F (6), CALR (1), MPL (1), triple-negative (1)) participated in the study. NGS was performed on the Illumina platform on DNA from peripheral blood at baseline and after 24 months of combination therapy. Data were analyzed using Biomedical Genomics workbench and VarSeq. Variants with coverage <100x, variant allele frequency (VAF) <1%, introns and germline variants, and SNPs with minor allele frequency >1% were excluded. A mut with VAF <1% in either a pre- or post-treatment sample was retained if VAF was >1% in the paired sample. However, a mut with a number of mutated reads below 7 was considered absent. Only pathogenic mut were reported. Statistical analysis was done using either chi-square test or Wilcoxon signed rank/rank sum test in R 4.0.2. A p-value <0.05 was considered significant. The ELN and IWG-MRT response criteria were used.Results: At baseline and 24 months, PV-pts had a mean number of mut of 2.3 and 2.4, and MF-pts 2.2 and 3.0, respectively. In all pts, 0 and 1 (0/4%) patient had no mut, 10 and 7 (40/28%) had 1 mut, 10 and 9 (40/36%) had 2 mut, and 5 and 8 (20/32%) had ≥ 3 mut at baseline and 24 months, respectively.In PV, 1 (6%) achieved CR and 3 (19%) achieved PR and 12 (75%) achieved NR. In MF, 4 (44%) achieved CR and 1 (11%) achieved PR and 4 either NR, PD, SD or CI. There was no association between pts achieving CR/PR or NR and median number of mut at baseline (CR/PR: 1, range 1-3) or (NR: 2, range 1-8), (p=0.28). Excluding driver mut, there was still no significant association.In all JAK2V617F positive pts, the median JAK2V617F allele burden (%JAK2V617F) decreased from 37% (range: 1.9-95) at baseline to 22.5% (range: 0-85) at 24 months. In pts achieving CR or PR (n=7), the reduction in %JAK2V617F was greater (median: 42% to 18%, p<0.05) compared with pts achieving NR (n=12) (median: 30% to 22%, p<0.005).Stratified according to molecular response (MR) (n=4) or non-MR (n=13), median % JAK2V617F at baseline was 40 (21-95) and 43 (42-70) in non-MR and MR, respectively, and 31 (11-85) and 13 (1.8-25) in non-MR and MR, respectively during treatment. At baseline, pts achieving MR had no non-driver mut and pts achieving non-MR had a median number of non-driver mut of 1 (range: 0-7) (p<0.02). Eight pts were not evaluable because they were either CALR or MPL positive, triple-negative, or had a baseline JAK2V617F ≤ 20%.In all pts, there were 30 non-driver mut in 15 genes at baseline and 40 non-driver mut in 19 genes at 24 months of therapy. At baseline and at 24 months, TP53 occurred in 6 and 6 (24%), TET2, ASXL1, RUNX1, or SF3B1 in 2 and 3 (8/12%), and CBL, DNMT3A, or SRSF2 in 2 and 2 (8%) pts, respectively. Eleven new non-driver post-treatment mut occurred in 9 pts and were more prevalent in pts with MF (6/9, 66%) compared to pts with PV (3/16, 19%), p<0.02. However, the VAF was low (median 1.2%, range 1.02% - 3.6%) and none of the new 11 post-treatment mut appeared in the same gene.Discussion and Conclusions: Earlier studies have shown an association between poor response to IFN and number of mut or high molecular risk mut. In this study, the presence of non-driver mut was associated with a significantly poorer molecular response. However, there was no association between the presence or type of non-driver mut and clinico-hematologic response. At 24 months, there was an evolution of subclones with a significantly higher number of new subclones appearing in pts with MF compared to pts with PV. However, the VAF was low and none of the acquired mut appeared in the same gene contradictory to another study showing an association between treatment-emergent mut in DNMT3A and treatment with IFN. In conclusion, these data show that a clinico-hematologic response is achievable during combination therapy despite the presence of non-driver mutations at baseline. DisclosuresBjørn: Novartis: Other: Ruxolitnib. Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board. OffLabel Disclosure:Interferon-alpha for treatment

Pulmonary Hypertension Independently Shortens Survival of Patients with Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are frequently associated with symptoms of fatigue, poor sleep, mood disorders, lightheadedness and shortness of breath. The cause of these symptoms is not well understood and is often attributed to blood count abnormalities or elevated inflammatory cytokines common in these disorders. There is a high degree of overlap between clinical manifestations of MPN and symptoms reported by pts with pulmonary hypertension (PH). Small studies have shown that pts with MPNs are at risk for PH but the extent to which PH contributes to clinical symptoms and prognosis in MPN pts is unknown.Methods: 300 sequential MPN pts undergoing transthoracic echocardiography (echo) between January, 2011 and April, 2017 were studied (Table 1). A second MPN cohort of 287 patients (NoEcho) was matched for age and diagnosis to the MPN Echo+ (Echo) cohort. Baseline demographics including age, gender, diagnosis, date of diagnosis, mutation profile, transformation, BMT, bleeding or thrombosis history, symptoms, cardiovascular risk factors, echo indication and survival were collected for all pts. To ensure accurate echo assessment of PH sequelae, RV assessment via both tricuspid annular plane systolic excursion (TAPSE) and peak tricuspid annular systolic velocity (S‘) were required for study inclusion. RV dysfunction (RVDYS) was defined by impairment of both TAPSE and S’. RV global longitudinal strain (GLS) was quantified as a secondary means of assessing RV function as a continuous variable. Pulmonary artery systolic pressure (PASP) was calculated based on tricuspid regurgitant velocity and inferior vena cava caliber; PH (PASP³35 mmHg) and advanced PH (PASP³50 mmHg) were defined via established criteria. LV systolic and diastolic function, geometry, and mass were quantified based on linear dimensions in parasternal long axis, consistent with quantitative methods previously validated. Echoes were interpreted in a high-volume laboratory, for which expertise and reproducibility have been documented. All cause mortality was assessed following echo.Results: PH was identified in the majority (56%; 135/239) of MPN pts who underwent echo (Table 2). PH was common among all MPN subtypes (46% CML, 58% ET, 57% PV, 63% MF, 100% CEL, 42% MPN/MDS and 79% MPN-NOS). Advanced PH was identified in 20% of all MPN pts and within each subtype (15% CML, 13% ET, 16% PV, 29% MF, 33% CEL, 25% MPN/MDS and 43% MPN-NOS). RVDYS was 8-fold more common (24% vs 3%; p=<0.001) among MPN pts with PH and the magnitude of RVDYS increased stepwise in relation to PASP (p<0.001), suggesting that PH was causally related to RV failure. Although impaired LV systolic function (LVEF<50%) was no more common in MPN pts with PH (17% vs 10%; p=0.10), LV hypertrophy (38% vs 14%, p=<0.001), LV mass (99 ± 31 vs 83 ± 23 g/m2, p<0.001), LV filling pressure (E/e': 14±6 vs 11±4; p<0.001), and frequency of LV diastolic dysfunction (any grade: 71 vs 52%; p=0.003) were all increased in pts with PH. Left atrium (LA) area was also increased in MPN pts with PH (24 vs 19 cm2; p=0.001), further suggesting a post-capillary cause for PH in this MPN population. Myocardial biopsy of an MPN patient with advanced PH demonstrated interstitial fibrosis in non-infarcted myocardium suggesting that fibrosis could play a role in LV diastolic dysfunction and PH in pts with MPNs.During median follow-up of 6.2 yrs (0.1 to 43 yrs), likelihood of death was higher for pts with advanced PH (odds ratio 3.1, p=0.004), or with RVDYS whether quantified by GLS (odds ratio 2.4, p=0.04) or TAPSE/S' (odds ratio 2.3, p=0.048). Cox Proportional Hazards model (parameterized for age, gender, MPN diagnosis, transformation and BMT) indicated that the Echo cohort was similar to the NoEcho control group. Pts with PH were more likely to experience fatigue than pts with a normal estimated PASP. Whereas dyspnea was no more common in pts with PH as a whole, pts with advanced PH were more likely to experience both fatigue and dyspnea compared to those with less severe PH or normal PASP. Cox proportional hazards modeling identified Advanced PH as an independent predictor of death in models that included Age, Gender, MPN Diagnosis, Transformation and BMT.Conclusions : PH with post-capillary hemodynamics is extremely common in pts with MPN and is associated with frequently reported symptoms such as fatigue. When advanced or when associated with RVDYS, PH independently confers increased mortality risk for MPN pts. [Display omitted] DisclosuresRitchie:Bristol-Myers Squibb: Other: Research funding to my institution; Astellas Pharma: Other: Research funding to my institution; Celgene: Consultancy, Other: Travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau.

Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.

Advanced Forms of the Myeloproliferative Neoplasms Are Associated with Chromosomal Abnormalities Involving 1q and 12q: Implicating MDM2 and MDM4 in Disease Progression

Inevitably patients (pts) with myeloproliferative neoplasms (MPNs) including polycythemia vera (PV) and essential thrombocythemia (ET) progress to more advanced forms of myelofibrosis (MF) and acute myeloid leukemia (AML). Loss of TP53 function plays a critical role in cancer biology yet, the MPNs are characterized predominately by wild type (WT) TP53 (Rampal et al, PNAS, 2014;111: 5401-10). The major protein regulators of TP53 are MDM2 and MDM 4. TP53 activity has been reported to be down-regulated in MPNs due to overexpression of MDM2 (Lu et al, Blood, 2012;120:3098-3105, Nakatake et al, Oncogene, 2012: 311: 323-33). Since the gene for MDM4 is located on chromosome 1q and the gene for MDM2 is located on chromosome 12q, we hypothesized that cytogenetic abnormalities involving these locations might lead to disease progression This hypothesis is supported by the recent observation that mice engineered to possess JAK2 V617F and lack TP53 are prone to develop AML as compared to JAK2 V617F+ mice with WT TP53 (Rampal et al, PNAS, 2014:111:5401-10). Gain of 1q (+1q) in the form of jumping translocations in pts with MPN was previously reported to be associated with transformation to AML (Najfeld et al, Br.J. Haematol., 2010; 151: 285-291).These studies were extended to 362 MPN pts enrolled in 5 different MPN-RC clinical trials involving pts with ET or PV (222), MF and MPN -Blast Phase (MPN-BP)(140) and 989 pts with MPNs, ET(177), PV (400), MF (385) and MPN-BP(27) who were studied at the Icahn School of Medicine at Mount Sinai (ISMMS) between 1986 and June 2017. 92 pts in the various MPN-RC trials (25.4%) and 344 (34.7%) of the pts from ISMMS were chromosomally abnormal. Among the abnormalities, 23 pts in the MPN-RC trials had either +1q/dup(1q)(n=16) or rearrangements of 12q (n=7) (6.3% of the total pts and 25.0% of the chromosomally abnormal pts). Similarly, the most frequently observed chromosomal abnormality at ISMMS associated with disease progression, either as a sole abnormality or as a subclone with other chromosomal abnormalities, was gain of 1q, identified in 59 pts (6.0% of total, 17.4% of the abnormal pts). Gain of 1q was observed in three forms: 1) as an unbalanced translocation with the breakpoint at 1q21. 2) a duplication of 1q region and/or 3) as a jumping +1q translocation. The most frequent unbalanced translocation identified was +der(9)t(1;9)(q21;q12) (24%) resulting in three copies of 1q and 3 copies of 9p. Rearrangement of 12q most frequently involved balanced translocations and/or inv(12q) with 50% of these pts having a breakpoint at 12q15 where the gene for MDM2 is located. Gain of 1q/dup(1q) and rearrangements of 12q were mutually exclusive with the exception of one patient with a translocation between chromosomes 1 and 12 [der(12)t(1;12)(q24;q15)]. Among PV/ET pts enrolled in MPN-RC trials, 1.3% (3/222) had +1q and 0.0% (0/222) had 12q abnormalities, while 11% (15/140) of pts enrolled in MF and MPN-BP treatment protocols had +1q and 5% (7/140) had 12q abnormalities. These findings indicate that +1q and structural abnormalities of 12q are associated with more advanced forms of MF and MPN-BP (p<0.001). In both, the MPN-RC and the ISMMS data bases, 42 cases with +1q and 13 cases with 12q abnormalities were identified where the driver mutational status was known. The JAK2 V617F mutation was observed in 83.3% (35/42)of pts with +1q while 23.1% (3/13) pts with 12q abnormalities were JAK2 V617F+.These data indicate that gain of 1q but not 12q abnormalities are associated with the JAK2 V617F mutation (p<0.001). In a limited number of pts (n=10) we were able to monitor the chronological acquisition of 1q and 12q abnormalities. The 1q abnormalities occurred 5-7 years following the initial cytogenetic evaluation of PV pts and the proportion of cells with gain of 1q increased over time being present in 100% of cells over the next 4-5 years of observation. By contrast, 12q abnormalities occurred exclusively in MF pts as the sole abnormality and appeared in 90-100% of the examined cells at presentation. In 5 MF pts with +1q, qPCR demonstrated upregulation of MDM 4 mRNA transcripts (86%) when compared to 5 pts without +1q. These data suggest that MPN disease progression is associated with specific chromosomal abnormalities that result in up regulation of MDM2 or MDM4 which further down-regulate p53 .These data provide the rationale for the use of MDM2/MDM4 antagonists as a means of halting MPN disease progression. DisclosuresMascarenhas:CTI Biopharma: Research Funding; Novartis: Other: DSMB member , Research Funding; Merck: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding; Promedior: Research Funding; Janssen: Research Funding.

Involvement of RUNX1 Pathway Is a Common Event in the Leukemic Transformation of Chronic Myeloproliferative Neoplasms (MPNs)

Introduction. Runt-related transcription factor 1 (RUNX1) is a transcription factor critical for normal and leukemic hematopoiesis. Three main isoforms, RUNX1 i-1a, i-1b and i-1c, are produced through alternative splicing of RUNX1. Unlike i-1b and i-1c, i-1a has a Runt homology domain (RHD) but lacks the transactivation domain (TAD), and inhibits transcriptional activity of -1b by competing with higher affinity for target gene sequences. Overexpression of i-1a in cell lines inhibits terminal myeloid differentiation. The i-1a is expressed at high levels in patients (pts) with acute myeloid leukemia (AML). Mutations of RUNX1 are common in CMML and MDS, whereas incidence and clinical correlates of RUNX1 mutations in MPNs are poorly known. Aim: To analyze the spectrum and clinical correlates of RUNX1 genetic abnormalities (RUNX1-abn), and the expression of RUNX-1a isoform, in a large series of pts with MPNs. Methods. 686 MPN pts (50 PV, 165 ET, 154 overt-PMF, 143 prefibrotic-PMF, 74 PPV-MF and 100 PET-MF, diagnosed according to 2016 WHO) were included in the study; of these, 625 were analyzed in chronic phase (CP) and 61 at leukemic transformation (LT; of a total of 106 LTs, 58%), including 29 cases with paired samples at diagnosis and LT. Established methods were used for JAK2, MPL and CALR genotyping. A high molecular risk (HMR) category was defined according to Vannucchi A, [Leukemia 2013]. Ion Torrent NGS platform was used to genotype all RUNX1 coding sequence. In 21/29 paired samples, a long reads genome sequencing by Oxford Nanopore technology (ONT) was performed. Copy number variation (CNV) analysis was performed with NanoGLADIATOR package [Magi A, Genome Biol 2019]. Differential expression of RUNX1 mRNA isoform1a and 1b was analyzed by qRT-PCR. Nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimates and log-rank test were used as appropriate. Results. Somatic RUNX1 mutations were identified in 31 pts (4.5%): 14/625 in CP (2.2%) and 17/61 in LT (27.9%; P&lt;.0001); 7/17 LT pts (41.1%) had two variants. 10/29 paired samples were RUNX1 mutated, in 8 of which mutations were acquired at LT. All mutations were heterozygous (median VAF 40%, range 0.4%-54%); 13 (34.2%) were frameshift, 5 (13.2%) nonsense and 20 (52.6%) missense. Most mutations clustered in RHD (26/38, 68.4%) and TAD (9/38, 23.7%). CNV involving RUNX1 was detected by ONT in 4/21 (19%) paired samples, including 2 gain and 2 loss, all acquired at LT; 2 pts had concurrent RUNX1 mutation. Overall, RUNX1abn, inclusive of mutations and CNV, occurred in 19/61 (31.1%) patients at LT. RUNX1-abn were more common in MPL (12.5%) and TN (7.5%) pts compared with JAK2 (4.3%) and CALR (2.0%) mutated (P=0.04), and were enriched in HMR category (61.3% vs 29.4% vs pts without RUNX1-abn; P=.001). We also analyzed CNV in a total of 47 selected genes included in the RUNX1 pathway (KEGG Orthology K08367); we found CNV in &gt;1 of these genes in 16/21 (76%) pts at LT vs 2/16 (12.5%) of CP pts. After median follow-up of 4.9y (range 0.1-36.2y) 273 pts died (39.8%). 106 pts (15.4%) progressed to AML after median of 4.7y (0.1-36.2y) from diagnosis. Incidence of LT differed significantly among diseases: 3 pts in PV (5.9%), 17 in ET (10.1%), 39 in overt-PMF (23.5%), 12 in prefibrotic-PMF (8.3%), 11 in PPV-MF (14.1%) and 24 in PET-MF (22.6%). RUNX1-abn were detected, respectively, in 100%, 53.0%, 23.1%, 41.7%, 36.4% and 12.5% of cases at either CP and/or LT. Leukemia-free survival (LFS) was shorter in pts harboring RUNX1-abn in CP compared to RUNX1-WT pt (2.8y vs NR; P&lt;.0001)(Fig 1A). Expression levels of i-1a and -1b were measured in 34 CP and 24 LT pts. Isoform-1a was significantly elevated in LT pts (mean 7.51±10.1) compared to CP (1.12±1.75; P=.009) (Fig 1B; lines represent the i-1a level rise from CP to LT in 4 paired samples). The i-1a expression levels were irrespective of RUNX1 mutated (mean 7.6±11.9) or unmutated (mean 9.2±6.3) status in LT pts (P=0.8). No difference of i-1b levels in CP (mean 0.9±0.7) versus LP (mean 1.6±1.4; P=0.08) was observed. The expression of i-1a was not dependent on gender, age, leukocyte or platelet count, hemoglobin and percentage of blast cells. Conclusions. These findings indicate that most MPN pts with LT have involvement of RUNX1 pathway through different mechanisms (mutations, CNV, altered isoform expression, genomic abnormalities of RUNX1 target genes), suggesting that a "RUNX1pathy" may represent a common event during leukemogenesis in MPNs. Figure 1 Disclosures Vannucchi: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; 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: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Exon-Level Genomic Abnormalities Are Frequent in Cytogenetically Normal Patients with Philadelphia-Chromosome Negative Myeloproliferative Neoplasms (MPNs)

Polycythemia vera (PV) and essential thrombocythemia (ET) exhibit an abnormal karyotype in 10%-28% of patients (pts) while the frequency increases in up to 90% of pts with advanced forms of myelofibrosis (PMF) and MPN accelerated/blast phase. Similarly, Klampfl et al. (Blood,118(1) 167-176) demonstrated that an increase in the number of genomic abnormalities detected by chromosomal microarrays (CMA) is associated with disease progression. Although genomic aberrations in cytogenetically normal pts with MPN have been previously reported, this study focused on identifying cryptic genomic alterations with exon-level precision to genomically characterize pts with MPN that were karyotypically normal at the time of CMA analysis. We prospectively evaluated a cohort of 151 cytogenetically normal pts (23% PV, 35% ET, 21% PMF, and 21% post PV/ET MF) using a high-resolution chromosomal microarray (CMA) platform that detects both regions of copy-neutral loss of heterozygosity (cnLOH) and copy number alterations (CNAs) with exon-level resolution (Agilent's cancer-specific 2x400k aCGH+SNP). In order to uncover regions harboring genes that may have biological and/or clinical implications, we identified the minimal overlapping regions (MORs) containing CMA abnormalities occurring in at least 4 pts, using the "GRanges" package (Bioconductor). Genomic aberrations were documented in 69% (n=104) of pts. Of the 238 CMA abnormalities identified, 52 were gains, 38 were losses, and 148 were regions of cnLOH. Next-generation sequencing (NGS) was performed in 97% of these pts and 65% had JAK2, 15% had CALR, and 2% had MPL mutations. The remaining pts (n=25) were triple-negative, and 12 pts harbored other non-driver gene mutations. Only CMA lesions were identified in 6 pts (4%) and both NGS and CMA could not genomically detect abnormalities in 7 pts (5%). The most frequent MOR mapping to a single gene harbored JAK2 cnLOH which occurred in 25% of pts, consistent with previous reports. A statistical difference in the JAK2V617F variant allele frequency (VAF) in pts with 9p cnLOH (VAF=80%) and without 9p cnLOH (VAF=18%) (p&lt;0.001) was observed. In pts with PV, a high JAK2 allele burden is associated with an aggressive phenotype (Vannucchi et al. Therapeutic Advances in Hematology 2010 2, 21-32). Alterations of 1q32.1 involving MDM4, a negative regulator of TP53, was the second most frequent MOR occurring in 14% of pts. We have recently demonstrated that gain of 1q results in overexpression of MDM4 and is associated with advanced forms of MPN (Marcellino et al, Blood Advances, 2018 2(24), 3581-3589). Four other recurrent MORs harboring a single gene, TAF15, RASA3, RHOC, and NCOR2 occurred in 8%, 5%, 4%, and 3% of pts, respectively. Each had intragenic gains. We previously reported alterations in these genes in the spleens of pts with MF (Zimran et al., Haematologica, 2018 (10)103). Our current results indicate that these same alterations are present in the bone marrow cells of cytogenetically normal MPN pts. Since these abnormalities are present in pts with advanced forms of MPN, alterations of these genes may represent markers for disease progression. Of the MORs harboring multiple genes, 3 regions involved 1p, 17q, and 18q in 4%, 3%, and 6% of pts, respectively. All pts had cnLOH of these regions and each harbored relevant genes that may be implicated in MPN; CDKN2C (1p), PPM1D (17q), and ASXL3(18q). Our results demonstrated that CMA abnormalities were present in 51% of pts with ET, 66% with PV, 87% with PMF, and 70%-100% with post ET/PV MF. CMA lesions were the sole alteration in 4% while 5% of chromosomally normal pts with an initial diagnosis of ET had neither somatic mutations nor CMA abnormalities. We provided evidence that recurrent genomic alterations can be identified in regions associated with disease progression, as well as novel regions harboring genes with potential pathobiological implications. We conclude that CMA is a vital tool in identifying cryptic genomic aberrations in cytogenetically normal pts with chronic phase MPN. Disclosures Hoffman: Merus: Research Funding. Ozbek:Viracor: Patents &amp; Royalties: Biomarker Patent. Mascarenhas:Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding; Promedior: Research Funding; Janssen: Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding. Kremyanskaya:La Jolla: Consultancy; Incyte, Celgene, Constellation, Protagonist.: Research Funding.

Real World Data on Obstetric (OC) and Maternal Complications (MC) Occurring in a Cohort of Patients with Ph Negative Myeloproliferative Neoplasms (MPN): Argentinian Multicentric Study

Introduction: Up to 20% of patients (pts) with essential thrombocythemia (ET) and up to 10-15% of pts with polycythemia vera (PV) are diagnosed before the age of 40 years and the percentage is even lower in pts with primary myelofibrosis (PMF). Pregnancy (Pcy) in MPN is associated with an increased incidence of thrombotic and bleeding events and obstetric complications with live birth rates of 50% to 70%. There are no evidence-based guidelines for the management of these pts and most of the data come from case series of few pts. There are no validated variables that can predict outcomes in pregnant women with MPN. Objetives: Primary: To learn the incidence of thromboembolic, hemorrhagic and OC in pregnant women with MPN in Argentina. Secondary: To evaluate parameters such as mutational status, platelet count, history of complications in previous Pcy, use of aspirin (ASA), low molecular weight heparin (LMWH) or interferon (IFN) and their relationship with obstetric outcomes. Materials and methods: Retrospective evaluation of medical records of pts with diagnosis of MPN and Pcy. Quantitative variables were expressed as median and interquartile range (IQR) and qualitative variables as total number and percentage (%). Fisher's exact test was used to analyze variables and their association with events and Wilcoxon test for platelet counts. Results: A total of 30 Pcy in 23 women with a diagnosis of MPN were recorded, 20 Pcy in pts with ET, 4 Pcy in pts with PV and 6 MF pregnancies. Pcy was planned in 56.6% of cases. There were OC in 15/19 previous Pcy. The median age was 32.9 years (IC25-75% 30-36 years). Mutational status was assessed in 22 pts: 9/22 JAK2 +, 7/22 CALR +, 1/22 triple-negative, 1/22 JAK2-CALR- and MPL not performed, 6/22 JAK2- CALR and MPL without data. The overall number of live births and first trimester spontaneous abortions (SA) were 26(86%) and 4(13.3%) respectively, without cases of second (2T) and third trimester (3T) fetal loss or stillbirths. There was a case of neonatal death in a patient with MF. We detected 13 obstetric events (ET: 9/20, PV: 2/4, and MF 2/6): 4 SA, 5 cases of fetal growth retardation, 1 partial placental abruption and 3 placental hematomas. There was no significant association between JAK2 mutational state, history of complications in previous Pcy, platelet count, use of ASA, LMWH or IFN and obstetric outcomes. Two of the pts who had SA presented smoking as a cardiovascular risk factor. There was heterogeneity among hematologist regarding therapeutic management: ASA was indicated in 24 Pcy and LMWH in prophylactic doses during Pcy in 15. In 21/25 cases LMWH was indicated as postpartum prophylaxis, only in 3/21 it was extended for 6 weeks(wks). Before Pcy 17 cases received cytoreductive treatment: 2 with HU and 1 with anagrelide that were discontinued at the time Pcy was confirmed, 5 with peg IFN, and 9 conventional IFN. During Pcy 8 continued with cytoreductive treatment (6/9 conventional IFN, 2/9, pegIFN, including 1 patient previously receiving HU) and 5 initiated IFN (1/5 conventional, 4/5 pegIFN). Platelet counts in pts who did not require cytoreduction dropped from 600 x 109/L (290-1596 x 109/L) at the beginning of Pcy to 470 (240-1548) x 109/L, p=0.0068, and 423 (240-843) x 109/L, p=0.0005, in the 2T and 3T, respectively, with non-significant increase after delivery to 494 x 109/L (122-1600 x 109/L). No cases of maternal thrombosis and 2 episodes of major postpartum bleeding who required surgical intervention one of them with hysterectomy were found.The median time of delivery was at 38 wks of gestation (IC25-75% 36.5-38.5). Vaginal delivery was performed in 6 Pcy, caesarean section (CS) was required due to emergency in 2, because of lack of fetal progression in 2, and 16 were scheduled CS. The median birth weight was 2900g (IC25-75% 2500-3300g). No congenital malformations in live births were detected. Conclusions: Pcy is a rare but high-risk event in MPN pts with 11/30 OC in this cohort. The live birth rate was high compared to the literature. The risk of MC was low, without thrombotic complications and 2/30 episodes of major postpartum bleeding. There was no correlation between mutational status, platelet count or treatments received and obstetric outcomes. There was a significant reduction in the number of platelets in pts with thrombocytosis without requiring cytoreduction. The high percentage of pts who finished their pregnancy with scheduled CS is highlighted. Disclosures No relevant conflicts of interest to declare.

Multi-Lineage Dysplasia Assessment By Immunophenotype in Myeloproliferative Neoplasms (MPN): Correlation with Disease' Variant, Clinical Features and Molecular Genetics

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 &gt;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 &gt;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 &lt; 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.

Thrombotic episodes in myeloproliferative neoplasms: A population based study.

e18554 Background: Myeloproliferative neoplasms (MPN) are complicated with high incidence of thrombotic episodes, often requiring chronic anticoagulation in high risk patients (pts). To improve our knowledge on risk stratification, we undertook a large population study on National Inpatient Sample (NIS) to investigate the demographic and health factors associated with venous thromboembolism (VTE) and acute coronary syndrome (ACS) in pts with MPN. Methods: In this retrospective cohort study using the most recent 2016 NIS, we included all pts with discharge diagnosis (dx) of MPN with or without current or prior dx of VTE and ACS using appropriate ICD-10 codes. Demographic and health characteristics were compared between groups. Multivariate logistic regression was done to compare length of stay and cost of care. STATA 13.0 was used for statistical analysis. Results: Among 162,435 pts with MPN, 3750 (2.3%) had VTE and 2820 (1.73%) had ACS. MPN pts with VTE had significantly higher age (mean 61.3 years) compared to those without VTE (mean 59.3 years). Smoking, obesity, and JAK2 mutation were significantly more common in MPN with VTE group. Pts with MPN and ACS were significantly older than those without ACS (67.85 vs. 59.2 years). These pts were mostly males and more likely to have hypertension, diabetes, smoking, dyslipidemia, obesity, and chronic kidney disease as comorbidities. Total hospital cost was significantly higher in MPN pts with VTE and ACS. Conclusions: In agreement with prior reports, age, smoking, and presence of JAK-2 mutation seem to be an important prognostic factors for both VTE and ACS in MPN. Other common comorbidities mandate critical consideration in assessment of thrombotic risk in MPN pts. [Table: see text]

Pan-Myeloid Leukemia Analysis: Machine Learning-Based Approach to Predict Phenotype and Clinical Outcomes Using Mutation Data

Pan-Myeloid Leukemia Analysis: Machine Learning-Based Approach to Predict Phenotype and Clinical Outcomes Using Mutation Data

Comorbidities Such As Thromboembolic Events Significantly Worsen Patient-Reported Quality of Life (QoL) and Symptoms in Myeloproliferative Neoplasms (MPN) - Data from the Bioregistry of the German Study Group for MPN (GSG-MPN)

Introduction: Ph-negative myeloproliferative neoplasms (MPN) represent a heterogeneous group of hematological malignancies which differ in various aspects such as clinical manifestation, underlying genetic aberrations, cytomorphological features and life expectancy. However, across all subtypes, patients (pts) with MPN often suffer from severe symptoms, resulting in an impairment of the quality of life (QoL).Methods: The German Study Group for MPN (GSG-MPN) Bioregistry is a non-interventional prospective study including pts of at least 18 years with diagnosis of Ph-negative MPN according to WHO criteria (2008) having provided written informed consent. The Bioregistry study also includes assessment of QoL at baseline and on an annual basis, with all pts completing the standardized MPN-SAF-TSS questionnaire (German version) and an additional item indicating pts' subjective overall QoL on an 11-point Likert scale. Total scores range from 0 to 90 and were calculated if at least 6 items were answered (Emanuel RM et al., J Clin Oncol. 2012; 30 (33): 4098-103.)). Clinical variables, as documented in the registry, included comorbidities, reported symptoms as assessed by the physician, bleeding, and thromboembolic events (TEE). For statistical analysis, standard descriptive methods, Spearman correlation coefficient, Wilcoxon test/Kruskal-Wallis test for significance testing, and Kendall´s tau-b statistics were used.Results: 1,403 pts who had completed at least six items of the QoL assessment at baseline were included in this analysis. Median age at diagnosis was 58 years (interquartile range [IQR] 22), 98% were Caucasian, 50% were female. 494 pts were diagnosed with essential thrombocythemia (ET, 35%), 444 pts with polycythemia vera (PV, 32%), 302 pts with primary myelofibrosis (PMF, 22%), 83 pts with MPN-unclassifiable (MPNu, 6%), 43 pts with post-ET-myelofibrosis (pET-MF, 3%) and 37 pts with post-PV-myelofibrosis (pPV-MF, 3%). The most common complaint reported via the MPN-SAF-TSS was fatigue, occurring in more than 80% of the pts in all entities except MPNu (77%). More than 50 % of pts in each entity reported to suffer from early satiety, night sweats, concentration problems, or overall impairment of QoL. Table 1 summarizes all 9 symptoms and overall QoL from the questionnaire categorized by entity. Interestingly, the pts suffering from PET-MF reported the highest symptom burden, while PPV-MF pts showed the lowest overall symptom burden (median total QoL score of 23 vs. 16; p=0.01). The strongest correlations among the different symptoms were seen for fatigue and overall QoL (Spearman´s rho 0.57, p<0.001) as well as concentration problems and overall QoL (Spearman´s rho 0.33, p<0.001). Furthermore, the impact of variables such as age, comorbidities and TEE on QoL was assessed. Abdominal discomfort increased with age (rho = -0.14, p<0.001). A history of TEE before baseline assessment correlated significantly with fatigue scores (Spearman rho= 0.07, p<0.01) and with concentration problems (rho=0.07, p<0.01). With an increasing number of TEE, scores for both of these items worsened over time (p< 0.01, respectively). Moreover, MPN-total score (MPN-TSS) was higher in pts with more comorbidities (Median: 18 (IQR:23), and 25 (27) for pts with <3 versus ≥3 comorbidities, respectively, p= 0.017). Next, we compared data on 5 of the pts symptoms (reported in the questionnaire) to their assessment by the treating physician (only 5 items were available both in the questionnaire and in our registry database) in order to understand whether the “physician´s opinion” is congruent with the patient´s reported outcome in the questionnaire. While there were clear associations between the two data sources, there were also significant discrepancies, e.g., the physician did not indicate fatigue in about 20% of pts with self-assessed fatigue score of >=6 points. The most concordant symptom was night sweats (further details in table 2).Conclusions: Most MPN pts suffer from a significant symptom burden which impairs their QoL. TEE influence fatigue and concentration problems. The perception of symptoms (particularly with respect to fatigue) differs between pts and treating physician which suggests that questionnaires should be used on a routine basis in order to faithfully reflect patient´s degree of suffering from MPN and/or treatment. [Display omitted] DisclosuresIsfort:Amgen: Other: i.e. travel support; Mundipharma: Other: i.e. travel support; Roche: Other: i.e. travel support; Incyte/Ariad: Consultancy; Pfizer: Consultancy, Honoraria, Other: i.e. travel support; BMS: Honoraria; Novartis: Consultancy, Honoraria, Other: i.e. travel support; Alexion: Other: i.e. travel support; Hexal: Other: i.e. travel support. Stegelmann:Novartis: Consultancy, Honoraria. Al-Ali:Gilead: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Alexion: Honoraria; Novartis: Consultancy, Honoraria, Other: Travel support, Research Funding; Celgene: Honoraria, Other: Travel support, Research Funding. Goethert:BMS: Consultancy, Honoraria, Other: i.e. travel support; Incyte: Consultancy, Honoraria, Other: i.e. travel support; Pfizer: Consultancy, Honoraria; Novartis: Honoraria; Proteros Biostructures: Honoraria; AOP Orphan: Other: i.e. travel support. Haenel:Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria; Amgen: Honoraria. Platzbecker:Celgene: Research Funding. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

Outcomes of Patients with Myeloproliferative Neoplasms (MPN) after Interferon-Alpha (IFN) Therapy Discontinuation

▪Background:We and others have shown that IFN therapy provides several benefits to MPN patients (pts) including molecular responses and possible long-term remissions off cytoreductive therapy (CRT). However, little is known about outcomes after IFN discontinuation, and the clinical relevance of achievement of molecular response is disputed.Methods:We collected all cases of MPN pts followed in our institution who received IFN therapy since 2000, and who discontinued treatment. Cumulative incidences of relapse, hematological transformation and thrombosis were based on competing risks estimator. Survival curves and event free survival (EFS, i.e. time to first event including transformation, thrombosis and death) curves from IFN start were plotted based on the Kaplan Meier method. Univariate logistic models were used to predict complete hematological response (CHR) without any CRT; Cox models were used to predict EFS, and cause-specific hazard of relapse. Multivariable models were then fitted, with a stepwise selection procedure. All tests were two-sided with p-values ≤0.05 denoting statistical significance.Results:A total of 149 out of 333 IFN treated MPN pts had discontinued therapy at time of analysis. 78 had polycythemia vera (PV), 61 essential thrombocythemia (ET), and 10 myelofibrosis (MF). Reasons for choosing IFN in these 149 pts were young age in 75 (50%), resistance / intolerance to previous therapies in 40 (27%), pregnancy in 4 and other in 30. Median age at IFN start was 49.5 years [interquartile range, IQR: 39-60] and median [IQR] time since diagnosis of MPN was 2 years [0.6-6.8]. 64 pts had a history of vascular event.123 pts were JAK2V617F+, 15 had CALR, 5 MPL and 1 JAK2 exon 12 mutations, and 5 were triple-neg. Among 47 pts tested by NGS, 15 had additional mutations.Best response to IFN was CHR in 123 (83%), partial response (PR) in 16, and failure in 10 pts. Reasons for IFN discontinuation were toxicity in 79 (53%), prolonged hematological CHR in 52 (35%), and other in 18. Median IFN treatment duration was 28.3 months (mos) [10-45]. At discontinuation, 114 (77%) pts were in CHR, 17 (11%) in PR, and IFN failed in 18 (12%).At IFN start, median mutant allele burdenwere 25% [IQR: 15-48] and 38% [34-43] for JAK2 and CALR resp. At IFN discontinuation, median %JAK2 and %CALR had decreased to 10% [3-25], and 13.5% [6-43], resp.At last follow up, 109 (73%) pts were alive in CHR for a median time after IFN discontinuation of 42 mos [23-58], including 46 (42%) without CRT after 33 mos [19-53]. Based on a multivariable logistic model, the CHR without CRT was associated with mutant allele burden at IFN discontinuation (OR=0.95, 95%CI, 0.95-0.96, P=0.034), stopping for toxicity reason (OR= 0.82, 95%CI, 0.75-0.89, P= 0.009), and cumulative dose of IFN (OR=1.01/mg, 95%CI, 1.003-1.02, P= 0.012).Hematological relapse occurred in 49 pts. IFN was restarted in 25 of them and all achieved hematological response, including 72% CHR. Based on a multivariable Cox model, the cause-specific hazard of relapse was decreased in ET (HR= 0.4, 95%CI, 0.2-0.8; P= 0.013) while increased with the baseline WBC count (HR= 1.07, 95%CI, 1.02-1.14, P= 0.011).Ten pts had a thrombotic event after IFN, with a 5 year cumulative incidence of 10% (95%CI, 4-16%). A total of 11 pts transformed to MF, 3 to MDS / AML, and 2 from ET to PV; the 5-year cumulative incidence of transformation was 3.8% (95%CI, 0.4-7.2%). Six patients had died (4 after transformation). Overall, 25 patients developed a thrombosis, transformation or death. The 5- and 10-year EFS were 92% (95%CI, 87-97) and 72% (95%CI, 62-84), resp. In multivariate model, only time between diagnosis and IFN start (HR=1., 95%CI, 1.002-1.016, P=0.011) and mutant allele burden at IFN discontinuation (HR=1.03, 95%CI, 1.016-1.048, P<0.0001) remained jointly associated with the outcome. Pts with an allele burden ≥ 25% at discontinuation had a higher hazard of event (HR= 3.4, 95%CI, 1.3-9.3, P=0.0145).Conclusion: This study allowed for the first time identification or several parameters associated with major outcomes after IFN therapy. In particular, a lower mutant allele burden at IFN discontinuation was associated with persistent complete hematologic response without therapy and longer event-free survival, indicating a clinical relevance in obtaining a deeper molecular response. Our results also show that late onset of IFN therapy after MPN diagnosis is associated with poorer outcome. DisclosuresKiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.