bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of AKT and Enhanced Resistance to Apoptosis Induced by Surface IgM Ligation in ZAP-70-Positive Chronic Lymphocytic Leukemia B Cells.
Authors: Chen Liguang , Meraz Gabriel A. , Kipps Thomas J. 
Published: April 2020

Abstract
Signaling through the B-cell receptor (BCR) for antigen has been implicated to play a role in the pathogenesis and/or progression of chronic lymphocytic leukemia (CLL). Previously we found that BCR ligation on CLL cells that expressed ZAP-70 induced significantly higher levels of phosphorylation in p72Syk, BLNK, and phospholipase C-gamma than did BCR ligation on CLL cells that lacked expression of this protein tyrosine kinase. We hypothesized that CLL B cells that express ZAP-70 also may have enhanced levels of AKT activation following BCR ligation that is associated with increased resistance to apoptosis. CLL B cells were stimulated via surface IgM ligation and monitored at various time points for phosphorylation of cytoplasmic signaling molecules via immunoblot analyses and for viability by flow cytometry after staining with propidium iodide and DiOC6 to detect changes in mitochondrial membrane potential associated with apoptosis. We found that CLL cells that expressed ZAP-70 experienced significantly higher levels of AKT phosphorylation within 10 minutes following BCR-ligation than did ZAP-70-negative CLL cells. Over time a significantly higher proportion of ZAP-70-negative CLL cells were induced to undergo apoptosis by BCR-ligation than similarly treated CLL cells that were ZAP-70 positive. The differences between these two groups in leukemia cell viability over time after BCR ligation could be abrogated by addition of LY294002, a PI3-K inhibitor, to the CLL cell cultures 30 minutes prior to surface IgM ligation. We conclude that ZAP-70-positive CLL cells are relatively resistant to apoptosis induced by surface IgM ligation under the experimental conditions used, a characteristic that is associated with enhanced phosphorylation of AKT and activation of AKT-dependent pathways following BCR ligation. These studies support a model proposing that BCR ligation induces signaling that results in enhanced growth and/or survival of CLL cells that express ZAP-70 relative to that of leukemia cells that lack expression of this tyrosine kinase. Because expression of ZAP-70 typically is associated with expression of unmutated Ig V genes in CLL, the improved signaling afforded by expression of this tyrosine kinase may account in part for the greater tendency for disease progression observed in patients with CLL cells that use unmutated Ig V genes.
Expression of Specialized Error-Prone DNA Polymerases in Chronic Lymphocytic Leukemia (CLL).
Authors: Crespo Marta , Gine Eva , Muntanola Ana , Villamor Neus , Montserrat Emili , Bosch Francesc 
Published: April 2020

Abstract
Specialized DNA polymerases are low fidelity enzymes required to bypass DNA lesions that are also involved in somatic hypermutation. Deregulation of these enzymes can lead both to an increased mutation rate and to an enhanced translesion synthesis capability. DNA polymerase Kappa (DNA pol Kappa) has been found to promote tumorigenesis and to be overexpressed in some cancers. Expression levels of specialized DNA polymerases Iota, Eta, Zeta and Mu were analyzed in primary B-cells from 12 CLL patients (6 with IgVH mutated genes [M] and 6 with unmutated [UM] genes). Expression of DNA pol Kappa was also assessed in 27 CLL samples (13 M and 14 UM), in B-cell lines derived from pre- and post-germinal center malignancies (NALM-6 [preB-acute lymphoblastic leukemia], JVM-2 [prolymphocytic leukemia], Granta, NCEB and REC [mantle-cell lymphoma] and Daudi [Burkitt’s lymphoma] and in normal B-cells from peripheral blood and tonsil. Expression of DNA polymerases was measured by Quantitative RT-PCR using Ramos (a constitutively hypermutating Burkitt’s lymphoma cell line) as a reference, considering an arbitrary unit of 1 for each gene. Although somatic hypermutation is absent in UM CLL cells, there were no differences in the expression of the DNA polymerases analyzed according to the mutational status. Expression of DNA pols Iota and Zeta was similar in CLL cells and Ramos (mean value: 0.95 and 1.3, respectively), whereas expression of DNA pol Mu was slightly higher (mean 3.24) and DNA pol Eta was underexpressed (mean 0.19). In contrast, among all the hematological malignancies analyzed, significant overexpression of DNA pol Kappa was only found in CLL samples (mean value; 8.43;range: 1.42–19.47). Induction of AID expression by stimulating CLL cells with CD40 ligand and IL-4 did not modulate DNA pol Kappa expression. Finally, expression of DNA pol Kappa in normal B cells from peripheral blood was similar to that in Ramos (0.87) and slightly higher in B cells from tonsil (2.02). In conclusion, no differences in DNA polymerases were observed between M and UM CLL. As previously described in some solid tumors, CLL cells showed an increased expression of DNA pol Kappa, regardless the mutational status of the IgVH genes, suggesting that the expression of this polymerase is not related to the process of somatic hypermutation that occurs in some CLL cells. Taken together, these results support the concept that DNA pol Kappa may behave as an oncogene in CLL and that its regulation may be crucial for maintaining genomic stability.
Thalidomide (T) in Combination with Fludarabine (F) as Initial Therapy for Patients (pts) with Treatment Naïve Chronic Lymphocytic Leukemia (CLL): Preliminary Results of a Phase I/II Clinical Trial.
Authors: Chanan-Khan Asher Alban , Miller Kena C. , Marshall Patricia , Padmanabhan Swaminathan , Brady William , Bernstein Zale P. , Wallace Paul , Czuczman Myron S. 
Published: April 2020

Abstract
Introduction: The importance of VEGF and TNF-a and their interaction with other cytokines in the pathogenesis of CLL has recently been identified. These cytokines in the tumor microenvironment (ME) form a network of growth promoting signal(s) that regulate the CLL cell survival and progression. T is an immunomodulatory agent, reported to exert antitumor activity through down regulation of VEGF, TNF-a and IL-6. We hypothesized that targeting the tumor ME with T will interrupt the growth promoting signals and predispose CLL cells to enhanced proapoptotic activity of F with resultant improvement of clinical responses. In this abstract we report our findings to date of this phase I/II clinical trial.
Patients and Methods: Treatment naïve pts requiring therapy for CLL were eligible for this study. T was started at D1 and continued for 6 months while F (25mg/m2 x 5 D every 4 wks) was given for 6 cycles starting on D7. Three doses of T (100,200,300 mg) were studied in phase I and as no dose response curve or DLT was noted, 200mg of T was selected for the phase II evaluation for concerns of cumulative toxicity of T. Low-dose coumadin (1 or 2 mg) was used for prophylaxis of venous thromboembolism (VTE).
Results: To date 20 pts (15M, 5F; median age 66, range 38–76 yrs) have been enrolled. Of these 11 pts had limited (0-II) stage and 9 pts had advance Rai stage (III/IV) disease. Pts who completed the intended 6 months of T were evaluable for response. Six pts were removed from study for toxicity (2VTE - 1 in the 1st week of therapy prior to F infusion, 2nd during the 4th cyc; 1 Hep C reactivation presumably secondary to F, 1 secondary to early death from advance disease, 1 non-compliance, and 1 early termination for poor tolerance to therapy), without evidence of disease progression. Thirteen pts completed 6 months of therapy and are available for response. Among the evaluable pts 9 (69.2 %) achieved a complete response (CR) and 4(30.7%) were noted to have a partial remission (PR). While all pts who completed 6 months of T achieved a major response, the ORR in the intent-to-treat population was 65% with median duration of response 12+ (range 3 – 18+) months. Fatigue, thrombocytopenia, and anemia were the most common side effects, noted in 75%, 65%, and 55% of the 20 patients, respectively.
Conclusion: T when combined with F results in a higher ORR when compared to F alone based on historical data. The combination FT was well tolerated with manageable toxicity. Completion of this ongoing phase II study will identify the role of this new combination in pts with CLL.
In Vivo Evaluation of Immunomodulating Effects of Lenalidomide (L) on Tumor Cell Microenvironment as a Possible Underlying Mechanism of the Antitumor Effects Observed in Patients (pts) with Chronic Lymphocytic Leukemia (CLL).
Authors: Chanan-Khan Asher Alban , Padmanabhan Swaminathan , Miller Kena C. , Pera Paula , DiMiceli Laurie , Ersing Noreen , Wallace Paul , Rickert Michael , Porter Carl 
Published: April 2020

Abstract
Introduction: L is a more potent analogue of thalidomide with antitumor activity reported in MDS and multiple myeloma. Clinical anti-leukemic activity of L is reported for the first time by our group in pts with CLL. The underlying mechanism of its antitumor activity remains undetermined. We investigated the effect of L on the tumor microenvironment and studied the modulation of soluble cytokines and immune cells (T and NK cells) in pts receiving L.
Patients and Methods: CLL pts enrolled on the clinical study with L were eligible. Pre and post (day 7) samples were obtained for evaluation of changes in serum cytokine and immune cell environment. Malignant cells were also obtained to investigate the in vitro antitumor activity of L prior to initiating treatment on clinical trial.
Results: With Anexin V staining for evaluation of apoptosis induction, in vitro testing of pts samples (n=10) showed only a modest increase in apoptosis at 200mg of L - levels clinically not achievable. Yet same pts treated with L on clinical study showed significant antitumor response, suggesting the mechanism to be possibly related to modulation of the tumor microenvironment. In evaluation of the tumor cytokine microenvironment (n= 10) we noted significant L induced increase in IL-10 (n=6), IL-8 (n=8), IP-10 (n=10), IL-8 (n=8), TNF-alpha (n=4) and decrease in PDGF (n=5) and RANTES (n=5). While evaluation of the immune cell repertoire we observed an absolute increase in T-cell as well as NK-cell after treatment with L.
Conclusion: Our in vitro evaluation does not suggest a direct apoptotic effect of L on the malignant CLL cells and thus support the hypothesis that the anti-leukemic effect noted in the clinical trial (reported separately) is most likely from in vivo modulation of the tumor microenvironment as is demonstrated from changes in the cytokine milieu and the cellular immune response. Collectively these changes may be responsible for the immune modulating properties of L and the resultant anti-CLL activity in pts.
Immune Therapy for Chronic Lymphocytic Leukemia Induces the Antibody Response Against a Novel Tumor-Associated Antigen, the Orphan Tyrosine Kinase Receptor ROR1.
Authors: Fukuda Tetsuya , Rassenti Laura Z. , Kipps Thomas J. 
Published: April 2020

Abstract
Transduction of Chronic lymphocytic leukemia (CLL) CLL cells with replication-defective adenovirus encoding CD154 (Ad-CD154) enhances the capacity of such cells to function as antigen presenting cells. In a clinical trial patients received multiple infusions of autologous CLL cells transduced ex vivo with Ad-CD154 to examine the safety and efficiency of Ad-CD154 gene therapy. Treated patients were found to mount antibody responses against the recombinant adenovirus and cellular immune responses against autologous CLL cells. We examined the antibody response generated in some of the treated patients against CLL cells. Pre and post treatment sera were incubated with CLL cells followed by detection with human anti-IgG antibody via flow cytometry. We found that 3 out of 6 patients examined developed IgG antibodies after therapy that could bind CLL cells. Microarray analyses of CLL samples identified a relatively small number of genes that were differentially expressed in CLL cells compared with normal B cell subsets or neoplastic B cells of other B cell malignancies. These CLL signature genes are candidates TAAs of CLL. One such a CLL signature gene encodes ROR1, a surface receptor tyrosine kinase. Surface expression of ROR1 was on CLL cells, but not on normal lymphocytes. We used two methods to detect the anti-ROR1 activity. One was the flow cytometric analysis using Chinese hamster ovary cell transfected with ROR1 cDNA. Another strategy used an ELISA with plates coated with recombinant ROR1protein. Both methods revealed that patients who developed anti-CLL autoantibodies following gene therapy also developed IgG antibodies reactive with ROR1. As such, ROR1 appears to be a candidate TAA that may be targeted by immune responses induced by Ad-CD154 gene therapy.
RhG-CSF Mobilized and Apheresis-Collected Endothelial Progenitor Cells for Therapeutic Vasculogenesis.
Authors: Korbling Martin , Reuben James M. , Gao Hui , Lee Bang-Ning , Giralt Sergio A. , Khouri Issa F. , Saliba Rima M. , Champlin Richard E. , Estrov Zeev 
Published: April 2020

Abstract
Endothelial progenitor cells (EPCs) have been identified as part of hematopoietic tissue-derived progenitor cells that are mobilized into the peripheral blood (PB) as a result of tissue injury. It therefore seems likely that circulating EPCs have therapeutic potential by aiding in the neovascularization of ischemic tissue. It was the purpose of this study to provide clinical data on the availability of circulating EPCs at steady-state and after rhG-CSF mobilization, and their collection by leukapheresis. Eight healthy donors underwent rhG-CSF (10 μg/kg) treatment over 4 days. Continuous-flow leukapheresis (COBE Spectra, Version 4.7) was performed on the fourth day of rhG-CSF treatment. Blood samples were drawn before starting rhG-CSF treatment, before apheresis, and from the apheresis collection bag. The total cell numbers collected per apheresis were based on processing a median of 17 L [10.7–20.1] blood volume or approximately three times the donor’s total blood volume. Blood and apheresis samples were analyzed by flow cytometry for EPC surface markers CD34, CD133, VEGFR-2, and by forming EPC colonies (CFU-EPC). The Wilcoxon matched-pairs signed-ranks test was used to compare the distributions at various sampling points.
All CD133 subsets were CD14 negative to exclude differentiated monocytes or macrophages. The median steady-state PB concentrations of circulating CD34+CD133+VEGFR-2+ cells was 0.9/μl [0–3], or 1.7/μl [0.9–4] for CD34+133−VEGFR-2+ EPCs. After 4 days of rhG-CSF mobilization treatment the PB CD34+CD133+VEGFR-2+ subset concentration increased by a median of 8-fold [p 0.01], and the CD34+133−VEGFR-2+ subset concentration by a median of 10-fold [p 0.01] over baseline. The median PB concentration of circulating CFU-EPCs increased by 10-fold [p 0.02]. The median absolute number of CD34+CD133+VEGFR-2+ and CD34+CD133-VEGFR-2+ cells collected by leukapheresis per kg of body weight was 0.845 x 106 [0.12–2.55] and 1.54 x 106 [0.06–2.4], respectively. A small population of CD133+34− VEGFR-2+ cells was identified in steady state and mobilized PB, and in the apheresis collect (0.11 x 106/kg [0–0.26]). CD34− progenitor cells expressing CD133 are believed to represent a primitive progenitor cell population containing SCID-repopulating cells. VEGFR-2 coexpression of those immature cells may define a circulating cell population that contributes to vasculogenesis.
Our data suggest that clonogenic circulating EPCs can be mobilized by rhG-CSF and collected by continuous-flow leukapheresis generating large numbers of EPCs, specifically in the range of 60 x 106 EPCs per leukapheresis procedure for a 70 kg individual. Circulating EPCs represent a novel blood cell component that can be clinically used in large quantities, either unmanipulated or EPC-selected, for therapeutic vasculogenesis.
The Induction of Apoptosis by Histone Deacetylase Inhibition in Chronic Lymphocytic Leukemia (CLL) Cells Is Mediated through the TNF-Related Apoptosis-Inducing Ligand (TRAIL) Pathway.
Authors: Szwajcer David , Gibson Spencer B. , Johnston James B. 
Published: April 2020

Abstract
Background and Objectives: Histone Deacetylase Inhibitors (HDIs) have been shown to induce cell cycle arrest, differentiation and apoptosis in a variety of myeloid and lymphoid malignancies while being relatively nontoxic to normal cells. HDIs have also been shown to sensitize CLL cells to TRAIL-induced apoptosis. In this study we investigated the effects of two HDIs, trichostatin (TSA) and valproic acid (VPA), alone and in combination with fludarabine (FLU) on apoptosis in the BJAB lymphoblastoma B-cell line and in primary CLL cells.
Materials and Methods: Cell viability was assessed by the MTT (3-(3,5-dimethylthiazol-2yl)- 2,5 diphenyl tetrazolium bromide) colorimetric assay, following 4 days of continuous drug treatment. Apoptosis was assessed by staining cell suspensions with acridine orange and quantitating apoptotic cell populations using fluorescence microscopy. Protein expression for the TRAIL receptors, DR4 and DR5, was determined by western blotting.
Results: As determined by the MTT assay, the mean concentrations of TSA and VPA to reduce cell viability by 50% (IC50) in CLL cells were 0.23 uM and 650uM, respectively. TSA at 0.5uM and VPA at 2mM induced 60% apoptosis following 48 hours of drug exposure in both BJAB and CLL cells, as measured by acridine orange staining. Addition of DR4:FC, which sequesters TRAIL away from its receptors, decreased TSA- and VPA-induced apoptosis in BJAB and CLL cells by 9 and 20% respectively, indicating activation of the TRAIL apoptotic pathway by these HDIs. In addition, exposure to TSA at a concentration of 1uM for 24 hours, up regulated the expression of DR5 in BJAB cells, but this was not observed with VPA at a concentration of 1.5mM, after up to 48 hours of treatment. Neither HDI influenced DR4 protein expression in these cells. Preliminary results show that VPA potentiated FLU-induced apoptosis by 20% in both BJAB and CLL cells.
Conclusions: Both TSA and VPA induce apoptosis in BJAB and CLL cells by activation of the TRAIL apoptotic pathway. For TSA, this effect may be potentiated by up regulation of DR5 expression. VPA potentiates FLU-induced apoptosis in BJAB and CLL cells, suggesting a therapeutic role for the combination of HDIs and nucleoside analogues in the management of CLL. Ongoing studies are determining whether this effect is specific for CLL cells, as compared to normal lymphocytes, and the optimum scheduling for synergy.
Identification of Candidate Tumor Suppressor Genes Silenced Epigenetically in Mantle Cell Lymphoma.
Authors: Kawamata Norihiko , Saitoh Takayuki , Sakajiri Sakura , Koeffler Phillip H. 
Published: April 2020

Abstract
Many tumor suppressor genes are silenced by epigenetic mechanisms in human cancers, including mantle cell lymphoma (MCL). In this study, we have used a variety of research tools to screen for genes that are epigenetically silenced in MCL. Changes in the global gene expression profile of the MCL cell line, Jeko1, were analyzed after treatment with the combination of the demethylating agent, 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, suberoyl anilide bishydroxamide, by DNA microarray technique. By screening over 22,000 genes, we identified 26 candidate tumor suppressor genes, expression of which were enhanced by the treatment, in the MCL line. Basal expression of these 26 genes were low in Jeko1 cells. The treatment enhanced the expression more than 2 folds and the enhancement was also confirmed by real-time PCR. Methylation status of these 26 genes were examined by bisulfite sequencing and/or combined bisulfite and restriction enzyme digestion assay in Jeko1 cells. We found hypermethylation of a CpG island in the middle of the INPP5F gene. We also found the hypermethylation of that region of INPP5F in normal peripheral blood. We also examined expression levels of these 26 genes in normal mantle cells by real-time PCR and found only 11 genes showed high levels of transcription in laser-dissected normal mantle cells. We examined expression of these 11 genes in eight MCL clinical samples by real-time PCR and found that only three genes, INPP5F, DUSP10 and FGD2 showed very low expression levels. We conclude that expression of INPP5F, DUSP10 and FGD2 genes were suppressed in MCL cells although the expression of these genes are high in normal mantle cells. INPP5F is a inositol phosphatase and could be involved in PI3K pathway. DUSP10 is a dual specific phosphatase and could be involved in JNK pathway. FGD2 is a RAS-GAP gene and could be involved in RAS pathway. These three genes may be candidate tumor suppressor genes in MCL and further functional analysis is ongoing.
Pre-Infusion Characteristics of the Predominant Cord Blood Unit Correlate with Hematopoietic Engraftment in the Setting of Non-Myeloablative Double Cord Blood Transplant (DCBT).
Authors: Haspel Richard L. , Kao Grace S. , Yeap Beow , Spitzer Thomas R. , Ritz Jerome , Antin Joseph H. , Ballen Karen 
Published: April 2020

Abstract
It is well established that delayed engraftment following transplant of single cord blood unit is mainly due to low CD34 cell dose. Protocols utilizing sequential infusion of two cord blood units increase the total dose of CD34 cells and lead to more rapid hematopoietic engraftment. One of the two cord bloods usually predominates. We assessed the effect of the pre-infusion variables of each cord blood unit on engraftment. Twenty-one patients with hematologic malignancies underwent non-myeloablative conditioning with fludarabine, mephalan and antithymocyte globulin. Two cord bloods, each a minimum 4/6 HLA match with the recipient and each other, were then infused sequentially 1 to 6 hours apart. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Chimerism analysis of peripheral blood leukocytes was performed by PCR amplification of short tandem repeat loci. Four patients with less than 4 weeks of chimerism data due to death or failed engraftment were excluded. The logrank test, using median values as the cutoff, was used to determine the relationship between pre-infusion variables and time to engraftment. All exact p-values are based on two-sided tests. In all patients, a single cord blood predominated by 3 months post-infusion. In 13 out of 17 transplants (76%), the first cord blood infused predominated (p=.05). The median times for engraftment were 41 days (range: 21–55 days) for platelets &gt;20000 (P20), 65 days (range: 34–91 days) for platelets &gt; 100000 (P100) and 18 days (range: 15–34 days) for ANC&gt;500. Total (cord1 + cord2) CD34 cells/kg (median: 1.9 x 105; range: 6.3 x 104 to 1.0 x 106) and total nucleated cells/kg (median: 4.02 x 107; range 2.98 x 107 to 5.27 x 107) infused did not affect engraftment. However, patients receiving greater than 1.1x 105 CD34/kg in the predominant unit (median: 1.1 x 105; range: 3.0x104 to 8.7x105) but not losing unit (median: 6.92 x 104; range: 8.3.0x103 to 7.4x105) had faster P100 (p=.042 versus p=0.54). The median storage time of the cord blood units infused was 1374 days (range: 272–3047 days). Faster P100 was observed when the predominant unit had a shorter storage time relative to the losing unit (median difference between units: 392 days, p=.03). Our study demonstrated that in the setting of non-myeloablative sequential DCBT, the majority of predominant units engrafted is the first one infused. The CD34 dose and storage time of the predominant unit are factors that can affect engraftment. Although only a small number of patients were studied, these results suggest that the capacity of stem cell niche may be limited and the quality of the cord blood unit occupying this niche can affect the speed of hematopoietic engraftment.