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Patients with oligomonocytic chronic myelomonocytic leukemia (OM-CMML) are currently classified according to the 2017 World Health Organization myelodysplastic syndromes classification. However, recent data support considering OM-CMML as a specific subtype of chronic myelomonocytic leukemia (CMML), given their similar clinical, genomic, and immunophenotypic profiles. The main purpose of our study was to provide survival outcome data of a well-annotated series of 42 patients with OM-CMML and to compare them to 162 patients with CMML, 120 with dysplastic type (D-CMML), and 42 with proliferative type (P-CMML). OM-CMML had significantly longer overall survival (OS) and acute myeloid leukemia-free survival than did patients with CMML, considered as a whole group, and when compared with D-CMML and P-CMML. Moreover, gene mutations associated with increased proliferation (ie, ASXL1 and RAS-pathway mutations) were identified as independent adverse prognostic factors for OS in our series. We found that at a median follow-up of 53.47 months, 29.3% of our patients with OM-CMML progressed to D-CMML, and at a median follow-up of 46.03 months, 28.6% of our D-CMML group progressed to P-CMML. These data support the existence of an evolutionary continuum of OM-CMML, D-CMML, and P-CMML. In this context, we observed that harboring more than 3 mutated genes, carrying ASXL1 mutations, and a peripheral blood monocyte percentage >20% significantly predicted a shorter time of progression of OM-CMML into overt CMML. These variables were also detected as independent adverse prognostic factors for OS in OM-CMML. These data support the consideration of OM-CMML as the first evolutionary stage within the proliferative continuum of CMML., This study was supported in part by grants from ISCI II-FEDER FIS PI16/0153, FIS PI17/313, FIS PI19/0005, 2017SGR205, and 2017SGR437, and Xarxa de Banc de Tumors de Catalunya. more...

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults worldwide. Although genome-wide association studies (GWAS) have uncovered the germline genetic component underlying CLL susceptibility, the potential use of GWAS-identified risk variants to predict disease progression and patient survival remains unexplored. Here, we evaluated whether 41 GWAS-identified risk variants for CLL could influence overall survival (OS) and disease progression, defined as time to first treatment (TTFT) in a cohort of 1039 CLL cases ascertained through the CRuCIAL consortium. Although this is the largest study assessing the effect of GWAS-identified susceptibility variants for CLL on OS, we only found a weak association of ten single nucleotide polymorphisms (SNPs) with OS (p < 0.05) that did not remain significant after correction for multiple testing. In line with these results, polygenic risk scores (PRSs) built with these SNPs in the CRuCIAL cohort showed a modest association with OS and a low capacity to predict patient survival, with an area under the receiver operating characteristic curve (AUROC) of 0.57. Similarly, seven SNPs were associated with TTFT (p < 0.05); however, these did not reach the multiple testing significance threshold, and the meta-analysis with previous published data did not confirm any of the associations. As expected, PRSs built with these SNPs showed reduced accuracy in prediction of disease progression (AUROC = 0.62). These results suggest that susceptibility variants for CLL do not impact overall survival and disease progression in CLL patients, Horizon 2020 856620, Instituto de Salud Carlos III Spanish Government, Marie Curie Actions PI17/02256 PI20/01845, Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades y FEDER PY20/01282, United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) P50 CA97274 R01 CA92153 more...

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

Simple Summary Chromothripsis, a genomic event that generates massive chromosomal rearrangements, has been described in 1-3% of CLL patients and is associated with poor prognostic factors (e.g., TP53 abnormalities and genomic complexity). However, previous studies have not assessed its role in CLL patients with complex karyotypes. Herein, we aimed to describe the genetic characteristics of 33 CLL patients with high genomic complexity and chromothripsis. Moreover, we analyzed the clinical impact of chromothripsis, comparing these patients against a cohort of 129 patients with complex karyotypes not presenting this catastrophic event. Nine cases were also assessed via the novel cytogenomic methodology known as optical genome mapping. We confirmed that this phenomenon is heterogeneous and associated with a shorter time to first treatment. Nonetheless, our findings suggested that TP53 abnormalities, rather than chromothripsis itself, underlie the dismal outcome. Chromothripsis (cth) has been associated with a dismal outcome and poor prognosis factors in patients with chronic lymphocytic leukemia (CLL). Despite being correlated with high genome instability, previous studies have not assessed the role of cth in the context of genomic complexity. Herein, we analyzed a cohort of 33 CLL patients with cth and compared them against a cohort of 129 non-cth cases with complex karyotypes. Nine cth cases were analyzed using optical genome mapping (OGM). Patterns detected by genomic microarrays were compared and the prognostic value of cth was analyzed. Cth was distributed throughout the genome, with chromosomes 3, 6 and 13 being those most frequently affected. OGM detected 88.1% of the previously known copy number alterations and several additional cth-related rearrangements (median: 9, range: 3-26). Two patterns were identified: one with rearrangements clustered in the region with cth (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (6/9). Cases with cth showed a shorter time to first treatment (TTFT) than non-cth patients (median TTFT: 2 m vs. 15 m; p = 0.013). However, when stratifying patients based on TP53 status, cth did not affect TTFT. Only TP53 maintained its significance in the multivariate analysis for TTFT, including cth and genome complexity defined by genomic microarrays (HR: 1.60; p = 0.029). Our findings suggest that TP53 abnormalities, rather than cth itself, underlie the poor prognosis observed in this subset. more...

Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical–biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations ( more...

Chromosomal translocations in chronic lymphocytic leukemia (CLL) are very rare, and therefore systematic analysis of large series of cases is needed to allow the identification of recurrent rearrangements, breakpoints involved, and target genes. The aims of the present study were to identify new translocations and their clinical impact and to establish their frequency in a large cohort of 2843 CLL patients. By conventional cytogenetics 250 translocations were identified in 215 (7.5%) patients, 186 (74%) were apparently balanced and 64 (26%) were unbalanced. All chromosomes were involved in translocations, except Y chromosome. The chromosomes more frequently translocated were in decreasing frequency chromosomes 14, 18, 13, 17, 1, 6, 2, 3, 8, and 11. Translocations were found in the karyotypes either as the unique chromosomal abnormality (27%), associated with another alteration (24%), or as a part of a complex karyotype (CK, 48%). A large proportion of rearranged breakpoints involved genes related to CLL such as IGH (14q32), RB1, MIR15A, MIR16-1 (13q14), BCL2 (18q21), IGL (22q11.2), TP53 (17p13), IRF4 (6p25-p23), ATM (11q22), and CDK6 (7q21). Overall, 76 novel CLL translocations were identified, including a recurrent t(8;11)(p21;q21-23). Whole-genome sequencing and/or copy-number microarray data of 24 cases with translocations confirmed all rearrangements, enabled refinement of 3 karyotypes and all breakpoints at gene level. The projected survival and time to first treatment significantly decreased linearly with the number of translocations. In summary, this study allowed to establish the frequency of translocations (7.5%) and to identify new translocations in a cohort of 2843 CLL patients. more...

Dysregulated NK cell-mediated immune responses contribute to tumor evasion in chronic lymphocytic leukemia (CLL), although the NK cell compartment in CLL-like monoclonal B-cell lymphocytosis (MBL) is poorly understood. In healthy individuals, human cytomegalovirus (HCMV) induces the expansion of NK cells expressing high levels of CD94/NKG2C NK cell receptor (NKR) specific for HLA-E.We analyzed the expression of NKG2A, NKG2C, ILT2, KIR, CD161, and CD57 in 24 MBL and 37 CLL. NKG2C was genotyped in these patients and in 81 additional MBL/CLL, while NKG2C gene expression was assessed in 26 cases. In 8 CLL patients with increased lymphocytosis (≥20 × 10NKR distribution did not significantly differ between MBL and CLL patients, although they exhibited reduced NKG2CReduced percentages of CD94/NKG2C more...

Introduction: Dysregulated NK cell-mediated immune responses contribute to tumor evasion in chronic lymphocytic leukemia (CLL), although the NK cell compartment in CLL-like monoclonal B-cell lymphocytosis (MBL) is poorly understood. In healthy individuals, human cytomegalovirus (HCMV) induces the expansion of NK cells expressing high levels of CD94/NKG2C NK cell receptor (NKR) specific for HLA-E. Methods: We analyzed the expression of NKG2A, NKG2C, ILT2, KIR, CD161, and CD57 in 24 MBL and 37 CLL. NKG2C was genotyped in these patients and in 81 additional MBL/CLL, while NKG2C gene expression was assessed in 26 cases. In 8 CLL patients with increased lymphocytosis (≥20 × 109 /L), tumor HLA-E and HLA-G expression was evaluated. Results: NKR distribution did not significantly differ between MBL and CLL patients, although they exhibited reduced NKG2C+ NK cells compared with a non-CLL group (4.6% vs 12.2%, P = .012). HCMV+ patients showed increased percentages of NKG2C+ NK cells compared with HCMV- (7.3% vs 2.9%, P = .176). Frequencies of NKG2C deletions in MBL/CLL were similar to those of the general population. Low/undetectable NKG2C expression was found among NKG2C+/- (45%) and NKG2C+/+ (12%) patients. CLL cases with increased lymphocytosis displayed especially reduced NKG2C expression (1.8% vs 8.1%, P = .029) and tumor cells with high HLA-E (>98%) and variable HLA-G expression (12.4%, range: 0.5-56.4). CLL patients with low NKG2C expression ( more...

MYC rearrangements (MYC-R) confer unfavorable prognosis to large B-cell lymphomas (LBCL). Because of the low incidence of such genetic alteration, surrogates to screen MYC-R may be useful in daily practice. Previous studies suggested that clone 1A9-1 of LMO2 loss may be a good predictor for the presence of MYC-R in LBCL. The present study examines the utility of LMO2 clone SP51. For this purpose, we have analyzed 20 Burkitt lymphomas and 325 LBCL. Among them, 245 cases were studied prospectively using whole tissue sections, and 100 retrospectively by tissue microarrays. The cohort of CD10-positive prospective cases achieved the best results. Lack of LMO2 SP51 expression predicted the presence of MYC-R with high specificity, accuracy, positive and negative predictive value (PPV/NPV), and positive and negative likelihood ratios (PLR/NLR). Compared with MYC protein expression, LMO2 SP51 obtained significantly higher specificity, accuracy, PPV, and PLR (94%, 91%, 85%, and 14.33 vs 73%, 77%, 56%, and 3.26, respectively), and similar NPV and NLR (92% and 0.22 vs 95% and 0.12). Compared with LMO2 clone 1A9-1, the sensitivity of LMO2 SP51 was lower (79% vs 89%). We conclude that LMO2 SP51 may be a useful marker to screen MYC-R in CD10-positive LBCL. more...

Background. CLL-like monoclonal B-ceII lymphocytosis (MBL) is considered a requisite precursor of CLL, with 1-2% of subjects annually progressing to CLL requiring therapy. The role of immune alterations leading to and operating in MBL and controlling progression to CLL is not well characterized. Since an increased frequency of immune-related conditions associated with immune dysfunction exists prior to CLL diagnosis (Landgren et al, Br J Haematol 2007 and Blood 2007; Andersen et al, Leukemia 2020), immune alterations likely exist in MBL. We have examined the serum protein profiles of MBL and CLL in search of clues linking immune dysfunction with malignant transformation. Objectives. 1. Characterize the serum proteomic profiles of MBL individuals, IGHV-mutated CLL (M-CLL) and IGHV-unmutated CLL (U-CLL) patients, and age-matched healthy controls (HC). 2. Compare the serum proteomic profiles between the groups. 3. Assess the effect of IGHV mutation status on serum proteomic profiles in CLL. Methods. A total of 12 MBL, 12 M-CLL, 12 U-CLL and 12 age-matched HC were studied. Patients were cared for at Northwell Health, New York (n=25) and Hospital del Mar, Barcelona (n=11). All patients were treatment naive except for one U-CLL patient who was treated one year beforehand. Serum samples were collected, and their protein levels measured employing SOMAmers (modified single-stranded DNA aptamers; SomaLogic) to quantify relative levels of 1,310 proteins. P-values Results. Overall, the levels of 862 proteins differed significantly between groups: MBL vs. HC: 10 downregulated (d) and 24 upregulated (u); M-CLL vs. HC: 206d and 6u; U-CLL vs. HC: 54d and 40u; M-CLL vs. MBL: 384d and 5u; U-CLL vs. MBL: 74d and 24u; and M-CLL vs. U-CLL: 35d and 0u. IPA highlighted a role for the pro-inflammatory HMGB1 pathway in several comparisons. First, an activated HMGB1 pathway was predicted in MBL compared to HC, together with activation of phagocytes, and an inhibition of the systemic immune suppressor TGF-β. Second and consistent with the former, U-CLL patients displayed an activated HMGB1 pathway compared to HC, along with other signs of immune stimulation (activated NFkB and Th1 pathways, maturation of dendritic cells, and inhibition of TGF-β) and leukemic progression (activated progression of tumor, and leukocyte extravasation). Third and contrary to the above, the HMGB1 pathway was inhibited when comparing M-CLL to HC, in line with a global immune suppression signature (inhibited PRR, GM-CSF, FcεRI, IL1, IL8, TNF, Th1, STAT3 and NFkB pathways, in addition to inhibited cell movement, viability and activation). Notably, inhibition of immune pathways was predicted for both M-CLL and U-CLL compared to MBL (diminished TNF and IL6 signaling, and reduced cell movement), although the greatest differences were seen for M-CLL vs. MBL comparison, including blockade of the HMGB1 pathway in M-CLL. Finally, the M-CLL vs. U-CLL comparison suggested inhibited INFγ, IL2, IL3, IL4, NFkB, and decreased T lymphocyte stimulation and movement of tumor cells in M-CLL patients. Conclusions. An increased pro-inflammatory signature with involvement of the HMGB1 pathway was identified in MBL and U-CLL compared with HC, whereas the opposite was seen for M-CLL. Since MBL most often exhibit mutated IGHV (91% of cases in our cohort), these findings suggest immune stimulation as a characteristic feature in the pre-leukemic and U-CLL leukemic stage that surprisingly is not operative in M-CLL. Consistent with this, M-CLL displayed a global immune suppression (HMGB1 pathway inhibition), whereas U-CLL exhibited signs of immune stimulation (HMGB1 pathway activation) compared to HC which may relate to distinct capabilities of the two subtypes to interact with the microenvironment. Lastly, the increased inflammatory signature identified in MBL, which are mainly IGHV-mutated, was lessened in CLL, mainly in M-CLL patients. This is consistent with a decreased influence of immune imbalance and the HMGB1 pathway associated with IGHV-mutated clonal expansions. Acknowledgments. GB is supported by a grant from Fundación Alfonso Martín Escudero. Disclosures Allen: Alexon: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees. Rhodes: Conquer Cancer Foundation Young Investigator Award: Other: Grant/Research Support; AbbVie, Genentech, Pharmacyclics, TG Therapeutics: Other: Consultant. more...

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. more...

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation. more...

Oligomonocytic chronic myelomonocytic leukemia (OM-CMML) is defined as those myelodysplastic syndromes (MDSs) or myelodysplastic/myeloproliferative neoplasms, unclassifiable with relative monocytosis (≥10% monocytes) and a monocyte count of 0.5 to 94% classical monocytes (MO1s) and CD56 and/or CD2 positivity in peripheral blood monocytes, was similar to overt CMML. The MO1 percentage >94% method showed high accuracy for predicting CMML diagnosis (sensitivity, 90.7%; specificity, 92.2%), even when considering OM-CMML as a subtype of CMML (sensitivity, 84.9%; specificity, 92.1%) in our series of 233 patients (39 OM-CMML, 54 CMML, 23 MDS, and 15 myeloproliferative neoplasms with monocytosis and 102 reactive monocytosis). These results support the consideration of OM-CMML as a distinctive subtype of CMML. more...

Recent evidence suggests that complex karyotype (CK) defined by the presence of =3 chromosomal aberrations (structural and/or numerical) identified by chromosome banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges towards routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with =5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcome, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and or TP53 mutations, TP53abs) and the expression of somatically hypermutated (M-CLL) or unmutated (U-CLL) immunoglobulin heavy variable genes (IGHV). Thus, they contrasted CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs and IGHV gene somatic hypermutation status, we propose a novel hierarchical model where patients with high-CK exhibit the worst prognosis, while M-CLL lacking CK or TP53abs as well as CK with +12,+19 show the longest overall survival. In conclusion, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with =5 chromosomal aberrations emerges as prognostically adverse, independently of other biomarkers. Prospective clinical validation is warranted before finally incorporating high-CK in risk stratification of CLL. more...

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. more...

On behalf of Grupo Español de Leucemia Linfática Crónica (GELLC) and Grupo Cooperativo Español de Citogenética Hematológica (GCECGH)., We analyzed the features of chronic lymphocytic leukemia (CLL) with multiple abnormalities (MA) detected by FISH. A local database including 2095 CLL cases was used and 323 with MA (15.4%) were selected. MA was defined by the presence of two or more alterations (deletions of 13q14 (13q-), 11q22 (11q-), 17p13 (17p-) or trisomy 12 (+12)). The combination of 13q- with 11q- and 13q- with 17p-, accounted for 58.2% of the series, in contrast to 11q- with 17p- (3.7%). Patients carrying MA since diagnosis presented a short time to first therapy(TTFT) (27 months) and overall survival (OS) (76 months). The combinations including 17p- had a shorter OS (58 months) than the ones without 17p- (not reached, p =.002). Patients with a complex-FISH were the ones with worse OS (34 months). MA imply poor prognosis when they emerge at diagnosis, probably due to the high incidence of bad prognosis markers, which may be a reflection of a more complex karyotype. more...

The prognosis of chronic lymphocytic leukemia (CLL) patients displaying trisomy 12 (+12) remains unclear. In this study, we analyzed the influence of the proportion of cells with +12, and other clinical and biologic factors, in time to first therapy (TTFT) and overall survival (OS), in 289 patients diagnosed with CLL carrying +12. Median OS was 129 months. One hundred seventy-four patients (60.2%) presented +12 in more...

Deletion of 13q14 as the sole abnormality is a good prognostic marker in chronic lymphocytic leukemia (CLL). Nonetheless, the prognostic value of reciprocal 13q14 translocations [t(13q)] with related 13q losses has not been fully elucidated. We described clinical and biological characteristics of 25 CLL patients with t(13q), and compared with 62 patients carrying interstitial del(13q) by conventional G-banding cytogenetics (CGC) [i-del(13q)] and 295 patients with del(13q) only detected by fluorescence in situ hybridization (FISH) [F-del(13q)]. Besides from the CLL FISH panel (D13S319, CEP12, ATM, TP53), we studied RB1 deletions in all t(13q) cases and a representative group of i-del(13q) and F-del(13q). We analyzed NOTCH1, SF3B1, and MYD88 mutations in t(13q) cases by Sanger sequencing. In all, 25 distinct t(13q) were described. All these cases showed D13S319 deletion while 32% also lost RB1. The median percentage of 13q-deleted nuclei did not differ from i-del(13q) patients (73% vs. 64%), but both were significantly higher than F-del(13q) (52%, P more...

INTRODUCTION. Chromosome banding analysis (CBA) is the gold standard to identify complex karyotypes (CK; ≥3 chromosomal aberrations in the same clone). CK are predictors of poor prognosis and treatment refractoriness in patients with chronic lymphocytic leukemia (CLL). Patients with CK (15% at diagnosis) constitute a heterogeneous subgroup with highly variable clinical course. Recent studies that aim to refine CK definition in CLL suggest that ≥5 is the number of anomalies detected by CBA that better predicts an impaired outcome (Baliakas et al, 2019). Molecular techniques as genomic microarrays also detect genomic complexity (GC). A recent multicentric ERIC study (Leeksma et al, ASH 2017) identified that patients with ≥5 copy number alterations (CNA) detected by microarrays are associated with an adverse outcome. However, risk stratification regarding genomic complexity assessed by CBA and microarrays has not been compared. OBJECTIVES. 1. To compare genomic complexity in CLL defined by CBA vs microarrays; 2. To compare risk stratification based on genomic complexity measured by both techniques. METHODS. The study cohort included 293 CLL patients from 16 European institutions (67% males) with available CBA result at diagnosis or prior to first treatment. The cohort was enriched in patients with CK (n=153, 52%). Tumor DNA extracted from peripheral blood (n=254) or bone marrow samples (n=39) obtained at the time of CBA was hybridized to CGH-arrays (n=12) and SNP-arrays (n=281) platforms. Clinically relevant aberrations [11q-, +12, 13q-, 17p-] and CNA ≥5Mb were considered for the anomaly count. Three risk groups were defined using previously suggested cut-off points for CBA and microarrays [non-CK/low-GC: 0-2; low/intermediate-CK/GC: 3-4; high-CK/GC: ≥5 (Baliakas et al, Leeksma et al)]. Groups obtained by both methods were compared and correlated with other clinical and biological data. Time to first treatment (TTT) of patients categorized according to the number of alterations detected by CBA and microarrays was analyzed. RESULTS. Median number of abnormalities detected was 3 (range: 0-19) by CBA and 2 (range: 0-18) by microarrays. When stratified according to previously defined criteria, a moderate agreement was observed between both techniques (κ=0.483, p Regarding the prognostic value of genomic complexity and considering the number of abnormalities detected as a continuous variable, CBA and microarrays showed a similar concordance index (C-index) for TTT (0.615 vs 0.609, respectively). When considering all the abnormalities independently of their size or when lowering the cutoff to 1Mb for those non-CLL abnormalities, similar impact on TTT was observed (C-index=0.593 vs 0.616). The three risk groups defined by each method showed significant differences on TTT (Figure 1, p CONCLUSIONS. 1. CBA and microarrays are helpful techniques for assessing genomic complexity in CLL patients; 2. Risk categories established by both methods have a significant impact on TTT although they show a moderate agreement; 3. Discordant cases are being investigated to refine genomic complexity criteria equivalent by both techniques. ACKNOWLEDGEMENTS. 17SGR437, GLD17/00282, FPU17/00361 Disclosures Rigolin: AbbVie: Speakers Bureau; Gilead: Speakers Bureau; Gilead: Research Funding. Gimeno:JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Bosch:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: 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; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Cuneo:Amgen: Honoraria; Abbvie: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. more...

Background. Dysregulated NK-cell responses have been reported in chronic lymphocytic leukemia (CLL) patients, but little is known about the NK cell compartment in CLL-like monoclonal B cell lymphocytosis (MBL). Human cytomegalovirus (HCMV) infection induces an adaptive reconfiguration of the NK cell compartment characterized by the differentiation and persistent expansion of a subset displaying the CD94/NKG2C NK receptor (NKR). Moreover, a deletion of the NKG2C (KLRC2) gene has been reported to modulate the magnitude of the NK cell repertoire redistribution. Little is known about the expression of NKG2C in CLL and MBL patients. Aims. To analyse the distribution of NKR, with special attention to NKG2C, in MBL and CLL patients, assessing the relation of the NK cell immunophenotype with clinical features. Methods. The study cohort included 61 patients, 24 were diagnosed with clinical MBL and 37 were treatment-naïve CLL (32/37 Binet A). The expression of NKG2C, NKG2A, ILT2 (LIR1, LILRB1), CD161, CD57 and KIRs (identified with a cocktail of monoclonal antibodies) was assessed by flow cytometry in peripheral blood NK cells. The NKG2C (KLRC2) genotype was analysed in a larger representative MBL/CLL cohort (n=135). Results. The proportions of NK cells were reduced in CLL patients compared to MBL (median 5.5% vs. 10%; P=0.003), whereas their absolute numbers were increased (median 0.85x109/L vs. 0.57x109/L; P=0.002). No significant differences between MBL and CLL were detected regarding the distribution of the different NKR: NKG2C (median: 2.7 vs. 5.9%, respectively), NKG2A (31.4 vs. 30.8%), ILT2 (18.0 vs.15.8%), KIRs (54.4 vs. 52.7%), CD161 (16.1 vs. 16.4%) and CD57 (40.4 vs. 38.9%). Though a reduced NKG2C expression was noticed in both entities, it was specially marked in patients with a greater (>30x109 cells/L) lymphocytosis (1.4 vs. 7.7%, P=0.016). The proportions of NKG2C+ NK cells in HCMV+ patients (85%, 47/55) as compared to HCMV- individuals were not significantly different (6.3% vs. 2.9%, respectively). HCMV+ patients showed a significantly lower NKG2C expression when compared with two independent age-matched cohorts of HCMV+ non-CLL/-MBL individuals, including 43 non-metastatic breast cancer patients (4.2% vs. 15.3% , P Conclusions. 1. MBL and CLL exhibited low proportions of NKG2C+ NK cells. This immunophenotype was particularly evident in CLL patients with increased lymphocytosis and could not be explained by differences in HCMV seropositivity, NKG2C zygosity nor age. 2. Additional studies are required to define the mechanism(s) and putative implications of the reduced NKG2C expression in these lymphoproliferative disorders. Acknowledgements. PI11/1621; PI15/437; 2017/SGR437; Fundació La Caixa; Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Abrisqueta:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. Bosch:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding. more...

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. more...

INTRODUCTION The diagnosis of CMML according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establishing an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia is frequent but not always present and cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present ≥1 mutation in TET2, SRSF2 or ASXL1, the use of NGS panels is not widespread. The study of PB monocyte subsets by flow cytometry (FC) has gained interest for CMML diagnosis. The increase of classical monocytes (Mo1) upper 94% presents a high sensitivity (Sn) and specificity (Sp) for CMML diagnosis (Sn 90.6, Sp 95.1; Selimoglu-Buet et al, Blood 2015). The 94% threshold was validated in two studies (Talati C et al, Blood 2017; Tarfi S et al, Blood Cancer J 2018). However, some controversies have recently appeared in the literature. Picot T detected the 95% cutoff as the one with the best Sn (100%) and Sp (97%) (Picot T et al, Front Oncol 2018). Hudson CA found that the presence of < 1.13% (Sn 100, Sp 96) of non-classical monocytes (Mo3) was the best predictor for CMML diagnosis (Hudson CA et al, Am J Clin Pathol 2018). With the exception of the study of Tarfi S, based on 47 CMML, the rest presented a very low number of patients (Talati C: 29; Picot T: 15; Hudson CA: 16) and therefore a bias could be expected specially when studying the Sn of the proposed methods. Moreover, the different series assessing the "monocyte assay" have no molecular data and therefore this could diminish the accuracy of the results since some patients may have received misdiagnoses. The aim of our study was to assess the Sn and Sp of different thresholds of Mo1 and Mo3 in a large series with well-annotated clinical, cytogenetic and molecular data. Moreover, we assessed whether the study of CD2 and CD56 monocyte expression in combination with the %Mo1 >94 test improves the detection of the disease. METHODS 50 CMML, 12 MDS, 11 MPN with ≥1x109/L monocytes and 79 reactive monocytosis with ≥1x109/L monocytes (N = 152) were prospectively studied from 02/2016 to 07/2019. We studied PB monocyte subsets by FC: Mo1 (CD14bright/CD16-), Mo2 (CD14bright/CD16+) and Mo3 (CD14dim or -/CD16bright). In addition, we assessed the expression of CD56 and CD2 in monocytes (positivity ≥ 20%). Finally, targeted NGS of the entire exonic sequence of 25 genes recurrently mutated in myeloid malignancies was performed (VAF sensitivity: 2%). Chi-Square or Fisher exact tests were used as appropriate. ROC curves were developed to explore optimal cutoffs in terms of sensitivity (Sn) and specificity (Sp). Moreover, we plotted the AUC of the subset of Mo1 and Mo3. Finally, the Youden index (YI) was used to detect the threshold of Mo1 and Mo3 with the best balance between Sn and Sp. RESULTS AND DISCUSSION The Sn and Sp of the Mo1>94% test in our series were similar to those reported by the French group (GFM). Our Sn and Sp were 90% and 92% respectively with a YI of 82. The Sn and Sp of the Mo1>93% were 94% and 84% with a YI of 78. Finally, the 95% cutoff proposed by Picot T et al showed a Sn of 81% and a Sp of 96% with a YI of 77. Therefore, the 94% cutoff presented the best balance between Sn and SP of the different thresholds assessed. The Mo3 threshold of 1.13% proposed by Hudson CA et al showed a Sn of 67% and a Sp of 95% with a YI of 62. The best Mo3 cutoff in our series was established in 3.18% with a Sn of 90% and Sp of 83%. The YI of this threshold was 73. The AUC for the percentage (%) of Mo1 (0.937, IC 95%: 0.89-0.99) was better than the AUC of the % of Mo3 (0.924, IC 95%: 0.88-0.97) reinforcing the use of %Mo1 as the item with the best discriminative power for CMML diagnosis. The AUC of the percentage of Mo1 population was similar to that reported by the GFM (Figure 1). The Sn and Sp for CD56 expression in monocytes was 67% and 91% respectively, while CD2 expression showed a Sn of 38% and a Sp of 99%. Finally, the presence of at least one of the following: Mo1 >94%, CD56+ or CD2+ presented the highest Sn (98%) and a Sp of 84%. This method may be a very good screening test due to the low false negative rate expected. This combined approach showed the best balance between Sn and Sp (YI: 82). CONCLUSIONS Our study supports the utility of the Mo1 >94% test as the best flow cytometry assay for establishing accurate diagnoses in CMML. The combined assay of Mo1, CD56 and CD2 may be of high utility as a screening test. Figure 1 Disclosures Bellosillo: Qiagen: Consultancy, Speakers Bureau; TermoFisher Scientific: Consultancy, Speakers Bureau. more...

INTRODUCTION The 2017 WHO classification requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB) for the diagnosis of CMML. Recently, Geyer et al. defines oligomonocytic CMML (O-CMML) as those MDS cases with relative monocytosis (≥10% monocytes) and monocyte count 0.594% is a highly sensitive and specific diagnostic marker for CMML. In the extent of our knowledge, there are no data about PB monocyte subset distribution by FC in O-CMML. Moreover, CD2 and CD56 expression is common in CMML and rarely observed in MDS, the group where O-CMML are currently included. Furthermore, we compared: the molecular profile; cytogenetic abnormalities; cytopenias; BM dysplasia; BM blast and monocyte percentage; PB monocyte percentage, and monocyte and leukocyte counts. METHODS 50 CMML and 33 O-CMML from a single institution were prospectively studied from 02/2016 to date. Table 1 summarizes morphologic, cytogenetic, molecular and clinical findings. We studied PB monocyte subsets by FC: Mo1 (CD14bright/CD16-), Mo2 (CD14bright/CD16+) and Mo3 (CD14dim or -/CD16bright). In addition, we assessed the expression of CD56 and CD2 in monocytes (positivity ≥ 20%). Finally, targeted NGS of the entire exonic sequence of 25 genes recurrently mutated in myeloid malignancies was performed (VAF sensitivity: 2%). Chi-Square, Fisher exact or Man-Whitney U tests were used as appropriate. RESULTS AND DISCUSSION The Mo1 percentage (%) was significantly inferior in O-CMML (P=0.007), but it is noteworthy that median and mean of Mo1% in O-CMML were upper the cutoff of 94% (median: 96.1 vs 98.1; mean: 94.7 vs 96.9). Moreover, the % of patients with >94% Mo1 was no significantly different when comparing O-CMML and CMML although a clear trend was observed (72% vs 90%; P=0.082). This result is impressive since, as previously reported, the specificity of the Mo1 >94% test is around 90-95% and only 5-10% of false positive rate (FP) should be expected. However, in O-CMML a 72% of FP was observed since following 2017 WHO recommendation these patients should be considered as MDS. No differences were observed neither in the % of patients showing CD56+ monocytes (65.6% vs 66.7%; P=0.923) nor in the % of them showing CD2+ (28.1% vs 37.5%; P=0.53) when comparing O-CMML and CMML. We observed no significant differences in platelet count, hemoglobin, BM dyserythropoiesis, BM dysgranulopoiesis, BM dysmegacaryopoiesis, BM blast %, percentage of abnormal karyotypes, and Spanish cytogenetic risk stratification. The main differences were observed in leukocyte count, monocyte count, PB monocyte %, BM monocyte %, and BM promonocyte percentage. Table 1. There were no differences in the number of mutated genes or in the number of mutations between CMML and O-CMML (Table 1). As expected, TET2 and SRSF2 were the most frequently mutated genes in both groups. Moreover, no significant difference was observed in the presence of TET2/SRSF2 co-mutation, the gene signature of CMML (32% vs 26% in CMML). The genes mutated at a frequency >10% in O-CMML were: TET2 (79%), SRSF2 (36%), SF3B1 (29%), ZRSR2 (25%), DNMT3A (15%), and ASXL1 (14%). The genes mutated at a frequency >10% in CMML were: TET2 (81%), SRSF2 (28%), ASXL1 (23%), CBL (23%), SF3B1 (16%), and NRAS (14%). Only two genes were mutated at a significant different frequency: CBL (4% vs 23% in CMML, P=0.041) and ZRSR2 (25% vs 7% in CMML, P=0.043). As expected, CMML showed a higher % of RAS pathway mutations (CBL, NRAS or KRAS) since these have been associated with proliferative features (4% vs 40%, P=0.001). This is especially evident in proliferative CMML in which genes associated with proliferation are present at higher frequencies: CBL (4% vs 39% in CMML, P=0.01), NRAS (0 vs 23% in CMML, P=0.029) and ASXL1 (14% vs 62% in CMML, P=0.004). A significant lower percentage of O-CMML with ZRSR2mut presented Mo1 >94% (33% vs 86%, P=0.024). As shown, O-CMML without ZRSR2mut showed this feature in a similar percentage than CMML (86% vs 90%). At a median follow-up of 31.2 months, 19% of O-CMML evolved to CMML showing a median time to evolution of 34 months. CONCLUSION Our data support the diagnosis of O-CMML as a distinctive subtype of CMML. Table 1 Disclosures Bellosillo: Qiagen: Consultancy, Speakers Bureau; TermoFisher Scientific: Consultancy, Speakers Bureau. more...

Introduction: MYC rearrangements (MYCr) occur in 5 to 15% of diffuse large B-cell lymphomas (DLBCL) and 20 to 35% of high-grade B-cell lymphoma, NOS (HGBL-NOS), are a defining criterion of the category HGBL with rearrangements of MYC and/or BCL2/BCL6 (HGBL, with MYCr and BCL2/BCL6), and may be present in 90% of Burkitt lymphoma. The current WHO classification considers cytogenetic techniques as the appropriate tool to detect MYCr but does not define how to approach to the identification of such alteration. As the global incidence of MYCr in large B-cell lymphomas (LBCL) is low, it is necessary to clarity whether FISH or other cytogenetic methods have to be applied to all LBCL or only in selected cases. We previously identified LMO2 as a potential surrogate marker of MYCr in LBCL (Colomo L, Am J Surg Pathol 2017). Our aim with this study is to confirm this observation and evaluate the clinical impact of this marker in the survival of patients with LBCL. Methods: We have prospectively studied between September 2014 and July 2019 a new series of 180 LBCL including patients with DLBCL, HGBL, with HGBL, with MYCr and BCL2/BCL6, HGBL-NOS and transformed low-grade lymphomas into DLBCL (tDLBCL) diagnosed according to WHO criteria. LMO2 (clone 1A9-1), MYC (clone Y69) and a common immunohistochemistry (IHC) panel of B and T-cell markers have been used for the histological categorization of the cases, using whole tissue sections. The cutoff for LMO2 and MYC were 30% and 40%, respectively. MYC and BCL6 genes were studied using break apart probes, and BCL2 gene using dual-color dual-fusion probes (IGH/BCL2), all from Vysis-Abbott. We have statistically correlated the loss of expression of LMO2 and the overexpression of MYC with the presence or absence of MYCr. Moreover, we performed survival analyses assessing the clinical impact of LMO2 in a series of 162 LBCL patients (112 DLBCL, 20 HGBL, with MYCr and BCL2/BCL6, 4 HGBL-NOS and 26 tDLBCL). The survival series included cases diagnosed before 2014 with IHC and FISH data. Results: The prospective series included 132 patients with DLBCL (78M/52F; median age 67 years, range 35-95), 9 HGBL, with MYCr and BCL2/BCL6 (5M/4F; median age 67 years, range 42-85), 4 HGBL-NOS (2M/2F; median age 58 years, range 42-89), and 35 tDLBCL (31 transformed follicular lymphomas, 3 marginal zone lymphoma and 1 lymphoplasmacytic lymphoma; 23M/20F; median age 64 years, range 40-82). LMO2 and MYC were expressed as follows, respectively: 84/130 (65%) and 46/132 (35%) in DLBCL; 1/9 (11%) and 8/9 (89%) in HGBL, with MYCr and BCL2/BCL6; 0/4 and 3/4 (75%) HGBL-NOS; 25/34 (73%) and 7/33 (21%) tDLBCL. MYCr were identified in 9/132 (7%) DLBCL; all HGBL, with MYCr and BCL2/BCL6; 4/4 HGBL-NOS; 7/35 (20%) tDLBCL. The table shows the comparisons between LMO2 and MYC protein expression for the identification of the presence of MYCr in the series of LBCL. Whereas in the whole series LMO2 and MYC had similar results, among CD10-positive cases, LMO2 had better results than MYC and identified better the presence of MYCr than MYC protein expression. The 5-year progression-free survival (PFS) according the diagnostic categories was 59% for DLBCL, 28% for HGBL, with MYCr and BCL2/BCL6, 25% for HGBL-NOS and 22% for tDLBCL (P=0.015). In addition, PFS was significantly lower for the presence of MYCr (26% vs 53%, P=0.02) and MYC IHC expression (35% vs 53%, P=0.005), and showed a positive trend for LMO2 loss of expression (39% vs 52%, P=0.1). The 5-year overall survival (OS) according the diagnostic categories was 67% for DLBCL, 23% for HGBL, with MYCr and BCL2/BCL6, 50% for HGBL-NOS and 77% for tDLBCL (P Conclusions: LMO2 detection by IHC is a useful tool to detect MYCr in aggressive LBCL, particularly in CD10-positive cases. Moreover, LMO2 protein expression captures the prognostic significance of the different diagnostic histological categories and the presence of MYCr in this group of lymphomas. Disclosures Sanchez-Gonzalez: Alexion: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Amgen: 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; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Salar:Celgene: Consultancy; Gilead: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. more...

INTRODUCTION: Chronic lymphocytic leukemia (CLL)-like monoclonal B cell lymphocytosis (MBL) is considered a precursor of CLL. It is found in 5-10% of elderly healthy individuals and shows a progression rate to CLL requiring therapy of 1.1% per year. A balance between microenvironmental factors and intrinsic properties of the emerging B cell clone may be decisive for the transition from MBL to CLL, although biomarkers of progression remain unknown. The objective is to describe biological markers (B cell gene expression profiles and serum cytokine levels) that predict progression from MBL to CLL. METHODS: Gene expression profiles of clonal B cells from 14 MBL subjects (median age: 76 years, clonal B cells: 0.5-4.3 x109/L) were evaluated. With a median follow-up from analysis of 59 months (range: 10-77), 3 cases (21.4%) had progressed to CLL Binet stage A at last follow-up (clonal lymphocytosis >5x109/L, range: 6.2-7.9). Clonal B cells (CD19+CD5+) were isolated from peripheral blood by immunomagnetic methods (Miltenyi Biotec). Extracted RNA (RIN>7) was hybridized to GeneChip Human Gene 2.0 ST arrays (Affymetrix). Gene expression profiles were compared between MBL cases that progressed to CLL (P-MBL, n=3) and non-progressive MBL cases (NP-MBL, n=11). Differential gene expression was evaluated employing linear models for microarrays in R, and genes with P1.5 or 5x109/L, range: 6.4-17.3). Clonal B cells and cytokine levels were compared between P-MBL (n=5) and NP-MBL (N=36). For cytokine levels, the optimal cut-off values to stratify MBL cases according to their progression risk were assessed using the maxstat R package, whereas for clonal B cells a cut-off value of 3.9 x109/L was considered according to the results obtained by Kostopoulos et al (Blood Cancer J, 2017). The effect of different covariates on progression-free survival was evaluated using log-rank test. Cox proportional hazards regression models were performed to assess their independent prognostic value. P RESULTS: A total of 455 genes were differentially expressed (250 upregulated and 205 downregulated in P-MBL). IPA predicted an inhibition of apoptosis as well as proteins with tumor suppressor activity (SMARCA4) in P-MBL, besides enhanced bioenergetic processes (transmembrane potential of mitochondria) and anti-inflammatory features (activation of IL13 pathway and decreased chemotaxis of phagocytes and granulocytes) (Table 1). P-MBL displayed increased clonal B cells (4.2 vs. 1.7 x109/L, P=0.003) and levels of IL10 (1.15 vs. 0.9 pg/mL, P=0.087) as well as diminished levels of IL6 (2.04 vs. 3.75 pg/mL, P=0.041). MBL cases with ≥3.9 x109/L clonal B cells, ≥1.08 pg/mL of IL10 and ≤2.04 pg/mL of IL6 had an increased risk of progression to CLL (P CONCLUSIONS: 1. P-MBL cases showed an inhibition of the apoptotic pathway and an activation of bioenergetic processes, which may account for the increased clonal B cells observed in this group. 2. P-MBL exhibited enhanced anti-inflammatory features, including augmented levels of the anti-inflammatory cytokine IL10. 3. Increased clonal B cells and IL10 levels predicted a higher risk of progression to CLL, suggesting that an augmented proliferative rate of clonal B cells together with a supporting tumor microenvironment are required for progression from MBL to CLL. ACKNOWLEDGEMENTS. PI11/01621, PI15/00437, 2017/SGR437, Fundació La Caixa, Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Pharmacyctics: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: 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; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. more...

INTRODUCTION: CXCL9, CXCL10 and CXCL11 (CXCL9-10-11) are closely related cytokines that specifically bind to their receptor CXCR3. They act inducing chemotaxis, proliferation and/or cytotoxicity of CD4+ Th1 and cytotoxic T cells, which express CXCR3. Although the CXCL9-10-11/CXCR3 axis promotes immune activation, their pro- or anti-tumor effects in chronic lymphocytic leukemia (CLL) remain controversial. The aims of this study are: 1. To investigate serum levels of CXCL9-10-11 and the protein expression of their receptor CXCR3, as well as Th1 and cytotoxic gene expression signatures and protein expression of the cytotoxic molecules granzyme B and perforin in peripheral blood (PB) CD4+ T cells of controls, CLL-like monoclonal B-cell lymphocytosis (MBL) and CLL Binet stage A patients. 2. To assess the correlations between all previous parameters. 3. To evaluate Th1, cytotoxic and PD1+ T cell populations during disease progression. METHODS: Samples from 52 MBL subjects, 61 untreated CLL patients (Binet stage A/B [CLL-A/CLL-B]: 53/8) and 31 age-matched controls were employed. Serum levels (pg/mL) of CXCL9-10-11 were measured in 24 controls, 41 MBL and 44 CLL-A patients using Human CXCL9/MIG Quantikine ELISA Kit (R&D Systems) and U-PLEX Platform (Meso Scale Discovery). In addition, cryopreserved PB mononuclear cells from 8 controls, 11 MBL, 10 CLL-A and 8 CLL-B were studied by flow cytometry. Anti-CD3, anti-CD4, anti-granzyme B, anti-perforin, anti-CXCR3 and anti-PD1 antibodies, FVS510 and Fixation/Permeabilization Kit were used for cell staining (BD Biosciences). Protein expression of CXCR3, granzyme B, perforin and PD1 (measured as percentage of positive cells in PB CD4+ T cells) was assessed using FACSCanto II cytometer (BD Biosciences). In addition, purified CD4+ cells from PB (purity≥90%) were isolated by immunomagnetic methods (Miltenyi Biotec) to analyze gene expression in 9 controls, 13 MBL and 14 CLL-A patients. Extracted RNA (RIN>7) was hybridized to GeneChip Human Gene 2.0 ST arrays (Affymetrix). Differential gene expression was evaluated with linear models in R, and genes with P-value1.5 were considered differentially expressed. Linear regression and Pearson correlations were calculated to evaluate the relationship between the different components of the CXCL9-10-11/CXCR3 axis (Figure 1). P-values RESULTS: MBL subjects showed significantly increased CXCL9-10-11 serum levels as well as CXCR3 protein expression, Th1 and cytotoxic gene expression and perforin protein expression in CD4+ T cells compared to controls. A similar trend was obtained for CLL-A versus controls, although the differences for CXCL10 and cytotoxic proteins did not achieve statistical significance despite their increased levels (Table 1). CXCL9 and CXCL10 serum levels were significantly and strongly correlated with CXCR3 protein expression in CD4+ T cells (r=0.746, P-value=0.001 and r=0.716, P-value=0.002, respectively). Very strong positive and significant correlations were also detected between CXCR3 protein expression and Th1 gene expression in CD4+ T cells (correlation coefficients >0.8 for 6/7 genes). Significant positive correlations were also observed between CXCR3 protein expression and cytotoxic genes as well as granzyme B protein (Table 2). Protein expression of CXCR3 and cytotoxic molecules were similarly increased in the different stages of the disease. However, CLL-B patients displayed an increased percentage of CD4+ T cells expressing PD1 (around 7% in MBL and CLL-A versus 16% in CLL-B), although significance was not achieved (Table 1). CONCLUSIONS: 1. The increased levels of the different components of the CXCL9-10-11/CXCR3 axis in MBL and CLL-A, together with the strong correlations observed, point to an important activation of this molecular pathway in the first stages of the disease. 2. Correlations between CXCR3 and Th1/cytotoxic genes/proteins suggest that the increased Th1/cytotoxic features of CD4+ T cells in MBL and CLL-A are triggered by CXCL9-10-11/CXCR3 stimulation, and might be considered as a potential target for CLL immunotherapy. 3. The lower percentage of PD1+ CD4+ T cells in MBL/CLL-A may allow efficient effector Th1/cytotoxic responses at these stages of the disease. ACKNOWLEDGEMENTS. PI11/01621, PI15/00437, 2017/SGR437, Fundació La Caixa, Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: Abbvie: Speakers Bureau; JANSSEN: Consultancy, Speakers Bureau. Rai:Genentech/Roche: Membership on an entity's Board of Directors or advisory committees; Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. Bosch:Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. more...

Recurrent cytogenetic abnormalities in chronic lym- phocytic leukemia (CLL), namely deletions of chromo- somes 11q, 13q, 17p and trisomy 12 (+12), define sub- groups of patients with different clinical behavior and response to treatment. 1 We and others previously report- ed a minor proportion of CLL cases with co-existing tri- somies of chromosomes 12 and 19 who share specific clinico-biological characteristics. 2-4 However, since the cohort was small, no definitive conclusions could be drawn. Here, we analyzed a large, multi-institutional series. We confirm and significantly extend previous observations through the identification of subgroups of +12 CLL cases harboring particular concurrent trisomies demonstrating distinctive clinico-biological profiles. We analyzed an unselected cohort of 4486 CLL patients with available classic cytogenetic (n=4285) or high-density 250K single nucleotide polymorphism (SNP)-array (n=201) data. We identified 712 cases (16% of the cohort) carrying +12. 5 Median time from diagnosis to cytogenet- ic/SNP analysis was 1.5 months (range 0-194); the major- ity of cases included in survival analysis were untreated prior to testing (94%). The study was approved by the local Ethics Review Committees. Details of the study cohort and the methodologies used are provided in the Online Supplementary Appendix. more...

Summary Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p-) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p- CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p- CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27–98) years at the time of fluorescence in situ hybridization analysis. After 17p- documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta2-microglobulin concentration (P more...

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. more...

INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Presence of dysplasia is frequent but not always present. Recently, Geyer et al. described olygomonocytic CMML (O-CMML) as those MDS cases with relative monocytosis (≥10% monocytes) and monocyte count 0.5-94% of total monocytes is a highly sensitive and specific diagnostic marker for CMML. We assessed peripheral monocyte subsets by FC in 11 O-CMML cases and compared those with 20 CMML cases, 14 D-CMML and 6 proliferative CMML (P-CMML). In addition, we studied the aberrant expression of CD56, CD2 and CD7 in monocytes. As mentioned, O-CMML may be considered an early-phase of D-CMML and some D-CMML may progress to P-CMML, an entity with an ominous prognosis. We compared the percentage of Mo1 and non-classical monocytes (Mo3) among O-CMML, D-CMML and P-CMML in order to evaluate if a progressive accumulation of Mo1 and a progressive decrease in Mo3 could be appreciated among these entities. In our view, Mo1 could be considered as a marker of tumor burden, while Mo3, formerly considered as a specific type of dendritic cell, could be related with immunosurveillance. In order to reinforce this hypothesis we evaluated if the reduction in Mo3 would be also accompanied by a decrease in plasmocytoid dendritic cells (pDC). METHODS Twenty CMML and 11 O-CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. We performed FC study of monocyte subsets in PB describing Mo1 (CD14bright/CD16-), intermediate monocytes (Mo2) (CD14bright/CD16+) and Mo3 (CD14dim or -/CD16bright). In addition, we assessed the expression of CD56, CD2 and CD7 in monocyte population and quantified pDC (CD123bright/HLA-DR+). Comparisons were evaluated by Chi-Square test, Man-Whitney U-test or by Kruskall-Wallis test as appropriate. RESULTS & DISCUSSION 1) 6/11 (55%) O-CMML showed an increase in the fraction of Mo1>94% of total monocytes. In contrast, 12/14 (86%) D-CMML and 6/6 (100%) P-CMML showed a percentage of Mo1>94% of total monocytes. Considering together all overt CMML, 18/20 (90%) presented Mo1>94% of total monocytes. This result was almost identical to that reported in the original study by Selimoglu-Buet. 2) The median percentage of Mo1 and Mo3 monocytes was statistically different among these three entities (Mo1, p=0,005; Mo3, p=0,002). Table 2. Interestingly, the median percentage of Mo1 (% Mo1) was significantly lower in O-CMML when compared to P-CMML (p=0,004) and a clear trend was observed when compared to D-CMML. In the same way, % Mo1 was significantly lower in D-CMML when compared to P-CMML (p=0,017). Moreover, the median percentage of Mo3 (% Mo3) was significantly higher in O-CMML when compared to P-CMML (p=0,002) and a clear trend was observed when compared to D-CMML. In the same line, % Mo3 was significantly higher in D-CMML when compared to P-CMML (p=0,002). Likewise, the median percentage of pDC (% pDC) was significantly higher in O-CMML when compared to P-CMML (p=0,004). A clear trend was observed when O-CMML was compared with D-CMML, and D-CMML with P-CMML. These data reinforce the hypothesis that progression from O-CMML to D-CMML and P-CMML could be guided by a progressive accumulation of Mo1 ("tumor burden increase") and by a progressive reduction of Mo3 and pDC ("immunosurveillance decrease"). 3) Expression of CD56, CD2 and CD7 in monocytes is depicted in Table 3. No aberrant expression of CD7 was observed in any case. In contrast, CD56 expression was observed in 9/11 O-CMML, 7/14 D-CMML and 5/6 P-CMML. Considering together all overt CMML, 12/20 expressed CD56. CD56 expression in monocytes is a common finding in CMML and has been rarely described in other myeloid disorders. This may be interpreted as another indicator that O-CMML is in the continuum of CMML. CONCLUSIONS O-CMML presents flow cytometric immunophenotypic characteristics in line with overt CMML. These data support that O-CMML is in the biological continuum of overt CMML. Disclosures No relevant conflicts of interest to declare. more...

INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establish an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia, mainly dysgranulopoiesis, is frequent but not always present in CMML. Cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present at least one mutation in TET2, SRSF2 or ASXL1 genes, the use of NGS panels is not widespread. Furthermore, mutations in these genes are among the most frequently observed in age-related clonal hematopoiesis. Therefore, complementary techniques are required to support the diagnosis of this entity. The study of the peripheral monocyte subsets by flow cytometry (FC) has gained special interest due to a high sensitivity and specificity for the diagnosis of CMML (S = 90.6%, E = 95.1%, Selimoglu-Buet et al., Blood, 2015). An increase in the fraction of classical monocytes (Mo1) to >94% of total monocytes is an event frequently observed in CMML. There are no specific bone marrow (BM) FC panels for the diagnosis of CMML and very few have been validated for the diagnosis of MDS. "Ogata score", the only multicenter validated score in MDS, has not been applied in CMML. The aim of our study was to evaluate the usefulness of FC in PB and BM for the diagnosis of CMML. METHODS Twenty-two CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. Diagnostic procedure consisted of morphological, cytochemical (Perls, myeloperoxidase, nonspecific esterase), cytogenetic and FC studies in BM, and morphological and FC studies in PB. "Ogata Score" was applied in BM samples (Table 2). Aberrant coexpression of CD2, CD7 and CD56 in BM monocytes was assessed. Immunophenotypic maturation profile of the monocytic elements in BM distinguishes: promonocytes (CD34-/CD117-/CD64++/CD14- or dim/CD45+/HLA-DR+++), mature monocytes (CD34-/CD117-/CD64++/CD14++/CD45++/HLA-DR++) and mature monocytes in terminal stage (CD300e+). In PB, the monocytes FC subsets (Mo1, Mo2 and Mo3) were studied, as well as the aberrant coexpression of CD2, CD7 and CD56 (Table 3). RESULTSThe presence of ≥2 aberrations in Ogata Score predicted properly the diagnosis of CMML in all patients analyzed (100% sensitivity). Due to the study design, we could not obtain results about specificity.An increase in Mo1 (classical monocytes) > 94% was detected in 18/20 patients (Table 3). This method predicted the diagnosis of CMML with a sensitivity of 91%, a result almost identical to the original study (Selimoglu-Buet et al., Blood, 2015).A good positive correlation was established between the percentage of BM promonocytes detected by morphology and by FC (Rho Spearman 0.61, P = 0.003).A negative correlation was found between the percentage of promonocytes by FC in MO and the expression of CD56 (Rho Spearman -0.612, P = 0.002). Similarly, CD56+ CMML presented a percentage of promonocytes by FC significantly lower than the CD56- CMML group (median: 24.5% (14-40) vs. 41% (23-71), P = 0.005). The expression of CD56 seems to be related to a more mature immunophenotypic profile of the monocytic population. On the other hand, the correlation between the percentage of CD56+ monocytes in BM and PB was almost perfect (Rho Spearman 0.928, P CONCLUSION Our findings support the usefulness of flow cytometry in bone marrow and peripheral blood for the diagnosis of CMML. Disclosures No relevant conflicts of interest to declare. more...