ALBERT

Description: Introduction: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by expansion of the granulocytic, erythrocytic, and megakaryocytic lineages in the bone marrow and peripheral blood, and in most cases, by the presence of a JAK2 mutation. Survival of patients with PV is decreased compared with age-matched controls, and this is mainly due to thromboembolic complications followed by progression to post-PV myelofibrosis and acute leukemia. While no curative treatment exists, cytoreductive treatment with hydroxyurea (HU) or ropeginterferon is approved in EU for first-line therapy, and ruxolitinib (RUX) is approved in EU and US for second-line therapy in patients with HU intolerance or resistance. The current futility analysis assesses the efficacy of ruxolitinib in newly-diagnosed PV treated within the Ruxo-BEAT trial. Methods: This clinical trial entitled "Ruxolitinib versus Best Available Therapy in patients with high-risk Polycythemia Vera or high-risk Essential Thrombocythemia" (Ruxo-BEAT; NCT02577926) is a multicenter, open-label, two-arm phase-IIb trial with a target population of 380 pts with PV and ET. Patients in first-line PV and in first and later lines ET are randomized in a 1:1 manner to receive either RUX or best available therapy (BAT). Crossover from BAT to RUX is possible in eligible patients after 6 months. Patients with PV in the RUX arm receive a starting dose of 10 mg bid and may increase their dose up to 20 mg bid. Primary endpoint is the rate of complete clinicohematologic response rate (CHR) at month 6 as defined by Barosi et al Blood 2009. Secondary endpoints include differences in the absence of phlebotomies, spleen size, patient-reported outcomes, and survival. This is a pre-specified futility analysis of RUX in the PV arm, after 50 PV patients had been enrolled. Of the 50 patients, 28 patients with newly-diagnosed PV were randomized into the RUX arm and were analyzed (a maximum of 6 weeks of HU, anagrelide, or interferon therapy was allowed). The PV arm would have to be closed if no favorable trend were observed for RUX for any of the following variables: (1) improvement (decrease) in the hematocrit level during 6 months of treatment, (2) improvement (decrease) of the JAK2V617F allele burden during 6 months of treatment, or (3) improvement of one of the following three symptom variables assessed by physician´s judgement or via MPN Symptom Assessment Form (MPN-SAF) during 6 months of treatment: pruritus, night sweats, or bone pain. Differences between screening (Hct) or baseline (all other variables) and end of month 6 (all variables) were calculated using Fisher´s exact test (for physician-assessed pruritus and night sweats) or the Wilcoxon matched-pairs signed rank test (all other variables). Results: 28 patients received RUX for at least 6 months. After 6 months, the mean hematocrit level decreased from 45.9+/-5.6% to 41.0+/-5.0% (mean+/-SD) (p=0.0003). The number of phlebotomies calculated per year decreased from 4.2+/-3.9% to 0.96+/-2.1 (p=0.0009). Mean JAK2V617F allele burden decreased from 50.2+/28.4% to 44.0+/-28.5% (p=0.0039). The percentage of patients, as assessed by the physician, with pruritus or night sweats decreased from 41% to 26% (trending with p=0.13), and from 30% to 11% (p=0.02), respectively. The points reported by patients themselves on the MPN-SAF survey for pruritus decreased from 2.7+/-3.0 to 1.3+/-1.5 (p=0.0095) and there was a strong trend for reduction of night sweat points (from 3.1+/-3.6 to 1.6+/-2.4; p=0.0579), while the points for bone pain remained unaltered (2.0+/-2.8 to 1.4+/-2.2; p=0.215). Conclusion: Treatment with ruxolitinib in first line PV is efficient regarding the above-mentioned endpoints. Recruitment of our trial will be ongoing. In order not to weaken the study´s statistical power, comparison of both arms was not performed. Disclosures Koschmieder: Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Isfort:Mundipharma: Other: Travel reimbursement; Amgen: Other: Travel reimbursement; Hexal: Other: Travel reimbursement; BMS: Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement; Roche: Other: Travel reimbursement; Alexion: Other: Travel reimbursement. Schafhausen:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Equity Ownership, Honoraria. Griesshammer:Novartis: Consultancy, Honoraria, Speakers Bureau. Platzbecker:Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Döhner:CTI Biopharma: Consultancy, Honoraria; Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Jost:Abbvie: Consultancy, Patents & Royalties: Royalty payments for the drug compound ABT-199, Research Funding; Bohringer: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Celgene: Other: Travel Support; Novartis: Research Funding. von Bubnoff:Novartis: Research Funding. Stegelmann:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Crysandt:Amgem: Other: travel grant; celgene: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees. Gezer:AMGEM: Membership on an entity's Board of Directors or advisory committees. Brümmendorf:Merck: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Research Funding. OffLabel Disclosure: Ruxolitinib as first-line treatment in newly-diagnosed PV

Description: Acute myeloid leukemia (AML) induces profound impairment of healthy hematopoiesis. The production deficit in the bone marrow (BM) leads to development of peripheral anemia, thrombocytopenia and neutropenia, which is a major cause of AML-associated morbidity and mortality. Despite much progress in understanding of AML biology, the mechanisms by which AML blasts interact with elements of normal hematopoiesis to cause cytopenia are unclear. Conventional wisdom has it that blasts infiltrate the marrow and displace normal hematopoiesis. If this concept were to be true, there should be a strong correlation between BM blast count and peripheral cytopenia. Surprisingly, analysis of 223 patients with newly diagnosed AML at a tertiary referral center revealed lack of correlation between initial BM blast count [% of cellularity] and hemoglobin level (ρ=-0.11, P=0.12), platelet count (ρ=-0.00, P=0.53) and absolute neutrophil count (ρ=0.13, P=0.06). This indicates that mechanisms other than displacement of normal hematopoiesis dictate the severity of cytopenia in AML patients. Hematopoiesis is tightly regulated by cytokines. Among them, thrombopoietin (TPO) acts through its receptor c-Mpl as the master regulator of megakaryopoiesis, but also exerts upstream effects on hematopoietic stem and progenitor cells (HSPC). TPO levels are controlled by receptor-mediated scavenging by cells carrying c-Mpl on the surface, with platelets representing the lion's share in a healthy organism. This negative feedback loop results in strong negative correlation between serum TPO concentration and platelet count in the steady state. When we examined this relationship in our AML cohort, TPO levels did not follow the expected negative correlation with platelet counts (ρ=-0.10, P=0.59). Comparison with historic controls with thrombocytopenia induced by chemotherapy for non-hematopoietic malignancy revealed that the lack of correlation was driven by AML cases with severe thrombocytopenia that had lower than expected levels of TPO in the serum. As HSPC are known to express c-Mpl, we hypothesized that HSPC-derived AML blasts may also express the receptor and cause insufficiency of hematopoiesis by means of receptor-mediated TPO scavenging. To test this hypothesis, we compared c-Mpl expression on blasts in AML cases with severe thrombocytopenia and low TPO concentration (potential scavenger cases) to cases with TPO levels adequate for the degree of cytopenia. Both surface flow cytometry and qPCR demonstrated higher c-Mpl expression in potential scavenger cases (3.1-fold, P=0.02). To determine whether this difference in expression translates into increased serum TPO clearance, we incubated AML blasts with high (c-Mpl+) and low (c-Mpl-) receptor expression in serum containing recombinant human TPO at a concentration of 100 pg/mL. After 2h, TPO clearance reached 45 pg per 106 cells in wells with c-Mpl+ blasts, compared to only 4 pg per 106 cells in wells with c-Mpl- blasts (P=0.02). This confirms the hypothesis that AML blasts can lower TPO levels by virtue of their c-Mpl expression. Validation studies in an independent, multi-center Dutch-Belgian-Swiss cohort of 437 AML cases confirmed lack of correlation between initial BM blast count and cytopenia. Ranked gene list correlation analysis of whole genome microarray data proved significant enrichment of the MPL transcript in patients with severe thrombocytopenia when compared to patients with average platelet counts (rank 27/20'589, FDR

Description: Clonal hematopoiesis of indeterminate potential (CHIP) was recently identified as a major risk factor for development of both hematologic malignancies and atherosclerotic cardiovascular disease in humans. The most commonly mutated gene in CHIP, DNMT3A, is a de novo DNA methyltransferase. The second most commonly mutated gene is TET2, an enzyme which can lead to loss of DNA methylation, and thus is thought to have an opposing biochemical function to DNMT3A. Surprisingly, mutations in both genes lead to convergent phenotypes, such as clonal expansion of mutated stem cells, increased risk of malignant transformation, and increased risk of coronary heart disease. A molecular mechanism linking CHIP and cardiovascular disease has been explored only for loss of function mutations in the Tet2 gene (Jaiswal et al., NEJM 2017; Fuster et al., Science 2017). Here we tested the ability of null mutations in Dnmt3a to contribute to atherosclerosis in hypercholesteremic mice. We further explored the biological basis for this association through gene expression analyses and single-cell RNA sequencing. To model cardiovascular disease associated with DNMT3A-mutated CHIP, atherosclerosis-prone Ldlr-/- mice received bone marrow from Dnmt3a+/+ mice (WT), or from Dnmt3a-/- mice (KO) and WT mice in a 1:9 ratio to mimic a typical variant allele fraction observed in human CHIP. Mice then consumed a high-fat, high-cholesterol diet (HFD), and underwent assessment of atherosclerosis. At 9 weeks, mice that had received 10% Dnmt3a-/- bone marrow displayed an average lesion size that was 40% larger compared to mice receiving control marrow only (p=0.04). The increase in lesion size resembles that we previously observed in mice receiving Tet2-/- marrow (Jaiswal et al., NEJM 2017). De novo DNA methylation by Dnmt3a can alter gene expression. To elucidate how such changes may accelerate atherosclerosis, we first performed transcriptome analysis using bulk RNA sequencing of cholesterol-stimulated bone marrow derived macrophages (BMDM) from either WT or KO mice. BMDMs lacking Dnmt3a showed significantly augmented expression of genes belonging to the CXC chemokine cluster, Cxcl1, Cxcl2 and Cxcl3, as well as increases in mRNAs encoding canonical pro-inflammatory cytokines Il1b and Il6. These changes mirrored those we saw in macrophages lacking Tet2 (Jaiswal et al., NEJM 2017). We next asked how transcriptomic changes observed using the ex vivo BMDM system translated into the in vivo lesional environment. Single-cell RNA sequencing (10X Genomics) was performed on atherosclerotic aortae from mice that had been competitively transplanted with WT, Dnmt3a-/-, or Tet2-/- marrow at a 1:9 ratio. Clustering demonstrated broad changes in lesional immune cell composition in mice harboring CHIP. Lack of either Tet2 or Dnmt3a substantially expanded the myeloid compartment, containing cells that drive atherogenesis. A reciprocal reduction mainly affecting T lymphocyte populations accompanied this expansion. Within the myeloid cell compartment, Dnmt3a-/- or Tet2-/- donor cells, but not WT donor cells, gave rise to a distinct lesional macrophage population. These cells expressed markers associated with tissue-resident macrophages (Mrc1, Lyve1, F13a1), but also highly expressed several inflammatory mediators (Cxcl1, Pf4, Ccl2, Ccl7, Ccl8), and a characteristic set of transcription factors (Jun, Fos, Egr1). Overall, the present study reveals broad changes to the lesional cellular composition and transcriptome induced by the most common CHIP mutations, and provides novel insight into the mechanisms by which CHIP accelerates atherosclerosis. Despite exerting opposite catalytic functions, lack of Dnmt3a or of Tet2 function lead to a myriad of similar downstream transcriptomic and cellular changes. These results indicate that mutations in Dnmt3a and Tet2 accelerate atherosclerosis through convergent mechanisms. Disclosures No relevant conflicts of interest to declare.

Description: Key Points AML-associated peripheral blood cytopenia is independent of bone marrow blast content, but strongly predicted by MPL expression on blast cells. MPLhi blasts scavenge TPO from serum, causing insufficient cytokine levels.

Description: Background: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, ineffective erythropoiesis, dysplastic megakaryocyte hyperplasia, and extramedullary hematopoiesis. MF includes primary MF (PMF), post-polycythemia vera MF (post-PV-MF), and post-essential thrombocythemia MF (post-ET-MF). Clinical presentation is heterogeneous, marked by splenomegaly, progressive anemia, and constitutional symptoms. The median survival in patients with high-risk disease is approximately 2 years. Hematopoietic Stem Cell Transplant (HSCT) is a potentially curative therapy, however due to considerable morbidity and mortality rates, HSCT is not appropriate for most patients, including elderly patients with intermediate-II and high-risk disease. The Janus kinase (JAK) inhibitor ruxolitinib is approved in the US and EU for the treatment of patients with intermediate or high-risk MF, including PMF, post-PV-MF, and post-ET-MF. In clinical studies, treatment with ruxolitinib has been shown to reduce spleen volume by International Working Group (IWG) criteria in approximately 28% to 42% of patients and improve constitutional symptoms of MF in approximately 46% of patients (Verstovsek, J Hematol Oncol. 2017). Ruxolitinib provides symptomatic improvement, however, does not target the malignant clone or appreciably reduce the degree of fibrosis; some patients experience disease progression and leukemic transformation while on therapy (Versotvsek, NEJM. 2010; Harrison, NEJM. 2012; Kremyanskaya, Br J Hem. 2014). Moreover, ruxolitinib is associated with AEs including anemia and thrombocytopenia, which can lead to discontinuation. Approximately 50% of patients treated with ruxolitinib discontinued treatment within 3 years and 73% at 5 years (Verstovsek, Haematologica. 2015; Verstovsek, J Hematol Oncol. 2017; Cervantes, Blood. 2013; Harrison, Leukemia. 2016). Median overall survival in patients who discontinue ruxolitinib is 14-16 months, highlighting the need for novel therapies targeting alternative pathways in the setting of failure or intolerance of JAK inhibitor therapy (Newberry, Blood. 2017). The tumor suppressor protein p53 is the master regulator of cell-cycle arrest and apoptosis in response to cellular stress or DNA damage. Murine double minute 2 (MDM2) is a key regulator of p53, inhibiting its activity via ubiquitination, nuclear export, and direct inhibition of transcriptional activity. Increased MDM2 protein expression has been observed in MF CD34+ cells, suggesting that MF might be sensitive to MDM2 inhibition (Lu M, Blood. 2017). KRT-232 is a potent and selective, oral, small molecule drug that targets MDM2 and prevents MDM2-mediated p53 inhibition, allowing p53 to mediate tumor cell-cycle arrest and apoptosis. In MF, TP53 is observed to be wild-type in 96% of MF patients, suggesting MDM2 inhibition could be a successful therapeutic strategy in this disease (Raza, Am J Hematol. 2012). KRT-232 has been investigated as monotherapy and in combination with trametinib or dabrafenib in phase I studies of AML and melanoma; the most common treatment-related adverse events (TRAEs) observed were nausea, diarrhea, vomiting, decreased appetite, anemia, leukopenia, thrombocytopenia, and fatigue. The majority of TRAEs were grade 1 or 2. Methods: KRT-232 is being evaluated in an open-label phase 2 study in patients with MF who relapsed on or are refractory to JAK inhibitors (Figure). Up to 247 patients ≥ 18 years of age, with ECOG performance status ≤ 2, with high-, intermediate-2, or intermediate-1 risk disease by Dynamic International Prognostic System (DIPSS), and failure of prior treatment with JAK inhibitors will be enrolled. The study will be conducted in 2 parts. Part A will identify the recommended dose and schedule by testing varying doses and schedules across 7 treatment cohorts. Part B will evaluate safety and efficacy using the recommended dose and schedule from Part A. The primary endpoint of the study is to determine spleen response at week 24; secondary endpoints include improvement in MPN-SAF Total Symptom Score (weeks 24 and 48), red blood cell (RBC) transfusion independence, and rates of complete remission and partial remission (IWG-ERT and ELN) at week 24. This trial is enrolling at multiple sites in the United States and Europe (NCT03662126, EudraCT: 2018-001671-21). Disclosures Garcia-Delgado: Hospital Virgen De La Victoria Malaga: Employment; Novartis: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Jourdan:Novartis: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria. Pluta:Freelight Poland: Honoraria; Sandoz: Honoraria; Servier: Honoraria; Jansen-Cilag: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria; Specialistic Hospital in Brzozow,Dept of Haematooncology Ks.Bielawskiego 18 36-200 Brzozow, Poland: Employment; Teva: Honoraria; Roche Poland: Membership on an entity's Board of Directors or advisory committees; Jansen Cilag Poland: Membership on an entity's Board of Directors or advisory committees. Ewing:Novartis: Honoraria, Other: Meeting attendance sponsorship ; Bristol Myers-Squibb: Other: Meeting attendance sponsorship . Khan:Amgen: Consultancy; Celgene: Consultancy; Incyte: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. Jost:Novartis: Research Funding; Celgene: Other: Travel Support; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Abbvie: Consultancy, Patents & Royalties: Royalty payments for the drug compound ABT-199, Research Funding; Bohringer: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau. Rothbaum:Kartos Therapeutics: Employment, Patents & Royalties: Pending; Quogue Bioventures LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. McGreivy:Kartos Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Verstovsek:Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. OffLabel Disclosure: Yes, KRT-232 is an investigational small molecule MDM2 inhibitor. This trial-in-progress abstract describes a registered clinical trial that will evaluate the safety and efficacy of KRT-232 for patients with myelofibrosis.

Description: Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-RKT is expressed by immune cells, including monocytes and macrophages. Among monocytes and macrophages, distinct subsets can be distinguished based on cell surface markers and pathophysiological function. We investigated expression of Plg-RKT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14++CD16+ human monocytes and Ly6Chigh mouse monocytes expressed the highest levels of Plg-RKT and bound significantly more plasminogen compared with the other respective subsets. Proinflammatory human macrophages, generated by polarization with lipopolysaccharide and interferon-γ, showed significantly higher expression of Plg-RKT compared with alternatively activated macrophages, polarized with interleukin-4 and interleukin-13. Directional migration of proinflammatory monocytes was plasmin dependent and was abolished by anti–Plg-RKT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6Chigh monocyte recruitment was observed in Plg-RKT−/− compared with Plg-RKT+/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-RKT in vivo. Our data demonstrate higher expression of Plg-RKT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity.

Description: Key Points Humanized cytokine KI mice support engraftment of human favorable-risk AML. Engraftment and gene-enrichment analysis suggest M-CSF dependency of inv(16) AML.

Description: The ETV6-ABL1 fusion gene, as consequence of a t(9;12)(q34;p13), is a rare but recurrent genetic aberration found in chronic or blast phase of eosinophilia-associated myeloproliferative neoplasms (MPN-eo) and de novo acute B-cell lymphoblastic leukemias (B-ALL) or lymphoblastic T-cell lymphomas (T-LBL). Here, we sought to evaluate a) relevant clinical characteristics, b) treatment options and c) survival in 7 ETV6-ABL1 positive patients (male, n=4; median age 46 years; range 20-61). Cytogenetic analyses revealed a conventional reciprocal translocation t(9;12)(q34;p13) in 3 cases, an ins(12;9)(p13;q34q22) and a normal karyotype in one patient each, and a complex karyotype in 2 patients. In all cases, ETV6-ABL1 was confirmed by FISH analysis and/or RT-PCR. Histopathological diagnoses of the hypercellular bone marrow (BM) included atypical chronic myeloid leukemia (n=3), MPN-eo (n=3, concomitant T-LBL in one patient) or chronic myelomonocytic leukemia (n=1). In peripheral blood, all patients presented with left shifted leukocytosis (median 84 x 109/l, range 21-143) and significant (〉1.5 x 109/l) eosinophilia (median 6.1 x 109/l, range 2.0-7.1) but without increased blast cells. Splenomegaly was present in 60% of patients. After a median of 3 months (range 0-6) from diagnosis, all patients were treated with a TKI (imatinib, n=5; dasatinib, n=1; nilotinib, n=1) at standard doses. On dasatinib (#2) or nilotinib (#3), 2 patients achieved a complete hematologic remission (CHR) within 3 months and complete cytogenetic remission (CCR) after 5 months (#3) or complete molecular remission (CMR) after 18 months (#2), respectively (Figure). On imatinib (n=5), 2 of 5 patients (#4 and #5) achieved a CHR within 3 months with loss of CHR after 9 (#4) and 5 months (#5), respectively. Patient #4 developed a myeloid sarcoma. After local radiation, he received an allogeneic stem cell transplant (SCT) but died 8 months later due to GvHD while in CMR. Patient #5 switched to nilotinib and achieved a CCR and CMR after 3 and 10 months, respectively. Patient #1 has not achieved a significant response after 4 months on imatinib. Patient #6 showed progressive disease within 2 months on imatinib, but achieved a rapid CHR and durable CCR and CMR on dasatinib after 13 and 14 months, respectively. Patient #7 presented with a classical myeloid/lymphoid (T-LBL) neoplasm with eosinophilia (MLN-eo) according to the WHO classification. On imatinib, a regression of lymphadenopathy, but persistence of leukocytosis and eosinophilia was observed. With increasing leukocytosis and reappearance of lymphadenopathy, the patient was switched to dasatinib (9 months), followed by nilotinib (2 months). The responses were only partial and transient and the patient died 23 months after diagnosis with myeloid blast phase (secondary acute myeloid leukemia). Overall, after a median treatment time of 22 months (range, 3-58), 4 patients are in CCR (n=1, patient #3) or CMR (n=3; patients #2, #5, #6) while on a second generation TKI and two patients (#4, #7) have died. On imatinib, none of 5 patients achieved a CCR or CMR. We conclude that a) cytogenetic analysis is an important tool for the identification of potential TK fusion genes such as ETV6-ABL1, b) ETV6-ABL1 is a candidate for incorporation into the WHO-defined subcategory ´MLN-eo´, c) patients with MPN-eo can achieve durable CHR, CCR or CMR on TKI with second generation TKI being more effective than imatinib d) close monitoring by cytogenetics, FISH and RT-PCR is recommended for early identification of inadequate response or resistance. Figure (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Figure. (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Disclosures Somervaille: Novartis: Consultancy, Honoraria; Imago Biosciences: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Predicting minimal residual disease (MRD) levels in tyrosine kinase inhibitor (TKI)-treated chronic myeloid leukemia (CML) patients is of major clinical relevance. The reason is that residual leukemic (stem) cells are the source for both, potential relapses of the leukemicclone but also for its clonal evolution and, therefore, for the occurrence of resistance. The state-of-the art method for monitoring MRD in TKI-treated CML is the quantification of BCR-ABL levels in the peripheral blood (PB) by PCR. However, the question is whether BCR-ABL levels in the PB can be used as a reliable estimate for residual leukemic cells at the level of hematopoietic stem cells in the bone marrow (BM). Moreover, once the BCR-ABL levels have been reduced to undetectable levels, information on treatment kinetics is censored by the PCR detection limit. Clearly, BCR-ABL negativity in the PB suggests very low levels of residual disease also in the BM, but whether the MRD level remains at a constant level or decreases further cannot be read from the BCR-ABL negativity itself. Thus, also the prediction of a suitable time point for treatment cessation based on residual disease levels cannot be obtained from PCR monitoring in the PB and currently remains a heuristic decision. To overcome the current lack of a suitable biomarker for residual disease levels in the BM, we propose the application of a computational approach to quantitatively describe and predict long-term BCR-ABL levels. The underlying mathematical model has previously been validated by the comparison to more than 500 long-term BCR-ABL kinetics in the PB from different clinical trials under continuous TKI-treatment [1,2,3]. Here, we present results that show how this computational approach can be used to estimate MRD levels in the BM based on the measurements in the PB. Our results demonstrate that the mathematical model can quantitatively reproduce the cumulative incidence of the loss of deep and major molecular response in a population of patients, as published by Mahon et al. [4] and Rousselot et al. [5]. Furthermore, to demonstrate how the model can be used to predict the BCR-ABL levels and to estimate the molecular relapse probability of individual patients, we compare simulation results with more than 70 individual BCR-ABL-kinetics. For this analysis we use patient data from different clinical studies (e.g. EURO-SKI: NCT01596114, STIM(s): NCT00478985, NCT01343173) where TKI-treatment had been stopped after prolonged deep molecular response periods. Specifically, we propose to combine statistical (non-linear regression) and mechanistic (agent-based) modelling techniques, which allows us to quantify the reliability of model predictions by confidence regions based on the quality (i.e. number and variance) of the clinical measurements and on the particular kinetic response characteristics of individual patients. The proposed approach has the potential to support clinical decision making because it provides quantitative, patient-specific predictions of the treatment response together with a confidence measure, which allows to judge the amount of information that is provided by the theoretical prediction. References [1] Roeder et al. (2006) Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights and clinical implications, Nat Med 12(10):1181-4 [2] Horn et al. (2013) Model-based decision rules reduce the risk of molecular relapse after cessation of tyrosine kinase inhibitor therapy in chronic myeloid leukemia, Blood 121(2):378-84. [3] Glauche et al. (2014) Model-Based Characterization of the Molecular Response Dynamics of Tyrosine Kinase Inhibitor (TKI)-Treated CML Patients a Comparison of Imatinib and Dasatinib First-Line Therapy, Blood 124:4562 [4] Mahon et al. (2010) Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 11(11):1029-35 [5] Rousselot 
et al. (2014) Loss of major molecular response as a trigger for restarting TKI therapy in patients with CP- CML who have stopped Imatinib after durable undetectable disease, JCO 32(5):424-431 Disclosures Glauche: Bristol Meyer Squib: Research Funding. von Bubnoff:Amgen: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Saussele:ARIAD: Honoraria; Novartis: Honoraria, Other: Travel grants, Research Funding; Pfizer: Honoraria, Other: Travel grants; BMS: Honoraria, Other: Travel grants, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding. Guilhot:CELEGENE: Consultancy. Mahon:NOVARTIS PHARMA: Honoraria, Research Funding; BMS: Honoraria; PFIZER: Honoraria; ARIAD: Honoraria. Roeder:Bristol-Myers Squibb: Honoraria, Research Funding.

Description: Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic neoplasms driven partly by the loss of differentiation and theblockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML. While the regulatory factors that maintain HSPC proliferation and differentiation under normal conditions are well understood, significantly less is known about how LIC fate is regulated. As many hematopoietic disorders are characterized by the overproduction of pro-inflammatory cytokines, we hypothesized that necroptosis controlled cytokine secretion and inflammatory cell death might influence AML development. We therefore addressed the role of MLKL and XIAP in AML and tested whether deletion of Mlkl or Xiap would affect disease progression. Here we show that MLKL limits oncogene-mediated leukemogenesis by promoting the inflammatory cell death of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (HSCs) in experimental mice. Upon oncogenic stress MLKL-dependent necroptosis and subsequent inflammasome activation were triggered, promoting the production of IL-1β, a potent stimulator of HSPC differentiation and maturation, thus, suppressing the emergence of LICs and limiting leukemogenesis. In a murine bone marrow transplantation model of AML the absence of MLKL accelerated AML development significantly. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartments to contain LICs. The survival advantage of Mlkl-/- HSPCs became apparent in colony-forming assays and liquid cultures specifically within the CMP and ST-HSC compartments. Sorted ST-HSCs from Mlkl-/- produced more GEMM colonies than WT, the colony type harboring the multipotential myeloid progenitor cells, and both ST-HSCs and CMPs retained significantly more lineage-negative cells in liquid culture. In addition, Mlkl-/- colonies showed a reduction in propidium iodide (PI)-positive dead cells compared with WT colonies. Importantly, WT cells showed caspase activation and produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Mlkl deficiency. We also observed reduced expression of MLKL in leukemic cells on both mRNA and protein level, implying that suppression of cell death was beneficial for the survival of LICs. In contrast, deletion of Xiap did not alter survival or differentiation of leukemic cells when compared with WT cells. Furthermore, XIAP was not differentially expressed on mRNA or protein level compared with WT, indicating that XIAP does not play a critical role in leukemogenesis. In agreement with the murine data, gene expression analysis from primary leukemia cells from two large patient cohorts newly diagnosed with AML showed significantly lower expression of MLKL, but not XIAP, in a variety of AML subtypes compared to healthy controls. Overall, our data demonstrate a key role for MLKL-mediated cell death and activation of the inflammasome in AML and represents a novel tumor-suppressive mechanism. Disclosures Peschel: MophoSys: Honoraria.