Abstract: Abstract 529 The nucleoporins (NUP) are a family of proteins, which form the building blocks of the nuclear pore complex. Translocations involving NUP family members NUP214 and NUP98 have been reported in acute myeloid leukemia (AML). DEK/NUP214 (also known as DEK/CAN, t(6;9)) is a known translocation in AML that is highly associated with FLT3/ITD and adverse outcome (see presentation by Moraleda P. et al). By Whole Transcript Sequencing (RNA Seq) we identified 2 cases of NUP98/NSD1 fusions by patients with cytogenetically normal (CN-) AML, both with FLT3/ITD. Recent studies by Hollink I. et al demonstrated high prevalence of FLT3/ITD in patients with NUP98/NSD1 translocations and its association with adverse outcome. Given the significant overlap between DEK/NUP214, NUP98/NSD1 fusions and FLT3/ITD, we studied the prevalence of these fusion transcripts in patients with FLT3/ITD to define the prognostic significance of genomic alteration and their contribution to clinical outcome. Pretreatment samples from patients with FLT3/ITD (N=117) as well as those with CN-AML (N=166) treated on COG-AAML0531 underwent evaluation for NUP98/NSD1 fusion transcripts RT-PCR. Fusion transcripts, as well as the break point junction was verified by Sanger sequencing. Presence of NUP98/NSD1 transcript was correlated with disease characteristics and clinical outcome in patients with FLT3/ITD. In patients with FLT3/ITD (N=117) NUP98-NSD1 was detected in 15 patients (13%). Demographics and disease characteristics of FLT3/ITD patients were compared between those with and without NUP98/NSD1. There were no significant differences in the median age (11 vs. 13 years, p=0.19) or blast % (85% vs. 80%, p=0.23) at diagnosis between patients with and without NUP98/NSD1. Mutations in NPM1 and CEBPA were not detected in those with dual FLT3/ITD and NUP98/NSD1; however, WT1 was significantly more common in FLT3/ITD patients with NUP98/NSD1 than in those without it (43% vs. 13%, p=0.01). Complete remission (CR) rates were compared in FLT3/ITD patients with and without NUP98/NSD1. CR rate in those with and without NUP98/NSD1 was 28% vs. 73% (p=0.002). Moreover, FLT3/ITD patients harboring NUP98/NSD1 were also more likely to have post-induction minimal residual disease (MRD) than those without NUP98/NSD1 (75% vs. 40.6%, p=0.03). In addition to patients with NUP98/NSD1, eight additional patients had NUP214/CAN translocation (i.e., t(6;9)), and cumulatively, translocations involving these two NUP genes accounted for 20% of patients with FLT3/ITD. CR rates in FLT3/ITD-positive patients with and without NUP translocations was determined. Those with NUP98 or NUP214 translocations had a CR rate of 40% compared to 74% in those without NUP translocations (p= 0.001). Further, the presence and prognostic significance of NUP98/NSD1 was then evaluated in CN-AML. The prevalence NUP98/NSD1 in this patient population was 7.8%. Of note, seventy-nine percent of the CN-AML patients with NUP98/NSD1 harbored FLT3/ITD as well. The CR rate for CN-AML harboring NUP98/NSD1 was significantly lower than in those without it (50% vs. 79.5%, p=0.03). When analyzing CN-AML patients who harbored both NUP98/NSD1 and FLT3/ITD, only 33% achieved CR, whereas all patients who with NUP98/NSD1 without FLT3/ITD achieved CR. Although the CR rate does not appear to be affected, AML patients with FLT3-ITD have higher relapse rates and therefore inferior outcome. We now show that among patients with FLT3/ITD, those with concurrent NUP fusions have a very low-rate of CR and high post-induction MRD than those without this fusion. The high prevalence of FLT3/ITD among NUP patients is likely not random and it is conceivable that NUP and FLT3/ITD have cooperating functional consequences that lead to a distinct leukemic phenotype and enhanced drug resistance. In conclusion, the presence of NUP in FLT3-ITD patients identifies a distinct subgroup of patients with a very high-rate of induction failure. FLT3-ITD patients should be further categorized according to the presence of NUP to improve risk-stratification of pediatric and young adult AML patients and help clinicians to identify those at very high-risk for induction failure. New therapy strategies are needed for this subgroup of patients with highly resistant disease. Disclosures: Loken: Hematologics, Inc: Employment, Equity Ownership. Pardo:Hematologics Inc: Employment.

Abstract: Background: Both cytogenetic and molecular abnormalities have been identified in acute myeloid leukemia (AML) that provide the framework for diagnostic classification and risk-stratification schemes. However, there are currently only a small number of informative prognostic markers, and it is a recurrent clinical observation that this limited battery fails to accurately predict outcome for many patients. This highlights the need for additional and refined tools to characterize disease risk in AML. Patients and Methods: For gene discovery, gene expression array data from 211 pediatric patients with AML were queried. Subsequently, expression of the gene of interest was quantified in cryopreserved pretreatment specimens from patients enrolled on the AAML03P1 and AAML0531 trials: AAML03P1 was a pilot phase 3 study investigating the feasibility of combining gemtuzumab ozogamicin (GO) with intensive chemotherapy in pediatric patients up to age 30, whereas AAML0531 was a phase 3 study on 〉 1,000 patients testing the value of GO addition to chemotherapy in a randomized fashion. Taqman-based quantitative reverse-transcriptase PCR was used to quantify mRNA expression of the gene of interested and the housekeeping gene, b-glucuronidase (GUSB). Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa), high risk (-5/5q-, monosomy 7, or FLT3-ITD with high allelic ratio), or standard risk (all other patients with cytogenetic/molecular data). Results: Multimerin-1 (MMRN1), a member of the elastin microfibrillar interface protein (EMILIN)/multimerin family that may mediate cellular adhesion via integrin receptors, was identified in gene expression array data as a gene whose expression varied widely, with expression levels possibly related to patient outcomes. To study this possible relationship further, we quantified MMRN1 mRNA in 183 participants of AAML03P1 and correlated expression levels with clinical outcome. MMRN1 expression varied 〉 80,000-fold relative to GUSB. Patients with the highest MMRN1 expression (4th quartile, corresponding to a relative MMRN1 expression of 〉 0.5) indeed had inferior event-free survival (EFS; P 〈 0.002) and higher relapse risk (P 〈 0.004), suggesting that high MMRN1 expression could serve as adverse prognostic factor in pediatric AML. To further validate these findings, we quantified MMRN1 expression in 750 participants of AAML0531, and correlated expression levels with clinical outcome and disease characteristics. In 740 of the 750 patient specimens, MMRN1 was detectable, varying 〉 130,000-fold relative to GUSB. Using a cut-off for high MMRN1 expression as defined in the AAML03P1 cohort (i.e. relative MMRN1 〈 0.5 [n=590] vs ≥0.5 [n=160] ), we found that patients with high MMRN1 expression had a lower response rate after the first course of induction therapy (67% vs. 77%, p=0.013) and more likely minimal residual disease after this initial chemotherapy cycle (43% vs. 26%, p=0.001). They also had inferior 5-year overall survival (OS; 44±9% vs. 69±4%, p 〈 0.0001), lower 5-year EFS (32±8% vs. 54±4%, p 〈 0.0001), and higher 5-year relapse risk (57±10% vs. 35±5%, p 〈 0.0001). At least partially, these differences could be attributed to associations between MMRN1 expression and disease characteristics. Specifically, patients with high MMRN1 expression were less likely to have low-risk disease (P 〈 0.001), and more likely had standard-risk (P 〈 0.001) or high-risk (P 〈 0.001) disease compared to those with low MMRN1 expression. However, after adjustment for disease risk, age, and treatment arm, high MMRN1 expression remained statistically significantly associated with inferior OS (hazard ratio [HR]=1.37 [95% confidence interval: 1.05-1.86] p=0.022), and a trend toward lower EFS (HR=1.25 [0.98-1.59] p=0.077) in multivariate cox models. Conclusion: This study identifies high MMRN1 expression as a novel adverse prognostic factor in pediatric AML. Although the association between adverse outcome and high MMRN1 expression is at least partly due to the higher proportion of adverse-risk features among AML patients with high MMRN1 expression, high MMRN1 expression continued to be associated with inferior survival even after adjustment of disease risk. Disclosures No relevant conflicts of interest to declare.

Abstract: Despite documented success of IST in tx of aplastic anemia (AA), a significant minority of pts remain refractory to tx and most clinical responses are incomplete. Due to comorbidities, HSCT may not be available for all refractory pts and until recently anabolic steroids were the mainstay of salvage tx. With the approval of the novel cMpl agonist eltrombopag (E), first in refractory and now in the upfront setting for SAA, the tx paradigm has changed. Clonal evolution from AA to MDS remains a serious complication. With the advent of E, a valid question proposed is whether the growing use of E would improve recovery rates thereby decreasing the rate of clonal evolution or if stimulation of growth factor receptors by E would potentially increase progression to sMDS. At our institution, 308 AA pts were managed from 1998-2018. Of these, 35 (median age 67 yrs, f/u 42 mo, F: M 17:18) were tx with E for IST-refractory AA (50-150 mg PO QD), with median tx duration of 19 mos; in contrast 202 pts tx in the non-E era received ~2 tx lines (median age 40 yrs, f/u 60 mo, F: M 99:103). Response was evaluated by sustained improvement in blood counts and transfusion independence after 12 wks of tx. This was attained in 66% (23/35). 14% (5/35) had stable disease w/intermittent transfusions (1 underwent HSCT). Among non-responders 1 developed PNH prompting tx w/eculizumab, 1 received HSCT and 2 progressed to MDS/AML. In non-E SAA pts, ORR to 1st tx was 61%. 18 progressed to hemolytic PNH, 20 to MDS/AML and 49 to HSCT. The MDS/AML evolution rate from SAA in pre-E era has been~15% in 10yrs(Socié, G et al. 2000). Our analysis was confirmatory, with median time to progression of 4 yrs. This implies that no firm estimates can be made on the impact of E on MDS progression on clinical grounds alone. Somatic mutations (SM) typical of MDS have been detected in AA/PNH. Studies show that SM present at evolution to MDS can be detected at AA dx in 30% pts. Clearly, some of the SM seen in AA constitute a progression risk, while others likely reflect clonal hematopoiesis. We studied clonal kinetics using deep NGS to identify early signs of clonal evolution/eradication and subsequently predict MDS risk. Reported is our retrospective analysis using a case control approach. 35 E-tx pts were matched with 32 non-E tx pts using response rates and clinical parameters. Serial samples were obtained at start and end of tx for both groups. 4/35 E pts had a chromosomal abnormality at dx(t[10;18],+Y,+12 and del11q, i(7)(q10); latter 3 resolved on f/u). In non-E pts, 3 had cytogenetic abnormality at dx (del16q,+15,+Y; latter two acquired + 6 and -7). Due to close PNH association we evaluated E related size of PNH clone. PNH granulocyte expansion was seen in 11/35 after E and in 5/32 non-E pts. In the E group, 2/35 progressed to MDS/AML compared to 7/32 in the non-E group (p=.08). Similarly there was no difference in PNH progression (p=.36). Serial NGS analysis for both groups revealed a ≥1 SM per pt (BCOR, PIGA, ASXL1, DNMT3A, etc.) in 14 E- pts vs 4 in control group (U2AF1, NF1,PHF6,SAMD9L). Average VAF of BCOR was 19% and PIGA was 24% in E and it was 34% in non E. At the end of observation period, 22/35 (E) had ≥1 SM vs 12/32 in control. NGS post tx noted that 5/35 w/single SM (CEBPA, EZH2, SAMD9L, U2AF1, TP53) had minimal expansion of their clone. Only the pt w/EZH2 acquired 2 additional clones of RUNX1 and U2AF1. The pt w/TP53 developed MDS. 6 pts expanded their original clone (BCOR, ASXL1/U2AF2, PIGA, VHL, NRAS, PIGA) of which the latter 4 acquired 1 additional SM (ZRSR2, PIGA, ASXL1, BCORL1). 1 pt had regression of the RIT1 clone, but acquired a BCOR. 2 others had transient clones of BCOR and DNMT3A at dx w/acquisition of ASXL1. Of those w/baseline SM, 8/21 acquired new SM, of which 1 developed AML w/ gain of DNMT3A, ASXL1 and U2AF1. In the controls, 2 pts w/NF1 and SAMD9L expanded their original clone and acquired ASXL1/SETBP1 and TP53 respectively, w/both having MDS/AML progression. 1 pt had a decrease in U2AF1 and another had a transient PHF6 clone. Pts without baseline SM acquired ~2 SM during the disease course. Many of these are the same SM classically found in MDS/AML. To date, there are no data to support an increased clonal evolution risk with use of E in AA pts either on clinical grounds or by molecular testing. However, presence of certain somatic mutations (CBL, SETBP1, DTA andRUNX1) strongly associated w/MDS/AML progression may warrant caution in the application of E or at least close molecular monitoring. Disclosures Thota: Incyte: Speakers Bureau. Nazha:MEI: Consultancy. Carraway:Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: Background: AML and MDS are heterogeneous myeloid neoplasias (MN) characterized by varied pathogenic mechanisms and associated with mixed clinical outcome. Five-year overall survival rates for younger AML patients (pts) are between 40-50% and for elderly pts, a dismal 10%. Until recently, molecular information has informed risk stratification for MN patients, but targeted therapies were limited (i.e ckit, flt3 inhibitors). Clinical characterization of IDH1/2mutant MN is of particular interest since the recent availability of specific IDH1/2inhibitors (i.e., enasidenib and ivosidenib). Datasets describing the outcome for patients with MN harboring IDH1/2mutations are critical to inform the potential utility of these novel IDH inhibitors as single agents or in combination with standard of care. Ongoing clinical trials are investigating the addition of IDH inhibition for upfront AML therapy. In this retrospective study, we combined cohorts from three institutions to enable reporting of the largest cohort (N=425) of IDH mutated MN pts in order to establish a baseline for therapeutic outcomes in an era which pre-dates the availability/addition of IDH inhibitors. Methods:We identified 425 patients from three large academic centers with a confirmed diagnosis of IDH1/2mutant AML (n=387), MDS (n=29) or MPN (n=9). Blood and bone marrow samples were analyzed for the presence of recurrent somatic mutations using NGS based multi-gene targeted sequencing panels. Demographic and disease related variables were annotated for initial treatment, response to therapy (IWG criteria) and subsequent survival details (overall survival, progression free survival). Time to first relapse and duration of remission was collected for 107 patients to date. Comprehensive response assessment details were available for 234 patients. Results: Of the 425 patients, 165 had a mutation inIDH1and 264 had a mutation in IDH2(82% IDH2R140, 18% R172K). Four cases had co-occurring mutations in IDH1 & IDH2. IDH1mutant cases were younger than IDH2mutant cases (62 vs. 65 years, p=0.05), predominantly male (50% vs.38%, p=0.01) and more likely to have intermediate risk AML (68% of IDH1/IDH2mutant cases had normal karyotype). A majority of these patients were treated with cytarabine-based intensive chemotherapy (n=362). Hypomethylating agents (n=29) or other less intensive therapies (n= 27) were also used. The overall response rate (ORR) to initial therapy was 65%. Response rates were similar for patients with both IDH1and IDH2mutation (i.e, 66% to any therapy). Response to intensive chemotherapy was 68% and 64% in IDH1and IDH2mutant cases respectively. As expected IDH1/2 mutant younger pts ( 〈 60 years) had a higher response rate to intensive chemotherapy (81% and 80 % respectively). The ORR for HMA therapy was 73% (64% IDH1, 80% IDH2mutants). 34% of IDH1mutants and 30%IDH2mutant patients underwent allogeneic stem cell transplantation. We have analyzed the impact of co-occurring somatic mutations on response to intensive chemotherapy. Non-responders to intensive therapy were enriched for RUNX1(26% vs.7%, p=0.002), SRSF2(34% vs.19%, p=0.045), and ASXL1 (18% vs.9%, p=0.11) mutations and were less likely to have NPM1 (24% vs. 51%, p=0.001) mutation. The median overall survival for IDH1 and IDH2 mutant MN caseswas 16.6 and 19.1 months, respectively. Conclusions: IDH1/2 mutated young AML patients appear to have chemo-sensitive disease. Despite excellent initial responses to intensive and non-intensive chemotherapy, the overall survival for IDH mutated pts was poor with shorter than expected remissions. Co-occurring mutations in RUNX1 and SRSF2 appeared to confer therapeutic resistance. Ongoing combination and maintenance strategies with targeted IDH1/2 inhibitors in conjunction with traditional therapies offer the potential to improve upon these outcomes in IDH1/2mutant MN. Future studies exploring the impact of early transplantation on overall survival for IDH1/2mutated MN are needed Disclosures Thota: Incyte: Speakers Bureau. Carraway:Novartis: Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Griffiths:Pfizer, Inc.: Research Funding; Novartis, Inc.: Research Funding; Celgene, Inc: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Alexion Inc.: Honoraria, Research Funding. Nazha:MEI: Consultancy. Gerds:CTI Biopharma: Consultancy; Apexx Oncology: Consultancy; Celgene: Consultancy; Incyte: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Wang:Amgen: Consultancy; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: Abstract 3555 Acute myeloid leukemia (AML) is characterized by various molecular abnormalities impairing differentiation and promoting proliferation. Survivin, a small inhibitor-of-apoptosis protein encoded by the BIRC5 gene, plays a critical role in apoptosis, cell division and proliferation. Relatively small studies in AML have previously shown expression of Survivin and its splice variants correlates with patient survival, white cell count and cytogenetics. Recent data have also demonstrated that Survivin is a transcriptional target of the FLT3-STAT pathway and regulates proliferation of FLT3-mutated AML. We performed complete molecular and expression characterization of the Survivin (BIRC5) gene in childhood AML. The entire coding sequence of the BIRC5 gene was sequenced in diagnostic specimens from 100 children with AML. No disease associated alterations were identified in the 100 patients tested. We further performed quantitative Survivin mRNA expression in 306 patients as well as evaluated for Survivin transcript splice variants in 96 patients with de novo AML treated on two multi-centre clinical trials (CCG-2961 and AAML03P1) to test the hypothesis that Survivin expression would correlate with both FLT3 mutational status and clinical outcome. Quantitative Survivin expression in AML blasts from diagnostic samples was performed by RT-PCR using TaqMan. When normalized to Survivin expression in normal peripheral blood mononuclear cells, Survivin expression 〉 1.0 correlated with an immature phenotype (FAB M0 12% vs. 1%, P = 0.001), monosomy 7 (11% vs. 1%, P = 0.008) and risk group. 26% of high Survivin expressors ( 〉 1.0) were high risk vs. 12% of low expressors (P = 0.024). Conversely, 37% of low expressors (≤1.0) were low risk compared to 16% of high expressors (P = 0.011). Correlation of Survivin expression level with clinical outcome failed to show association with remission status or clinical outcome. Splice variants were identified by amplification of the entire BIRC5 transcript and fragment length analysis of the amplified products. Three distinct splice variants were identified with the most prominent product (peak 2), identified as Survivin-wt, expressed in all samples tested. Expression of the smaller isoform (peak 1) and larger isoform (peak 3) varied from patient to patient. Ratio of peak 3 (P3) to peak 1 (P1) product was determined for each patient (P3/P1 ratio) as a measure of expression of the two variants. Alternate variant ratio varied from 0 (primarily P1 product) to 〉 10 (primarily P3 product). Clinical and laboratory characteristics and outcome was evaluated in patients with isoform ratio ≥1 vs. those with lower ratio. A P3/P1 ratio ≥1 was present in 24% of patients (n = 22) and was associated with high WBC count (median WBC 69.4 vs. 19.4 x103/uL, P = 0.025), M5 FAB phenotype (36 vs. 12%, P = 0.02) and +8 cytogenetics (46% vs. 0 %, P 〈 0.001). In support of our initial hypothesis, there was a trend toward a lower P3/P1 ratio ( 〈 1) in patients wild-type for FLT3 (78% vs. 55%, P = 0.054). The correlation between P3/P1 and clinical outcome was of most interest. Patients with a P3/P1 ratio ≥1 had significantly lower complete remission rates and higher rates of induction failure after both course 1 (45% vs. 88%, P 〈 0.001 and 23% vs. 3%, P = 0.009 respectively) and course 2 of induction therapy (57% vs. 80%, P = 0.044 and 24% vs. 5%, P = 0.026 respectively). Consistent with the poor response to induction, patients with a P3/P1 ratio ≥1 had dramatically inferior 5 year overall (36 vs. 60%, P = 0.011) and event-free survival (23 vs. 53%, P = 0.001) than those with P3/P1 〈 1. When analyzed by risk-group, this correlation remained significant in standard and high-risk patients but not low-risk patients. In summary, these data suggest that the presence of a high P3/P1 ratio of alternate Survivin splice variants is strongly associated with refractory disease and inferior outcome for children with de novo AML. The biological reasons underpinning these findings are yet to be delineated, but it is plausible the ratio of alternate Survivin splice variants promotes the anti-apoptotic functions of Survivin, reducing the ability of cytotoxic chemotherapy to induce cell death. Disclosures: No relevant conflicts of interest to declare.

Abstract: Background Achieving a complete remission (CR) in patients with newly diagnosed acute myeloid leukemia (AML) after induction chemotherapy with cytarabine and an anthracycline (7+3) remains an important treatment goal associated with better overall survival (OS). Approximately 25-30% of younger, and up to 50% of older patients (pts) fail to achieve CR. AML pts with residual leukemia at day 14 receive a second cycle of the same regimen; whether these pts have worse survival than pts not requiring re-induction is unclear. Information on pts with primary refractory AML and the best treatment strategy in this setting are limited. Methods Pts with newly diagnosed AML treated at our institution between 1/2000 and 1/2015 were included. Pts received standard induction chemotherapy with cytarabine for 7 days and an anthracycline for 3 days (7+3). Bone marrow biopsies were obtained at day 14 and a second cycle of the same regimen (7+3 for younger adults, 5+2 for older adults) was given to pts with residual leukemia (blasts 〉 5%). All responses were assessed at day 30 +/- 5 days post induction. Response was defined as CR and CR with incomplete hematologic recovery (CRi) or platelet recovery (CRp) per International Working Group (IWG) 2003 response criteria. Cytogenetic risk stratifications were based on CALGB/Alliance criteria. OS was calculated from the time of diagnosis to time of death or last follow up. A panel of 62 gene mutations that have been described as recurrent mutations in myeloid malignancies was used to evaluate whether genomic data can be used to predict response. Results: Among 227 pts with AML, 123 received 7+3 and had clinical and mutational data available. Median age was 60 years (range, 23-82). Median baseline WBC was 8.2 X 109/L (range, 0.3-227), hemoglobin 8.9 g/L (range, 4.7-13.8), platelets 47 X 109/L (range, 9-326), and BM blasts 46% (range, 20-95). Cytogenetic risk groups were: favorable in 12 (10%), intermediate in 68 (56%) [normal karyotype in 44 (36%)], and unfavorable in 42 (34%). A total of 93 pts (76%) responded, 69 (74%) received 1 cycle of induction and 24 (26%) required re-induction at day 14 due to residual leukemia. A total of 39 pts (32%) received allogeneic stem cell transplant (ASCT): 18 (46%) from a matched sibling donor, 16 (41%) from a matched unrelated donor and 5 (13%) had an umbilical cord transplant. With a median follow up of 13.5 months, the median OS for the entire group was 13 months (m, range, 0.1-120). The median OS for pts who failed 1-2 cycles of 7+3 was significantly worse than pts who responded (median 2.6 vs 16.9 m, p = 0.002). When pts undergoing ASCT were censored, the median OS was 2.3 vs 9.9 m, p= 0.003, respectively. Overall, 33 pts (27%) had residual leukemia at day 14 and received re-induction, 24 (72%) achieved a response at day 30+/- 5 days. The median OS for pts who received re-induction was inferior compared to pts who did not (10.1 vs. 16.1 months, p= 0.02). When pts who received ASCT were censored, the OS was similar (8.5 vs. 7.4 months, p = 0.49, respectively). Among the 30 pts with persistent disease following induction therapy at day 30, 11 (37%) died from induction complications, 6 (20%) received salvage therapy with mitoxantrone/etoposide/cytarabine, 3 (10%) received high dose cytarabine, 2 (7%) received azacitidine, and 8 (27%) received best supportive care. Among pts who received salvage chemotherapy 56% achieved CR and proceeded with ASCT. Two pts had ASCT with residual leukemia and relapsed within 3 m of ASCT. Pts who received ASCT after induction failure had a significantly better OS compared to non-transplant pts (median OS 22.0 vs. 1.4 months, p 〈 0.001, respectively); however, this benefit was only seen in pts who had ASCT in CR. We then investigated if genomic mutations can predict response or resistance to chemotherapy. Out of the 62 genes tested, only a TP53 mutation was associated with resistance, p = 0.02. Further, pts with TP53 mutations had significantly inferior OS compared to TP53 wild type regardless of ASCT status (1.4 vs 14.8 m, p 〈 0.001) Conclusion: Pts with newly diagnosed AML who fail induction chemotherapy with a 7+3 regimen have a poor outcome. Re-induction with the same regimen at day 14 for residual leukemia converted most non-responders to responders, but was associated with worse OS. ASCT improves outcome only in pts who achieve CR with salvage therapy. TP53 mutations predicted resistance to chemotherapy with 7+3. Disclosures Carew: Boehringer Ingelheim: Research Funding. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract: Genetic alterations in the Nucleoporin (NUP) family of genes are involved in myeloid leukemogenesis and are associated with poor prognosis. We previously showed that NUP98-NSD1 is prevalent in acute myeloid leukemia (AML) and is highly associated with FLT3-ITD and dismal outcome. As genetic alterations in the NUP family are frequently cryptic by conventional karyotyping, their incidence has been underestimated. The COG/NCI TARGET AML initiative has performed comprehensive genome-wide characterization of diagnostic specimens from 200 pediatric AML cases in order to identify novel genetic lesions with prognostic and therapeutic significance. The interrogation of the whole genome and RNA sequencing data generated by this initiative identified numerous fusion transcripts involving the NUP family of genes, including NUP98-NSD1, NUP98-KDM5A, NUP98-HOXA9, NUP98-HMG3, NUP98-HOXD13, NUP98-PHF23, NUP98-BRWD3, CLINT-NUP98 and DEK-NUP214. All computationally identified NUP fusions were verified by orthogonal methodology and high-throughput screening assay was developed for frequency determination. The verified NUP fusions were screened in children treated on COG AAML0531 and AAML03P1 to define their prevalence, clinical characteristics and association with clinical outcome. The impact of NUP fusions was initially evaluated in patients with cytogenetically normal AML (CN-AML). NUP fusions were observed in 14.5% (35 of 242) patients: NUP98-NSD1 (N=21), DEK-NUP214 (N=3), NUP98-HMG3 (N=3), NUP98-HOXD13 (N=2), NUP98-PHF23 (N=2) and NUP98-KDM5A (N=4). The NUP fusions NUP98-BRWD3, NUP98-HOXA9 and CLINT-NUP98 were not found in CN-AML patients. Demographics and disease characteristics of CN-AML patients with and without NUP fusions were compared. Although patients of Asian descent comprised only 7% of the study population, they harbored significantly higher number of NUP fusions (29% vs 5%, P =0.002). Among those of Asian descent with CN-AML, 35% harbored a NUP fusion. We also noted an inverse association between NUP fusions and African-Americans where NUP fusions were not identified in any of African-American patients (P =0.031). NUP fusions were correlated with other common mutations in AML. NPM1 (9% vs 28%, P =0.007) and CEBPA (6% vs 19%, P =0.06) were rare in patients with NUP fusions, whereas FLT3/ITD (62% vs 34%, P =0.002) and WT1 (32% vs 8%, P 〈 0.001) were significantly more prevalent in patients harboring NUP fusions. Patients with NUP fusions had a significantly lower complete remission (CR) rate (53% vs. 77%, P =0.004) and 5-year event free survival (EFS, 32% vs 53%, P =0.003) than those without N UP fusions. Given the high co-occurrence of NUP fusions and FLT3-ITD, we investigated the prevalence and clinical correlation of NUP fusions in all FLT3-ITD-positive patients. The prevalence of NUP fusions in FLT3-ITD patients was 26% (43 of 164). The CR rate was lower in patients co-expressing the NUP fusion and FLT3-ITD (40% vs 71%, P 〈 0.001) than in those with FLT3-ITD alone. In addition, minimal residual disease (MRD) was more common in patients co-expressing NUP fusions and FLT3-ITD (68% vs 42%, P =0.008) than in those with FLT3-ITD alone. Finally, patients co-expressing FLT3-ITD and NUP fusions had a 5-year EFS of 28% vs 35% (P =0.093) for those with FLT3-ITD only. Next, we investigated the prevalence of NUP fusions in specific cytogenetic groups and found that NUP fusions were rare in patients with core binding factor and were not observed in patients with MLL rearrangements. In this study we report on the discovery, verification and frequency validation of NUP fusions, a new class of genetic alterations in AML. We demonstrate that NUP fusionsare common in pediatric patients and patients with CN-AML harboring NUP fusions have poor outcome and are more likely to have post-induction MRD than those without thesefusions. Furthermore, there is a high co-occurrence of FLT3-ITD and NUP fusions and patients harboring both genetic lesions have a lower CR rate and high post-induction MRD than those with FLT3-ITD alone. NUP fusions define a new subgroup of pediatric AML patients with an overall poor prognosis. AML harboring NUP fusions likely share similar mechanisms of leukemogenesis and targeting these genetic lesions will likely improve outcome in a significant subset of pediatric AML patients. Disclosures No relevant conflicts of interest to declare.

Abstract: SF3B1 is a splicing factor gene whose mutations are pathognomonic of MDS with ring sideroblasts. Because of the ubiquitous importance of splicing, a major barrier in targeting cells with spliceosomal mutations is the discovery of agents decreasing the competitiveness of mutant cells while preserving the integrity of wild type cells. To date no specific therapies are FDA approved for SF3B1 mutant (SF3B1MT) MDS and few agents are in early clinical testing. We describe a novel targeted approach to drug development for SF3B1MT malignancies. Our investigative strategy started with a high throughput drug screen. We introduced K700E mutation into myeloid cells using CRISPR/Cas9. We then subjected K562+/K700E and matched-parental K562 cells to high throughput drug screen of a library of 3,000 mechanistically annotated, non-redundant, bioactive compounds. Top hits were validated by dose response testing (8 concentrations in half-log dilutions). Our interest focused on compounds with cytostatic activity towards K562+/K700E cells. Among these, a 4-pyridyl-2-anilinothiazole (PAT) showed preferential inhibition of growth in K562+/K700E cells with an IC50 of 3uM. Dose response showed that K562+/K700E cells were significantly sensitive to PAT with a growth inhibition of 20%, 32%, 51%, 65%, 95% at .3uM (P=.01), 1uM (P=.002), 3uM (P 〈 .0001), 10uM (P 〈 .0001), and 20uM (P=.01). High doses (10, 20uM) were toxic to parental cells. PAT treatment did not induce growth arrest in other myeloid cells (THP1, MOLM13FLT3, OCI-AML3DNMT3A, SIG-M5TET2/DNMT3A, K562PHF6) including cells with mutations in other splicing factors (K562U2AF1, K562LUC7L2). PAT induced similar effects in primary SF3B1MT MDS cells at 3uM while it did not induce significant growth inhibition in primary MDS cells with other mutations, including other spliceosomal mutations (e.g.,U2AF1) (N=6). In normal bone marrow cells (N=6), complete growth arrest of erythroid and myeloid cells was observed at high doses (20uM). Using PAT as our lead, we employed a fragment based reiterative medicinal chemistry approach to synthesize selective compounds and improve therapeutic index. Libraries were constructed following Lipinski rules with ease of synthetic construction in mind. Pilot libraries were constructed via the classic Hantsch thiazole synthesis which involves condensation of α-halo ketones with substituted thioureas. This enterprise investigated SAR modifications of PAT by considering features such as regiochemistry and basicity of the nitrogen of the pyridine ring in the head region; replacement of the spacer 2,4-disubstituted thiazole ring with heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); alternatives for the aniline of the tail region e.g., sulfonamide, amide, and substituted amine linkers and substituted aromatic and aliphatic ring structures for the phenyl substituent. A major challenge in drug development of agents targeting spliceosomal mutations is the identification of key clusters of aberrantly spliced genes restored by drug treatment, e.g., identification of a pharmacodynamic endpoint that could be used to prove that that the drug reached its target. SF3B1 mutations induce aberrant 3′-ss selection by promoting use of alternative branch points. To identify biomarkers of splicing changes to screen our libraries, K562+/K700E and parental cells were treated with PAT (3uM) and RNA libraries were subjected to RNA Seq. The splicing pattern of parental cells was used as reference. We identified 328 cassette exons (±25% splicing inclusion difference, pFDR 〈 .05) in K562+/K700E cells of whom the splicing of 22 exons was partially or completely restored upon treatment. Among these, PAT treatment restored the splicing of exons in sensitive 3′-ss sequences (ENOSF1), RNA binding SR-related factors (ACIN1), zing fingers (ZC3H7A) already associated with SF3B1 downregulation, histone modifiers (HMGN3), mitochondrial genes (TMEM126B), proto-oncogenes (CREB1) and heat shock proteins (DNAJC24). Ongoing experiments include tests of the ability of PATs to restore the splicing of misspliced exons and its efficacy in reducing the percentage of SF3B1MT cells in xenografts of K562+/K700E and primary SF3B1MT cells and Sf3b1+/K700E mice. In sum, we described novel classes of compounds that inhibit the expansion of SF3B1MT cells by restoring the splicing defects intrinsically associated with SF3B1. Disclosures Carraway: Novartis: Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Abstract: Background: Myocyte enhancer factor 2C (MEF2C) was initially identified as essential transcription factor for cardiac muscle development. However, subsequent studies have indicated that MEF2C plays a much broader biological role, including in the normal hematopoietic system. Recent studies have now identified MEF2C as cooperating oncogene in acute myeloid leukemia (AML) and suggested a contribution to the aggressive nature of at least some subtypes of AML. These findings raised the possibility that MEF2C could serve as marker of poor-risk disease and, therefore, have prognostic significance in AML. To test this hypothesis, we retrospectively quantified MEF2C expression in participants of the AAML0531 trial and correlated expression levels with disease characteristics and clinical outcome. Patients and Methods: AAML0531 (NCT00372593) was a multicenter phase 3 study that determined the addition of gemtuzumab ozogamicin to intensive chemotherapy among 1,022 eligible patients aged 〈 30 yearswith newly diagnosed de novo non-APL AML, excluding those with bone marrow failure syndromes, juvenile myelomonocytic leukemia, or Down syndrome (if ≤3 years of age) between 2006 and 2010. Cryopreserved pretreatment ("diagnostic") specimens from patients enrolled on AAML0531 who consented to the biology studies and had bone marrow samples were available were included in this study. Total RNA from unsorted specimens was extracted, quantified, and subjected to quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using TaqMan primers to determine expression of MEF2C and, for normalization, the housekeeping gene, β-glucuronidase (GUSB). Patient samples were run in duplicate, and the ΔΔCT method quantified as 2(-ΔΔCT) was used to determine the expression levels of MEF2C relative to GUSB. Results: In all 751 available patient specimens, MEF2C mRNA was detectable and varied 〉 3,000-fold relative to GUSB (0.0091-29.1272 [median: 0.7978]). Patients with the highest relative MEF2C expression (4th quartile) less likely achieved a complete remission after one course of chemotherapy than the other patients (67% vs. 78%, P=0.005). They also had an inferior overall survival (P=0.014; at 5 years: 55±8% vs. 67±4%), inferior event-free survival (P 〈 0.001; at 5 years: 38±7% vs. 54±4%), and higher relapse risk than patients within the lower 3 quartiles of MEF2C expression (P 〈 0.001; at 5 years: 53±9% vs. 35±5%). Of note, exploratory multiple cutpoint analyses for overall and event-free survival indicated that the most statistically significant results were centered around the Q4 cutpoint region, supporting our approach of comparing patients with the highest quartile of relative MEF2C expression with those having lower relative MEF2C expression. Importantly, MEF2C expression was strongly associated with cytogenetic and molecular abnormalities. Specifically, patients with high MEF2C expression less likely had CBF translocations (inv(16): P=0.007, and t(8;21): P 〈 0.001) or normal karyotype AML (P 〈 0.001); conversely, they were more likely to have leukemia with monosomy 7 (P 〈 0.001) and abnormalities involving 11q23 (P 〈 0.001). Furthermore, patients with high MEF2C less likely had a FLT3/ITD (P =0.018) or a mutation in either NPM1 (P=0.010) or CEBPA (P =0.002). Consistently, patients with high MEF2C expression less likely had low-risk disease (16% vs. 46%, P 〈 0.001) and more likely had standard-risk disease (68% vs. 42%, P 〈 0.001) than those with lower MEF2C expression. Indeed, after adjustment for disease risk, age, FAB category, and treatment arm, high MEF2C expression was no longer statistically significantly associated with inferior overall survival (hazard ratio [HR]=0.99 [95% confidence interval: 0.72-1.36] , P=0.929), inferior event-free survival (HR: 1.14 [0.86-1.49], P=0.365), or higher relapse risk (HR: 1.32 [0.91-1.92] , P=0.137), suggesting that MEF2C cooperates with additional pathogenic abnormalities. Conclusion: High MEF2C expression identifies a subset of AML patients with adverse-risk disease features and poor outcome. These findings provide the rationale for therapeutic targeting of MEF2C transcriptional activation in AML. Disclosures Walter: AstraZeneca, Inc.: Consultancy; Covagen AG: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen, Inc.: Research Funding; Pfizer, Inc.: Consultancy; Amphivena Therapeutics, Inc.: Consultancy, Research Funding.

Abstract: Abstract 2388 Acute myeloid leukemia (AML) is characterized by genomic abnormalities that impair differentiation and promote proliferation. There are known karyotypic alterations and somatic mutations associated with relapse risk, but are rarely predictive of response to induction chemotherapy. miRNAs are epigenetic regulators of cell cycle progression and proliferation and their altered expression can play roles in malignancy and cancer cell behavior. Aberrant expression of miRNAs has been implicated in AML pathogenesis and unique expression profiles have been established in specific subsets. Altered miR-181a and miR-155 expression was reported to correlate with specific cytogenetic and molecular characteristics and with clinical outcome. We inquired whether expression of miR-155 and miR-181a correlates with disease characteristics and clinical response in childhood AML. We evaluated diagnostic specimens from 175 AML patients with normal karyotype (NK) treated on COG AML trial AAML0531 for expression of the miRNAs by quantitative TaqMan MicroRNA Assays normalized against normal marrow. There was a 4 log-fold variation in miR-181a, and a 3 log-fold variation in miR-155 expression levels. Patients were divided into 4 quartiles (Q1-Q4) with Q1 consisting of patients with the lowest, and Q4 with the highest expression levels of the specific miRNA. We then correlated disease characteristics and clinical response across quartiles. There was no association of miR-181a or miR-155 expression with age, gender or race. For miR-155, patients in Q4 had a higher diagnostic WBC% than those in Q1-Q3 (60.4 vs. 23.3, p=0.003). Diagnostic bone marrow blast% for Q4 vs. Q1-Q3 was 83% vs. 65% (p=0.004). There was no correlation between miR-181a expression, for either diagnostic WBC (p=0.117) or bone marrow blast % (p=0.237). High miR-155 expression was accompanied by increased prevalence of FLT3-ITD mutations. 78% of patients in Q4 were FLT3-ITD+ vs. 27% in Q1-Q3 (p 〈 0.001). miR-155 expression was not associated with mutations in CEBPA (p=0.76), NPM1 (p=0.98) or WT1 (p=0.57) genes. With the high prevalence of FLT3-ITD, elevated miR-155 expression was associated with high-risk disease, where 50% in Q4 had high-risk disease vs. 12% in the Q1-Q3 (p 〈 0.001). High miR-155 expression was inversely associated with low-risk disease.18% in Q4 had low-risk disease vs. 40% in Q1-Q3 (p=0.009). miR-155 expression was correlated with response to induction chemotherapy. Of patients in Q4, 58% achieved a morphologic complete remission (CR) vs. 81% in Q1-Q3 (p=0.003). As FLT3-ITD is highly prevalent in Q4, we inquired if miR-155 expression correlated with response to induction therapy in FLT3-ITD- patients. FLT3-ITD- patients in Q4 had a 44% CR rate vs. 86% in Q1-Q3 (p=0.008), suggesting that in addition to its association with FLT3-ITD, miR-155 expression may independently provide data on potential response to chemotherapy. Evaluation of miR-181a expression demonstrated that prevalence of CEBPA mutations increased with increasing miR-181a expression and 40% of patients in Q4 were CEBPA+ vs. 7% in Q1-Q3 (p 〈 0.001). There was no association between miR-181a expression and FLT3-ITD (p=0.727), NPM1 (p=0.466) or WT1 (p=0.701) mutations. High miR-181a expression was highly associated with low-risk disease, with 57% in Q4 categorized as low risk vs. 27% in Q1-Q3 (p 〈 0.001). miR-181a expression was not associated with response to induction chemotherapy (CR 80% in Q4 vs. 74% in Q1-Q3, p=0.43). Remission induction rate was re-evaluated after exclusion of CEBPA+ patients. Expression levels had no effect on CR rate (p=0.62). However, of patients in Q4, 71% had evidence of minimal residual disease (MRD) vs. 36% in Q1-Q3 (p=0.003). The correlation of miR-181a expression and MRD remained significant even after exclusion of FLT3-ITD, NPM1 and CEBPA mutations (MRD of 86% vs. 31%, p=0.01). We demonstrate significant association of miR-155 and miR-181a expression with specific disease characteristics and clinically significant mutations in pediatric AML. We also show that elevated expression is highly predictive of induction failure, providing potential biomarkers for identifying patients at high risk of poor response prior to therapy. miRNA expression could provide clinically significant information for use in therapeutic allocation. Validation of this finding in a larger cohort of pediatric and adult patients is underway. Disclosures: Loken: Hematologics, Inc: Employment, Equity Ownership. Pardo:Hematologics Inc: Employment.