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Morphologic interpretation is the standard in diagnosing myelodysplastic syndrome (MDS), but it has limitations, such as varying reliability in pathologic evaluation and lack of integration with genetic data. Somatic events shape morphologic features, but the complexity of morphologic and genetic changes makes clear associations challenging. This article interrogates novel clinical subtypes of MDS using a machine-learning technique devised to identify patterns of cooccurrence among morphologic features and genomic events. We sequenced 1079 MDS patients and analyzed bone marrow morphologic alterations and other clinical features. A total of 1929 somatic mutations were identified. Five distinct morphologic profiles with unique clinical characteristics were defined. Seventy-seven percent of higher-risk patients clustered in profile 1. All lower-risk (LR) patients clustered into the remaining 4 profiles: profile 2 was characterized by pancytopenia, profile 3 by monocytosis, profile 4 by elevated megakaryocytes, and profile 5 by erythroid dysplasia. These profiles could also separate patients with different prognoses. LR MDS patients were classified into 8 genetic signatures (eg, signature A had TET2 mutations, signature B had both TET2 and SRSF2 mutations, and signature G had SF3B1 mutations), demonstrating association with specific morphologic profiles. Six morphologic profiles/genetic signature associations were confirmed in a separate analysis of an independent cohort. Our study demonstrates that nonrandom or even pathognomonic relationships between morphology and genotype to define clinical features can be identified. This is the first comprehensive implementation of machine-learning algorithms to elucidate potential intrinsic interdependencies among genetic lesions, morphologies, and clinical prognostic in attributes of MDS.

Key Points 5-hydroxymethylcytosine and TET2 messenger RNA (mRNA) downregulation are common in myelodysplastic syndromes irrespective of TET2 mutations.TET3 mRNA expression levels are associated with distinct clinical outcomes in myelodysplastic syndromes with and without TET2 mutations., Decrease in DNA dioxygenase activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general downregulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, whereas 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was upregulated in MDS and inversely correlated with TET2 expression in wild-type cases. Although TET2 was reduced across all age groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influenced the clinical phenotype of TET2 deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers.

Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR β and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.

BACKGROUND : Unexplained cytopenia (UC) and low-risk myelodysplastic syndrome (MDS) are distinguished mainly by morphologic dysplasia, which sometimes shows inter-observer discrepancy. We hypothesized that gene mutations are strong prognostic factors for these low-risk patients. METHODS : We enrolled patients from four medical centers with unexplained cytopenia of at least one lineage. Diagnosis of low-risk MDS was made according to WHO 2016 classification and a revised international prognostic scoring system (R-IPSS) score of ≤ 3.5. DNA was extracted from bone marrow or blood and sequenced by targeted next generation sequencing (NGS). RESULTS : One hundred twenty-one patients were recruited: 25% with UC and 75% with low-risk MDS. Complete blood counts were similar, but low-risk MDS patients carried higher numbers of mutations (1 vs. 0; p=0.04) than UC patients. Overall, the most frequent mutated genes were TET2 (14.6%), SF3B1 (12.2%) and ASXL1 (9.7%). Survival rates of low-risk MDS patients vs. UC patients were not significantly different. UC patients and low-risk MDS patients without genetic abnormalities showed superior 5-year progression free survival compared to MDS patients with mutations (100% vs. 76.0%; p=0.005). Overall, ASXL1 mutations were associated with decreased 4-year overall survival compared to wild-type (59% vs. 31%; p=0.01). In a multivariate analysis, ASXL1 and DNMT3A mutations in low-risk MDS patients were associated with a higher risk of disease progression with hazard ratios of 7.88 (95% CI 1.76-35.32, p=0.01) and 7.45 (95% CI 1.61-34.46, p=0.01), respectively. CONCLUSION : Mutation detection is important for proper risk stratification of patients presenting with idiopathic cytopenia. Micro-abstract : Diagnosis of myelodysplastic syndromes is mainly based on cytopenia(s) and morphologic criteria which sometimes show equivocal results. We demonstrate utilities of gene mutations in patients with idiopathic cytopenia in terms of diagnosis and prognostication aspects.

Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. Immunogenetic patterns associated with self-antigenic presentation remain unclear. Herein we analyzed the molecular landscape of HLA complexes and T-cell receptor (TCR) repertoires of a large cohort of IAA patients and controls. We show that antigen binding sites of class II HLA molecules in IAA are characterized by a high level of structural homology, only partially explained by specific risk allele profiles, implying reduced binding capabilities compared to controls. Few amino acids within the synapsis HLA-DRB1-antigen-TCR, are identified as strongly associated with IAA phenotype. Those structural patterns may affect TCR repertoires, promoting immunological cross-reactivity and autoimmunity. These findings inform on the immunogenetic risk associated with IAA and on general pathophysiological mechanisms potentially involved in autoimmunity.Key pointsClass II human leukocyte antigen (HLA) loci in idiopathic bone marrow failure (BMF) syndromes are characterized by low functional divergence and decreased peptide binding capabilities, only partially explained by enrichment in risk alleles.A superstructure at the interface with the peptide binding site of DRB1 locus, potentially involved in the presentation of self-antigenic specificities, can be identified in BMF patients.This immunogenetic pattern may contribute to decrease T-cell receptor repertoire diversity, expand autoreactive T-cell clones and increase autoimmune propensity in BMF.

Extranodal NK/T cell lymphomas (ENKTCLs) are aggressive Epstein-Barr virus-associated T/NK neoplasms that predominantly affect Asians. To explore the causative somatic events, we conducted a comprehensive genetic analysis of 19 ENKTCL patients by whole-genome (

Clinical impact and mechanistic contributions to leukemogenesis are difficult to assign to less common somatic mutations. However, the genetics of inherited syndromes can often be helpful in discerning the biological functions and mechanistic consequences of genes in other diseases. PHF6 (Xq26.2) encodes a protein consisting of two PHD-type zinc finger domains with activity in transcriptional regulation. PHF6 translocations were originally described in T-ALL and its mutations were later observed also in CML and adult AML. Germline (GL) PHF6MT cause Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked disorder characterized by intellectual disability and, to date, only a few BFLS cases were found to develop lymphoma or T-ALL. While regularly encountered in myeloid neoplasia (MN), the impact and functional meaning of PHF6 are not well established. To determine the incidence, distribution and molecular context of PHF6MT we studied a large cohort of patients with MN (n=8617) collected from our institution and public series. 1 Overall, 73% of patients were AML (pAML 69%; sAML 4%), MDS (22%) and MDS/MPN (5%) with a median age at diagnosis of 67 ys (18-100). We detected 149 patients (2%) carrying at least 1 PHF6MT with 11 harboring more than 1 hit. Four patients carried -X in addition to PHF6MT (2 males; 2 females). Majority of patients (68%) carried frameshift del/ins and nonsense. Mutations were scattered across all coding region with a slightly enrichment (47%) in the second PHD domain (239-330 aa) including the frequent R274Q/X (n=17). Common hits mainly affected arginine residues essential for DNA binding capacity (R129X n=9, R116X=7, R319X=5, R225X=3) followed by other hits (I314T=6, Y301X and C20fs=4 each). Of note, R116X, R225X, R274X, C280Y, H329R and Y303* lesions overlapped with the T-ALL PHF6MT spectrum while no overlap was found with GL mutations found in BFLS. Overall, 75% of all PHF6MT carriers were males and carried mostly (80%) truncating lesions. Compared to mutational frequencies observed in other X-linked genes, truncating PHF6MT behaved similarly to those in ZRSR2 (78%), STAG2 (73%) and BCOR (62%). Conversely, BCORL1MT, KDM6AMT and PIGAMT were evenly distributed between genders. When evaluating mutational characteristics in males and females, no differences were found in sex-adjusted median variant allelic burden of PHF6MT (54.8 vs 51%) nor its mRNA expression levels suggesting locus inactivation. PHF6MT tended to be older than PHF6WT patients (72 vs 68 ys; P= .05) and had mostly (63%) AML followed by MDS (23%) and MDS/MPN (14%). OS was similar between PHF6MT and PHF6WT patients (P= .16). Expression analyses showed that PHF6 loss leads to deregulation of chromatin and transcriptional factor genes. Indeed, in our cohort the most comutated genes were transcriptional factors and chromatin modifiers genes such as RUNX1 (26/149, 17%), ASXL1 (23/149, 15%) and TET2 (17/149, 11%). Of note, this group characterized by the triple ASXL1, RUNX1, TET2 mutational configuration clustered in one of the genomic groups previously identified (GC-3) 1 but the presence of these lesions did not worsen the OS as compared to PHF6MT without this mutational constellation. A low frequency of SF3B1MT (4%) was also noted confirming the enrichment of PHF6MT in AML rather than in low risk MDS. Further, 12% (14 males; 4 females) of PHF6MT patients had X-mutation mosaicism as shown by concomitant hits in BCOR (n=8), ZRSR2 (4), STAG2 (5), KDM6A (1). PHF6MT were equally founder lesions (30%; 44/149) and subclonal (34%; 50/149) whereas the rest was indistinguishable by VAF discrimination (co-dominant). The most common subclonal mutations were U2AF1 (14%, 6/44), IDH1/2 (9%, 4/44) and RUNX1 (7%, 3/44). When PHF6MT were subclonal, the founder hits were in TET2 (14%, 7/50), DNMT3A and RUNX1 (12%, each 6/50) genes. Given the high frequency of RUNX1MT in PHF6MT we investigated whether RUNX1 and PHF6 might be correlated. Transcriptomic analysis of 6246 patients (from 9 public studies) 2 showed a direct linear correlation (AdjR2= .03, P=5.55e-05) between the expression of the two genes. Our study is the largest to date to investigate the genetic landscape of PHF6MT in MN and highlights a strong connection of PHF6 with transcriptional regulation and chromatin genes. Ongoing scDNA-seq will clarify whether these mutations were acquired in distinct clones helping in dissecting the clonal hierarchy of PHF6MT cases. Disclosures Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Advani: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenics: Research Funding; Immunogen: Research Funding; OBI: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

The pathogenesis of idiopathic aplastic anemia (IAA) involves a human leukocyte antigen (HLA)-restricted T-cell autoreactivity against unknown antigens preferentially distributed on early hematopoietic stem and progenitor cells (HSPCs). Genetically acquired GPI-anchor and HLA deficiency have been both linked to clonal immune evasion from T-cell pressure. We hypothesized that, in analogy to anti-tumor adaptive immune evasion, pathophysiology of immune escape in IAA originates together with a broader dysfunction of antigen presentation/processing machinery and immune regulatory proteins, beyond HLA molecules, as an effect of immune pressure under T-cell attack. This initial immune reaction would produce up-modulation of these pathways, ultimately promoting the acquisition of mutations and expansion of immune resistant clones. To test this hypothesis, we first performed single-cell RNAseq analysis in HSPCs in IAA patients at disease manifestation, 1 which showed signatures of dysfunction of antigen presentation machinery, with up-regulation of most of the HLA molecules, proteasome subunits and endoplasmic reticulum related organelle transporters. Strikingly, DRB1 was among the top 3 genes upregulated in IAA patients compared to controls (q-values 1.23E-35; Fig.1A), underscoring the etiological impact that antigen presentation via this locus has in the initiation of autoimmune process. Mild upregulation was also seen in DQB1 and B loci (q-values 4.7E-07 and 2.1E-10, respectively). We then studied molecular escape mechanisms by genotyping 204 IAA and PNH patients, with either a targeted or whole genome sequencing (WGS) platform. By application of a newly in-house developed bioinformatic pipeline, we detected somatic aberrations in HLA region involving both class I and II alleles in 36% of IAA patients including point mutations, frameshift insertions or deletions and copy number variations inducing allelic loss. B*14:02 and A*02:01 emerged as the most commonly mutated class I alleles with a few hotspot mutations identified, particularly in exon 1 (c.19C>T, p.R7X, Fig.1B,C), confirming previous reports. 2,3 In class II, DQB1 and DPA1 loci were frequently targeted by fine mutational events, while more complex allelic loss phenomena interested prevalently DRB1 and DQB1 loci. Those aberrations were identified at diagnosis (35%), during disease follow-up (33%) or at the time of malignant evolution (27%), with higher clonal size in specimens collected during the course of the disease (median VAF 3% [2-27%] at diagnosis, 8% [2-98%] at follow-up, and 2.2% [2.0-6.1%] at evolution). Of 41 patients with at least one HLA aberration and characterized with an extended genotypic study, only 6 harbored also >1 somatic myeloid mutation (14%), versus 30/90 (33%) not affected by somatic hits in HLA (p=.026; Fig.1D). HLA aberrant cases also showed lower number of somatic myeloid mutations (OR=0.44; p=.0262) with driver hits rarely present (Fig.1E). In terms of PIGA mutations, an increased PIGA mosaicism was observed in the HLA mutant group, underlying that both processes have similar pathophysiologic origin as a product of the immune selection pressure (OR: 1.55 [95%CI 1.1-4.2], p=.0201). We then investigated, through WGS of 53 patients, the presence of somatic mutations in other immune genes which could be triggered by immune pressure. Hence, in 47% of the cases we were able to find pathogenic or likely pathogenic hits in genes encompassing proteasome complex, vesicle trafficking, transactivators and interferon regulatory factors, including CREBBP, TAP1, CIITA, PSMC5, PSMB4 and IRF9 (Fig.1F), whose pathogenicity was computationally assessed through recently implemented somatic classifiers. 4 Those hits were not mutually exclusive neither with HLA nor with PIGA mutations, however their VAF was significantly lower compared to concurrent HLA and PIGA lesions, underscoring their lower driver potential within the immune escape environment compared to PNH and HLA-lacking clones. Altogether our results describe the diversity of molecular and immune events taking place in IAA and PNH. Our study suggests that following initial immune insult, clonal architecture of residual hematopoiesis can be dominated by multiple modes of immune escape, agonistically participating to a mechanism of "adaptive" clonal recovery, likely in opposition to the "maladaptive" malignant progression. Figure 1 Figure 1. Disclosures Maciejewski: Alexion: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

Introduction/Methods: Enzymes that modify histone H3 at lysine 27 (H3K27) to thereby regulate gene transcription (epigenetic enzymes) are recurrently inactivated by deletion and/or mutation in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemias (AML). Frustrating understanding of mechanisms is that writers and erasers of the same modification, e.g., methyltransferases that create, and demethylases that remove, H3K27 trimethylation (H3K27me3), a repression ('off') mark, are recurrently inactivated. Moreover, acetyltransferases that write H3K27 acetylation (H3K27ac), an 'on' mark mutually exclusive with H3K27me3, are also recurrently inactivated. One clue to underlying mechanisms is that MDS/MPN/AML present with diverse lineage and maturation phenotypes - perhaps these emerge from, or select for, the diverse epigenetic enzyme mutations. We therefore identified genes upregulated with specific myeloid lineage-commitment, maturation and function fates (~500 genes each) and then examined distributions of H3K27me3 and H3K27ac at these gene-loci in: (i) embryonic stem cells (ESC); (ii) hematopoietic stem and progenitor cells (HSPC); (iii) mature myeloid cells (monocyte [mono], pro-erythroblast, megakaryocyte [MK]); and (iv) AML cells. Results: Terminal-myeloid programs underwent substantial remodeling to gain H3K27ac 'on' mark from ESC/HSPC to mature myeloid (Fig.1A). Providing a mechanism for this, the H3K27 acetyltransferases EP300 and CREBBP were recruited into the RUNX1/SPI1 myeloid-lineage master transcription factor (MTF) hub by cooperation between their transcription activating domains. Mutated/translocated RUNX1, or mutated-NPM1 that cytoplasmically dislocated SPI1, disrupted this cooperation, and reverted hub content to default recruitment of histone deacetylases (HDAC) instead. Demonstrating cause-effect, inhibiting these HDAC renewed AML cell maturation to terminal lineage-fates. Meanwhile, MYC-target (proliferation) genes have high baseline H3K27ac in ESC and HSPC and do not require major remodeling during ontogeny (Fig.1A). An H3K27ac remodeling requirement for lineage-maturation but not proliferation/housekeeping explains selection pressure for inactivating mutations in EP300 or CREBBP, that we found in ~1.2% of MDS/MPN/AML in our (n=690) and other series. Consistent with pan-lineage-maturation needs for H3K27ac, the mutations were found in all lineage sub-types. H3K27me3 'off' mark was mostly erased from myeloid programs in HSPC, but was greater at MK vs erythroid, and also at mono vs granulocyte genes (Fig.1B). This implied more need for H327me3 demethylase (KDM6A/UTX) for HSPC commitment into MK vs erythroid, or mono vs granulocyte, lineages. Accordingly, KDM6A was most upregulated in MK and mono-lineage cells (Fig.1C), and myeloid-conditional Kdm6a knockout decreased platelets and increased red cells in the spleen - reported by others: https://doi.org/10.1182/blood.V128.22.1467.1467. RUNX1-ETO has been shown to specifically impede granulocytic but not mono differentiation - https://www.nature.com/articles/2403396 and KDM6A inactivating mutations were significantly more likely to occur secondary to RUNX1-ETO (4-9% BEAT and AMLSG case series) vs other cytogenetics (0.005%). Selection for KDM6A secondary mutations could thus be to channel myeloid precursors toward lineages most efficiently impeded by primary mutations. Although H3K27me3 was substantially erased at all myeloid-commitment and terminal programs (except MK) in HSPC vs ESC, subsequent lineage-commitment and maturation entailed rewriting H3K27me3 at preceding HSPC and alternate lineage-fate programs, including at MTF genes for alternate fates (Fig.1B, D). Primary MDS/MPN/AML and AML cell lines with inactivating mutations/deletions in H3K27 methyltransferase EZH2 thus displayed aberrant co-expression of lineage MTFs and gene expression programs of normally mutually exclusive lineages (Fig.1E-G). Conclusion. Epigenetic remodeling requirements vary by myeloid lineage and maturation stage. Thus, epigenetic enzyme mutations are selected by, and cause, lineage-context of transformation. This knowledge can guide choice of specific epigenetic enzyme inhibitors to remedy the lineage-maturation defects that drive and define myeloid malignancies. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The prevailing theory in del(5q) is that haploinsuffciency (HI) stemming from deletion and not simply LOH (loss of heterozygosity) is the culprit in clonal evolution. To date no haploinsufficient gene has been found to be the leukemogenic factor conveying growth advantage, but various other genes have been found to be important for phenotypic features or for propensity to acquire subsequent specific lesions. RPS14 is an example of such a gene, particularly in patients (pts) with isolated del(5q), responsible for macrocytic anemia and erythroid dysplasia and a propensity for acquisition of TP53 mutations. We hypothesized that RPS14 downmodulation and its consequences may be more common than del(5q) and it is frequent pathophysiologic feature in MDS. We first analyzed the genomic and expression profile of 170 pts with del(5q) and 825 diploid for 5q. We developed a new analytic pipeline to identify the most HI genes present in a large number of del(5q) pts. Genes within CDR (common deleted region) were classified as HI from the linear model fit if (i) clonality vs. gene expression slope from the isolated del(5q) was negative and FDR After applying model-based sparse clustering approach on all cohort, we obtained 7 clusters (Figure 1). As expected, del(5q) cases clustered together and showed consistent HI of 5q marker gene expression. Cluster-1 (n=146) included almost all del(5q) cases, except for 8 "mis-categorized" patients. It was characterized by low risk MDS (LR-MDS), presence of anemia/neutropenia and low mutational burden, with TP53 being the most commonly mutated gene and the only cluster with CSNK1A1 mutations. The remaining non-del(5q) patients were grouped in 6 clusters. Diploid cluster-2 (n=133) featured a normal karyotype, frequent ASXL1 and TET2 mutations, and profound down-modulation of RPS14 in all the patients included in the cluster (vs. other diploid pts). While the median RPS14 expression in cluster-1 (del(5q) cluster, with 50% adjusted clonality) was 7.29 (range 4.68-8.82 Log 2CPM), cluster-2 exhibited a median RPS14 expression of 6.12 Log 2CPM (range: 4.91-7.31 Log 2CPM). Clusters-3, -4, -5 (n=138, 90, 94, respectively) included most of the high risk MDS (HR-MDS). Cluster-3 was enriched for thrombocytopenia and SRSF2 mutations; cluster-4 for anemia, thrombocytopenia and ASXL1 and SRSF2 mutations. Cluster-5 was characterized by pancytopenia and frequent ASXL1 mutations and CK (complex karyotype). Cluster-6 (n=66) and -7 (n=233) contained the majority of non-del(5q) LR-MDS. When we analyzed the RPS14 expression in these clusters based on the RPS14 expression in cluster 2 we found 13% (n=18), 21% (n=19), 9% (n=8), 14% (n=9), 7% (n=16) of low RPS14 expressors in cluster-3, -4, -5, -6, -7, respectively. Cluster-2 showed a similar percentage of patients with anemia, and thrombocytopenia vs. Cluster-1 (69 vs. 50%, 23 vs. 30%; respectively). The mutational profile included a higher frequency of mutations for SRSF2 (29 vs. 0%), NRAS/KRAS (22% vs. 4%), ASXL1 (40 vs. 15%), TET2 (35 vs. 15%), and JAK2 (17 vs. 6%). These results indicate a more proliferative molecular spectrum of RPS14 downregulated cluster-2 than del(5q)-cluster-1, but RPS14 downmodulation did not lead to acquisition of TP53 mutations (4% vs. 76%). Considering all non-del(5q) RPS14 low expressors (n=186), only 3% of the cases had TP53 mutations. Since TP53 and CSNK1A1 mutations were characteristic of cluster-1 we studied interactions with HI RPS14. HI RPS14 in del(5q) and diploid low expressors showed a decreased expression of CDKN1A (P In conclusion, RPS14 expression defect is more widespread than del(5q) in MDS. However, only del(5q) RPS14 HI pts are prone to harbor TP53 and CSNK1A1 mutations; a group of diploid pts with low RPS14 and CSNK1A1 expressions might mimic some del5q features and could potentially respond to similar treatments. Figure 1 Figure 1. Disclosures Diez-Campelo: Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Carraway: AbbVie: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

In recent decades, there has been a substantial increase in recognition of germline (GL) predisposition to MN. Some congenital blood disorders predispose to subsequent myeloid neoplasia (MN). Severe congenital neutropenia (SCN) and Fanconi anemia serving as prominent examples. There is emerging evidence that heterozygous variants of disease recessive conditions may constitute risk alleles for late developing MN. However, their low penetrance may preclude identification of such alterations. Our previous analysisof allelic burden among selected combined potential GL predisposition genes to MN identified that the myeloperoxidase (MPO) gene (17q22-23) carried the highest risk of pathogenic alterations (Li, S.T, Leukemia, 2020). GL MPO mutations cause MPO deficiency syndrome, one of the most common inherited disorder associated with phagocyte defects and variable clinical penetrance. Despite the high prevalence, inherited MPO deficiency patients are asymptomatic and may remain unrecognized. We investigated 3280 MN and bone marrow failure syndromes (BMF) pts including 1138 MDS, 260 MDS/MPN, 1661 AML and 221 AA for presence of MPO mutations. In total, 38 different germline MPO mutations were identified in 143 cases. With a stringent bioanalytic pipeline, 28 pathogenic/likely pathogenic variants from 100 MN patients were included in the study. Germline MPO variants were significantly enriched in MN compared to control population (294 vs. 125 per 10 4 individuals; PC) followed by R569W (13/100), M519fs* (13/100) and Y173C (6/100) with none being biallelic. While no differences were found in distribution of -7/7q, del5q, or del20q, MPO variants carriers harbored less tri-8 compared to wild type counterparts (2% vs. 12%; P=.008). Only FLT3 and NRAS were significantly associated with MPO mutations. We then investigated the effects of MPO deficiency on hematopoietic function in murine model using competitive repopulation assays, whereby the difference in CD45.1/CD45.2 isotypes transplanted in to ROSA26 (tdTomato-EGFP) recipients assayed by flow cytometry allowed distinction of the 2 grafts. Mpo-/- cells gained over time proliferative advantage over normal murine bone marrow cells. Reverse combinations (graft mix vs recipient) also replicated this result. This effect was not due to increased HSC content in Mpo-/- marrow as there were no significant differences in LSK, CMP and MEPs cells. We then investigated the clonogenic consequences of Mpo-/- cells in the setting of H 2O 2 induced oxidative stress, after exposing to hydrogen peroxide. Mpo-/- cells increased clonogenic potential after second serially replating. This prompted us to investigate an MPO inhibitor, AZD5904 in human leukemia cells with different MPO expression (HL-60, high; K562, low). High MPO expressors retained higher cell viability following H 2O 2 and MPOi addition compared to those without MPOi (85±10 vs.59±8, P In conclusion, for the first time we demonstrated that germline MPO variants constitute risk alleles for MN evolution and replicated the potential replicative advantage of MPO deficient cells in murine model and potential mechanisms of the MPO deficiency in vitro including activation of non-homologous DNA repair response and error-prone DNA repair favoring replication. Repeated cycles of stress hematopoiesis e.g., due to increased infection rate may provide conditions in which MPO variants contribute to the risk of MN. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy.

Downregulation of class I human leukocyte antigen (HLA)-restricted antigen presentation has been identified as mechanism of immune-escape in many malignant and non-malignant disorders. In idiopathic aplastic anemia (AA), evolution of immune-privileged paroxysmal nocturnal hemoglobinuria (PNH) clones has been attributed to immune escape due to deficiency of GPI-anchored protein in the context of T-cell mediated autoimmunity. However, other mechanisms of clonal selection may also operate with or independently of PNH. Our group first described the presence of both somatic uniparental disomy (UPD) and microdeletions of the HLA region leading to loss of heterozygozity (LOH) and/or haploinsuffciency.1 Later the proof-of-concept of somatic mutations in HLA class I was provided.2 Mechanistically, HLA LOH leads to loss of an allele involved in the presentation of immune-dominant peptides, while haploinsufficiency may decrease the presentation threshold. Moreover, the general level of individual structural diversity of HLA molecules may determine the ability to present diverse targets, eventually derived from auto-antigens, and functionally would operate in the opposite direction to HLA LOH. In this scenario, we hypothesize that defects in both class I and II HLA loci may constitute different patterns of immune escape, reducing respectively CD8+ and CD4+ related activation and thus contributing to rescue hematopoietic stem cells from the immune attack. Furthermore, our idea is that the immune-escape environment may be related to the grade of HLA evolutionary divergence (HED), a metric that, accounting for the degree of structural diversity within a particular locus, represents an indirect measure of the antigenic landscape that the hematopoietic target cell is able to present (see abstract #:142693). Using a deep targeted HLA NGS panel and a newly developed in-house bioinformatic pipeline (characterized by stringent criteria for alignment, preprocessing and variant calling in the HLA region, based on the IPD IMGT/HLA database, Fig.A), we studied a large cohort of patients with idiopathic bone marrow failures (AA n=75, AA/MDS=10). In addition, we determined the impact of inter-loci HED on the probability to acquire somatic hits in HLA genes. Overall, 29 somatic HLA mutations were found in 16 patients (18%) at a median VAF of 11% (range: 2-93%):12 in class I (41%) and 17 in class II (59%), with 5 patients carrying mutations in both classes (Fig.B, C, D). The majority of those events (N=21, 72%) occurred in subjects also harbouring a PNH clone of small size (12 out 16 patients, median PNH clone size 1% [range:1-46%]). Most mutated loci were A and C for class I and DQB1 for class II (Fig. C, D); 9 mutations were identified as missense, with disruptive changes, 7 were intronic indels while 13 hits were localized in 5' or 3' untranslated regions (UTRs) (Fig.E, F). Through a computational prediction of the HLA regulatory domains involved in the UTR aberrations, we identified domains essential for the binding of GATA-1, RXRbeta, SP-1 and NFKB. The impairment of those regions may affect the transcription of HLA complexes. AA HLA mutant cases had more frequently a severe disease at diagnosis (severe AA: 81% vs. 60%, respectively in HLA mutated vs non mutated cases) and were in most part responders to immunosuppressive therapy (complete/partial responses: 75% vs 50% in HLA mutated vs non mutated patients). Within the AA/MDS group instead HLA mutations were found in 4 out of 10 patients (40%), including of note three -7/del7q cases. Using Pierini and Lenz algorithm3 to determine inter-class HED, we found that HLA mutations tended to occur more often in patients with a high inter-class mean HED (94% vs 72% in non mutated group, p=.001, Fig. G), consistent with the idea that higher structural diversity of HLA molecules may induce more pervasive auto-immune responses, stronger immune pressure and ultimately the establishment of immune-escape mechanisms. In summary, our results indicate the importance of class-I and -II HLA loci somatic hits as markers of autoimmunity and thereby the severity of the immune selection pressure, configuring possibly alternative mechanisms of immune-escape, in addition to immune privileged PNH clones. This environment may ultimately facilitate leukemic clonal expansion in AA-MDS setting. Disclosures Patel: Alexion: Other: educational speaker. Peffault De Latour:Apellis: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Severe aplastic anemia (sAA) is bone marrow (BM) failure syndrome characterized by immune mediated BM hypoplasia and pancytopenia usually treated with immunosuppressive therapy (IST) or allogeneic BM transplant. Despite the successes of IST in treating acquired, idiopathic sAA, most of the available treatment options only convert severe into non-severe forms of disease. Recently, eltrombopag (Epag), a small molecule thrombopoietin receptor (TPOR) agonist has been introduced into the routine treatment of de-novo and refractory AA with robust clinical response. Epag treatment showed remarkable tri-lineage responses in AA, suggesting an effect on hematopoietic stem and progenitor cells (HSPCs) amplification. The use of hematopoietic growth factors in AA that increases HSPCs has been associated with justified fears of facilitating AA evolution to MDS. Similarly, Epag has parallel effects on the amplification of HSPCs and there has been recent signals that long term treatment of Epag may increase the MDS risk. Using an in-house developed highly stable cell free high throughput screening of bioactive small molecule chemical library, we identified Epag as one of the most potent inhibitor of TET2. TET2 is the most abundant DNA dioxygenase in HSPCs. The loss-of-function mutations of TET2 frequently occurs in myeloid neoplasia. Here using in vitro and in vivo model system we report that effect of Epag, in part, is mediated by its ability to inhibit TET-dioxygenase activity. We demonstrate that Epag directly binds to TET2 in the presence of Fe2+ and inhibits its dioxygenase function in cell free system. In cell culture model we observed that nearly 40% of Epag partitioned into to nucleus suggesting its potential TET2 inhibitory role in cells. To test if TPOR signaling activation by Epag has any impact on TET activity we engineered murine cell lines BaF3 and 32D by introducing human TPOR. Irrespective of the status of TPOR-JAK-STAT pathway activation, as reflected in STAT5 phosphorylation, Epag demonstrated a robust TET-inhibition activity. However, in similar experiments peptide agonist of TPOR or another small molecule TPOR agonist Avotrombopag did not have any TET inhibitory effects. Consistent with these observations, Epag but not TPO or Avotrombopag inhibited TET activity in murine BM. Thus, Epag mediated TET2 inhibition in these cells are specific and independent of TPOR signaling. Consistent with previous reports, we observed that Epag treatment can significantly expand murine HSPCs even in the absence of TPOR signaling activation. The hematopoietic activity of Epag in murine system is dependent on its ability to inhibit TET2 as confirmed by the lack of Epag effects on Tet2-/- HSPCs. We further confirmed that TET-inhibition by Epag is central for its ability to expand HSPCs in murine model under in vivo settings using BM transplant experiments. We transplanted a mixture of cells derived from Tet2+/+ Pep Boy (CD45.1, 95%) and Tet2-/- C57BL/6 (CD45.2, 5%) into lethally irradiated recipient PepBoy mice. Epag treatment of mice reconstituted with the mixture of BM cells increases the fraction of Tet2+/+ BM cells only. Interestingly, Epag mimic loss of Tet2 in vivo as observed in the expansion of myeloid compartments of Tet2+/+ fractions reflected in CD11b-CD11c+, CD11b+CD11c-, CD11b+CD11c-Ly6C+Ly6G- (monocytes) and CD11b+CD11c-Ly6ClowLy6G+ (neutrophils). We did not observe any significant change in B220+, CD4+ or CD8+ lymphoid populations. On the contrary, we observed a growth restrictive effect of Epag treatment on Tet2-/- myeloid cells. Consistent with our hypothesis that Epag inhibits TET-dioxygenase activity, we observed hypermethylation in the mononuclear cells derived AA patients (n= 16) after Epag treatment. This was further confirmed in ex vivo Epag treatment of BM cells derived from healthy donor. Epag treatment significant increase clonogenic expansion of normal BM cells but not TET2 mutant myeloid cells. In summary, here we demonstrate that Epag is a potent inhibitor of TET-dioxygenase and its biological consequences are independent of TPOR-JAK-STAT activation. Epag treatment mimics TET2 loss of function in AA cells, however it restricts clonal expansion of TET2 mutant myeloid cells. Disclosures Patel: Alexion: Other: educational speaker. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

HLA evolutionary divergence (HED) is a continuous metric that quantifies the breadth of the immunopeptidome that can be presented by an individual's HLA allotype. High HED scores have been shown to be associated with better outcome after immune check point inhibitor therapy in solid tumors.1 However, the role of HED has never been assessed in autoimmune disorders, and may have an impact on disease susceptibility and outcomes. Here we explored the impact of HED on clinical features of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). The immunological landscape of those diseases is characterized by an HLA-restricted CD8+ and CD4+ mediated autoimmunity which leads to hematopoietic stem cell destruction, with a crucial role played by immune-dominant PNH clones and immune escape mechanisms likely associated with the down-regulation of the HLA-antigen presentation. Since higher HED scores reflect a wider antigenic spectrum and potentially more diverse peptide presentation capabilities, we hypothesize that this context not only may elicit T-cellular responses by auto-antigen presentation but also that HLA structural diversity can impact on disease phenotype and have a role within the definition of an immune-dominant (higher HED) or immune-escape (lower HED) environment. Using an NGS-based platform targeting the entire HLA locus, we have sequenced DNA samples of a cohort of 98 well-annotated AA, PNH, or AA/PNH patients. We then calculated HED at the classical HLA class I and II loci (A, B, C, DRB1, DQA1, DQB1, DPB1, DPA1), and investigated its impact on disease course and outcomes (Fig. A, B). Homozygosity for at least 1 out of 8 loci was frequently found (70%), especially for class II, however only 2% of patients had >1 homozygous locus). HLA-B and HLA-DQA1 genotypes demonstrated the highest HED among HLA-I and II loci, respectively (Fig. C). While for class I loci no difference was detected across diagnostic subgroups, mean HED for class II loci was significantly lower for PNH patients as compared to AA (6.031 vs 7.623 respectively p=0.047). Analysis of HED metrics across several binary outcomes within logistic regression models demonstrated that higher HED at class I and class II loci was generally associated with unfavorable disease characteristics and response outcomes. Specifically, high mean HED in class I and particularly in B and C loci tracked with disease severity (severe/very severe AA according to modified Camitta criteria: OR= 1.36 [95%CI 1.12, 1.70], p=0.003),2,3 whereas higher mean HED in class II was associated with refractoriness to immunosuppression [OR=1.18 (95%CI: 1.01-1.42), p=0.041]. In particular, a strong relationship was found for DQB1 locus [p=0.032, OR=1.12 (95%CI: 1.01-1.25)] in severe AA patients. Moreover, high HED in DPB1 was associated with early disease presentation [OR= 1.32 (95%CI 1.02, 1.80), p=0.047]. No correlation was found with overall survival. In summary, our results indicate that higher HED scores correlate with severity, early disease onset and refractoriness to immune-suppression in AA patients. High HED may increase the probability of presenting self-peptides that elicit dominant autoimmune T cell responses. Consistent with this line of reasoning, it is not surprising that lower HED (for class II HLA) is found in PNH subjects and that disruptive HLA somatic mutations in AA are preferably found in loci with increased structural diversity, consistent with the idea that less diversity may produce more immune-escape (see Abstract #: 142501). Our results suggest that HED may be useful for risk stratification of patients with immune-mediated bone marrow failure disorders, and more generally, that the ability to present a wider antigenic peptide spectrum may produce a more pervasive autoimmune response. Disclosures Patel: Alexion: Other: educational speaker. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago.

Unlike leukemic driver mutations, PIGA mutations produce an escape phenotype in the context of immune-mediated bone marrow failure such as aplastic anemia (AA). Another way to create clinical advantage will be to disable HLA-mediated cytotoxic T cell recognition. Determinants of cytotoxic T cell response might include some accessories glycosylphosphoinositol (GPI)-linked moieties but the main stimulus is likely to be provided by HLA-presented antigenic peptides. Somatic hits in HLA genomic region (microdeletions, uniparental disomies [UPDs] of HLA locus on 6p and later mutations) have been previously assessed in AA patients [1-3]. Mechanistic analogy to immune-privileged GPI-anchor protein deficiency in PNH due to PIGA mutations[4] or deletion[5] of PIGA locus are obvious. We stipulate that HLA mutations may contribute to the intrinsic expansion of PNH clones under immune pressure being: i) additive to the effects of PIGA mutations in creating immune escape or ii) redundant and thus less frequent in PNH clones as in patients without PIGA mutation. Using a deep targeted-sequencing panel covering HLA classical loci, and applying an in-house newly developed pipeline for the study of the HLA region (AbstractID#142501), we detected class I/II HLA somatic mutations of 10 patients with PNH. An integrative mutational analysis of PIGA and myeloid genes was then performed in order to comprehensively evaluate the role of HLA somatic hits within the scenario of PNH clonal evolution. At the time of this submission HLA sequencing was completed for a total of 35 patients but full analysis is available for the first 10 cases. Overall, of these 10 PNH patients 20 samples were analyzed from sorted GPI(+) and GPI(-) myeloid fractions (mean purity >95%). Median age at diagnosis was 36 years (11-66) while median PNH granulocyte clone size at time of sampling was 76% (5.11-99). A total of 41 PIGA mutations (Fig.1A) were detected solely in the GPI(-) fraction (mean VAF 58%), with 8 patients harboring clonal mosaicism as previously described.[6] Six somatic mutations of HLA class I (N=3, Fig.1B) and class II (N=3, Fig.1C) loci were found in 4 patients (67% detected on GPI(+) and 33% on GPI(-) fraction) at a low VAF (mean 3.36%). All these events were insertions or deletion of one or more bases. Class I mutations were located in intron 5, exon 3 and 3' untranslated regions (UTR). Class II were found instead in exon 2 (N=2) and intron 4. A functional and topographical annotation based on IPD-IMGT/HLA database suggested that exonic mutations were disruptive, impairing the bio-functionality of antigen presentation site. The detected intronic mutations instead impair HLA moiety assembling within cellular membrane, possibly altering splicing of the transmembrane domain. Moreover, a computational prediction of the regulatory domains involved in the 3'UTR aberration, showed a possible involvement of the miRNA has-miR-4524a-3p binding site, potentially affecting HLA post-transcriptional regulation. Of note, in 1 patient (UPN 9, Fig.1D) we did not find any PIGA, PIGT or HLA mutation. Finally, myeloid gene mutations analysis revealed the presence of a subclonal ASXL1 mutation in 1/10 patients in the GPI(-) fraction. Of note, this patient (UPN 1) had older age and showed 12 different somatic PIGA hits. This finding is probably explicable with the scenario of PIGA as the initial ancestral event accompanied by secondary mutations previously shown by our group as occurring in 10% of PNH cases in the course of disease evolution.[7-8] In summary, somatic HLA class I/II mutations can be found in patients with PNH. HLA mutations can occur in GPI(+) cells in subclonal fashion but also in GPI(-) cells. The latter clonal mosaicism indicates that various mechanisms of immune escape may play a role. Subclonal HLA mutations may impact the immune pressure on PNH clone dynamics, reflecting an alternative immune escape pathway in patients without PNH clone. (Fig.1E) In addition, detection of occasional "myeloid" hits suggests that various modes of PNH clone maintenance and expansion may be operative. We will present at ASH analysis of a full cohort of these patients including properly powered clinical correlations. Figure 1 Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Somatic mutations in components of the RNA-splicing machinery (LUC7L2, PRFP8, SF3B1, SRSF2, U2AF1, ZRSR2) are detected in several subtypes of myeloid malignancies with some of them delineating a district a clinico-morphologic phenotype. RNA-splicing defects exist also in cases without splicing factor mutations suggesting that myelodysplastic syndrome (MDS) is a "spliceopathy" in which the presence of mutations represent the most extreme pole in the mis-splicing spectrum. Although some studies have shown the effects of individual splicing factor mutations on a variety of downstream targets, no key genes have been identified. Literature reports have shown that cells from patients with myeloid malignancies are highly susceptible to alterations in splicing factors with studies presenting an epistasis among RNA-splicing factor mutations (Blood.2020006868). The dilemma is whether this epistasis has a null effect or suppresses/ activates splicing factors in the context of double hits. More recently large scale genomic studies have led to the discovery of cells carrying mutations in more than one splicing factor. We took advantage of a molecular characterized collection of an in-house MDS and AML patients (n=1,742) and publicly available data of AML (n=2,302; Beat AML Master Trial, The Cancer Genome Atlas, German-Austrian Study Group) to describe the co-existence of a chimeric splicing factor phenotype and its clonal architecture. We identified a total of 40 patients carrying at least two hits in the most common components of the RNA-splicing machinery (PRPF8, SF3B1, SRSF2, U2AF1, ZRSR2). No patient harbored two hits in the same gene. In total, we detected 149 somatic mutations. A higher frequency of mutations was found in SRSF2 (60%; 24/40) followed by SF3B1 (48%; 19/40), ZRSR2 (42%; 17/40), U2AF1 (30%; 12/40) and PRPF8 (12.5%; 5/40). Other splicing factors were found mutated more rarely as in the case of LUC7L2 which was present in only 2 patients. Mutations occurred at common hotspots in SF3B1 (K666; K700; R625), SRSF2 (P95), U2AF1 (S34; Q157) and were missense, frameshift, and splice mutations in PRPF8 and frameshift in ZRSR2. Other splicing factor mutations were less frequently observed to co-occur (LUC7L2 with SF3B1 or SRSF2). In total, double splicing factor mutations were mostly enriched in male. Seventy-six% of ZRSR2 mutant patients were male. Notable the frequency of double splicing factor mutant patients was higher in AML compared to MDS and MDS/MPN (53% vs. 25% vs. 22). Thirty-two% of double mutant patients had sAML. Most common co-occurrent genes in this cohort included TET2 (32%; 13/40), RUNX1 (22%; 9/40) and ASXL1/ STAG2 (18%; 7/40 for both). The two splicing factors that were more concomitantly mutated were SF3B1/SRSF2. In fact, concomitant SF3B1/SRSF2 were found in 22% (9/40) of the patients. As a whole, SF3B1/SRSF2 average variant allele frequency (VAF)was similar (43% vs. 47%). In this group of patients, molecular hits were not preferential with half of the patients carrying one or the other as first clone. The second most concurrent mutation with SRSF2 after SF3B1 was ZRSR2 (18%; 7/40). SRSF2 was less frequently co-mutated with U2AF1 (4 patients) or PRPF8 (3 patients). U2AF1 was found co-mutated with SRSF2 and ZRSR2 (4 patients, each) and SF3B1 (3 patients). One patient with sAML had a triple splicing factor configuration (PRPF8 VAF: 29%, SF3B1 VAF: 26%, U2AF1 VAF: 20%). In terms of conventional cytogenetics, half of the patients carried normal karyotype. Among the patients with abnormal karyotype, the most common abnormality was +8 (12%; 5/40) with 4 patients having isolated +8. One patient was hemizygous carrying -17 and PRPF8 mutation. Two patients had del(7q) and del(5q) abnormalities each. The most frequent mutation in the SF3B1/SRSF2 mutant patients was RUNX1 (44%; 4/9); in two cases, SRSF2 was the dominant/ founder clone. Three patients with SF3B1/SRSF2 also carried STAG2 while two patients had TET2 and FLT3. In summary, we describe a subset of myeloid malignancies with a complex mutational associations involving splicing factor genes. The presence of these mutations might uncover novel biological clues in the mechanisms by which splicing factors drive the phenotypes of MDS and/or AML which might not represent only a random association but define new ways by which splicing factors might silence the activity of one versus another. Disclosures No relevant conflicts of interest to declare.

Germline alterations causing inherited myeloid neoplasia (MN) have become prominent in the 2016 WHO classification. Increasing evidence reveals that many patients with MN (MDS and AML) may indeed harbor unsuspected predisposing or direct driver germline alterations. Penetrance of such alterations may be variable and family history unreliable or absent. Our previous screening of a selection of genes known to be involved in the pathogenesis of human cancers directly or indirectly according to the difference in the allelic burden in 690 MN patients (Li, S.T, Leukemia, 2020) we identified the myeloperoxidase (MPO) gene as having the highest burden of pathogenic alterations. MPO (17q22-23) encodes a heme-containing enzyme exclusively expressed in myeloid cells. MPO is known for its role in host defense against common microbial infections. In the presence of H2O2, MPO catalyzes the formation of hypohalous acids which are potent oxidants and strong bactericides. Many alterations in MPO have been found in hereditary MPO deficiency, an autosomal recessive syndrome with variable clinical penetrance. MPO deficiency syndrome is characterized by increased susceptibility to infections. To date, formal cancer association has not been established. In particular due to relatively mild phenotype, hereditary deficiency may remain unrecognized. Inspired by results of an initial screen we investigated MPO mutations in 3262 patients with various bone marrow failure syndromes (AA/PNH, 213) and MN (AML, 1655; MDS, 1133; MDS/MPN, 161; MPN, 100). In a subset of patients (20%), paired normal and tumor samples were sequenced in order to confirm germline origin. Germline MPO mutations were identified in 105 cases with 52% of them in AML. The most common recurrent mutation was c.2031-2A>C (42%; 44/105) (3' splice site of intron11) followed by I642L/F 15% (16/105), R569W 14 % (15/105), M519fs 13% (14/105), R460Q 7% (7/105), Y173C 5% (5/105), and R480H 2% (2/105). None of the variants were biallelic. The expected frequency of overall MPO variants in our cohort was higher than gnomAD (0.26% vs. 0.18%; P= .15×10-3) with odds ratio of 1.4. Notably, MPO mutations were significantly enriched in patients with age < 60 (2.2%; odds ratio =12.1, 95%CI=9.3-17.3; PC, R569W, M519fs, Y173C) contributed to this significant enrichment. Co-occurring lesions of germline MPO mutants (MPOMT) were then investigated: DNMT3A, SF3B1, TP53, NRAS were commonly observed in MPOMT compared to MPO WT-MN (20 vs. 14%, 17 vs. 13%, 16 vs. 8%, and 12 vs. 7%). Normal karyotype was found in 47% of carriers without enrichment for complex, -7/del(7q), del(5q), del(20q) or +8 karyotypes compared to WT. We compared RNA expression patterns between patients with pathogenic MPO mutations and those WT for MPO. Examination of genes significantly overexpressed in MPOMT showed strong enrichment in pseudoperoxidases hemoproteins involved in oxygen transport-globin (P=10-3) and heme biosynthesis (P=.012) (Fig.1C). The overexpression of hemoglobin/myoglobin genes in MPOMT suggests an excessive compensatory process due to the lack of true peroxidase activity. The fact that MPO has microbicidal activity led us to investigate the consequences of MPO mutations. Using shotgun whole-microbiome sequencing of circulating DNA, we examined the overall bacterial load in each patient. We found that MPO pathogenic mutation carriers had significantly higher bacterial burden than non-carriers (Fig.1D). The high bacterial burden of MPO mutants possibly promoted chronic inflammation, known to correlate with cancer pathogenesis. Indeed, we found that the presence of pathogenic MPO mutations was correlated with decreased survival in AML (P = 1.5 x 10-5). In sum, this is the first cohort showing germline MPO variants as predisposing alterations in MN. MPO variants might cause partial or total MPO deficiency resulting in chronic inflammatory responses by excessive compensation of immunity against invading microbes. This chronic inflammation stages can increase proliferation of HSC or myeloid progenitors and give rise to DNA replication associated mutations. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Somatic mutations of X-chromosomal PIGA gene in hematopoietic stem cells are the key molecular events in the pathogenesis of PNH. PIGA mutations lead to impaired biosynthesis of glycosylphosphoinositol (GPI)-anchor. Resultant lack of GPI-anchored proteins (GPI-AP) leads to a clonal expansion of PNH clone and increased sensitivity of PNH red cells (RBCs) to complement-mediated hemolysis. Based on immunofluorescent staining, the severity of GPI-APs deficiency on the RBCs can be distinguished in: PNH type-1 (TI) with a normal expression; PNH type-2 (TII) with a partial reduction; and PNH type-3 (TIII) PNH with a total absence of CD55 and CD59.1 Fractions of these RBC types are believed to be a result of the molecular spectrum of PIGA mutations ranging from missense in various amino acids positions to truncations and locus deletions, which can be concurrent in some patients. Consequently, the presence of TII and TIII cells indicates the overall severity of PIGA defect and thus individual phenotypic subtypes can be defined as predominantly TII or TIII according to the dominant clonal defect. We hypothesize that PIGA genotype may shape PNH phenotype trajectory with regard to the severity of GPI-AP deficiency and clinical course. For instance, incomplete TII RBCs PNH phenotype should result in slower expansion due to incomplete selection advantage as compared to complete loss of PIGA function indicted by TIII RBCs, typical of hemolytic PNH. However, the process of "GPI-AP painting", a passive transfer from normal to TIII RBC could result in the presence of pseudo TII RBCs with intermittent phenotype. Here, using deep targeted sequencing, we correlated PNH flow cytometric phenotype with the type of PIGA mutations. For improved resolution, our study focused on patients with large clones and hemolytic PNH. Among 295 diagnosed patients who had a PNH clone (>0.5% of PNH granulocytes), we identified 60 (median age 37 years, range 5-84) who fulfilled the criteria for hemolytic PNH (PNH clone size >20% and LDH>x2.5 ULN), tested before complement blocker therapy. Whole blood DNA was subjected to multi-amplicon deep NGS using primers covering all exons of PIGA gene.2 Accordingly, 13/60 (21%) were classified as having TII and 47/60 (79%) TIII PNH. Overall, median granulocyte clone size was 71.5% (21.5-99) with no differences between TII/TIII subtypes (p=0.872) as well as it was for baseline characteristics such as sex ratio, blood counts and previous history of aplastic anemia. A total of 137 PIGA mutations were found, 27 in TII and 110 in TIII dominant patients. (Fig.1A-B) Overall, >1 and >2 mutations (clonal mosaicism) were found in 85% and 57% of patients, respectively. In cases negative by NGS of PIGA gene (9/60, 89% TIII PNH), no mutations were found even after sorting to purity of GPI(+) cells. Frameshift (39%, n=54) and missense (30%, n=41) mutations were most common, followed by splice site (16%, n=22), non-sense (11%, n=15) and non frameshift insertion/deletion (4%, n=5). Although PIGA mutations are spread throughout the entire coding region, TIII patients harbouring non-frameshift hits had more often truncating mutations mapping within the first 200bps across the PIGA domain. We demonstrated that missense PIGA hits were enriched in TII PNH, while frameshift deletions in TIII dominant patients (p=.0021 and .0201, respectively- Fig.1C). Moreover, when only dominant clone was analyzed, missense and frameshift mutations were strong predictors of PNH RBC phenotype, defining TII RBC (p=.0002) and TIII RBC patients (p=.02), respectively. (Fig.1D-E) When patients harbouring 1 unique somatic hit were studied (16/60, 27%), missense mutations accounted only for 19% of cases. Indeed, in an internal cohort of 89 AA patients with small PNH granulocytes clones ( PIGA missense and truncating mutations dictate the susceptibility of PNH RBCs to complement. We show that while missense mutations may still lead to a deficient but not totally absent GPI-AP synthesis, truncating mutations seem to be responsible for an entirely disruptive phenotype, causing the complete absence of GPI-APs on RBC membrane surface. Sequencing of other genes in the GPI synthetic pathway (AbstractID# 139335) may help to identify other somatic hits explaining PNH phenotype of cases lacking PIGA. Figure Disclosures Patel: Alexion: Other: educational speaker. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Germline DDX41 mutations were recently reported to cause MDS/AML and donor-derived leukemia after transplantation. While previously described in Western countries, DDX41 variants have not been reported in a Southeast Asian population. We performed targeted sequencing of blood or bone marrow samples from 109 Thai patients with myeloid malignancies. Among the 109 patients (75 MDS, 8 MPN, 11 MDS/MPN and 15 AML), the most frequent mutations were in ASXL1 (17.4%), TET2 (16.5%) and SRSF2 (12.8%), respectively. DDX41 variants were detectable in six (5.5%) cases. Four patients exhibited three presumable germline DDX41 mutations: p.S21fs (n = 2), p.F235fs (n = 1), and p.R339H (n = 1). While p.S21fs was previously reported in myeloid neoplasm, the latter two variants have not been described. Two of these cases harbored concomitant probable germline/somatic DDX41 mutations (p.S21fs/p.R525H and p.R339H/p.K494T), while the other two patients carried only somatic mutations (p.R525H and p.F438L). The p.K494T and p.F438L variants have not been previously reported. In patients with DDX41 alterations, the diagnoses were MDS with excess blasts (4), secondary AML (1) and low-risk MDS (1). In conclusion, we identified DDX41 variants in Thai patients with myeloid malignancies in which these variants could be used to assess predisposition to MDS in Southeast Asia.

Curative potential of allogeneic hematopoietic cell transplantation (aHCT) in myeloid malignancies is principally related to the graft versus leukemia (GvL) effect exerted by donor-derived immune effectors on leukemic cells. However, different pathways may drive post-transplant relapse, including perturbation/attenuation of T cell-mediated GvL responses. For example, decreased expression of HLA alleles has been described in post-aHCT relapse.1 This could be due to down-regulation of HLA or 6p HLA locus deletion. However, the occurrence of chromosome 6p uniparental disomy (UPD) along with del6p (first described by our group in the context of aplastic anemia)2 suggests that HLA loss of heterozygosity (LOH) and inherent loss of one allele (presenting immunodominant peptides) may be a significant contributor to leukemic relapse either directly, causing decreased HLA expression or as additive effect to HLA down-regulation. In relapses after haploidentical or mismatched aHCT, LOH of mismatched allele has been described.3 Here, we hypothesize that not only copy neutral LOH or haploinsufficient HLA expression but also defective function due to HLA mutations may lead to immune escape from GvL. Such a mechanism would involve the loss of an allele responsible for the presentation of an immunodominant antigenic peptide. Moreover, unlike relapse due to the acquisition of additional myeloid mutations, HLA mutant relapse would also acquire resistance to donor lymphocyte infusion (DLI) therapy. In order to dissect the immunogenomic mechanisms leading to leukemic immune-evasion, we performed a comprehensive genetic analysis of specimens sequentially collected from a cohort of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) relapsed after aHCT. Specifically, we applied a deep-targeted NGS panel to study HLA region along with 173 genes known to have a role in leukemogenesis and cancer ontogeny. So far, 57 paired/serial biospecimens from 25 transplanted patients have been analyzed (25 samples at AML/MDS diagnosis, 25 at the moment of relapse after aHCT and 7 samples at relapse after chemotherapy) Overall, we found the acquisition of 8 disruptive HLA somatic mutations in 6 patients at post-transplant relapse (24%), 4 in class I and 4 in class II loci. None of those events were found in samples at diagnosis or at post-chemotherapy relapse, suggesting the possibility of an "immune-escape relapse". Those somatic hits accounted for 4 intronic indels, 1 frameshift insertion, one splicing site and 2 point mutations in 3′ and 5′ untranslated regions (UTRs). Median variant allele frequency (VAF) was 17% (range 2-58%). Of note is that all HLA mutant patients received a matched aHCT (4 from related and 2 from a 10/10 matched unrelated donors) suggesting that this mechanism is independent from the deletion and the loss of an immune privileged mismatched allele. Interestingly, median time to relapse was 514 (range 119-935) days for HLA-mutated patients vs 126 (62-543) for HLA wild type cases (p=.00042), consistent with the hypothesis that the establishment of immune-tolerance, and the presence of a GvL effect (less likely in early relapses) are required for the selection of those mutations. When somatic genotype of these patients prior and after transplant was studied, we found that post-aHCT relapses were associated in most cases with a new genomic configuration with either loss of previous events or acquisition of new subclonal mutations in myeloid or cancer related genes (in 17/25 cases). However no difference was seen in terms of number and patterns of new events among the HLA mutated and HLA wild type patients. Results shown here represent an important proof-of-concept for the role played by somatic mutations in HLA genes in the setting of post-aHCT AML/MDS relapses. These events, in analogy to the deletion or copy neutral LOH, may promote immune escape relapse resistant to immunologic manipulations and may coexist or be an alternative pathway to the progression/relapse characterized by acquisition of myeloid subclonal driver mutations. In that context, HLA mutations would be considered facilitator lesions. Disclosures Hsi: CytomX: Consultancy, Honoraria; Eli Lilly: Research Funding; Abbvie: Research Funding; Miltenyi: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Hamilton:Syndax Pharmaceuticals: Consultancy, Honoraria. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC). Majhail:Incyte: Honoraria; Mallinckrodt: Honoraria; Nkarta Therapeutics: Honoraria; Anthem, Inc.: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Mutations in tumor suppressor genes and oncogenes are both potentially therapeutically actionable in acute myeloid leukemia (AML). The Wilms' Tumor 1 (WT1) gene is located on 11p13 and encodes a zinc finger transcription factor which has been found to be overexpressed and mutated in AML. In normal development, WT1 is only expressed in a small subset of hematopoietic stem cells. While its overexpression suggests an oncogenic role, the invariable presence of mutations in the cysteine-histidine zinc finger domains indicates a tumor suppressor function, similar to that in WAGR syndrome/11p deletion syndrome in which it was first discovered. Like its unknown function in AML, the clinical significance and genetic associations of WT1 mutations have been also controversial. Although studies of WT1 mutations in AML have been conducted, the lack of solid clinical and molecular characterization of large WT1-mutant (WT1MT) AML cohort has hampered its definition. In this study, we took advantage of a compendia of genomic results from Cleveland Clinic and publicly available data of 2188 AML patients (primary (p)AML, n= 1636; secondary (s)AML, n= 433; therapy-related (t)AML, n= 119, excluding cases with acute promyelocytic leukemia, MLL-rearrangement, and core-binding factor AML). While several reports only focused on cytogenetic normal AML (CN-AML), which represented 61% of our cohort, we additionally included all other cytogenetic risk groups. In total, WT1 mutations were detected in 5% (114/2188) of patients. WT1 mutations were enriched in pAML (85%) compared to sAML (11%) and tAML (4%). Thirty-nine patients (13%) carried more than 1 WT1 mutation. WT1MT were younger [59 vs 64 years, P=0.0002] and more often females (55% vs 45%, P=0.03) as compared to WT1 wild type (WT1WT) patients. Univariate analyses of baseline parameters showed that WT1MT AML had a more proliferative phenotype with a higher WBC [15.1 vs 9.5 x109/L, P=0.03] and bone marrow blast percentages [73 vs 59%, P=0.002] and with lower platelet counts [44 vs 56 x109/L, P=0.008] compared to WT1WT cases. In the WT1MT cohort, 70% had a normal karyotype, with complex karyotype being significantly less frequent vsWT1WT patients [4 vs 16%, P=0.001]. The most common cytogenetic abnormalities in WT1MT patients included +8 (8%) followed by -9/del(9q) (3%) and -7/del(7q) (3%). Only 1 patient carried inv(3)/t(3;3) or -17/del(17p). In sum, no statistical differences in cytogenetics were found between WT1MTvsWT1WT AML patients. Next, identified mutational signatures of WT1MT patients. A panel of 44 myeloid genes and their hotspot configurations were selected according to their relevance in AML. In comparison to WT1WT AML patients, multivariate analyses showed that WT1MT patients had higher odds of biallelic CEBPA (12 vs 3%; P=0.009) and FLT3 internal tandem duplication mutations (FLT3ITD, 31 vs 16%; P=0.01) but lower odds of SRSF2 mutations (2 vs 9%, P=0.04). Since FLT3ITD has been previously described to be associated with WT1 mutations, we also focused on investigating whether mutations in the tyrosine kinase domain (TKD) were frequent in WT1MT as well. Although we found increased percentages of FLT3TKD (11%) among the WT1MT patients compared to WT1WT cohort (8%), this difference did not reach statistical significance. To uncover multifactor lesions (cytogenetic and/ or additional molecular lesions) of prognostic importance, we performed survival analyses. Although the combination of WT1 mutations and FLT3TKD shortened overall survival (OS) by 2-times in WT1MT patients vsWT1WT cases with FLT3TKD (23.7 vs 45.9 months), this result was not significant (P=0.1). In addition, the concurrent presence of other cytogenetic and molecular features didn't reveal significant impact on OS. In sum, using an adequately powered cohort, our study of the genomic landscape of WT1MT AML patients identified its genomic associations and their clinical and prognostic inferences. The application of advanced machine learning methods to large datasets of WT1MT AML patients might be crucial to capture the complex genomic interactions of WT1 gene in AML. Disclosures Carraway: BMS: Consultancy, Other: Research support, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau. Nazha:MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Jazz: Research Funding. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

The loss of chromosome 7 (monosomy 7, -7) or its long arm [del(7q)] is a common karyotypic abnormality found in MDS and are associated with a poor prognosis. However recent studies may suggest a difference in survival outcomes. These lesions occur both independently and in a complex karyotype (CK). The leukemogenic features of -7/del7q can be explained through loss of heterozygosity or haploinsufficiency (HI) of key deleted genes. In addition, the phenotype of del(7q) and -7 might be further modified by the clonal architecture determining the mode of molecular evolution. Based on the hypothesis that a deletion will exert its biologic consequences in genes with HI expression and by co-associated mutations, a comprehensive analysis of genes on chr.7 was performed. Within a cohort of 8,161 patients with myeloid neoplasms (MN), we identified 511 with -7 (6%) and 143 with del(7q) (2%). In 29% of cases, del(7q) was found as an isolated alteration, while 90% of CK had del(7q); -7 showed similar percentages of isolated cases but was less common in CK (20%, 55%; respectively) cases. Del(7q) was significantly (P The most common co-associated molecular lesions with -7/del(7q) were: TP53 (31%), DNMT3A (13%), TET2 (13%), RUNX1 (12%), ASXL1 (11%), del(5q) (25%) and CK (54%). Del(7q) vs. -7 differed in the frequency of TP53 (39 vs. 29%; P=.03), DNMT3A (19 vs. 11%; P=.03), and IDH1/2 (13 vs. 52%; P Comparing the -7/del(7q) clone (calculated using allelic imbalance bio-analytic pipeline) to co-associated mutations allow us to identify the rank of -7/del(7q) in the clonal hierarchy: -7/del(7q) was an ancestral event in 58% and a secondary hit in 42% of the patients. When -7/del(7q) was the dominant hit, patients had fewer associated mutations, while in cases with secondary -7/del7q somatic mutations of TP53 (60%), IDH1/2 (30%), and DNMT3A (20%) were the ancestral hits suggesting a different clonal architecture according to -7/del(7q) clone size. Expression data was available for 49 of -7/del(7q) and 643 diploid cases for chr.7. Of the 694 informative genes on chr.7: 147 were deleted in all patients according to CNV analysis. We restricted the analysis to 15 uniformly HI genes selected because the expression inversely correlated with the level of 7q ploidy (ACTR3B, FASTK, GSTK1, IMPDH1, LRRC4, MEST, MGAM, NUP205, PRRT4, SLC37A3, SSBP1, TMEM209, TNPO3, ZC3HC1, ZNF277). Expression for these genes was then analyzed following normalization by -7/del7q clonal size. Unsupervised clustering grouped 72% of -7/del(7q) in one expression cluster. Classical -7/del(7q) signature genes fulfilled these criteria and with uniform HI in all cases. However, when we also included CUL1, CUX1, EZH2, KMT2C, and LUC7L2 to this analysis, the precision of -7/del(7q) clustering was 75% and defined a subcluster (#6). We were also able to identify cases previously misclassified (n=9) which were dispersed into 3 other clusters (14%, 8%, 3%) while 4% (n=23) of diploid cases were also misclassified and re-assigned into cluster 6. When we restricted the analysis to -7 cases only, a signature of 6 unique genes (C7orf43, CDK14, POLM, STAG3L5P, TRIM4) were found. Interestingly only POLM was on 7p, while the rest were on 7q22.1. In sum, we describe an integrated genomic and transcriptomic analysis of -7/del(7q) and we present data showing a complex heterogeneity of genes with HI expression which suggest that the clinical picture of patients carrying -7/del(7q) might be the result of a combination of clonality and HI. Disclosures Patel: Alexion: Other: educational speaker. Sekeres:Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. 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.

Somatic TP53 mutations are found in 10% of adult patients with MDS and de novo AML and in up to 20% of patients with therapy-related myeloid neoplasms. TP53 status is associated with complex karyotype (CK), aberrations of chromosome 5 and poor survival. Moreover, mutated TP53 (TP53MT) may be an indication for hematopoietic cell transplantation, but also predictive of relapse following the procedure, making this particular category of myeloid neoplasms (MN) a conundrum of clinical management. Unlike other tumor suppressor genes, missense (ms) mutations within the DNA-binding domain (DBD) are the most common genetic alterations in TP53 gene representing up to 80% of somatic hits, with involvement of canonical hotspots (R175H, Y220C, M237I, R248Q, R273H, R282W) in around 30% of cases. A loss-of-function (LOF) dominant-negative effect (DN) may explain the ability of TP53MT to interfere with wild type (WT) functions. Moreover, TP53 germ line (GL) mutations are responsible for Li-Fraumeni syndrome, and GL contamination may also exist in adult MN. Here we comprehensively characterize TP53MT MNs to better dissect the role of specific mutational configurations and identify the selective forces affecting outcomes in this poor prognostic MN category. A total of 764 TP53 mutations were found in 632 MN patients. Ms mutations were the most common (75%) followed by frameshift (11%), splice site (7%), nonsense (5%) and insertion/deletions (2%), with 20% of patients harboring more than 1 lesion. Topographical annotation revealed that ms mutations typically (98%) occurred within the DBD (residues 102-292) and only 2% occurred outside this region (vs. 28% in case of truncating mutations, p We then hypothesized that truncating (frameshift/nonsense/deletion) alterations require additional hits as the presence of one allele may be partially protective. Consequently, the VAF for these lesions may be a result of a UPD in a smaller fraction of cells; biallelic truncation hits thus may be truly biallelic rather than clonally mosaic, which can be demonstrated only by single cell DNA sequencing. In contrast, DN mutations in canonical hotspots decrease the function beyond 50% of the activity, with further inactivation would having less of an effect, thus exerting less selection pressure for acquisition of additional lesions. Indeed, second truncating hits (including UPD and del(17p)) were common (30%), while none of the dominant ms hits had a double-mutant hotspot configuration (vs. 14% of non-canonical ms double mutant), and these canonical dominant hits were less likely to be paired with del(17p) or truncating mutations (8%). Only 25% of CK had a WT configuration of TP53, consistent with our theory that dominant ms hits were more likely to be present without del(17p). It is possible that the inability to assert clear survival differences according to the number or types of TP53 lesions may be due to an inability to resolve the intraclonal configuration of mutations using VAF calculations. We also conclude that non-canonical ms mutations (many of them classified as VUS) may have a variable impact, with functional consequences ranging from those that are less severe than truncations to various degrees of negative dominance. Analyses of the impact of ms mutations on TP53 tetramers (which may contain various doses of mutant vs WT monomer), will shed further light on the biology of TP53MT MN. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Takeda/Millenium: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; ASTEX: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC). Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Neomorphic mutations in IDH1/2 producing R-2-Hydroxyglutrate (R-2HG), are common in myeloid malignancies and in various solid cancers. A diffuse hypermethylated status is the biological consequence of the R-2HG-mediated inhibition of several α-ketoglutarate (αKG)-dependent enzymes including DNA dioxygenases TET1, TET2 and TET3.1,2 Specifically, the inhibition of TET2, either induced by the interaction with R-2HG or by direct genomic silencing (as in case of TET2 loss of function mutations) is responsible for the block of the DNA cytosine demethylation pathway, inducing changes in expression patterns, (e.g. decreasing expression of tumor suppressor genes) and impairing execution of differentiation programs. Analysis of genomic data from a Cleveland Clinic (CCF) cohort of AML/MDS patients combined in a meta-analytic fashion with BeatAML3 and Tumor Cancer Genome Atlas (TCGA) cohorts (1119 profiled patients) showed that IDH1/2 mutations are mutually exclusive (only 3% [N=4/106] of AML IDH1/2 mutated cases had TET2 mutations, expected to be at a frequency of 18% [N=110/585] in IDH1/2 wild type cases, p=.000125). In this scenario we suggest that the loss of TET2 activity due to mutations prevents the expansion of IDH1/2 mutant myeloid neoplasms (MNs) because of phenotypic redundancies inducing synthetic lethality. With this premise we stipulated that a critical level of DNA dioxygenase activity exists and thus cells with low TET2 activity will not tolerate further inhibition by R-2HG. Here we propose to apply pharmacologic inhibition of TET2 to produce an additive effect on DNA dioxygenases to investigate whether this will result in a synthetic lethality of IDH1/2 mutant cells. Specifically we hypothesize that TET-dioxygenase inhibition may be implemented as a possible therapeutic strategy in neomorphic IDH1/2 mutant MNs. To explore this hypothesis we conducted a series of in vitro experiments in different isogenic cell lines expressing either mutant or wild type IDH1 or IDH2, that were simultaneously mutant, wild type (WT) or knock down (KD) for TET2 (TF1-IDH2R140Q, K562-IDH1R132C both WT for TET2 gene, and K18-IDH1R132CTET2KD and SIGM5-IDH1R132C TET2MT, both with a doxycycline inducible promoter for mutant IDH1). First we found that the doxycycline induction of ectopic IDH1R132C expression led to R-2HG increase (~10,000-fold over the baseline) and induced cell death in TET2-deficient cells (experiments conducted in SIGM5-IDH1R132C cells showing 70% of decrease in cell growth after five days of IDH induction with doxycycline), confirming the cytotoxic effect of cellular R-2HG. We then tested in IDH1/2MT cells sensitivity towards TETi76, a specific TET inhibitor designed on R-2HG scaffold (with more than 200 fold potency compared to R-2HG in cell-free assays of 5-hydroxy-methyl cytosine [5hMC] production).4 This compound showed particular selectivity towards inhibition of DNA dioxygenases when a set of 23 other dioxygenase inhibitors were screened. Most importantly, consistent with our hypothesis, TETi76 preferentially inhibited the proliferation of IDH1/2MT cells either following doxycycline-induction both in TET2WTand TET2 deficient models (K562 TET2WT, K18 TET2kD, SIGM-5 TET2MT cell lines), or in models not carrying the inducible promoter (TF1 TETWT) (Growth inhibition: 20-25% in IDHWT vs 70-80% in IDHMT cell lines after 72h of co-culture with TETi76 treatment for concentrations ranging between 1 and 5 µM. P-value range: 0.04-0.001 in pairwise comparisons with untreated controls). Overall, our findings are consistent with the idea that neomorphic IDH1/2MT phenocopies loss of function TET2MT, through R-2HG, down-modulating pathways fundamental for cell homeostasis, division and differentiation. If a residual TET-activity is needed for the function of IDH1/2MT cells, the complete block of the residual activity appears to inevitably disrupt this phenotype impairing cell growth and proliferation. This is also in agreement with the paucity of TET3 and TET1 mutation in the context of TET2MT carriers. In summary, results shown here represent an important proof of concept that the increased inhibition of DNA dioxygenase activity, instead of being more leukemogenic, can be synthetically lethal. Our observations may have implications with regard to the therapy of IDH1/2 mutated neoplasms including AML and MDS Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Context: Myeloid neoplasia (MN) phenotype and outcome associates with the clonal architecture of SF3B1 mutations (SF3B1MT). Objective: To explore the implications of the clonal architecture of SF3B1MT in MN. Design/Setting: Clinical characteristics, mutational profile, and the clonal hierarchy of MN patients were reviewed at The Cleveland Clinic. Genomic studies were conducted by whole-exome and targeted deep sequencing. Patients: A cohort of 5102 patients was studied for detailed clinical and molecular annotation. Main outcome measures: To describe whether the clonal nature of SF3B1MT might alter the cellular trajectories in patients with MN. Results: The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone analysis. We first assigned clonal hierarchy to SF3B1MT 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 Conclusions: Our study of the clonal architecture of SF3B1MT highlights that clonal progression of cases with MN harboring SF3B1MT might be inferred by the rank of additional genetic lesions cooperating with SF3B1.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare subtype of peripheral T-cell lymphoma affecting younger patients and associated with hemophagocytic lymphohistiocytosis. To clarify the molecular pathogenesis of SPTCL, we analyzed paired tumor and germline DNAs from 13 patients by whole-exome sequencing. All cases were Asians and were phenotypically sporadic with no family history of SPTCL. Consistent with a recent report, germline mutations in HAVCR2, encoding T-cell immunoglobulin mucin 3 (TIM3), were identified in 11 of 13 (85%) cases. All mutated cases were primary SPTCL, whereas the 2 cases without mutation were secondary SPTCL associated with underlying diseases, including viral infection and autoimmune disease. Ten patients harbored homozygous p.Y82C mutations, and 1 showed compound heterozygous mutations (p.Y82C and p.T101I). Both missense mutations altered highly conserved residues located in the extracellular immunoglobulin variable–like domain. According to the Genome Aggregation Database of >138 500 general individuals, both mutations were documented with minor allele frequencies < 0.007, indicating remarkable enrichment of these HAVCR2 alleles in SPTCL. SPTCL cells also harbored somatic mutations (6.2 per patient) that are frequently identified in genes associated with epigenetic regulation and signal transduction. In conclusion, individuals harboring biallelic HAVCR2 (TIM3) germline mutations were highly susceptible to sporadic SPTCL, which was also associated with clonal somatic mutations.

Background Gram--negative bacteremia in acute leukemia patients with neutropenia is associated with high morbidity and mortality rate. Appropriate antibiotic for empirical treatment is crucial; however, antibiotic selection is challenging especially in setting with high prevalence of infections with multidrug-resistant (MDR) organisms. Data on associated factors of MDR Gram-negative bacteremia in this population is limited. Methods A retrospective study was conducted in King Chulalongkorn Hospital, Bangkok, Thailand. Medical records of patients aged ≥15 years with acute leukemia who were hospitalized in our institute and had neutropenic fever between 1 January 2001 and 31 December 2016 were reviewed. Demographic data, causative organisms, treatment and outcomes were documented. Episodes of MDR Gram-negative bacteremia were compared with non-MDR group. Associated risk factor was assessed by multivariate logistic regression Results From total 405 admission records of 227 acute leukemia patients, 587 episodes of neutropenic fever occurred with 131 episodes of Gram-negative bacteremia. Majority (81.68%) were bacteremia without source of infection. Most common causative pathogens were E. coli, P. aeruginosa and K. pneumoniae, respectively. Sixty episodes of Gram-negative bacteremia (53.57%) were caused by MDR pathogen. Associated factor for MDR Gram-negative bacteremia was prior colonization or infection with MDR Gram-negative bacteria within 3 months (P = 0.049). Subgroup analysis in patients with non-lactose fermenter bacteremia showed higher rate of intensive care unit (ICU) admission and in-hospital death in MDR group (P = 0.03 and P = 0.004). Conclusion Prior colonization or infection by MDR Gram-negative bacteria within 3 months was associated with MDR Gram-negative bacteremia in acute leukemia patients with neutropenic fever. Thoroughly review of previous culture data and active screening for colonization may increase chance of appropriate empirical antibiotics. Disclosures All authors: No reported disclosures.

Genomic advances have initiated a new era of precision oncology. Targeted therapeutics can rationally select potentially responsive patient populations and exclude resistant patients based on molecular mechanisms of action. The application of immunomodulatory drugs (IMiDs) evolved from unselective usage to the identification of del(5q) as a target, which then led to mechanistic clues and clarification of its mode of action. Lenalidomide (LEN) has been used in non del(5q) patients with a variable response rate, but the molecular underpinnings for LEN efficacy in this setting are not known. Genomic analyses (Negoro et al., Leukemia 2016) have provided only a marginal improvement in response prediction, suggesting that other factors might be relevant in sensitizing cells to LEN. The effects of LEN are related to its binding affinity to cereblon (CRBN), the substrate adaptor of the CRL4CRBN E3 ubiquitin ligase, a cullin-ring ligase consisting of damaged DNA-binding protein1(DDB1), cullin4a (CUL4A), and regulator of cullins-1 (ROC1). LEN selectively activates CRL4CRBN E3 ubiquitin ligase to selectively degrade CSNK1A1 leading to synthetic lethality in patients with del(5q) that are haploinsufficient (HI) for CSNK1A1. However, LEN and the new generation compounds pomalidomide and iberomide (CC-220) also trigger recruitment and degradation of the zinc finger transcription factors IKAROS (IKZF1, 7p12.2) and AIOLOS (IKZF3, 17p12-q21.1) through CRBN, and therefore promote their proteosomal degradation. The synthetic lethality of these immunomodulator agents relies on expression levels of AIOLOS and IKAROS. Response to those agents depends strictly on low expressions of AIOLOS and IKAROS sensitizing certain cell types (e.g., myeloma). We hypothesized that levels of LEN sensitivity might correlate with expression levels of its targets in selected disease subtypes of myeloid neoplasia (MN). We studied a large cohort of patients with MN (n=3,328) and we identified 122 patients with -7 and 194 patients with del(7q). We noted that for those cases with informative RNA-seq expression, IKAROS exhibited haploinsufficient (HI) mRNA levels in 67% of patients with -7 and 40% in del(7q), as defined based on levels Our preliminary results suggest that selective application of new generation IMiDS targeting IKAROS have clinical potential in patients with -7. Supportive data from in vitro cellular models will be comprehensively presented. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmacutical: Research Funding; Daiichi Sankyo: Consultancy; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee. 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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. 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.

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.