Abstract: The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody–drug conjugate in vitro and in vivo.

Abstract: Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: 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; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Abstract: There are few reports of transferrin saturation (TfSat) and serum ferritin (SF) phenotypes and HFE C282Y and H63D genotypes in native Americans. We compared initial screening data of 645 native American and 43,453 white HEIRS Study participants who reported a single race/ethnicity and who did not report a previous diagnosis of hemochromatosis or iron overload. Each underwent TfSat and SF measurements without regard to fasting, and HFE C282Y and H63D genotyping. Elevated measurements were defined as: TfSat & gt;50% (men), & gt;45% (women); and SF & gt;300 ng/mL (men), & gt;200 ng/mL (women). Mean TfSat was lower in native American men than in white men (31% vs. 32%, respectively; p = 0.0337), and lower in native American women than in white women (25% vs. 27%, respectively; p & lt;0.0001). Mean SF was similar in native American and white men (153 μg/L vs. 151 μg/L; p = 0.8256); mean SF was lower in native American women than in white women (55 μg/L vs. 63 μg/L, respectively; p = 0.0015). The respective percentages of native American men and women with elevated TfSat or elevated SF were similar to those of white men and women. The respective mean TfSat and SF values of native American and white participants with genotype HFE wt/wt were similar. The C282Y allele frequency was 0.0340 in native Americans and 0.0683 in whites (p & lt;0.0001). The H63D allele frequency was 0.1150 in native Americans and 0.1532 in whites (p = 0.0001). We conclude that the screening TfSat and SF phenotypes of native Americans do not differ greatly from those of whites. The respective allele frequencies of HFE C282Y and H63D are significantly lower in native Americans than in whites.

Abstract: Abstract 1011 Background. CMR was introduced in London to assess myocardial iron loading in 1999 and some of these patients now have 10 years of follow-up, most with contemporary CMR determinations. The impact of long-term monitoring of myocardial iron loading in thalassemia major (TM) on the proportion of patients with increased myocardial iron (cT2* 〈 20ms) and on patterns of mortality has not been previously described in a longitudinal cohort over this duration. Patients and Methods. All patients regularly attending two London thalassemia centres, who received their first CMR Jan 1999 - Dec 2000 were analyzed as a cohort. Patients underwent initial CMR at the Royal Brompton Hospital and received CMR follow up (FU) either there or at the Heart Hospital (UCLH). 132 patients were identified as having received a CMR in 1999–2000. A minimum 7 years CMR FU was required for inclusion in the long-term CMR analysis. 109 patients had at least 7 years of CMR follow up (range 7.0–10.6 years, median 9.2). The median age at 1st CMR was 27.9 years (range 7.7 – 49.5 years). At baseline, patients were receiving chelation with deferoxamine (DFO) monotherapy (70%), deferiprone (DFP) monotherapy (21%), or a combination of these agents (9%). At latest FU, patients were receiving DFO (32%), deferasirox (DFX) (28%), DFP (22%), or combined DFP and DFO therapy (18%). Results: Improvement in cardiac iron: In 1999–2000, 60% of TM patients had cT2* values ≤20ms and 17% had cT2* values 〈 10ms. By contrast, at long term FU, only 23% now have cT2* ≤20ms, 7% have cT2* values 〈 10ms (p 〈 0.001). Changes to chelation therapy: 31% of patients stayed on the same chelator; 33% had 1 chelator switch, 26% 2 switches and 11% 3 or more switches. 18 switches in chelation therapy were due to side-effects (12 DFP, 5 DFX, 1 DFO). There were 9 breaks in chelation therapy during pregnancy in 8 different women. The proportions of patients with T2* 〈 20ms fell significantly for those who remained on DFO or DFP monotherapies throughout, or who changed chelation modalities on only one (p=0.002) or two (p=0.02) occasions. Patients who received had 3 or more switches did not show a improvement in this respect. The latter group was also the only subset that showed significant deterioration in myocardial iron (p 〈 0.001). Mortality rates: the overall mortality rate for the initial cohort was 1.65 per 1000 patient years (95% CI 0.71 – 3.24); median age at death 35.6 years (range 27.3–48.4). This is a substantial improvement in the mortality index compared with the UK thalassemia registry data, of 4.3 per 1000 patient years during the period 2000–2003 (Modell et al, JCMR, 2008). The incidence rate ratio is 0.387 (95% CI 0.11–0.961), p 〈 0.05, with patients in our cohort 61% less likely to die than those in the 2000–2003 cohort. Causes of death: there were 8 deaths during the FU period: 3 with complications of hepatitis C (all with cT2* 〉 20ms), 3 with sepsis (2 with cT2* 〈 10ms and impaired ejection fraction, 1 with cT2* of 18ms), 1 with breast cancer, 1 with sudden unexplained death (cT2* 〉 20ms). Thus in only 2 patients could excessive cardiac iron loading be considered a causal/contributory factor. There was no significant difference in the baseline cT2* between those who died and those currently still alive (p= 0.2), meaning that death as a drop-out cause does not explain iron loading trends over FU. Chelators at death: DFO (4), DFP (2), DFX (1), combination (1). Conclusions: Over a decade we have seen an almost 3 fold fall in the proportion of patients with myocardial iron overload. Mortality has become substantially lower and cardiac iron overload is no longer the leading cause of mortality. In addition to CMR, this decade has seen the advent of two new oral iron chelators and many patients switched chelation regimen, sometimes several times, during the follow up period. Whilst the contribution of the individual components of this practice to the improved outcome cannot be concluded without randomized studies, it is clear that this modern management of TM is associated with reduced TM mortality. Disclosures: Off Label Use: Deferiprone is off label in the USA but licensed in Europe. Shah:Novartis: Honoraria, Speakers Bureau; Apotex/ Swedish Orphan: Honoraria. Pennell:Siemens: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Cardiovascular Imaging Solutions: Director of CVIS, Equity Ownership. Porter:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract: FMS-like Tyrosine Kinase 3 (FLT3) is a receptor tyrosine kinase that promotes growth and survival of hematopoietic stem and progenitor cells. Mutations in the gene encoding FLT3 are found in 20-30% of acute myeloid leukemias (AML), with approximately 25% of all AMLs containing an internal tandem duplication (ITD) in the juxtamembranal region. FLT3-ITD AML is associated with poor prognosis, with a 5 year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. As such, FLT3 inhibitors have been developed to treat FLT-ITD AML. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming. A Phase II study of quizartinib (AC220) showed few complete remissions in AML patients treated with AC220 as a single agent. Additionally, patients who relapse after FLT3 inhibitor treatment can develop FLT3-inhibitor resistant clones, many of which acquire a D835Y substitution. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse for FLT3-ITD AML patients. Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. After initial biochemical and functional analysis, NCGC-2327 emerged as our lead compound, and further optimized with improved solubility, stability, and permeability properties suitable for in vivo utility (NCGC-1481). Both compounds have excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 〈 5.08 x 10-10 M). AC220, NCGC-2327, and NCGC-1481 treatment of primary human FLT3-ITD-mutant AML cells show EC50 at subnanomolar concentrations (0.5 nM, 0.4 nM, and 0.1 nM respectively) as determined by CellTiter Glo proliferation assays. To ensure that our compounds were selectively effective against FLT3-ITD-mutant AML, primary human NRas-mutant AML cells treated with the inhibitors revealed an EC50 outside of the tested range ( 〉 30 µM). To assess the ability of the compounds to induce apoptosis, we treated FLT3-ITD-mutant AML cells with AC220, NCGC-2327, NCGC-1481, or Crenolanib (a FLT3 inhibitor that can inhibit FLT3-ITD-D835Y) for 72 hours. NCGC-148-treated cells showed the greatest levels of apoptosis (AnnexinV+) as compared to Crenolanib, AC220, and NCGC-2327 (P 〈 0.01). Consistent with the viability assays, NCGC-1481 treatment showed the greatest inhibition of leukemic progenitor function in methylcellulose (Vehicle: 189 ± 39, Crenolanib: 151 ± 32, AC220: 36 ± 3, NCGC-2327: 22 ± 3, and NCGC-1481: 3 ± 2) (P 〈 0.01). Relapse and resistance is a primary concern for patients treated with AC220, therefore we investigated leukemic subclonal resistance in vitro after treatment with Crenolanib, AC220, NCGC-2327, or NCGC-1481. FLT3-ITD-mutant AML cells were treated for 72 hours, washed and then allowed to recover in the absence of the compounds for one week. Subclonal recovery was assessed by measuring cell viability (AnnexinV+) and leukemic progenitor function (methylcellulose) for up to 1 week post treatment. NCGC-2327 and NCGC-1481 delayed, and in some instances prevented, subclonal recovery as compared to AC220 or Crenolanib treatment. NCGC-2327 and NCGC-1481 show comparable potency to current FLT3 inhibitors (i.e., AC220 and Crenolanib) in regards to inhibition of FLT3 signaling, proliferation, and induction of apoptosis in FLT3-ITD-mutant AML. However, NCGC-2327 and NCGC-1481 are exquisitely effective at preventing subclonal recovery of FLT3-ITD-mutant AML as compared to both AC220 and Crenolanib. Taken together, these findings suggest our novel FLT3 inhibitors show promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract: Approximately 25% of all acute myeloid leukemias (AMLs) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with poor prognosis, with a five-year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse in FLT3-ITD AML patients. Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. We have previously shown that our current lead compound (CTDS-004) has excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 〈 5 x 10-10 M). Additionally, CTDS-004 potently inhibits proliferation and progenitor function, and induces apoptosis of FLT3-ITD positive human AML cell lines in comparison to the second generation FLT3 inhibitors, AC220 and Crenolanib. Importantly, CTDS-004 exhibits minimal effects on the function and viability of normal human CD34+ BM cells. The most important distinction between CTDS-004 and current FLT3-ITD inhibitors is that CTDS-004 retains its efficacy and prevents subclonal recovery of FLT3-ITD AML cells that have become refractory to AC220 or Crenolanib treatment. To gain insight into the mechanistic basis for the superior response of FLT3-TID AML to CTDS-004 as compared to AC220, we performed RNA sequencing of MLL-AF9 FLT3-ITD cells after 6 and 12 hours of treatment with AC220 or CTDS-004 (IC10: 0.3 and 0.1 nM, respectively). Although both compounds target FLT3-ITD, there were striking distinctions in gene expression changes after treatment with AC220 and CTDS-004, highlighting the differences in the biological impact and cytotoxic effect of these compounds. Our lead compound has promising pharmacokinetic and pharmacodynamics properties in vivo, therefore, we tested CTDS-004 in a xenograft model of human AML. NRGS mice (NOD.Cg-Rag1tm1MomIL2rgtm1WjlTg[CMV-IL3,CSF2,KITLG]1EAv/J) were transplanted with human MLL-AF9 FLT3-ITD leukemic cells (2 x 105 cells/mouse) via tail vein injection. Starting on day 10 post-engraftment, the mice were treated with CTDS-004 (30 mg/kg; n=5), AC220 (15 mg/kg; n=5), or PBS (n=5) via intrape ritoneal injection on a cycle of five days of daily injection followed by two days of rest for the duration of the experiment. Kaplan-Meier survival analysis revealed that CTDS-004 is extremely effective at prolonging survival in the human FLT3-ITD AML xenograft model. There was a significant increase in median survival for CTDS-004 and AC220 treatment groups compared to the PBS control group (Median survival post-xenograft: PBS: 28 days, AC220: 54 days, CTDS-004: 53 days; p=0.0002). Taken together, these findings suggest our novel FLT3 inhibitor shows promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: AML is a highly aggressive hematologic malignancy. Patient (pt) outcomes are affected by disease-related factors including cytogenetic findings and gene mutations, as well as pt-related factors, such as age and race. Younger pts have superior survival: ~50% of pts diagnosed as AYAs (18-39 years) may be cured of their disease. However, the impact of race on the outcome and associated disease profiles in this pt population are unknown. Methods: We compared survival and molecular profiles of 655 Non-Hispanic Black and Non-Hispanic White (hereafter referred to as Black, n=89 and White, n=566) AYA AML pts treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols based on standard intensity cytarabine/anthracycline induction therapy between 1986 and 2016. Three hundred ten pts were analyzed molecularly via targeted sequencing of 81 genes. Additionally, we performed integrated genomic profiling (whole-exome sequencing and transcriptome sequencing) and measured residual disease (MRD) in serial samples of 4 Black pts who relapsed with their disease. Results: A comparison of clinical characteristics of AYA AML pts by race revealed almost identical age and sex distribution, and we found no significant differences between clinical features at diagnosis. With regard to genetic profiles, 42% of White pts were cytogenetically normal, whereas only 18% of Black pts had cytogenetically normal AML (CN-AML; p & lt;0.001). The abnormal karyotypes in Black pts more often contained abnormalities associated with core-binding factor (CBF) AML (39% v 25%, p=0.01; Fig. 1A). White pts had more known pathogenic NPM1 variants (29% v 9%, p=0.01), whereas Black pts had a higher incidence of ZRSR2 pathogenic variants (9% v 0.4%, p=0.004) and tended to have pathogenic KRAS variants more often (12% v 5%, p=0.11; Fig. 1B). Black AYA AML pts had worse outcomes including a higher early death rate (ED, defined as death within 30 days of diagnosis; 11% v 2%, p & lt;0.001), a trend towards lower complete remission (CR) rate (73% v 82%, p=0.06) and a shorter overall survival (OS; median, 1.5 v 3.1 years [y], p=0.002). Notably, this survival disparity was almost exclusively driven by pts aged 18-29 y: Black pts had a higher ED rate (16% v 3%, p=0.002), a lower CR rate (66% v 83%, p=0.01) and shorter OS (median, 1.3 v 10.2 y, p & lt;0.001) but not disease-free survival (DFS; p=0.16) than White pts aged 18-29 y. In contrast, there were no significant differences in these outcome metrics between Black and White pts aged 30-39 y (Fig. 2). Among all pts consolidated with intensive chemotherapy (n=566), multivariable analysis revealed Black race as an independent prognosticator of shorter DFS (p=0.04) and OS (p & lt;0.001). These differences in OS were also significant when we included pts who received allogeneic transplantation in 1st CR (n=655; p & lt;0.001). 18-29 y old Black pts with any non-CBF AML had very poor OS compared to White pts (5-y rates, 12% v 45%, p & lt;0.001). CBF-AML pts aged 18-29 y tended to have an inferior OS compared with White pts (5-y rates, 41% v 44%, p=0.10). To gain insights into the genetic features of Black AYA AML pts at different stages of the disease, we performed integrated genomic profiling on paired leukemic samples from diagnosis and relapse of 4 Black AYA pts. In all pts, the original dominant leukemic clone persisted and was dominant at relapse (Fig. 3). This suggests that the leukemic clone persists during treatment with conventional cytotoxic chemotherapy. This observation was further supported by MRD detection of NPM1 mutations in NPM1-mutated pts at time of morphologic CR. Conclusion: Black AYA AML pts present with distinct molecular features, including very high frequencies of CBF AML, and low frequency of NPM1. Pts aged 18-29y account for the race-associated survival disparity, especially non-CBF pts who have dramatically poor survival. On the one hand, the lower CR rates combined with persistence of dominant clones at relapse suggest reduced response to induction chemotherapy, and suggests the need for different treatment intensities and/or modalities in this pt cohort. On the other hand, high early death rates are indicative of delay in diagnosis and care, including health inequities, calling for systematic changes particularly for this population. Figure 1 Figure 1. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Mims: Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Aptevo: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Glycomemetics: Research Funding; Kartos Pharmaceuticals: Research Funding; Xencor: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Blum: Leukemia and Lymphoma Society: Research Funding; Syndax: Honoraria; AmerisourceBergen: Honoraria; Abbvie: Honoraria; Celyad Oncology: Research Funding; Nkarta: Research Funding; Forma Therapeutics: Research Funding; Xencor: Research Funding. Larson: Rafael Pharmaceuticals: Research Funding; Epizyme: Consultancy; Astellas: Consultancy, Research Funding; Gilead: Research Funding; CVS/Caremark: Consultancy; Takeda: Research Funding; Novartis: Research Funding; Cellectis: Research Funding. Stone: Onconova: Consultancy; Boston Pharmaceuticals: Consultancy; Innate: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; AbbVie: Consultancy; GlaxoSmithKline: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Janssen: Consultancy; Arog: Consultancy, Research Funding; Aprea: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Macrogenics: Consultancy. Paskett: Pfizer: Research Funding; Merck: Research Funding. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Eisfeld: Karyopharm (spouse): Current Employment.

Abstract: Background: AML is a disease affecting predominantly older patients (pts), but does occur across the entire age spectrum; younger adults [age & lt;60 years (y)] have better outcomes. Using 2 large datasets (from Germany and the US), we sought to identify whether mutational frequencies, cytogenetic aberrations or outcome measures would demonstrate unique patterns to independently assort populations by age. Methods: We analyzed the molecular profiles of 2,823 adult AML pts enrolled onto clinical frontline protocols of 2 large cooperative study groups from 2 continents [US, Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology (Alliance), n=1743; Germany, AML Cooperative Group [AMLCG], n=1080] between 1986 and 2016. Treatment of all pts included intensive induction therapy, whereas pts enrolled on CALGB/Alliance protocols precluded allogeneic transplantation in 1 st complete remission. Pts in both cohorts were profiled for molecular features via targeted sequencing platforms. Frequencies of mutations genes and selected cytogenetic findings were then calculated in both datasets for the group of pts aged 18-24 y and for older pts by 5-year intervals until the age of 74 y and for pts older than 75 y. We also analyzed survival outcomes of 1,669 AML pts younger than 60 y using the same age intervals up to age 59 y. Results: Our side-by-side analysis shows remarkable congruence of results between German and US pt populations. Selected AML-associated gene mutations (mutation frequency ≥4%) and recurrent cytogenetic abnormalities followed 3 basic distribution patterns across the age spectrum (Fig. 1A): group 1 with increasing frequency with increasing age [ASXL1, BCOR, IDH1/2, RUNX1, SRSF2, TET2, TP53; complex karyotype and cytogenetically normal AML (CN-AML)]; group 2 with decreasing frequency with increasing age (CEBPA, EZH2, FLT3-TKD, GATA2, KIT, KRAS, PTPN11, NRAS, WT1; inv(16), t(8;21) and 11q23/KMT2A rearrangements) and group 3 with non-linear frequency distribution, which included the 3 most common AML-associated gene mutations (NPM1, DNMT3A, FLT3-ITD), SF3B1 and mutations in the cohesin complex genes (RAD21, SMC1A, SMC3, STAG2) (Fig. 1A). Notably, within the first 2 distribution groups, there seem to be no obvious age that could serve as a cut point separating age groups that are markedly different with regard to their molecular patterns. Particularly, this includes an age group that is commonly used for pt cohort definitions such as pts aged 18-39 y referred to as adolescent and young adults (AYA) or even treatment decisions and eligibility (eg, ages 60 or 65 and older for consideration as elderly AML). With respect to pt outcomes, expectedly, there was almost linear shortening of overall survival (OS) as age increased (p & lt;.001; Fig. 1B, Table 1). Within the European LeukemiaNet (ELN) genetic-risk groups, there was also an age-associated shortening in OS rates (Table 1): in favorable and intermediate risk pts the 5y-OS declined over the age range (favorable risk pts; US, p=.002; GER, p & lt;.001; intermediate risk pts, US, p & lt;.001; GER, p=.009, Table 1). Adverse risk pts had less variability in survival outcome across age (US, p=.004; GER, p=.22). Thus, while ELN criteria risk stratifies each age group, age itself is an important qualifier with regards to OS within each ELN group given the wide survival range among the age group. Again, there were no distinct outcome changes at certain age groups, further supporting the consideration of age as a continuum in AML for both biology and risk stratification. Conclusions: To our knowledge, this is the first large scale depiction of mutational patterns in AML inclusive of the entire adult age spectrum. Our international study demonstrates that patterns of individual mutations based on age are remarkably consistent between countries, and defy assortment based on typical age conventions. Given the continuous distribution of either increasing or decreasing frequency of many mutations, there are distinctly different mutational profiles for the youngest pts compared with older pts, however choosing a precise cut-off, such as age 39 for AYA pts or 59 for consideration as "younger AML", does not seem to be supported by our analyses. This observation supports a more personalized approach that also considers molecular subgroups in clinical practice instead of the age rigidity set in many clinical trials. *shared first: M.C.,K.L.; #last: T.H.,AK.E. Figure 1 Figure 1. Disclosures Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hiddemann: F. Hoffmann-La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Mims: Glycomemetics: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Aptevo: Research Funding; Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Xencor: Research Funding; Kartos Pharmaceuticals: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Blum: Celyad Oncology: Research Funding; Forma Therapeutics: Research Funding; Xencor: Research Funding; Nkarta: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie: Honoraria; AmerisourceBergen: Honoraria; Syndax: Honoraria. Larson: Epizyme: Consultancy; Astellas: Consultancy, Research Funding; Gilead: Research Funding; CVS/Caremark: Consultancy; Takeda: Research Funding; Novartis: Research Funding; Rafael Pharmaceuticals: Research Funding; Cellectis: Research Funding. Stone: Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Onconova: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Innate: Consultancy; GlaxoSmithKline: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Boston Pharmaceuticals: Consultancy; Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Arog: Consultancy, Research Funding; Aprea: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Macrogenics: Consultancy. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Metzeler: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Daiichi Sankyo: Honoraria; Astellas: Honoraria; AbbVie: Honoraria; Pfizer: Consultancy; Celgene/BMS: Consultancy, Honoraria, Research Funding. Eisfeld: Karyopharm (spouse): Current Employment.

Abstract: Background: AML is a clinically and molecularly heterogeneous disease associated with poor survival. Multiple disease-related factors including cytogenetic findings and gene mutations, as well as patient-related factors, such as demographics and African American (AA) heritage, have been identified that impact on pt outcomes. However, with recent improved survival it is unknown whether racial health disparities persist. Moreover, we are not aware of a large study that assessed possible race-associated molecular differences. Thus, the goals of our study were to 1) analyze the outcomes of adult AML pts in a nationwide population study, including possible impacts of sociodemographic, financial and racial disparities and 2) characterize molecular features of AA compared with those of Caucasian AML pts. Methods: For a nationwide population analysis, the Surveillance Epidemiology End Results (SEER) Program of the National Cancer Institute was used to identify 11,190 adults aged 18-60 years (y) diagnosed with AML (excluding acute promyelocytic leukemia) between 1986 and 2015. To characterize molecular features we performed targeted sequencing of 81 genes in 1,339 AML pts treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology (Alliance) protocols based on standard intensity cytarabine/anthracycline induction followed by consolidation between 1986 and 2016. No Alliance pt received an allogeneic stem cell transplant in 1st complete remission (CR). Results: The associations between demographic parameters and risk of death among SEER registry AML pts are shown in Table 1. While there was a slightly higher risk of death for men (HR 1.09) and a lower risk of death for pts with a higher median household income ( & gt;79.6k vs & lt;54.4k, HR 0.85), the strongest factor affecting survival of AML pts was self-reported pt race, after accounting for other variables in the model. Specifically, AA AML pts had a higher risk of death compared with that of white AML pts (HR 1.28), with 3-year (y) overall survival (OS) rates of 32% and 41%, respectively (P & lt;.001; Figure 1A). To evaluate if this race-associated survival disparity also persists in pts treated in the setting of clinical trials, we analyzed the survival of pts similarly treated on Alliance protocols. Although there was no difference in CR rates between AA and Caucasian Alliance AML pts, AA pts had inferior disease-free survival (DFS; median, 0.8 y vs 1.4 y, P=.02) and OS (median, 1.2 y vs 1.8 y, P=.02) compared with Caucasian pts, indicating that access to similar treatments might not alleviate racial survival disparities (Figure 1B). To assess whether any race-associated pretreatment features may help explain the different outcomes, we analyzed the clinical, cytogenetic and gene mutation features of the Alliance cohort. AA pts less often had normal cytogenetics (38% vs 51%, P=.01) and had a lower frequency of prognostically favorable NPM1 mutations (25% vs 38%, P=.04) and higher frequencies of spliceosome gene mutations (24% vs 12%; P=.009) than Caucasian pts. Multivariable analyses for outcome in AA pts did not identify any molecular features associated with achievement of CR or DFS. However, AA pts harboring FLT3-ITD or IDH2 mutations had shorter OS compared with wild-type pts (FLT3-ITD, HR=1.95, P=.03; IDH2, HR=2.17, P=.008). Notably, other well-established mutational features known to associate with outcome (e.g., NPM1 or TP53 mutations) did not impact on survival. Lastly, we performed uni- and multivariable outcome analyses for OS in our Alliance pts. Remarkably, being NPM1-mutated and not being AA were the only positive prognostic factors associated with longer OS in the final risk model (NPM1 mut vs wt, HR=0.72, P & lt;.001; Caucasian vs AA, HR=0.72, P=.03). Conclusion: Self-reported AA race is the most important pt-associated factor associated with poor survival in AML pts & lt; 60 y of age based on SEER. Survival analyses in Alliance pts identify AA race as independent poor survival prognosticator in AML pts besides established molecular markers. . This disparity must be urgently addressed to ensure improved outcomes for AA AML pts, and larger studies to establish molecular risk profiles are needed. Support: U10CA180821, U10CA180882 U24CA196171, https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224 Disclosures Bhatnagar: KaryoPharm Therapuetics: Research Funding; Cell Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Mims:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy. Walker:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Powell:Genentech: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Novartis: Research Funding. Kolitz:Magellan: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Stone:Biolinerx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Other; Aztra-Zeneca: Consultancy; Jazz: Consultancy; Argenix: Other; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Pfizer: Consultancy; Gemoab: Consultancy; Syndax: Consultancy, Research Funding; Takeda: Other: DSMB; Macrogenics: Consultancy; Trovagene: Consultancy; Syntrix: Other: DSMB; Abbvie: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Arog: Consultancy, Research Funding; Syros: Consultancy; Stemline: Consultancy. Byrd:Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Acerta Pharma: Research Funding; Syndax: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Kartos Therapeutics: Research Funding; Vincera: Research Funding; Novartis: Research Funding. Eisfeld:Karyopharm: Current Employment, Current equity holder in publicly-traded company; Vigeo Therapeutics: Consultancy.