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1
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Cano-Pérez E, García-Díaz G. Performance of CpG-oligonucleotide DSP30 and interleukin-2 in the cytogenetic study of mature B-cell neoplasms: A systematic review and meta-analysis. Crit Rev Oncol Hematol 2025; 209:104659. [PMID: 39954875 DOI: 10.1016/j.critrevonc.2025.104659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Revised: 02/10/2025] [Accepted: 02/12/2025] [Indexed: 02/17/2025] Open
Abstract
Traditional B-cell mitogens often fail to promote effective cell division in vitro for mature B-cell neoplasms, hindering chromosome analysis. The combination of DSP30 and IL-2 (DSP30/IL-2) has been suggested as a better alternative. This review evaluates DSP30/IL-2 efficiency using a systematic review and meta-analysis of 20 studies. Studies comparing the successful culture rate (SCR) and/or abnormalities detection rate (ADR) of DSP30/IL-2 against traditional mitogens and/or fluorescence in situ hybridization (FISH) were included. Subgroup analyses were performed for cases of chronic lymphocytic leukemia (CCL) and other B-cell neoplasms (OBCN). The findings show no significant difference in SCR between DSP30/IL-2 and traditional mitogens, but DSP30/IL-2 significantly increases ADR. However, DSP30/IL-2's ADR is lower than that of FISH. Analyses by CLL and OBCN subgroups showed similar results. Overall, DSP30/IL-2 is a superior alternative for cytogenetic studies in mature B-cell neoplasms.
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Affiliation(s)
- Eder Cano-Pérez
- Biology program, Universidad de Cartagena, Cartagena, Colombia.
| | - Génesis García-Díaz
- Clinical Biochemistry Department, Faculty of Chemical Sciences, Universidad Central de Ecuador, Quito, Ecuador; Cytogenetics Department, Centro de Investigación y Desarrollo Biotecnológico Gen & Biolab, Quito, Ecuador
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2
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Sciaccotta R, Gangemi S, Penna G, Giordano L, Pioggia G, Allegra A. Potential New Therapies "ROS-Based" in CLL: An Innovative Paradigm in the Induction of Tumor Cell Apoptosis. Antioxidants (Basel) 2024; 13:475. [PMID: 38671922 PMCID: PMC11047475 DOI: 10.3390/antiox13040475] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Revised: 04/09/2024] [Accepted: 04/13/2024] [Indexed: 04/28/2024] Open
Abstract
Chronic lymphocytic leukemia, in spite of recent advancements, is still an incurable disease; the majority of patients eventually acquire resistance to treatment through relapses. In all subtypes of chronic lymphocytic leukemia, the disruption of normal B-cell homeostasis is thought to be mostly caused by the absence of apoptosis. Consequently, apoptosis induction is crucial to the management of this illness. Damaged biological components can accumulate as a result of the oxidation of intracellular lipids, proteins, and DNA by reactive oxygen species. It is possible that cancer cells are more susceptible to apoptosis because of their increased production of reactive oxygen species. An excess of reactive oxygen species can lead to oxidative stress, which can harm biological elements like DNA and trigger apoptotic pathways that cause planned cell death. In order to upset the balance of oxidative stress in cells, recent therapeutic treatments in chronic lymphocytic leukemia have focused on either producing reactive oxygen species or inhibiting it. Examples include targets created in the field of nanomedicine, natural extracts and nutraceuticals, tailored therapy using biomarkers, and metabolic targets. Current developments in the complex connection between apoptosis, particularly ferroptosis and its involvement in epigenomics and alterations, have created a new paradigm.
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Affiliation(s)
- Raffaele Sciaccotta
- Hematology Unit, Department of Human Pathology in Adulthood and Childhood “Gaetano Barresi”, University of Messina, Via Consolare Valeria, 98125 Messina, Italy; (R.S.); (G.P.); (L.G.)
| | - Sebastiano Gangemi
- Allergy and Clinical Immunology Unit, Department of Clinical and Experimental Medicine, University of Messina, Via Consolare Valeria, 98125 Messina, Italy;
| | - Giuseppa Penna
- Hematology Unit, Department of Human Pathology in Adulthood and Childhood “Gaetano Barresi”, University of Messina, Via Consolare Valeria, 98125 Messina, Italy; (R.S.); (G.P.); (L.G.)
| | - Laura Giordano
- Hematology Unit, Department of Human Pathology in Adulthood and Childhood “Gaetano Barresi”, University of Messina, Via Consolare Valeria, 98125 Messina, Italy; (R.S.); (G.P.); (L.G.)
| | - Giovanni Pioggia
- Institute for Biomedical Research and Innovation (IRIB), National Research Council of Italy (CNR), 98164 Messina, Italy;
| | - Alessandro Allegra
- Hematology Unit, Department of Human Pathology in Adulthood and Childhood “Gaetano Barresi”, University of Messina, Via Consolare Valeria, 98125 Messina, Italy; (R.S.); (G.P.); (L.G.)
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3
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Macrophage- and BCR-derived but not TLR-derived signals support the growth of CLL and Richter syndrome murine models in vivo. Blood 2022; 140:2335-2347. [PMID: 36084319 DOI: 10.1182/blood.2022016272] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 09/01/2022] [Indexed: 11/20/2022] Open
Abstract
A large amount of circumstantial evidence has accumulated suggesting that Toll-like receptor (TLR) signals are involved in driving chronic lymphocytic leukemia (CLL) cell proliferation, but direct in vivo evidence for this is still lacking. We have now further addressed this possibility by pharmacologically inhibiting or genetically inactivating the TLR pathway in murine CLL and human Richter syndrome (RS) patient-derived xenograft (PDX) cells. Surprisingly, we show that pharmacologic inhibition of TLR signaling by treatment with an IRAK1/4 inhibitor delays the growth of the transplanted malignant cells in recipient mice, but genetic inactivation of the same pathway by CRISPR/Cas9-mediated disruption of IRAK4 or its proximal adaptor MyD88 has no effect. We further show that treatment with the IRAK1/4 inhibitor results in depletion of macrophages and demonstrate that these cells can support the survival and enhance the proliferation of both murine Eμ-TCL1 leukemia and human RS cells. We also show that genetic disruption of the B-cell receptor (BCR) by CRISPR/Cas9 editing of the immunoglobulin M constant region gene inhibits the growth of human RS-PDX cells in vivo, consistent with our previous finding with murine Eμ-TCL1 leukemia cells. Finally, we show that genetic disruption of IRAK4 does not result in negative selection of human CLL cell lines xenografted in immunodeficient mice. The obtained data suggest that TLR signals are unlikely to represent a major driver of CLL/RS cell proliferation and provide further evidence that signals from macrophages and the BCR promote the growth and survival of CLL and RS cells in vivo.
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4
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Heerema NA, Muthusamy N, Zhao Q, Ruppert AS, Breidenbach H, Andritsos LA, Grever MR, Maddocks KJ, Woyach J, Awan F, Long M, Gordon A, Coombes C, Byrd JC. Prognostic significance of translocations in the presence of mutated IGHV and of cytogenetic complexity at diagnosis of chronic lymphocytic leukemia. Haematologica 2021; 106:1608-1615. [PMID: 32414849 PMCID: PMC8168513 DOI: 10.3324/haematol.2018.212571] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2018] [Accepted: 05/08/2020] [Indexed: 11/09/2022] Open
Abstract
Mutations of the IGH variable region in patients with chronic lymphocytic leukemia (CLL) are associated with a favorable prognosis. Cytogenetic complexity (>3 unrelated aberrations) and translocations have been associated with an unfavorable prognosis. While mutational status of IGHV is stable, cytogenetic aberrations frequently evolve. However, the relationships of these features as prognosticators at diagnosis are unknown. We examined the CpG-stimulated metaphase cytogenetic features detected within one year of diagnosis of CLL and correlated these features with outcome and other clinical features including IGHV. Of 329 untreated patients, 53 (16.1%) had a complex karyotype (16.1%), and 85 (25.8%) had a translocation. Median time to first treatment (TFT) was 47 months. In univariable analyses, significant risk factors for shorter TFT (p3.5, log-transformed WBC, unmutated IGHV, complex karyotype, translocation, and FISH for trisomy 8, del(11q) and del(17p). In multivariable analysis, there was significant effect modification of IGHV status on the relationship between translocation and TFT (p=0.002). In IGHV mutated patients, those with a translocation had over 3.5 times higher risk of starting treatment than those without a translocation (p.
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Affiliation(s)
- Nyla A. Heerema
- Department of Pathology, The Ohio State University Wexner Medical Center
| | - Natarajan Muthusamy
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Qiuhong Zhao
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Amy S. Ruppert
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | | | - Leslie A. Andritsos
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Michael R. Grever
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Kami J. Maddocks
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Jennifer Woyach
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Farrukh Awan
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Meixiao Long
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Amber Gordon
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - Caitlin Coombes
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
| | - John C. Byrd
- Department of Hematology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
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5
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Chen J, Moore A, Ringshausen I. ZAP-70 Shapes the Immune Microenvironment in B Cell Malignancies. Front Oncol 2020; 10:595832. [PMID: 33194762 PMCID: PMC7653097 DOI: 10.3389/fonc.2020.595832] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 09/17/2020] [Indexed: 12/12/2022] Open
Abstract
Zeta-chain-associated protein kinase-70 (ZAP-70) is a tyrosine kinase mainly expressed in T cells, NK cells and a subset of B cells. Primarily it functions in T cell receptor (TCR) activation through its tyrosine kinase activity. Aberrant expression of ZAP-70 has been evidenced in different B cell malignancies, with high expression of ZAP-70 in a subset of patients with Chronic Lymphocytic Leukemia (CLL), associating with unfavorable disease outcomes. Previous studies to understand the mechanisms underlying this correlation have been focused on tumor intrinsic mechanisms, including the activation of B cell receptor (BCR) signaling. Recent evidence also suggests that ZAP-70, intrinsically expressed in tumor cells, can modulate the cross-talk between malignant B cells and the immune environment, implying a more complex role of ZAP-70 in the pathogenesis of B cell malignancies. Meanwhile, the indispensible roles of ZAP-70 in T cell and NK cell activation also demonstrate that the autologous expression of ZAP-70 in the immune environment can be a central target in modulation of tumor immunity. Here we review the evidences of the link between ZAP-70 and tumor immunology in the microenvironment in B cell malignancies. Considering an emerging role of immunotherapies in treating these conditions, understanding the distinct molecular functions of ZAP-70 in a broader cellular context could ultimately benefit patient care.
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Affiliation(s)
| | | | - Ingo Ringshausen
- Department of Haematology, Jeffrey Cheah Biomedical Centre, Wellcome Trust/MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom
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6
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Schulze-Edinghausen L, Dürr C, Öztürk S, Zucknick M, Benner A, Kalter V, Ohl S, Close V, Wuchter P, Stilgenbauer S, Lichter P, Seiffert M. Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia. Cancers (Basel) 2019; 11:E822. [PMID: 31200555 PMCID: PMC6627891 DOI: 10.3390/cancers11060822] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 05/30/2019] [Accepted: 06/05/2019] [Indexed: 12/11/2022] Open
Abstract
Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn-/- mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn-/- chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.
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Affiliation(s)
- Lena Schulze-Edinghausen
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Claudia Dürr
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Selcen Öztürk
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Manuela Zucknick
- Oslo Centre for Biostatistics and Epidemiology, Department of Biostatistics, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 0372 Oslo, Norway.
| | - Axel Benner
- Division of Biostatistics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Verena Kalter
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Sibylle Ohl
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Viola Close
- Internal Medicine III, University of Ulm, 89081 Ulm, Germany, and Cooperation Unit Mechanisms of Leukemogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Patrick Wuchter
- Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden-Württemberg-Hessen, 68167 Mannheim, Germany.
| | - Stephan Stilgenbauer
- Internal Medicine III, University of Ulm, 89081 Ulm, Germany, and Department of Internal Medicine I, Saarland University, 66421 Homburg, Germany.
| | - Peter Lichter
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
- German Cancer Research Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
| | - Martina Seiffert
- Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
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7
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Gupta R, Li W, Yan XJ, Barrientos J, Kolitz JE, Allen SL, Rai K, Chiorazzi N, Mongini PKA. Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins. THE JOURNAL OF IMMUNOLOGY 2019; 202:2924-2944. [PMID: 30988120 DOI: 10.4049/jimmunol.1801142] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Accepted: 03/13/2019] [Indexed: 12/25/2022]
Abstract
Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.
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Affiliation(s)
- Rashmi Gupta
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
| | - Wentian Li
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
| | - Xiao J Yan
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
| | | | - Jonathan E Kolitz
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
| | - Steven L Allen
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
| | - Kanti Rai
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and.,Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549
| | - Nicholas Chiorazzi
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030.,Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and.,Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549
| | - Patricia K A Mongini
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030; .,Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549
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8
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Gupta R, Yan XJ, Barrientos J, Kolitz JE, Allen SL, Rai K, Chiorazzi N, Mongini PKA. Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2018; 201:1570-1585. [PMID: 30068596 PMCID: PMC6103916 DOI: 10.4049/jimmunol.1800591] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Accepted: 06/26/2018] [Indexed: 12/30/2022]
Abstract
Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rβ (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.
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Affiliation(s)
- Rashmi Gupta
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
| | - Xiao J Yan
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
| | - Jacqueline Barrientos
- Department of Medicine, North Shore University Hospital-Long Island Jewish Medical Center, Northwell Health, Manhasset, NY 11303
| | - Jonathan E Kolitz
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
- Department of Medicine, North Shore University Hospital-Long Island Jewish Medical Center, Northwell Health, Manhasset, NY 11303
- Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
| | - Steven L Allen
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
- Department of Medicine, North Shore University Hospital-Long Island Jewish Medical Center, Northwell Health, Manhasset, NY 11303
- Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
| | - Kanti Rai
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
- Department of Medicine, North Shore University Hospital-Long Island Jewish Medical Center, Northwell Health, Manhasset, NY 11303
- Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
| | - Nicholas Chiorazzi
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030
- Department of Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and
- Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549
| | - Patricia K A Mongini
- The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030;
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9
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Crassini K, Shen Y, O'Dwyer M, O'Neill M, Christopherson R, Mulligan S, Best OG. The dual inhibitor of the phosphoinositol-3 and PIM kinases, IBL-202, is effective against chronic lymphocytic leukaemia cells under conditions that mimic the hypoxic tumour microenvironment. Br J Haematol 2018; 182:654-669. [PMID: 29978459 DOI: 10.1111/bjh.15447] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2018] [Accepted: 05/14/2018] [Indexed: 12/15/2022]
Abstract
Despite significant advances in treatment, chronic lymphocytic leukaemia (CLL) remains an incurable disease. Ibrutinib and idelalisib, which inhibit Bruton Tyrosine kinase (BTK) and phosphoinositol-3 (PI3) kinase-δ respectively, have become important treatment options for the disease and demonstrate the potential of targeting components of the B-cell receptor-signalling pathway. IBL-202 is a dual inhibitor of the PIM and PI3 kinases. Synergy between the pan-PIM inhibitor, pPIMi, and idelalisib against a range of haematological cell lines and primary CLL cells supports the rationale for preclinical studies of IBL-202 in CLL. Importantly, IBL-202, but not idelalisib, was cytotoxic against CLL cells under in vitro conditions that mimic the hypoxic tumour microenvironment. The significant effects of IBL-202 on CD49d and CXCR4 expression and migration, cycle and proliferation of CLL cells suggest the drug may also interfere with the migratory and proliferative capacity of the leukaemic cells. Collectively, these data demonstrate that dual inhibition of the PIM and PI3 kinases by IBL-202 may be an effective strategy for targeting CLL cells, particularly within the environmental niches known to confer drug-resistance.
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Affiliation(s)
- Kyle Crassini
- Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia
| | - Yandong Shen
- Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia.,School of Molecular Biosciences, University of Sydney, Sydney, Australia
| | | | | | | | - Stephen Mulligan
- Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia.,School of Molecular Biosciences, University of Sydney, Sydney, Australia
| | - O Giles Best
- Northern Blood Research Centre, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, Australia.,School of Molecular Biosciences, University of Sydney, Sydney, Australia
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10
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Crassini K, Shen Y, Stevenson WS, Christopherson R, Ward C, Mulligan SP, Best OG. MEK1/2 inhibition by binimetinib is effective as a single agent and potentiates the actions of Venetoclax and ABT-737 under conditions that mimic the chronic lymphocytic leukaemia (CLL) tumour microenvironment. Br J Haematol 2018; 182:360-372. [DOI: 10.1111/bjh.15282] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Accepted: 02/16/2018] [Indexed: 12/19/2022]
Affiliation(s)
- Kyle Crassini
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
| | - Yandong Shen
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
- School of Molecular Biosciences; University of Sydney; Sydney Australia
| | - William S. Stevenson
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
| | | | - Chris Ward
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
| | - Stephen P. Mulligan
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
- CLL Australian Research Consortium (CLLARC); Kolling Institute of Medical Research, St Leonards; Sydney Australia
- School of Molecular Biosciences; University of Sydney; Sydney Australia
| | - O. Giles Best
- Northern Blood Research Centre; Kolling Institute of Medical Research; Royal North Shore Hospital; St Leonards Sydney Australia
- CLL Australian Research Consortium (CLLARC); Kolling Institute of Medical Research, St Leonards; Sydney Australia
- School of Molecular Biosciences; University of Sydney; Sydney Australia
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11
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Affiliation(s)
- Byron B. Au-Yeung
- Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA
| | - Neel H. Shah
- Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
| | - Lin Shen
- Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, University of California, San Francisco, California 94143, USA;,
| | - Arthur Weiss
- Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, University of California, San Francisco, California 94143, USA;,
- Howard Hughes Medical Institute, University of California, San Francisco, California 94143, USA
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12
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Tao W, Hurst BL, Shakya AK, Uddin MJ, Ingrole RSJ, Hernandez-Sanabria M, Arya RP, Bimler L, Paust S, Tarbet EB, Gill HS. Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses. Antiviral Res 2017; 141:62-72. [PMID: 28161578 PMCID: PMC5572660 DOI: 10.1016/j.antiviral.2017.01.021] [Citation(s) in RCA: 84] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Revised: 01/28/2017] [Accepted: 01/31/2017] [Indexed: 11/28/2022]
Abstract
The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.
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Affiliation(s)
- Wenqian Tao
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX 79409, USA
| | - Brett L Hurst
- Department of Animal, Dairy and Veterinary Sciences and the School of Veterinary Medicine, Utah State University, Logan, UT 84322, USA
| | | | - Md Jasim Uddin
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX 79409, USA
| | - Rohan S J Ingrole
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX 79409, USA
| | - Mayra Hernandez-Sanabria
- Center for Human Immunobiology, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA
| | - Ravi P Arya
- Center for Human Immunobiology, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA
| | - Lynn Bimler
- Center for Human Immunobiology, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA
| | - Silke Paust
- Center for Human Immunobiology, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA
| | - E Bart Tarbet
- Department of Animal, Dairy and Veterinary Sciences and the School of Veterinary Medicine, Utah State University, Logan, UT 84322, USA
| | - Harvinder Singh Gill
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX 79409, USA.
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13
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Pozzo F, Bittolo T, Vendramini E, Bomben R, Bulian P, Rossi FM, Zucchetto A, Tissino E, Degan M, D'Arena G, Di Raimondo F, Zaja F, Pozzato G, Rossi D, Gaidano G, Del Poeta G, Gattei V, Dal Bo M. NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components. Leukemia 2017; 31:2407-2415. [PMID: 28321119 DOI: 10.1038/leu.2017.90] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Revised: 03/13/2017] [Accepted: 03/16/2017] [Indexed: 12/24/2022]
Abstract
In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.
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Affiliation(s)
- F Pozzo
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - T Bittolo
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - E Vendramini
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - R Bomben
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - P Bulian
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - F M Rossi
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - A Zucchetto
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - E Tissino
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - M Degan
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - G D'Arena
- Department of Onco-Hematology, IRCCS 'Centro di Riferimento Oncologico della Basilicata', Rionero in Vulture, Italy
| | - F Di Raimondo
- Division of Hematology, Ferrarotto Hospital, Catania, Italy
| | - F Zaja
- Clinica Ematologica, Centro Trapianti e Terapie Cellulari 'Carlo Melzi' DISM, Azienda Ospedaliera Universitaria S Maria Misericordia, Udine, Italy
| | - G Pozzato
- Department of Internal Medicine and Hematology, Maggiore General Hospital, University of Trieste, Trieste, Italy
| | - D Rossi
- Hematology, Institute of Oncology Research and Oncology Institute of Southern Switzerland, Bellinzona, Switzerland
| | - G Gaidano
- Department of Translational Medicine, Division of Hematology, University of Eastern Piedmont, Novara, Italy
| | - G Del Poeta
- Division of Hematology, S Eugenio Hospital and University of Tor Vergata, Rome, Italy
| | - V Gattei
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
| | - M Dal Bo
- Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
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14
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Koczkodaj D, Filip AA. Chromosome Preparation for Chronic Lymphoid Malignancies. Methods Mol Biol 2016; 1541:33-41. [PMID: 27910012 DOI: 10.1007/978-1-4939-6703-2_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/21/2023]
Abstract
Conventional cytogenetics is invariably one of the most important methods used in diagnostics of chronic lymphoproliferations. It complements fluorescence in situ hybridization (FISH) and molecular analysis. Presence of particular chromosomal alterations in chronic lymphocytic leukemia enables patients' stratification into appropriate cytogenetic risk groups and influences treatment decisions. In other non-Hodgkin lymphomas cytogenetic analyses are employed also in minimal residual disease assessment.As lymphocytes in chronic lymphoid malignancies are characterized by low proliferation rate in vitro, it is critical to induce their division in the culture properly. Here, we describe methods of lymphocyte isolation from patient's samples, conditions of cell culture, and the most commonly used mitogens for B- and T-lymphocytes in hemato-oncologic analyses.
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Affiliation(s)
- Dorota Koczkodaj
- Department of Cancer Genetics, Medical University of Lublin, Radziwiłłowska 11, Lublin, 20-080, Poland.
| | - Agata A Filip
- Department of Cancer Genetics, Medical University of Lublin, Radziwiłłowska 11, Lublin, 20-080, Poland
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15
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Samuel J, Jayne S, Chen Y, Majid A, Wignall A, Wormull T, Najeeb H, Luo JL, Jones GDD, Macip S, Dyer MJS. Posttranscriptional Upregulation of p53 by Reactive Oxygen Species in Chronic Lymphocytic Leukemia. Cancer Res 2016; 76:6311-6319. [PMID: 27634759 DOI: 10.1158/0008-5472.can-16-0843] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Accepted: 08/09/2016] [Indexed: 11/16/2022]
Abstract
Chronic lymphocytic leukemia (CLL) cells multiply and become more resistant to immunochemotherapy in "proliferation centers" within tissues, whereas apoptosis occurs in the periphery. Various models recapitulate these microenvironments in vitro, such as stimulation with CD154 and IL4. Using this system, we observed a 30- to 40-fold induction of wild-type p53 protein in 50 distinct human CLL specimens tested, without the induction of either cell-cycle arrest or apoptosis. In contrast, the mRNA levels for p53 did not increase, indicating that its elevation occurred posttranscriptionally. Mechanistic investigations revealed that under the conditions studied, p53 was phosphorylated on residues associated with p53 activation and increased half-life. However, p53 protein induced in this manner could transcriptionally activate only a subset of target genes. The addition of a DNA-damaging agent further upregulated p53 protein levels, which led to apoptosis. p53 induction relied on the increase in intracellular reactive oxygen species observed after CD154 and IL4 stimulation. We propose that chronic oxidative stress is a characteristic of the microenvironment in B-cell "proliferation centers" in CLL that are capable of elevating the basal expression of p53, but to levels below the threshold needed to induce arrest or apoptosis. Our findings suggest that reactivation of the full transcriptional activities of p53 in proliferating CLL cells may offer a possible therapeutic strategy. Cancer Res; 76(21); 6311-9. ©2016 AACR.
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Affiliation(s)
- Jesvin Samuel
- Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.,Mechanisms of Cancer and Ageing Lab, University of Leicester, Leicester, United Kingdom.,Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | - Sandrine Jayne
- Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom.,Department of Cancer Studies, University of Leicester, Leicester, United Kingdom
| | - Yixiang Chen
- Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.,Mechanisms of Cancer and Ageing Lab, University of Leicester, Leicester, United Kingdom.,Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | | | - Alice Wignall
- Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.,Mechanisms of Cancer and Ageing Lab, University of Leicester, Leicester, United Kingdom.,Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | - Timothy Wormull
- Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.,Mechanisms of Cancer and Ageing Lab, University of Leicester, Leicester, United Kingdom.,Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | - Hishyar Najeeb
- CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom.,Department of Cancer Studies, University of Leicester, Leicester, United Kingdom.,Department of Clinical Biochemistry, College of Medicine, University of Duhok, Kurdistan Regional Government, Iraq
| | - Jin-Li Luo
- CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | - George D D Jones
- CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom.,Department of Cancer Studies, University of Leicester, Leicester, United Kingdom
| | - Salvador Macip
- Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom. .,Mechanisms of Cancer and Ageing Lab, University of Leicester, Leicester, United Kingdom.,Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom
| | - Martin J S Dyer
- Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester, United Kingdom.,CRUK Leicester Centre, University of Leicester, Leicester, United Kingdom.,Department of Cancer Studies, University of Leicester, Leicester, United Kingdom
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16
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Crassini K, Shen Y, Mulligan S, Giles Best O. Modeling the chronic lymphocytic leukemia microenvironment in vitro. Leuk Lymphoma 2016; 58:266-279. [PMID: 27756161 DOI: 10.1080/10428194.2016.1204654] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Microenvironments within the lymph node and bone marrow promote proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Successful treatment of CLL must therefore target the leukemic cells within these compartments. A better understanding of the interaction between CLL cells and the tumor microenvironment has led to the development of in vitro models that mimic the mechanisms that support leukemic cell survival and proliferation in vivo. Employing these models as part of the pre-clinical evaluation of novel therapeutic agents enables a better approximation of their potential clinical efficacy. In this review we summarize the current literature describing how different aspects of the tumor microenvironment have been modeled in vitro and detail how these models have been employed to study the biology of the disease and potential efficacy of novel therapeutic agents.
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Affiliation(s)
- Kyle Crassini
- a Northern Blood Research Centre , Kolling Institute of Medical Research, Royal North Shore Hospital , Sydney , Australia
| | - Yandong Shen
- a Northern Blood Research Centre , Kolling Institute of Medical Research, Royal North Shore Hospital , Sydney , Australia
| | - Stephen Mulligan
- a Northern Blood Research Centre , Kolling Institute of Medical Research, Royal North Shore Hospital , Sydney , Australia.,b Chronic Lymphocytic Leukemia Research Consortium (CLLARC) , Australia
| | - O Giles Best
- a Northern Blood Research Centre , Kolling Institute of Medical Research, Royal North Shore Hospital , Sydney , Australia.,b Chronic Lymphocytic Leukemia Research Consortium (CLLARC) , Australia
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17
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Holmes PJ, Peiper SC, Uppal GK, Gong JZ, Wang ZX, Bajaj R. Efficacy of DSP30-IL2/TPA for detection of cytogenetic abnormalities in chronic lymphocytic leukaemia/small lymphocytic lymphoma. Int J Lab Hematol 2016; 38:483-9. [PMID: 27565124 DOI: 10.1111/ijlh.12513] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2015] [Accepted: 03/14/2016] [Indexed: 11/29/2022]
Abstract
INTRODUCTION Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia in the Western Hemisphere. Cytogenetic abnormalities in CLL are used for diagnosis, prognosis and treatment. However, detecting these is difficult because mature B cells do not readily divide in culture. Here, we present data on two mitogen cocktails: CpG-oligonucleotide DSP30/Interleukin-2 (IL-2) and DSP30/IL-2 in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS We analysed 165 cases of CLL with FISH and cytogenetics from January 2011 to June 2013. In 2011, three cultures were set-up: unstimulated, DSP30/IL-2-stimulated and TPA-stimulated. In 2012-2013, two cultures were set-up: unstimulated and stimulated with TPA/DSP30/IL-2. RESULTS In 2011, FISH had a detection rate of 91% and cytogenetics using DSP30/IL2 had a detection rate of 91% (n = 22). In 2012-2013, FISH had a detection rate of 79% and cytogenetics using TPA/DSP30/IL-2 had a detection rate of 98% (n = 40). The percentage of cases with normal FISH but abnormal cytogenetics increased from 9% in 2011 to 21% in 2012-2013. The TPA/DSP30/IL-2 cultures in 2012-2013 detected more novel abnormalities (n = 5) as compared to DSP30/IL-2 alone (n = 3). CONCLUSIONS TPA/DSP30/IL2 was as good as or better than DSP30/IL2 alone. TPA/DSP30/IL-2 offers a high detection rate for CLL abnormalities with a single stimulated culture and may increase detection of clinically significant abnormalities.
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Affiliation(s)
- P J Holmes
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA
| | - S C Peiper
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA
| | - G K Uppal
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA
| | - J Z Gong
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA
| | - Z-X Wang
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA
| | - R Bajaj
- The Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, PA, USA.
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18
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Toll-like receptors signaling: A complex network for NF-κB activation in B-cell lymphoid malignancies. Semin Cancer Biol 2016; 39:15-25. [DOI: 10.1016/j.semcancer.2016.07.001] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Revised: 07/06/2016] [Accepted: 07/07/2016] [Indexed: 11/17/2022]
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19
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Patients with chronic lymphocytic leukaemia (CLL) differ in the pattern of CTLA-4 expression on CLL cells: the possible implications for immunotherapy with CTLA-4 blocking antibody. Tumour Biol 2015; 37:4143-57. [PMID: 26490985 PMCID: PMC4844645 DOI: 10.1007/s13277-015-4217-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Accepted: 10/12/2015] [Indexed: 12/28/2022] Open
Abstract
Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67+ cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.
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20
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CTLA-4 affects expression of key cell cycle regulators of G0/G1 phase in neoplastic lymphocytes from patients with chronic lymphocytic leukaemia. Clin Exp Med 2015; 16:317-32. [PMID: 26003188 PMCID: PMC4969362 DOI: 10.1007/s10238-015-0360-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 05/14/2015] [Indexed: 10/31/2022]
Abstract
Previously, we showed that cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is overexpressed in chronic lymphocytic leukaemia (CLL) and its expression is correlated with the expression of the major regulators of G1 phase progression: cyclins D2 and D3, and cyclin-dependent kinase inhibitory protein 1 (p27 (KIP1) ). In the present study, we blocked CTLA-4 on the surface of both CLL cells and normal B lymphocytes to investigate the impact of CTLA-4 on the expression of the mentioned G1 phase regulators. We found that in CLL patients and in healthy individuals, the median proportions of cyclin D2-positive cells as well as cyclin D3(+) cells significantly decreased following CTLA-4 blockade. Moreover, CTLA-4 blockade led to an increase in the median frequencies of p27 (KIP1) -positive cells, although this increase was marked only in CLL patients. Our study showed that CTLA-4 affects the expression of the key regulators of G1 phase progression in CLL cells as well as in normal B lymphocytes and may contribute to a better understanding of the role of CTLA-4 in the regulation of G1 phase progression.
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21
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Porakishvili N, Vispute K, Steele AJ, Rajakaruna N, Kulikova N, Tsertsvadze T, Nathwani A, Damle RN, Clark EA, Rai KR, Chiorazzi N, Lydyard PM. Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor. Mol Med 2015; 21:46-57. [PMID: 25611435 DOI: 10.2119/molmed.2014.00265] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2014] [Accepted: 01/05/2015] [Indexed: 12/23/2022] Open
Abstract
Chronic lymphocytic leukemia (CLL) development and progression are thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR) and environmental signals for survival and expansion including toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either prosurvival Bruton tyrosine kinase (BTK)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT-mediated, or proapoptotic p38 mitogen-activated protein kinase (p38MAPK)-mediated signaling pathways, while selective immunoglobulin M (sIgM) ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pretreatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the prosurvival BTK/PI3K/AKT toward the proapoptotic p38MAPK pathway. Thus preengaging CD180 could prevent further prosurvival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients.
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Affiliation(s)
- Nino Porakishvili
- Faculty of Science and Technology, University of Westminster, London, United Kingdom
| | - Ketki Vispute
- Faculty of Science and Technology, University of Westminster, London, United Kingdom
| | | | - Nadeeka Rajakaruna
- Faculty of Science and Technology, University of Westminster, London, United Kingdom
| | - Nina Kulikova
- Faculty of Science and Technology, University of Westminster, London, United Kingdom.,Javakhishvili Tbilisi State University, Georgia
| | | | - Amit Nathwani
- UCL Cancer Institute, University College London, London, United Kingdom
| | - Rajendra N Damle
- The Feinstein Institute for Medical Research, Manhasset, New York, United States of America
| | - Edward A Clark
- University of Washington, Seattle, Washington, United States of America
| | - Kanti R Rai
- The Feinstein Institute for Medical Research, Manhasset, New York, United States of America
| | - Nicholas Chiorazzi
- The Feinstein Institute for Medical Research, Manhasset, New York, United States of America
| | - Peter M Lydyard
- Faculty of Science and Technology, University of Westminster, London, United Kingdom
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22
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Differential expression of Toll-like receptor (TLR) and B cell receptor (BCR) signaling molecules in primary diffuse large B-cell lymphoma of the central nervous system. J Neurooncol 2014; 121:289-96. [PMID: 25391967 DOI: 10.1007/s11060-014-1655-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2014] [Accepted: 10/26/2014] [Indexed: 12/22/2022]
Abstract
Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a distinct and aggressive lymphoma that is confined to CNS. Since, central nervous system is barrier-protected and immunologically silent; role of TLR/BCR signaling in pathogenesis and biology of CNS DLBCL is intriguing. Genomic mutations in key regulators of TLR/BCR signaling pathway (MYD88/CD79B/CARD11) have recently been reported in this disease. These observations raised possible implications in novel targeted therapies; however, expression pattern of molecules related to TLR/BCR pathways in this lymphoma remains unknown. We have analyzed the expression of 19 genes encoding TLR/BCR pathways and targets in CNS DLBCLs (n = 20) by Nanostring nCounter™ analysis and compared it with expression patterns in purified reactive B-lymphocytes and systemic diffuse large B cell lymphoma (DLBCL) (n = 20). Relative expression of TLR4, TLR5, TLR9, CD79B and BLNK was higher in CNS DLBCLs than in control B-lymphocytes; where as TLR7, MALT1, BCL10, CD79A and LYN was lower in CNS DLBCLs (P < 0.0001). When compared with systemic DLBCL samples, higher expression of TLR9, CD79B, CARD11, LYN and BLNK was noted in CNS DLBCL (>1.5 fold change; P < 0.01). The B cell receptor molecules like BLNK and CD79B were also associated with higher expression of MYD88 dependent TLRs (TLR4/5/9). In conclusion, we have shown over expression of TLR/BCR related genes or their targets, where genomic mutations have commonly been identified in CNS DLBCL. We have also demonstrated that TLR over expression closely relate with up regulation of genes associated with BCR pathway like CD79B/BLNK and CARD11, which play an important role in NF-kB pathway activation. Our results provide an important insight into the possibility of TLR and/or B-cell receptor signaling molecules as possible therapeutic targets in CNS DLBCL.
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23
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Liaw FPS, Lau LC, Lim AST, Lim TH, Lee GY, Tien SL. CpG Oligonucleotide and Interleukin 2 stimulation enables higher cytogenetic abnormality detection rates than 12-o-tetradecanolyphorbol-13-acetate in Asian patients with B-cell chronic lymphocytic leukemia (B-CLL). Int J Hematol 2014; 100:545-53. [PMID: 25301672 DOI: 10.1007/s12185-014-1681-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2014] [Revised: 09/19/2014] [Accepted: 09/21/2014] [Indexed: 10/24/2022]
Abstract
The present study was designed to compare abnormality detection rates using DSP30 + IL2 and 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Asian patients with B-CLL. Hematological specimens from 47 patients (29 newly diagnosed, 18 relapsed) were established as 72 h-DSP30 + IL2 and TPA cultures. Standard methods were employed to identify clonal aberrations by conventional cytogenetics (CC). The B-CLL fluorescence in situ hybridization (FISH) panel comprised ATM, CEP12, D13S25, and TP53 probes. DSP30 + IL2 cultures had a higher chromosomal abnormality detection rate (67 %) compared to TPA (44 %, p < 0.001). The mean number of analyzable metaphases and abnormal metaphases per slide was also higher (p < 0.005, p < 0.001, respectively). Culture success rate, percentage of complex karyotype, and percentage of non-clonal abnormal cell were not significantly different (p > 0.05). Thirteen cases with abnormalities were found exclusively in DSP30 + IL2 cultures compared to one found solely in TPA cultures. DSP30 + IL2 cultures were comparable to the FISH panel in detecting 11q-, +12 and 17p- but not 13q-. It also has a predilection for 11q- bearing leukemic cells compared to TPA. FISH had a higher abnormality detection rate (84.1 %) compared to CC (66.0 %) with borderline significance (p = 0.051), albeit limited by its coverage. In conclusion, DSP30 + IL2 showed a higher abnormality detection rate. However, FISH is indispensable to circumvent low mitotic indices and detect subtle abnormalities.
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Affiliation(s)
- Fiona Pui San Liaw
- Cytogenetics Laboratory, Department of Pathology, Singapore General Hospital, Academia, Level 9, Diagnostics Tower, 20 College Road, Singapore, 169856, Singapore,
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Brejcha M, Stoklasová M, Brychtová Y, Panovská A, Štěpanovská K, Vaňková G, Plevová K, Oltová A, Horká K, Pospíšilová Š, Mayer J, Doubek M. Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: a prospective analysis. Leuk Res 2013; 38:170-5. [PMID: 24246692 DOI: 10.1016/j.leukres.2013.10.019] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2013] [Revised: 10/20/2013] [Accepted: 10/22/2013] [Indexed: 11/25/2022]
Abstract
Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p=0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period.
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Affiliation(s)
- Martin Brejcha
- Department of Hematology, Hospital Novy Jicin, Czech Republic
| | - Martina Stoklasová
- Laboratory of Medical Genetics - Department of Cytogenetics, AGEL Research and Training Institute - Novy Jicin Branch, AGEL Laboratories, Czech Republic
| | - Yvona Brychtová
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Anna Panovská
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Kristina Štěpanovská
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Gabriela Vaňková
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Karla Plevová
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic; Central European Institute of Technology, Masaryk University, Brno, Czech Republic
| | - Alexandra Oltová
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Kateřina Horká
- Laboratory of Medical Genetics - Department of Cytogenetics, AGEL Research and Training Institute - Novy Jicin Branch, AGEL Laboratories, Czech Republic
| | - Šárka Pospíšilová
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic; Central European Institute of Technology, Masaryk University, Brno, Czech Republic
| | - Jiří Mayer
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic; Central European Institute of Technology, Masaryk University, Brno, Czech Republic
| | - Michael Doubek
- Department of Internal Medicine - Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic; Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
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25
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Shi M, Cipollini MJ, Crowley-Bish PA, Higgins AW, Yu H, Miron PM. Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation. Am J Clin Pathol 2013; 139:662-9. [PMID: 23596118 DOI: 10.1309/ajcp7g4vmyzjqvfi] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.
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Affiliation(s)
- Min Shi
- Departments of Pathology, UMASS Memorial Medical Center, Worcester, MA
| | | | | | - Anne W. Higgins
- Departments of Pathology, UMASS Memorial Medical Center, Worcester, MA
- Hospital Laboratories, UMASS Memorial Medical Center, Worcester, MA
| | - Hongbo Yu
- Departments of Pathology, UMASS Memorial Medical Center, Worcester, MA
| | - Patricia M. Miron
- Departments of Pathology, UMASS Memorial Medical Center, Worcester, MA
- Hospital Laboratories, UMASS Memorial Medical Center, Worcester, MA
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López de Frutos L, Álvarez Y, Armengol G, Caballín MR. New mitogens in cultures for multiple myeloma cytogenetic analysis. Leuk Lymphoma 2013; 54:2548-50. [PMID: 23418873 DOI: 10.3109/10428194.2013.777066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Laura López de Frutos
- Department of Cytogenetics, Balagué Center Special Techniques Laboratory, Hospitalet de Llobregat , Barcelona , Spain
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27
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Rütgen BC, Willenbrock S, Reimann-Berg N, Walter I, Fuchs-Baumgartinger A, Wagner S, Kovacic B, Essler SE, Schwendenwein I, Nolte I, Saalmüller A, Escobar HM. Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model. PLoS One 2012; 7:e40078. [PMID: 22761949 PMCID: PMC3386195 DOI: 10.1371/journal.pone.0040078] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2012] [Accepted: 06/05/2012] [Indexed: 11/24/2022] Open
Abstract
Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.
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Affiliation(s)
- Barbara C. Rütgen
- Central Laboratory, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Saskia Willenbrock
- Small Animal Clinic and Research Cluster of Excellence ‘REBIRTH’, University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
| | - Nicola Reimann-Berg
- Small Animal Clinic and Research Cluster of Excellence ‘REBIRTH’, University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
| | - Ingrid Walter
- VetBioBank, VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Andrea Fuchs-Baumgartinger
- Institute of Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Siegfried Wagner
- Small Animal Clinic and Research Cluster of Excellence ‘REBIRTH’, University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
| | - Boris Kovacic
- Department of Biomedical Sciences, Translational Oncology, Institute for Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Sabine E. Essler
- Department of Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Ilse Schwendenwein
- Central Laboratory, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Ingo Nolte
- Small Animal Clinic and Research Cluster of Excellence ‘REBIRTH’, University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
| | - Armin Saalmüller
- Department of Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Hugo Murua Escobar
- Small Animal Clinic and Research Cluster of Excellence ‘REBIRTH’, University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany
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The miR-17∼92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes. Leukemia 2012; 26:1584-93. [PMID: 22343732 DOI: 10.1038/leu.2012.44] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17∼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17∼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P ≤ 0.05). Consistently, transfection with miR-17∼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70(high) (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17∼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.
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Xiaoxia Z, Weihua N, Qingyong Z, Fengli W, Yingying L, Xiaxia S, Zhonghui L, Guixiang T. Maltose-binding protein isolated from Escherichia coli induces Toll-like receptor 2-mediated viability in U937 cells. Clin Transl Oncol 2011; 13:509-18. [PMID: 21775279 DOI: 10.1007/s12094-011-0689-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
BACKGROUND Stimulation of Toll-like receptors (TLRs) by microbial products has been utilised to potentiate immune responses against haematologic malignancies. The maltose-binding protein (MBP) of Escherichia coli could induce the activation of immune cells via TLR4. The aim of the present study was to investigate whether TLRs mediated the biological effects of MBP on U937 and Jurkat cells in vitro. METHODS We observed the effect of MBP on U937 and Jurkat cells by using the WST, cell cycle analysis and morphological observation. Further, cells were stimulated with MBP for indicated times and doses, and detected by RT-PCR, western blotting, immunohistochemistry and immunofluorescence staining to investigate the mechanisms involved in cell viability. RESULTS MBP enhanced the viability of U937 and Jurkat cells, and the effects were blocked by anti-TLR2, but not anti-TLR4 in U937 cells. Further studies confirmed that MBP was able to directly bind to U937 and Jurkat cells and modulate TLR expression. The effects of MBP depended on the activation of NF-κB and MAP kinase in U937 and Jurkat cells. CONCLUSIONS Our results demonstrated that MBP could directly promote U937 cell viability via TLR2. It suggested that MBP may be used as an adjuvant for participating in the immunotherapy of haematologic malignancies.
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Affiliation(s)
- Zhao Xiaoxia
- Department of Immunology, Jilin University, 2 Xinmin Avenue, Changchun, Ji Lin 130021, People's Republic of China
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30
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Reimann-Berg N, Murua Escobar H, Kiefer Y, Mischke R, Willenbrock S, Eberle N, Nolte I, Bullerdiek J. Cytogenetic analysis of CpG-oligonucleotide DSP30 plus Interleukin-2-Stimulated canine B-Cell lymphoma cells reveals the loss of one X Chromosome as the sole abnormality. Cytogenet Genome Res 2011; 135:79-82. [PMID: 21811057 DOI: 10.1159/000330126] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.
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Affiliation(s)
- N Reimann-Berg
- Small Animal Clinic and Research Cluster REBIRTH, University of Veterinary Medicine Hannover, Germany. nicola.reimann-berg @ tiho-hannover.de
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31
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Fabricius D, Breckerbohm L, Vollmer A, Queudeville M, Eckhoff SM, Fulda S, Strauss G, Debatin KM, Jahrsdörfer B, Meyer LH. Acute lymphoblastic leukemia cells treated with CpG oligodeoxynucleotides, IL-4 and CD40 ligand facilitate enhanced anti-leukemic CTL responses. Leukemia 2011; 25:1111-21. [PMID: 21527935 DOI: 10.1038/leu.2011.87] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the majority of patients initially respond to upfront chemotherapy, relapses with poor prognosis occur in approximately 20% of cases. Thus, novel therapeutic strategies are required to improve long-term survival. B-cell precursor (BCP)-ALL cells express low levels of immunogenic molecules and, therefore, are poorly recognized by the immune system. In the present study, we investigated the effect of various combinations of potent B-cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of primary BCP-ALL cells and a series of BCP-ALL cell lines. The combination of CpG, IL-4 and CD40L was identified as most effective to enhance expression of immunogenic molecules on BCP-ALL cells, resulting in an increased capacity to induce both allogeneic and autologous cytotoxic T lymphocytes (CTL). Importantly, such CTL exhibited significant anti-leukemic cytotoxicity not only towards treated, but also towards untreated BCP-ALL cells. Our results demonstrate that the combination of CpG with other B-cell stimulators is more efficient than CpG alone in generating immunogenic BCP-ALL cells and anti-leukemic CTL. Our results may stimulate the development of novel adoptive T cell transfer approaches for the management of BCP-ALL.
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Affiliation(s)
- D Fabricius
- Department of Pediatrics, University of Ulm, Ulm, Germany.
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Rocha CK, Praulich I, Gehrke I, Hallek M, Kreuzer KA. A rare case of t(11;22) in a mantle cell lymphoma like B-cell neoplasia resulting in a fusion of IGL and CCND1: case report. Mol Cytogenet 2011; 4:8. [PMID: 21457541 PMCID: PMC3077317 DOI: 10.1186/1755-8166-4-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2010] [Accepted: 04/01/2011] [Indexed: 12/20/2022] Open
Abstract
The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease. Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas.
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Increased expression of Toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma. Arch Oral Biol 2011; 56:877-84. [PMID: 21333270 DOI: 10.1016/j.archoralbio.2011.01.010] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2010] [Revised: 01/19/2011] [Accepted: 01/21/2011] [Indexed: 11/21/2022]
Abstract
OBJECTIVE Toll-like receptor-9 (TLR-9), a new member of the interleukin-1 receptor superfamily, was recently found to have a high level of expression in many carcinoma specimens. The objective of this study was to examine the TLR-9 expression and its role in tumour cell proliferation in oral squamous cell carcinoma. MATERIALS AND METHODS Western blot and immunohistochemistry were used to detect TLR-9 protein in oral squamous cell carcinoma (OSCC) Tca-8113 cell lines and clinical specimens (n=60). The relationship between TLR-9 expression and clinicopathologic features was analysed. Cell proliferation and inflammatory chemokines secretion were tested by MTT and ELISA methods respectively. RESULTS Results showed that TLR-9 expression level was higher in OSCC tissues than in paired adjacent normal tissues (P<0.01), and the expression level of TLR-9 was significantly associated with tumour size (P=0.001), tumour clinical stage (P=0.003) and Ki-67 expression (P<0.01). In vitro results also suggested that stimulation of Tca-8113 cells with TLR-9 agonist CpG-ODN could significantly increase tumour cell proliferation as well as subsequent IL-1α and IL-6 secretions (P<0.01), which could be partially inhibited by usage of anti-TLR-9 protein. CONCLUSIONS It was therefore hypothesized that increased expression of TLR-9 may be of great value in assessing the development of OSCC, and could be used as a new target for OSCC prevention and therapy in future.
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Muthusamy N, Breidenbach H, Andritsos L, Flynn J, Jones J, Ramanunni A, Mo X, Jarjoura D, Byrd JC, Heerema NA. Enhanced detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide in combination with pokeweed mitogen and phorbol myristate acetate. Cancer Genet 2011; 204:77-83. [PMID: 21494579 PMCID: PMC3073597 DOI: 10.1016/j.cancergen.2010.12.006] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Reproducible cytogenetic analysis in CLL has been limited by the inability to obtain reliable metaphase cells for analysis. CpG oligonucleotide and cytokine stimulation have been shown to improve metaphase analysis of CLL cytogenetic abnormalities, but is limited by variability in the cytokine receptor levels, stability and biological activity of the cytokine in culture conditions and high costs associated with these reagents. We report here use of a novel, stable CpG, GNKG168 along with pokeweed mitogen (PWM) and phorbol 12-myristate 13-acetate (PMA) for conventional cytogenetic assessment in CLL. We demonstrate that the combined use of GNKG168+PWM/PMA increased the sensitivity of detection of chromosomal abnormalities compared to PWM/PMA (n=207, odds ratio=2.2, p=0.0002) and GNKG168 (n=219, odds ratio=1.5, p=0.0452). Further, a significant increase in sensitivity to detect complexity ≥3 with GNKG168+PWM/PMA compared to GNKG168 alone (odds ratio 8.0, p=0.0022) or PWM/PMA alone (odds ratio 9.6, p=0.0007) was observed. The trend toward detection of higher complexity was significantly greater with GNKG168+PWM/PMA compared to GNKG168 alone (p=0.0412). The increased sensitivity was mainly attributed to the addition of PWM/PMA with GNKG168 because GNKG168 alone showed no difference in sensitivity for detection of complex abnormalities (p=0.17). Comparison of fluorescence in situ hybridization (FISH) results with karyotypic results showed a high degree of consistency, although some complex karyotypes were present in cases with no adverse FISH abnormality. These studies provide evidence for potential use of GNKG168 in combination with PWM and PMA in karyotypic analysis of CLL patient samples to better identify chromosomal abnormalities for risk stratification.
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Affiliation(s)
- Natarajan Muthusamy
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | | | - Leslie Andritsos
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | - Joseph Flynn
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | - Jeffrey Jones
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | - Asha Ramanunni
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | - Xiaokui Mo
- Center for Biostatistics, The Ohio State University, Columbus, OH
| | - David Jarjoura
- Center for Biostatistics, The Ohio State University, Columbus, OH
| | - John C. Byrd
- Division of Hematology, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH
| | - Nyla A. Heerema
- Department of Pathology, The Ohio State University, Columbus, OH
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Heerema NA, Byrd JC, Dal Cin PS, Dell' Aquila ML, Koduru PRK, Aviram A, Smoley SA, Rassenti LZ, Greaves AW, Brown JR, Rai KR, Kipps TJ, Kay NE, Van Dyke DL, Chronic Lymphocytic Leukemia Research Consortium. Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) Study. CANCER GENETICS AND CYTOGENETICS 2010; 203:134-40. [PMID: 21156225 PMCID: PMC3018693 DOI: 10.1016/j.cancergencyto.2010.07.128] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 06/11/2010] [Accepted: 07/08/2010] [Indexed: 11/30/2022]
Abstract
Cytogenetic abnormalities are important prognostic indicators in CLL. Historically, only interphase cytogenetics was clinically useful in CLL, because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL cells. The CLL Research Consortium tested the effectiveness and reproducibility of CpG-ODN stimulation for detecting chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with the traditional pokeweed mitogen plus 12-O-tetradecanoylphorbol-13-acetate (PWM+TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control sample and 12 CLL samples showed that, excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Abnormal clones in CLL were more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. With CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture, but did enhance detection of abnormalities and complexity in CLL. Because karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful for identifying this additional prognostic factor in CLL.
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Affiliation(s)
- Nyla A Heerema
- The Ohio State University, 1645 Neil Ave, 129 Hamilton Hall, Columbus, OH 43210, USA.
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36
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B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells. Leukemia 2010; 24:1885-92. [DOI: 10.1038/leu.2010.176] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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LMP-420: a novel purine nucleoside analog with potent cytotoxic effects for CLL cells and minimal toxicity for normal hematopoietic cells. Leukemia 2010; 24:1580-7. [DOI: 10.1038/leu.2010.150] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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38
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Antiapoptotic effect of interleukin-2 (IL-2) in B-CLL cells with low and high affinity IL-2 receptors. Ann Hematol 2010; 89:1125-32. [PMID: 20544350 DOI: 10.1007/s00277-010-0994-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2010] [Accepted: 05/11/2010] [Indexed: 10/19/2022]
Abstract
Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins.
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Muzio M, Bertilaccio MTS, Simonetti G, Frenquelli M, Caligaris-Cappio F. The role of toll-like receptors in chronic B-cell malignancies. Leuk Lymphoma 2010; 50:1573-80. [PMID: 19672768 DOI: 10.1080/10428190903115410] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Toll-like receptors (TLR) are key players in host defence from infection. They recognize a specific set of molecular patterns of microbial origin, immediately trigger an innate immune response, and bridge innate and adaptive immunity. TLR have also been shown to play a role in tumor development. In this context, chronic B-cell malignancies are an interesting example as clonal B lymphocytes remain responsive to and dependent on stimuli originating from the microenvironment which then become crucial for maintaining and propagating the disease. Emerging evidences suggest that, among other microenvironmental elements, TLR ligands may play a role in the pathogenesis of chronic B-cell lymphoid malignancies. Conceivably, their manipulation may find a place in specific settings of treatment of these tumors.
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Affiliation(s)
- Marta Muzio
- Laboratory of Lymphoid Malignancies, Division of Molecular Oncology, San Raffaele Scientific Institute, Milano, Italy
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40
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Chen L, Xu M, Wang ZY, Chen BW, Du WX, Su C, Shen XB, Zhao AH, Dong N, Wang YJ, Wang GZ. The development and preliminary evaluation of a new Mycobacterium tuberculosis vaccine comprising Ag85b, HspX and CFP-10:ESAT-6 fusion protein with CpG DNA and aluminum hydroxide adjuvants. ACTA ACUST UNITED AC 2010; 59:42-52. [PMID: 20298499 DOI: 10.1111/j.1574-695x.2010.00660.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ag85b and HspX of Mycobacterium tuberculosis (Mtb) (H37Rv) were expressed and purified in this study. These two proteins were combined with another fusion protein CFP-10:ESAT-6 (C/E) (Ag), then mixed with the adjuvants CpG DNA and aluminum hydroxide and used to vaccinate mice and guinea pigs challenged with Mtb (H37Rv). The number of spleen lymphocytes secreting Ag85b, HspX and C/E-specific interferon-gamma were significantly higher in the Ag+Al+CpG group than in the Ag and CpG groups. The combination of Ag, Al and CpG induced the highest concentrations of anti-Ag85b, anti-HspX and anti-C/E immunoglobulin G in mouse serum. Mouse peritoneal macrophages from the Ag+Al+CpG group secreted significantly higher levels of interleukin-12 compared with macrophages from the other groups. The total mean liver, lung and spleen lesion scores and bacterial loads in the spleen in guinea pigs vaccinated with Ag+Al+CpG were lower than those of the other groups, but no significant difference was found. These results show that the mixture of Ag85b, HspX and C/E with a combination of CpG and aluminum adjuvants can induce both humoral and cellular immune responses in mice, whereas it plays only a small role in the control of disease progression in guinea pigs challenged with Mtb.
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Affiliation(s)
- Lei Chen
- School of Public Health, Shandong University, Ji'nan, China
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41
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Put N, Konings P, Rack K, Jamar M, Van Roy N, Libouton JM, Vannuffel P, Sartenaer D, Ameye G, Speleman F, Herens C, Poirel HA, Moreau Y, Hagemeijer A, Vandenberghe P, Michaux L. Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study. Genes Chromosomes Cancer 2009; 48:843-53. [PMID: 19582829 DOI: 10.1002/gcc.20691] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B-cell mitogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin-2 (IL-2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL-2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL-2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL-2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL-2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.
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Affiliation(s)
- Natalie Put
- Centrum voor Menselijke Erfelijkheid, Katholieke Universiteit Leuven, Leuven, Belgium
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42
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Weiner GJ. CpG oligodeoxynucleotide-based therapy of lymphoid malignancies. Adv Drug Deliv Rev 2009; 61:263-7. [PMID: 19168102 DOI: 10.1016/j.addr.2008.12.006] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/15/2008] [Indexed: 01/22/2023]
Abstract
Preclinical and early clinical trials indicate synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG ODN) have potent immunostimulatory effects. CpG ODN are being explored as immune adjuvants in vaccination strategies and as potential treatments for a wide variety of disorders including cancer and asthma. Therapeutic approaches designed to take advantage of this potent class of agents are based largely on the ability of CpG ODN to activate professional antigen presenting cells (APCs) that express the target receptor - Toll-Like Receptor 9 (TLR9). B-cell malignancies are unique in that the malignant cells themselves express TLR9. CpG ODN can have a direct effect on the malignant B cells and lead to activation induced cell death. CpG ODN also alter the phenotype of target malignant B cells as indicated by upregulation of MHC, immunostimulatory molecules, and antigens that serve as targets for other approaches to lymphoma immunotherapy such as CD20. B cell malignancies are also relatively sensitive to the cytokines that are produced by dendritic cells in response to CpG ODN. Thus, B cell malignancies appear to be uniquely sensitive to CpG ODN because of both the direct and indirect effects the CpG ODN on target cells and the sensitivity of B cell malignancies to an immune response. Preclinical studies support further exploration of the potential of CpG ODN as a component of therapy for lymphoid malignancies. Ongoing clinical trials are exploring the potential of CpG ODN, both alone and in combination with other agents.
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Affiliation(s)
- George J Weiner
- Holden Comprehensive Cancer Center at the University of Iowa, Department of Internal Medicine, Iowa City, 52242, USA.
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Lejeune M, Rybicka JM, Chadee K. Recent discoveries in the pathogenesis and immune response toward Entamoeba histolytica. Future Microbiol 2009; 4:105-18. [PMID: 19207103 DOI: 10.2217/17460913.4.1.105] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Entamoeba histolytica is an enteric dwelling human protozoan parasite that causes the disease amoebiasis, which is endemic in the developing world. Over the past four decades, considerable effort has been made to understand the parasite and the disease. Improved diagnostics can now differentiate pathogenic E. histolytica from that of the related but nonpathogenic Entamoeba dispar, thus minimizing screening errors. Classically, the triad of Gal-lectin, cysteine proteinases and amoebapores of the parasite were thought to be the major proteins involved in the pathogenesis of amoebiasis. However, other amoebic molecules such as lipophosphopeptidoglycan, perioxiredoxin, arginase, and lysine and glutamic acid-rich proteins are also implicated. Recently, the genome of E. histolytica has been sequenced, which has widened our scope to study additional virulence factors. E. histolytica genome-based approaches have now confirmed the presence of Golgi apparatus-like vesicles and the machinery for glycosylation, thus improving the chances of identifying potential drug targets for chemotherapeutic intervention. Apart from Gal-lectin-based vaccines, promising vaccine targets such as serine-rich E. histolytica protein have yielded encouraging results. Considerable efforts have also been made to skew vaccination responses towards appropriate T-helper cell immunity that could augment the efficacy of vaccine candidates under study. Thus, ongoing efforts mining the information made available with the sequencing of the E. histolytica genome will no doubt identify and characterize other important potential vaccine/drug targets and lead to effective immunologic strategies for the control of amoebiasis.
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Affiliation(s)
- Manigandan Lejeune
- University of Calgary, Department of Microbiology & Infectious Diseases, Calgary, AB, T2N 4N1, Canada.
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44
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Toll-like receptors and their role in carcinogenesis and anti-tumor treatment. Cell Mol Biol Lett 2008; 14:248-72. [PMID: 19096763 PMCID: PMC6275910 DOI: 10.2478/s11658-008-0048-z] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2008] [Accepted: 12/09/2008] [Indexed: 01/07/2023] Open
Abstract
Toll-like receptors (TLRs) have been described as major components of the innate immune system, recognizing the conserved molecular structures found in the large groups of pathogens called pathogen-associated molecular patterns (PAMPs). TLR expression is ubiquitous, from epithelial to immunocompetent cells. TLR ligation triggers several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators but also anti-inflammatory and anti-tumor cytokines. The result of this activation goes beyond innate immunity to shape the adaptive responses against pathogens and tumor cells, and maintains host homeostasis via cell debris utilization. TLRs have already become potent targets in infectious disease treatment and vaccine therapy and in neoplastic disease treatment, due to their ability to enhance antigen presentation. However, some studies show the dual effect of TLR stimulation on malignant cells: they can be proapoptotic or promote survival under different conditions. It is therefore crucial to design further studies assessing the biology of these receptors in normal and transformed cells. The established role of TLRs in human disease therapy is based on TLR7 and TLR4 agonists, respectively for the novel treatment of some types of skin cancer and for the anti-hepatitis B virus vaccine. Some clinical trials involving TLR agonists as potent enhancers of the anti-tumor response in solid tumors have begun.
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45
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Muzio M, Scielzo C, Bertilaccio MTS, Frenquelli M, Ghia P, Caligaris-Cappio F. Expression and function of toll like receptors in chronic lymphocytic leukaemia cells. Br J Haematol 2008; 144:507-16. [PMID: 19036098 DOI: 10.1111/j.1365-2141.2008.07475.x] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Mature B-cells can recognize microbial antigens via B-cell-receptor (BCR) in a specific way and via Toll-like receptors (TLR) in a costimulatory manner. A wealth of information is gathering on the possible role of antigenic stimulation in the natural history of Chronic Lymphocytic Leukaemia (CLL). However little is known regarding the repertoire and function of TLR in CLL cells. The TLR family includes 10 different transmembrane proteins devoted to recognize specific pathogen-associated molecular patterns and to alarm immunocompetent cells to trigger an immune response. Here, we studied fresh leukaemic cells for the expression pattern of TLR1 to TLR10, NOD1, NOD2 and SIGIRR (also known as TIR8). CLL cells were found to express several pattern recognition receptors including TLR1, TLR2, TLR6, TLR10, NOD1 and NOD2. The specific TLR expressed by CLL cells were functional. Leukaemic cells, upon stimulation with TLR1/2/6 ligands, such as bacterial lipopeptides, activated the nuclear factor-kappaB signalling pathway, expressed CD86 and CD25 activation molecules, and were protected from spontaneous apoptosis. These findings further support the hypothesis that CLL cells resemble antigen-activated B-cells and suggest a potential role of TLR in modulating CLL cell response in the context of specific antigen recognition.
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Affiliation(s)
- Marta Muzio
- Department of Oncology, Unit and Laboratory of Lymphoid Malignancies, Istituto Scientifico San Raffaele and Università Vita-Salute San Raffaele, Milano, Italy
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46
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Struski S, Gervais C, Helias C, Herbrecht R, Audhuy B, Mauvieux L. Stimulation of B-cell lymphoproliferations with CpG-oligonucleotide DSP30 plus IL-2 is more effective than with TPA to detect clonal abnormalities. Leukemia 2008; 23:617-9. [PMID: 18830262 DOI: 10.1038/leu.2008.252] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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47
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Abstract
The humoral immune system senses microbes via recognition of specific microbial molecular motifs by Toll-like receptors (TLRs). These encounters promote plasma cell differentiation and antibody production. Recent studies have demonstrated the importance of the TLR system in enhancing antibody-mediated defense against infections and maintaining memory B cells. These results have led the way to the design of vaccines that target B cells by engaging TLRs. In hematologic malignancies, cells often retain B cell-specific receptors and associated functions. Among these, TLRs are currently exploited to target different subclasses of B-cell leukemia, and TLR agonists are currently being evaluated in clinical trials. However, accumulating evidence suggests that endogenous TLR ligands or chronic infections promote tumor growth, thus providing a need for further investigations to decipher the exact function of TLRs in the B-cell lineage and in neoplastic B cells. The aim of this review is to present and discuss the latest advances with regard to the expression and function of TLRs in both healthy and malignant B cells. Special attention will be focused on the growth-promoting effects of TLR ligands on leukemic B cells and their potential clinical impact.
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48
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Andritsos LA, Johnson AJ, Lozanski G, Blum W, Kefauver C, Awan F, Smith LL, Lapalombella R, May SE, Raymond CA, Wang DS, Knight RD, Ruppert AS, Lehman A, Jarjoura D, Chen CS, Byrd JC. Higher doses of lenalidomide are associated with unacceptable toxicity including life-threatening tumor flare in patients with chronic lymphocytic leukemia. J Clin Oncol 2008; 26:2519-25. [PMID: 18427150 PMCID: PMC4312490 DOI: 10.1200/jco.2007.13.9709] [Citation(s) in RCA: 123] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
PURPOSE Lenalidomide is a novel therapeutic agent with uncertain mechanism of action that is clinically active in myelodysplastic syndrome (MDS) and multiple myeloma (MM). Application of high (MM) and low (MDS) doses of lenalidomide has been reported to have clinical activity in CLL. Herein, we highlight life-threatening tumor flare when higher doses of lenalidomide are administered to patients with CLL and provide a potential mechanism for its occurrence. PATIENTS AND METHODS Four patients with relapsed CLL were treated with lenalidomide (25 mg/d for 21 days of a 28-day cycle). Serious adverse events including tumor flare and tumor lysis are summarized. In vitro studies examining drug-induced apoptosis and activation of CLL cells were also performed. RESULTS Four consecutive patients were treated with lenalidomide; all had serious adverse events. Tumor flare was observed in three patients and was characterized by dramatic and painful lymph node enlargement resulting in hospitalization of two patients, with one fatal outcome. Another patient developed sepsis and renal failure. In vitro studies demonstrated lenalidomide-induced B-cell activation (upregulation of CD40 and CD86) corresponding to degree of tumor flare, possibly explaining the tumor flare observation. CONCLUSION Lenalidomide administered at 25 mg/d in relapsed CLL is associated with unacceptable toxicity; the rapid onset and adverse clinical effects of tumor flare represent a significant limitation of lenalidomide use in CLL at this dose. Drug-associated B-cell activation may contribute to this adverse event. Future studies with lenalidomide in CLL should focus on understanding this toxicity, investigating patients at risk, and investigating alternative safer dosing schedules.
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MESH Headings
- Adult
- Antineoplastic Agents/administration & dosage
- Antineoplastic Agents/adverse effects
- Apoptosis/drug effects
- Dose-Response Relationship, Drug
- Flow Cytometry
- Humans
- Lenalidomide
- Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
- Leukemia, Lymphocytic, Chronic, B-Cell/mortality
- Leukemia, Lymphocytic, Chronic, B-Cell/pathology
- Male
- Middle Aged
- Neoplasm Recurrence, Local/drug therapy
- Survival Rate
- Thalidomide/administration & dosage
- Thalidomide/adverse effects
- Thalidomide/analogs & derivatives
- Tumor Cells, Cultured
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Affiliation(s)
- Leslie A Andritsos
- Division of Hematology-Oncology, Department of Medicine, The Ohio State University, Columbus, OH, USA
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Abstract
The mammalian target of rapamycin mTOR is a central element in an evolutionary conserved signalling pathway that regulates cell growth, survival and proliferation, orchestrating signals originating from growth factors, nutrients or particular stress stimuli. Two important modulators of mTOR activity are the AKT and ERK/MAPK signalling pathways. Many studies have shown that mTOR plays an important role in the biology of malignant cells, including deregulation of the cell cycle, inactivation of apoptotic machinery and resistance to chemotherapeutic agents. The development of several mTOR inhibitors, in addition to rapamycin, has facilitated studies of the role of mTOR in cancer, and verified the antitumour effect of mTOR inhibition in many types of neoplasms, including lymphomas. Clinical trials of rapamycin derivatives in lymphoma patients are already in development and there are encouraging preliminary results, such as the substantial response of a subset of mantle cell lymphoma patients to the rapamycin analogue temsirolimus. Based on results obtained from in vitro and in vivo studies of the mTOR pathway in lymphomas, it seems that better understanding of mTOR regulation will reveal aspects of lymphomagenesis and contribute to the development of more powerful, targeted therapies for lymphoma patients.
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50
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Dicker F, Schnittger S, Haferlach T, Kern W, Schoch C. Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: a study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression. Blood 2006; 108:3152-60. [PMID: 16840733 DOI: 10.1182/blood-2006-02-005322] [Citation(s) in RCA: 156] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Abstract
Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgVH status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.
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Affiliation(s)
- Frank Dicker
- MLL Munich Leukemia Laboratory GmbH, Max-Lebsche-Platz 31, 81377 Munich, Germany.
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