bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Randomized Trial of Valacyclovir Versus Valganciclovir To Prevent CMV Reactivation in Patients with CLL Receiving Alemtuzumab.
Authors: O’Brien Susan , Ravandi-Kashani Farhad , Wierda William G. , Giles Francis , Thomas Deborah , Huang Xuelin , Riehl Todd D. , Tarrand Jeffrey J. , Brandi O’Neal , Kantarjian Hagop M. , Keating Michael J. 
Published: April 2020

Abstract
Alemtuzumab is an effective treatment for patients with CLL, but is an immunosuppressive antibody that depletes normal T-cells as well as B-cells. Prophylaxis for HSV and PCP is standard with this agent. About 25% of patients will experience CMV reactivation, manifested by fever and antigenemia. We conducted a randomized trial wherein patients being treated with an Alemtuzumab-containing regimen received either prophylaxis with valacyclovir 500 mg po daily, or valganciclovir 450 mg po b.i.d. CMV antigen was monitored every 2 weeks for 12 weeks. The study design planned to enroll 128 patients, but stopping rules for early termination were met. Forty-six patients were entered, six were inevaluable. Median age was 58 years (range 25–83); median number of prior therapies was 2 (0–10). Diagnoses included CLL (29), T-PLL (3), HCL (1), ATLL (1), marginal zone leukemia (1), LGL leukemia (2), ALL (1), T-cell lymphoma (2). Of patients with active CLL (some received treatment for MRD), Rai stage was I–II = 13, III–IV = 10. Patients received varying Alemtuzumab-containing regimens including single agent (5), or combined with: Rituximab (2), Pentostatin (6), FCR (23), or Hyper-CVAD (4). Seven of 20 patients enrolled on the valacyclovir arm experienced symptomatic CMV reactivation with the number of CMV antigen containing cells ranging from 19–651. None of the 20 patients randomized to valganciclovir experienced symptomatic CMV reactivation (p=0.004). One patient receiving valganciclovir had 6 CMV antigen + cells at 2 weeks but was asymptomatic and continued on prophylaxis; repeat testing at 4 weeks showed 0 Ag + cells. Given the heterogeneity of regimens and the frequency of myelosuppression expected, it is uncertain as to whether valganciclovir increased the incidence of myelosuppression. In conclusion, this agent was highly effective for prophylaxis of CMV in patients receiving Alemtuzumab. Further studies may address whether the use of this antibiotic with single-agent Alemtuzumab increases the incidence of myelosuppression and whether a lower dose of valganciclovir (450 mg daily) would be as effective in prophylaxis.
Treatment of Refractory Autoimmune Hemolytic Anemia in B-CLL with Alemtuzumab (Humanized CD52 MAb).
Authors: Lundin Jeanette , Celsing F. , Karlsson C. , Osterborg A. 
Published: April 2020

Abstract
Alemtuzumab is a humanized monoclonal antibody that targets the CD52 antigen. CD52 is abundantly expressed on leukemic B-CLL cells (approximately 4 x 105 binding sites/B-CLL cell) and to a lesser degree on normal B and T cells, which may result in pronounced immunosuppression. Treatment with alemtuzumab has achieved an ORR of 33% in patients with fludarabine refractory B-CLL (Keating et al. J Clin Oncol.2002;20:205–213) and &gt;80% in patients with symptomatic but previously untreated B-CLL (Lundin et al. Blood.2002;100:768–773). Progressive B-CLL is frequently complicated by autoimmune complications such as autoimmune hemolytic anemia (AIHA), which is estimated to occur in 10% to 20% of patients. We report here on 5 patients with advanced B-CLL and 1 with hairy cell leukemia (earlier diagnosed as B-CLL), all of whom developed severe AIHA resistant to conventional therapy (eg, corticosteroids, rituximab, chemotherapy, etc.) that received alemtuzumab as salvage treatment for AIHA. The median age of patients was 66 years (range, 59–77 years). At the start of alemtuzumab therapy, all patients had disease classified as Rai stage III or IV, were heavily pretreated with chemotherapy, and had severe transfusion dependent DAT-positive AIHA, which was resistant to conventional AIHA therapy, with a median Hb of 7.8 g/dL. The alemtuzumab dose was quickly escalated to 30 mg SC tiw (n = 3) or IV (n = 3), and continued for a median treatment period of 4 weeks (range, 3–8 weeks). Of 6 patients who received alemtuzumab, 5 responded with a &gt;2 g/dL rise in Hb concentration, which eliminated the need for transfusion after a median of 3 weeks (range, 2–8 weeks). At the end of therapy, the median Hb concentration in the responding patients was 122 g/dL (range, 100–125 g/dL). Alemtuzumab was well tolerated with only minor “first-dose” reactions. However, 1 patient had a CMV reactivation, as indicated by a fever of unknown origin that responded rapidly to foscarnet. All patients were alive at a median follow-up time of 5 months, and none of the responding patients experienced another episode of AIHA. We conclude that alemtuzumab may be an important therapeutic option for patients with severe, refractory CLL-related AIHA, who have not previously responded to conventional treatment.
A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s.
Authors: Li Zhe , Klusmann Jan-Henning , Godinho Frank J. , Lee Hee-Won , Reinhardt Dirk , Orkin Stuart H. 
Published: April 2020

Abstract
Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.
Dissecting the Molecular Mechanisms of Mutant C/EBPalpha (p30) in Normal and Malignant Human Hematopoiesis.
Authors: Cammenga Jorg , Niebuhr Birte , Horn Stefan , Delwel Ruud , Stocking Carol 
Published: April 2020

Abstract
Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.
Reduced Binding of C/EBPα to Myeloid Specific Promoters with Altered Gene Expression in the Presence of PML/RARα.
Authors: Wagner Katharina , Krauter Jürgen , Görlich Kerstin , Drescher Bettina , Johansen Lisa M. , Radomska Hanna S. , Duprez Estelle , Ganser Arnold , Tenen Daniel G. 
Published: April 2020

Abstract
The hallmark of acute promyelocytic leukemia is the reciprocal translocation t(15;17) resulting in the fusion protein PML/RARα . According to the current concept of the pathogenesis of APL, PML/RARα interferes with RARα target gene expression. Until now, the crucial target genes have not been found. The transcription factor C/EBPα is essential for myeloid development and mutations have been found in 10% of AML patients. No mutations of C/EBPα in APL have been described. However, using the U937 cell line with a Zinc-inducible PML/RARα (U937PR9), a decrease of C/EBPα binding to the G-CSF-R promoter in the presence of PML/RARα was detected in EMSA as well as chromatin immunoprecipitation. This finding indicates, that the function of C/EBPα might be impaired in APL. To test whether the effect is limited to the G-CSF-R promoter, C/EBPα binding to other myeloid promoters was analyzed upon Zn-induction of PML/RARα . In those experiments decreased binding of C/EBPα to other promoters of myeloid specific genes, such as neutrophil elastase, was detected. In contrast, no effect of Zn-induction on the binding of Sp3 to the thymidine-kinase promotor was found. Next, the functional importance of the alteration in binding was demonstrated. In the presence of PML/RARα , decreased G-CSF-R RNA expression as well as G-CSF-R surface expression was found in U937PR9 cells. In addition, the RNA expression of elastase was reduced after Zn-induction. To investigate the effect of PML/RARα on C/EBPα function in t(15;17) positive cells, a knockdown of PML/RARα by lentivirally delivered shRNAs in NB4 cells was used. After lentiviral transduction, a marked decrease of PML/RARα RNA and protein was found. Consistent with the results in U937PR9 cells a 5–6 fold increase of elastase RNA expression and a 2 fold increase in G-CSF-R-RNA was observed after PML/RARα knockdown. Taken together, decreased binding of C/EBPα to promoters of myeloid specific genes was found in the presence of PML/RARα . This alteration in binding was associated with a reduction of RNA expression. Decreasing PML/RARα with siRNA had the opposite effects. Currently, the mechanism for this effect is being investigated.
Identification of a MEF Gene Mutation in a Familial Hemophagocytic Lymphohistiocytosis Patient That Decreases MEF Transcriptional Activity.
Authors: Lacorazza H. Daniel , Lopiccolo Serena , Rubenstein Eric , Liu Yan , Nunes Juliana , Hasle Henrik , Fadeel Bengt , Ericson Kim , Henter Jan-Inge , Nimer Stephen D. 
Published: April 2020

Abstract
Familial hemophagocytic lymphohistiocytosis (FHL) is generally an autosomal recessive disease of early childhood characterized by uncontrolled T cell and macrophage activation leading to a fatal accumulation of activated T lymphocytes and histiocytes in different tissues. Immune regulation is severely impaired in these patients (both CTL and NK cells) and defects in perforin gene expression, an important component of the lytic granules, were the first defects identified in these patients. So far, FHL genetics have identified a locus at chromosome 9q22 (not molecularly defined yet), at chromosome 10q21-22 (where mutations in perforin gene have been described in 20% of FHL patients), at chromosome 17q25 (where mutations in the Munc13-4 gene have been found) and most recently at chromosome 6q24 (where mutations in the syntaxin-11 gene have been found). We described previously that MEF-null mice, which lack the ETS-transcription factor also known as ELF-4 (located at Xq26.1), have impaired NK cell development and function, and no NK cell perforin gene expression. We also showed that MEF regulates the human perforin proximal promoter via two ETS binding sites and showed that MEF is bound to the perforin promoter in vivo, using chromatin immunoprecipitation. Subsequently, we initiated a study of MEF gene integrity in male patients that have been studied and lacked perforin gene mutations. In addition to a known polymorphism in exon 9 (503 C→T) present in approximately 30% of unrelated patients and leukemic cell lines, we found a nucleotide change in exon 9 of MEF (408, C→T) in a male FHL patient from Denmark. The mutation found in the son, who was diagnosed with FHL and received a BMT at 12 years of age, was also seen in the patient’s grandmother, mother and one sister (heterozygous). Mutation 408 leads to an amino acid change (Val→Met) in a region located between the Ser/Thr and Pro rich domains in the C-terminal portion of MEF. We generated a MEF-mutant (MEFVal→Met) protein; it has a reduced capacity to activate the perforin gene promoter (only 30–40% of the wild type). MEFVal→Met does not have a dominant negative effect, when co-transfected with wild type protein, which could account for the FHL phenotype found in the male only. We are further examining the biology of this mutation in MEF-null cells to see if it can contribute to FHL pathology. Transcription factor abnormalities, such as the MEF mutation in this family, should be evaluated to help explain the pathogenesis of FHL in different patients.
C/EBP Is Deregulated by PML-RAR in Acute Promyelocytic Leukemia.
Authors: Guibal Florence , Radomska Hanna S. , Johansen Lisa M. , Tenen Daniel G. 
Published: April 2020

Abstract
Acute promyelocytic leukemia (APL) cells are blocked at the promyelocyte stage of myeloid differentiation. The majority of APL cells display the t(15;17) reciprocal chromosomal translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa). Cells harboring this reciprocal translocation can be induced to differentiate after treatment with all-trans retinoic acid (at-RA) both in vivo and in vitro. During normal hematopoiesis, differentiation is regulated by several key transcription factors. One of them, CCAAT/enhancer binding protein alpha (C/EBPa), controls expression of genes regulating normal myeloid differentiation. Its disruption leads to a block of granulocytic differentiation. We thus hypothesize that C/EBPa could be deregulated in APL and therefore participate in the pathogenesis of APL. Using the U937PR9 cell line, which expresses an inducible PML-RARa, we observed that expression of PML-RARa induced a decrease of both C/EBPa mRNA and protein, leading to decreased C/EBPa DNA binding activity. Using a transient transfection assay with a C/EBPa promoter construct in presence or absence of PML-RARa, we are able to demonstrate that PML-RARa can repress C/EBPa promoter activity. This repression is specific to the fusion protein, as both PML and RARa have no effect upon the C/EBPa promoter. A computer search of the C/EBPa promoter sequence did not exhibit any evident RARE binding site, and therefore we are currently mapping the site(s) responsible for this repression. In conclusion, PML-RARa down regulates C/EBPa expression; this down regulation could participate in the pathogenesis of APL. This hypothesis is also supported by the observation that at-RA treatment of APL cell lines (NB4 and HT93) induces a rapid restoration of both C/EBPa RNA and protein. Thus, a decrease in both C/EBPa expression and activity could contribute to the differentiation block of APL cells by deregulating the normal myeloid differentiation program.
Prevalence of Heparin-Dependent Platelet Antibodies in Children after Cardiopulmonary Bypass Surgery.
Authors: Punzalan Rowena C. , Hanson Sheila J. , Ghanayem Nancy , Curtis Brian R. , Murkowski Kathleen , Havens Peter L. , McFarland Janice G. 
Published: April 2020

Abstract
Heparin is used during cardiopulmonary bypass (CPB) surgery in children. Upon exposure to heparin, heparin-dependent platelet antibodies (HDPA) may form against complexes of platelet factor 4 and heparin on platelet surfaces. Heparin-induced thrombocytopenia (HIT) or heparin-induced thrombocytopenia and thrombosis (HITT) may then ensue. Among adult CPB patients, up to 50% demonstrate HDPA, although only 2% develop HIT and 1% develop HITT. In previous studies done among non-CPB newborns and pediatric intensive care patients, the incidence of HDPA has been zero to minimal.
Objective: To determine prevalence of HDPA among children undergoing CPB who are exposed to unfractionated heparin for &gt;120 hours after CPB.
Methods: We designed a prospective pilot study. All patients ≤ 12 years old who were to receive heparin during CPB were eligible. The presence of HDPA was assessed by heparin-platelet factor 4 enzyme-linked immunosorbent assay (ELISA); positive and equivocal results were confirmed by serotonin release assay. Blood samples were obtained at the time of cessation of heparin or after 10 days on heparin, whichever came first.
Results: Thirty patients were enrolled: 15 were aged 2–19 days (median 6 days); 15 were aged 1.2 to 50 months (median 4.5 months). One patient had borderline positive and 1 had positive results by heparin-platelet factor 4 ELISA; both had negative results by serotonin release assay; neither developed clots. Eighteen patients developed thrombocytopenia, which is common after CPB, including the 2 with equivocal results by ELISA. Six patients, all with negative ELISAs, developed symptomatic thromboses.
Conclusion: There were no HDPA identified among children who received unfractionated heparin for CPB. The specificity of heparin-platelet factor 4 ELISA among children should be assessed.
Absence of P21 Gene Augments Proliferation of Mesenchymal Stem Cells.
Authors: Tsai Anthony Y. , Filice Stephanie C. , Javazon Elizabeth H. , Badillo Andrea T. , Flake Alan W. 
Published: April 2020

Abstract
Bone marrow derived mesenchymal stem cells (MSCs) have the capacity to differentiate into cells of the various mesodermal lineages. Efficiency of engraftment, however, remains a significant obstacle in MSC transplantation. P21 is a G1 checkpoint regulator and cyclin-dependent kinase inhibitor. In its absence, hematopoieitc stem cell (HSC) proliferation increases under normal homeostatic conditions. The aim of the study is to evaluate the effects of p21 deficiency on the proliferative capacity of MSCs. Therefore, MSCs from p21 KO and wildtype mice were maintained in culture in hypoxic and normoxic conditions. Cell counts and respective days in cultures were recorded from which population doubling (PD) times were calculated and compared. 1:1 Competition assays were performed between p21 KO MSCs transduced with DsRed and eGFP transduced WT MSCs as well as between DsRed transduced p21 KO MSCs and non-transduced WT MSCs. Osteogenic, chondrogenic, and adipogenic differentiation assays were performed on both cell populations using standard protocols. Sca-1, CD34, CD45, CD90, MHC Class I and II and other surface antigens were assessed using FACS analysis. In normoxic conditions, the p21 KO MSCs went through 25.68 population doublings in 60 days versus 25.43 population doublings in 103 days for the p21 WT MSCs. Under hypoxic conditions, the KO MSC population doubled 23.3 times in 58 days versus 14.78 times in 60 days for the WT MSCs. All population doubling times were taken from passage 10 cells. When the DsRed transduced KO MSCs (96.6-98.5% DsRed+) was placed in a 1:1 competition assay with eGFP transduced WT MSCs (99.3-99.9% eGFP+) or non-transduced WT MSCs (99.9–100% eGFP−), the normoxic results are as shown in Table 1. Similarly, the competition assay result between DsRed transduced KO MSCs (86.9–94.1% DsRed+) and eGFP transduced WT MSCs (99.7–100% eGFP+) or non-transduced WT MSCs (100% eGFP−) for the hypoxic condition are shown in Table 2. Both the p21 KO and wildtype MSCs populations were able to differentiate into ostenogenic, adipogenic, and chondrogenic lineages. No significant surface marker differences were observed between the 2 populations on FACS analysis. Our results clearly showed that p21 deficient MSCs have increased proliferative ability in vitro compared to normal MSCs. These findings have implications for expansion of MSC populations in vitro , and for the enhancement of competitive capacity of MSCs following in vivo administration.
Table 1 - Normoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 6 64.2±3.7% 34.5±4.3% 70.3±0.4% 19.7±0.6% Day 11 84.0±4.5% 13.6±4.6% 92.0±0.8% 6.4±0.7% Table 2 - Hypoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 4 70.3±1.7% 30.0±1.7% 83.0±0.8% 18.6±0.9% Day 8 75.7±0.7% 25.8±0.7% 81.3±1.2% 20.6±1.2%