Patnaik, Mrinal M.

Background MDS is a heterogeneous disease and the revised International Prognostic Scoring System (IPSS-R) is utilized in prognostication. The percentage (%) of blasts in the bone marrow is determined in the aspirate morphologically. Though the former is the standard of care the blast percentage is also reported by flow cytometry and biopsy which can many times be inconsistent. We previously presented the utilization of biopsy based blast percentage which showed meaningful prognostic groups compared to aspirate. In this study we compare the blasts as reported by the aspirate and flow cytometry in MDS-EB in calculating IPSS-R. Methods The MDS database was reviewed for cases of MDS-EB after due IRB approval at the Mayo clinic. We calculated IPSS-R scores based on the aspirate blast % (IPSS-RAsp) and flow blast% (IPSS-Rfl). The aspirate blast percentage was reported morphologically. Suboptimal aspirates were excluded from the study. The flow blast percentage was determined by immunophenotyping. The overall survival (OS) was determined by IPSS-RAsp and IPSS-RFl. OS estimates were calculated by Kaplan-Meier curves and log-rank testing using JMP v.13. Uno's concordance statistic was used to compare the 2 risk scoring systems. Results Of 1322 patients, 431 (33%) cases were identified with MDS-EB out of which 120 (29%) cases had blasts reported in the aspirate and flow. Based on aspirate MDS EB1: 54% (n=65), MDS EB2 46% (n=55). The hematological, cytogenetic and R-IPSS categories were compared between MDS-EB1 and MDS- EB 2. The blast percentage and hemoglobin levels was significantly different between MDS-EB1 and EB2 as seen in table 1, however the IPSS-R risk groups were not significantly different. The flow cytometry was concordant with aspirate in 66/120 (55%) cases. Out of the dis-concordant cases only 20% (11/54) was upstaged by flow cytometry with most of the patients being down staged as expected by the techniques used in processing the blood and hence not reliable when reported low (Figure 1). The OS outcomes based on the IPSS- R asp, IPSS-Rfl areshown in figure 2A,2B .The p value with aspirate based R-IPSS was more significant than flow cytometry based R-IPSS (p= 0.0007 vs 0.0174). We compared the two models for observed OS differences using the Uno model which was not statistically significant. (p= 0.6) Conclusions Both models did not show a difference which is likely due to the very small sample size. However flow cytometry did down stage more patients when disconcordant and may have less value in that setting. It would be ideal to compare all 3 models aspirate, biopsy and flow cytometry however we did not have enough number of patients to do the comparison. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Background: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in adult populations include several rare hematologic malignancies with similar clinicopathologic features that have a significant impact on patient QOL and lead to increased risk of infection, hemorrhage, anemia and transformation to AML (e.g. CMML, aCML, MDS/MPN-RST, and MDS/MPN-U). The only approved therapies for any of the MDS/MPNs are DNA methyltransferase inhibitors (DNMTis) for patients (pts) with CMML. Due to both the rarity and the heterogeneity of MDS/MPNs, it has been challenging to study in dedicated, prospective studies and thus refining treatment strategies has been difficult and optimal salvage treatments in pts who fail DNMTis have also not been rigorously studied. ABNL-MARRO (A Basket study of Novel therapy for untreated MDS/MPN and Relapsed/Refractory Overlap Syndromes) is an international cooperation providing the framework for clinical trials for MDS/MPN, and explore clinical-pathologic biomarkers. Study Design and Methods: The first study within ABNL-MARRO, ABNL MARRO 001 (AM-001), is an open label, randomized phase 1-2 study that will test 3 novel oral treatment combinations in MDS/MPNs, with each of 3 treatment arms including a novel targeted agent and ASTX727, a fixed dose combination of the DNMTi decitabine and the cytidine deaminase inhibitor cedazuridine to improve decitabine oral bioavailability. The novel agents combined with ASTX727 are: a selective JAK1 inhibitor itacitinib, pan PIM inhibitor INCB053914 and LSD1 inhibitor, INCB059872. Each combination will be tested first in a safety run-in (Phase 1b) to determine the RP2D and schedule of each combination therapy using a 3+3 de-escalation design, and a maximum of four dose decrements will be allowed. After the RP2D and schedule has been determined for a given novel drug combination, efficacy of that combination therapy in MDS/MPN pts will then be evaluated in phase 2 using a Simon's Two-Stage design to allow early discontinuation of any futile treatment regimen and to pursue potentially beneficial combinations in larger cohorts of pts. While each of the targeted agents either has undergone or is currently undergoing safety testing in combination with the DNMTi in myeloid diseases, clear rates of expected responses in MDS/MPN are not known. Retrospective studies of DAC or AZA reveal overall response rates (ORR) from 10-56% in CMML (Helbig et al, 2019; Duchman et al, 2018; Coston et al, 2019). The ORR from the ASTX727 single agent phase I study was 30%, but this included only 14% CMML (remainder MDS pts) and 45% of pts with previous treatment with AZA or DAC (Savona et al, 2019). Finally, and importantly, there are insufficient clinical trial data available for pts non-CMML MDS/MPN syndromes. Thus, the criteria for the Simon's Two-Stage design for each arm are based on best estimation from results of above studies. Accordingly, the null hypothesis that the ORR, as per the MDS/MPN Proposed Response Criteria (Savona et al, 2015; combined CR+MR+PR+Clinical benefit - CB), is 35%, will be tested against a one-sided alternative. In the first stage, 14 DNMTI-treatment naïve (TN) pts will be accrued in each AM-001 Arm and will receive treatment at the RP2D and schedule of the novel targeted agent and of ASTX727, as determined in the phase 1. If there are 5 or fewer responses in these 14 pts before the 7th cycle of therapy, the Arm will be stopped for futility. Otherwise, 30 additional pts (including both TN subjects and subjects relapsed after/refractory to other DNMTI-containing therapies) will be accrued for a total of 44. The null hypothesis will be rejected if 21 or more responses are observed in 44 pts before the 7th cycle of therapy. This design yields a type I error rate of 0.05 and power of 80% when the true response rate is 55%. The probability of early termination and expected sample size of any arm is 64% and 25 pts, respectively, assuming low (35% RR) efficacy Beyond the primary objectives of the study to evaluate the safety and efficacy of novel treatment combinations in MDS/MPN, the study will establish the ABNL MARRO infrastructure that leverages the expertise of the MDS/MPN international working group (IWG) for future prospective studies; will forge innovative scientific research that will improve our understanding of pathogenetic etiologies; and will inform the clinical application of diagnosis, risk stratification tools, and response assessments in MDS/MPNs. Disclosures Savona: AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties. Fenaux:Aprea: Research Funding; Jazz: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Padron:Incyte: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Sanz:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman - La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen - Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Santini:Menarini: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Acceleron: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Honoraria. OffLabel Disclosure: ASTX727, LSD1 inh, PIM inh, JAK1 inh in MDS/MPN

Background: CMML is a heterogeneous disease with overlapping features of MDS and MPN. Several risk scores have been proposed in CMML, including CPSS (Such Blood 2013) that identifies 4 risk groups (low, intermediate-1 [int-1], int-2 and high risk) with median overall survival (OS) ranging from 12 months to > 5 years. HSCT is the only curative treatment in CMML, with an expected 20-40% post-transplantation OS. Current guidelines, common to MDS and CMML recommend rapidly transplanting higher-risk CMML and delaying transplant in lower-risk disease (de Witte Blood 2017). Retrospective transplant studies have shown better outcome in lower-risk CMML (Liu et al BBMT 2017). Whether this translates into a survival benefit compared to a delayed transplant strategy requires prospective studies. Multistate models based on retrospective data from transplant and non-transplant patients (pts) have addressed similar issues in MDS (Koreth JCO 2013, Della Porta Leukemia 2017). Through a large collaborative study, we address the question of optimal timing for HSCT in CMML patients through a similar large international collaborative study. Method: We retrospectively selected pts from 2 registries: International MDS/MPN Working Group (IWG cohort, Padron Blood Cancer J 2015) and EBMT with the following criteria: WHO-defined CMML, age ≤ 70y, ECOG 0-2, diagnosis after 2000, available CPSS at the time of diagnosis. Different states were considered in higher- (CPSS int-2 and high) and lower- risk (CPSS low/int-1) pts: diagnosis, AML transformation, transplantation and death. IWG data was used to estimate transition probabilities from diagnosis to AML, transplantation and death and from AML to transplantation and death. EBMT data was used for transplantation to death transitions (Figure 1). Results: 719 and 403 pts were identified in the IWG and EMBT registry, respectively (resp). Median age was 64 (range 16-70) in IWG and 58 (19-70) years in EBMT cohort. Patients were male in 69% and 67% of IWG and EBMT cohorts, respectively (resp). CPSS was low in 22% and 13%, int-1 in 31% and 31%, int-2 in 40% and 45% and high in 8% and 11% of the IWG and EBMT pts, resp. In the IWG cohort, the 1 year cumulative incidence of transformation into AML were 7.3% and 18.2% in lower and higher risk patients, resp. Among the 719 pts from IWG, 102 received HSCT. At the time of diagnosis the expected life time of higher risk (int-2 and high) patients was 25.2 months while it was 44.1 months in lower risk (int-1 and low) patients. Table 1 reports gain or loss in expected life time (until 60 months after diagnosis) for transplantation at different intervals from diagnosis (within 6, 12, 18 and 24 months) for patients who have survived and not transformed to AML until the prediction time point, taking into account CPSS at diagnosis. Expected life time is also given separately for higher risk pts after transformation into AML. There was a modest gain of life expectancy with transplantation in higher risk patients (CPSS int-2 and high) while it was not the case in lower risk patients. The survival gain in higher risk pts with HSCT rose from 1.11 at 6 months to 3.4 months at 24 months. There was also a survival benefit of HSCT in higher risk pts who were transformed to AML during their follow-up which was even higher in terms of gain of months: from 9.89 month if HSCT performed within 6 months to 14.25 months if performed within 18 months. Of note, the loss of survival in lower risk patients who underwent HSCT decreased over time: 11.1 months if HSCT is performed within 6 months and 3.7 months if HSCT is performed within 24 months, probably owing to transition to higher-risk CMML at this stage in some pts, a transition that could not be captured in the present dataset. Conclusion: For the first time, using a multistate model, we could provide evidence that the transplant benefit in CMML is restricted to higher-risk pts, in line with findings in MDS and current guidelines. More analyses will be performed to analyze other subgroups of patients, especially regarding their risk (other classification than CPSS, the role of somatic mutation…). More studies are needed to analyze the role of pre HSCT treatment and its potential impact on post-transplant outcome. Disclosures Robin: Novartis Neovii: Research Funding. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Fenaux:Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Aprea: Research Funding. Kroeger:Riemser: Research Funding; Novartis: Honoraria, Research Funding; Medac: Honoraria; DKMS: Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria; Sanofi-Aventis: Honoraria. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Finke:Riemser: Honoraria, Other: research support, Speakers Bureau; Neovii: Honoraria, Other: research support, Speakers Bureau; Medac: Honoraria, Other: research support, Speakers Bureau. Komrokji:Agios: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; Incyte: Consultancy. Killick:JAZZ: Honoraria; Celgene: Honoraria; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; ALEXION: Honoraria. Blaise:Molmed: Consultancy, Honoraria; Sanofi: Honoraria; Pierre Fabre medicaments: Honoraria; Jazz Pharmaceuticals: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Patients who develop therapy-related myeloid neoplasms (t-MN) have dismal outcomes. Previous studies reported the incidence and risk factors associated with t-MN development. Lenalidomide, in the setting of oral, but not intravenous, melphalan is associated with a higher risk of t-MN (Palumbo et. al, Lancet Oncology, 2014). We carried out this study to evaluate the clinical and pathologic features of t-MN, therapies employed, and factors that predict long-term survival after diagnosis. Patients and methods: We identified patients who received the first ASCT 1998-2016 at our institution. t-MN was defined per the WHO 2016 classification. Median overall survival (OS) was calculated from the time of t-MN diagnosis to last follow-up or death. Statistical analyses were performed using SAS (JMP v14.1) or GraphPad Prism (v7). Results: Out of 2115 patients that underwent at least one ASCT, 53 (2.5%) developed t-MN. Thirty-five of 53 (66%) patients who developed t-MN had received lenalidomide. Among 2062 patients that did not develop t-MN, 916 (44.4%) patients received lenalidomide. Lenalidomide exposure was associated with development of t-MN (χ2 with Yate's correction 8.9, p=0.003). Ten patients were excluded from further analyses due to lack of follow up. Clinical characteristics are shown in Table 1a (N=43). Median age at t-MN diagnosis was 70 years (range 44-79). Median time from ASCT to t-MN was 5 years (range 1-15). After a median follow-up of 70 months (95% CI, 38-134), the median OS was 12 months (95% CI, 9-17, Figure). Primary causes of death were t-MN (71%), MM (12%), both (6%), and other including infection, GVHD, and unknown (12%). Seven (16%) had t-AML and 36(84%) had t-MDS. Three (42%) of 7 patients with t-AML had pure erythroid phenotype. At the time of last follow-up, 9 (21%) were alive. Seven (17%) underwent two ASCT, 16 (36%) received more than 2 years cumulative dose of lenalidomide. Median number of cycles of alkylator therapy including high-dose melphalan (HDM) used for ASCT was 2 (range 1-6). On univariate analysis, factors predicting OS from t-MN diagnosis were ≥ 2 alkylator vs. < 2 cycles (11 vs. 27 months, p=0.02), ≥10% vs. <10% blast at the time of t-MN diagnosis (5.5 vs. 17 months, p=0.01), and the presence of complex karyotype (CK) vs. not (11 vs. 17 months, p=0.03). There was no difference in survival among t-MDS patients who transformed to t-AML vs. those who did not, those who received 1 vs. 2 ASCT, and those with lenalidomide duration ≥ 2 vs. <2 years. On multivariate analysis, the number of alkylator therapies and CK, but not % blasts predicted OS from t-MN diagnosis (Figure). Eight (19%) patients proceeded to receive an autologous (n=4) or allogeneic (n=4) transplant for t-MN (Table 1b). There was no survival advantage for patients who underwent SCT for t-MN, compared to those who did not (17 vs. 10 months, p=0.3). Similarly, there was no survival advantage for patients who received allo SCT compared to auto SCT (21 vs. 16 months, p=0.7). Survival for t-MDS and t-AML was also not different (15 vs. 6 months, p=0.2). There is only one long-term survivor in the transplant group (s/p allogeneic SCT). Four of seven (57%) patients died of progressive t-MN, 1 died of progressive MM, progressive MM and t-MN, and TRM each. Conclusions: Lenalidomide, in the setting of HDM, is associated with an increased risk of t-MN. Increased exposure to alkylators, a higher tumor burden, and the presence of CK predict poor survival in t-MN. As HDM/ASCT followed by lenalidomide maintenance remains the standard of care for eligible patients, minimizing exposure to other alkylator regimens may be prudent. While considered the standard, only a minority undergo transplant; and post-transplant outcomes remain poor. Studies to identify prognostic genetic and epigenetic risk factors are ongoing. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND: CMML is a myelodysplastic/myeloproliferative neoplasm with a median survival of 32 months and no therapies that improve its natural history. We have previously demonstrated that CMML bone marrow mononuclear cells (BMNCs) are hypersensitive to GM-CSF and that the GM-CSF axis is a viable therapeutic target (Padron et al., Blood 2013). Lenzilumab is a novel, humaneered IgG1κ monoclonal antibody, with high affinity for human GM-CSF that has activity in preclinical models of CMML. We report a Phase 1 clinical trial testing the safety and preliminary efficacy of this agent in CMML. METHODS: The study was approved by scientific and ethical review boards. This was a multicenter Phase 1 study designed to evaluate the safety and determine the recommended phase 2 dose of lenzilumab in subjects with CMML. Dose escalation proceeded using a standard 3+3 study design to determine the maximum tolerated dose (MTD). Three dose cohorts included 200 mg, 400 mg, and 600 mg, were given IV on day 1 and 15 of cycle 1 and then only on day 1 of subsequent 28-day cycles. Key inclusion criteria included a WHO-defined diagnosis of CMML and a platelet count greater than 20 x103 cells/dL. Response was evaluated utilizing the MDS/MPN International Working Group Criteria (Savona Blood 2015). Pharmacokinetic analysis and pharmacodynamics were evaluated by pSTAT5 by flow cytometry. RESULTS: Between July 2016 and June 2018, a total of 15 patients were enrolled. The median age at study entry was 74 years (range 52-85) and 80% were male. Nine patients were classified as CMML-0, 3 as CMML-1, and 3 as CMML-2. Seventy three percent of patients had normal cytogenetics or -Y. The most commonly mutated genes at screening included TET2 60%, ASXL1 53%, SRSF2 47%, and RAS pathway (i.e. NRAS or CBL) mutations 40%. Nine patients were previously treated with hypomethylating agents and/or experimental therapies, 3 were treated with hydroxyurea only, and 3 were untreated. The mean Hgb was 9.7g/dL (7.6-14g/dL), the mean platelet count was 147 x103 cells/dL (16-942 x103 cells/dL), and 66% of cases were MPN-CMML by the French-American-British classification at study entry. Three patients were enrolled at each dose level and an additional 6 patients were enrolled at 600mg as planned. Consistent with prior studies of lenzilumab, no dose limiting toxicities were identified and no treatment emergent grade 3 or 4 toxicities were reported. The mean duration on therapy was 221.8 days (14-787 days) and the majority of patients discontinued study drug because of disease progression or lack of response (69%). Five of 15 (33%) patients enrolled achieved clinical benefit by MDS/MPN IWG criteria with 3 platelet responses, 1 neutrophil response, and 1 spleen response. An additional patient had bone marrow myeloblast reduction from 6% to 1% which allowed that patient to undergo allogeneic stem cell transplantation. Clinical response was not statistically associated with somatic mutations or changes in pSTAT5 between screening and cycle 3. However, 3 of 4 patients with NRAS mutation achieved clinical benefit or had clinical meaningful bone marrow myeloblast reductions. CONCLUSION: Lenzilumab is well tolerated in patients with CMML, with no grade 3 or 4 treatment emergent adverse events or DLTs reported. Durable clinical benefit was achieved in 33% of patients and one patient was bridged to allogenic transplant, providing proof of concept that GM-CSF inhibition has activity in CMML. The favorable safety and activity profile of lenzilumab warrants future evaluation as part of a combination regimen targeted to specific subtypes more likely to respond, including patients with NRAS mutations. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Komrokji:celgene: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; DSI: Consultancy; Incyte: Consultancy. Lo:Humanigen: Employment. Durrant:Humanigen: Employment. Chappell:Humanigen: Employment. Ahmed:Humanigen: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

Introduction: Germline RUNX1 mutations/deletions result in a Familial Platelet Disorder with propensity to Myeloid Malignancy (FPDMM); an autosomal dominant condition characterized by thrombocytopenia, qualitative platelet defects, and myeloid clonal evolution (MDS and AML). The advent of next generation sequencing (NGS) has allowed detection of clonal cytopenias of unclear significance (CCUS) prior to bone marrow (BM) morphological changes. Somatic RUNX1 mutations (RUNX1MT) can be seen in myeloid malignances, including MDS/MPN overlap syndromes, with an indeterminate prognostic impact. We carried out this study to define the landscape of clonal evolution in FPDMM patients and to compare germline RUNX1MT with somatic mutations. Methods After IRB approval, families with RUNX1MT FPDMM (detected by germline sequencing and deletion/duplication assays) and patients with 2016, WHO-defined MDS/MPN overlap syndromes (except JMML) were included in the study. Index patients with FPDMM underwent BM biopsies and in select cases NGS at diagnosis and then as indicated for follow up, or at disease progression. All MDS/MPN overlap syndrome patients underwent a 29-gene panel NGS assay on BM samples obtained at diagnosis by previously described methods (Patnaik et al., BCJ 2016). Results Eight index patients with FPDMM were included; median age at diagnosis 58 (range, 0-71) years, 3 (38%) males (Figure A). Four (50%) had intragenic deletions involving multiple exons in 3 cases and 2 nucleotides in 1 case, 1 (13%) each had missense, frameshift and splice site mutations, while 1 (13%) patient had a whole gene deletion (Figure C). Notably, in 4 (100%) of 4 patients with large deletions, NGS testing was negative for RUNX1 abnormalities. Four (50%) index patients presented with a myeloid neoplasm; JAK2, SRSF2, ASXL1 mutant CMML, MDS/MPN-U with trisomy 8, AML with del7q and normal karyotype AML (negative NGS). Three (38%) index patients had CCUS at diagnosis with TET2 (67%) being the most common acquired mutation, and others including SRSF2, EZH2, and SF3B1. Cumulatively, myeloid clonal evolution was seen in 7 (88%) patients. Three (38%) index patients (AML-2, CMML-1) underwent allogeneic HCT, and at last follow up were in remission. Median age at myeloid clonal evolution was 61 (range, 4-71) years; median overall survival (OS) from date of myeloid clonal evolution was 12 (range, 7-53) months. For the comparative analysis, 499 patients with MDS/MPN overlap syndromes were included; 382 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T (MDS/MPN with ring sideroblasts and thrombocytosis), 17 (3%) with aCML (atypical CML), and 52 (10%) with MDS-MPN-U (unclassifiable). Median age at diagnosis was 71 (range, 18-99) years, and 329 (66%) were male. RUNX1MT were detected in 57 (11%) patients; 43 (11%) with CMML, 5 (30%) with aCML, and 9 (17%) with MDS-MPN-U. No RUNX1MT were found in MDS-MPN-RS-T. Overall, 62 RUNX1MT were identified in 57 patients, with 52 (39 CMML, 4 aCML, 9 MDS-MPN-U) and 5 (4 CMML, 1 aCML) patients harboring 1 and 2 RUNX1MT, respectively. RUNX1MT spanned the entire coding sequence without specific hotspots (Figure B): 27 (44%) were missense, 2 (3%) involved the splicing sites, 5 (8%) were nonsense and 28 (45%) were frameshift. There was only one RUNX1MT (R273K), seen in both germline and somatic patients (Figures B and C). Compared to wild type patients, RUNX1MT patients with MDS/MPN overlap syndromes had significantly lower platelet counts (median 72 vs 117 x109/L, p<0.0001), without any other phenotypic differences. In the somatic RUNX1MT cohort, co-occurring mutations included ASXL1 (54%), SRSF2 (54%), TET2 (49%), and NRAS (28%), with NRAS (28% vs 13%, p=0.0028), and SRSF2 (54% vs 39%, p=0.0218) mutations clustering with RUNX1MT. Median OS of the entire cohort was 29 (range, 0-170) months. On univariate analysis, while there was a trend for an inferior OS in RUNX1MTvs wild type patients (20 vs 29 months, p=0.0523), the leukemia-free survival was clearly inferior (p=0.0175, Figure D). Conclusion RUNX1 MT FPDMM is a germline predisposition syndrome with a high rate of myeloid clonal evolution (88%), including clonal cytopenias (38%). Germline RUNX1MT have minimal overlap with somatic mutations seen in myeloid malignancies, with NGS testing often missing RUNX1 deletions. Somatic RUNX1MT in myeloid malignancies are significantly associated with thrombocytopenia and an inferior leukemia-free survival. Figure Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Chronic myelomonocytic leukemia (CMML) belongs to the group of "mixed myelodysplastic/myeloproliferative neoplasm". There is currently no effective chemotherapy to treat CMML. Approximately 30% of CMML cases evolve to acute myeloid leukemia (AML) soon after their initial diagnosis, contributing to the poor prognosis of CMML patients. Ras pathway genes are frequently mutated in CMML patients (NRAS:11%; CBL:10%; KRAS:8%). Our whole exome sequencing of CMML patient samples and targeted sequencing of CMML patient-derived myeloid colonies showed that oncogenic NRAS could serve as an initiating or progression mutation. Consistent with human genetics, all the oncogenic Nras mice we and others characterized (NrasG12D/+, NrasG12D/G12D, and NrasQ61R/+), develop myeloproliferative (MP)-CMML like phenotypes.However, oncogenic NRAS alone is insufficient to drive CMML progression or transformation to AML. Additional sex combs-like 1 (ASXL1) is a human homolog of fly Asx. Mutations in ASXL1are predominantly nonsense and frequently identified in all myeloid malignancies (e.g. 40% in CMML). ASXL1mutations predict inferior overall survival in univariable analysis in multiple large CMML cohort studies and are significantly associated with NRASmutations in CMML patients. Therefore, we hypothesize that loss of Asxl1 cooperates with oncogenic Nrasto drive CMML progression. To test our hypothesis, we used Vav-Cre to drive oncogenic Nras expression and/or Asxl1 deletion in hematopoietic system. We refer these mice as NrasG12D/+, Asxl1-/-and NrasG12D/+;Asxl1-/-(NA) mice. Consistent with the literature, Asxl1-/-mice are normal at 6-week old. NrasG12D/+ mice showed mild CMML-like phenotypes, including enlarged spleen, increased white blood cell and monocyte counts, and expanded LSKs (Lin-Sca-1+c-Kit+) and myeloid progenitors (MPs). Compared to NrasG12D/+ mice, NA mice showed more sever phenotypes that were associated with hyperactivated ERK signaling in MPs at both basal level and upon GM-CSF stimulation. Our new NrasG12D/+mice developed a fully penetrant MP-CMML with the median survival of ~430 days. Loss of Asxl1 significantly shortened the median survival of NrasG12D/+ mice to 220 days. Half of NA mice displayed CMML transformation to AML. The transformed AML was serially transplantable to recipients. To investigate the mechanism(s) underlying NA-driven CMML/AML, we performed RNA-Seq analysis in Lin- c-Kit+BM cells from age-matched control, Asxl1-/-, NrasG12D/+, and moribund NA mice with CMML or AML phenotypes. We found 844 differentially expressed genes (DEG) in AML cells compared to control cells (FDR<0.05 and fold change>2). Among these genes, 341 are common between NA and published Nf1+/-;Asxl1+/-AML cells, including AP-1 complex genes (Jun, Junb, Jund and Fosb) that are crucial for malignant propagation in several AML subtypes. The remaining 503 genes are unique in NA AML, including Flt3, one of the mostly mutated genes in AML. These data suggest that our NA AML model demonstrates some distinct mechanisms from Nf1+/-;Asxl1+/-model. In-depth analysis of RNA-seq data is still ongoing. We also examined multiple histone marks using Western blot in BM cells of 6-week old mice. Our data showed that levels of H3K4me3, H3K4me1, and H3K27me3 were downregulated in Asxl1-/-cells, whereas H3K27Ac level was comparable to that of control cells. In contrast, H3K27Ac level was upregulated in NrasG12D/+and NA cells, while levels of H3K4me3, H3K4me1, and H3K27me3 were indistinguishable from those of control cells. BRD4 is a member of the BET family of bromodomain-containing proteins that binds to acetylated histones to activate gene transcription. Thus, we hypothesized that Ras/MEK/ERK signaling and BRD/BET are potential targets to inhibit AML progression. To test this hypothesis, we treated the AML cells and BMT recipients using trametinib (a MEK inhibitor approved by FDA to treat melanoma) and GSK525762 (a pan BET inhibitor under clinical development). We demonstrated thatcombined treatment significantly inhibited the growth of leukemia cells in vitro and prolonged the survival of recipients in vivo. It was more effective than single agent alone. Overall, we established a novel CMML/AML mouse model, which represents a significant group of CMML patients with poor prognosis. Our study provides a strong rational to treat these patients with combined MEK and BET inhibition. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.