Myeloproliferative Neoplasia Research Articles

PP-32 - Morbidity symptoms of myeloproliferative neoplasia

PP-32 - Morbidity symptoms of myeloproliferative neoplasia

Morbidity Symptoms of Myeloproliferative Neoplasia

Morbidity Symptoms of Myeloproliferative Neoplasia

Roles of JAK2 in Aging, Inflammation, Hematopoiesis and Malignant Transformation.

Clonal alterations in hematopoietic cells occur during aging and are often associated with the establishment of a subclinical inflammatory environment. Several age-related conditions and diseases may be initiated or promoted by these alterations. JAK2 mutations are among the most frequently mutated genes in blood cells during aging. The most common mutation within the JAK2 gene is JAK2-V617F that leads to constitutive activation of the kinase and thereby aberrant engagement of downstream signaling pathways. JAK2 mutations can act as central drivers of myeloproliferative neoplasia, a pre-leukemic and age-related malignancy. Likewise, hyperactive JAK-signaling is a hallmark of immune diseases and critically influences inflammation, coagulation and thrombosis. In this review we aim to summarize the current knowledge on JAK2 in clonal hematopoiesis during aging, the role of JAK-signaling in inflammation and lymphocyte biology and JAK2 function in age-related diseases and malignant transformation.

Tratamiento actual de las neoplasias mieloproliferativas: frente a tres escenarios

Tratamiento actual de las neoplasias mieloproliferativas: frente a tres escenarios

Thrombosis and Bleeding in Philadelphia Negative Myeloproliferative Neoplasia: Incidences and Risk Factors

Objectives: Thrombohemorrhagic events developing in the Philadelphia negative myeloproliferative neoplasms (MPN) may worsen the clinical course of the disease and increase the morbidity and mortality. The aim of this study is to assess critical risk factors of the hemostatic complications in MPN.
 Methods: The patients who were diagnosed as MPN based on world health organization 2016 criteria are included into the study. The categorical and continuous data were compared with Chi-square and independent sample t-test. The statistical significance threshold was considered as p <0.05.
 Results: 181 MPN patients were evaluated in this study. The number of the patients with PV, ET, and PMF were 89, 50 and 42 respectively. At the time of diagnosis, the median age was 54 years. There were 50 patients who had thrombosis at the time of diagnosis, and 22 patients have developed thrombosis during the follow-up. Venous thrombosis (VT) is more frequent than arterial thrombosis. The smoking habit was statistically higher in the patient with MPN who developed thrombosis (p=0.006). The most frequent hemorrhagic complication was gastrointestinal bleeding. Platelet levels of patients with complications during the course of MPH were found to be lower than those without complications (p <0.001).
 Discussion: The hemostatic complication rates due to MPN were 39% thrombosis and 13% bleeding. While the cigarette usage was significantly higher in the thrombosis group, advanced age, male sex, MPN type, JAK-2 positivity were similar between the groups.
 Conclusion: Further experimental and clinical studies should focus on the pathobiology and proper clinical management of the MPN patients with hemostatic complications.

Safety and Efficacy of Haploidentical Peripheral Blood Stem Cell Transplantation for Myeloid Malignancies Using Post-transplantation Cyclophosphamide and Anti-thymocyte Globulin as Graft-versus-Host Disease Prophylaxis

Haploidentical stem cell transplantation (haploSCT) has greatly improved access to curative treatment for myeloid malignancies in patients without suitable matched sibling/unrelated donors. We investigated the safety and efficacy of haploSCT after reduced intensity conditioning (RIC) with anti-thymocyte globulin (ATG), post-transplant cyclophosphamide (PTCy), and cyclosporine to prevent rejection and graft-versus-host disease (GVHD). In this study, 47 patients received RIC using fludarabine, busulfan, and total body irradiation (200 cGy). Unmanipulated peripheral blood grafts were used. GVHD prophylaxis included ATG (4.5 mg/kg day–3 to −1), PTCy (50 mg/kg/day day +3, +4), and cyclosporine from day +5. The median follow-up was 15 months (range 3–27). Thirty one (66%) patients had acute myeloid leukemia (AML), 10 (21%) had high-risk myelodysplastic syndrome, and 6 (13%) had a myeloproliferative neoplasia. Median age was 60 years (range 22–73). The d+100 cumulative incidences of grade II–IV and III–IV acute GVHD were 17% (95% confidence interval (CI) 7.9–29.1) and 6.4% (1.6–15.9), respectively. The cumulative incidence of moderate-severe chronic GVHD at 1 year was 15.2% (95% CI 6.5–27.1). Overall survival (OS) and relapse-free survival (RFS) were 55.2% (95% CI 39.5–68.4) and 49.5% (95% CI 34.2–63), respectively. Nonrelapse mortality (NRM) for all patients at 1 year was 37.1% (95% CI 23.2–51.1). Infection was the main cause of death (26%). For AML, 1-year OS, RFS, and NRM were 64.1% (95% CI 43.3–78.9), 54.5 (95% CI 34.6–70.7), and 26.8% (95% CI 12.3–43.6), respectively. In conclusion, unmanipulated haploidentical peripheral blood stem cells (PBSC) transplantation following RIC and dual in vivo T-cell depletion results in a low incidence of acute and chronic GVHD for patients diagnosed with myeloid malignancies.

PB2198 CRUCIAL AGE FOR JAK2 V617F MUTATION: GENDER DIFFERENCES

Background:The prevalence of JAK2 V617F mutation of Ph‐negative myeloproliferative neoplasias (MPN) increases with age. The dependence of the JAK2 V617F mutation prevalence and the rate of increasing its burden from the patients sex was discussion [Patterson‐Fortin J, et al., 2017; Stein BL et al., 2010]. There is monotonically increasing curve of somatic mutations and oncological morbidity as a usual presentation of growth graphs. However, the using of the population threshold model of the age dynamics of oncological diseases makes it possible to set of critical points of inflection (second order phase transition) and to quantify the intensity of the increase in the burden of disease [Landau LD, Lifshitz EM, 2013; Soukhovolsky VG et al., 2015].Aims:Assessment of the intensity of the JAK2 V617F mutation prevalence on the age and sex dependence using the model of the “second order phase transition”.MethodsThe data base of the Krasnoyarsk branch of the National Hematology Research Center from Jan 2012 to Feb 2019 examination has been used. The JAK2V617F allele burden was measured in whole blood samples by an allele‐specific quantitative polymerase chain reaction using of the “Myeloskrin JAK2 kit” (Formula of gene ltd). The relationship between the q (rate of JAK2 V617F detection) and the 1/T indicator (“reverse age”) in each age‐old five‐year cohort on 1154 men and 958 women referred with suspicion of MPN was studied. The values of q2 should be close to zero as according to the model of second order phase transitions for physical systems until reaching a certain critical patients age Tc and the relationship between the q2 and 1/T variables will be described by the equation q2 = a‐b/T with an angular coefficient b characterizing the increase in the intensity of the mutational burden with age after reaching the critical age Tc.Results:Using of our model led to transforms the monotonous growth curve of mutation prevalence with age. The value of q2 is close to zero at high values of 1/T (young patients) and for smaller values of 1/T (more older age) the relationship between q2 and 1/T becomes linear (figure). The hypothesis of the statistical significance differences of regression for male and female was tested by Fisher test for the significance level α=5%. Hypothesis of the absence of differences between the two regressions is not rejected. The critical age of patients transition to the phase with a high risk of mutation development corresponds for male Tc = 48.8 years and for female Tc = 36.0 years. Intensity of further accumulation of the mutation burden in male b = 44.35 ± 12.86, in female b = 27.52 ± 4.57.Summary/Conclusion:The “second order phase transitions model” it possible to introduce in the population analysis the indicators of the “critical age” Tc and the intensity of accumulation of the burden of mutations b. This estimate are not significantly different for male and female for our sample. The calculated critical age turned out to be significantly lower than the median of the age of diagnosis of MPN (53 and 57 years). The proposed model will be useful for analyzing the characteristics of the population of MPN in different cohorts, as well as in different social and environmental conditions of life.image

PF404 TUMOR‐DERIVED EXOSOMES MAY BE USED AS NEW INFORMATIVE TOOL FOR THE DETECTION OF BONE MARROW RESIDUAL CML LEUKEMIC CELLS ACTIVITY

Background:Ph+ Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by BCR‐ABL1 fusion‐gene, derived from the t (9;22) translocation. Tyrosine Kinase Inhibitors (TKIs) target BCR‐ABL1 protein, induce molecular response in the most of patients, prolong the survival and allow the treatment free remission. Many studies have demonstrated the persistence of leukemic cells in the bone marrow (BM) niche, even after treatment. The conventional minimal residual disease monitoring for CML patients is based on the RT‐qPCR quantification of BCR‐ABL1 transcript in the peripheral blood (PB) cells. However, it is not able to appreciate the activity of residual CML leukemic cells in the BM, responsible of the molecular relapse in the patients undergoing TKIs discontinuation. Exosomes, carrying different information of the cell of origin, have garnered considerable attention in cancer research thanks to the discovery of their messenger role in cellular communication. Moreover, there are some evidences regarding the possibility to isolate the different exosomes sub‐populations targeting the antigens on their surface.Aims:In this study we aimed to explore the feasibility of the tumor‐derived exosomes fraction enrichment in CML patients in chronic phase (CP) and treated with TKIs. Ph‐ patients and healthy subjects served as controls.Methods:BCR‐ABL1 transcript was quantified following on PB cells both conventionally, according to the International Scale, and by dPCR, given the last published evidences of its accuracy and sensitivity. The exosomes enrichment was performed by a specific kit based on immuno‐selection for a pan‐cancer antigen. The quantification of BCR‐ABL1exosomal transcript was performed by dPCR. In addition, Y4 transctript, an exosomal housekeeper, was quantified in order to assess the exosomal RNA quantity.Results:The possibility of the tumor‐derived exosomes enrichmentwas confirmed by the quantification of Y4 and the immuno‐selection of the exosomes resulted specific thanks to the negative results obtained in healthy controls. In Ph‐controls and in CML patients we were able to isolate tumor exosomes (Y4 positive). Moreover, the presence of BCR‐ABL1 transcript in all CML exosomal samples highlighted, for the first time, the presence of active leukemic cells in CML patients in CP.Summary/Conclusion:These results suggest that tumor derived exosomes could be consider as a new toolfor the identification of active leukemic cells and for the assessment of an innovative monitoringfocused on the biological meaning of exosomes in CML.image

PB2236 APPLICATION OF HETERODUPLEX ANALYSIS FOR CARL MUTATION DETECTION

Background:CARL exon 9 somatic mutations are highly specific to confirm the diagnosis of essential thrombocythemia (ET) and myelofibrosis (MF). Moreover, the cases of CALR mutations in the absence of JAK2 mutations when polycythemia vera (PV) or idiopathic erythrocytosis (IE) is diagnosed are reported in the literature. CALR mutations are concentrated in exon 9 and they involve various deletions and insertions as well as their combinations that lead to the frameshifting. Nowadays over 50 different CALR mutations associated with chronic myeloproliferative neoplasia (CMN) have been identified and classified. To identify all possible variants, it is necessary to use sequencing. However, due to the high cost of sequencing, developing a two‐stage algorithm for detect mutations in CARL exon 9 using inexpensive screening is of immediate practically necessity. We have previously proposed a two‐stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening test by heteroduplex analysis (Subbotina T et al, Haematologica 2017).Aims:The aim of this study was to demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non‐denaturing PAGE as the preliminary screening test for detection of CARL exon 9 somatic mutations.Methods:6 DNA samples of patients with different phenotypic form of CMN and different level of CARL mutation allele burden were included in this study. The informed consents from these patients were obtained. The presence of the CARL mutations and allele burden levels were identified by pyrosequencing method with PyroMark Q24 (Qiagen, Germany) and by Sanger's DNA sequencing with AB3500 (USA). The PCR with the additional stage of formation heteroduplexes was performed using the Real‐time PCR kit (Syntol, Russia) and CFX 96 Real Time System (Biorad, USA). PCR products were analyzed by electrophoresis in 8% PAGE.Results:The ET or MF diagnoses for five of six patients were confirmed by bone marrow trephine biopsies histological examination. It is important to note one out of six patients had a confirmed diagnosis of PV, which is a rare event. The results of electrophoresis on non‐denaturing PAGE are reported in Figure 1. All six patients have six different indel mutation variants in the 9 exon of the CARL: с. 1154_1155insGTGTC; с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52); c.1154_1155insTTGTC; c.1092_1143del (51); c.1154_1155insTTGTC, accordingly. The minimum number of inserted nucleotides were 5 and the maximum number of falling nucleotides were 52. Variants of CARL mutations for № 1, 4, 5 and 6 patients have been already described. Patients № 2 and № 3 carry new (yet included in the COSMIC website) mutations in CARL: с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52). The allele burden level ranged from 25% to 50%.Summary/Conclusion:The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non‐denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming pyrosequencing or sanger's sequencing. The two‐stage approach allows to optimize the algorithm of the CARL exon 9 mutation detection and to improve the efficiency of testing for patients suspected of having CMN.image

Long-Term Survival in the Concomitant Evolution of Two Cancers: The Idiopathic Myelofibrosis and the Clear Cell Renal Carcinoma; A Case Report

Introduction: The association of chronic myeloproliferative neoplasia with urogenital cancer is rare (1.6% - 1.9%), with the death risk of 1.21 - 2.28 times higher in patients with solid cancer and chronic myeloproliferative disease. Case Presentation: We present the case of a patient diagnosed with triple-negative idiopathic myelofibrosis treated and monitored at the Department of Hematology of the Clinical Emergency County Hospital, Brasov, Romania. After five years of idiopathic myelofibrosis, which was treated with hydroxyurea, a second solid neoplasia was identified as left renal cell carcinoma in Robson stage I. The complete remission of the renal carcinoma was achieved with the left nephrectomy sustained for a duration of approximately four years, overlapping with the partial remission of myelofibrosis under treatment with ruxolitinib. The recovery of the renal carcinoma significantly influenced myelofibrosis therapy and subsequently led to the patient’s development. The global survival, in this case, is 124 months for myelofibrosis and 64 months for the concomitant evolution of the two cancers. Conclusions: Despite a number of peculiarities (age at diagnosis and complications during the treatment of two cancers), the patient had a prolonged survival.

Comprehensive clinical, molecular and histopathological analysis of bone marrow in chronic myeloproliferative neoplasia

ObjectivesTo analyze the possible correlation between molecular changes in the JAK2, MPL and CALR genes, the morphological pattern of bone marrow and the clinical-hematologic profile of patients. Patients and methodsWe conducted a retrospective study that included 140 patients diagnosed with Philadelphia-negative myeloproliferative neoplasia (Ph-MPN) in a single center. ResultsIn essential thrombocythemia (ET), the patients with the JAK2 V617F mutation presented more leucocytes and neutrophils than patients who presented the CALR mutation, who had more platelets and a greater need for cytoreductive therapy. These findings support the fact that the mutational state in ET appears to define subtypes of patients with substantially different clinical courses and prognoses. In myelofibrosis, the mutational state appears to influence the histopathological changes found in the bone marrow biopsy, which did not occur in polycythemia vera or ET.

Análisis integrado clínico, molecular e histopatológico de la médula ósea en las neoplasias mieloproliferativas crónicas

ObjectivesTo analyse the possible correlation between molecular changes in the JAK2, MPL and CALR genes, the morphological pattern of bone marrow and the clinical-haematologic profile of patients. Patients and methodsWe conducted a retrospective study that included 140 patients diagnosed with Philadelphia-negative myeloproliferative neoplasia (Ph-MPN) in a single centre. ResultsIn essential thrombocythaemia (ET), the patients with the JAK2 V617F mutation presented more leucocytes and neutrophils than patients who presented the CALR mutation, who had more platelets and a greater need for cytoreductive therapy. These findings support the fact that the mutational state in ET appears to define subtypes of patients with substantially different clinical courses and prognoses. In myelofibrosis, the mutational state appears to influence the histopathological changes found in the bone marrow biopsy, which did not occur in polycythaemia vera or ET.

Rigosertib ameliorates the effects of oncogenic KRAS signaling in a murine model of myeloproliferative neoplasia.

Aberrant signaling triggered by oncogenic or hyperactive RAS proteins contributes to the malignant phenotypes in a significant percentage of myeloid malignancies. Of these, juvenile myelomonocytic leukemia (JMML), an aggressive childhood cancer, is largely driven by mutations in RAS genes and those that encode regulators of these proteins. The Mx1-cre kras+/G12D mouse model mirrors several key features of this disease and has been used extensively to determine the utility and mechanism of small molecule therapeutics in the context of RAS-driven myeloproliferative disorders. Treatment of disease-bearing KRASG12D mice with rigosertib (RGS), a small molecule RAS mimetic that is in phase II and III clinical trials for MDS and AML, decreased the severity of leukocytosis and splenomegaly and extended their survival. RGS also increased the frequency of HSCs and rebalanced the ratios of myeloid progenitors. Further analysis of KRASG12D HSPCs in vitro revealed that RGS suppressed hyperproliferation in response to GM-CSF and inhibited the phosphorylation of key RAS effectors. Together, these data suggest that RGS might be of clinical benefit in RAS-driven myeloid disorders.

AACR White Paper: Shaping the Future of Cancer Prevention - A Roadmap for Advancing Science and Public Health.

The recent pace, extent, and impact of paradigm-changing cancer prevention science has been remarkable. The American Association for Cancer Research (AACR) convened a 3-day summit, aligned with five research priorities: (i) Precancer Atlas (PCA). (ii) Cancer interception. (iii) Obesity-cancer linkage, a global epidemic of chronic low-grade inflammation. (iv) Implementation science. (v) Cancer disparities. Aligned with these priorities, AACR co-led the Lancet Commission to formally endorse and accelerate the NCI Cancer Moonshot program, facilitating new global collaborative efforts in cancer control. The expanding scope of creative impact is perhaps most startling-from NCI-funded built environments to AACR Team Science Awarded studies of Asian cancer genomes informing global primary prevention policies; cell-free epigenetic marks identifying incipient neoplastic site; practice-changing genomic subclasses in myeloproliferative neoplasia (including germline variant tightly linked to JAK2 V617F haplotype); universal germline genetic testing for pancreatic cancer; and repurposing drugs targeting immune- and stem-cell signals (e.g., IL-1β, PD-1, RANK-L) to cancer interception. Microbiota-driven IL-17 can induce stemness and transformation in pancreatic precursors (identifying another repurposing opportunity). Notable progress also includes hosting an obesity special conference (connecting epidemiologic and molecular perspectives to inform cancer research and prevention strategies), co-leading concerted national implementation efforts in HPV vaccination, and charting the future elimination of cancer disparities by integrating new science tools, discoveries and perspectives into community-engaged research, including targeted counter attacks on e-cigarette ad exploitation of children, Hispanics and Blacks. Following this summit, two unprecedented funding initiatives were catalyzed to drive cancer prevention research: the NCI Cancer Moonshot (e.g., PCA and disparities); and the AACR-Stand Up To Cancer bold "Cancer Interception" initiative.

Treosulfan, Fludarabine and Cytarabine As Conditioning before Allogeneic Hematopoietic Stem Cell Transplantation

Background: The combination of treosulfan witth fludarabine was successfully introduced into toxicity-reduced conditioning regimens for hematopoietic stem cell transplantation (HCT). However, the risk of post-HCT relapse remains of concern. Here we report for the first time on the results of an individual treatment approach with treosulfan, fludarabin and cytarabine as conditioning for allogeneic HCT in patients with AML, MPN or MDS.Methods: 22 patients were treated with fludarabine 30 mg/m² given on day -6 to day -2, treosulfan 14 g/m² administered on days -4 to day -2 and cytarabin 2g/m² given on days -6 and -5. GvHD-prophylaxis consisted of cyclosporine A and methotrexate or MMF. In addition, antithymocyte globulin was applied in case of an unrelated donor. One patient received bone marrow and the remaining patients received peripheral blood stem cells from matched related donors (9%), matched unrelated donors (73%) or mismatched unrelated donors (18%). All patients were considered to have high risk of relapse because of unfavourable cytogenetic features and/or insufficient or missing response to previous treatment. Three patients (14 %) with CML after blast crisis received the combination because of the reported high relapse rate (46%) after three-day scheduled conditioning with treosulfan [1]. In addition, patients were considered to be ineligible for myeloablative standard conditioning because of multi-morbidity (n = 4; 18% with HCT-CI >2) and/or age >55 years (n = 14; 64%).Results: The median age of patients was 59 (35-68) years. Patients suffered from acute myeloid leukemia (n = 14, 64%), myeloproliferative neoplasia (n = 6, 27%) or myelodysplasic syndrome (n = 2, 9%). The conditioning regime was well tolerated and nearly all patients engrafted and achieved complete donor-type chimerism, except for one who died very early from sepsis. Another patient with underlying myelofibrosis suffered from secondary graft failure on day 100. Two patients developed aGVHD °III/ IV. None of the patients suffered from veno-occlusive disease or severe chronic GVHD. Overall survival and event-free survival at one year reached 60.2% and 59.6%, respectively. Six patients died from infectious disease, two from relapse and one patient from acute GVHD °IV.Conclusion: The combination of cytarabine with the established conditioning of treosulfan and fludarabine is feasible in patients with high risk of relapse and ineligible for myeloablative standard conditioning.Holowiecki J, Giebel S, Wojnar J et al. Treosulfan and fludarabine low-toxicity conditioning for allogeneic 334 haematopoietic stem cell transplantation in chronic myeloid leukaemia. Br J Haematol 335 2008; 142(2): 284-92. DisclosuresHilgendorf:Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding. Frietsch:Deutsche Krebshilfe: Research Funding. Scholl:Abbivie: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Deutsche Krebshilfe: Research Funding; Alexion: Other: Travel support. Hochhaus:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Pfizer: Research Funding. Casper:Medac: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding.

The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML

Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML.We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %.In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression.Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis.Results:HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1.Conclusions:HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. DisclosuresNo relevant conflicts of interest to declare.

Treatment Patterns and Healthcare Resource Utilization in Patients with Relapsed/Refractory Acute Myeloid Leukemia and a Subset with IDH1-Mutation: A United States Medical Chart Review Study

▪Introduction: In the United States (US), acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Patients with relapsed or refractory AML (R/R) have a particularly poor prognosis, and more effective and less toxic treatments are urgently needed. The gene encoding isocitrate dehydrogenase 1 (IDH1) is mutated in 6-10% of AML patients. Drugs that target the altered enzyme have shown significant clinical activity in the R/R setting, with the first such agent, ivosidenib, recently receiving FDA approval. We conducted a retrospective chart review to evaluate current treatments and healthcare resource use in an R/R AML population, with a sub-analysis focusing on the population with IDH1 mutations (mIDH1). Findings from this study will help to better understand the future impact of targeted IDH1 inhibitors in this difficult-to-treat patient population.Methods:US-based hematologists and oncologists were recruited from existing physician panels to extract information from eligible patients' medical charts. Each physician extracted information, including patient demographics, medical history, AML treatment, and AML-related healthcare resource use. Eligible patients must have been initially diagnosed with R/R AML and tested for mIDH1 between January 2012 and June 2017. Patients were excluded if they had ever been enrolled in a clinical trial for an IDH1 inhibitor. The index date was defined as the date of initial R/R diagnosis and information was collected through end of follow-up, defined as the end date of care, date of death, or date of data collection, whichever occurred first. Patient characteristics, treatments, and AML-related healthcare resource use were described. mIDH1 patients were oversampled to achieve the targeted sample size of 300 patient charts, evenly split between mIDH1-positive and IDH1 wild type (wt) patients. As a result, findings reported for the overall patient population were reweighted to reflect an 8.0% prevalence of mIDH1.Results:Data were collected from 182 oncologists/hematologists, providing chart information for 304 patients (154 mIDH1-positivepatients and 150 mIDH1-negativepatients). Median follow-up time for all patients was 11.1 months. After reweighting, the mean age for the overall patient population was 57.7 years old and most patients were first diagnosed with de novoAML (92.8%), as opposed to secondary AML (7.2%). The population had a higher percentage of relapsed patients than primary refractory patients (73.7% versus 26.3%) and a higher percentage of patients treated in a community-based setting than an academic-based setting (64.2% versus 35.8%).After the initial diagnosis of R/R AML, 90.9% of all patients received some type of active treatment, with intensive chemotherapy being the most common initial treatment (65.0%), followed by less-intensive chemotherapy regimens (19.3%), and hypomethylating agents (14.9%). 44.8% and 11.0% of patients also received a second- or third-line treatment regimen, respectively. 10.4% of patients received a stem cell transplant during the follow-up period.There was significant health resource utilization in this population. 82.0% of patients had at least one inpatient admission during the follow-up period, with the overall population averaging 0.28 admissions per-patient-per-month. The most common reason for AML-related inpatient admissions was chemotherapy administration. 66.4% of patients were hospitalized for treatment administration, 31.8% for infections, and 27.1% for febrile neutropenia. In addition, 39.6% of patients had at least one emergency room (ER) visit and 14.2% of patients had at least one intensive care unit (ICU) admission. The treatment and healthcare resource use burden was similarly high for the subset of mIDH1-positive patients. No significant differences were found between the mIDH1-positive and IDH1 wtpatients.Conclusions:Using retrospective medical chart data, this study highlights current treatment and healthcare resource use for adult patients with R/R AML. There is a high burden associated with R/R AML due to the health issues associated with rapidly progressive disease and the toxicity of available treatments, which require inpatient admissions. As more targeted drugs become available for patients in the R/R setting, such as ivosidenib, it will be of interest to re-evaluate the burden and clinical outcomes in the R/R AML population. DisclosuresGriffin:RXi Pharmaceuticals: Consultancy; Analysis Group: Consultancy; Myeloproliferative Neoplasia Foundation: Other: Grant ; Lilly Pharmaceuticals: Other: Grant; Sun Pharmaceuticals: Consultancy; Novartis Pharma: Other: Grant, Patents & Royalties: Royalties ; Astellas Pharma: Consultancy. Storm:Agios Pharmaceuticals: Employment. Wilhelm:Agios Pharmaceuticals: Employment. Boscoe:Agios Pharmaceuticals: Employment. Macaulay:Analysis Group, Inc.: Employment. Zhou:Celgene Corporation: Research Funding; Analysis Group, Inc.: Employment. Faust:Analysis Group, Inc.: Employment. Cheung:Analysis Group, Inc.: Employment.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cell Type-Specific Deregulation of Polypyrimidine Tract- Binding Proteins (PTBPs) Drive Aberrant Splicing in Multiple Myeloma (MM) and Acute Myeloid Leukemia (AML)

Genome-wide transcriptome profiling detected an increased splicing alterations in MM and AML. While these malignancies are derived from different cell linages, their tumor cells acquire similar aberrant splicing (AbSp), mostly intron retentions. To delineate AbSp mechanism in MM/AML, we focused on PTBPs (1/2/3) that play a critical role in intron excision. We have previously reported deregulated expression of splicing factors (SFs) in MM/AML patient and healthy donor (HD) bone marrow (BM). As MM progressed, PTBP1/2 progressively increased, and PTBP3 gradually decreased (ASH 2017). In AML, PTBP2/3 upregulation and PTBP1 downregulation were detected in patient samples in which increased intron retentions were identified by genome-wide splicing analysis (CCR 2015).Here, these findings were validated by TaqMan assays for PTBPs in 48 MM and 325 AML patient samples, 16 MM/AML cell lines, and in plasma cells (PCs) and CD34+cells from 14 HDBM. Results were consistent with differential expressions of PTBPs in MM/AML previously analyzed. Upregulation of PTBP1/2 and PTBP2/3 proteins were detected in MM and AML cell lines, respectively. PTBP1/2 upregulation was pronounced when MM cell lines were cocultured with BM stromal cells (MMBMSC) derived from MM patients' BM. Importantly, we detected increased proliferation and decreased apoptosis in MM/AML cell lines overexpressing PTBPs. These effects were most evident after coculturing MM cell lines with MMBMSC as compared to HDBMSC.To evaluate PTBP effects on AbSp, we knocked down and/or overexpressed PTBPs in MM/AML cell lines and assessed PDL1 splicing in MM, and NOTCH2 and FLT3 splicing in AML. PDL1 is spliced in ~ 30% of 90 MM patients; while NOTCH2/FLT3/CD13 are spliced in 78%/50% of 387 AML patients, respectively. After PTBP1/2 knockdown in MM cells we detected 4- to 11-fold downregulation of PDL1 splice variants, with proportional upregulation of wild-type PDL1 levels; concurrently, MM cell proliferation was decreased and apoptosis increased. To evaluate MM specific splicing alterations in the context of the MM BM microenvironment, PDL1 splicing, and PTBP1/2 mRNA/protein expressions, were monitored in MM cell lines cocultured with MMBMSC or HDBMSC. We observed time-dependent PDL1 splice variant upregulation, and higher levels of PTBP1/2 in MM cells. We also noted time-dependent PDL1 variant expression switching in association with PTBP1/2 deregulation in the BM microenvironment. RNA-seq analysis and western blotting showed that MMBMSC culture with tumor cells increased intron retention, and altered SF expressions, including PTBPs in BMSC.We next monitored PTBP effects on splicing in AML cells by evaluating NOTCH2 and FLT3 splicing in an TF1, an AML cell line that overexpresses PTBP2/3. RT-PCR analysis showed association between PTBP3 overexpression and NOTCH2 and FLT3 AbSp in TF1 cells. For further validation, we developed an ex vivo splicing assay composed of an FLT3/CD13 splicing cassette with a GFP reporter, which allows for evaluation of splicing events by flow cytometry (FACS) and microscopy. In this assay, cells overexpressing PTBP2/3 caused FLT3/CD13 minigene splicing similar to that detected in AML patients. Also, by RT-PCR, we showed that overexpression of PTBPs caused intron retention, that was confirmed by cloning and sequencing of PCR products and consistent with the FACS analysis and microscopy.Finally, we have tested effects of PTBPs using in vivo AML models (BMT and xenograft). In the BMT model, animal median survival was 48 days post-BMT for MLL-AF9/PTBP3 and 56 days in the control group. In the xenograft model, animal median survival was 66, 94, & 106 days after injection of TF1-PTBP3, TF1-PTBP2, and TF1 cells in mice, respectively (P>0.0001). These studies suggest that PTBP3 overexpression in partnership with the MLL-AF9 promotes occurrence of AML in mice. Tumor RNA samples harvested from these animals were subjected to RNA-seq analysis, which showed increased AbSp association with PTBP3 overexpression.Our studies indicate that deregulated PTBP1/2 expression in MM and of PTBP2/3 in AML drive time-dependent AbSp (intron retention) and splice variant switching, which in MM is induced by culture with BMSC; and conversely, that analogous changes are induced in BMSC cultured with tumor cells. They define role of deregulated expression of the PTBPs in MM/AML pathogenesis, and suggest novel targets for therapeutic intervention. DisclosuresStone:Pfizer: Consultancy; Jazz: Consultancy; Fujifilm: Consultancy; Merck: Consultancy; Cornerstone: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Novartis: Consultancy, Research Funding; Sumitomo: Consultancy; Ono: Consultancy; Orsenix: Consultancy; Otsuka: Consultancy; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Argenx: Other: Data and Safety Monitoring Board; Arog: Consultancy, Research Funding; Astellas: Consultancy. Griffin:Astellas Pharma: Consultancy; Novartis Pharma: Other: Grant, Patents & Royalties: Royalties ; Analysis Group: Consultancy; Sun Pharmaceuticals: Consultancy; RXi Pharmaceuticals: Consultancy; Lilly Pharmaceuticals: Other: Grant; Myeloproliferative Neoplasia Foundation: Other: Grant . Anderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy.