ABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.
Subject(s)
Apoptosis/drug effects , CD47 Antigen/agonists , Membrane Potential, Mitochondrial/drug effects , Peptides/pharmacology , Animals , CD47 Antigen/metabolism , Calcium/metabolism , Cell Death/drug effects , Cell Line, Tumor , Female , Humans , Kaplan-Meier Estimate , Leukemia, Experimental/drug therapy , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Mice, Inbred BALB C , Peptides/chemistry , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Thrombospondin 1/chemistryABSTRACT
Trisomy 12 (tri12) is the second most frequent chromosomal aberration (15%-20%) in chronic lymphocytic leukemia (CLL). Tri12 confers an intermediate prognosis but is a heterogeneous entity. We examined whether additional mutational or chromosomal alterations might impact tri12 patient outcomes. This retrospective study, carried out by the French Innovative Leukemia Organization, included 188 tri12 patients with comprehensive information on immunoglobulin heavy chain (IGHV) gene status, karyotypic/FISH abnormalities, and NOTCH1, TP53, SF3B1, and MYD88 mutations. The main cytogenetic abnormalities associated with tri12 were del(13q) (25%), additional trisomies (14%) (including tri19 (10%) and tri18 (4%)), 14q32 translocations (10%), del(17p) (6.5%), del(14q) (4%), and del(11q) (4%). Unmutated (UM) IGHV, NOTCH1, and TP53, mutations were identified in respectively 66%, 25%, and 8.5% of cases. Multivariate analyses showed that additional trisomies (HR = 0.43, 95% CI = 0.23-0.78, P = .01) were associated with a significantly longer time to first treatment in Binet stage A patients and with a lower risk of relapse (HR = 0.37, 95% CI = 0.15-0.9, P = .03) in the overall tri12 population. Binet stage B/C, TP53 disruption, and UM IGHV status were associated with a shorter time to next treatment, while Binet stage B/C (HR = 4, 95% CI = 1.6-4.9, P = .002) and TP53 disruption (HR = 5, 95% CI = 1.94-12.66, P = .001) conferred shorter overall survival in multivariate comparisons. These data indicate that additional cytogenetic and mutational abnormalities, and particularly additional trisomies, IGHV status, and TP53 disruption, influence tri12 patient outcomes and could improve risk stratification in this population.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/epidemiology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Trisomy/genetics , Aged , Chromosomes, Human, Pair 12/genetics , Cytogenetic Analysis , DNA Mutational Analysis , Female , France/epidemiology , Humans , Immunoglobulin Heavy Chains/genetics , Male , Middle Aged , Retrospective StudiesABSTRACT
Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Mutation , Neoplasm Recurrence, Local/genetics , Salvage Therapy/methods , Ataxia Telangiectasia Mutated Proteins/genetics , High-Throughput Nucleotide Sequencing , Humans , Kaplan-Meier Estimate , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Neoplasm Recurrence, Local/diagnosis , Phosphoproteins/genetics , Prognosis , Prospective Studies , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/genetics , Treatment Outcome , Tumor Suppressor Protein p53/geneticsABSTRACT
OBJECTIVES: This study investigates whether achieving complete remission (CR) with undetectable minimal residual disease (MRD) after allogeneic stem cell transplantation (allo-SCT) for chronic lymphocytic leukemia (CLL) affects outcome. METHODS: We retrospectively studied 46 patients transplanted for CLL and evaluated for post-transplant MRD by flow cytometry. RESULTS: At transplant time, 43% of the patients were in CR, including one with undetectable MRD, 46% were in partial response, and 11% had refractory disease. After transplant, 61% of the patients achieved CR with undetectable MRD status. By multivariate analysis, reaching CR with undetectable MRD 12 months after transplant was the only factor associated with better progression-free survival (P = 0.02) and attaining undetectable MRD, independently of the time of negativity, was the only factor that correlated with better overall survival (P = 0.04). CONCLUSION: Thus, achieving undetectable MRD status after allo-SCT for CLL is a major goal to improve post-transplant outcome.
Subject(s)
Flow Cytometry/methods , Hematopoietic Stem Cell Transplantation , Leukemia, Lymphocytic, Chronic, B-Cell , Allografts , Disease-Free Survival , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Male , Middle Aged , Neoplasm, Residual , Survival RateABSTRACT
In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.
Subject(s)
Cell Line , Mast Cells/pathology , Mastocytosis, Systemic/genetics , Proto-Oncogene Proteins c-kit/genetics , Animals , Blotting, Western , Cell Line/cytology , Cell Line/immunology , Cell Line/metabolism , Cell Separation , Flow Cytometry , Heterografts , Humans , Immunoglobulin E/immunology , Immunoglobulin E/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , TransfectionABSTRACT
BACKGROUND: Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. METHODS AND FINDINGS: In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. CONCLUSIONS: Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.
Subject(s)
Apoptosis/drug effects , B-Lymphocytes/metabolism , CD47 Antigen/metabolism , Drug Resistance, Neoplasm , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Peptides/pharmacology , Phospholipase C gamma/metabolism , Aged , Aged, 80 and over , Animals , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Male , Mice , Mice, Inbred NOD , Middle Aged , Peptides/therapeutic use , Thrombospondin 1/therapeutic useABSTRACT
Hepatitis C virus (HCV) infection increases the risk of B-cell non-Hodgkin lymphomas (B-NHL). Antiviral treatment (AT) can induce hematological responses in patients with marginal zone lymphomas (MZL). The ANRS HC-13 Lympho-C study aimed at a better understanding of the impact of AT on HCV associated B-NHL. This multicentric study enrolled 116 HCV-positive patients with B-NHL between 2006 and 2012. Cytological and histological samples were collected for centralized review. At lymphoma diagnosis, median age was 61 years and gender ratio M/F was 1. Cytohistological distribution was marginal zone lymphoma (MZL) n = 45 (39%), diffuse large B-cell lymphoma (DLBCL) n = 45 (39%), and other types n = 26 (22%). MZL patients had more frequent detection of rheumatoid factor (68% vs. 35%; P = 0.001) and more frequently mixed cryoglobulinemia (74% vs. 44%; P = 0.021) than patients with DLBCL. Among patients receiving AT, a sustained virologic response was achieved in 23 of 38 (61%) patients with MZL and in 9 of 17 (53%) with DLBCL (P = 0.42). Three-year overall survival (OS) and progression-free survival were 78% 95%CI [63-88] and 64% [48-76], respectively, without difference between cytohistological groups. Outcome analysis showed a favorable association between OS and AT in all patients (P = 0.05) and in the subgroup of MZL patients only (P = 0.04). Our data support that AT improves the outcomes of HCV-associated NHLs. The impact of new AT regimen with protease inhibitor needs to be investigated in this setting. [clinicalTrials.gov Identification number NCT01545544]
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Antiviral Agents/therapeutic use , Hepatitis C/drug therapy , Lymphoma, B-Cell, Marginal Zone/drug therapy , Lymphoma, Large B-Cell, Diffuse/drug therapy , Adult , Aged , Aged, 80 and over , Cryoglobulinemia/physiopathology , Female , Hepatitis C/complications , Hepatitis C/mortality , Hepatitis C/pathology , Humans , Lymphoma, B-Cell, Marginal Zone/complications , Lymphoma, B-Cell, Marginal Zone/mortality , Lymphoma, B-Cell, Marginal Zone/pathology , Lymphoma, Large B-Cell, Diffuse/complications , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Male , Middle Aged , Rheumatoid Factor/blood , Survival Analysis , Treatment OutcomeABSTRACT
Whether sex chromosome loss (SCL) is an age-related phenomenon or a cytogenetic marker of hematological disease is unclear. To address this issue in chronic lymphocytic leukemia (CLL), we investigated 20 cases with X or Y chromosome loss detected by conventional cytogenetics (CC). The frequency of SCL was low in CLL (2.3%). It was the sole abnormality, as detected by CC, in 10/20 (50%) patients. Fluorescence in situ hybridization (FISH) analyses confirmed SCL in all patients tested, present in 5-88% of cells (median: 68%). Deletions of 13q were observed by FISH in 16/20 (80%) patients. Compared with CLL without SCL, SCL was significantly associated with 13q deletion, especially when bi-allelic (P = 0.04). Co-hybridization analyses showed that SCL could be a concomitant, primary or secondary change, or be present in an independent clone. FISH analyses were performed on blood sub-populations isolated by Ficoll or flow cytometry. Comparing mononuclear cells (including CLL cells) and polynuclear cells separated by Ficoll, a maximum of 2% of polynuclear cells were found with SCL, whereas mononuclear cells exhibited a significantly higher loss frequency (range: 6-87%) (P = 0.03). Comparing B-cells (including CLL cells) and T-cells sorted by flow cytometry, the proportion of B-CD19+ cells with SCL was significantly higher (range: 88-96%) than that observed in T-CD3+ cells (range: 2-6%) (P = 0.008). We conclude that SCL has to be considered as a clonal aberration in CLL that may participate in the oncogenic process.
Subject(s)
Aneuploidy , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Sex Chromosome Aberrations , Adult , Age Factors , Aged , Aged, 80 and over , Chromosomes, Human, Pair 13/genetics , Clone Cells , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphocyte Subsets/pathology , Male , Middle AgedABSTRACT
Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS.
Subject(s)
Chromosomes, Human, Pair 14/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Receptor, Notch1/genetics , Trisomy/genetics , Adult , Aged , Aged, 80 and over , Chromosomes, Human, Pair 12/genetics , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , MutationABSTRACT
Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/classification , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Molecular Diagnostic Techniques/methods , Molecular Targeted Therapy , Receptors, Antigen, B-Cell/genetics , Amino Acid Sequence , Gene Rearrangement, B-Lymphocyte/genetics , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Models, Biological , Molecular Sequence Data , Molecular Targeted Therapy/methods , Molecular Targeted Therapy/trends , Receptors, Antigen, B-Cell/metabolism , Somatic Hypermutation, Immunoglobulin/geneticsABSTRACT
Chronic lymphocytic leukaemia (CLL) is a heterogenous disease characterized by the accumulation of neoplastic CD5+/CD19+ B lymphocytes. The spreading of the leukaemia relies on the CLL cell's ability to survive in the blood and migrate to and proliferate within the bone marrow and lymphoid tissues. Some patients with CLL are either refractory to the currently available therapies or relapse after treatment; this emphasizes the need for novel therapeutic strategies that improving clinical responses and overcome drug resistance. CD38 is a marker of a poor prognosis and governs a set of survival, proliferation and migration signals that contribute to the pathophysiology of CLL. The literature data evidence a spatiotemporal association between the cell surface expression of CD38 and that of other CLL antigens, such as the B-cell receptor (BCR), CD19, CD26, CD44, the integrin very late antigen 4 (VLA4), the chemokine receptor CXCR4, the vascular endothelial growth factor receptor-2 (VEGF-R2), and the neutrophil gelatinase-associated lipocalin receptor (NGAL-R). Most of these proteins contribute to CLL cell survival, proliferation and trafficking, and cooperate with CD38 in multilayered signal transduction processes. In general, these antigens have already been validated as therapeutic targets in cancer, and a broad repertoire of specific monoclonal antibodies and derivatives are available. Here, we review the state of the art in this field and examine the therapeutic opportunities for cotargeting CD38 and its partners in CLL, e.g. by designing novel bi-/trispecific antibodies.
ABSTRACT
Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Humans , B-Lymphocytes/metabolism , Fatty Acids/metabolism , Fatty Acids/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Metabolic Reprogramming , Mitochondria/metabolismABSTRACT
Chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Aiolos, a member of the Ikaros family of zinc-finger transcription factors, plays an important role in the control of mature B lymphocyte differentiation and maturation. In this study, we showed that Aiolos expression is up-regulated in B-CLL cells. This overexpression does not implicate isoform imbalance or disturb Aiolos subcellular localization. The chromatin status at the Aiolos promoter in CLL is defined by the demethylation of DNA and an enrichment of euchromatin associated histone markers, such as the dimethylation of the lysine 4 on histone H3. These epigenetic modifications should allow its upstream effectors, such as nuclear factor-κB, constitutively activated in CLL, to gain access to promoter, resulting up-regulation of Aiolos. To determine the consequences of Aiolos deregulation in CLL, we analyzed the effects of Aiolos overexpression or down-regulation on apoptosis. Aiolos is involved in cell survival by regulating the expression of some Bcl-2 family members. Our results strongly suggest that Aiolos deregulation by epigenetic modifications may be a hallmark of CLL.
Subject(s)
Epigenesis, Genetic , Ikaros Transcription Factor/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Adult , Aged , Aged, 80 and over , Apoptosis/genetics , Apoptosis/physiology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Base Sequence , Cell Survival/genetics , Cell Survival/physiology , Chromatin/genetics , Chromatin/metabolism , CpG Islands , DNA Methylation , DNA Primers/genetics , Female , Gene Expression , Gene Knockdown Techniques , Humans , Ikaros Transcription Factor/antagonists & inhibitors , Ikaros Transcription Factor/metabolism , In Vitro Techniques , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Models, Biological , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA, Small Interfering/genetics , Subcellular Fractions/metabolismABSTRACT
Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70(-) and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70(+) and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70(-)) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease.
Subject(s)
Amyloidosis/drug therapy , Boronic Acids/administration & dosage , Protease Inhibitors/administration & dosage , Pyrazines/administration & dosage , Bortezomib , Female , Humans , Immunoglobulin Light-chain Amyloidosis , MaleABSTRACT
Long-term responses have been reported after autologous stem cell transplantation (ASCT) for chronic lymphocytic leukemia (CLL). We conducted a prospective, randomized trial of ASCT in previously untreated CLL patients. We enrolled 241 patients < 66 years of age with Binet stage B or C CLL. They received 3 courses of mini-CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone) and then 3 courses of fludarabine. Patients in complete response (CR) were then randomized to ASCT or observation, whereas the other patients were randomized to dexamethasone, high-dose aracytin, cisplatin (DHAP) salvage followed by either ASCT or 3 courses of fludarabine plus cyclophosphamide (FC). The primary end point was event-free survival (EFS). After up-front treatment, 105 patients entered CR and were randomized between ASCT (n = 52) and observation (n = 53); their respective 3-year EFS rates were 79.8% and 35.5%; the adjusted hazard ratio was 0.3 (95% CI: 0.1-0.7; P = .003). Ninety-four patients who did not enter CR were randomized between ASCT (n = 46) and FC (n = 48); their respective 3-year EFS rates were 48.9% and 44.4%, respectively; the adjusted hazard ratio was 1.7 (95% CI: 0.9-3.2; P = .13). No difference in overall survival was found between the 2 response subgroups. In young CLL patients in CR, ASCT consolidation markedly delayed disease progression. No difference was observed between ASCT and FC in patients requiring DHAP salvage.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Stem Cell Transplantation , Adolescent , Adult , Age Factors , Aged , Cisplatin/administration & dosage , Cyclophosphamide/administration & dosage , Cytarabine/administration & dosage , Dexamethasone/administration & dosage , Disease-Free Survival , Doxorubicin/administration & dosage , Female , Humans , Male , Middle Aged , Neoplasm Staging , Prednisone/administration & dosage , Survival Rate , Time Factors , Transplantation, Autologous , Vincristine/administration & dosageABSTRACT
Waldenström's macroglobulinemia is a disease of mature B cells, the genetic basis of which is poorly understood. Few recurrent chromosomal abnormalities have been reported, and their prognostic value is not known. We conducted a prospective cytogenetic study of Waldenström's macroglobulinemia and examined the prognostic value of chromosomal aberrations in an international randomized trial. The main aberrations were 6q deletions (30%), trisomy 18 (15%), 13q deletions (13%), 17p (TP53) deletions (8%), trisomy 4 (8%), and 11q (ATM) deletions (7%). There was a significant association between trisomy of chromosome 4 and trisomy of chromosome 18. Translocations involving the IGH genes were rare (<5%). Deletion of 6q and 11q, and trisomy 4, were significantly associated with adverse clinical and biological parameters. Patients with TP53 deletion had short progression-free survival and short disease-free survival. Although rare (<5%), trisomy 12 was associated with short progression-free survival. In conclusion, the cytogenetic profile of Waldenström's macroglobulinemia appears to differ from that of other B-cell lymphomas. Chromosomal abnormalities may help with diagnosis and prognostication, in conjunction with other clinical and biological characteristics.
Subject(s)
Chromosome Aberrations , Waldenstrom Macroglobulinemia/genetics , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Chlorambucil/therapeutic use , Chromosome Deletion , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 4/genetics , Chromosomes, Human, Pair 6/genetics , Female , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Karyotype , Male , Middle Aged , Prognosis , Prospective Studies , Trisomy , Vidarabine/analogs & derivatives , Vidarabine/therapeutic use , Waldenstrom Macroglobulinemia/drug therapy , Waldenstrom Macroglobulinemia/pathologyABSTRACT
STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies.
Subject(s)
Bone Marrow Transplantation/adverse effects , Disease Models, Animal , Hematologic Neoplasms/genetics , Mutation/genetics , Myeloproliferative Disorders/genetics , STAT3 Transcription Factor/genetics , Animals , Hematologic Neoplasms/diagnosis , Humans , K562 Cells , Mice , Mice, Inbred C57BL , Myeloproliferative Disorders/diagnosisABSTRACT
The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.
Subject(s)
Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Genes, Immunoglobulin Heavy Chain/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Receptors, Antigen, B-Cell/genetics , Complementarity Determining Regions/genetics , Humans , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
OBJECTIVE: To characterize the clinical features of ocular Whipple's disease (WD) and determine the long-term prognosis after antibiotic treatment. DESIGN: Retrospective case series. PARTICIPANTS: Medical records of patients referred between January 1993 and December 2010 were reviewed for chronic corticosteroid-resistant uveitis or neuro-ophthalmologic findings consistent with WD. Eleven patients (male/female = 9/2) were included in this study. METHODS: Diagnosis was based on cytologic examination and molecular analysis of samples (cerebrospinal fluid, vitreous, duodenum, or any involved lymph node). It was based on cytology before the routine use of polymerase chain reaction (PCR) and on both cytology and molecular biology for more recent patients. Long-term antibiotic therapy included oral trimethoprim-sulfamethoxazole (TMP-SMX) and rifampin, TMP-SMX alone, rifampin alone, or tetracycline alone. MAIN OUTCOME MEASURES: (1) Demographic and clinical characteristics of patients with positive PCR for Tropheryma whipplei or periodic acid-Schiff-positive macrophages in the vitreous and (2) long-term prognosis after antibiotic treatment. RESULTS: Mean age at diagnosis was 63 years (range, 51-73 years). Average time between the onset of the disease and diagnosis was 2 years (range, 1 month to 11 years). Mean follow-up was 7.2 years (range, 0.25-18 years). Ophthalmologic findings consisted of chronic uveitis (9 patients), isolated bilateral optic disc swelling (1 patient), and Parinaud syndrome (1 patient). All patients had PAS-positive macrophages, and 6 patients had a positive PCR for T. whipplei. Nine patients were treated with TMP-SMX and rifampin. One patient treated with only tetracycline relapsed and was successfully treated with TMP-SMX. No major side effects were reported. Intraocular inflammation and neurologic manifestations were controlled in all cases. At the end of follow-up, 2 patients were off treatment, 2 patients had a neurologic relapse after treatment interruption, and 5 patients were still taking TMP-SMX. One patient was taking only rifampin. Two patients were lost to follow-up. CONCLUSIONS: Ocular WD seems to be a neurologic manifestation of WD. Trimethoprim-SMX with rifampin is an efficient treatment, and prolonging treatment for at least 1 year is recommended. Long-term low-dose antibiotic therapy may reduce the rate of relapse, neurologic involvement, and death.
Subject(s)
Eye Infections, Bacterial/diagnosis , Eye Infections, Bacterial/drug therapy , Tropheryma/isolation & purification , Whipple Disease/diagnosis , Whipple Disease/drug therapy , Aged , Anti-Infective Agents/therapeutic use , Cerebrospinal Fluid/microbiology , Drug Resistance , Drug Therapy, Combination , Enzyme Inhibitors/therapeutic use , Eye Infections, Bacterial/microbiology , Female , Fluorescein Angiography , Follow-Up Studies , Glucocorticoids/therapeutic use , Humans , Lymph Nodes/microbiology , Male , Middle Aged , Polymerase Chain Reaction , Prognosis , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Retrospective Studies , Rifampin/therapeutic use , Tetracycline/therapeutic use , Trimethoprim, Sulfamethoxazole Drug Combination/therapeutic use , Tropheryma/genetics , Uveitis/drug therapy , Vitreous Body/microbiology , Whipple Disease/microbiologyABSTRACT
In somatic cells, eroded telomeres can induce DNA double-strand break signaling, leading to a form of replicative senescence or apoptosis, both of which are barriers to tumorigenesis. However, cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis, enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction, and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs, 2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably, these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions, or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together, our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction.