bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of MicroRNA (miR) miR-15a/miR-16-1 Downregulates Expression of BCL-2 Protein in Chronic Lymphocytic Leukemia.
Authors: Chen Liguang , Tang Li , Calin George , Croce Carlo M. , Kipps Thomas J. 
Published: October 2019

Abstract
MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002).
These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.
Activation of NFAT and NFkB Transcription Factors upon B Cell Receptor (BCR) Stimulation Is Restricted to Evolutive Chronic Lymphocytic Leukemia (CLL) Cases.
Authors: Chevallier Nathalie S. , Deglesne Pierre-Antoine , Beitar Taoufik , Baran-Marszak Fanny , Letestu Remi , Vlad Amalia , Lefebvre Valerie , Clavereau Isabelle , Cymbalista Florence , Varin-Blank Nadine 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which BCR signaling capacity plays a central role in disease progression. Previously, we have shown that BCR engagement allows the identification of two groups of patients with a strong correlation between the in vitro cell survival response and the biological criteria (IgVH somatic hypermutations, ZAP 70 expression ) or the clinical stage. One group, corresponding to non evolutive patients, showed absence of a substantial BCR signaling (Non responder). In contrast, BCR signaling promoted in vitro cell survival in the group of Responder cases corresponding to evolutive patients. In the latter only, response to BCR ligation was associated with induction of NFkB and NFAT regulated genes, such as Cyclin D2 and CD23.
Since NFAT and NFkB transcription factors, regardless of the patients clinical status, have been described as constitutively nuclear in B-CLL cells, the aim of our study was to investigate whether the functional implication of these factors was different between our two groups. First, we confirmed by confocal microscopy and band shift analysis that localization of NFAT and NFkB was uniformely nuclear independently of the CLL status and the BCR response capacity. By RQ-PCR , we also investigated the expression of the various isoforms of NFAT (NFAT1, 2, 3 and 4) and showed that their expression profile was similar to normal B cells. Despite the nuclear localization of NFkB and NFAT, DNA binding capacity was only enhanced in cases responsive to BCR stimulation. Finally, using specific Tat-peptide inhibitors of NFAT and NFkB nuclear transport upregulation of cyclin D2 and CD23 was abrogated and survival advantage was lost in responder cases thereby confirming the crucial role of these factors upon BCR stimulation. However, in non responder cases in which no CD23 or cyclin D2 induction was present, this inhibition had no impact on cell survival upon BCR stimulation . Nonetheless, these cells retained the ability to respond to ionomycin or PMA reflecting functional PKC/calcium-calcineurin signaling pathways. Altogether, our results showed that NFAT and NFkB were activated in responder cells only despite their homogenous expression in all cases. In conclusion, the inability of the cells from stable B-CLL cases to activate the NFAT and NFkB pathways upon BCR signaling is not related to a functional defect or abnormal expression of these factors but likely reflects an early signaling defect. Moreover, our results show that the use of peptide inhibitors may restore a stable profile in B-CLL cells from evolutive patients.
Comparison of the ZAP70+ and ZAP70− B-CLL Kinome: Higher Kinase Activity in ZAP70+ B-CLL Cells.
Authors: Wang Miao , Rosati Stephano , Parikh Kaushal , Peppelenbosch Maikel , Daenen Simon , Kluin P.M. , Visser Lydia 
Published: October 2019

Abstract
B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical course. The expression of ZAP70 can serve as a novel prognostic marker in B-CLL, but the function of ZAP70 in the disease is still unknown. A high throughput analysis of protein kinases allows a comprehensive detection of signal transduction events in CLL. The goal of this study was to detect the activity of kinases in B-CLL cells and compare the kinase activity of (IgM positive) B-CLL cells immediately after stimulation through IgM. We compared 2 cases of ZAP70+ and 2 cases of ZAP70− untreated B-CLL patients. Tumor cells were isolated from lymph node cell suspensions, purified by negative selection (> 95% purity), and cross linked with biotin labeled anti-IgM F(ab)2 (10 μ g/mL) followed by avidin (10 μ g/mL) for 10 minutes at 37°C. Whole cell lysates in combination with 32 P-ATP were overlaid on a kinomics chip, which consists of 1024 peptides in triplicate (including 48 peptides as internal controls) and can detect the activity of 207 different kinases.
We found significant differences of the activity of 74 kinases between ZAP70+ and ZAP70− cases, with a higher phosphorylation of 93 peptides in the ZAP-70+ group. Phosphorylation of ZAP70 by kinase LCK was up regulated with a factor 2.7 in the ZAP-70+ B-CLL group. The kinases SYK, LCK, Fyn, HPK1, CAMK2A, PRKAR1A and PKC all showed a higher activity in the ZAP70+ group. These kinases are mainly involved in the B cell receptor pathway and indicate that ZAP70+ B-CLL cells are more activated than the negative group. The kinases CDC2, CDK2, PLK, and ABL also showed a higher activity in the ZAP70+ B-CLL cases, indicating a higher proliferation rate. PTK6 and LIMK1 which also had a higher activity in the ZAP70+ group are oncogenic kinases and play a fundamental role in transformation and migration.
Crosslinking of the BCR via IgM resulted in changes in the activity of 73 kinases, 55 showing a lower activity. Since ZAP70 expression can be induced through activation in normal B-cells, it is possible that the already highly activated status of the ZAP70+ cells causes a fast dampening after IgM stimulation. In the ZAP70− group only a minimal change was detected upon IgM cross linking. After 10 minutes stimulation with anti-IgM, the activity of ZAP70 itself as assessed by alterations of the phosphorylation status of its substrate HIP-55 was down regulated in exclusively the ZAP70+ cases. HIP55 possibly connects ZAP70 with both the BCR and apoptosis pathways.
In summary, ZAP70 is not only a prognosis marker of B-CLL, but might also play a key role in survival, proliferation, activation and transformation of B-CLL cells.
Introduction of Constitutivelly Active Akt in Primary CLL B-Cells Results in Upregulation of Mcl-1, Bcl-XL and Cyclin D3 and Promotes Leukemic Cell Survival.
Authors: Longo Pablo , Gobessi Stefania , Laurenti Luca , Sica Simona , Leone Giuseppe , Efremov Dimitar G. 
Published: October 2019

Abstract
The PI3K/Akt and Raf/MEK/ERK pathways are key regulators of various cellular responses, including proliferation, survival, differentiation, migration and malignant transformation. These pathways are activated in chronic lymphocytic leukemia B-cells by a number of survival or growth stimulatory signals, such as immobilized anti-IgM antibodies, interleukin-4, phorbol-ester, CXCL12, or stimulatory CpG oligonucleotides. Moreover, enhanced activation of Akt has been implicated in the pathogenesis of the CLL-like disorder that develops in mice transgenic for the TCL1 oncogene. To further delineate the relative contribution of the PI3K/Akt and Raf/MEK/ERK pathways in regulating leukemic cell growth and survival, we introduced constitutively active Akt or constitutively active MEK2 in primary CLL B-cells by nucleofection. Expression of constitutively active Akt consistently promoted survival, as evidenced by a higher percentage of Annexin V/PI negative cells after 48 hours in culture (median 52%) compared to samples transfected with control vector (median 31%). Immunoblot analysis of several important antiapoptotic proteins revealed that enforced activation of Akt upregulates Mcl-1 and Bcl-xL, whereas no changes were observed in the levels of Bcl-2. Expression of constitutively active Akt also induced an increase in size and granularity of the leukemic cells, indicating increased metabolic activity. These changes were associated with significant induction of cyclin D3, indicating that activation of Akt is required for both leukemic cell survival and cell cycle progression. In contrast, introduction of constitutively active MEK2 induced sustained activation of ERK, but showed only a modest increase in the percentage of viable CLL B-cells (median 36%) and no significant changes in the levels of any of the investigated antiapoptotic proteins. These experiments provide direct evidence that sustained activation of Akt promotes leukemic cell survival and upregulates Mcl-1, an antiapoptotic protein that has been associated with resistance to chemotherapy in patients with CLL.
CD21 Expression Is Inversely Correlated with the Ability of CLL Cells To Induce Tyrosine Phosphorylation in Response to Anti-IgM.
Authors: Pepper Chris , Alghazal Suhair , Brennan Paul , Rowntree Clare , Lin Thet Thet , Pratt Guy , Fegan Chris 
Published: October 2019

Abstract
B-cell chronic lymphocytic leukaemia (CLL) cells have a differential capacity to signal through the B-cell receptor (BCR) following ligation with anti-IgM. This difference in signaling capacity has been suggested to contribute to the clinical disparity between the two subsets. However, as there appears to be no obvious correlation between response to IgM ligation and surface IgM expression, CD20 expression or CD79b expression it may be that the differential signaling response in these two distinct subsets is not caused by the BCR itself. In this study we investigated the expression of the BCR co-receptor complex (CD19, CD21 and CD81) in a cohort of CLL patients (n = 60) in order to determine whether expression of the co-receptor was associated with known CLL prognostic markers; IgVH gene mutational status, ZAP-70 and CD38 expression. In addition, we examined the relationship between co-receptor expression and the capacity of CLL cells to respond to anti-IgM cross-linking of the BCR. CLL samples showed significantly lower expression of both CD19 and CD21 when compared to normal B-lymphocytes (P <0.0001 and P<0.0001 respectively). In addition, samples derived from patients with unmutated IgVH genes showed significantly lower expression of CD19 and CD21 but not CD81 when compared to samples derived from patients with mutated VH genes (P = 0.04, P = 0.005 and P = 0.5 respectively). In contrast, none of the co-receptor molecules (CD19, CD21, CD81) were associated with CD38 expression (P = 0.82, P = 0.6, P = 0.64 respectively) or ZAP-70 expression (P = 0.2, P = 0.13 and P = 0.4 respectively). We next compared CD21 expression with levels of tyrosine phosphorylation (pre- and post-IgM ligation of the BCR). The results showed that there was no correlation between CD21 expression with either basal or IgM-stimulated tyrosine phosphorylation (P = 0.84 and P = 0.24 respectively). However, there was a significant inverse correlation with the percentage change in tyrosine phosphorylation following BCR cross-linking (P = 0.01). These data suggest that CLL cells with higher CD21 expression have a diminished magnitude of response to BCR ligation supporting the hypothesis that CLL cells with higher CD21 expression undergo receptor densitisation. Conversely, patients with lower CD21 expression may avoid receptor densitisation and consequently have a higher capacity for signaling following ligation of the BCR. Taken together, our data indicate a role for the BCR co-receptor in the signaling capacity of CLL cells and hence in the clinical course of the disease.
Humoral Immune Responses Against the Oncofetal-Antigen/Immature Laminin Receptor (OFA/iLR) in Patients with Chronic Lymphocytic Leukemia.
Authors: Friedrichs Birte , Siegel Sandra , Schmitz Norbert , Zeis Matthias 
Published: October 2019

Abstract
The 37 kD oncofetal antigen/immature laminin receptor (OFA/iLR) is a strongly overexpressed tumor antigen on malignant cells of numerous tumor entities including several hematological malignancies but it is not present on the cell surface of normal differentiated tissues. OFA/iLR is widely expressed on chronic lymphocytic leukemia (CLL) cells while absent on different normal B-cell subsets. The aim of this study was to evaluate and characterize humoral immune responses towards OFA/iLR in patients with CLL. 38 healthy individuals and 68 untreated patients with chronic lymphocytic leukemia were analysed for the presence of anti-OFA/iLR-antibodies. Sera were screened in an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM anti-OFA/iLR-antibodies. Detectable IgG or IgM responses to OFA protein were present in 32 of 68 patients (47,0%) but only in 4 of 38 (10,5%) healthy donors. IgG subtype analysis revealed a predominant IgG1 and IgG3 response. Utilizing 42 peptides deduced from the complete OFA/iLR amino acid sequence, 24 (35,3%) samples with significant anti-OFA/iLR IgG and/or IgM titers were also reactive with at least one OFA/iLR epitope. Patient-derived anti-OFA/iLR antibodies were capable of recognizing and selectively killing OFA/iLR expressing CLL cells in complement-mediated and antibody-dependent cellular cytotoxicity (ADCC) assays. Kaplan-Meier plots for progression-free survival (PFS) revealed a tendendy for longer PFS in patients with detectable humoral responses compared patients with negative ELISA (p=0,0165).
In addition, sera from eight CLL patients who had undergone allogeneic hematopoetic stem cell transplantion (allo-HSCT) were analysed. Six out of eight patients showed high values for IgG or IgM anti-OFA/iLR-antibodies after allo-HSCT as long as remaining in complete remission suggesting a potential role of anti-OFA/iLR-directed Graft-versus-Leukemia effects. For the first time, these data suggest that anti-OFA/iLR antibodies might be involved in the immunological control of OFA/iLR expressing CLL cells in vivo.
Early Clinical and Pharmacokinetic Results of Lower Dose Fludarabine and Cyclophosphamide, and High Dose Rituximab (FCR-Lite) for Patients with Untreated Chronic Lymphocytic Leukemia (CLL).
Authors: Tarhini Ahmad A. , Land S. , Meisner D. , Pietragallo L. , Sulecki M. , Lim F. , Kiefer Gauri J. , Pinkerton R. , Obsorn J. , Sukaly S. , Schaefer P.M. , Foon Kenneth A. 
Published: October 2019

Abstract
Introduction
Standard FCR therapy in untreated CLL patients (F-25 mg/m2 d1–3 q 4wk; C-250 mg/m2 d 1–3 q 4wk; R-500 mg/m2 d1 q 4wk for 6 cycles) was recently reported to have complete remission (CR) of 70% and overall responses (OR) of 95% (J Clin Oncol2005;23:4079). The major toxicity was grade 3/4 neutropenia during 52% of courses. One approach to decrease neutropenia without compromising efficacy could be by reducing the doses of F and C and increasing the dose of R.
Methods
We conducted a phase II study for previously untreated advanced CLL patients treated with FCR-Lite (F-20mg/m2 d1–3 q 4 wk; C-150 mg/m2 d1–3 q 4 wk; R-500mg/m2 d1 and d14 q 4wks; maintenance R-500 mg/m2 q 3 months until progression). A Simon two-stage design was used where 15 patients were accrued in the first stage and because of acceptable toxicity and response rate in stage I an additional 35 patients will be treated. The primary endpoint was response rate.
Results
Twenty eight patients (19 male, 9 female), age 36–85 years (median 58) were treated with a total of 154 FCR-Lite courses. All 28 patients were evaluable for toxicity. Grade 3/4 neutropenia occurred during 12 (8%) courses. There was one episode of neutropenic fever. Grade 3/4 thrombocytopenia occurred during 4 (3%) courses. Grade III anemia occurred during 2 (1%) courses. One patient had pneumonia. Among the 21 evaluable patients for response, the CR rate was 86%, PR rate was 14% with an OR rate of 100%. Seventeen of the CR patients were tested by flow cytometry and had no evidence of CD5+/CD19+ cells in their bone marrow after therapy. Preliminary data from pharmacokinetics performed on 16 patients showed time dependent increase in trough and peak antibody concentrations throughout treatment (Table 1).
Conclusions
Our results in the first 28 patients suggest FCR-Lite is highly effective with considerably less grade 3/4 neutropenia than standard FCR. Complete responders had no detectable CD5+/CD19+ cells in their bone marrow following FCR-Lite.
Table 1. Serum Rituximab Concentrations Before and After Infusion (N=16 patients) Infusion No. Concentration (μg/ml) (Mean) C1,d1, before 0 C1,d1, after 116.1 C1,d14, before 10.0 C1,d14, after 207.8 C2,d1, before 33.2 C2,d1, after 231.4 C3,d14, before 125.3 C3,d14, after 345.5 C4,d1, before 173.2 C4,d1, after 390.8
Oligonucleotide Microarray Analysis of Gene Expression Profiles in Chronic Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Oh Sukjoong , Lee Young Ju , Mee Kwon Soo , Park Eunkyung , Cho Sungbum , Kim In Ho , Yoon Sung Soo , Kim Joo Han , Park Seonyang 
Published: October 2019

Abstract
Chronic graft-versus-host disease (GVHD) is one of the most serious long-term complications following allogeneic hematopoietic stem cell transplantation. The pathophysiology of chronic GVHD is poorly understood. Certain genes may affect the outcome by modulating tissue injury with the alloimmune reaction. We assessed the gene profiles of 21 transplant recipients with oligonucleotide microarray (CodeLink®) containing 20,142 probes. All patients received hematopoietic stem cells from HLA-matched sibling donors except one who’s HLA was mismatched in one A locus. All patients were in complete remission and in complete chimerism state. Among the 21 patients, 11 had chronic GVHD and 10 not. RNAs were extracted from peripheral blood mononuclear cells of the transplant recipients. Sample-wise hierarchical clustering of the whole gene expression profile showed tendency of GVHD patients aggregating in the dendrogram. In GVHD patients, 141 genes were revealed up-regulated by SAM method. Gene ontology annotation of these genes indicated that up-regulated genes were related with proteolysis, lipid biosynthesis, phosphate metabolism and tissue development. A total of 461 pathways were examined to find the pathways showing differential expression patterns between GVHD and non-GVHD patients. The results showed that 227 pathways were statistically significant. These pathways included many immune-related processes, including T cell receptor signaling pathway, T helper cell surface molecules pathway, and HIV induced T cell apoptosis. With the 17 selected classifier genes, prediction accuracy of PAM algorithm was 86%. We could identify differentially expressed genes and pathways in chronic GVHD patients using oligonucleotide microarrays. We also found that extracted classifier genes showed relatively high prediction accuracy. Gene expression profile data may provide new insights into biological mechanism underlying GVHD and reveal disease-associated biomarkers of potential therapeutic targets.