Features Of Chronic Lymphocytic Leukemia Research Articles

Linking aberrant chromatin features in chronic lymphocytic leukemia to transcription factor networks

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF. CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B‐cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease.

Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

One of the cardinal features of chronic lymphocytic leukemia (CLL) is its association with a profound immunosuppression. NK cell function is markedly impaired in CLL patients, who show a significant dysregulation of the expression of activating and inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2, also termed LIR-1, LILRB1) in the regulation of NK cells in CLL. Our results show that ILT2 expression was significantly decreased on leukemic cells and increased on NK cells of CLL patients, particularly in those with advanced disease and with bad prognostic features, such as those carrying chromosome del(11q). The immunomodulatory drug lenalidomide may regulate the expression of ILT2 and its ligands in CLL since it significantly increased the expression of ILT2 and partially reestablished the expression of its ligands on leukemic cells. Furthermore, lenalidomide significantly increased the activation and proliferation of NK cells, which was strongly enhanced by ILT2 blockade. Combining ILT2 blockade and lenalidomide activated NK cell cytotoxicity resulting in increased elimination of leukemic cells from CLL patients. Overall, we describe herein the role of an inhibitory receptor involved in the suppression of NK cell activity in CLL, which is restored by ILT2 blockade in combination with lenalidomide, suggesting that it may be an interesting therapeutic strategy to be explored in this disease.

Splicing modulation sensitizes chronic lymphocytic leukemia cells to venetoclax by remodeling mitochondrial apoptotic dependencies.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eμ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.

Characterizing the kinetics of lymphocytosis in patients with chronic lymphocytic leukemia treated with single-agent ibrutinib

Increased absolute lymphocyte count (ALC) is a key feature of chronic lymphocytic leukemia (CLL) but is also observed during treatment with B-cell receptor pathway inhibitors including ibrutinib, a first-in-class inhibitor of Bruton’s tyrosine kinase. In patients with CLL treated with single-agent ibrutinib in two multicenter, open-label, randomized, phase 3 studies (RESONATE-2, NCT01722487; RESONATE, NCT01578707), lymphocytosis was observed in 77 of 136 (57%) patients treated in first-line and 133 of 195 (69%) relapsed/refractory patients. On treatment, lymphocytosis resolved in 95% of patients in the first-line and 94% in the relapsed/refractory setting. The median duration of lymphocytosis was 12 and 14 weeks in the first-line and relapsed/refractory settings, respectively. Lymphocytosis is a common and predictable pharmacodynamic effect of ibrutinib treatment, and in the absence of other signs of progression, does not represent disease progression. Lymphocytosis resolves in the majority of patients and does not require interruption or discontinuation of ibrutinib therapy.

Morphological, immunophenotypic, and genetic features of chronic lymphocytic leukemia with trisomy 12: a comprehensive review.

Chronic lymphocytic leukemia is an extremely heterogeneous disease and prognostic factors such as chromosomal abnormalities are important predictors of time to first treatment and survival. Trisomy 12 is the second most frequent aberration detected by fluorescence in situ hybridization at the time of diagnosis (10–25%), and it confers an intermediate prognostic risk, with a median time to first treatment of 33 months and a median overall survival of 114 months. Here, we review the unique morphological, immunophenotypic, and genetic characteristics of patients with chronic lymphocytic leukemia and trisomy 12. These patients carry a significantly higher expression of CD19, CD22, CD20, CD79b, CD24, CD27, CD38, CD49d, sIgM, sIgk, and sIgλ and lower expression of CD43 compared with patients with normal karyotype. Circulating cells show increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and of the adhesion molecule CD323. Patients with chronic lymphocytic leukemia and trisomy 12 frequently have unmutated IGHV, ZAP-70 positivity, and closely homologous stereotyped B-cell receptors. They rarely show TP53 mutations but frequently have NOTCH1 mutations, which can be identified in up to 40% of those with a rapidly progressive clinical course.

Transcriptional mechanism of vascular endothelial growth factor-induced expression of protein kinase C\u03b2II in chronic lymphocytic leukaemia cells

A key feature of chronic lymphocytic leukaemia (CLL) cells is overexpressed protein kinase CβII (PKCβII), an S/T kinase important in the pathogenesis of this and other B cell malignancies. The mechanisms contributing to enhanced transcription of the gene coding for PKCβII, PRKCB, in CLL cells remain poorly described, but could be important because of potential insight into how the phenotype of these cells is regulated. Here, we show that SP1 is the major driver of PKCβII expression in CLL cells where enhanced association of this transcription factor with the PRKCB promoter is likely because of the presence of histone marks permissive of gene activation. We also show how vascular endothelial growth factor (VEGF) regulates PRKCB promoter function in CLL cells, stimulating PKCβ gene transcription via increased association of SP1 and decreased association of STAT3. Taken together, these results are the first to demonstrate a clear role for SP1 in the up regulation of PKCβII expression in CLL cells, and the first to link SP1 with the pathogenesis of this and potentially other B cell malignancies where PKCβII is overexpressed.

The Association of Dyslipidemia With Chronic Lymphocytic Leukemia: A Population-Based Study.

Metabolic syndrome (MetS) is a risk factor for development of cancer. Because aberrant lipid metabolism is a pathogenic feature of chronic lymphocytic leukemia (CLL), our objective was to determine if CLL patients have a higher prevalence of MetS preceding diagnosis and to determine the impact of lipid-lowering medications on survival. We conducted a population-based case-control study in Ontario, Canada, using administrative databases of adults age 66 years and older to compare the prevalence of MetS preceding CLL with age- and sex-matched control subjects. Logistic regression was used to study the association between MetS and its components to CLL. The Kaplan-Meier method and Cox Regression were used to investigate survival. All statistical tests were two-sided. We identified 2124 persons with CLL and 7935 control subjects from January 1, 2000, to December 31, 2005, with follow-up until March 31, 2014, three years from the date of last contact with the health care system, or death. The mean age was 75.6 years, 20.2% had diabetes, 35.8% had hypertension, and 17.6% had dyslipidemia. In multivariable analysis, dyslipidemia (odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.11 to 1.44, P < .001) and hypertension (OR = 1.12, 95% CI = 1.01 to 1.25, P = .03) were associated with the development of CLL, whereas MetS and diabetes were not. Lipid-lowering medication was associated with a statistically significant improved survival in patients with CLL (HR = 0.53, 95% CI = 0.47 to 0.61, P < .001). We demonstrate a higher prevalence of dyslipidemia preceding a diagnosis of CLL compared with control subjects, supporting preclinical data. Lipid-lowering medications appear to confer a survival advantage in CLL. Prospective studies are needed to confirm these results and test their potential as therapeutic applications.

Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy

AbstractGenetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS. Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment.

Characteristics of chronic lymphocytic leukemia in Senegal.

BackgroundChronic lymphocytic leukemia (CLL) is a mature B-cell neoplasm characterized by the expansion of CD5-positive lymphocytes in peripheral blood. While CLL is the most common type of leukemia in Western populations, the disease is rare in Africans. Hence, clinical and laboratory data and studies of CLL in Sub Saharan populations have been limited. The aims of this study were to analyze the characteristics of senegalese patients with CLL at the time of the diagnosis and to identify the correlation between clinical characteristics (Binet stage) with age, gender, laboratory parameters and chromosomal abnormalities.MethodsIn this study, we investigated the clinical and laboratory characteristics of CLL in Senegal. A total of 40 patients who had been diagnosed with CLL during the period from July 2011 to April 2015 in Senegal were evaluated. Cytology and immunophenotype were performed in all patients to confirm the diagnosis. The prognosis factors such as Binet staging, CD38 and cytogenetic abnormalities were studied. The statistical analysis was performed using STATA version 13 (Stata college station Texas). Each patient signed a free and informed consent form before participating in the study.ResultsThe mean age was 61 years ranged from 48 to 85. There were 31 males and only 9 females (sex ratio M : F = 3,44). At diagnosic, 82.5 % of the patients were classified as having advanced Binet stages B or C. The prognosis marker CD38 was positive in 28 patients. Cytogenetic abnormalities studied by FISH were performed in 25 patients, among them, 68 % (17 cases) had at least one cytogenetic abnormality and 28 % had 2 simultaneous cytogenetic abnormalities.ConclusionAfricans may present with CLL at a younger age and our data suggest that CLL in Senegal may be more aggressive than in Western populations.

Reactivation of Smac-mediated apoptosis in chronic lymphocytic leukemia cells: mechanistic studies of Smac mimetic.

Dysfunctional apoptotic machinery is a hallmark feature of chronic lymphocytic leukemia (CLL). Accordingly, targeting apoptosis regulators has been proven a rational approach for CLL treatment. We show that CLL lymphocytes express high levels of XIAP, cIAP1, and cIAP2 compared to normal lymphocytes. Smac mimetic, Smac066, designed to bind to BIR3-domain of IAPs, induce apoptosis in primary CLL cells (n=71; p<0.0001), irrespective of prognostic markers. Apoptosis was mediated by diminished levels of IAPs (XIAP-p=0.02; cIAP-p<0.0001) and increased activation of caspases-8, −9, −3. The caspase-cleavage was in direct association with the levels of apoptosis (r2=0.8 for caspases-8, −9, −3). Correlative analysis revealed a direct relationship between reduction in IAPs and degree of apoptosis (r2=0.6 (XIAP); 0.5 (cIAP2)). There was a strong association between apoptosis, IAP-degradation, and concurrent caspase-activation. Pan-caspase inhibitor Z-Vad-fmk reversed the degradation of Mcl-1, but not IAPs suggesting that smac066 is selective to IAPs, however, Mcl-1 degradation is through caspase-mediated cleavage. Immunoprecipitation experiments revealed physical interaction between caspase-3 and XIAP that was disrupted by smac066. Importantly, XIAP and cIAP2 were markedly induced in bone-marrow and lymph-node microenvironments, providing a basis for IAP antagonists as anti-tumor agents in CLL. Smac066 synergized with ABT-737, revealing a mechanistic rationale to jointly target BH3 and BIR3 domains.

Clinical characteristics of chronic lymphocytic leukemia occurring in chornobyl cleanup workers.

The recently demonstrated radiation-induction of chronic lymphocytic leukemia (CLL) raises the question as to whether the amount of radiation exposure influences any of the clinical characteristics of the disease. We evaluated the relationship between bone marrow radiation doses and clinical characteristics and survival of 79 CLL cases diagnosed during 1986-2006 in a cohort of 110 645 male workers who participated in the cleanup work of the Chornobyl nuclear accident in Ukraine in 1986. All diagnoses were confirmed by an independent International Hematology Panel. Patients were followed up to the date of death or end of follow-up on 31 October 2010. The median age at diagnosis was 57 years. Median bone marrow dose was 22.6 milligray (mGy) and was not associated with time between exposure and clinical diagnosis of CLL (latent period), age, peripheral blood lymphocyte count or clinical stage of disease in univariate and multivariate analyses. Latent period was significantly shorter among those older at first exposure, smokers and those with higher frequency of visits to the doctor prior to diagnosis. A significant increase in the risk of death with increasing radiation dose was observed (p = 0.03, hazard ratio = 2.38, 95% confidence interval: 1.11,5.08 comparing those with doses ≥22 mGy to doses <22 mGy). After adjustment for radiation dose, survival of CLL cases was significantly shorter among those with younger age at first exposure, higher peripheral blood lymphocyte count, more advanced clinical stage of disease and older age at diagnosis (all p < 0.05). This is the first study to examine association between bone marrow radiation doses from the Chornobyl accident and clinical manifestations of the CLL in Chornobyl cleanup workers. The current study provides new evidence on the association of radiation dose and younger age at first radiation exposure at Chornobyl with shorter survival after diagnosis. Future studies are necessary with more cases in order to improve the statistical power of these analyses and to determine their significance. Copyright © 2016 John Wiley & Sons, Ltd.

Perturbation of the normal immune system in patients with CLL

AbstractImmune dysregulation is a cardinal feature of chronic lymphocytic leukemia (CLL) from its early stage and worsens during clinical observation, even in absence of disease progression. Although the mechanisms remain unclear, new insights are emerging into the complex relationship between the CLL clone and its immune environment. T cells are increased in early-stage disease and show progressive accumulation and exhaustion. The mechanisms that drive this expansion may include auto-antigens involved in the original clonal expansion. In addition, chronic viral infections such as cytomegalovirus generate huge virus-specific immune responses, which are further expanded in CLL. Attention is now focused largely on the direct immunosuppressive properties of the tumor. Remarkably, CLL clones often have features of the recently described regulatory B cells producing immunosuppressive IL-10. Better knowledge of the regulatory properties intrinsic to CLL cells may soon become more important with the switch from chemotherapy-based treatments, which trade control of CLL with further impairment of immune function, to the new agents targeting CLL B-cell receptor–associated signaling. Treatment with these new agents is associated with evidence of immune recovery and reduced infectious complications. As such, they offer the prospect of immunologic rehabilitation and a platform from which to ultimately replace chemotherapy.

Strategies targeting apoptosis proteins to improve therapy of chronic lymphocytic leukemia

A typical feature of chronic lymphocytic leukemia (CLL) is the impaired ability of the leukemic cells to execute their apoptotic suicide program. Various strategies have been developed to restore apoptosis in CLL cells ex vivo. This article reviews the strategies targeting proteins that directly regulate the mitochondrial pathway of apoptosis and caspase activation: (i) inhibiting the expression or activity of prosurvival proteins of the Bcl-2 and IAP (inhibitor of apoptosis protein) families, which are overexpressed in CLL cells and (ii) upregulating proapoptotic BH3-only members of the Bcl-2 family (which are antagonists of the prosurvival members). Preclinical and clinical data have revealed that inhibiting the activity of prosurvival Bcl-2 proteins with BH3 mimetics (so-called because they mimic BH3-only proteins) is an attractive strategy for CLL therapy. Recent results suggest that the development of BH3 mimetics capable of directly activating the apoptosis effectors Bax and Bak may also be envisaged.

MDM2 promotor polymorphism and disease characteristics in chronic lymphocytic leukemia: results of an individual patient data-based meta-analysis.

A number of single nucleotide polymorphisms have been associated with disease predisposition in chronic lymphocytic leukemia. A single nucleotide polymorphism in the MDM2 promotor region, MDM2SNP309, was shown to soothe the p53 pathway. In the current study, we aimed to clarify the effect of the MDM2SNP309 on chronic lymphocytic leukemia characteristics and outcome. We performed a meta-analysis of data from 2598 individual patients from 10 different cohorts. Patients' data and genetic analysis for MDM2SNP309 genotype, immunoglobulin heavy chain variable region mutation status and fluorescence in situ hybridization results were collected. There were no differences in overall survival based on the polymorphism (log rank test, stratified by study cohort; P=0.76; GG genotype: cohort-adjusted median overall survival of 151 months; TG: 153 months; TT: 149 months). In a multivariable Cox proportional hazards regression analysis, advanced age, male sex and unmutated immunoglobulin heavy chain variable region genes were associated with inferior survival, but not the MDM2 genotype. The MDM2SNP309 is unlikely to influence disease characteristics and prognosis in chronic lymphocytic leukemia. Studies investigating the impact of individual single nucleotide polymorphisms on prognosis are often controversial. This may be due to selection bias and small sample size. A meta-analysis based on individual patient data provides a reasonable strategy for prognostic factor analyses in the case of small individual studies. Individual patient data-based meta-analysis can, therefore, be a powerful tool to assess genetic risk factors in the absence of large studies.

Poly(ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro

DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL.

Differential Distribution Of Recurrent Gene Mutations In Subsets Of Chronic Lymphocytic Leukemia Patients With Stereotyped B-Cell Receptors: Results From A Multicenter Project Of The European Research Initiative On CLL In A Series Of 2482 Cases

▪A remarkable feature of chronic lymphocytic leukemia (CLL) is the existence of quasi-identical, stereotyped B-cell receptor immunoglobulins (BcR IGs), strongly supporting an antigen-driven pathway to CLL development. Subsets of cases with distinct stereotyped BcRs collectively account for almost one-third of all CLL. Furthermore, just a handful of major stereotyped subsets represent a substantial fraction of the entire cohort and, perhaps more importantly, an even larger fraction of clinically aggressive CLL. In several major subsets, stereotypy extends from shared primary IG sequences to shared clinical and biological features, including immune signaling, mRNA and miRNA expression, DNA methylation, and genomic aberrations. Regarding the latter, recent evidence indicates that different subsets display distinct profiles of recurrent gene mutations, even when limiting the analysis to subsets with similar IGHV gene mutational status. However, it should be emphasized that even the largest subsets account for only ∼3% of the cases with available IGHV-D-J sequence information, indicating that for meaningful conclusions to be reached, large patient cohorts are essential. Here, taking advantage of a series of 2482 CLL cases consolidated in the context of a multicenter collaboration coordinated by ERIC, the European Research Initiative on CLL, we systematically explored the genetic background of stereotyped subsets. Our main focus was on recurrent mutations in the NOTCH1 (entire exon 34 or targeted analysis for del7544-45), TP53 (exons 4-9), SF3B1 (exons 14-16), BIRC3 (exons 6-9) and MYD88 (exon 5) genes. Overall, 1313 cases (52.9%) carried mutated IGHV genes (M-CLL), whereas the remaining 1169 cases (47.1%) carried unmutated IGHV genes (U-CLL). Cases were sub-classified into the following major subsets: (i) U-CLL: #1, n=72; #3, n=25; #5, n=11; #6, n=22; #7, n=37; #8, n=20; (ii) M-CLL: #4, n=32; #77, n=12; #148, n=20; and, (iii) subset #2 (IGHV3-21, variable mutational status), n=57. Mutations in the MYD88 and BIRC3 genes were relatively rare, with no clear bias to any subset. With regards to the other three genes, only a single mutation in the TP53 gene was identified in a total of 80 M-CLL subset cases. Among U-CLL subsets and clinically aggressive subset #2, we noted asymmetric mutation frequencies, summarized as follows. (1) TP53 mutations were (a) enriched in subsets #3 and #7 (frequency >10%) and, in contrast, absent in subsets #5 and #6, though all these subsets utilize the IGHV1-69 gene; (b) enriched in subset #1 (9%) and, interestingly, subset #99, a less populated subset that is highly similar to subset #1 (2/4 cases positive for TP53 mutations); (c) absent in subset #2; and, (d) relatively infrequent in subset #8 (5%), the latter known to display the highest risk for Richter's transformation among all CLL. Differences between these subsets showed a trend for statistical significance (p=0.09). (2) NOTCH1 mutations exhibited (a) increased frequency in subsets #1 (28%) and #8 (25%); (b) among IGHV1-69 expressing subsets, lower frequencies in subsets #3 (8%), #5 (10%) and #7 (3%) compared to subset #6 (25%); and (c) intermediate frequency (9%) in subset #2 (p=0.0078 for comparison between subsets). (3) SF3B1 mutations were (a) significantly (p<0.001) enriched in subsets #2 (47.6%), #3 (40%) and #7 (25%) compared to all other major subsets (subset #1: 7.5%; subset #5: 0%; subset #6: 7.7%; subset #8: 0%); and, (b) positive in 3/7 cases (42.8%) of subset #169, a minor subset sharing remarkable IG sequence similarities to subset #2. In conclusion, in the largest study thus far conducted, we confirm and significantly extend recent observations indicating that different CLL stereotyped subsets display distinct genetic makeup. On these grounds, we propose that differential modes of immune signaling in the context of subset-biased antigen-IG interactions may be associated with the acquisition and/or selection of certain genomic aberrations in various stereotyped subsets, ultimately underlying clinical aggressiveness through distinct mechanisms. Finally, our findings suggest that sequence similarities between different subsets (e.g. #1 and #99, #2 and #169) likely reflect similar pathobiology, underscoring the relevance of the molecular sub-classification of CLL based on BcR stereotypy. Disclosures:Stamatopoulos:Roche: Research Funding.

Characteristics Of Chronic Lymphocytic Leukemia (CLL) In Senegal. Clinical Features, Cytology, Immunophenotype, Cytogenetic Abnormalities and Altered Expression Of Micro-RNA

The incidence of CLL in Africa is not known for several reasons, among them the difficulty to access the diagnosis since there is only clinical features and cytology which are available in several countries in francophone Africa, especially in Senegal. The WHO criteria which have been published since 2001 from the first revised WHO classification of tumours of haematopoitic and lymphoid tissues cannot be applied since there is no facility for immunophenotyping and cytogenetic. Moreover, no research in place can be set up without any precise diagnosis.In this study, we have developed the flow cytometry technique in the laboratory of Immunology in CHU Le Dantec in Dakar to obtain the immunophenotype looking at the expression of Kappa/lambda light chains, CD19, CD22, CD20, FMC7, CD5, CD10, CD23, CD38. Peripheral blood smears were fixed according to the technique for FISH to detect the following cytogenetic abnormalities: trisomy12, deletion 13q14, deletion 11q22-23, deletion 17p. RNA to test by qRT-PCR the expression of microRNA mir15a, mir16-1, mir181a, mir181b, and mir 34a and 34b was obtained from peripheral blood lymphocytes.Twelve cases of CCL aged ranging from 50 to 80 year old were characterized at clinical level according to the Binet's system: 1 stage A, 5 stage B, 6 stages C. In most of cases (9 cases) hyperlymphocytosis of small lymphocytes with clumped chromatin and scanty cytoplasm was very high (122.000 to 336 000/mm3). The “Matutes” score obtained by immunophenotyping was 5 or 4 for 11 patients in favor of typical CLL. The expression of CD38 which is a marker of unfavorable prognosis is positive in 9 patients. Nine patients had cytogenetic abnormalities: 3 trisomy 12, 6 del 13q14, 2 del 11q22-23, 1 del17p. Four patients had two simultaneous cytogenetic abnormalities (tri 12, tri 13 ; del13q14, del11q22-23 ; del13q24, del17p ; del13q24, tri12).The analysis of mi-RNA showed high expression of mir15a and mir 16-1 and low value of mir181a and mir 181b which were described in aggressive CLL ; however the expression of mir 34a and mir34b was also high.This study shows the aggressiveness of CLL in Senegal, probably due to the delay of the diagnosis. We have also demonstrated the possibility to set up immunophenotype and the possibility of a precise diagnosis of lymphoproliferative disorder in place, in western Africa and the series will be extended. Moreover we have also demonstrated the possibility to construct translational research project from samples characterized in the University Hospital in Dakar gathering the cases from different sanitary structures in the country.Such experiment would be a model for epidemiologic, clinico-biologic and translational research studies in order to set up in the country the capacity building for diagnosis and research. Disclosures:No relevant conflicts of interest to declare.

In Chronic Lymphocytic Leukemia PD-1 Is Expressed Independently From PDCD1 Gene Polymorphisms and Does Not Influence BCR Signaling

BackgroundThe programmed death-1 (PD-1, CD279) is a negative lymphocytic immunoreceptor responsible for peripheral tolerance. Through inhibition of TCR/BCR signaling PD-1 impairs activation and effectors functions of T and B lymphocytes, where it is expressed upon activation. PD-1 was described on exhausted T cells and it is aberrantly expressed in several malignancies, where a PD-1 pathway was related to a potential tumor escape mechanism from the immunosurveillance. Previously, we characterized PD-1 as a novel phenotypic feature of chronic lymphocytic leukemia (CLL) cells. PD-1 was up-regulated on both transcript and surface protein levels in CLL patients in comparison to healthy volunteers (HVs). Moreover, in vitro activated CLL cells in conditions mimicking microenvironment of proliferation centres up-regulated PD-1 on their surface. AimsDespite aberrant expression in CLL patients, PD-1 mechanism and function in CLL is unclear. To investigate PD-1 influence on BCR signaling in CLL cells we analyzed expression and phosphorylation of SYK, LYN, and ZAP-70 tyrosine kinases. To verify a relation of PD-1 expression to microenvironment in vivo, we analyzed PD-1 expression in mononuclears from 149 peripheral blood (PBMC) and 140 bone marrow (BMMC) CLL samples, representing accumulation and proliferation disease compartment respectively and 30 healthy PBMC samples. Finally, we characterized polymorphism of PDCD1 gene in 114 CLL patients and 150 HVs. MethodsQuantitative reverse transcriptase PCR was performed for the PD-1 transcript assessment in 182 CLL patients (148 PBMC, 140 BMMC). Expression and phosphorylation of tyrosine kinases were analyzed with western blot (for SYK and LYN) and flow cytometric methods (for ZAP-70 and PD-1) in CD19+ magnetically separated cells from 12 CLL patients. For each kinase two separate phosphorylation sites were analyzed, responsible either for activation or inhibition respectively: Y525/526 and Y323 for SYK, Y507 and Y396 for LYN, Y315/319 and Y292 for ZAP-70. Five single nucleotide polymorphism sites (SNPs) were analyzed by restriction fragments length polymorphism (RFLP-PCR) in group of 114 CLL patients and 150 HVs. ResultsThe median level of PD-1 transcripts in CLL patients was higher in comparison with HVs (p<0.0001), but there was no difference between blood and bone marrow compartments. The expression and phosphorylation of LYN and SYK were not dependent on PD-1 expression in 12 CLL patients, with PD-1 range from 4.2%. to 67.53%. Flow cytometric analysis showed that ZAP-70 expression was not related to the PD-1 level. Median percentages of cells with phosphorylated ZAP-70 activation/inhibition sites were low (Y315/319: 0.62%, Y292: 0.5%). Phosphorylation of Y292 showed strong negative correlation with PD-1 level (r2=-0.8, p=0.0019). Analysis of PDCD1 polymorphism showed that neither of SNPs frequency (loci: rs36084323, rs11568821, rs2227981, rs2227982, rs41386349) was different in CLL group compared to HVs. Particular substitutions were not related to the PD-1 expression level. Frequencies of haplotypes in CLL and HVs showed no significant difference. Linkage disequilibrium comparison of CLL and HVs revealed complete dependency of rs2227981/rs2227982 (p=0.03) and rs2227981/rs36084323 (p=0.03) loci in CLL patients. Summary / ConclusionPD-1 expression is strongly up-regulated in CLL patients regardless microenvironment of leukemic cells. No PD-1 influence on expression and activation of BCR related kinases might suggest impaired or alternative function of PD-1 pathway in CLL cells. Characterization of PDCD1 polymorphism revealed no association of PD-1 SNPs with CLL, therefore, PD-1 up-regulation might not be driven by genetic alternations. Disclosures:No relevant conflicts of interest to declare.

The genomic landscape of chronic lymphocytic leukemia: clinical implications

A precise understanding of the genomic and epigenomic features of chronic lymphocytic leukemia (CLL) may benefit the study of the disease’s staging and treatment. While recent reports have shed some light on these aspects, several challenges need to be addressed before translating this research into clinical practice. Thus, even the best candidate driver genes display low mutational rates compared to other tumors. This means that a large percentage of cases do not display clear tumor-driving point mutations, or show candidate driving point mutations with no obvious biochemical relationship to the more frequently mutated genes. This genomic landscape probably reflects either an unknown underlying biochemical mechanism playing a key role in CLL or multiple biochemical pathways independently driving the development of this tumor. The elucidation of either scenario will have important consequences on the clinical management of CLL. Herein, we review the recent advances in the definition of the genomic landscape of CLL and the ongoing research to characterize the underlying biochemical events that drive this disease.

Expression of Bcl2L12 in chronic lymphocytic leukemia patients: association with clinical and molecular prognostic markers

Dysregulation of apoptosis is a distinctive feature of chronic lymphocytic leukemia (CLL), although a unique mechanism underlying apoptosis resistance of CLL B lymphocytes has not been identified yet. Aberrant expression as well as genetic and epigenetic alterations of numerous genes involved in different pathways of apoptosis regulation has been described in CLL. Here, we report the expression analysis of Bcl2L12 (Bcl2-like 12), a novel apoptotic gene belonging to Bcl2 family, in 58 Serbian CLL patients. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis revealed a significant overexpression of Bcl2L12 mRNA in CLL samples compared to non-leukemic samples, implying its role in the pathogenesis of the disease. Receiver operating characteristic (ROC) analysis showed that Bcl2L12 expression efficiently discriminates CLL cases from healthy controls. However, relatively homogenous Bcl2L12 mRNA expression among patients did not reflect their clinical characteristics (with the exception of lactate dehydrogenase status and time from diagnosis to treatment) and failed to show association with the most informative prognostic markers, namely the mutational status of rearranged immunoglobulin heavy chain variable region genes, CD38 and lipoprotein lipase gene (LPL) expression.