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Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G

Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro. Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor. Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. Figure 1 Figure 1. Disclosures Jerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4+ and CD8+ T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4+ T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related. This work has been supported by the following grants: PI11/01621, PI15/00437, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; 2014/SGR585, Generalitat de Catalunya; Fundació La Caixa; H2020 No. 692298 project “MEDGENET, Medical Genomics and Epigenomics Network” by the EU; H2020 “AEGLE, An analytics framework for integrated and personalized healthcare services in Europe”, by the EU.

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 Next we transfected cultured primary cells from two c.7541_7542delCTNOTCH1mut and 5 NOTCH1wt patients. Figure 1b shows the cell viability assay after 48 h of the transfection, highlighting a marked reduction in viable cells in NOTCH1mut cases in each silencing experiment, (siRNA_Control: 77% NOTCH1sirna: 71 %, CCND2siRNA: 73% and CCND1: 72%) which was not paralleled in NOTCH1wt cases (siRNA_Control: 81% NOTCH1sirna: 80 %, CCND2siRNA: 84% and CCND1: 78 %). Figure 1C shows how the apoptosis index increased in every silencing experiment compared with NOTCH1mut siRNA pool control. However, for therapeutic purposes the accurate approach should increase apoptosis, leading the cells to a controlled death, and reduce necrosis, which is responsible of inflammation and collateral damage. The optimal apoptosis/necrosis ratio in our experiments, it is shown by the silencing of CCDN2 in NOTCH1mut (ratio A/N=5,15) where percentage of necrotic cells was markedly reduced (fig.1d). Conclusions: We found a marked upregulation of cyclins governing the transition from G1 to S phase of the cell cycle in patients with CLL and NOTCH1 mutation compared with those wild type cases for this lesion. In addition, silencing of cyclin D2 in the NOTCH1mut CLL subset was able to reach a similar degree of apoptosis than silencing NOTCH1, with a more favourable reduction of necrosis, emerging as a suitable candidate for a therapeutic target. Disclosures Jerez: Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION The use of new drugs in chronic lymphocytic leukemia (CLL) has allowed to revert the poor prognosis conferred by both the alterations of TP53 (deletion and mutation) and the absence of somatic hypermutation (HMS) of the heavy chain gene of immunoglobulins (IGHV). These alterations are part of the essential pre-treatment tests of the CLL according to the International Workshop on CLL Guidelines (iwCLL, Hallek et al, 2018). Three independent methodologies are required for these analyses, consequently time- and cost-consuming. Next-generation sequencing (NGS) could integrate all three types of studies into one. AIM To design a NGS panel for the detection of genetic alterations with diagnostic, prognostic and predictive value in CLL, as well as other alterations with clinical relevance. PATIENTS AND METHODS A capture panel (SureSelect QXT, Agilent) of 170Kb was designed covering the coding region of 17 genes, 11 regions of copy number alteration (CNAs), 2 translocations and the HMS-IGHV. A total of 83 samples of patients with CLL from three centers were sequenced. The effectiveness of the panel to detect already known alterations was evaluated in two groups of samples: 46 samples that presented any essential alteration of the iwCLL, including mutations of TP53, del (11q), +12, del (13q), del (17p), and the HMS status of the IGHV (group 1), and in 19 other samples with ≥1 different known cytogenetic / molecular alteration (group 2). Four healthy individuals were included as control. The samples were sequenced in a MiSeq (Illumina, average coverage of 600x, paired-end reading of 126bp). The analysis was carried out using a specific algorithm developed by Dreamgenics bioinformaticians, confirming the alterations with Integrative Genome Viewer (IGV). RESULTS The NGS panel allowed the identification of 214 mutations, 92 CNA regions and 83 IGHV genes. The concordance of results between the NGS panel and conventional methods was 100% (78/78) for mutations, 92% for CNAs (172/187) and 90% for use of IGHV (57/63). Considering the alterations of group 1, the agreement was 100% in mutations of TP53 (26/26), 100% of del (11q) (41/41), 98% +12 (41/42), 98% of del (17p) (42/43), and 79% of del (13q) (33/42). Two of the discrepancies in CNAs were cases identified by NGS but FISH negative [del (17p) and del13q)], probably because they are small deletions ( CONCLUSIONS This panel of NGS allows to detect> 93% of the essential genetic alterations of the iwCLL, and> 94% of the total genetic alterations analyzed. The results are preliminary but promising, although validation of the methodology is required. Disclosures No relevant conflicts of interest to declare.