scholarly journals Higher gene expression variability in the more aggressive subtype of chronic lymphocytic leukemia

2014 ◽  
Author(s):  
Simone Ecker ◽  
Vera Pancaldi ◽  
Daniel Rico ◽  
Alfonso Valencia
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cellular Communication ◽  
Lymphocytic Leukemia ◽  
Resistance To Therapy ◽  
Expression Variability ◽  
Disease Subtype ◽  
Aggressive Subtype

Background: Chronic Lymphocytic Leukemia (CLL) presents two subtypes which have drastically different clinical outcomes. So far, these two subtypes are not associated to clear differences in gene expression profiles. Interestingly, recent results have highlighted important roles for heterogeneity, both at the genetic and at the epigenetic level in CLL progression. Results: We propose to use gene expression variability across patients to investigate differences between the two CLL subtypes. We find that the most aggressive type of this disease shows higher variability of gene expression across patients and we elaborate on this observation to produce a method that classifies patients into clinical subtypes. Finally, we find that, overall, genes that show higher variability in the aggressive subtype are related to cell cycle, development and inter-cellular communication, probably related to faster progression of this disease subtype. Conclusions: There are strong relations between disease subtype and gene expression variability linking significantly increased expression variability to phenotypes such as aggressiveness and resistance to therapy in CLL.

Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 560-560 ◽  
Author(s):  
Ma. Reina Improgo ◽  
Adam Kiezun ◽  
Yaoyu Wang ◽  
Lillian Werner ◽  
Petar Stojanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Transcriptional Activity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Rt Pcr ◽  
Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Subnetwork-based analysis of chronic lymphocytic leukemia identifies pathways that associate with disease progression

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2639-2649 ◽  
Author(s):  
Han-Yu Chuang ◽  
Laura Rassenti ◽  
Michelle Salcedo ◽  
Kate Licon ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Disease Progression ◽  
Expression Profiles ◽  
Treatment Strategies ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Cancer Evolution ◽  
Sample Collection ◽  
Over Time

Abstract The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous. Several prognostic factors have been identified that can stratify patients into groups that differ in their relative tendency for disease progression and/or survival. Here, we pursued a subnetwork-based analysis of gene expression profiles to discriminate between groups of patients with disparate risks for CLL progression. From an initial cohort of 130 patients, we identified 38 prognostic subnetworks that could predict the relative risk for disease progression requiring therapy from the time of sample collection, more accurately than established markers. The prognostic power of these subnetworks then was validated on 2 other cohorts of patients. We noted reduced divergence in gene expression between leukemia cells of CLL patients classified at diagnosis with aggressive versus indolent disease over time. The predictive subnetworks vary in levels of expression over time but exhibit increased similarity at later time points before therapy, suggesting that degenerate pathways apparently converge into common pathways that are associated with disease progression. As such, these results have implications for understanding cancer evolution and for the development of novel treatment strategies for patients with CLL.

scholarly journals Gene Expression Profiling of B Cell Chronic Lymphocytic Leukemia Reveals a Homogeneous Phenotype Related to Memory B Cells✪

2001 ◽  
Vol 194 (11) ◽  
pp. 1625-1638 ◽  
Author(s):  
Ulf Klein ◽  
Yuhai Tu ◽  
Gustavo A. Stolovitzky ◽  
Michela Mattioli ◽  
Giorgio Cattoretti ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Variable Region ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cell Subpopulations

B cell–derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that &gt;50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of ∼12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (&lt;30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5+ B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.

Multi-Parameter Phenotypic Analysis of Members of Chronic Lymphocytic Leukemia Clones Identifies Distinct Proliferative and Resting/Re-Entry Compartments with Discrete Gene Expression Profiles.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 668-668 ◽  
Author(s):  
Carlo Calissano ◽  
Rajendra N Damle ◽  
Xiao J. Yan ◽  
Wentian Li ◽  
Sonia Marsilio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Signaling ◽  
Expression Profiles ◽  
Cell Movement ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Phenotypic Analysis ◽  
Ki 67 ◽  
And Migration

Abstract Abstract 668 Background. In vivo studies of deuterium (2H) incorporation into newly-synthesized DNA of chronic lymphocytic leukemia (B-CLL) B cells indicate that the disease is dynamic, with ongoing birth and death of individual members of a leukemic clone. Therefore, a detailed phenotypic analysis might define the “life cycle” of subpopulations of leukemic cells within a B-CLL clone, thereby identifying recently-born members and members born earlier. Such an extended phenotype could enable structural and genetic characterization of the intra-clonal heterogeneity that birth and death create, and thereby help understand the biology of B-CLL cells and define novel cell surface markers or marker combinations as therapeutic targets. Methods. Our previous studies indicated that B-CLL clones could be subdivided into three populations based on relative densities of expression of CXCR4 and CD5. The CXCR4dimCD5bright subpopulation is enriched in cells with 2H-labeled, newly-synthesized DNA and contains significantly more Ki-67+ and MCM6+ cells than CXCR4intCD5int cells within the same clone. These latter cells in turn are enriched for the same parameters compared to the CXCR4brightCD5dim fraction. We have now identified other markers associated with the CXCR4dimCD5bright subpopulation (“proliferative compartment”) and the CXCR4brightCD5dim (“resting/re-entry compartment”) in B-CLL. PBMC from 20 B-CLL cases were subjected to multi-parameter immunofluorescent, flow cytometric analyses to define the expression of CD11a, CD20, CD23, CD27, CD38, CD47, CD49d, CD52, or Fcƒ×RII on the CD19+CXCR4dimCD5bright and CD19+CXCR4brightCD5dim B-CLL cell subpopulations. Companion studies were performed on Ki-67+ and Ki-67- subpopulations. Finally, gene expression profiling was performed on CXCR4dimCD5bright, CXCR4intCD5int, and CXCR4brightCD5dim subsets of 12 B-CLL cases using the Illumina HumanWG-6 platform. Results. Compared to CXCR4brightCD5dim cells within a clone, the CXCR4dimCD5bright compartment contained significantly more cells expressing CD23, CD52 (both p<0.0001), CD11a (p<0.001), CD20, CD38 (both p<0.01) and CD49d (p<0.05). In addition, this same fraction expressed significantly higher densities of Fcƒ×RIIb (p<0.0001), CD23, CD52 (both p<0.01), and CD11a (p<0.05). Gene expression profiles comparing the two subsets revealed over 1,309 significant differences. Functional activities associated with these genes differed dramatically between the two compartments. Of the top 25 over-expressed genes in the CXCR4dimCD5bright proliferative compartment, 11 related to proliferation (e.g., NAPSA, MARCKS, CCND2), 10 were involved in cell signaling and activation (e.g., GPR183, ADAP1), and 4 related to cell movement and migration (e.g., CXCR3). In the CXCR4brightCD5dim resting/re-entry compartment, 12 of the 25 most overexpressed genes were involved in apoptosis and cell death (e.g., RXR2, HRK), 7 in cell signaling (e.g., ADARB1, RAB37) and 5 in cell movement and migration (e.g., MDK, IRS2). Conclusions. These studies defined a reciprocal, extended surface membrane phenotype that distinguishes cells of the proliferative and the resting/re-entry compartments in B-CLL. This phenotype is based both on the percent of expressing cells and on the density of expression of well-defined surface molecules. In addition, gene expression analyses identified genes differentially expressed between the two compartments. These data support the notion that cells in these two compartments are a phenotypic continuum, with the former having divided recently and trafficking from the location where division occurred and the latter having divided earlier, being less robust, and attempting to traffic back to a lymphoid microenvironment to obtain survival signals. We suggest that targeting these two populations with appropriate monoclonal antibodies, many of which are currently in clinical trials in B-CLL and other B-cell lymphoproliferative disorders, may have a beneficial effect. Although this approach may not immediately diminish the bulk of the leukemic mass, it would preferentially eliminate the small fraction of cells that sustains a B-CLL clone and the fraction of cells that maintain a B-CLL clone after receiving survival signals, thereby permitting the remaining cells to die spontaneously. Disclosures: Allen: Antisoma: Research Funding.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 976-976 ◽  
Author(s):  
John C. Riches ◽  
Ajanthah Sangaralingam ◽  
Shahryar Kiaii ◽  
Tracy Chaplin ◽  
Demet Cekdemir ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Family Member ◽  
Cell Function ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Sign in / Sign up

Export Citation Format

Share Document