Levine, Ross L.

Background: NGS of myeloid mutations is an integral part of AML clinical decision-making. There is currently no information regarding concordance between NGS panels in AML using samples from the same patient across various platforms in different diagnostic laboratories. To study this important question, we analyzed NGS of myeloid mutations in diagnostic samples from The Beat AML Master Trial (BAMT) for newly diagnosed older AML patients, and compared variant calls made between institutional laboratories enrolling the study subject with those made by Foundation Medicine (FM), the central laboratory used for treatment assignment in this precision medicine trial. Methods: We identified newly diagnosed AML patient samples (peripheral blood (PB) and/or bone marrow (BM)) from 2 lead institutions in the BAMT(Ohio State, OSU and Oregon Health and Sciences University, OHSU) that were analyzed by both the institutional and by FM from Nov 15, 2016 to Apr 15, 2019. Samples sent to both laboratories >3 days apart were excluded. Samples were analyzed at the institutional laboratories using their respective NGS mutational panels and by FM using the FoundationOne®Heme(FMH) NGS panel which utilizes capture based sequencing. The OSU NGS assay utilizes sequencing on Illumina MiSeq. The OHSU NGS assay employs semiconductor-based sequencing (Ion Torrent PGM platform). The variant allele frequency (VAF) sensitivity for detection for all 3 laboratories range from 1-2%. We evaluated the ability to identify mutations in 8 genes : FLT3, IDH1/ 2, NPM1, TET2, DNMT3A, WT1 and TP53 used in treatment assignment in theBAMT. A detection cutoff of 2% was used to define the presence or absence of a mutation. Overall, agreement was defined as the number of times the local and central laboratories made the same call divided by the total number of patients. Sensitivity was defined as the number of present calls made locally divided by the number of present calls made centrally, and specificity as the number of absent calls made locally divided by the number of absent calls made centrally. The overall kappa statistic, controlling for institution, provided another measure of agreement between local and central calls, where a value of 1 indicates perfect agreement. Results: 194 patient samples were identified using methods above and analyzed locally at the screening institution (125 at OSU, 69 at OHSU) and centrally at FM. Type of tissue analyzed for variants between local site and FM were 59 PB, 129 BM, and 6 with BM/PB mismatch. Overall agreement in presence/absence calls between local and central results for each of the 8 genes was over 95% (Table 1). There was perfect agreement for NPM1. The sensitivity was above 94% for all genes except TP53 (88.6%) and WT1 (63.6%). Failure to detect a mutation locally was primarily due to reporting of all TP53 variants, including variants of unknown significance (VUS) (5) by FM as agreed upon in the study protocol, detection at low levels below local site sensitivity cutoff (1), detection of variants in a portion of gene not covered at the local site(1)and possible artifact (1). For the WT1 gene, discordance in 5 samples included VUS (3) reported by FM ,a variant detected in a portion of the gene not covered at the local site(1).and difference in leukemic tissue analyzed with mutation not detected by the central laboratory on a PB sample, and present at the institutional lab on a BM sample; affecting the overall agreement and specificity but not sensitivity. Specificity was at least 98% for each of the 8 genes. Finally, most discrepancies in reported mutations in FLT3 (n=2), IDH1 (n=1), IDH2 (n=2), DNMT3A (n=4) and TET2 (n=5) were due to reporting of VUS in one laboratory and not by another. Conclusion: Detection of pathogenic myeloid mutations using orthogonal assays showed a high degree of concordance for genes used in therapeutic assignment on the BAMT.The small number of discordant results, in TP53 and WT1, were attributed to the reporting of VUS. This study illustrates the importance of quality control and standardization as NGS continues to be widely utilized in AML for clinical decision making, with a variety of platforms across multiple laboratories. Our next steps involve evaluating the differences in VAFs reported between local and central laboratories when a given mutation is identified, as well as the potential reasons for observed differences and clinical implications of known pathogenic mutations vs putative VUS. Disclosures Borate: Daiichi Sankyo: Consultancy; AbbVie: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Pfizer: Consultancy. Vergilio:Foundation Medicine: Employment; Roche Holding AG: Equity Ownership. Stein:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Patel:France Foundation: Honoraria; Dava Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Astellas: Research Funding; Abbvie: Research Funding; AI Therapeutics: Research Funding; Forma: Research Funding; Incyte: Research Funding; Kite: Research Funding; Takeda: Research Funding. Stock:Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Schiller:Amgen: Other, Research Funding; Astellas: Research Funding; Biomed Valley Discoveries: Research Funding; Bristol Myer Squibb: Research Funding; Celgene: Research Funding, Speakers Bureau; Constellation Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J&J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding; Agios: Research Funding, Speakers Bureau. Blum:AmerisourceBergen: Consultancy; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Astellas,: Research Funding; Xencor: Research Funding; Forma: Research Funding. Kovacsovics:Pfizer: Research Funding; Jazz: Consultancy; Novartis: Research Funding; Abbvie: Research Funding; Amgen: Consultancy, Research Funding. Foran:Agios: Honoraria, Research Funding. Druker:Pfizer: Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding. Byrd:Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Prelude Therapeutics: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees. Mims:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: The Philadelphia chromosome negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), carry a propensity to evolve into an accelerated phase (MPN-AP, 10-19% blasts in the blood or marrow) and blast phase (MPN-BP, ≥20% in the blood or marrow). Such disease evolution carries a dismal prognosis with a median survival of 3-5 months, and thus the development of innovative treatment strategies represents an urgent unmet clinical need. Responses in patients with MPN-AP/BP to both hypomethylating agents and single-agent ruxolitinib (RUX) have been reported. In vitro, the combination of RUX and decitabine (DEC) are synergistically active in cells derived from patients with MPN-BP and from a murine model of MPN-BP. These observations led us to explore the safety of the combination in MPN-AP/BP in a phase I study. An MTD for RUX in combination with DEC 20mg/m2/d x 5 days was not reached in this study and the recommended Phase II dose of RUX was selected as 25mg BID for the first cycle of therapy followed by 10mg BID in subsequent cycles with a goal to minimize myelosuppression and maintain ambulatory administration (Rampal et al ASH 2016). Here we report the final results of the phase 2 combination of RUX + DEC in patients with MPN-AP/BP (NCT02076191). Methods: We conducted a multicenter, open label, phase II trial in patients with MPN-AP/BP, following a previous diagnosis of ET, PV or PMF. Patients enrolled were treated with the RPTD dose of RUX + DEC on a 28-day cycle until progression of disease, unacceptable toxicity or patient/physician withdrawal. Adverse events (AEs) were assessed using the NCI CTCAE v4.0. The primary endpoint was response rate by cycle 6 using modified Cheson criteria: CR required 0% peripheral blood blasts, ANC >1000/uL, hemoglobin ≥10g/L, and platelets ≥100x109/L; CRi required 0% peripheral blood blasts with incomplete count recovery; and PR required ≥50% decrease in peripheral blood blasts regardless of blood counts. Results: A total of 25 patients were accrued to study. The median age was 71 years (46.7-85.6). 10 patients (40%) had MPN-AP and 15 patients (60%) had MPN-BP. 6 patients (24%) had been previously been treated with single-agent RUX, and 2 patients (8%) had been treated with single-agent DEC. Median palpable spleen size was 7cm (range 0 - 20cm). The median number of cycles of treatment received on study was 4 (range 1 - 20). The most common grade 3/4 non-hematologic AEs observed were pneumonia (16%), febrile neutropenia (16%) and infection (8%). The most common grade 3/4 hematologic AEs were anemia (16%), neutropenia (12%) and leukopenia (8%). The most common reasons for study discontinuation were AEs in 9 patients (36%), disease progression in 5 (20%) patients and hematopoietic cell transplantation in 2 (8%) patients. Eighteen (72%) patients were evaluable for response. The overall response rate (CR+CRi+PR) was 61% (11/18 patients). CRi was observed in 2 patients (11%), PR in 9 patients (50%) and no response in 7 patients (39%). The median time to CR/PR was 3.4 mos (95% CI: 2.1 - not estimated). The median peripheral blood blast count reduction was 80.1% (range 71.4-100%) (Figure 1A). The median reduction in spleen size by palpation was 70.5% (range 0-100%) (Figure 1B). Median overall survival for all patients was 7.6 months (95% CI: 4.3 - Not estimated); 9.7 months (95% CI: 4.3 - Not estimated) for MPN-BP and 5.8 months (95% CI: 3.6 - Not estimated) for MPN-AP (Figure 2). Three patients (17%) were able to proceed to HCT. The results of ongoing genomic, histopathologic and cytokine profiling studies will be available for presentation at the meeting. Conclusions: The combination of RUX+DEC has activity in patients with MPN-AP/BP. The drug combination is generally well tolerated and can be administered as an outpatient, a distinct advantage over intensive induction therapies that have not shown significant benefit in these patients. Additional work is ongoing to assess the genomic and clinical variables predictive of response with this combination therapy approach. Given the tolerability and efficacy, this regimen may be of particular benefit to patients who are not candidates for intensive therapies. Disclosures Rampal: Constellation: Research Funding; Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Jazz: Consultancy, Honoraria. Mascarenhas:Celgene: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Novartis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Promedior: Research Funding. Wang:Jazz: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Novartis: Speakers Bureau. Gerds:Celgene: Consultancy; Apexx Oncology: Consultancy; Incyte: Consultancy; CTI Biopharma: Consultancy. Heaney:Roche: Consultancy, Research Funding; Incyte: Research Funding; Blueprint: Research Funding; Novartis: Research Funding; Decipheral: Research Funding. Abboud:Agios: Honoraria; Jazz: Honoraria, Speakers Bureau. Kremyanskaya:Incyte: Research Funding. Odenike:ABBVIE: Honoraria, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI/Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotherapy Science: Research Funding; Agios: Research Funding; Celgene: Research Funding; NS Pharma: Research Funding; Janssen: Research Funding; Astex: Research Funding; Gilead Sciences: Research Funding. Levine:Roche: Consultancy, Research Funding; C4 Therapeutics: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; Novartis: Consultancy; Isoplexis: Equity Ownership. Mesa:Novartis: Consultancy; NS Pharma: Research Funding; Genentech: Research Funding; UT Health San Antonio - Mays Cancer Center: Employment; CTI Biopharma: Research Funding; Gilead: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; Incyte Corporation: Research Funding; Promedior: Research Funding. Dueck:Phytogine: Employment; Bayer: Employment; Pfizer: Honoraria. Hoffman:Incyte: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Merus: Research Funding; Summer Road: Research Funding.

Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Abstract Background: Eastern Cooperative Oncology Group trial E1900 (E1900) showed that induction therapy with a high daily dose of daunorubicin (90 mg/m2) (DNR 90) improves survival in younger patients (pts) (<50 yrs) and intermediate (int) cytogenetic risk AML, but at 2 years of follow-up no benefit was seen in older pts (50-60 yrs), or in those with unfavorable cytogenetic risk or FLT3-ITD mutant AML (Fernandez et al. N Engl J Med 2009). Here we update results of E1900 with longer follow-up, focusing on the benefit of DNR 90 in cytogenetic and common molecular subgroups. Methods: Overall survival (OS) was measured from randomization for induction therapy to death from any cause (censored at last contact). Hazard ratios (HR) for death were computed using univariate and multivariable Cox proportional hazards models; multivariable Cox models were adjusted for sex, age, hemoglobin level, leukocyte count, platelet count, and cytogenetic profile. All conclusions regarding the impact of DNR 90, unless noted, are similar based on univariate and multivariable analysis. Results: Overall, 657 pts were enrolled with a median follow-up of 80.1 months. The HR for death in the DNR 90 group as compared with the standard-dose daunorubicin (45 mg/m2) (DNR 45) group was 0.74 (p=0.001). Pts <50 yrs benefited from DNR 90 (p = 0.002) while those >=50 yrs were not proven to benefit (p = 0.12). Pts with favorable (fav) and int. cytogenetic risk benefited from DNR 90 (p = 0.03 and p = 0.02, respectively). A benefit for pts with unfavorable cytogenetic risk was seen on multivariable analysis (p = 0.04). Impact of DNR 90 by mutation status: The 3 most common mutations were FLT3-ITD (24%), NPM1 (26%), and DNMT3A (24%). AML pts with any of these 3 mutations benefited from DNR 90 (p = 0.009, p = 0.002, and p = 0.02, respectively). FLT3-ITD pts who received DNR 90 had a 4-yr OS of 31%. Benefit was seen in pts age 50-60 with FLT3-ITD or NPM1 mutation (p = 0.02 and p = 0.04, respectively). No benefit of DNR 90 was seen in a small cohort of pts with MLL-PTD (p = 0.06). Benefit of DNR 90 in FLT3-ITD, NPM1, and DNMT3A mutant AML was confirmed in the int. cytogenetic risk group. Impact of DNR 90 on prognostic impact of NPM1: The presence of an NPM1 mutation conferred an improvement in OS in the DNR 90 group which was not seen in the DNR 45 group (p = 0.01 vs p = 0.3). This finding was confirmed in the int. cytogenetic risk group. Conclusion: With median follow-up of over 6 years on E1900, we confirm that DNR 90 improves outcome in pts with fav/int cytogenetic risk, DNMT3A or NPM1 mutant AML, or age < 50 (Patel et al. N Engl J Med 2012). Additionally, we now demonstrate that DNR 90 additionally benefits pts with FLT3-ITD AML, and pts with unfavorable cytogenetic risk, regardless of age. Moreover, we show that the favorable prognostic impact of the NPM1 mutation is only present when pts receive DNR 90. Given the benefit of DNR 90 across all cytogenetic risk groups as well as common molecularly defined subgroups of AML, DNR 90 should be the standard for all pts up to age 60 who are candidates for induction chemotherapy. Table HR for death by AML cohort. Subgroup N Univariate Model DNR 45 DNR 90 HR (DNR 90/DNR 45) & 95% CI Wald P All patients (n=657) Overall 330 327 0.74 (0.61, 0.89) 0.001 Age < 50 yrs³ 50 yrs 188 142 172 155 0.66 (0.50, 0.85) 0.81 (0.62, 1.06) 0.002 0.12 Cytogenetic Favorable Intermediate Unfavorable 38 232 59 51 212 63 0.51 (0.28, 0.93) 0.76 (0.61, 0.96) 0.79 (0.54, 1.16) 0.03 0.02 0.22 FLT3-ITD WT MUT 215 83 241 64 0.74 (0.59, 0.92) 0.61 (0.42, 0.89) 0.008 0.009 MLL-PTD WT MUT 290 16 296 15 0.70 (0.58, 0.86) 0.46 (0.21, 1.04) 0.0004 0.06 NPM1* WT MUT 180 65 192 65 0.70 (0.55, 0.89) 0.50 (0.32, 0.78) 0.003 0.002 DNMT3A WT MUT 177 61 194 58 0.66 (0.52, 0.85) 0.62 (0.41, 0.94) 0.001 0.02 * Statistically significant test of interaction (p<0.2) Figure 1A: Overall Survival by NPM1 Mutation Status and Treatment Arm Figure 1A:. Overall Survival by NPM1 Mutation Status and Treatment Arm Figure 1B: Overall Survival of FLT-ITD Mutant AML Patients by Treatment Arm Figure 1B:. Overall Survival of FLT-ITD Mutant AML Patients by Treatment Arm Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 851 Clinical studies have demonstrated that acute myeloid leukemia (AML) is heterogeneous with respect to presentation and to clinical outcome, and many studies have shown that cytogenetics can be used to improve prognostication. More recently, it has been shown that FLT3, NPM1, and CEBPA mutations can be used to further risk stratify AML patients. Although recent studies have identified novel, recurrent somatic mutations in AML in genes including TET2, ASXL1, IDH1, IDH2, and PHF6, their prognostic value has not yet been evaluated among patients treated on a large phase III clinical trial. Here we report full-length DNA resequencing of FLT3, NPM1, CEBPA, H/K/NRAS, KIT, WT1, TET2, ASXL1, IDH1/2, TP53, RUNX1, PTEN, and PHF6 in pre-treatment genomic DNA from 398 patients with de novo AML younger than 60 years of age enrolled in the ECOG E1900 protocol. We could identify a clonal alteration, defined as a somatic mutation or cytogenetic abnormality, in 91.2% of all patients; 42% had 1 somatic alteration, 36.4% had 2 alterations, 11.3% had 3 alterations and 1.5% had 4 or more somatic alterations. Mutational Analysis: We identified somatic mutations in FLT3 (37% total; 30% ITD, 7% TKD), NPM1 (14%), CEBPA (10%), TET2 (10%), NRAS (10%), WT1 (10%), KIT (9%), IDH2 (8%), IDH1 (6%), RUNX1 (6%), ASXL1 (4%), PHF6 (3%), KRAS (2.5%), TP53 (2%), and PTEN (1.5%) (Figure 1A). We then performed correlation analysis to determine the frequency of different mutational combinations in AML patients. In addition to previously identified mutational correlations (FLT3 and NPM1, KIT and core binding factor leukemia), we found that FLT3 and ASXL1 mutations were mutually exclusive in this large cohort (p =0.0008). In addition, we found that IDH1/IDH2 mutations were mutually exclusive of both TET2 (p=0.02), and WT1 (p=0.01) mutations consistent with overlapping roles in AML pathogenesis (Figure 1B). Integration of mutational data with response data and outcome: Mutations in ASXL1 (CR rate 33.3% versus 66.5% for ASXL1-WT patients; p= 0.009) were enriched in patients who did not respond to induction chemotherapy. We found that mutations in FLT3 (p=0.0005), ASXL1 (p=0.005; Figure 1C), and PHF6 (p=0.02) were associated with reduced OS. Notably, the adverse effects of ASXL1 and PHF6 mutations on outcome were restricted to patients treated with standard dose daunorubicin (p<0.01 for each mutational cohort); ASXL1 and PHF6 mutational status did not affect outcome in patients treated with high-dose daunorubicin, demonstrating that increased intensity induction chemotherapy can improve outcomes in genetically defined poor risk AML subsets. We also found that mutations in CEBPA (p= 0.04) and in IDH2 (p=0.003) were associated with improved OS; the favorable impact of IDH2 mutations on outcome was exclusive to patients with IDH2 R140Q mutations (Figure 1D). Within the subset of patients with cytogenetically defined intermediate risk AML, we found, as expected, that FLT3-ITD mutations were associated with reduced OS and CEBPA mutations were associated with improved OS. Importantly, we found that IDH2 R140Q mutations were associated with improved OS (p=0.003) in patients with intermediate risk AML regardless of FLT3 mutational status. In contrast, TET2 mutations (P=0.0002) and PHF6 (p<0.0001) were associated with reduced OS in intermediate risk, FLT3-WT, AML. Summary: These data provide important clinical implications of genetic alterations in AML and provides insight into the multistep pathogenesis of adult AML. We identified novel mutations that impact response to induction chemotherapy and predict overall outcome, including ASXL1, PHF6, TET2 and IDH2, suggesting these mutations can be used to improve AML risk stratification and therapeutic decisions in AML patients 18–60 years of age. Most importantly, we identify TET2, IDH1/2, and WT1 mutations as mutually exclusive somatic alterations that form a novel mutational complementation group that contributes to AML pathogenesis. Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3862 Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs). Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion. Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML. Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort. Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR. Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant. The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328). We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree. Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. Figure 1. Figure 1. Disclosures Loren: Merck: Research Funding. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 3070 Background: EZH2 and ASXL1 mutations were recently described in a spectrum of myeloid malignancies; mutational analysis of small patient cohorts has suggested the highest mutational frequency in myelofibrosis (MF) and chronic myelomonocytic leukemia (CMML). The current study seeks to determine i) EZH2 and ASXL1 mutational frequencies in WHO-defined subcategories of MF, CMML and blast-phase myeloproliferative neoplasm (MPN), ii) if these mutations are mutually exclusive of TET2, IDH, JAK2 and MPL mutations and iii) clinical correlates of ASXL1 and EZH2 mutations in primary MF (PMF) and CMML. Methods: The study population included 94 patients: 46 PMF, 22 post-polycythemia vera/essential thrombocythemia MF (post-PV/ET MF), 11 blast-phase MPN and 15 CMML (10 CMML-1 and 5 CMML-2). High throughput DNA resequencing was used to screen archived bone marrow for EZH2, ASXL1, TET2, IDH, JAK2 and MPL mutations. Results: ASXL1 mutations were identified in all disease categories, including PMF (13%), post-PV/ET MF (23%), blast phase MPN (18%), and CMML (20%). We identified somatic mutations in TET2 in 15%, 14%, 18%, and 13% of PMF, post-PV/ET MF, blast phase MPN, and CMML, respectively. By contrast, mutations in EZH2 and IDH1/2 were less frequent. EZH2 mutations were seen in 3 out of 46 PMF patients (7%) and were not observed in patients with post-PV/ET MF or blast phase MPN. Mutations in IDH1/2 were restricted to blast-phase MPN (36%) and PMF (7%). No mutations in EZH2 or IDH1/2 were seen in CMML. Although we identified frequent TET2 and ASXL1 mutations, we only identified one patient with concurrent mutations in both genes. Three ASXL1 mutation-positive patients also had mutations in EZH2 or IDH and one patient had concurrent ASXL1, TET2 and IDH mutations. In addition, 7 ASXL1, 7 TET2, and 1 IDH mutated patients were JAK2V617F-positive. MPL mutations were also documented in all three mutation categories. All EZH2- and ASXL1-mutated PMF patients displayed normal karyotype and none underwent leukemic transformation during follow-up. Furthermore, mutated versus unmutated patients, in both instances, were not significantly different in age and sex distribution or clinical characteristics. The 3 EZH2-mutated PMF patients died after 29, 48 and 67 months from the time of mutation analysis. In univariate analysis, the presence of mutant ASXL1 in PMF was associated with worse survival (p=0.06) but the borderline significance was lost during multivariable analysis that included risk stratification according to DIPSS (Passamonti et al. Blood 2010; 115: 1703–1708). The 3 ASXL1 mutated CMML cases were alive after 40, 34 and 12 months from time of mutation analysis and none of them had progressed to acute leukemia; karyotype was normal in two of the patients and showed isolated trisomy 8 in one. Conclusions: ASXL1 mutations are as frequent as TET2 mutations in MF and CMML. In contrast, EZH2 mutations are infrequent and cluster with PMF. ASXL1 and EZH2 mutations are not mutually exclusive events, seem to be associated with normal karyotype and do not appear to be leukemogenic or prognostically detrimental in PMF or CMML. Disclosures: No relevant conflicts of interest to declare.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.