ALBERT

Description: Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Background: De-ubiquitinating enzyme BAP1, functionally related to ASXL1, is mutated in various hereditary cancers and its deletion is associated with the appearance of myelodysplastic/myeloproliferative features in mice. BRCA1 is known to drive homologous recombination, playing a critical role in preserving genomic integrity. Finding an impaired DNA-damage via in chronic myelomonocytic leukemia (CMML) would open the synthetic lethality strategy to MDS/MPN diseases, moreover if the already targeted hypermethylation mechanism is not involved. Methods: BAP1 and BRCA1 expressions levels were quantified by RT-qPCR. Methylation detection was performed on 57 CMML patients and 27 controls using Methylation Cancer Panel I from Illumina (San Diego, CA) interrogating 2 CpG sites at the 5´promoter region of BAP1. We developed a sandwich-type ELISA assay to measure the grade of ubiquitination of BRCA1. Fifty micrograms of total protein from blood-sorted monocytes, granulocytes and lymphocytes lysate of 19 patients were used. Total BRCA1 was captured in precoated microtiter and an anti-mono and poly ubiquitin conjugates monoclonal antibody (HRP-linked FK2) was added. A second ELISA assay determines the nanogrames of BRCA1 protein quantity in those 50 µg of sample lysate. It allows to determine the ratio of ubiquitylated BRCA1 light units/total BRCA1 (uBRCA1/BRCA1), which measures in a quantitative manner the percentage of BRCA1that is ubiquitylated in a given sample. Results: Samples of 175 patients were included in the study: CMML=61; MDS=34; AML=50; CML=30 and 10 controls. CMML showed the lowest values (65% compared with the controls), significantly lower than the other groups, except for CML patients: CMML vs MDS, p=0.001; CMML vs AML, p

Description: Thymic-independent peripheral expansion of CD8+ cells derived from the graft in the initial stage of post-HSCT immune recovery is a well-known physiological event. Nevertheless, the description of symptomatic LGL leukemias and aggressive malignant cases in this setting may generate uncertainty, mostly in those cases in which the cytotoxic T lymphocyte expansion CTLe persists beyond the early transplantation period. We aimed to assess the nature of CTLe in adults during the post-alloHSCT period in a series of 154 patients with a long term surveillance. We studied the longitudinal kinetics of those expansions, their relation to clinical events, and their phenotypic and molecular features, including recently reported CTL leukemia-STAT3 mutations. In our study, trying to adhere to the WHO annotation of T-LGL, we considered two definitions for a CTL expansion: an absolute increase (≥ 2000 x109/L), and a relative expansion (a CD8/CD4 ratio ≥ 1.5), persisting more than six months in both cases. Persistent relative CTLe cases are frequent (49%) and related with timoglobulin prophylaxis (p≤0.001), acute graft versus host disease (GVHD, p=0.02), reduced intensity conditioning (p=0.04) and fungal and viral infections in the early post-HSCT. No differences in the number of serious infectious events from day 180 was found. Absolute CTLe are scarce (9%), related with chronic GVHD and absence of relapses. TCR rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. We studied in a cross sectional manner with an extended immunophenotypic panel 17 patients: 5 patients with an absolute CTLe and 12 cases with a relative CTLe. A similar cytotoxic T αβ-effector phenotype was observed in all cases, with slight differences in the expression of CD25, CD16 and 1a. One patient with a relative CTLe expressed CD56 intensely: his ratio normalized at day 730 and no immune-related events were recorded. DNA stored during the post-alloHSCT setting was available from 68/75 relative CTLe patients (14/14 absolute CTLe cases). All of them went through molecular TCR rearrangement and STAT3 exon 21 mutations determination. In the relative CTLe cohort, TCR rearrangement was described as clonal, oligoclonal or polyclonal in 77%, 16% and 7%, respectively. Regarding absolute CTLe patients, TCR rearrangement was described as clonal in all the patients (n=14) of this subset. To increase the sensibility of the Sanger PCR, it was performed on DNA from CD3+ sorted cells in 54 out of 68 cases. No STAT3 mutation could be found in the CD3+ sorted fraction of relative or absolute defined CTLe. Not using an absolute threshold would establish a diagnosis of a persistent CTL expansion in 49% of our cohort of allo-transplanted patients. Additional diagnostic tools, as an effector phenotype, the presence of a NK marker or a monoclonal TCR rearrangement would not reduce significantly that percentage: CD57 was invariably expressed in CTLe cases, and 80% of our patients with expansions showed a TCR monoclonal pattern. STAT3 mutations resulting in persistent proliferation of CTL clones are a frequent event in large granular lymphocytic leukemia, and those clones have also been described in autoimmunity-driven disorders as acquired aplastic anemia and hypocellular myelodysplastic syndromes. We establish in this study the absence of exon 21 STAT3 mutations in the persistent CTL expansions found in a large series of patients with a long-term post-alloHSCT surveillance. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period, supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Disclosures No relevant conflicts of interest to declare.

Description: Background: We and others have reported on the impact of recurrent somatic mutations not only in the multistep pathogenetic process, but also in the clinical heterogeneity of chronic lymphocytic leukemia (CLL) patients. Immunophenotyping, as part of the diagnostic workout, is used for assessing clonality, as a differential diagnosis tool, and to examine the expression of molecules associated with a worse prognosis. Recently, NOTCH1 mutations have been linked to low CD20 levels in CLL and with a relative resistance to anti-CD20 immunotherapy in vitro. But to date, there is limited information on the correlation between cell surface marker expression and the presence of somatic mutations in CLL. The aim of this study was to evaluate potential associations between an extended phenotypic panel and the mutational status of 13 recurrently mutated genes in CLL detected by deep sequencing. Patients and Methods: To this end, we performed targeted NGS sequencing of blood samples, collected at diagnosis, from 131 CLL patients. Every patient underwent, at baseline, a flow cytometry characterization with a panel including (sIg)λ, (sIg)κ, CD19, CD5, CD11b, CD81, CD10, CD79b, CD29, CD38, FMC7, CD22, CD45, CD103, CD11c, CD25, ZAP70, CD11a, and CD24. We designed a TruSeq Custom Amplicon panel (Illumina, Inc. San Diego, CA, USA) containing 13 genes and covering 28.099 bases. The average amplicon size was 238 base pairs and ~ 99.1% of the regions were covered on both strands. Paired-end sequencing (2x250 bp) was performed with MiSeq v2.2 chemistry, and a mean depth of 998 reads/base within the regions of interest was obtained. Raw data were analyzed with IlluminaonJboard Real Time Analysis (RTA v.2.4.60.8) software and MiSeq Reporter. Results: With a median age of 68 y.o. (range, 33-95) and a slight male predominance, the median follow up time of our cohort was 43 months (24-104). We found that 47/131 (35%) patients harbored at least one mutation, with NOTCH1 (n = 13, 10%), ATM (n = 10, %), TP53 (n = 8, %), and SF3B1 (n = 8, 5.5%), as the most frequently mutated genes. Those patients with a NOTCH1 mutation showed a lower CD25 expression (25 mean fluorescence intensity units (MFIu)) than those without a mutation (45 MFIu), p=0.001. In addition, a higher expression of CD5 (265 vs. 219 MFIu, p= 0.02), of the monoclonal light chain (90.5 vs. 58.6 MFIu, p=0.03), and a higher percentage of CD38+ cells in the CD19+CD5+ compartment (37% vs. 19%, p=0.006) were significantly associated with the presence of, at least, one somatic mutation. We could not validate the recently reported association between the presence of NOTCH1 mutations and a low expression of CD20. In our cohort, the MFI expression in NOTCH1 mutated and non-mutated patients was 176 and 135 units, respectively (p=0.2) In the multivariate Cox analysis, the presence of a somatic variant in TP53 and a higher percentage of positive CD38 cells in the tumour population showed both a worse overall survival and shorter time to first treatment. The independence of these two variables was also supported by not finding a significative difference percentage of CD38 positive cells between TP53 mutated and non mutated cases (p=0.5). Conclusions: The associations described herein suggest potential pathogenic pathways in CLL, in particular the CD25-NOTCH1 axis, with a significative inferior expression of CD25 when activating NOTCH1 mutations are present. The relationship found between these two variables, with an inversed direction to that found in physiological conditions, has also been shown in the setting of NOTCH1-mutated acute lymphoblastic leukemia, emerging as a potential targetable pathway in this subset of CLL patients. Disclosures Maciejewski: Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Description: Background and Aim: Azacitidine have shown clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs, encoding for immunogenic proteins which are normally expressed in the testicles and trophoblastic cells of the ovary, have been shown to undergo derepression after the use of demethylating agents in cancer cell line models. We took advantage of the unique model of Aza-treated high risk MDS to in vivocharacterize candidates for immunotherapy following hypometilating therapy. Methods:Targeted RNA-Seq (tRNA-Seq) designed to capture 214 CTA genes was performed in 19 MDS or CMML patients at day 0 and at day +28 after first AZA cycle. In 10 patients the analysis was extended to third and/or four cycles. Sequencing, coverage, mapping and alignment was performed using the Ion Torrent Browser Suite. We used LIMMA package or empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes. A homogeneous treatment schedule with AZA (75 mg/m2/day x 7 days) was received by each patient.The response criteria were from IWG 2006, including a bone marrow and cytogenetic reevaluation after 6 cycles. The main candidates selected from de tRNA-seq experiment were characterized at protein level in cell lysate and/or plasma by Western Blot. Primary antibodies used from Thermo Fisher were: ADAM29, DDX53, PRAME, B-TUBULINA, FAM46D, B-ACT, TMPRSS12, MAGEB4, GAPDH, MAGEA1, MAGEA2, TSPY2, Cxorf48, DNAJB8, NY-ES0-1, CT83/Cxorf61 and TFDP3 from Santa Cruz Biotechnology. We used either cell lysate or plasma to study the protein levels in non-reducing SDS-PAGE at different acrylamide concentration depending on the protein size. ImageJ was used to quantify protein levels. Results:The median age of the cohort was 69 (range 48-81 years). The cohort consisted of 16 MDS and 3 CMML patients. MDS Patients were stratified based on IPSS as low or intermediate-1 (n=14) and intermediate-2 or high (n=2) risk groups and CMML patients based on a CPSS of intermediate-2 (n=3). Five patients were classified as complete responders achieving a complete cytogenetic and/or marrow remission (CR). Regarding the tRNA-Seq, on average, we achieved 3.888.308 mapped reads per sample (p25-p75, 1.2x106 - 5.9x106) which represents a sufficient depth for digital gene expression profiling of 214 genes. CTAs re-expressed significantly after one AZA cycle in complete responders were TFDP3 (FC=6,4), ZNF645 (FC=2), MAGEB4 (FC=3,1), MAGEA5 (FC=2), DDX53 (FC=2), VENTPX1 (FC=5,5), TSPY2(FC=3,4) and TSYP3 (FC=3,5). Other potential candidates with significantly re-expression in any responders vs. non responder were:SLCO6A1, ADAM29, DDX53, PRAME, FAM46D, TMPRSS12, MAGEA1, MAGEA2, Cxorf48, DANJB8, NYESO-1 and CT83. In the western-blot experiments, protein expression, longitudinally measured in peripheral blood lysate and/or plasma showed a parallel dynamic to gene expression in the case of TFDP3and DDX53. Conclusions:In this study, targeted RNA-seq is shown as effective and accurate tool to detect weakly expressed transcripts as CTAs. In this work, TFDP3 and DDX53, validated at proteomic level, emerge as most promising candidates for immunotherapy in combination with hypomethylating agents in MDS patients. Disclosures No relevant conflicts of interest to declare.

Description: Background: The 2016 reviewed classification of the World Health Organisation (WHO) defines a group of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including the chronic myelomonocytic leukemia (CMML), the myelodyplastic syndrome with ring sideroblasts and thrombocytosis (MDS-RS-T) and the MDS/MPN unclassifiable (MDS/MPN-U). The presence of typical clinical characteristics of MDS and MPN hinders the diagnosis and the prognosis of MDS/MPN-U. Aim: The objective of this study was to compare the clinical characteristics and the prognosis of a series of patients with MDS/MPN such as CMML, MDS-RS-T and MDS/NPM-U from the Spanish registry of MDS. Method: We analized 107 patients diagnosed with MDS/MPN (MDS-RS-T, MDS/NPM-U and CMML) according to the 2016 WHO classification. A comparison of the clinical characteristics, overall survival (OS) and cumulative incidence of progression (CIP) was performed. Results: Median (range) age was 74 (23-93) years and 68/107 (64%) were males. The number of patients in each group was: MDS-RS-T (n=45), MDS/MPN-U (n=29) and CMML (n=33). The main clinical characteristics of the three groups are described in Table 1. There were significant statistical differences in hemoglobin and lactate dehydrogenase levels and leukocyte, monocyte and platelet counts between the three groups. With a median (range) of follow-up of 3.1 (0.3-19.3), 3.7(0.7-10.4) and 4 (1.8-8.5) years for MDS-RS-T, MDS/MPN-U, and CMML, respectively, the OS (95%CI) at 5 years was significantly better in patients with MDS-RS-T (61% [42%; 80%]) compared to MDS/MPN-U and CMML patients (21% [1%; 41%] and 19% [3%; 35%], p=0.002) (Figure 1). The CIP (95%CI) at 5 years between the three groups was significantly different: MDS/MPN-U and CMML (40% [18%; 61%] and 32% (14%-52%, respectively) vs. MDS/RS-T 8% [0.4%; 30%]) (p=0.005) (Figure 2). Conclusions: 1) In this series of patients with MDS/MPN (MDS-RS-T, MDS/MPN-U and CMML) according to the 2016 WHO classification clinical characteristics were similar except for hemoglobin and lactate dehydrogenase levels and leukocyte, monocyte and platelet counts. 2) Patients with MDS-RS-T had longer OS and less CIP than those with MDS/MPN-U and CMML; 3) The prognosis of MDS/MPN-U and CMML were similar. Supported by grants from: AGAUR 9015-470120/2015, 2017-SGR288 (GRC), CERCA Program from Generalitat de Catalunya, and "La Caixa" Foundation. Disclosures No relevant conflicts of interest to declare.

Description: Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure. Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score 〉30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded. Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%). Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. Table. Table. Disclosures Díez-Campelo: Celgene: 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.

Description: Background and Aim: Chronic lymphocytic leukemia (CLL) is an incurable neoplasm with considerable clinical heterogeneity that is underpinned by distinct genetic changes. This poses a great challenge to the development of effective treatment, as responses to novel therapies may vary depending upon the biological subtype of disease. NOTCH1 has emerged as the most commonly mutated gene in CLL at diagnosis, its frequency rises with progression, and the variant c.7541_7542delCT accounts for approximately 80% of cases. NOTCH1-mutated (NOTCH1mut) CLL subset represents a subtype that responds poorly to anti-CD20 drugs, has worse outcomes, and a proliferative profile. We aimed, among the proliferative signals, to study the D-type cyclins that govern cell cycle entry in a NOTCH1mut subset which lacks an accurate targeted therapy. Methods: We performed targeted sequencing on 289 CLL cases with a TruSeq Custom Amplicon panel from Illumina, containing 13 genes recurrently mutated in CLL. Paired-end sequencing reached a mean depth of 938 reads/base. Out of 30 NOTCH1mut patients, RNA from the CD19+ fraction was available in 25 cases. Cyclins D1, D2, D3 and cyclin dependent kinases CDK4 and CDK6 expression was measured by RT-qPCR in 25 NOTCH1mut and 57 NOTCH1wild type (NOTCH1wt). CD19+ B-CLL cells from 7 NOTCH wt and 2 NOTCH1mut patients were cultured in Expansion Human Media supplemented with CD40L, IL-4 antibiotics and inactivated BFS. On day ten 2,5x105 B cells were added to a combination of ON-TARGETplus SMART pool siRNA NOTCH1, pool siRNA CCND1, SMART pool siRNA CCND2, or ON-TARGETplus siCONTROL as negative control. Electroporation was performed with Neon Transfection System in 10 ul tips using settings of 1400 volts, 10ms width, and 3 pulses. Transfected cells were double stained with Annexin V-FITC conjugate and PI in isotonic solution, and then analyzed by FC. Viable cells were defined as double negative cells. Results: A total of 82 CLL patients, 25 NOTCH1mut and 57 NOTCH1wt were included in the RT-qPCR study. Figure 1a shows fold change expression and p value comparing NOTCH1mutvs.NOTCH1wt of CDK4 (FC= 11,1; p

Description: Background and Aim: The entity defined by the WHO 2017 classification as myeloid neoplasms with germinal predisposition without preexisting disorder or organ dysfunction is particularly interesting within myelodysplastic syndromes (MDS) for three main reasons: i) in myeloid disease derived from congenital bone marrow failure, therapeutic strategies (e.g., type of conditioning regimen) are defined; it is not the case within the group of young patients with MDS harbouring germinal variants, actually a group candidate for allogeneic transplantation of hematopoietic progenitors, ii) its incidence exceeds the cases secondary to congenital bone marrow failure, and iii) the implications of genetic counseling to patients and relatives are yet to be defined and have not been addressed at the time of diagnosis of MDS. Methods: Whole exome sequencing (WES) was performed on 118 tumour and 73 paired germinal DNA from patients of 16 Spanish Group of MDS (GESMD) centers, diagnosed with de novo MDS between 16-60 years old without previous organ dysfunction. WES libraries were sequenced on a HiSeq4000-NovaSeq6000-Illumina platform, mean number of reads per sample was 144,429,985 with a Phred Quality Score 〉30 in 94% of bases and a 100x average depth. To identify potential germline-predisposing mutations, a selection tool incorporating 279 genes associated with cause or predisposition to bone marrow failure or cancer was implemented. The analysis of the variants was carried out by means of an in house pipeline: filtering out intronic, synonymous, and those variants with minor allele frequency (MAF) in the general population 〉1% (ExAC, 1000 Genomes-phase3, TOPmed), and requiring the presence both in tumour and germline DNA with a VAF〉37%. In 45 cases without germline material the last requirement was substituted by not being reported as somatic in COSMIC in any cancer. To determine pathogenicity we followed conservative criteria: a CADD Phred score ≥20 and to be considered deleterious in, at least, three out of six used algorithm. Results: In 118 patients, the median age at diagnosis was 47 years with the following WHO 2017 diagnoses: 12% MDS-SLD, 9% MDS-RS, 34% MDS-MLD, 30% MDS-EB, 3% MDS-del(5q), 1%MDS-U, 11% CMML. We found deleterious/pathogenic germ variants in 68 of 118 patients. Strikingly, we found a higher frequency than expected, for this specific subset, in genes not yet considered in the category of myeloid neoplasms with germline predisposition: MSH6 (n=5;4.2%), ATR (n=5;4.2%), ERCC6L2 (n=4;3.4%), PMS2 (n=2; 1.6%), MLH1 (n=3;3.5%), HCLS1 (n=2;1.7%), ITGB3 (n=2;1.7%), LYST (n=4; 3.4%), SAMD9 (n=1;0.8%), MSH2(n=1;0.8%). In genes already considered in the category of myeloid neoplasms with germline predisposition: MPL (n=2;1.7%), DDX41 (n= 2;1.7%), RUNX1 (n=1; 0.8%), ANKRD26 (n=1;0.8%). We also detected 10 cases with deleterious germline variants in genes related to Fanconi anemia (BRIP1, FANCC, FANCD2, FANCG, FANCM, SLX4, XRCC2, RAD51C and BRCA2), 2 cases with a germline variant in a Shwachman-Diamond gene DNAJC21 (1.7%), and 3 cases with germline variant in a telomere biology gene (RTEL1, CTC1 and TERT). We then focused on the characterization of the variants found in 5 genes not considered to date as predisposing to MDS: MSH6, MSH2, MLH1, ATR and PMS2 involved in the instability of microsatellites and whose alteration determines genomic instability and predisposition to a different number of solid tumors. The frequency of patients carrying these variants (13.5%) is much higher than the frequency of DDX41 (1.7%) or CEBPA (not found in our cohort), the two genes currently considered in the category with germline predisposition without a preexisting disorder or organ dysfunction. The sixteen patients carrying these mutations were characterized by a median age of 47 (16-60) years with the following diagnoses, 33% MDS-MLD, 27% MDS-EB1 and 40% CMML, presenting up to 40% with decrease cellularity in the bone marrow. Conclusions: We describe, for the first time, a high frequency of germinal variants in genes that drive genomic instability by modulating the microsatellite pathway, in young adults diagnosed with MDS without previous organ dysfunction. Their frequency and high pathogenicity index warrant functional validation experiments and pose them as potential candidates to be included in future classifications and to be considered in clinical, therapeutic and genetic counseling strategies. Disclosures Díez-Campelo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sanz:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman - La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen - Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.