Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to secondary acute myeloid leukemia (sAML). However, the mutational dynamics and clonal evolution underlying disease progression are poorly understood at present. In order to elucidate the mutational dynamics of pathways and genes occurring during the evolution to sAML, next-generation sequencing was performed on 84 serially paired samples of MDS patients who developed sAML (discovery cohort) and 14 paired samples from MDS patients who did not progress to sAML during follow-up (control cohort). Results were validated in an independent series of 388 MDS patients (validation cohort). We used an integrative analysis to identify how mutations, alone or in combination, contribute to leukemic transformation. The study showed that MDS progression to sAML is characterized by greater genomic instability and the presence of several types of mutational dynamics, highlighting increasing ( STAG2 ) and newlyacquired ( NRAS and FLT3 ) mutations. Moreover, we observed co-operation between genes involved in the cohesin and Ras pathways in 15-20% of MDS patients who evolved to sAML, as well as a high proportion of newly acquired or increasing mutations in the chromatin-modifier genes in MDS patients receiving a disease-modifying therapy before their progression to sAML.

Background Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high‐risk alterations such as del(11q)/ATM‐mutations and del(17p)/TP53‐mutations have not been established. Methods We integrated next‐generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response. Results Targeted sequencing analysis of the co‐occurrence of high‐risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co‐occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9‐edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition. Conclusions Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

Background: Chromosome 14q32 rearrangements involving the immunoglobulin heavy chain gene (IGH) affect less than 5% of chronic lymphocytic leukemia (CLL) patients. Their clinical course is aggressive and the outcome, worse than other CLL subtypes (Cavazzini et al, 2008; Gerrie et al, 2012). However, the biology of CLL showing IGH rearrangements (CLL-IGHR) is not completely defined. The identification of novel recurrent mutations in CLL by next generation-sequencing (NGS) has offered a more comprehensive view into the genomic landscape of the disease and improved the prognostication of CLL. Thus, mutational analysis might be especially useful in those patients with uncertain prognosis, such as those carrying IGH rearrangements. Aim: To analyze the mutational profile of CLL-IGHR patients by targeted NGS in order to improve our understanding of the genetic underpinnings of this subgroup. Methods: The study was based on 899 CLL patients, well characterized at cytogenetic, biological and clinical level, forty-two of them (4.7%) showing IGH rearrangements. Targeted NGS was performed in 231 CLL samples: 117 with 13q deletion, 27 with 11q deletion, 26 trisomy 12, 42 showing IGH rearrangements and the remaining 19 without any cytogenetic alteration. CD19+ B cells were isolated and DNA extracted. SureSelectQXT targeted enrichment technology and a custom-designed panel (MiSeq, Illumina), including 54 CLL-related and recurrent mutated genes, was carried out. The panel yielded 100x or greater coverage on 97% of the genomic regions of interest and the mean coverage obtained was 600x. Mutations were detected down to 3% allele frequency. Results: The mutational analysis of CLL-IGHR patients identified a total of 72 mutations in 32 genes. Seventy-one percent of patients (30/42) harbored at least one mutation. The most frequently mutated genes in this cohort were NOTCH1 (28.6%), POT1 (14.3%), TP53 (9.5%), SF3B1 (7%), BRAF (7%), EGR2 (7%), IGLL5 (7%) and MGA (7%), followed by BCL2, HIST1H1E and FBXW7 (4.8%), uncommonly mutated genes in CLL at these frequencies (Table 1). In fact, mutations in NOTCH1, BRAF, EGR2, BCL2, HIST1H1E and FBXW7 were significantly associated with CLL-IGHR patients (p=0.013, p=0.003, p=0.021, p=0.038, p=0.038 and p=0.021 respectively). In terms of time to the first therapy (TFT), CLL-IGHR had an intermediate-negative impact (median TFT=24 months) compared to the presence of cytogenetic alterations associated with good prognosis such as 13q deletions (median TFT>120 months; p<0.0001) (Figure 1A). Furthermore, the presence of mutations in the most frequently mutated genes (NOTCH1, POT1, TP53, SF3B1 or BRAF) within patients with IGH rearrangements had a negative clinical impact in the TFT and allowed us to refine the prognosis of this subgroup. Thus, the median TFT of patients with mutations was 1 month while the median TFT of patients without mutations was 14 months (p=0.014) (Figure 1B). A total of 17 out of 42 CLL-IGHR patients (40.5%) carried the t(14;18). Interestingly, patients with t(14;18) were characterized by: 1) A lower mutation frequency (average of mutations/patient=1.05) than the rest of rearrangements with unknown partners (average=2.16; p=0.039), and 2) The presence of mutations in BCL2 (11%) and HIST1H1E (11%). By contrast, CLL-IGHR without BCL2 rearrangement showed mutations in POT1 (20%), TP53 (16%), SF3B1 (12%) and BRAF (12%). Moreover, t(14;18) was significantly associated with good prognosis markers such as the mutated status of the variable region of the immunoglobulin genes (IGHV-M) (p=0.002). However, there was no significant difference in terms of TFT between patients with t(14;18) and patients with other IGH rearrangements (p=0.27). Conclusions: CLL patients with IGH rearrangements showed: i. A high gene mutation frequency; ii. A distinct mutational profile, with recurrent mutations in POT1, EGR2, BRAF, IGLL5 and MGA genes; iii. An adverse clinical outcome refined by the negative effect of genetic mutations. iv. Patients with t(14;18) presented a lower mutation frequency than the rest of rearrangements, carrying mutations in BCL2 and HIST1H1E, and associated with good-prognosis markers such as IGHV-M. Funding:PI15/01471; CIBERONC CB16/12/00233; FEHH-Janssen(MHS); JCyL(MQÁ) Disclosures Mateos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme UDP-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied three patients from two unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed four variants affecting GALE, three of them previously unreported (Pedigree A: p.Lys78ValfsX32 and p.Thr150Met; Pedigree B: p.Val128Met and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease of N-acetyl-lactosamine (LacNAc), demonstrating a hypoglycosylation pattern, reduced surface expression of GPIbα-IX-V complex, and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients' derived megakaryocytes demonstrated normal ploidy and maturation but decreased proplatelet formation due to the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand Factor were also demonstrated. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasized the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P&lt;0.001). In a second experiment, HG3-del(11q), HG3-del(11q) TP53MUT and HG3-del(11q) ATMMUTTP53MUT cells were injected. Strikingly, HG3-del(11q) TP53MUT cells were able to outcompete HG3-del(11q) cells in spleen and bone marrow (P&lt;0.001; P&lt;0.001). By contrast, HG3-del(11q) ATMMUTTP53MUT cells failed to engraft neither in spleen nor bone marrow, being outcompeted by the other injected cells (P&lt;0.001), providing biological insights on the mutual exclusivity of these genetic events in CLL (Fig 1B). We next assessed whether these cell models could predict responses of these combined abnormalities to BTK and PI3K inhibitors. We observed that HG3-del(11q) TP53MUT and HG3-TP53MUT cells showed partial response to ibrutinib and idelalisib, although the IC50 values were still higher than the ones observed in HG3WT clones, especially with idelalisib (27.4 and 20.6 vs. 1.8 uM, respectively). Nonetheless, we found that HG3-del(11q) TP53MUT cells were highly sensitive to novel preclinical drugs that have been shown to be effective in TP53 deficient cells such ATR inhibitors, with an IC50 value comparable to HG3WT cells (mean IC50 0.55 vs. 0.67 uM) (Fig 1C). In summary, we show that mutations in TP53 can appear in a subset of monoallelic del(11q) CLL cases, conferring a synergistic clonal advantage in vivo, and therefore a dismal clinical impact on the OS of this CLL subgroup. In addition, the biological basis of mutual exclusivity of biallelic ATM and TP53 alterations in CLL was assessed, underscoring the importance of the number of alleles affected by these alterations, and establishing novel pre-clinical models for the study of the biology and therapeutic response of concurrent genetic abnormalities in the disease. Funding: PI18/01500 FI19/00191 CD19/00222 *MQA CPC equal contr Disclosures No relevant conflicts of interest to declare.

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Introduction Inherited thrombocytopenias (ITs) are a heterogeneous group of rare platelet disorders. which lead not only to increased bleeding, but also to syndromic forms. ITs are caused by genetic alterations in megakaryopoiesis-related genes. In the last years, whole-exome sequencing (WES) has allowed the identification of novel genes involved in IT. Aim To perform the molecular, clinical and platelet characterization of two unrelated families with syndromic IT, to unveil the underlaying alteration leading to the disease. To explore the functional role of the identified alterations during megakaryocytic (Mk) differentiation. Methods WES was performed in two unrelated non-consanguineous families with lifelong severe macrothrombocytopenia (MCT), bleeding, and extra-hematological manifestations. Bleeding score (BS) was recorded by ISTH-BAT. Platelet phenotyping included platelet count (P), blood film, aggregometry (LTA) and flow cytometry (FC). UDP-galactose-4-epimerase enzymatic activity was measure by HPLC/MS/MS. In vitro functional studies were performed through overexpression of GALE genetic variants in human K562 cell line to elucidate its role in Mk differentiation, by measuring cell ploidy and expression of CD41, CD61 and CD42b surface markers after 7-days of PMA treatment. Results Family pedigrees are shown in Figure 1. Three patients (A.II.1, A.II.2 and B.II.1) were referred due to lifelong severe MCT and moderate-severe bleeding tendency (Figure 1). Moreover, they presented mental retardation, mitral insufficiency, and increased bilirubin levels. Blood film revealed enlarged, giant, and grey platelets (A.II.1: 36%, 6% and 54%, respectively; A.II.2: 56%, 4%, 34%, respectively; B.II.1: 32%, 46%, 12%). LTA showed moderate/severe impaired aggregation with ADP, TRAP-6, CRP, epinephrine, arachidonic acid, and ristocetin. FC confirmed null secretion of alpha and dense granules in A.II.1, A.II.2 and reduced levels in B.II.1 (7.8%, 8.1%, 28.3% respectively, vs. 51.7% control platelets with ADP 10µM; 10.8%, 7.8%, 36.7% respectively, vs. 95.6% control platelets with TRAP6 25µM). WES revealed that both pedigrees carried compound heterozygous variants in GALE (NM_001127621.2): c.230_231insTGTT; p.Lys78Valfs*32 (exon 3), and c.449C&gt;T; p.Thr150Met (exon 5) in A.II.1 and A.II.2 patients; and, c.668T&gt;C, p.Leu223Pro (exon 7), and c.382G&gt;A, p.Val128Met (exon 5) in B.II.1 (Figure 1). Enzymatic activity of the GALE-encoded protein UDP-galactose-4-epimerase was severely reduced in the affected patients: both A.II.1, A.II.2 patients had 1.3 μmol/h/g hemoglobin (control: 8.8 μmol/h/g hemoglobin), and B.II.1 patient had 0.6 μmol/h/g hemoglobin (control: 8 μmol/h/g hemoglobin). Furthermore, in vitro overexpression assays between wild-type GALE and p.Thr150Met, p.Leu223Pro and p.Val128Met variants, confirmed a delayed maturation of Mks upon PMA treatment (at 3, 5 and 7 days). characterized by a significant reduction in the expression of the megakaryocytic surface markers CD41, CD61 and CD42b. Conclusion WES has allowed us to identify pathogenic variants in GALE, which were associated with syndromic IT characterized by severe macrothrombocytopenia. Patients harboring these pathogenic variants presented moderate to severe bleeding tendency associated with cardiovascular and neurological abnormalities. Regarding the platelet phenotype, the presence of giant and grey platelets and the absence of both platelet granules were the most remarkable features reported. Moreover, these GALE variants led to an alteration in in vitro Mk maturation, supporting the thrombocytopenic phenotype observed in patients. Funding ISCIII (PI17/01966, PI 17/01311, PI20/00926), GRS (GRS2061A/19, GRS2135/A/2020), Fundación Séneca (19873/GERM/15), Fundación Mutua Madrileña (AP172142019), Premio López Borrasca (2019), Grupo Trabajo Patología Hemorrágica-SETH (2020). Figure 1 Figure 1. Disclosures Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. The clinical efficacy of lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs. Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence. Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR &lt; 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b). Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)