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Proper diagnostic distinction of bone marrow failure syndromes can often be challenging. In particular, for older patients with idiopathic aplastic anemia (AA), differential diagnosis includes myelodysplastic syndrome (MDS), which can atypically present in a hypocellular form. In addition to blasts and overt dysplasia, the presence of chromosomal abnormalities and a spectrum of somatic mutations may be revealing. Both clonal cytogenetic aberrations and somatic mutations most typically correspond to a clonal myelodysplasia, but clonal somatic mutations have also recently been found in AA. True driver myeloid mutations are uncommon in AA. Marrow hypocellularity in AA and occasionally in MDS patients points toward a similar immune mechanism responsible for deficient blood cell production and indicates that cytopenias in early hypocellular MDS might be treated with immunosuppressive modalities. Primary hypocellular MDS has to be distinguished from post-AA secondary MDS, most commonly associated with del7/7q. Post-AA MDS evolves at the rate of about 10% in 10 years, but recent observations suggest that widespread use of eltrombopag may influence the risk of progression to MDS. This complication likely represents a clonal escape, with founder hits occurring early on in the course of AA. A similar mechanism operates in the evolution of paroxysmal nocturnal hemoglobinuria (PNH) in AA patients, but PNH clones are rarely encountered in primary MDS.

Genetic mutations (somatic or germline), cytogenetic abnormalities and their combinations contribute to the heterogeneity of acute myeloid leukemia (AML) phenotypes. To date, prototypic founder lesions [e.g., t(8;21), inv(16), t(15;17)] define only a fraction of AML subgroups with specific prognoses. Indeed, in a larger proportion of AML patients, somatic mutations or cytogenetic abnormalities potentially serve as driver lesions in combination with numerous acquired secondary hits. However, their combinatorial complexity can preclude the resolution of distinct genomic classifications and overlap across classical pathomorphologic AML subtypes, including de novo/primary (pAML) and secondary AML (sAML) evolving from an antecedent myeloid neoplasm (MN). These prognostically discrete AML subtypes are themselves nonspecific due to variable understanding of their pathogenetic links, especially in cases without overt dysplasia. Without dysplasia, reliance is mainly on anamnestic clinical information that might be unavailable or cannot be correctly assigned due to a short prodromal history of antecedent MN. We explored the potential of genomic markers to sub-classify AML objectively and provide unbiased personalized prognostication, irrespective of the clinicopathological information, and thus become a standard in AML assessment. We collected and analyzed genomic data from a multicenter cohort of 6788 AML patients using standard and machine learning (ML) methods. A total of 13,879 somatic mutations were identified and used to predict traditional pathomorphologic AML classifications. Logistic regression modeling (LRM) detected mutations in CEBPA (both monoallelic "CEBPAMo" and biallelic "CEBPABi"), DNMT3A, FLT3ITD, FLT3TKD, GATA2, IDH1, IDH2R140, NRAS, NPM1 and WT1 being enriched in pAML while mutations in ASXL1, RUNX1, SF3B1, SRSF2, U2AF1, -5/del(5q), -7/del(7q), -17/del(17P), del(20q), +8 and complex karyotype being prevalent in sAML. Despite these significant findings, the genomic profiles of pAML vs. sAML identified by LRM resulted in only 74% cross-validation accuracy of the predictive performance when used to re-assign them. Therefore, we applied Bayesian Latent Class Analysis that identified 4 unique genomic clusters of distinct prognoses [low risk (LR), intermediate-low risk (Int-Lo), intermediate-high risk (Int-Hi) and high risk (HR) of poor survival) that were validated by survival analysis. To link each prognostic group to pathogenetic features, we generated a random forest (RF) model that extracted invariant genomic features driving each group and resulted in 97% cross-validation accuracy when used for prognostication. The model's globally most important genomic features, quantified by mean decrease in accuracy, included NPM1MT, RUNX1MT, ASXL1MT, SRSF2MT, TP53MT, -5/del(5q), DNMT3AMT, -17/del(17p), BCOR/L1MT and others. The LR group was characterized by the highest prevalence of normal cytogenetics (88%) and NPM1MT (100%; 86% with VAF>20%) with co-occurring DNMT3AMT (52%), FLT3ITD-MT (27%; 91% with VAF In conclusion, the heterogeneity inherent in the genomic changes across nearly 7000 AML patients is too vast for traditional prediction methods. Using newer ML methods, however, we were able to decipher a set of prognostic subgroups predictive of survival, allowing us to move AML into the era of personalized medicine. Disclosures Advani: OBI: Research Funding; Abbvie: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Immunogen: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; Takeda: Research Funding. Ravandi:Abbvie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; BMS: Consultancy, Honoraria, Research Funding. Carraway:Novartis: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Jazz: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kantarjian:Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Abbvie: Honoraria, Research Funding; Aptitute Health: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Adaptive biotechnologies: Honoraria; Ascentage: Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria. Kadia:Pfizer: Honoraria, Research Funding; Novartis: Honoraria; Cyclacel: Research Funding; Ascentage: Research Funding; Astellas: Research Funding; Cellenkos: Research Funding; JAZZ: Honoraria, Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Pulmotec: Research Funding; Abbvie: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Research Funding. Sekeres:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Background. While childhood leukemia and bone marrow failure disorders always raise suspicions of a familial trait, recognition of germ line (GL) contribution to the pathogenesis of adult presentation of these disorders may be difficult because of the absent and unreliable family history, long disease anticipation and incomplete penetrance due to various factors, including competing mortality and interaction with environmental factors. The search for such GL alterations may involve unbiased whole exome sequencing or genome wide analysis studies approaches or, as we conducted here, rely on identification of GL variants in a rationally selected panel of genes previously involved in leukemia and bone marrow failure (BMF). Methods. We hypothesize that, in analogy to pediatric disease, pathogenic GL alterations may exist also in a subset of adult patients with BMF and myeloid neoplasia (MN). Such lesions may contribute to the clinical features and the preferential occurrence of somatic hits leading to disease manifestation. We analyzed genomic data of 350 patients with BMF syndromes (AA, PNH, AA/PNH) and a cohort of 2,827 patients with MN (MDS, MDS/MPN, and AML) disorders. Our analysis included variant calling, stringent exclusion of artifacts and selection of GL variants (GLVs). These were then subjected to a bioanalytic pipeline and classified as tier-1, tier-2 and variants of uncertain significance according to their potential pathogenic importance. Tier-1 was defined as structurally predicted pathogenic mutations with known disease association, frameshift/nonsense mutations and highly recurrent missense mutations with low frequency in the general population. Tier-2 was defined as missense mutations with a high general population frequency (≥.01 but Results. In the BMF cohort, 10% had tier-1 and 44% had tier-2 GLVs. Remarkably, 27 patients (pts) (7%) had concomitant tier-1 and tier-2 mutations in different genes, among those, 5 were biallelic (SBDS, FANCM, BARD1, TERT, PALB2) and the rest were compound heterozygous. When we focused on tier-1 lesions, FA GLVs were most prevalent in this cohort comprising 18/350, including FANCA (n=3), BRCA1 (n=2) BRCA2 (n=2) and 11 other FA gene (affected once). In addition, we found also 60 tier-2 FA gene mutations. Surprisingly, telomerase-associated GLVs fulfilling tier-1 criteria were found only in 7 patients [CTC1 (n=4), TERT, TINF2, and WRAP53 (1 each)] and tier-2 in 18 patients. The rest of the tier-1 GLVs were [NF1 (n=4), SBDS (n=3) SAMD9L (n=2), BARD1, BCOR, BLM, CBLC, CHEK2, MLH1, MRE11A, XRCC3 (1pt each). Further analysis of the BMF cohort assessing disease outcomes revealed that among 40 pts, who progressed to MDS, 25% had tier-1 and 27% had only tier-2 GLVs. Reverse analysis showed that 27% of carriers of tier-1 GLVs progressed to MDS/AML as compared to only 10% of negative cases (P In summary. Our analysis indicates that a significant fraction of adult patients with BMF are carriers of predisposition variants with broad clinical and social implications. Disclosures Patel: Alexion: Other: educational speaker. Carraway:Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Up to 15% of AA patients (pts) treated conservatively with immunosuppression will evolve to myeloid neoplasia (MN), either MDS or AML, over a median time of 10 years regardless of response (0-18 years; n=238). The pathogenesis of MN secondary to AA is diverse and will often include antecedent clonal facilitating events that herald progression. Minor clones have been described in AA, some of which are not contributory to later evolution while other may result in subsequent progression. MDS evolution in inherited bone marrow failure (BMF) syndromes suggests that germ line (GL) alterations can be predisposing. In addition, progression to MN may reflect immune escape due to selection pressure e.g., through acquisition of HLA mutations. Here, we studied the molecular landscape of MN arising from AA, to better understand its pathogenesis and ultimately to develop measures of early detection, prevention, and therapeutic strategies. Among 350 pts diagnosed with AA and PNH, 38 (11%) developed a secondary MN (sMN). Median age at AA/PNH diagnosis was 61 years (15-76). Almost all of pts who underwent transformation (89%) received a 1st line treatment consisting of ATG+CsA in 85% of cases (ORR 59%; 21% CR and 38% PR) and 47% received more than one form of treatment, suggesting a lack/incomplete response or relapse. MDS was the most frequent diagnosis at evolution (77%), followed by AML (21%) and MPN (2%). Myeloid evolution was less common in pts with moderate AA (7% vs 14% in severe) or in the presence of a PNH clone (21% vs. 52% in non-progressors, p=.0003). First we investigated GL alterations classified as Tier1 (9/38 pts) and Tier2 (11/38) based on their pathophysiological impact. Tier1 variants included NF1, CBLC, SBDS (n=2), and SAMD9L and overall were more frequently detected in del(7q) pts (76%, p=.0001). Tier2 included FA variants (BRCA2, FANCI, FANCD2; n=3). Of note, in sMN Tier1 variants were detected in 24% vs. 8% in non-evolved cases (p=.008) and none had concomitant Tier1/Tier2 configuration (0% vs. 9% in non-progressors, p=.05) or GATA2 variants. Cytogenetic abnormalities were most frequent at the time of MN progression in 83% of cases, with chr. 7 alterations present in 47% of cases (-7, 35%; del(7q),12%), followed by complex karyotype (CK, 13%), involving chr.7 in 75% of cases. By comparison, -7/del(7q) are present in 7.5% of cases of our internal cohort of primary MN (p=.0001), but no differences in -7 and del7q distribution were seen. A total of 148 somatic variants (myeloid and HLA panels) were found at the time of evolution in 34/38 sMN pts, with an average of 4.4 mutations/patient. ASXL1 (29% vs 14%, p=.02) and SETBP1 (15% vs 3%, p=.005) hits were more frequent in evolved cases while TET2 and TP53 mutations were less common as compared with pMN. Of note, sMN pts with CK harbored ASXL1 and TP53 mutations in 50% of cases. In a cross-sectional analysis of evolved cases studied at AA onset (n=17) and at myeloid evolution (n=35), somatic lesions in TET2, DNMT3A and ASXL1 genes were found in 5, 1 and 3 pts at baseline, respectively. If variants in TET2 and DNMT3A likely reflect antecedent CHIP, ASXL1 variants may have a role in driving myeloid progression as shown by the higher mutation rate in post AA cases. This hypothesis is further supported by the acquisition of subclonal chr7 abnormalities and by the overall higher clonal burden at sMN onset (median VAF 24% vs 43% respectively, p=.0001). When comparing pts with chr7 abnormalities with de novo counterpart, in sMN genes appeared most commonly mutated in ASXL1 (p=.02), SETBP1 (p=.0007), ETV6 (p=.02) and NF1 (p=.02), while TP53 mutations were less common.The intrinsic peculiarity of this -7/del(7q) sMN subset is also underlined by a different median survival time (12 vs 48 months in sMN vs pMN, respectively, p=.0002). The HLA mutational analysis available for 10 sMN cases showed the presence of somatic class I/II loci variants in 4/10 of progressors, including pts with chr7 abnormalities in 3/4 of cases. Of note, all class I HLA mutations were found in locus C. By comparison, in non-progressing AA pts HLA class I/II variants were found in 13% of pts. Our results demonstrate that AA progression to MN has distinct molecular characteristics. The presence of HLA mutations suggests that immune escape or immune selection may play a role, while the presence of GL predisposition variants shows that they not only may facilitate AA but also clonal evolution as described from classic congenital BMF. Disclosures Patel: Alexion: Other: educational speaker. Voso:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Carraway:Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Novartis: Consultancy, Speakers Bureau; Abbvie: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Lenalidomide (LEN) has established a new paradigm of targeted therapy in MDS. The mechanistic underpinnings of LEN efficacy are related to the synthetic lethality of this agent through its ability to bind cereblon (CRBN). LEN induces degradation of CK1α, which is encoded by the CSNK1A1 gene located on the del(5q) CDR, whereby haploinsufficient levels of this gene allow for selective toxicity to deletion mutants. Another common cytogenetic abnormality present in patients with myeloid neoplasia (MN) is -7/del(7q). To date no selective therapies exist for -7/del(7q), an urgent unfulfilled need, given the poor prognosis associated with this cytogenetic abnormality. We were interested to explore if this same notion of selective toxicity may be possible in del(7q) myeloid patients and sought to screen drugs for this focused population. From a large cohort of patients with MN (n=3,328), we found -7/del(7q) in 10% (n=316) of patients. We first identified a signature pattern of haploinsufficient genes on -7/del(7q) based on NGS. We then searched for haploinsufficient genes which, if targeted by investigational drugs, could provide a therapeutic window for selected MN subtypes in analogy to LEN in del(5q). For the purpose of our analysis, haploinsufficient expression was defined as 97) with sensitivity levels of KG-1 in the lower μM ranges. Our analysis also identified changes in death box-RNA-helicases (DDX41, DDX24,DDX54) and DNA binding proteins (CHD3), opening the possibility that MLN4924 might lead to degradation of key proteins implicated in the pathogenesis of MDS/ AML. The therapeutic index was confirmed by the absence of toxicity to normal CD34+ cells which were unresponsive to MLN4924 because they lack the expression of NEDD8-activating enzyme (NAE) (major target of MLN4924). In contrast to normal cells, NAE is highly expressed in myeloid cancer cells. In an ongoing dose-escalation study of MLN4924 plus AZA, 1 patient with -7 had stable disease after 6 cycles of therapy and 2 patients with del(7q) achieved complete and partial remissions. In sum, we propose that MN with -7/del(7q) abnormalities might represent a patient population genetically hypersensitive to synthetic lethality by neddylation inhibitors. Disclosures Hutter: MLL Munich Leukemia Laboratory: Employment. Advani:Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Genentech, Verastem: Consultancy; Takeda: Research Funding. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Savona:AbbVie: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Acute myeloid leukemia (AML) with t(8;21) or inv(16) chromosomal rearrangements are distinct heterogeneous disease entities within AML that are classified together as core binding factor AML (CBF-AML). Given the nature of the chromosomes involved, these rearrangements lead to the production of leukemogenic chimeric transcripts (RUNX1-RUNX1T1 and CBFB-MYH11) which disrupt the physiologic activity of the heterodimeric transcription factor CBF complex. Although CBF-AML patients have a favorable prognosis and good response to treatment compared to other AML subtypes, survival outcomes are not uniform. Indeed, 30-50% of patients with CBF-AML eventually relapse, and the 5-10 yr survival ranges between 55-61% for patients < 60 yr in MRC/NCRI AML trials. Studies have analyzed the clonal architecture of CBF-AML patients and identified cooperating mutations independently of receptor tyrosine kinases (FLT3, KIT) mutations while others have found a 30% occurrence of KIT mutations. Studies of murine models of Runx1 and Cbfb have demonstrated that inactivation of both genes does not lead to leukemia, suggesting that other factors are necessary to recapitulate the leukemia phenotype fully. Although mutations in RAS family of genes (NRAS/KRAS) are among the most frequently observed mutations described in CBF-AML [54% in inv(16) and 30% in t(8;21)], no associations between those mutations and survival outcomes have been found. Because of the lack of association between RASMT and clinical outcomes, their role in CBF-AML is still unknown. Here, we focused on dissecting the impact of RAS mutations (NRAS/KRAS; RASMT) on the clinical characteristics, survival outcomes, and the molecular associations among CBF-AML patients by evaluating the clonal succession of RASMT. In total, 284 CBF-AML patients were identified, in whom inv(16) and t(8;21) represent 61% (n=173) and 39% (n=111) of the cases, respectively. Thirty-five % (99/284) of the patients carried RAS mutations (NRAS=78; KRAS=21) with 8 patients harboring 2 mutations comprising NRAS, KRAS, or both NRAS/KRAS genes. RAS mutations were point mutations affecting known hotspots in NRAS and KRAS. Both RASMT and RASWT had a median age < 60 years (55 (14-83) vs. 49 (7-83) years, P=0.9) and sex was equally distributed among the two groups. Leukopenia, defined as white blood cell count In sum, our study of the frequency, clonal architecture and impact on survival of RASMT in CBF-AML patients points out the unique characteristics of this specific disease subgroup in which sole RASMT might represent, in addition to KITMT, a mutational lesion cooperating with inv(16) and t(8;21) rearrangements in driving leukemic evolution. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Glycomimetics: Consultancy, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Abbvie: Consultancy; Incyte: Speakers Bureau; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Daiichi Sankyo: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Acute promyelocytic leukemia (APL) is characterized by PML-RARA fusion caused by the t(15;17)(q24;q21) translocation. Although PML-RARA fusion explains the dedifferentiation in most of APL patients, it still does not entirely represent the unique cause of all the clinical manifestations of the disease failing to determine the full leukemic phenotype. Up to 40 % of APL patients have an additional chromosomal abnormality other than PML-RARA. Murine studies have reported that additional cytogenetic abnormalities and secondary somatic mutations (for instance FLT3-ITD) might contribute to leukemia progression. Indeed, mice expressing mutant PML-RARA develop definitive leukemia after one year, suggesting that additional hits are required for transformation. Moreover, no distinct genetic signature has been characterized by next generation sequencing (NGS). This confirms that no gene has been reproducibly identified. APL respond to all-trans retinoic acid (ATRA) in the great majority of patients. However, one quarter of APL develop ATRA resistance suggesting that additional secondary chromosomal abnormalities might be evolving resistance. Combination of low dose arsenic, modify certain epigenetics, with ATRA decreased resistance potential and improved response. In the line with other possible factors involved in ATRA resistance, is the broad nature of the targets of ATRA. A molecular core network of ATRA's targets has been clustered in differentiation, growth factors and nuclear receptors possibly cooperating with PML-RARA and additional chromosomal abnormalities. Herein, we aimed to characterize the gene mutations and chromosomal abnormalities playing key roles in cellular differentiation and epigenetic regulation and to correlate the occurrence of these alterations with treatment response and survival outcomes in APL. We took advantage of a large cohort of APL patients (n=145). Median age of the cohort was 50 yrs (19-85); equal gender distribution; median blood counts were: [WBC 6.2 x 109/L (0.4-155); 37% had leukopenia], hemoglobin [9.8 g/dL (2.7-16.2); 32% had anemia] and platelets [29 x 109/L (range of 0-228); 93% had thrombocytopenia]. In terms of karyotype, 15% of the patients carried +8, 7% had complex karyotyping (≥3 cytogenetic abnormalities), 2% had -7/del (7q) or del (12p), 1% had -17/del(17p), and 1 patient had -5. Mutational analysis of 30 genes panel, identified 141 mutations carried by 65% (94/145) of APL patients. The most frequent mutations were observed in FLT3-ITD (61/143; 43%), WT1 (26/139; 23%), and ASXL1 (7/136; 5%) genes. Less frequent mutations were found in 3.7% of CEBPA, KRAS, and NRAS genes as well as in CBL, EZH2, TET2 (3% each) genes. Additionally, we noted that all mutations were recurrent in specific functional pathways and patients carried mutations in more than 1 gene of the same pathway. Of note, cell signaling and proliferation genes (CBL, NRAS, KRAS, KIT, FLT3) were the most frequently mutated (77/141, 55%) and impacted OS (HR: 1.7, P=0.02). Moreover, transcriptional factors which are often mutated in AML (e.g. CEBPA, TP53, NPM1, RUNX1, WT1) as well as major determinants of cell's fate were markedly mutated (38/141, 27%) suggesting that genetic impairment of signaling and transcription might contribute to the lack of differentiation observed in APL phenotypes. Mutations in epigenetic genes and histone methyltransferases (ASXL1, BCORs, DNMT3A, EZH2, IDH1/2, TET2) were also found in 18/141 (13%) while genes regulating cell proliferation and RAS family (CBL, NRAS, KRAS, NPM1) were enriched in 16/132 (12%) of APL cohort. We then analyzed the genetic picture of remission (APLRm, n=131, 90%) and relapsed (APLR, 1sr relapsed to ATRA, n=14, 10%) patients. Acknowledging the low number of APLR, we observed that molecular mutations did not make a key difference in APLRvs. APLRm [except for a complete lack of mutations in epigenetic pathways (0% vs. 13%)]. Contrarily, specific cytogenetic abnormalities were more common in APLR compared to APLRm as the case of +8 (36% vs. 11%; P= .02) and -17/del(17p) (2/14 vs. 0/131; P= .008). In sum, our study demonstrates that PML-RARA might be accompanied by additional acquired chromosomal change with a variety of genetic mutations in key pathways driving cellular differentiation. These molecular/ cytogenetic associations could determine resistance to ATRA and overall APL patients' survival. Disclosures No relevant conflicts of interest to declare.

Therapy-related acute myeloid leukemia (t-AML) is a complex disease entity. It results from molecular abnormalities induced by chemotherapy, radiation and immunosuppressive therapies. As a group of diseases, t-AML may represent cases that progressed from therapy-related myelodysplastic syndromes (t-MDS) and "de novo" t-AML. The classification t-AML also includes patients (pts) whose AML is a second primary cancer e.g., due to a genetic predisposition to develop multiple, distinct cancers (those cases would be indistinguishable from the non-therapy-related AML). Origins of the disease may also vary: t-AML may evolve from clonal hematopoiesis of indeterminate potential (CHIP) that preceded the first cancer, as a de novo disease or as a disease which progressed from "de novo" CHIP. Comprehensive genomic analyses involving clonal hierarchy may reveal genetic patterns pointing towards a potential molecular pathogenesis. We applied targeted gene sequencing to analyze a large cohort of pts with AML (n=2696) for the presence of somatic mutations: comparator subtypes include primary AML (pAML, n=2133) and secondary AML (evolving from an antecendent MDS; sAML, n=446) to be compared to t-AML (n=117). These pts have had a history of other primary malignancies for which they received cytotoxic treatments including chemotherapy and/or radiation. t-AML pts were younger than pts with other AML types (median age: 60 years for t-AML vs. 65 and 69 for pAML and sAML). t-AML pts were more likely to have leukopenia compared to pAML (23% vs. 21%, P=0.7) but significantly less likely than pts with sAML (23% vs. 38% P=0.002). Normal cytogenetics were significantly less present in t-AML when compared to pAML (39% vs. 62% P In sum, cytogenetic and molecular features of t-AML were more similar to those of sAML rather than pAML. TP53 was the most commonly mutated gene and carried a dismal prognosis, possibly representing a selective growth advantage after receiving chemotherapy and/or radiation therapies for primary cancers. Disclosures Nazha: Novartis: Speakers Bureau; Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Incyte: Speakers Bureau; Jazz Pharmacutical: Research Funding; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Genetic studies have been early introduced for the classification of acute myeloid leukemia (AML), but specific molecular lesions [e.g., t(8;21), inv(16), t(15;17)] define only a fraction of patients. In the genomic era, many mutations and cytogenetic abnormalities and their combinations have been described in AML; their diversity contributes to the heterogeneity of pathomorphologic presentations. While certain mutations are restricted to individual classical morphologic subtypes, pathognomonic lesions are difficult to find. Therefore, to date, molecular mutations or cytogenetic abnormalities have been used to assess prognosis rather than define disease sub-entities according to molecular pathogenesis, likely a future standard in AML diagnosis. Balanced translocations are prototypic founder lesions, however, founder somatic vs. subclonal mutations have not been used to define subtypes of AML. Molecular classification may better reflect the pathogenesis of AML than morphologic/clinical subtypes e.g., secondary (sAML) vs. primary AML (pAML) or AML with normal (ncAML) vs. abnormal cytogenetics (acAML). This work describes the results of a large and comprehensive analysis of genomic landscape and cytogenetics in AML. Initially, 6788 cases were analyzed, including 4867 from our institutions (Cleveland Clinic Foundation and Munich Leukemia Laboratory, n=4862) fortified by publically deposited cases (n=1926) to define gene signatures and succession of mutations distinguishing pAML vs. sAML. While data for core-binding factor AML, MLL gene rearrangement leukemia, acute promyelocytic leukemia and therapy-related AML have also been analyzed (n=1094), we focused on remaining sAML (n=876) vs. pAML (n=4808) and within these entities (ncsAML, n=367; acsAML, n=509 vs. ncpAML, n=2856 and acpAML, n=1952). Although, sAML and pAML showed usual clinical differences, a reliable diagnostic algorithm could not be constructed using supervised analytic approaches and thus we aimed at identification of molecular biomarkers with prognostic and diagnostic value. Abnormal cytogenetics were present in 57% of AML including complex: 13%, +8; 11%: -5/del(5q): 8%; and -7del7q: 8%. Deep NGS for the 25 top mutated genes was applied and correlated with clinical and cytogenetic parameters (Fig.1A-B). We then defined/compared mutational landscape in acAML and ncAML separately. AcsAML showed more complex karyotypes (58% vs. 40% in acpAML, P Using combined data sets of mutations and cytogenetic abnormalities and clinical parameters, we then constructed supervised and unsupervised analyses to identify signatures of pAML vs. sAML: discordant cases between the molecular and the classic diagnostic assignment of sAML vs. pAML were investigated and a novel molecular classification irrespective of the pAML and sAML derivation has been created to include founder and secondary mutations combinations and concordance of the outcomes. Disclosures Advani: Abbvie: Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Daiichi Sankyo: Consultancy; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Incyte: Speakers Bureau; Abbvie: Consultancy; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Background. As compared to somatic mutations, our understanding of germline (GL) variants' contribution to myeloid neoplasia (MN) is less advanced. To date, only a limited number of leukemogenic GL alterations have been identified in adult cases, but WHO Classification of Leukemias does distinguish a separate entity of MN with GL predisposition. Conceptually, pathogenetic variants can be sub-classified according to their penetrance ranging from "leukemia genes" (e.g., CEBPA, NPM1, RUNX1) to "leukemia risk genes" (e.g., DKC, FANCA, SBDS). Methods. Using publicly available data we compiled a cohort of 600 MN for which whole exome sequencing was available for both tumor and GL DNA. With the aid of an extensive calling pipeline we investigated the presence of pathogenic GL variants in 677 genes known to be involved in cancer. Application of a stringent pathogenicity score and somatic contamination filters allowed for identification of 5880 variants (with frequency Results. Overall 14 rare allele homozygous alterations and compound heterozygotes were found in 7/23 Fanconi anemia (FA) genes (e.g., BRCA2, SLX4, FANCI, FANCD2, FANCA, ERCC4, and BRIP1). Biparental inheritance in FA genes occurred more than would be expected given the total number of rare variants in the patient cohort suggesting a disease predisposition for individuals with biparental rare FA gene variants constituting a non-clonal first hit with a long latency period. This finding prompted us to prospectively analyze the frequency of FA variants in an independent MN cohort of 729 [MDS, MDS/MPN and secondary AML (sAML)] and 268 marrow failure (BMF) cases (AA, PNH). We have defined Tier-1 GL variants as those known variants (described in various FA data bases), pathogenic variants according to ASHG and new frame-shift or missense variants scored deleterious by using >4/6 applied scoring algorithms. All other GL variants including possibly pathogenic according to ASHG were assigned to Tier-2 group. The analysis revealed a total of 39 mutations (Tier 1); 80 Tier 2 variants were not included in this stringent analysis. There were 13 frame-shifts and 17 stop-gain, and there was 1 case in which 2 genes or 2 alleles were affected. The most commonly affected gene was BRCA2 with 6/39 alterations (6 variants) followed my BRCA1 in 5/39 (5 variants), FANCD2 with 5/39 alterations (4 variants), FANCA/FANCB and FANCG each with 3/39 (3 variants), FANCC in 2/39 (2 variants to a total frequency of 3.2% (23/729) and 6% (16/268) in MN and BMF, respectively. If combined, the frequency of these variants in control cohort would be expected to be 0.09%. The average age of presentation of FA carriers was 62 vs. 67 yrs. in all other MN (P=.01). The average age of presentation in BMF with FA variant was 30 vs. 45 yrs (P=0.02) in the rest of BMF group. Remarkably, we have identified 33 patients with AA who progressed to MDS, mostly del(7q), within this group: 9/33 FA Tier-1 carriers were found among these patients vs. 16/238 in non-progressors. Conclusions. The prevalence of FA gene variants are enriched in patients with MN (primarily MDS and sAML) and in BMF (primarily AA and PNH) suggesting that the carrier status of these genes may not follow an autosomal recessive trait and that the presence of a healthy allele is not entirely protective. The impact of carrier configuration may be missed due to long latency, competing morbidities and the resultant incomplete penetrance. Mono-allelic forms under genomic insult and replicative stress conditions, such as emergent increased hematopoiesis, endogenous or exogenous DNA damaging toxins and other environmental factors, might be responsible for instability in DNA repair responses. The clinical implications of it includes responsiveness to chemotherapy and in fact the choice of chemotherapy, transplant conditioning and for tumor surveillance e.g., in patients with AA at risk for MDS progression. Disclosures Nazha: Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Abbvie: Consultancy; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Daiichi Sankyo: Consultancy; MEI: Other: Data monitoring Committee. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

TET2 mutations (TET2MT) occur around 40% of myeloid neoplasms (MN) and often constitute founder hits, as concluded from patient's clonal architecture and subclinical TET2MT clones in healthy individuals at risk for MN. The mechanisms of TET2MT in leukemogenesis involve at least two potential mechanisms: i) TET2 mutations could lead to stem cell expansion and altered differentiation and/or ii) they may convey a mutator phenotype with progression due to a higher rate of subsequent genetic hits. Both mechanisms are compatible with the weak driver function of TET2MT. Here, we studied whether TET2MT predispose to additional oncogenic mutations through faulty DNA repair,i.e., whether they produce a clonally acquired mutator phenotype. TET2MT lead to the inhibition of passive demethylation. Alternatively, they impair demethylation via excision repair of 5fC/5caC. Moreover, global 5mC accumulation may increase background C>T mutation rates via 5mC deamination linking TET2 to base excision repair machinery. We investigated this hypothesis because of the finding of increased mutability found in Tet2KO and Tet2kdin the mouse as determined by the numbers of coexisting subclonal hits in Tet2MTdisease (Pan F et.al., Nature Comm, 2017). Using WES analyses (n=435), we identified 95 cases with TET2MT stringently selected for high VAF and most damaging effects as confirmed by 2D-UPLC-MS/MS assay showing low levels of the TET2-dependent DNA oxidation products. To test whether these TET2MT indeed predisposed to additional mutations, we enumerated somatic SNV vs. TET2WT cases. Sequencing of MSH2, MSH3, MSH6, MLH1, CHEK2, and BRCA2 excluded the potential effects of alteration in these genes on our results. Similarly, the MMR gene expression were not significantly different among 20 TET2MT MDS, 71 TET2WT MDS pts, and 17 controls, indicating MMR downregulation was not the culprit. Consistent with our theory, TET2MT pts had a 1.5-fold increase in median WES SNV (p50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect. HeLa TET2kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. In mice, 1.4-fold increased mutagenicity at Tet2-dependent active demethylation sites was found, in a manner also suggestive of a defective MMR. TET2MT cases (n=5) were shown to be microsatellite-stable at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites. MOLM-13 TET2kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2kd renders cells vulnerable to DNA damage. In murine cells, by overexpressing Flag/V5-tagged TET2 in MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. Using HPRT mutability assay, we measured mutational frequency in the HPRT1 gene in control, TET2kd, MSH6kd,and TET2kd/MSH6kd HeLa cells. Moreover, HPRT1 mutational frequency in TET2kd HeLa cells increased 24-fold compared to parental controls while MSH6kd HeLa cells had a further 10-fold increase in HPRT1, likely reflecting the presence of TET2-independent mechanisms for MSH6-mediated MMR. Intriguingly, the dual TET2kd/MSH6kd HeLa cells had equivalent MSI and mutational frequencies to the MSH6kd HeLa cells, supporting MSH6-dependence for the mutator phenotype induced by TET2 loss. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2kd altered MSH6 nuclear co-localization. TET2 immunoprecipitation from human CD34+ cells demonstrated abundant MSH2 binding to TET2, although we were unable to co-precipitate MSH6 in the same experiment. In sum, we uncovered novel connections among TET2, MMR proteins, epigenetic modifications, and genomic instability. Given that MSH2/6 is known to preferentially bind 5hmC, TET2 may target MSH2/6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of pts with TET2MT-driven MN. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Myelodysplastic syndromes (MDS) ultimately will progress to a higher-risk form or acute myeloid leukemia. This evolution is accompanied by acquisition of genetic hits following ancestral mutations, with further subclonal diversification of the clonal architecture. Conceptually, therapeutic approaches targeting ancestral hits have the potential to eradicate MDS at early stages of ontogenesis. Founder SF3B1 mutations are frequent in MDS and therefore represent rational targets for drug development. During our drug discovery efforts we identified an existing drug that selectively inhibits the growth of SF3B1 mutant (SF3B1MT) cells. We consequently examined the effects of known compounds possibly arresting clonal expansion of SF3B1MT MDS cells. For our studies, we generated CRISPR/Cas9 knock-in human cells stably expressing the recurrent SF3B1 K700E mutation and a chromophore-tagged reporter (mRFP). Because of the high frequency of SF3B1 mutations and their effects on the splicing of a multitude of genes, we hypothesized that some of the corresponding downstream effects could be effectively targeted. Using luminescent cell viability assays and flow cytometry analysis, we screened a 3,000 compound library for selective sensitivity against isogenic SF3B1MT cells. A subset of this library contained 23 calcium2+ channel blockers (CCBs) of which one specific dihydropirimidine (DHP) showed the highest inhibitory activity against SF3B1MT cells. Several studies point towards a role of calcium signaling in MDS. Proteins active in calcium metabolism (GPR68, calpain, calpastatin) are involved in modulating sensitivity to drugs used in MDS (e.g., lenalidomide). Divalent substrates including Fe2+ may use CCs and it's plausible that our DHP can inhibit iron overload by blocking Fe2+ trafficking through L-type CCs. In this regard, CCBs have been shown to stimulate the activity of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Furthermore, it is known that the SF3B1 mutations reduce the expression of the iron transporter ABCB7, leading to increased iron accumulation. All of this led us to further evaluate the effects of DHP in vitro and in vivo. DHP had higher growth inhibitory activity against SF3B1MT cells when tested in vitro using 8 serial concentrations in half-log dilutions for a period of 3 days (20% and 60% growth inhibition at 1 and 3μM). Mixed competitive experiment of K700EmRFP and WTGFP cells treated with increased doses of DHP (1, 3, 5, 10, 20, 50 μM) reduced the competitiveness of K700EmRFP over the time inducing a 3-fold reduction at 50μM. K700EmRFP cells expressed half the amount of ABCB7 mRNA compared to WTGFP cells by RT-PCR. Therapeutic index provided by DHP was determined in vivo. Bone marrow cells of B6. Gt(ROSA)26Sortm1Sor/J (CD45.2) donors were transplanted in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients. Two weeks after transplantation, average engraftment (measured as percentage of CD45.2(+) donor cells) was 96% ± 0.02. DHP (10 mg/Kg) was orally administered. No decrease in the proportion of CD45.2(+) donor cells was seen post-treatment compared to pre-treatment (96±0.01 % vs. 96%±0.009). Similarly no change in the proportion of CD45.2(+) donor cells was observed in competitive repopulation experiments when B6. Gt(ROSA)26Sortm1Sor/J cells were mixed 1:1 with transgenic Sf3b1+/K700E cells (40%±0.13 vs. 44%±0.02). In contrast, preliminary experiments showed that DHP had an effect on reducing Sf3b1+/K700E allele burden in two chimeric mice (to 21% and 24%) compared to pre-treatment. DHP is structurally unique in comparison to other DHP-based CCBs; in that it possess a -CH20-CH2CH2NH2 moiety linked directly to the DHP scaffold that may in itself provide opportunities or modes for Fe2+ chelation as well as its L-type CCB activity. Our observations and structural analyses therefore provide impetus to explore this feature to possibly improve the drug's efficacy. In sum, we have demonstrated that the clonal growth of cells carrying SF3B1 mutations might be suppressed using the known L-type CCB DHP. This might represent a novel modulator of ATPase activity of ABC transporters in SF3B1MT expressing low ABCB7 levels. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Large granular lymphocytic leukemia (LGL) is a lymphoproliferative cytotoxic T cell (CTL) that typically presents in elderly population. The spectrum of the disease ranges from semi-reactive oligoclonal to clonal CTL expansion, and from silent to a chronic leukemia with para-neoplastic manifestation. However the description of the disease still preceded the new genetic landscape mapping techniques. In lieu of this, we analyzed the genetic landscape of LGL using a set of 33 genes. Interestingly, not only did the deeper analysis help with a better understanding of the complex CTL proliferative processes but indicated a suspected association with conditions such as clonal hematopoiesis of indeterminate potential (CHIP). Association of LGL with age related CH has been described in the past by us and others. Our results indicate that LGL is often associated with the presence of myeloid mutations, generally consistent with the most common typical CHIP mutations (DNMT3A, TET2, ASXL1). We then analyzed a set of 13 patients with coexistent MDS/LGL. This observation raises the question that LGL is, at least in some cases, a tumor surveillance response. All LGL patients were diagnosed using a stringent single center diagnostic criteria and at the time of diagnosis, were also checked for other conditions including MDS to prevent any risks of overlooked diagnoses. A previously well described diagnostic standard of >3/5 positive criteria was used; (i) presence of large granular lymphocytes (>500/μL, by differential counts from complete blood count) for >6 months; (ii) abnormal cytotoxic T lymphocytes expressing CD2, CD56 and CD57 and lacking CD28; (iii) preferential usage of a TCR Vβ family by flow cytometry; and (iv) TCR gene rearrangement by PCR; (v) bone marrow infiltration with LGL is another diagnostic criterion but a designated marrow biopsy is often not performed in clear LGL which are otherwise asymptomatic. Coexistent presence of MDS and LGL was confirmed in 13/240 (5%) of the patients. These patients displayed typical features of MDS including abnormal cytogenetics (8/13), dysmorphia of myeloid cells in the bone marrow (13/13), ringed sideroblasts (1/13) and the presence of highly clonal somatic myeloid mutations (8/13) with an average VAF of 28+3%. When compared to LGL only, the MDS/LGL series had 15% vs. 39% (P=0.014) STAT3/5b mutations. We detected the presence of mutant myeloid clones in 25% (41/161) of the patients with pure LGL with an average VAF of 35+ 2%. Suspecting that these mutations may be present early in MDS and CHIP, we compared the mutational spectrum of LGL against MDS (n=835) and CHIP. Indeed, an overlap between the typical CHIP mutations was noted with LGL; in addition the clonal burden in CHIP was observed to be lower, 13% vs. 33% in LGL indicating a more advanced myeloid disease in LGL. This could be due to overlapping demographics or through a common pathophysiologic link. Spliceosomal mutations, cohesion complex, PRC2 and RAS mutations were overrepresented in MDS and CHIP in comparison to LGL. Our results indicate that LGL is often associated with myeloid mutations that are also typical in CHIP. Though, age-related changes or other mutagenic stressors, including inflammatory changes could be contributing factors to both LGL and CHIP outgrowth, conversely LGL may evolve as a consequence of a tumor surveillance response to myeloid neoplasms or CHIP. While the coexistence of MDS and LGL has been described in the past, and our analysis corroborates this, we observed that LGL may also coexist with CHIP in some patients. The implication of this would be beneficial to our understanding of, not only, the individual diseases but could also impact care in CHIP survivorship. Disclosures Nazha: MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Tolero, Karyopharma: Honoraria; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Saunthararajah:EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties; Novo Nordisk: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Mutations (MT) in the 5' untranslated region (UTR) of ANKRD26 (A26) are implicated in ANKRD26- related thrombocytopenia (A26-RT), an autosomal dominant disorder of mild to moderate thrombocytopenia (TP) often presenting in adulthood, although severe and pediatric cases are reported. Erythrocyte and leukocyte counts are normal to increased, with unremarkable morphology. Platelet (plt) size is usually normal, as with ETV6- and RUNX1-mutated TP. Together, A26, ETV6, and RUNX1 germline (GL) MT comprise a separate 2016 WHO category of myeloid neoplasms (MN) with GL predisposition and preexisting platelet disorders. Normal plt size separates A26-RT from other familial TPs with giant plts. No consistent morphological plt aberrations have been reported. Bleeding history is absent or mild, and while TP is not life threating, 8-10% of patients (pts) develop a MN, including a 30-fold increased risk for AML relative to the general population. A26 is an inner membrane adaptor protein with 2 major domains: ankyrin repeats (ANKR) and coil-coil (C-C), both of which interact with signaling and cytoskeletal proteins. A26-RT MT are almost exclusively in the 5'UTR, however, rare A26 coding variants (A26-CV) are reported to segregate with familial TP. Still, some studies on A26-RT limit sequencing to the 5'UTR. As such, A26-CV are not well represented or described. We performed whole-exome sequencing (WES) on 195 pts with MN seen at Cleveland Clinic between 2004 and 2012, and downloaded WES .bam files from online sources, totaling 653 MN cases. Using a standard pipeline for discovery of rare GL variants ( Complete clinical description was available for 8 A26-CV pts, all diagnosed with MDS or MDS/MPN at med age of 64. Two pts had antecedent bruising 1 year prior to diagnosis (dx). No prior bleeding was noted. Three pts had prior TP, two of whom had bicytopenia and pancytopenia. First degree family history (FH) was positive for cancer in 6 pts (75%), including 2 pts with FH of hematologic neoplasms. One pt had a 2nd-degree relative with a non-malignant hematologic condition requiring transfusions. At dx, cytogenetics were normal and complex in 2 pts each (25%). Deletions in chromosomes 5, 17, 20, and Y were observed in 1 pt each, and monosomy 7 in 2 pts. On bone marrow aspirate, 6 pts (75%) had dysmegakaryopoiesis, found in interstitial patterns and clusters. Mild to moderate dyserythropoiesis was observed in 5 pts (63%), and 5 pts had dysgranulopoiesis. The med blast percentage was 1.5% (range 0-8%), with 5 pts having hypercellular marrow, med 65% (range 40-95%). On corresponding CBC, pts were anemic (med hemoglobin of 9.5 g/dL), with the majority (n=5) showing signs of erythroid dysplasia, and also thrombocytopenic (med plt of 99 k/μL), with giant plts observed in 3 pts. Two pts had both giant plts and abnormal erythroid morphology. Hypo and monolobate, hyposegmented, and hypogranular forms were observed in the 3 lineages, as well as detached nuclear lobes, budding, segmentation, and nuclear-cytoplasmic dyssynchrony. On next generation sequencing, co-occurring SRSF2 and ASXL1 MT were observed in 3 and 2 pts, respectively. In sum, we have identified 22 A26-CV in MN, suggesting a role in predisposition as with A26-RT. We have seen in our cohort that A26-CV pts present differently from those with A26-RT. They have a variable past medical history and limited FH of TP, are anemic, with multilineage dysplasia observed not just in bone marrow, but also on peripheral blood smear, especially in megakaryocytes and erythrocytes. This is not surprising, as A26 is expressed in both lineages. The presence of giant plts is noteworthy. The mechanism for hypomorphic A26-CV may differ from that of the A26 5' UTR, which increase A26 levels in late-stage megakaryopoiesis by abrogating RUNX1/FLI1 binding, leading to aberrant proplatelet formation. Given the plt size and presence of nuclear phenotypes, altered interactions with signaling and cytoskeletal proteins could be involved, and may represent a novel A26 phenotype. Further investigation and association of A26-CV with MN ontogeny is under way. Disclosures Mukherjee: Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau. Advani:Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Research Funding. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Gerds:Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Roche: Research Funding; Incyte: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Both founder and secondary mutations in AML and MDS have prognostic implications that may depend on their rank in subclonal hierarchy. Mutational profiles at relapse after chemotherapy may be different from relapse following alloHCT as a result of the graft-versus-leukemia (GVL) effect. We hypothesized that the molecular landscape of AML and MDS at the time of post-alloHCT relapse may (1) recapitulate the clonal hierarchy of the founder and secondary mutations at diagnosis, (2) be represented by founder mutations without secondary mutations, or (3) be characterized by acquisition of new mutations. Using bone marrow samples banked at diagnosis and at the time of relapse, we compared mutational characteristics using a targeted multi-amplicon deep NGS panel of the 176 most commonly mutated genes in myeloid neoplasia. Thirty patients (pts) with AML (n=22) or MDS (n=8) who relapsed after their first alloHCT were included as the "transplant group", and samples from diagnosis or pre-HCT relapse (if available), whichever was last, and at relapse were compared. A comparison "chemotherapy group" included 11 pts with AML (n=7) or MDS (n=4) who relapsed after achieving complete remission (CR) from chemotherapy were included to compare mutational changes from diagnosis to relapse. For this analysis, we selected the 27 most commonly observed somatic mutations. For the transplant group, median age at alloHCT was 59 years (range 32-73). Cytogenetics at diagnosis were normal in 47% and complex in 20%. At diagnosis, IDH2, NPM1, ASXL1, RUNX1, FLT3, and TP53 mutations were found in 13%, 13%, 10%, 10%, 7%, and 3% of pts, respectively. The majority received reduced-intensity conditioning (53%), bone marrow grafts (53%) from matched unrelated donors (55%). Sixteen patients (AML=15, MDS=1) were in CR at transplant. Median time from alloHCT to relapse was 114 days (median 28-935 days). The mutational profiles for all patients changed at post-alloHCT relapse. While the majority both gained and lost different mutations (60%), some just gained (27%) or lost mutations (13%) at relapse. The number of mutations gained ranged from 0-5 (median 1) and mutations disappeared ranged from 0-5 (median 2). The most common newly acquired mutation was FLT3 (17%) followed by DNMT3A (13%). Two cases (7%) showed re-acquisition of mutations that were present at diagnosis but disappearing after induction; 1 patient re-acquired FLT3, NPM1, and RIT1 mutations while another reacquired a TET2 mutation. Most commonly disappearing mutations at post-alloHCT relapse was IDH2 (10%). DNMT3A (20%) was the most common mutation that persisted through chemotherapy and alloHCT. For the chemotherapy group, median age at diagnosis was 59 (range 32-71). Cytogenetics at diagnosis were normal in 55% and complex in 18%. At diagnosis, NPM1, FLT3, TP53, and ASXL1, mutations were found in 27%, 18%, 18%, and 9%, respectively; none had IDH2 or RUNX1 mutations. Median time from diagnosis to relapse was 154 days (range 33-1,716 days). As in the post-alloHCT group, changes in the mutational profile were found in all patients. Six patients (55%) gained newly acquired mutations while 5 patients lost a mutation without acquiring a new mutation: 2 cases with TP53 loss and 1 case each of PTPN11, RIT1, and TET2 losses. The number of mutations gained and disappeared per patient varied (each median 1, range 0-3). The most commonly gained mutations were DNMT3A and TET2 (18% each). DNMT3A and GNAS were the most commonly persistent mutations at chemotherapy relapse (18% each). The biology of relapse following alloHCT is complex. Our results show that while clonal changes were always present at relapse following chemotherapy or following alloHCT, the mutational evolution was more diverse and complex at post-alloHCT relapse as compared to relapse after chemotherapy alone. This results signifies the importance of mutational profile test at relapse for the targeted therapy. Larger scale analyses can further elucidate somatic hits suggesting the mechanism of escape from the GVL. Disclosures Gerds: Incyte: Consultancy, Research Funding; Pfizer: Consultancy; Celgene Corporation: Consultancy, Research Funding; CTI Biopharma: Consultancy, Research Funding; Roche: Research Funding; Sierra Oncology: Research Funding; Imago Biosciences: Research Funding. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Mukherjee:McGraw Hill Hematology Oncology Board Review: Other: Editor; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Advani:Glycomimetics: Consultancy, Research Funding; Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding. Nazha:Jazz Pharmacutical: Research Funding; Abbvie: Consultancy; Daiichi Sankyo: Consultancy; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Novartis: Speakers Bureau. Majhail:Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy; Atara Bio: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Systematic application of NGS has led to the discovery of a myriad of somatic mutations. One important class of genes affected by mutations in myeloid neoplasia (MN) is involved in epigenetic regulation either directly by modifying DNA or by enzymes modifying histones such as histone methyltranferases and demethylases (e.g., EZH2, MLL, UTX). This work focuses on mutations of EP300, a histone acetyl transferase. EP300 (22q13.2) encodes the adenovirus E1A associated cellular p300 transcriptional co-activator protein with lysine acetyltransferase activity as does its paralog CBP (16p13.3). Ablation of EP300 in mice results in embryonic lethality. Heterozygous germline EP300 mutations were described in Rubinstein-Taybi syndrome (RBTS), a congenital neurodevelopmental disorder characterized by facial dysmorphology, distal limb abnormalities and mental retardation. In addition, RTBS patients appear to have a predisposition for childhood malignancies. Somatic EP300 mutations have been found in solid tumors and lymphoid neoplasia but have been less studied in MN. We analyzed DNA sequencing results of 2382 MN patients [AML (n=1646), MDS (n=344), MDS/MPN (n=79), and MPN (n=31)]. Germline status of lesions was confirmed using skin-biopsy or CD3+ derived DNA, when such specimens were available. New nonsense, deletion, and exceedingly rare, individual, missense mutations (4/6 applied scoring algorithms (CADD, PolyPhen2, SIFT, LRT, MutationAssesor and MutationTaster within Annovar) were included in downstream analyses. EP300 mutations were detected in 2% of patients (47/2382); 61% (28/46) were somatic and 39% (18/46) germline. Mutation types in somatic vs. germline were: missense (43 vs. 68%), nonsense (25 vs. 21%), splice site (14 vs. 0%), deletion (7 vs. 5%) and insertion (10 vs. 0%). No patients with hemizygous deletions were detected by SNP array. Median age of EP300 somatic and germline mutants were similar compared to EP300 wild type [63 (56-81) vs. 65 (29-79) vs. 65 (18/92) years]. For somatic EP300 mutants, 43% of cases (10/23) were characterized by normal and 22% complex cytogenetics without enrichment for -7/7q, del5q, del20q or +8. Of note, 67% of MDS patients (4/6) had a prior history of other malignancies treated with chemoimmunotherapy or radiation (lymphoma, prostate cancer). Using VAF-based bioanalytic methods, we were able to roughly recapitulate the clonal architecture and identify founder lesions. We compared the expected vs. observed VAFs according to the corresponding variability in read counts and a stringent binomial distribution algorithm. EP300 mutations were more likely to be dominant lesions (62.5%) while 37% were subclonal. Serial sample analysis was performed; EP300 mutant subclones expanded during progression of the diseases suggesting a tumor suppressor function of EP300. In normal hematopoiesis, EP300 expression increases proportionally with myeloid precursors' maturation. We found that EP300 mRNA levels were significantly lower in AML patients vs. healthy controls (log2CPM: 7.92 vs. 8.27, P=.03; Beat AML). One therapeutic approach would be to alleviate the effects of the mutations using HDACi. However, HDACi had equal sensitivity in EP300 mutant and WT cells in publicly available databases of drug sensitivity and also according to expression levels (low vs. high). Another alternative therapeutic approach is based on the potential synthetic lethality of a total loss of EP300 concluded from embryonic lethality in mice. Using in vitro proliferation assays we found that the HATi S7152 was more effective in growth inhibition (GI) in the EP300 mutant cells, Jurkat (M2118I) at 10 µM (GI: 84%) compared to EP300 wild type cells e.g., KG-1 (43%), K562 (54%), U937 (24%) and TF-1 (15%) at 10mM while NB-4 (L2393V) cells only reached a GI (27%) at 20µM. In sum, we characterized for the first time the spectrum of somatic and germline EP300 mutations in MN and the possible therapeutic implications of using HATi to modulate EP300 activity. Figure Disclosures Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Rationale: Dose-reduction of a nucleoside analog reduces cytotoxicity and hence can increase safety, but with less cytotoxicity-driven efficacy as a trade-off. Such an efficacy trade-off, however, may not apply to the nucleoside analog decitabine, because it has a molecular-targeted action of depleting DNA methyltransferase 1 (DNMT1) separable from cytotoxicity, and that occurs and is saturated at low concentrations. In fact, higher decitabine doses/concentrations, by causing cytotoxicity, limit feasible exposure times for this molecular effect that can cytoreduce p53-null, chemorefractory myeloid malignancies via terminal-differentiation instead of apoptosis, simultaneously sparing normal hematopoiesis. Hence, we designed decitabine application to avoid cytotoxicity and increase DNMT1-targeting and describe here results in 69 patients (25 of whom were previously described with shorter follow-up (NCT01165996)(J Clin Invest 2015; 125(3):1043-55]). Methods: IRB approved Myeloid Malignancy Registry (16-020) and Sample Repository protocols (5024) curated demographic, laboratory, intervention, outcome, and mutational data in myeloid malignancy patients after written informed consent; 69 of 1694 patients in the Registry received the alternative decitabine regimen: (i) Dose: 0.1-0.2 mg/kg (~3.5-5 mg/m2), verified to deplete DNMT1 without cytotoxicity in primate and human studies; (ii) Schedule: 1-2X/week, frequent and distributed, to increase S-phase dependent DNMT1-depletion; (iii) Route: subcutaneous, to avoid high peak concentrations.A standard starting dose of 0.2 mg/kg was reduced to 0.15 mg/kg if infection risk was high (e.g., ANC10% or for lack of response to 1X/week. Neutropenia from treatment was managed by interruption then resumption upon neutrophil recovery (same dose or lower by 0.05 mg/kg). Routine anti-emetic prophylaxis was not required. Results: Non-cytotoxic DNMT1-depletion was confirmed by bone marrow gH2AX and DNMT1 measurements (published for 1st25 patients). The 69 patients had MDS 54%, MDS/MPN 14%, MPN 17% and AML 15%. Their median age was 69 years (range 45-89). Prior therapies (77%) were 5-azacytidine 29%, lenalidomide 19%, erythropoietin 26%, hydroxyurea 7%, ruxolitinib 7%, cytarabine 4%. The side-effect was neutropenia (non-cytotoxic DNMT1-depletion skews myeloid differentiation away from GMP and to EMK), complicated by fever/infection in 31 patients (45%), with 1 septic death. Eight of these 31 patients (25%) had fever/infection before treatment. Blood count improvements meeting IWG criteria for response (hematologic improvement [HI]/complete remission [CR]) occurred in 30 patients (43%; CR 14%) (Fig 1A) and were durable(median treatment duration in HI/CR 82 weeks [range 14-347]). Treatment decreased bone marrow myeloblasts, even in cases without HI/CR (Fig 1B). HI/CR was achieved in MDS/AML containing monosomy 7, trisomy 8, complex cytogenetic abnormalities and/or multiple mutations (Fig 1C). Cytogenetics were normalized in 10/36 (28%) (Fig 1C). Patients with HI/CR had better overall survival (median 31 vs18 months) (p=0.036) (Fig 1D). Predictors of HI/CR were higher baseline neutrophils (median 3.04 vs 1.23 x109/L; p=0.002)and higher marrow cellularity (median 70 vs48%; p=0.044)(Fig 1E, F). Conclusion: Myeloid malignancies containing diverse genetic abnormalities responded sustainably (up to 6.5 years follow-up) to a decitabine regimen designed and demonstrated to be non-cytotoxic yet DNMT1-targeting, consistent with scientific data validating DNMT1 as a mutation-agnostic target that operates in a final common pathway of myeloid transformation. Although this single-agent regimen is non-curative, we postulate that avoidance of cytotoxicity, combined with target-validity, enabled sustained disease control/transfusion-freedom in several patients. Reported links between some mutations, e.g., in TET2, and decitabine response could be via higher neutrophils that in turn enable frequent decitabine exposures, a basic requirement for response. Also fundamental, HI/CR requires not just suppression of malignant clones but marrow capacity to support and recover functional hematopoiesis, suggesting why low baseline marrow cellularity predicted non-response. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. OffLabel Disclosure: Alternative dose and scheduling of Decitabine for myeloid neoplasms

Large granular lymphocytic leukemia (LGLL) is an indolent disease and often associated with autoimmune disorders such as rheumatoid arthritis. The association of humoral immune disorders resulting from abnormal B cell activity, may affect original LGLL pathogenesis, clinical presentation and management through modifying disease presentation, progression and/ or resistance to standard care. Coexistence of T cell LGL leukemia with B cell abnormalities has previously been identified in the literature although described in sporadic case reports. However, no large case series or cohorts have been collected so far to study the frequency of the B-cell dyscrasia (BCD) associated with LGLL and describe clinical/ hematological findings in patients with this co-association. Here, we conducted a retrospective review of patients diagnosed with LGL leukemia at The Cleveland Clinic Foundation to search for any associated BCDs. We then classified our population into 2 groups: LGLL with BCD vs. LGLL without BCD, and comprehensively compared them for baseline, clinical and molecular characteristics. A total of 244 T-LGL patients were collected and studied. All cases were uniformly diagnosed with LGLL if 3 out of 4 following criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) presence of abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. Molecular studies including targeted deep sequencing for STAT3mutations were performed. Bone marrow biopsy results were reviewed to exclude other conditions. Endpoints of the study were death or lost to follow up. In our cohort, we found a frequent manifestation of humoral immune system abnormalities. We identified coexisting BCD in 45% (109/ 244) of LGLL patients, of whom 28 (11.2%) had monoclonal gammopathy of unknown significance (MGUS), and 13 (5.2%) had chronic lymphocytic leukemia (CLL/SLL). Six LGLL patients had multiple myeloma (2.4%). Moreover, polyclonal hypergammaglobulinemia (n=28, 11.2%) or hypogammaglobulinemia (n=14, 5.6%) was reported in 42 LGLL-patients (16.8%). The frequency of other disorders of B-cell origin was also examined. The total incidence of B-cell abnormalities in our LGLL cohort was 45%. Indeed an heterogeneous appearance of other B-cell disorders was observed including mantle cell lymphoma (n=2), DLBCL (n=6), marginal zone lymphoma (n=3), Waldenstrom's macroglobulinemia (n=1), Burkitt's lymphoma (n=1), indolent lymphoma (n=1), Hodgkin's lymphoma (n=1), non-Hodgkin's lymphoma (n=3), neck lymphoma (n=1), and smoldering myeloma (n=2). Patient with LGLL-BCD were older as compared to the ones without (median age: 62 vs. 63 years; ≥60 years: 57% vs. 69%, respectively), although the difference was not statistically significant (P=0.07). Gender was equally distributed (male: 54%, n=132; female: 46%, n=112) in patients who developed BCD. Conventional cytogenetics showed that patients without BCD were more often associated with abnormal cytogenetics (24%, n=9) as compared to LGLL-BCD (9%, n=5). Interestingly, BCD was found in 55 men and 54 women in whom only 6 patients had NK-LGLL while the remaining (n=103 patients) had T-LGLL suggesting a higher association with LGLL of T- rather than of NK-cell origin. Leukopenia was observed in 25/109 patients, with average absolute lymphocytes of 4.18 k/µL and LGL count of 2333 k/µL. Blood count showed: neutropenia in 44, anemia in 65, and thrombocytopenia in 29 out of 109 LGLL patients with BCD. TCR rearrangements were seen in 74 while somatic STAT3 mutations were observed in 37 LGLL patients while more enriched (44%, n=52) in LGLL without BCD. The association of other autoimmune conditions e.g., rheumatoid arthritis, was not different between the two groups (15% vs. 16% in LGLL with BCD vs. without; P=0.8). In sum, our investigation shows that BCD were frequent in LGLL and coexisted in 45% of the patients, commonly in the form of MGUS, and/ or hypergamaglobulinemia. Perhaps, the co-association of B-cell pathology with LGLL suggests that the two diseases either share pathogenetic driving mechanisms to enhance both B cells and T cells clones or that immunological dysfunction in setting of B cell dyscrasia could trigger/potentiate LGL expansion and/or transformation in this context. Disclosures No relevant conflicts of interest to declare.

Myeloid malignancies can evolve from prior hematologic disorders, most commonly AML evolving from MDS. AA and PNH are not malignant conditions but they can evolve to myeloid malignancies with a lower frequency compared to MDS and other myeloid and myeloproliferative diseases. PNH can evolve to myeloid disorders with an incidence rate of 4-10%. Here we aim to revisit the subject of malignant evolution of PNH to myeloid disorders by analyzing the molecular background of PNH using modern NGS technologies. Clinical characteristics, demographics and mutational profiles of patients were collected at The Cleveland Clinic Foundation. In total a cohort of 243 patients evaluated for hemolytic PNH (n=83), AA (39) and AA/PNH (n=121) followed for a period of 22 years was evaluated. Inclusion criteria were: complete flow cytometric panels of PNH cells, patients without antecedent AA (pPNH, 45), patients evolving from AA with PNH clone >20% (sPNH, 38), karyotype (at diagnosis of PNH and transformation), clinical parameters including time to malignant progression, and molecular characterization resulting from NGS performed using various library preparation systems (TruSeq, TruSight, Nextera), comparison of molecular mutations with control cohorts (AA, 160; MDS, 835). The incidence rate of myeloid disorders in our PNH cohort was 3% (7/243). Among hemolytic PNH patients, 7 patients progressed to AML (n=1), MDS (n=5) or myelofibrosis (n=1). Median age was 48 yr (range, 24-80); M/F, 5/2. Median PNH clone size was 71% (range, 29-99). Three progressors were in the pPNH and 4 in the sPNH group. Time to malignant diagnosis was A total of 45 somatic mutations equally distributed in the 2 groups (pPNH, 22; sPNH, 23) and with similar VAFAVG (pPNH vs. sPNH 38% vs. 33%) were found. PNH showed a higher proportion of individuals with mutations compared to AA/PNH+AA (42 vs. 22% of cases with ≥1 mutation; P=.002) with a median VAF percentage significantly higher in PNH vs. AA/PNH+AA (40 vs. 19%; P55), 17% were equivocal (possibly hits biclonal with a VAFSUM between 45-55), while 13% were more likely a result of clonal chimerism (hits present in different clones; VAFSUM Clonal evolution of hemolytic PNH to MDS/AML is rare, but still occurs and it is accompanied by mutations in typical myeloid genes (BCOR, NPM1, TET2, U2AF1) which in permissive circumstances are capable to change the cell's fate favoring clonal evolution. Our results suggest that most PNH cases can carry additional mutations in the same clone and these mutations can be secondary hits, with PIGA mutations being the founder lesions. However even when mutations in myeloid genes are dominant, the phenotype of the patients is inferred by PIGA. This observation supports the nature of PNH as a monogenic disease with clinical manifestations resulting by PIGA mutations rather than by myeloid genes. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.