bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of the Antigen-Presenting Cell Function of Mantle Cell Lymphomas (MCL) by Novel Molecularly Based Immunotherapeutic Strategies.
Authors: Wang Hongwei , Cheng Fengdong , Noyes D. , Wright K. , Mohapatra S. , Tao J. , Schell M. , Sebti S. , Smith M. , Bhalla K. , Pinilla J. , Dessureault S. , Celis D. , Sotomayor E.M. ,   (Intr. by Eduardo M. Sotomayor)
Published: April 2020

MCL is an aggressive and incurable B-cell malignancy with an intrinsic characteristic to relapse after an initial good response to treatment. Manipulation of the immune system to unleash its well-known specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses in MCL. In previous studies in an A20 B-cell lymphoma murine model we have demonstrated that augmentation of the antigen-presenting cell function of the malignant B-cell is required for elicitation of an effective anti-lymphoma immunity1. Inhibition of Stat3 signaling, a negative regulator of inflammatory responses, and modulation of histone deacetylases function were identified as two novel targets to augment the immunogenicity of the malignant B-cell. In this study we determined therefore whether manipulation of these intracellular pathways in murine and human MCL cells could result in priming of antigen-specific T-cells and/or restoration of the responsiveness of tolerant T-cells. First, in vitro treatment of FC-muMCL1 cells - cell line derived from a MCL tumor that arise following pristine injection into Em-cyclin D1 transgenic mice- with increasing concentrations of the Stat3 inhibitors, Cucurbitacin I (CuI) or CPA-7 resulted in an enhanced presentation of OVA-peptide to naive CD4+ T-cells specific for a MHC class II restricted epitope of Ovalbumin (OT-II cells). Indeed, these antigen-specific T-cells produce higher levels of IL-2 and IFN-gamma compared to anti-OVA T cells that encountered cognate antigen in FC-muMCL1 cells treated with LPS alone. Similarly, we found that culture of the human MCL cells JEKO or Z138 with allogeneic human peripheral blood mononuclear cells in the presence of increasing concentrations of the Stat3 inhibitors also resulted in increased IL-2 production by T-cells. In the next set of experiments, we determined whether treatment of FC-muMCL1 cells with the hydroxamic acid analogue pan-HDAC inhibitor LAQ824 could influence their antigen-presenting capabilities and their ability to activate T-cell responses. Unlike, MCL cells treated with LPS alone, FC-muMCL1 cells treated with LPS and LAQ824 effectively prime antigen-specific CD4+ T-cells as determined by their production of both IL-2 and IFN-gamma in response to cognate peptide. Furthermore, in vitro treatment of Z138 MCL cells with LAQ824 also led to enhanced IFN-gamma production by allogeneic human PBMCs. Taken together, our findings points to inhibition of Stat3 signaling and inhibition of histone deacetylases as appealing molecularly based immunotherapeutic strategies to augment the immunogenicity of MCL cells.
Mid-Treatment Evaluation of 18-FDG-Positron Emission Tomography/Computed Tomography (PET) as Predictive Value of Response Assessment in Aggressive Non Hodgkin Lymphoma (NHL).
Authors: Pregno Patrizia , Chiappella Annalisa , Penna Daniele , Bellò Marilena , Benevolo Giulia , Botto Barbara , Freilone Roberto , Orsucci Lorella , Scaglia Sabrina , Bisi Giovanni , Vitolo Umberto , Gallo Eugenio
Published: April 2020

Introduction: The PET is mostly used in staging of NHL patients at diagnosis and as response assessment at the end of treatment. The evaluation by PET after few cycles of chemotherapy may be useful to predict chemosensitivity and then response, progression-free survival and overall survival in this subset of patients. In our study we introduced the PET in the mid-treatment evaluation (PET-2) of aggressive NHL disease.

Patients and methods: From June 2004 to December 2006, 25 consecutive aggressive NHL patients were evaluated for this study: 17 males and 8 females respectively with median age of 49 years (range 21–67). We included: 18 Diffuse Large B Cell, 1 anaplastic, 2 mantle cell, and 4 follicular grade III NHL. Patients characteristics were: 11/25 bulky disease; 20/25 intermediate or high IPI risk; 3/25 stage II, 3/25 stage III and 19/25 stage IV. All patients were staged according to standard imaging procedures completed by PET at the diagnosis and at the end of treatment. After 2 or 4 cycles of Rituximab-CHOP-like chemotherapy PET-2 was repeated in all of them.

Results: PET-2 demonstrated: 16/25 patients negative and 9/25 patients positive. The conventional and PET restaging performed at the end of treatment were 21/25 negative and 4/25 positive. Among the 16 patients PET-2 negative, 14/16 remained negative at the final PET evaluation and achieved CR, 2/16 became positive with demonstration of progression disease. Among the 9 patients PET-2 positive, 7/9 became negative with achievement of CR. The other 2/9 patients remained positive at the end of therapy: one of them was false positive because of parotid gland carcinoma without NHL involvement. Among 21/25 patients PET negative at the final evaluation, 20/25 patients remained in CR with a median follow-up of 18 months and only one patient negative at the PET-2 and at the final PET demonstrated disease progression. Finally, with a median follow-up of 18 months, FFS was 84% in all pts, 81% in PET-2 negative patients and 89% in PET-2 positive patients respectively.

Conclusions: The PET is an important imaging technique for staging and end-treatment evaluation in lymphoma disease, because it can better define CR patients. In Hodgkin disease several studies demonstrated that the early evaluation by PET is a crucial prognostic factor to test chemosensitivity and then to predict favourable outcome. In our study the mid-evaluation of response by this procedure had not so clear predictive value of response assessment, because patients, even if positive at PET-2, can achieve CR at the end of treatment. More large studies are needed to determine the real impact of on-course PET as response assessment in aggressive NHL patients.
EBV-Specific Cytotoxic T Lymphocytes by LMP2a- and EBNA3a-Specific T Cell Receptor Gene Transfer for Adoptive Therapy in EBV-Associated Hodgkin’s Disease and Post-Transplant Lymphoma.
Authors: Nguyen Tuan D. , Gelfort Haike , Sebelin-Wulf Kathrin , Schmetzer Oliver , Uckert Wolfgang , Pezzutto Antonio , Subklewe Marion
Published: April 2020

Adoptive transfer of polyclonal EBV-specific T cell lines has been used as prophylaxis and therapy in patients with EBV-associated malignancies. However, this strategy is time-consuming and demands difficult generation of lymphoblastoid cell lines (LCLs) and corresponding T cells for each individual patient. We applied an alternative strategy to confer T cell immunity against EBV-antigens by isolating EBV antigen-specific T cell receptors (TCRs) for transduction of primary human T cells for adoptive therapy. Previously, we have demonstrated the feasibility of using peptide-pulsed dendritic cells (DC) for generating high-affinity EBV antigen-specific T cell lines and T cell clones. Based on this strategy, T cell clones directed against LMP2a and EBNA3a were generated and functionally analyzed. Monospecificity was demonstrated by homogeneous double staining with CD8 and appropriate tetramers. High avidity of T cell clones (< 0.01 μM) was shown by peptide titration in an ELISPOT assay for IFN-γ secretion. In addition, the cytokine secretion profiles of some of the T cell clones were tested by cytokine bead array assay. High secretion levels of IFN-γ, IL-2 as well as TNF-α after stimulation with the EBNA3a- or LMP2a-peptide were shown for the corresponding T cell clones. Potent TCRs from one LMP2a-specific, HLA-A2-restricted and one EBNA3a-specific, HLA-B8-restricted T cell clone were isolated and cloned into the retroviral vector MP71. Transduction efficiency of TCR-deficient T cell lines was > 40% (TCR-LMP2a) and > 30% (TCR-EBNA3a) as measured by tetramer staining. Both TCR-LMP2a- and TCR-EBNA3a-redirected T cell lines were functional as indicated by NFAT-mediated luciferase expression upon TCR-MHC-peptide ligation. Primary human T cells were successfully transduced with TCR-LMP2a (∼ 12% tetramer-positive) and TCR-EBNA3a (∼ 3% tetramer-positive). Importantly, both TCRs conferred similar cytolytic activity against EBV-transformed B cell lines. Our data support the development of TCR-transduced T cells for adoptive transfer in EBV-associated malignancies, including Hodgkin′ s disease and nasopharyngeal carcinoma in which only subdominant EBV antigens are expressed. The feasibility and the therapeutic potential of TCR-transduced T cells for adoptive transfer have already been shown in a clinical phase I trial in patients with metastatic melanoma. We believe that redirecting human PBLs is a rapid and efficient tool toward adoptive transfer in EBV-associated malignancies.
Statins Impair Antitumor Effects of CD20 mAb by Inducing Conformational Changes of CD20.
Authors: Golab Jakub , Winiarska Magdalena , Bil Jacek , Wilczek Ewa , Wilczynski Grzegorz , Lekka Malgorzata , Engelberts Patrick J. , Mackus Wendy J.M. , Stoklosa Tomasz , Nowis Dominika , Makowski Marcin , Glodkowska Eliza , Issat Tadeusz , Mrowka Piotr , Lasek Witold , Gorska Elzbieta , Wasik Maria , Warzocha Krzysztof , Sinski Maciej , Gaciong Zbigniew , Dabrowska-Iwanicka Anna , Jakobisiak Marek , Parren Paul W.H.I.
Published: April 2020

A number of monoclonal antibodies (mAb) are presently in development or approved for CD20-directed immunotherapy. Ofatumumab, a human IgG1 anti-CD20 mAb, is currently being evaluated in phase III clinical trials for B-CLL and FL, and in phase II clinical trials for rheumatoid arthritis (RA). Rituximab, a chimeric IgG1 anti-CD20 mAb, has been approved for 1st line treatment of CD20-positive B cell lymphomas alone or in combination with chemotherapy, and in RA. Virtually all rituximab-treated patients relapse after single-agent treatment. The clinical efficacy of rituximab might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising anti-lymphoma effects. We studied the influence of statins on ofatumumab- and rituximab-mediated cytotoxicity. Surprisingly, B cells incubated with statins showed decreased rather than increased CD20 mAb-mediated complement-dependent cytotoxicity (CDC) in cell viability assays. However, cell lysis of statin-treated B cells remained higher when using ofatumumab in comparison to rituximab. Statins decreased CD20 immunostaining in flow cytometry but did not affect total cellular CD20 levels when compared to non-treated cells. Incubation of B cells with other cholesterol depleting agents (methyl-β-cyclodextrin (MβCD) and berberine) established that the presence of plasma membrane cholesterol and not lipid rafts may be required for CD20 mAb-mediated CDC. Cholesterol restitution reversed ofatumumab- and rituximab-mediated CDC and CD20 mAb staining. Statin incubation resulted in conformational changes of CD20 and impaired binding of CD20 mAb to the CD20 molecule as observed by atomic force microscopy. Freshly isolated cells of 4 B cell lymphoma patients were treated with the cholesterol-depleting agent MβCD, and CD20 mAb (B9E9) binding and rituximab-mediated CDC were significantly decreased (P<0.05 t-test, versus control) In addition, 5 hypercholesterolemia patients were treated with atorvastatin. Ofatumumab binding to freshly isolated B cells was found decreased in all patients upon statin treatment (P<0.01, paired t-test). Based on these data, statins may interfere with CD20 detection and anti-lymphoma activity of CD20 mAb and may have significant clinical implications as impaired mAb binding to CD20 conformational epitopes elicited by statins may delay diagnosis, postpone treatment or impair anti-lymphoma activity of CD20 mAb. MAb which are less sensitive to such changes may minimize possible effects of statins on mAb-mediated immunotherapy in these patients.
Improved Responses and Cures with Less Hematologic Toxicity Using Anti-CD20 Bispecific Antibody Pretargeted Radionuclides Compared to Directly Radiolabeled IgG in a NHL Model.
Authors: Goldenberg David M. , Sharkey Robert M. , Karacay Habibe , Chang Chien-Hsing , Rossi Edmund A. , McBride William J.
Published: April 2020

Directly radiolabeled anti-CD20 IgGs approved for the treatment of follicular and transformed non-Hodgkin lymphoma produce higher response rates than rituximab. However, these treatments result in severe and protracted hematologic toxicity, which is directly related to the slow blood clearance of these radiolabeled products. Pretargeting procedures separate radionuclide-targeting from the slow antibody-targeting step, and because the radiolabeled compound is small in size, it clears rapidly and thoroughly from the blood and body in just a few hours, minimizing radiation exposure. We have developed a novel recombinant and humanized Tri-Fab bispecific antibody (bsMAb) for pretargeting using the modular Dock-and-Lock procedure. This bsMAb divalently binds to CD20, with the monovalent Fab binding to a unique peptide hapten (HSG) that carries the therapeutic radionuclide. Nude mice bearing established (0.5 to 0.9 cm3) subcutaneous Ramos human B-cell lymphomas were given a single dose of either a 90Y-humanized anti-CD20 IgG (150 and 175 μCi; 50 μg) or a bsMAb-pretargeted 90Y-DOTA-HSG-peptide (150, 250, 500, and 700 μCi). Tissue counting data derived 24 h after injecting the radiolabeled product showed similar tumor uptake between the IgG and pretargeted peptide, but tumor/blood ratios were >1000:1 for the pretargeted peptide vs. 1:1 for the IgG. At both 90Y-IgG doses, blood counts decreased ∼90% for 2 weeks after treatment, while the pretargeted groups only decreased ∼60% at the 700 μCi dose to as low as a 25% decrease at the 250 μCi dose, with full recoveries occurring within 2 weeks of the nadir. The severe hematologic toxicity also resulted in 2/10 and 4/10 deaths within 2 weeks of the 150- and 175-μCi 90Y-IgG doses, respectively. In the remaining animals, after experiencing an initial response, all tumors progressed to ≥1.5 cm3 within 3 weeks of treatment. Only 1/10 animals given 500 μCi of the pretargeted dose showed tumor progression, with all others in this group and the 700-μCi-group showing no evidence of disease. Earlier studies have shown 40%, 59%, and 90% cure rates after 14 to 18 weeks evaluation with 150, 250, and 500 μCi, respectively, of the bsMAb-pretargeted 90Y-peptide (n = 10 to 22 animals). These studies demonstrate that bsMAb pretargeting significantly improves response rates, with durable cures and without the excessive hematologic toxicity commonly associated with directly radiolabeled antibodies.

(Supported in part from USPHS grant P01-CA103985.)
Development of a New Fully Human Anti-CD20 Monoclonal Antibody for the Treatment of B-Cell Malignancies.
Authors: Bornstein Gadi Gazit , Queva Christophe , Tabrizi Mohammad , VanAbbema Anne , Chavez Carlos , Wang Ping , Laing Naomi , Raja Sandhya , Wen Shenghua , Rong Xianhui , Green Larry L. , Yang Xiaodong , Blakey David
Published: April 2020

In spite of the widespread use of Rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop fully human antibodies with improved efficacy. Towards this end, using XenoMouse™ technology, a fully human IgG1 monoclonal antibody specific to human CD20 was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to Rituximab, in the Ramos human lymphoma cell line. In addition, mAb 1.5.3 was active in mediating complement dependent cytotoxicity (CDC) and elicited improved antibody-dependent cellular cytotoxicity (ADCC) relative to Rituximab in Ramos, Raji, and Daudi human B-lymphoma lines. To recapitulate various aspects of acquired resistance to Rituximab, as observed in a subpopulation of patients, Rituximab-resistant clones were established from lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior cytolytic activity against engineered Rituximab-refractory lymphoma clones, as well as across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Rituximab-sensitive cell lines and -refractory engineered lymphoma clones in vivo. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to Rituximab in a primate PD model. In contrast to Rituximab, mAb 1.5.3 is a fully human antibody and is thus anticipated to exhibit a longer serum half-life with minimal immunogenicity following repeated administration. In sum, these results demonstrate the superior anti-tumor activity of mAb 1.5.3 relative to Rituximab and its potential for improved clinical activity in the treatment of B-cell malignancies.
Activating Mutation of Heme Oxygenase (HO)-1: A Novel Disease Entity Characterized by Microcytic, Hemolytic Anemia, a Defect of Bilirubin Synthesis and Hereditary Hemophagocytic Lymphohistiocytosis.
Authors: Greil Johann , Verga-Falzacappa Mavi , Tsukahara Hirokazu , Behnisch Wolfgang , Heimpel Hermann , Schneider Marion , Janka Gritta , Claus Maren , Muckenthaler Martina , Kulozik Andreas E.
Published: April 2020

Primary hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition that is caused by an abnormal accumulation of activated macrophages. The common NK- and cytotoxic T-cell dysfunction in HLH has been related to genetic defects of vesicle transport and apoptosis. Here, we identify a novel form of hereditary HLH that is related to and possibly caused by a homozygous missense mutation (G139V) of the active centre of heme oxygenase-1 (HO-1). The clinical picture of this male patient is characterized by persistent, severe microcytic hemolytic anemia without hyperbilirubinemia since birth. In the second year of life, the clinical syndrome of HLH developed with persistent fever, marked hepatosplenomegaly, thrombocytopenia, hyperferritinemia, hypertriglyceridemia, and elevated levels of soluble CD25. Liver histology showed marked lympho- and erythrophagocytosis, which confirmed the diagnosis of HLH. The clinical syndrome responded well to immunosuppressive treatment with etoposide, dexamethasone and cyclosporine A. PRF1, UNC13-D and STX11 mutations were excluded. Functional analysis of NK-activity showed only slightly decreased killing activity with normal responses of NK-cells to cytokine stimulation. Persistent endothelial damage was indicated by a pronounced elevation of vWF and D-dimer concentrations. The persistently very low bilirubin levels despite marked hemolysis suggested a defect of hemoglobin degradation. HO-1 is the rate limiting enzyme of this pathway resulting in bilirubin synthesis. Normally, HO-1 is constitutively expressed at a low level but strongly induced by heme, and hypoxia. Functional analysis of HO-1 protein in the patient’s mononuclear blood cells showed a high constitutive expression that did not respond further to heme treatment. Sequence analysis revealed a homozygous GGT (Gly) to GTT (Val) point mutation of HO-1 codon 139. This mutation of the enzyme’s active center is known from in-vitro analyses to change enzyme activity from an oxygenase into a peroxidase. Consistent with this change in activity, quantitative analysis of reactive oxygen species (ROS) metabolites in the urine of this boy showed high amounts of bilirubin oxidative metabolites, 8-hydroxy-2′-deoxyguanosine and acrolein-lysine. In this report we thus identify a novel disease entity which is characterized by an activating mutation of HO-1 which results in a defect of hemoglobin degradation, bilirubin synthesis and in excessive oxidative stress and inflammation.
Intravenous, Intraperitoneal, and Subcutaneous Routes of Administering 64Cu-DOTA-HB22.7 Display Equivalent Tumor Targeting Ability.
Authors: Martin Shiloh M. , O’Donnell Robert T. , McKnight Hayes , Sutcliff Julie L. , Tuscano Joseph M.
Published: April 2020

BACKGROUND: Chemotherapy for Non-Hodgkin’s Lymphoma (NHL) is initially effective, but often limited by toxicity and resistance. New agents for the treatment of NHL are needed. HB22.7 is a monoclonal antibody that binds CD22 and has previously been shown to reduce human lymphoma xenograft volume in nude mice.

OBJECTIVES: (1) Develop a 64Cu-DOTA-HB22.7 antibody, for in vivo imaging and potential therapy of NHL. (2) Determine if tumor targeting of 64Cu-DOTA-HB22.7 is equivalent between intravenous (IV), intraperitoneal (IP), and subcutaneous (SQ) routes of administration.

METHODS: DOTA-HB22.7’s ability to bind CD22+ cells was assessed by flow cytometry. 64Cu-DOTA-HB22.7 was injected IV, IP, or SQ into xenograft-bearing nude mice and tumor targeting assessed by positron emission tomography (PET) and biodistribution assay. Pharmacokinetics were determined by measuring radioactivity of blood samples. Serum was analyzed by radio-TLC.

RESULTS: DOTA conjugation does not affect HB22.7’s ability to bind CD22. 64Cu-DOTA-HB22.7 demonstrates specific tumor targeting at 24 and 48 hrs. Targeting is equivalent regardless of route of administration. Pharmacokinetics demonstrate that 64Cu-DOTA-HB22.7 can access the bloodstream (and thus, target tumor) in IP or SQ injections. By 48 hrs, blood 64Cu levels are (a) equivalent, regardless of injection route and (b) below peak levels, indicating clearance from the circulation. Serum analysis shows that right-shifted TLC peaks, possibly 64Cu-DOTA-HB22.7 metabolites, are present only in the IV injected group at 48 hrs.

CONCLUSION: These findings establish 64Cu-DOTA-HB22.7 as an NHL-specific imaging agent and indicate its potential for use in radioimmunotherapy. These findings provide evidence that more accessible routes of administration can achieve equivalent targeting results, and may lead to more efficient and accurate administration of antibody-based therapeutics in mice.
Loss of Pak1 Corrects Multiple Gain of Function Phenotypes in Nf1+/− Mast Cells.
Authors: McDaniel Andrew , Clapp D. Wade , Chernoff J.
Published: April 2020

Neurofibromatosis type 1, caused by mutations in the tumor suppressor gene NF1, is the most common genetic disorder in man and is characterized by the development of tumors called neurofibromas. Studies in genetically engineered Nf1 knockout mice have demonstrated that loss of the Nf1 tumor suppressor in Schwann cells, the known tumorigenic cell in neurofibromas, is necessary but not sufficient for tumor formation. By using adoptive transfer, we have now established that haploinsufficiency of Nf1 in the hematopoietic microenvironment (specifically mast cells) is necessary and sufficient for tumor progression in a Nf1 conditional knockout model. It is known that mast cells promote tumor progression via the secretion of growth factors and proteinases. Thus molecules that target mast cell function may be useful therapeutically. We have previously demonstrated that haploinsufficiency of Nf1 results in hyperproliferation and hyperactivation of the mitogen-activated protein kinase (MAPK) pathway in bone marrow-derived mast cells in response to kit-ligand (kit-L). Cross-cascade signaling between phosphoinositide 3-kinase activated Rho-GTPases and the classical MAPK pathway has been shown to be responsible for these abnormal phenotypes. p21-activated kinases (Paks) are downstream mediators of Rho-GTPase proteins that have been implicated in immortalized cells as positive regulators of MAPK pathway members and as modulators of cell growth and cytoskeletal dynamics. Utilizing an intercross of Pak 1−/− mice with Nf1+/− mice, we studied how Pak1 in regulates cellular functions associated with haploinsufficiency at Nf1 in primary mast cells. Loss of Pak1 corrects the gains of function in proliferation and migration found in Nf1+/− mast cells back to wild type levels via differential regulation of MAPKs. Pak1 regulates proliferation via a Pak/Erk pathway, while a Pak1/p38 pathway is critical for migration. The increased release of preformed inflammatory mediators in Nf1 haploinsufficient mast cells was also abrogated by genetic disruption of Pak1. Pak1 was found to contribute to MAPK signaling by modulating both ERK and p38 activation. Loss of Pak1 reduces the hyperactivation of ERK and p38 found in Nf1+/− mast cells to that of wild type controls. Further, using a model that stimulates local proliferation of mast cells in vivo, we confirm that loss of Pak1 corrects the accumulation and degranulation of Nf1+/− mast cells in vivo to wild type levels. Thus, Pak1 is a novel mediator of Ras-dependent mast cell functions, and treatments that target Pak1 may be useful as a targeted therapy for treatment of neurofibromas.
Characterisation of Nilotinib Transport in Chronic Myeloid Leukaemia Cells.
Authors: Davies Andrea , Giannoudis Athina , Lucas Claire M. , Harris Robert J. , Manley Paul W. , Pirmohamed Munir , Clark Richard E.
Published: April 2020

We have previously shown that imatinib is a substrate for both the efflux transporter P-glycoprotein (ABCB1; MDR-1 gene product) and the influx transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). High hOCT1 and low ABCB1 expression levels in chronic myeloid leukaemia (CML) patients correlate with improved clinical outcome (Wang L. et al. Clinical Pharmacology and Therapeutics; epub June 13th 2007). The second generation tyrosine kinase inhibitor nilotinib is 30-fold more potent than imatinib and is effective in imatinib-resistant patients. However, its cellular transport has not been characterised. The CML cell line KCL22 was used as it has low basal hOCT1 expression. Drug transport studies of 14C-radiolabelled nilotinib (kind gift from Novartis) were performed using high expressing hOCT1 transfected KCL22 cells, with or without OCT1 and ABCB1 inhibitors. Further vectorial ABCB1 efflux studies were performed on confluent monolayers of ABCB1 transfected Type II Madin Darby canine kidney (MDCKII) cells. Nilotinib efflux was not modulated in the presence of the ABCB1 inhibitors PSC-833 (10μM), tariquidar (500nM) and verapamil (500μM), and no evidence of transport via ABCB1 was seen in the transfected ABCB1 MDCKII cells. Nilotinib influx was not modulated in hOCT1 over-expressing cells, or in the presence of OCT1 inhibitors prazosin (100μM) and amantadine (500μM). Nilotinib uptake was also not modulated by the hOCT1 substrate tetraethylamine (TEA) (5μM), however nilotinib inhibited TEA uptake. The distribution coefficient (logD) of nilotinib was 2.1, compared with 0.81 for imatinib, demonstrating high lipophilicity for nilotinib. Flow cytometry revealed greater suppression of phospho-CrKL (a surrogate marker for BCR-ABL blockade) with nilotinib after 1 hour when compared with imatinib, regardless of hOCT1 expression, indicating that nilotinib is effective at BCR-ABL suppression even at low hOCT1 levels, in contrast to imatinib. Nilotinib was not a substrate for the ABCB1 and hOCT1 transporters; however it may be a potential inhibitor of hOCT1. Therefore the level of expression of ABCB1 and hOCT1 in CML is unlikely to be an indicator of the clinical outcome for patients receiving nilotinib, in contrast to what we have shown for imatinib. Nilotinib may therefore be effective in patients who have low hOCT1 expression before start of therapy.
Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo.
Authors: Alder Jonathan K. , Georgantas Robert W. , Hildreth Richard L. , Kaplan Ian M. , Morisot Sebastien , Yu Xiaobing , McDevitt Michael , Civin Curt I.
Published: April 2020

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate here that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− FL cells into lethally irradiated wild-type (wt) mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. While the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.
Increased Risk of Osteopenic Fractures in Elderly Patients with Gaucher Disease.
Authors: Weinreb Neal J. , Costantini Lisa
Published: April 2020

Bone disease is a common cause of morbidity in Gaucher disease (GD). 60% of adult patients (pts) have decreased bone mineral density (BMD). However, in elderly pts, statistically “normal” BMD may not indicate functionally normal bone strength. Here we report serial dual energy x-ray absorptiometry (DXA) scores and fracture (Fx) occurrence for 29 GD pts age 57–88 (9M, 20F) treated with enzyme replacement therapy (ERT) with imiglucerase, often in conjunction with bisphosphonates.

Results: Lumbar (L) spine Z-scores were mostly normal during 9.2 ± 4.9 y on ERT: median −0.50; mean (SD) −0.05 (0.41); centiles 10–90: −1.81 - 1.21. Nonetheless, new Fxs occurred in 13 of 29 (44.8%) pts: hip (6), spine (4), other location (11), multiple sites (7). Fx occurred in 5/8 (62.5%) pts with splenectomy v 8/21 (38.1%) pts without and in 7/20 (35%) N370S homozygotes v 6/9 (67%) with other genotypes. 10y Fx probability: 42% (CI 17–55). L spine T-scores were < −1 for 18/29 pts, indicating persistent osteopenia/osteoporosis in 62%, and < −2.68 in 25%. New Fx occurred in 8/15 (53.0%) pts with T-scores ≤ the median of −1.60 v 5/14 (35.7%) with T-scores > −1.60. Pts with low T scores were predominantly women (13/15F v 7/14M) and had higher use of bisphosphonates (86.7% v. 35.7%). Fx were not restricted to pts with the lowest T-scores. Findings were similar for femoral neck T-scores.

Conclusions: Normal DXA Z-scores in elderly GD pts do not indicate low Fx risk. Low T-scores, as seen in our pts, do confer an important Fx risk. The 42% 10y Fx probability in our elderly GD pts is higher than expected for comparably aged individuals without GD. Because other risk factors such as Fx history, nutritional status, and concurrent illness were similar in our pts regardless of Fx status, cumulative pathophysiology from long-standing, untreated GD may independently increase Fx risk. Treatment with ERT may allow pts to achieve normal Z-scores. However, starting ERT late in life, even in conjunction with bisphosphonates, may not reverse established osteopenia/osteoporosis sufficiently to avoid future Fx. Therefore, detection of bone loss in young pts and early therapeutic intervention to achieve and maintain normal BMD is an important component of GD management.
Telomere Length Shortening in Langerhans Cell Histiocytosis.
Authors: Bechan Gitanjali I. , Meeker Alan K. , DeMarzo Angelo M. , Racke Frederick , Jaffe Ronald , Sugar Elizabeth , Arceci Robert J.
Published: April 2020

The purpose of this study was to analyze telomere lengths in Langerhans Cell Histiocytosis (LCH). LCH is a clonally derived, proliferative disorder of phenotypically immature CD1a+ Langerhans cells (LCs). The etiology of LCH is unknown and data supporting an immune dysregulatory disorder as well as a clonal neoplasm have been reported. Telomere shortening has been associated with cancers and premalignant lesions as well as being important in promoting chromosomal instability. We therefore conducted a study to quantitatively determine the telomere lengths in LCH. Tissue arrays of reactive lymph nodes, local, multisystem, and systemic LCH containing 9, 7, 12, and 10 specimens respectively were used. These tissue arrays included 2 specimens that had unaffected skin represented, providing an opportunity to examine non-pathologic LCs. The tissue arrays were first hybridized with a N-terminal covalently linked Cy3-labeled, telomere specific peptide nucleic acid (PNA) probe to stain telomeres. The slides were then processed for indirect immunofluorescence to identify LCs using an anti-CD1a primary antibody followed by an anti-mouse IgG fraction Alexa Fluor 488. Finally, slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Digital fluorescent telomere signals were visualized using a Nikon Eclipse E400 epifluorescence microscope and telomere lengths were analyzed with ImageJ software. A total of 1039 measurements were made from 38 individuals. A significant shortening of telomeres was demonstrated in LCH LCs of local, multisystem and systemic LCH in comparison to telomere lengths of reactive lymph node LCs and normal skin, which were not significantly different. To investigate whether telomere length shortening of LCH LCs is selective, we analyzed the telomere length in lymphocytes in LCH specimens and in the reactive lymph node specimens. There was no significant evidence of telomere shortening of lymphocytes in LCH compared to lymphocytes in reactive lymph nodes (p=0.1862). These data show significant telomere shortening in the LCs in all stages of disease involvement with more variability among local cases. These observations suggest that in most cases LCH may share important mechanisms of telomere shortening and survival with clonal pre-neoplastic disorders and cancer.
Protein Kinase C α Is Critically Involved in MyD88-Dependent Toll-Like Receptor 2-Mediated Activation of Dendritic Cells.
Authors: Springael Cecile , Langlet Christelle , Goldman Michel , Willems Fabienne , Aksoy Ezra
Published: April 2020

Introduction: Protein kinases C (PKC) isoforms are key regulators of several Toll-like receptor (TLR) signalling pathways. In the present study, we investigated the role of conventional PKCα in the MyD88-dependent TLR2-mediated activation of dendritic cells (DC).

Methods: We compared the functional and molecular responses of DC derived from wild type and PKCa deficient mice to TLR2 stimulation. We next investigated the consequences of conventional PKC (cPKC) inhibition during TLR2 stimulation in human monocyte-derived DC using the cPKC pharmacological inhibitor Gö6976. Finally, reporter gene assays were performed in human embryonic kidney (HEK) cells stably expressing TLR2 in the presence of dominant negative (DN) PKCα. Results: Bone marrow-derived DC from PKCα deficient mice showed an attenuated response to the TLR2/1 ligand Pam3CSK4 characterized by a statistically significant reduction in interleukin (IL)-12p40 and TNFα production. IL-6 synthesis was also reduced albeit not significantly. In western blot analysis, PKCα−/− DC displayed an impaired phosphorylation of the mitogen activated protein (MAP) kinases p38 and c-Jun NH2-terminal kinase (JNK) in response to Pam3CSK4. TLR2-mediated activation of the IκB kinases (IKK) α and β and inhibitor of κB (IκB) degradation were abolished in PKC deficient DC, suggesting an impaired nuclear factor (NF)-κB activation. In human monocyte-derived DC, inhibition of conventional PKC activity by the isoform specific inhibitor Gö6976 severely decreased cytokines production in response to TLR2 ligands. At the concentration of 1μM, Gö6976-treated DC exhibited impaired p38 and JNK activation. The NF-κB activation pathway was repressed with higher concentrations of the pharmacological inhibitor. Finally, in reporter gene assays, DN PKCα but not DN PKC𝛉 repressed AP-1 and NF-κB mediated promoter activity upon TLR2 engagement.

Conclusions: Our results demonstrate that PKCα is a key component of the MyD88-dependent TLR2 signalling pathway in dendritic cells and regulates the activation of NF-κB and MAP kinases pathways.
SNP Arrays Facilitate Genotyping of Non-Synonymous SNP in MDS To Identify Disease Susceptibility Loci.
Authors: Jankowska Anna M. , Przychodzen Bartlomiej P. , Gondek Lukasz P. , Maciejewski Jaroslaw P.
Published: April 2020

Myelodysplastic syndrome (MDS) is a clonal premalignant disease of hematopoietic stem cells characterized by cytopenias and predilection to acute myeloid leukemia (AML). While various exogenous factors (exemplified by chemotherapy-related MDS) constitute known risks for the development of MDS, it is possible that despite long latency, complex genetic traits contribute to MDS susceptibility. Such heritable factors include genes involving DNA repair, apoptosis, senescence, carcinogen catabolism and immune surveillance. Previously, disease association studies were mainly empiric and relied on rational selection of a very limited number of polymorphisms. Recent advances of SNP-array (SNP-A) technology allow for screening of a large number of SNPs. While some SNP-A utilize haplotype tags, custom arrays may specifically target known non-synonymous SNPs. We hypothesized that application of SNP-A genotyping may facilitate identification of potentially pathogenic SNPs. Such a screening approach is a hypothesis forming tool and our study is the first application of this technique to MDS. We have used the 13.9K non-synonymous Genotyping BeadChip (Illumina); DNA from 151 MDS patients (low risk: N=79, advanced: N=51, CMML1/2: N=21) and 99 controls (120 historical controls). In total, ∼2.4 mil genotypes were obtained. Genotype calls were computed and analyzed with Exemplar software. In the initial training Bonferroni correction was not applied. Instead as a hypothesis-forming approach we have ranked all SNPs according to their p value (automated analysis of multiplexed statistical evidence) and case/control ratio. We focused our search on the 100 highest ranking SNPs with a control frequency <5%. Globally, in the whole group 49 SNPs showed a p value of <.001, 75 SNPs present in ≤1% of controls and were found in >5% of cases. For example, the AA (rs3219484) variant of MUTYH, a gene involved in oxidative DNA repair was found in 19% of MDS vs. 2.6% of controls (p=9×10−8). To limit the impact of heterogeneity, subgroups of MDS were also analyzed separately. Among many interesting SNPs found, the AG genotype of (rs8192297) ANPEP was associated with RARS (35% vs. 15% in controls; p=.007). Similarly, the GA form of rs3730947 in DNA repair gene LIG1 was found in 11% of RARS patients (p=.00045), though it was absent in other MDS subtypes and controls. Similar, “enrichment” was observed in patients with CMML1/2, showing e.g., heterozygosity of WDR35 (rs1060742), ALPK2 (rs3809975) at the frequency of 45% and 30% (13% and 4% of controls; p=.0006, p=.0001). In sum, our study constitutes the first application of SNP-A genotyping to study susceptibility loci in MDS.
A Lack of Correlation between EVI1 Expression, MDS Subtype and Karyotype Abnormalities Suggests Multiple Pathways Involved in EVI1 Impact on Hematopoietic Cells in MDS Patients.
Authors: Cermak Jaroslav , Ing Ota Fuchs , Provaznikova Dana , Michalova Kyra , Mikulenkova Dana , Brezinova Jana , Klener Pavel
Published: April 2020

EVI1 proto-oncogene located on human chromosome 3q26 has been repeatedly described as an important gene involved in pathogenesis of human acute myeloid leukemia or myelodysplastic syndrome (MDS). EVI1 encodes a nuclear DNA binding protein and its abnormal expression in immature hematopoietic cells leads to altered proliferation and differentiation of both erythroid and myeloid lineages. In our study, an incidence and prognostic relevance of EVI1 expression was studied in a group of 62 patients with different subtypes of primary MDS. EVI1 expression was detected using semiquantitative RT-PCR on samples obtained repeatedly from bone marrow and peripheral blood of patients or healthy controls. The results for individual cDNAs corresponding to the amounts of individual EVI1 mRNAs were related to the amount of standard housekeeping constitutively expressed GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Abnormal EVI-1 expression was found in 12 out of 62 patients (19%). The distribution of positive patients according to the WHO classification was: RA-2, RCMD-3, 5q- syndrome-1, RAEB I.-3, RAEB II.-1, sAML -2, that represented 0% (RARS,CMML), 17% (RCMD,RAEB II.), 18% (sAML), 30%(RA,5q-syndrome) and 33% (RAEB I.) of all patients studied. Only 2 out of 12 patients with abnormal EVI-1 expression had abnormalities of chromosome 3. confirmed by mFISH, 2 patients had complex karyotype abnormalities involving chromosome 11. Five out of 50 negative patients had complex abnormalities including loss of chromosome 3. in 1 patient and abnormities of chromosome 11. in 2 patients. No significant difference in median survival and rate of leukemic transformation was observed between both the groups (25,0 months and 33% in EVI1 positive group vs. 15,3 months and 28% in EVI1 negative group, P=0,35). Nevertheless, in four EVI1 negative patients with RCMD or RAEB I. an abnormal EVI1 expression occurred repeatedly at the time of progression to RAEB I.or RAEB II. All these patients subsequently died on refractory AML. Our results suggest that abnormal EVI1 expression may play role in early MDS (eg. by impairment of erythropoiesis due to blocking of GATA-1 dependent transcription) as well as in advanced MDS (eg. by stimulation of proliferation and alteration of differentiation of myeloid precursors by inhibition of TGFβ or IFNα signalling pathway). This may be an explanation for limited prognostic relevance of abnormal EVI1 expression in MDS patients in our study. However, a novel occurrence of EVI1 expression in the course of the disease might serve as an additional adverse prognostic factor.
Different Patterns of AML1 Mutations between De Novo Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia.
Authors: Shih Lee-Yung , Liang Der-Cherng , Huang Chein-Fuang , Kuo Ming-Chung , Lin Tung-Liang , Fu Jen-Fen , Shih Yu-Shu , Wu Jin-Hou , Wang Po-Nan , Dunn Po , Tang Tzung-Chih , Chang Hung
Published: April 2020

Background: Transcription factor AML1/RUNX1 is essential for normal hematopoiesis. AML1 mutations have been described in radiation-associated and therapy-related myelodysplastic syndrome (MDS) and have rarely been reported in patients with chronic myelomonocytic leukemia (CMML).

Aims: We sought (1) to define the frequencies of AML1 mutations in de novo MDS and CMML, and (2) to compare the difference in mutation patterns between the two disorders.

Methods: AML1 mutations were examined on bone marrow samples obtained at initial diagnosis from 107 patients with de novo MDS (12 RCMD, 45 RAEB1 and 50 RAEB2) and 79 patients with CMML (53 CMML1 and 26 CMML2). Mutational analysis was performed by direct sequencing of all RT-PCR products amplified with 3 overlapping primer pairs which cover the entire coding sequences of AML1b gene from exon 1 through exon 8. Samples with abnormal sequencing results were subjected to repeated PCR and sequencing using genomic DNA with alternative primers.

Results: Seventeen of 107 patients with MDS (15.9%) had 20 AML1 mutations; 8 mutations were located in Runt homology domain (RHD) (exons 3–5) and 12 mutations were located in the non-RHD region (exons 6–8). The 20 AML1 mutations included 9 missense mutations, 5 nonsense mutations, 2 frameshift mutations, and 4 silent mutations. One patient has two missense mutations at RHD and another two had one missense mutation with one silent mutation. Thirty-one AML1 mutations were detected in 29 of 79 CMML patients (36.7%); 20 patients had 22 mutations located in the N-terminal part and the remaining 9 patients had one single mutation located in the C-terminal region. The patterns of the 31 mutations consisted of 8 missense mutations, 7 nonsense mutations, 15 frameshift mutations and 1 silent mutation. One CMML patient had two missense mutations in RHD and another patient had two frameshift mutations in RHD. Cloning analysis showed that the two mutations were on different alleles in both patients. The frequency of AML1 mutations was significantly higher in patients with CMML than in MDS (P=0.002). Mutations in N-terminal region occurred more frequently in CMML than in MDS (22/30 vs. 8/20, P=0.042). CMML patients had a higher frequency of frameshift mutations as compared with MDS patients (P=0.006). Except for a significantly lower platelet count in CMML patients with AML1 mutations, there were no differences in age, sex, blood counts, percentages of blasts in bone marrow or peripheral blood, subtypes of MDS or CMML, cytogenetic risk groups, time to AML transformation or overall survival between AML1(+) and AML1(−) in MDS and between AML1(+) and AML1(−) CMML patients.

Conclusion: Our results showed that AML1 mutations occurred frequently in both de novo MDS and CMML but with different mutation patterns.
Genome-Wide Analysis of DNA Copy Number in Multiple Myeloma Using High-Density SNP Arrays Reveals Clustering Patterns with Distinct Transcriptional Profiles.
Authors: Mosca Laura , Agnelli Luca , Kwee Ivo , Fabris Sonia , Ronchetti Domenica , Todoerti Katia , Lionetti Marta , Verdelli Donata , Nobili Lucia , Baldini Luca , Morabito Fortunato , Deliliers Giorgio Lambertenghi , Bertoni Francesco , Neri Antonino
Published: April 2020

Multiple myeloma (MM) is characterized by a high genomic instability that involves both ploidy and structural rearrangements. Nearly half of MM tumors are non-hyperdiploid and are frequently associated with 13q deletion and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32. The remaining tumors are hyperdiploid, showing low prevalence of both IGH translocations and chromosome 13 deletions. Our study was aimed at providing insights into the genomic heterogeneity associated with plasma cell neoplasms by defining the genome-wide pattern of genetic lesions in a representative and stratified panel of MM patients. To this end, genome-wide profiling data of 45 plasma cell dyscrasia patients (41 MM and 4 plasma cell leukemia) were generated on GeneChip® Human Mapping 50K Xba SNP arrays, and the local DNA copy number variations were calculated using the DNAcopy Bioconductor package. The patients were clustered using the non-negative matrix factorization (NMF) algorithm to identify, within the natural grouping of profiles, the strongest clusters on the basis of their genomic characteristics. We identified three consistent clusters, characterized byrecurrent gains of odd-chromosomes, suggestive of the hyperdiploid status (Group A),high frequency of chromosome 13 deletion and 1q gains (Group B), orhigh frequency of chromosomes 13, 14, 16 and 22 deletions and losses of 1p and 4p regions, together with some cases showing 1q gains (Group C).

To determine whether peculiar transcription fingerprints characterized these groups, gene expression profiles of 40 out of 45 corresponding samples generated on GeneChip® HG-U133A arrays were analyzed using the Prediction Analysis of Microarray (PAM) software. The multi-class analysis identified 229 transcripts (corresponding to 195 genes), which specifically marked the three groups. In particular, Group A was characterized by the overexpression of genes involved in the translational machinery or thought to be involved in MM pathogenesis such as the HGF, the tumor necrosis factor ligand TRAIL, DKK1, and c-KIT. Upregulation of the CKS1B gene was present in Group B and C, most likely reflecting the high frequencies of 1q gains in tumors within group B and C and its consequent deregulation. Group C was marked by the specific downregulation of genes mainly mapped to 1p arm: AMPD1, CSDE1, AKR1A1 and the PRKACB kinase, suggesting a relationship with the recurrent 1p loss within the group. Our data further supported the notion that structural abnormalities in multiple myeloma are associated with gene expression imbalances, and provide novel analytical approaches for the identification of genetic lesions and molecular patterns of the disease.