bluuebunny/crossref_metadata_2025_split huggingface dataset:
Feasiblity and Implication of Bidirectional Sequencing Using a Multiplex Framework 2 Region Primer for Somatic Mutation Analysis of the Immunoglobulin (IgH) Heavy Chain in Chronic Lymphocytic Leukemia (CLL).
Authors: Nelson Adin , Rijo Ivelise , Zhang Zhigang , Zelenetz Andrew D. , Noy Ariela 
Published: October 2019

Abstract
Background: A somatic mutation rate of >2% of the immunoglobulin (IgH) heavy chain gene, in comparison to germline DNA, is a positive prognostic indicator in chronic lymphocytic leukemia (CLL). Genomic DNA is amplified using the Biomed-2 multiplex PCR primer set, sequenced in the reverse direction only using a 3′ JH primer, and compared to the germline sequence in VBase, a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences. It is unknown whether forward sequencing using the multiplex 5′ Biomed-2 primers in a single reaction is feasible or whether doing so improves the accuracy of the mutational analysis.
Methods: DNA was extracted from peripheral blood or bone marrow mononuclear cells from patients with CLL using the QIAGEN QIAspin DNA mini-kit. The FR2 region of the IgH gene was amplified using tube B of the Biomed-2 multiplex PCR primer set, separated in 2% agarose gel, excised, and prepared for sequencing using the QIAGEN QIAquick gel extraction kit. Bidirectional DNA sequencing was performed with the full set of multiplex 5′ FR2 primers combined in one reaction for the forward sequence and in a second reaction with the Biomed-2 3′ JH primer for the reverse sequence. A consensus sequence was obtained and mutation rates were calculated based on the consensus sequence and the reverse sequence separately.
Results: The FR2 region was amplified from 30 CLL patients. 20 samples produced bidirectional consensus DNA sequences, 2 sequenced only with multiplex FR2 primers, 6 sequenced only with a JH primer, and 2 produced no sequences. Compared with unidirectional JH sequencing, bidirectional sequencing did not reassign any patient in the germline IgH group to the mutated category. In 15 patients, the uni- and bi-directional sequencing were identical. In 4 patients, a single basepair difference was noted. A random permutation test yields a two-sided p-value as 12.5%, indicating that no significant difference was detected between the uni- and bi-directional sequencing. Multiplex bidirectional sequencing did provide sequence information within the CDR3 region at the 3′ terminus of the amplimer which is partially truncated when using the JH primer alone.
Conclusion: Bidirectional sequencing does not provide a somatic IgH mutation rate that differs significantly from that obtained from the reverse sequence alone and does not likely influence the IgH mutational prognostic assignment. Nonetheless, the bidirectional consensus sequence adds bases in the CDR3 region which can be used for research applications such as the generation of patient-specific primers for PCR. It is also noteworthy that multiplex PCR using the Biomed-2 primers is feasible.
Protein Expression Profiling of Cytokines and Cytokine Receptors on Purified Chronic Lymphocytic Leukemia Cells.
Authors: Yu Zhifeng , Sun Baohua , Kantarjian Hagop M. , Amin Hesham M. , Sun Xiaoping 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) B-cells rapidly undergo apoptosis when cultured in vitro, which contrasts with their prolonged survival in vivo. Multiple cytokines and cytokine receptors are believed to work together to regulate the survival of CLL cells. The literature is conflicting as to whether the CLL cells themselves produce significant amounts of cytokines compared with normal B-cells and how the CLL cells respond to these cytokines. This discrepancy is largely due to the different experimental conditions that have been used whereby various amounts of exogenous cytokines were introduced into the experimental system from, for example, the serum used to supplement the culture medium and the lysate or conditioned medium of CLL cells where other types of mononuclear cells were not removed. The aim of the current study is to reveal the intrinsic production and secretion of cytokines and cytokine receptors in CLL cells when exogenous sources are minimized. We purified CD19+ cells by magnetic beads from peripheral blood mononuclear cells of five CLL patients who had stage I or II disease and had not received any therapy. CD19+ cells from healthy donors were used as control. We used a cytokine antibody array approach that simultaneously measured 174 cytokines and cytokine receptors. We determined both intracellular levels in purified CLL cells and secreted levels in serum-free conditioned medium. The intracellular levels of cytokines and cytokine receptors of the purified CLL cells and the normal B-cells were not significantly different. However, the secretion of interleukin-6 (IL-6) was 3.0 times lower (p = 0.038) and that of eotaxin was 2.2 times higher (p = 0.028) in CLL-conditioned medium than in normal B-cell-conditioned medium. We further studied the effect of IL-6 and anti-IL-6 antibody on the apoptosis of purified CLL B-cells in serum-free culture, but no significant change was found in the presence or absence of IL-6 or IL-6 antibody. Except for IL-6 and eotaxin, our results suggest that CLL cells and their normal counterparts produce and secrete similar amounts of cytokines and cytokine receptors in vitro and that the in vivo longevity of CLL cells may be due to the concerted effects of various molecules and cellular interactions in the microenvironment.
What Have We Learned from Measuring Health-Related Quality of Life in Patients with Chronic Lymphocytic Leukemia? A Systematic Review of Published Studies from 1990 to 2007.
Authors: Molica Stefano , Mauro Francesca , Vignetti Marco , Foà Robin , Efficace Fabio 
Published: October 2019

Abstract
While a large amount of health-related quality of life (HRQOL) studies has been conducted in patients with solid tumors, no data are available for patients with CLL. The aim of the present work was therefore to quantify and evaluate the quality of studies investigating patient’s self-reported HRQOL in CLL patients. A systematic literature search for studies meeting the criteria, published from 1990 to 2007, was undertaken on a number of databases. Candidate articles were mainly searched in PubMed and the Cochrane Central Register of Controlled Trials (CENTRAL). Meeting abstracts at ASCO and ASH conferences were also assessed. We used the following key terms to identify candidate articles “quality of life” and “CLL” or “lymphocytic leukemia” or “chronic leukemia”. All articles included in the review were independently evaluated based on a pre-defined set of methodological issues and criteria by two reviewers (SM and FE). These included a number of methodological and statistical quality criteria such as HRQOL missing data documentation, timing of assessment, rationale for instrument selection and discussion of outcomes in terms of clinical significance. In addition, HRQOL and traditional clinical outcomes were extracted and analyzed. More than 200 candidate articles were evaluated, but only 5 studies met the inclusion criteria for the review. The majority of articles were excluded as HRQOL was not measured by patient’s reported tools. Overall, some 1019 patients were involved in these studies. HRQOL was assessed in 3 prospective randomized controlled trials (RCTs) and 2 longitudinal non-randomized studies. Two of these 5 studies were only published as abstracts. The EORTC QLQ-C30 was the most frequently used measure being utilized in 4 out of 5 studies. Some methodological constraints were identified, mainly in terms of handling of HRQOL missing data and possible lack of sensitivity of the measure being used. The most robust evidence stemmed from a prospective RCT comparing Fludarabine (F) vs. Fludarabine + Cyclophopsphamide (FC) where HRQOL was assessed as a secondary endpoint. While there was no significant HRQOL difference between treatment arms over time, this study showed nonetheless that the newer FC combination had no detrimental effect on patient’s HRQOL compared with the standard F arm. This information definitely reinforces concomitant clinical findings which favor the more intensive FC treatment (e.g. in terms of clinical response and progression-free survival). Taken together, very limited conclusions can to date be drawn with regard to HRQOL issues in patients with CLL in view of the paucity of data in this area. There is urgent need of implementing HRQOL research in patients with CLL and of providing the scientific community with robust evidence-based data on the impact of new treatment approaches in these patients.
The Non-Death Domain Containing TNFR Member HVEM Induces Apoptosis and Chemokine Production on B-Chronic Lymphocytic Leukemia Cells.
Authors: Olive Daniel R. , Pasero Christine , Barbarat Bernadette , Truneh Alem , Just Sylvaine , Aurrand Therese , Rey Jerome , Costello Regis T. 
Published: October 2019

Abstract
B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia and it still remains incurable. New therapies are required for this disease characterized by failure of mature lymphocytes to undergo apoptosis. Members of tumor necrosis factor receptors (TNFR) family play important roles in cell activation, proliferation, differentiation and apoptosis. We focused our study on HVEM (Herpes virus entry mediator), devoid of intracytoplasmic death domain, which is known to costimulate T- and B-cell activation, and its ligand LIGHT. Surprisingly, we found that LIGHT, as well as some anti-HVEM antibodies, induced apoptosis of B-CLL cells. This apoptosis was associated with activation of caspase-3, -8 and -9, decrease in mitochondrial membrane potential, and upregulation of the pro-apoptotic protein Bax. Importantly, the efficiency of HVEM-cell killing compared favorably with that of the pan-B cell therapeutic monoclonal antibody Rituximab. In addition, HVEM induced upregulation of various cytokines and chemokines, and a major increase in IL-8 secretion. Thus, HVEM stimulation induces apoptosis of B-CLL cells and could potentially also participate in the recruitment of immune effectors, and may therefore be useful for clinical treatment of this malignancy.
Biologic Sequelae of CDC2 Inhibition in MM.
Authors: Fulciniti Mariateresa , Tassone Pierfrancesco , Hideshima Teru , Anderson Kenneth C. , Munshi Nikhil C. 
Published: October 2019

Abstract
Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.
An Anti-VEGF Antibody, in Combination with Bortezomib, Markedly Inhibits Tumor Growth in SCID-hu Models of Human Multiple Myeloma.
Authors: Campbell Richard A. , Sanchez Eric , Steinberg Jeffrey , Share Michael , Wang Joseph , Li Mingjie , Leuchter Richard , Chen Haiming , Shalitin Dror , Pang Shen , Said Jonathan , Plowman Gregory D. , Berenson James R. 
Published: October 2019

Abstract
Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. We previously evaluated the effects of single agent mouse/human anti-VEGF antibody G6.31 (Campbell et al, Blood (ASH Annual Meeting Abstracts), Nov 2006) in several of our mouse models of human MM. In this study, we evaluated the effects of the same anti-VEGF antibody in combination with bortezomib or lenalidomide. Severe combined immunodeficient (SCID) mice were implanted into the left superficial gluteal muscle with either a 2.0 – 4.0 mm3 fragment from a patient when she was bortezomib-sensitive, LAGκ-1A, or resistant, LAGκ-1B. The tumors were allowed to grow for 21 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups (n=10 mice/group). Treatment groups consisted of a control IgG antibody or anti-VEGF antibody administered via i.p. injection twice weekly at a dose of 2 mg/kg, bortezomib administered via intravenous injection at a dose of 0.25 or 0.5 mg/kg twice weekly, lenalidomide administered via i.p. injection at a dose of 50 mg/kg daily × 5 per week, anti-VEGF antibody (2 mg/kg) + bortezomib (0.25 or 0.5 mg/kg), and anti-VEGF (2 mg/kg) + lenalidomide (50 mg/kg). Mice receiving the combination therapy of anti-VEGF + bortezomib (0.5 mg//kg) antibody showed marked inhibition of tumor growth and reduction of paraprotein levels compared to mice receiving control antibody. Notably, this combination also produced much more marked anti-MM effects compared to bortezomib treatment alone, anti-VEGF antibody alone, or vehicle alone. This combination was well tolerated. In contrast, mice receiving the combination of anti-VEGF antibody + lenalidomide showed no significant differences in tumor volume or hIgG levels compared to single agent treatment or vehicle alone. The markedly improved anti-MM effects of the combination of bortezomib and anti-VEGF antibody compared to single agent treatment in this in vivo study of human MM is promising, and these results have provided the preclinical rationale for an ongoing randomized multi-center Phase II trial.
Elevated Pretransplant Serum Ferritin in Autologous Stem Cell Transplant.
Authors: Mahindra Anuj , Bolwell Brian , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Dean Robert , Andresen Steve , Kalaycio Matt , Sweetenham John , Copelan Edward 
Published: October 2019

Abstract
Iron overload is an adverse prognostic factor in patients who undergo allogeneic hematopoietic stem-cell transplantation (HSCT) for thalassemia and apparently in patients with acute leukemia and myelodysplastic syndrome as well. Iron overload has also been associated with susceptibility to infection following autotransplantation and with impaired immunity in other settings. We report here the results of a large study of consecutive patients undergoing autologous hematopoietic stem-cell transplantation (ASCT) for various hematologic malignancies to assess the influence of pretransplantation ferritin on outcomes following transplant. Pretransplantation ferritin, obtained within 100 days preceding transplant, was available on 397 patients undergoing autologous HSCT for various disorders (NHL=257, MM= 61, HD=58, AML=21) from 11/2/2000 to 12/28/2006.The median ferritin was 529.3 ng/ml (range, 12.8–4330). The median patient age was 52 (range, 19–77). Recursive partitioning analysis identified baseline ferritin >685 ng/ml as an adverse prognostic factor for survival as shown: ESTIMATED SURVIVAL BY PRETRANSPLANT FERRITIN LEVEL
Figure Figure
Age, gender, disease, disease status at transplant, interval from diagnosis to transplant, number of prior chemotherapy regimens, prior radiation therapy, ferritin, albumin, AST, Alkaline phosphatase, LDH, preparative regimen and CD 34+ dose were analyzed in a multivariable analysis. Elevated ferritin was an independent, adverse, significant prognostic factor for survival (p<0.001, HR=2.29),relapse-free survival (P<.001, HR=1.79) and relapse (p=0.006, HR=1.68). The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of ferritin. Elevated ferritin was also significantly predictive of a higher incidence of relapse mortality (p<0.001) as shown below: RELAPSE MORTALITY BY FERRITIN LEVEL
Figure Figure
Conclusions: Elevated ferritin adversely influences survival, relapse-free survival, and relapse mortality following autologous transplantation. Whether the increased number of deaths due to relapse is related to the established immunosuppressive affect of iron overload is unknown. Iron chelators have been shown to inhibit the growth of tumor cells in vitro and in vivo. Trials designed to analyze the benefit of iron chelation therapy prior to ASCT in patients with iron overload are warranted.
Reactive Oxygen Species Target the Degradation of the EVI1 Oncoprotein.
Authors: Shackelford David , Wang You , Waxman Samuel , Ren Ruibao 
Published: October 2019

Abstract
Aberrant expression of EVI1 has been frequently found in myeloid malignancies as well as in cancers of the ovary, lung, head and neck, cervix, and breast, and is associated with a poor patient survival. Targeted degradation of oncoproteins is an effective strategy for cancer therapy. The AML1/MDS1/EVI1 (AME) transcription factor fusion protein is a product of the human t(3;21)(q26;q22) translocation found as a secondary mutation in some cases of CML during the blast phase (CML-BP) and in patients with de novo and therapy-related myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Expression of AME in mouse bone marrow cells by retroviral transduction impairs hematopoiesis and eventually induces an acute myeloid leukemia (AML)-like disease in mice. Arsenic Trioxide (ATO) has been found to be an effective treatment for patients suffering from acute promyelocytic leukemia (APL). This is, at least in part, mediated by degradation of the PML/RARα oncoprotein that is associated with over 90% of APL. We have recently shown that ATO used at therapeutic levels also degrades AME. The ATO treatment induces differentiation and apoptosis in AME leukemic cells in vitro and causes decrease in peripheral leukemic cells and splenomegaly in vivo. ATO appears to target AME at both the EVI1 and MDS moieties of the protein for degradation via the ubiquitin-proteasome pathway and proteasome-independent mechanism, respectively. To investigate the mechanism of ATO induced degradation of EVI1 oncoproteins, we examined the effect of reactive oxygen species (ROS), on EVII expression, one of the downstream effectors of ATO-induced apoptosis. We found that EVI1 degradation correlates with the amount of ROS generated in cells. EVI1 can also be targeted for degradation by doxorubicin, a chemotherapeutic agent that is effective in treating AML and ovarian cancer and a strong ROS inducer. Interestingly, the antioxidant N-acetyl cysteine (NAC) abrogates the degradation of the EVI1 protein in the presence of doxorubicin. These results demonstrate that EVI1 can be targeted for degradation by ROS. ROS inducing agents could be used as a part of targeted therapy for EVI1-positive malignancies.
Development of Allosteric Inhibitors of the Interaction of AML1 and CBFβ for the Treatment of Leukemia.
Authors: Douvas Michael G. , Roudaiya L. , Grembecka J. , Speck N. , Bushweller J.H. 
Published: October 2019

Abstract
The protein-protein interaction between the subunits of the nuclear transcription factor core binding factor, RUNX1 (CBFα) and CBFβ, plays a critical role in hematopoiesis. Chromosomal rearrangements that target CBF genes are among the most common mutations in leukemia, including t(8;21) found in approximately 12% of cases of AML and resulting in the protein AML1-ETO. We describe the development of small molecule inhibitors of the interaction between RUNX1 and CBFβ. We used virtual screening to identify lead compounds. These leads were used to generate a library of compounds to explore the structure-activity relationships and optimize activity, resulting in the identification of low micromolar IC50 inhibitors of this protein-protein interaction. We confirmed by FACS FRET that inhibition also occurs in mammalian cells. HEK-293 cells were transfected with Cerulean-Runt domain and Venus-CBFβ. FRET emission from cells was monitored on a flow cytometer to assess binding of CBFβ and the Runt domain. Results with drug were compared to FRET between Runt and wild-type CBFβ and FRET between Runt and a mutant CBFβ with 2 point mutations abrogating binding to Runt. The small molecule inhibitor KG-3-275 inhibited CBFβ - Runt domain binding in a dose dependent manner (Table 1). Treatment of the t(8;21) leukemia cell lines Kasumi-1 and SKNO-1 results in inhibition of proliferation. SKNO-1 cells are inhibited in a dose-dependent manner by KG-3-275 but not by the weak inhibitor KG-1-253 (Fig 1). KG-3-275 does not inhibit the growth of renal tubular and hepatocellular cell lines (Fig 1). Kasumi-1 and SKNO-1 undergo apoptosis in a dose-dependent manner upon treatment with KG-3-275 (Table 2). Finally, KG-3-275 shows synergy with ATRA in increasing differentiation of Kasumi-1 cells as measured by CD11b expression, consistent with recent published results establishing a link between AML1-ETO and repression of RAR signaling (Fig 2). These data indicate drugs inhibiting CBF interactions hold promise as targeted agents in the treatment of CBF leukemias.
Fluorescence Resonance Energy Transfer Geometric Mean in HEK-293 Cells Cerulean-Runt domain + Venus-CBFb 4.07 +/− 0.1 Cerulean-RD + Venus-CBFb61/104 1.85 +/− .09 C-RD + V-CBFb + KG-3-275 200 mcm 3.05 +/− .15 C-RD + V-CBFb + KG-3-275 100 mcm 3.25 +/− 0.1 C-RD + V-CBFb + KG-3-275 50 mcm 3.45 +/− .25 C-RD + V-CBFb + KG-3-275 25 mcm 3.6 +/− 0.2 % Cells Annexin V/PI Negative at 72 Hrs Kasumi-1 0.25% DMSO 91.2 +/− 0.9 KG-3-275 25 mcm 91.2 +/− 1.2 KG-3-275 50 mcm 85.9 +/− 2.4 KG-3-275 100 mcm 66.7 +/− 6.7 SKNO-1 0.25% DMSO 77.6 +/− 3.4 KG-3-275 25 mcm 77.6 +/− 2.3 KG-3-275 50 mcm 63.7 +/− 3.6 KG-3-275 100 mcm 13.7 +/− 6.8 Cellular Proliferation as measured by MTTAssay at 72 Hrs Cellular Proliferation as measured by MTTAssay at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs