ALBERT

Description: Introduction Patients diagnosed with AML with NPM1mutation (NPM1mut AML) included in the European LeukemiaNet favorable genetic risk category (ELNfav, i.e., without FLT3-ITD or with a low allelic burden FLT3-ITD comutation [FLT3-ITD/FLT3wt 0.05) or MRD reappearance after molCR. All MFs were confirmed in a second sample collected at least 4 weeks apart from previous sample. For patients who present a MF, alloSCT was recommended, without a predefined indication of debulking previous salvage chemotherapy. Results Out of 145 patients with ELNfav NPM1mut AML (75 M/70 F; median age, 56, 18-74), 132 (91%) achieved CR after 1 (n=124) or 2 courses (n=8). After a median follow-up of 25 months, 2-year overall survival (OS), event-free survival (EFS), leukemia-free survival (LFS) of the entire cohort were 79.9% (±3.6), 71.8% (±4) and 79.4% (±3.8), respectively. Among patients with available complete MRD information (n=89), 33 patients developed an hematological and/or molecular failure (Mol/Hem failure), resulting into a Mol/Hem failure-free survival at 2 years of 62% (±5.6%); median time from CR to Mol/Hem failures was 8.2 months (2-43). Fourteen patients presented with an overt hematological relapse (hREL), preceded by a detectable MF in 8. Overall, 14 received salvage therapy in morphological CR, MRD(+) status (MF), and 14 For hREL. Among MF, 5 patients received HDAC-type salvage treatment (followed by alloSCT in 3) and 9 proceeded directly to alloSCT. In 5 additional MF patients with MRD persistence after consolidation, subsequent MRD monitoring showed decreasing MRD levels until complete clearance (n=4) or intermitent detection (n=1) and have not received further therapy. After salvage therapy, 12/14 (86%) patients with MF achieved molCR, and 10/14 (71%) treated for hREL achieved CR2 (MRD negative in 7, 50%), followed by alloSCT in 9. OS after Mol/Hem failure was 64.8% (± 8.4) at 2 years, 88.8 (±7.5%) in patients treated for MRD(+)-status (MF) and 32.1% (±13.6%) in patients treated with overt hREL (p

Description: BACKGROUND: The clinical course of splenic marginal zone lymphoma (SMZL) is usually indolent, but development of transformation to aggressive lymphoma (SMZL-T) is seen in 10 to 15% of the cases. Risk factors to predict SMZL-T are poorly defined. AIM: The aims of our study were: 1.- to assess the risk of transformation in a large series of SMZL patients; 2.- to analyze the prognostic factors for this event and 3.- to analyze clinical and biological features of SMZL-T. PATIENT AND METHODS: We identified 84 SMZL diagnosed at the HCB between 1994 and 2017 and 15 of them (18%) had developed histological transformation (SMZL-T). In addition, we reviewed 21 SMZL with transformation referred to HCB from other centers. Median age at diagnosis of low-grade SMZL in the 84 patients was 63 years (range, 32-91), and male/female ratio was 34/50. Complex karyotype (CK) defined as ≥3 chromosomal aberrations by conventional cytogenetics was observed in 22% and del(7q) in 59% of cases. The risk distribution according to the Splenic Marginal Zone Lymphoma Study Group simplified score HPLL/ABC was: low: 34, intermediate: 51 and high: 15. After a median follow-up of 80 months (range, 1-265), 37 of 84 (44%) patients have died, with a median overall survival (OS) of 146 months. To assess the risk of transformation and to identify predictive factors for such event, we exclusively evaluated the cohort of 84 patients from HCB. RESULTS: Fifteen of the 84 patients in the HCB cohort (18%) developed histological transformation. The cumulative incidence of transformation at 60 months from diagnosis was 15% (95% CI: 7 - 23) (Figure). In univariate analysis, variables predicting transformation were anemia, lymphopenia, thrombocytopenia, hypoalbuminemia, CK and high-risk scores of Intergruppo Italiano Linfomi (IIL) and HPLL (p

Description: Mantle cell lymphoma (MCL) is a rare B-cell-neoplasm with an aggressive clinical course characterized by the hallmark translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1. Furthermore, genomic profiling revealed numerous secondary genetic alterations and recurrent mutations contributing to MCL pathogenesis. Among them, mutations in the NOTCH1 gene have been described with a frequency of ~10% and are associated with significantly shorter survival rates. Therefore, further investigation of the biological impact of this mutation in MCL and its potential as a target for a specific inhibitory antibody therapy is of great interest. Here we investigated the role of the Notch receptor ligands Jagged1 (JAG1), Jagged2 (JAG2), Delta-like canonical Notch ligand 1 (DLL1) and Delta-like canonical Notch ligand 4 (DLL4) in activating the Notch1-signaling pathway in NOTCH1-mutated Mino and NOTCH1-wt Jeko1 MCL cell lines and evaluated the effects of the novel and specific monoclonal Notch1-antibody OMP-52M51. Mino cells are characterized by a single nucleotide substitution in exon 34 of the NOTCH1 gene, leading to truncation of the C-terminal PEST-domain. These mutations are known to remove the recognition site from the ubiquitin ligase degradation complex, resulting in increased stability and transcriptional activity of Notch1 upon ligand-stimulation. We confirmed the predicted truncation of Notch1 in Mino cell lysates and demonstrated that the expression of cleaved Notch1 was potently stimulated by recombinant DLL4 protein. In contrast, in the NOTCH1-wt cell line Jeko1, no stable overexpression of cleaved Notch1 could be observed upon any ligand-stimulation. Treatment of the NOTCH1-mutated cells with OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed transcriptional induction of well-known Notch1-target genes HES1 (hairy and enhancer of-split 1), DTX1 (deltex E3 ubiquitin ligase 1) and c-MYC as observed by real-time quantitative PCR. Gene expression profiling furthermore revealed that OMP-52M51 significantly impeded DLL4-induced upregulation of numerous genes involved in lymphoid biology and lymphomagenesis, as well as genes related to the cell cycle and the MAP-Kinase signaling pathway. Western blot analysis confirmed Notch1-antibody-mediated abrogation of enhanced expression of p-ERK and p-MEK upon DLL4-ligand-stimulation. Further investigations of functional effects revealed inhibitory effects of OMP-52M51 on enhanced tumor cell migration of DLL4-stimulated cells. As the DLL4-Notch-axis is known to be involved in regulating angiogenesis, we investigated the impact of activating NOTCH1-mutations in MCL on HUVEC tube formation and observed increased vessel sprouting upon DLL4-stimulation that could be abolished by treatment with OMP-52M51. Importantly, all these effects were specific for NOTCH1-mutated cells and did not occur in the NOTCH1-wt cell line Jeko1. In conclusion, we demonstrate for the first time that DLL4 is the most important ligand to activate Notch1 in MCL harbouring NOTCH1-mutations in the PEST-domain. Our findings indicate that enhanced Notch1 activity promotes a more aggressive behavior of the disease and specific antibody-inhibition of the Notch1-signaling pathway might provide an interesting therapeutic alternative for a subset of MCL patients, warranting further investigation. Disclosures No relevant conflicts of interest to declare.

Description: Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediatedby the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Gene expression profiling of 423 CLL cases, assessed by the computational GSEA analysis tool, identified enrichment of gene sets related to cytokines and inflammation in the subgroup carrying mutations in MYD88. Accordingly, CLL cells from individuals with MYD88 mutations showed higher p65 NF-κB activity and secreted higher basal levels of CCL2, CCL3 and CCL4 cytokines compared with unmutated cases. To assess the effects of this pathway in CLL, we stimulated these cells with a TLR agonists mix, containing Pam3CSK4, HKLM, FSL1 and ODN2006, which was able to induce cytokines secretion, increase in NF-KB activity and STAT3 signaling as well as cells proliferation and migration. Here, we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. ND2158 was able to efficiently decrease all TLR effects seen with the mix like STAT3 signaling and the secretion of CCL2, CCL3, CCL4, TNFα, IL1β, IL10 and IL6 cytokines (assessed by Luminex). NF-κB signaling was also modulated by the drug, as observed by the downregulation of IκBα phosphorylation protein level, p65 translocation to the nucleus and decreased p65 DNA binding activity. ND2158 also impaired CLL proliferation (checked by carboxyfluoresceinsuccinimidyl ester staining) and migration towards CXCL12 in MYD88-mutated and -unmutated CLL cases.ND2158 also induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. To further validate this results, we checked the effect of ND2158 in splenocytes from leukemic Eμ-TCL1 mice and similar antitumor effects were seen. In addition to the direct impact on tumor cells, we analyzed whether ND2158 also affects other immune cells that also signal via MYD88/IRAK4. Monocytes, broadly known for their importance in tumor supporting, were also modulated by TLR agonists, and a reduction in activation markers and cytokines secretion was achieved after ND2158 treatment. In vivo studies in Eμ-TCL1 leukemic mice showed a significant reduction in tumor load in CLL-affected organs (peripheral blood, spleen, lymph node and peritoneal cavity) associated with reduced PD-L1 expression levels in mice treated with 100 mg/kg/day of the IRAK4 inhibitor. A decrease in spleen weight and size was also detected after the treatment. Corroborating the in vitro studies,ND2158 also modulated tumor-supporting monocytes. The effect of ND2158 was also assessed in CD8+ effector T cells, where a low proliferative (Ki-67+ percentage), exhausted phenotype (increase in TIGIT, CD244, CD160 and LAG3 markers) and less functional population (increase in the immune checkpoint protein PD-1) was found. Based on our in vitro data, it seems that ND2158 targets both MYD88 mutated and unmutated CLL cases to a similar degree. Since CLL progression in the TCL1 mouse model has not been reported to be driven by an activating MYD88 mutation, our in vivo results support the notion that IRAK4 inhibition might also be effective in CLL patients with an unmutated MYD88 status. To overcome the potential negative impact of IRAK4 inhibition on T cells, the combination treatment with drugs that improve T cell function and avoid their rapid exhaustion should be considered. As we observed increased expression of several inhibitory receptors on CD8+ T cells in ND2158-treated mice, including PD-1 and TIGIT, blocking these receptors with antibodies might be a successful strategy to overcome loss of T cell function and improve therapy outcome. In summary, our data show the importance of TLR signaling in CLL development and suggest IRAK4 as a novel treatment target for CLL that targets both the malignant cells and the tumor-supportive inflammatory milieu. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered 〉80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A〉C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A〉C point mutation occurring at position 3 of the U1 snRNA gene (g.3A〉C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A〉C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A〉C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A〉C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

Description: Introduction The European LeukemiaNet (ELN) 2017 classification for acute myeloid leukemia (AML) stratifies patients in 3 risk categories, according to genetic features of the disease. ELN classification is commonly used to guide post-remission treatment; favorable risk patients do not seem to benefit from allogeneic hematopoietic transplantation (alloHCT) in first complete remission (CR1) while this procedure is highly recommended in adverse risk patients. Post-remission treatment in intermediate risk patient is still debated. This classification has not been prospectively validated. Herein we analyze the prognostic impact of ELN 2017 classification in patients treated with the same protocol (CETLAM-12), including a well-defined preplanned alloSCT policy according to genetic risk. Methods We analyzed characteristics and outcome of patients diagnosed with de novo AML, included in CETLAM-12 protocol for patients up to 70 years, with an available genetic characterization at diagnosis allowing an accurate ELN 2017 stratification. Genetic risk allocation was based on cytogenetic analysis and pre-specified RT-PCR of determined markers (including CBF-rearrangement, NPM1, FLT3, CEBPA, and MLL-PTD) performed in all patients, and targeted Next Generation Sequencing testing (available in 143 patients). CETLAM-12 protocol defines a genetic risk stratification in three groups, closely similar to that proposed by the ELN 2017 classification, and recommends a post-remission strategy based on this risk assessment. Patients from the favorable group received three courses of consolidation with high-dose cytarabine (HiDAC), whereas alloSCT in CR1 is strongly recommended for intermediate and adverse risk patients following one HiDAC-based consolidation course. Results We included in the study 813 patients (400F/413M; median age 56, 17-76); with a 37 months median estimated follow-up (range 0.1-92). The outcomes of the entire cohort are shown in table 1. Out of all patients, 641 could be classified according to the ELN 2017 classification, due to the presence of risk defining genetic features identified by any of described methods. In the group of .atients allocated in the favorable risk category (n=316; 49%), twenty-seven patients died during induction. Fifty-eight relapses were observed, mostly in patients with NPM1 mutation (n=41). AlloSCT was finally performed in CR1 in 84 patients (27%) due to MRD persistence or reappearance (n=40), overt hematological relapse (n=27), persistent aplasia following chemotherapy (n=3) and protocol deviation (n=14). In the group of patients allocated in the intermediate risk category (n=95; 15%), twelve patients died during induction. AlloSCT in CR1 was performed in 62 patients (67%), with 5 patients receiving an alloSCT in CR2. In the group of patients allocated in the adverse risk category (n=230; 36%), twenty-five patients died during induction. AlloSCT could be finally performed in CR1 in 138 patients (60%) and 88 relapses occurred (42 before alloSCT, 46 after alloSCT). Amongst ELN adverse risk patients, a subgroup with a significant worse outcome has been identified, defined by AML with complex karyotype +/- TP53 mutation or chromosome 3q26/MECOM-GATA2 rearrangement. This group (ELNadv+) presented a lower CR rate, with a higher relapse rate and fewer proportion of patients who receive a pre-planned alloSCT in CR1. OS and EFS of ELNadv+ is significantly lower than ELN adverse patients. In the multivariate analysis for OS including age, sex, WBC count at diagnosis and number of cycles to achieve CR, only age and ELNadv+ status showed independent prognostic value. Outcome data is summarized in table and figures attached. Conclusions The initial risk-adapted post-remission assignment planned in CETLAM-12 could be performed in the majority of patients. Despite this different proposed post-remission treatment, ELN risk classification was able to identify three groups of patients with a markedly different outcome. Interestingly, within the unfavorable ELN category, a very high-risk ELNadv+ subgroup can be distinguished, with a dismal outcome with current approach, warranting the implementation of innovative pre and post-transplant strategies aimed to prevent treatment failure. Figure Disclosures Tormo: MSD: Honoraria; Janssen: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Honoraria; Servier: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Salamero:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria; Daichii Sankyo: Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Sierra:Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Current diagnosis of chronic myelomonocytic leukemia (CMML) requires peripheral blood (pb) monocytosis ≥1×109/L. Accordingly, cases which fulfill other diagnostic criteria of CMML but not reaching the required pb monocytosis threshold would be classified as MDS or unclassifiable MPN/MDS according to WHO classification (Arber et al, Blood 2016) Recently, a group of authors (Geyer et al, Modern Pathology 2017) proposed the term oligomonocytic CMML (OM-CMML) for these patients with blood monocytes ≥10% of the WBCs, but only accounting for 0.5-1 × 109/L as an absolute value and fulfilling all other criteria of CMML and suggested that they should be managed as other patients with classical CMML despite lacking pb monocytosis ≥1×109/L. To address clinical value of this proposed newly entity, we analyzed the incidence, clinico-biological characteristics and outcome of a series of patients fulfilling the proposed criteria for OM-CMML from a single center with a long follow-up. Methods We included patients diagnosed between 1997 and 2019 who gathered the proposed criteria for OM-CMML (Geyer et al, Modern Pathology 2017). These patients were compared with a cohort of patients from the same study period diagnosed with classical CMML. Statistical analyses were performed using Rv3.1 and SPSS v20. Next generation targeted sequencing (NGS) was performed with Ion Ampliseq AML Research and Oncomine Myeloid Research Assay panel Results Overall, we included in the study 213 patients, including 35 (16%) who fulfilled the proposed criteria for OM-CMML. Median follow-up of alive patients was 42 months. In the OM-CMML group, 71% were males, median age was 74 years (51-92). OM-CMML patients presented at diagnosis with a lower leucocyte count (WBC) (median value, 4.6(2.2-7.5)x109/L vs 10(3-119)x109/L, p