bluuebunny/crossref_metadata_2025_split huggingface dataset:
Centrosome Aberrations in Chronic Myeloid Leukemia (CML) Are Correlated with Disease Progression and Chromosome Instability.
Authors: Giehl Michelle , Fabarius Alice , Zheng Chun , Frank Oliver , Hochhaus Andreas , Hafner Mathias , Hehlmann Rudiger , Seifarth Wolfgang 
Published: April 2020

Abstract
Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC).
Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies.
Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0).
Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.
Loss of IRF-4 Exacerbates the CML-Like Phenotype of IRF-8 Knockout Mice.
Authors: Schatz Jonathan , Ren Ruibao , Singh Harinder 
Published: April 2020

Abstract
IRF-4 and IRF-8 are related interferon regulatory factors that are required for the development and function of myeloid and lymphoid cells. Mice deficient in IRF-8 develop a myeloproliferative disorder with many similarities to human chronic myeloid leukemia (CML). Our group has shown that in B-lymphocyte development, IRF-4 and 8 function redundantly to control the pre-B to B transition. Though expression of IRF-4 has been demonstrated in myeloid cells, its role in these lineages has not been defined. We hypothesized that, similar to their role in B-cells, IRF-4 and 8 may to some extent function redundantly in myeloid cells. To test this hypothesis, myelopoiesis was analyzed in IRF-4/8 double knockout (DKO) mice. We found that from seven weeks of age, the DKO animals have a more aggressive leukemic phenotype than the IRF-8 KO mice. The white blood cell (WBC) counts of DKO animals were typically in the range of 40,000 to 80,000 cells per microliter, campared to 15,000 to 20,000 for age-matched IRF-8 KO animals. FACS analysis showed that the increased WBCs in the DKO animals was due to a massive expansion of mature granulcytes. The absolute granulocyte counts of DKO animals were typically 7-fold to 10-fold higher than those of IRF-8 KO animals. In addition, histologic preparations showed that by 15 weeks of age the spleens, lymph nodes, and bone marrow of DKO animals were invaded by large numbers of mature granulocytes and pseudo-Gaucher cells, with complete effacement of the normal micro-architecture. Age-matched IRF-8 KO animals showed invasion to a lesser degree, with preservation of many of the normal architectural features. In conclusion, IRF-4 is an important myeloid tumor suppressor, whose loss augments the myeloproliferative disease manifested in IRF-8 deficient mice. This suggests the two transcription factors, similar to their role in B-cell development, function redundantly to control the normal maturation of myeloid-lineage cells.
Intrinsic Regulation of the Interactions between the SH3 Domain of p85 Subunit of Phosphatidylinositol-3 Kinase and BCR/ABL Oncogenic Tyrosine Kinase.
Authors: Ren Shuyue , Xue Fan , Feng Jan , Skorski Tomasz 
Published: April 2020

Abstract
BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Here we show that SH3 domain of p85 (p85-SH3) pulls-down the p210BCR/ABL kinase from hematopoietic cell lysates. In addition, we characterize the p85-SH3 mutants, which abrogate or enhance this interaction. The results of pull-down assays of the p85-SH3 mutants seem to support the assumption that p85-SH3 interacts with the BCR/ABL protein network via the proline-rich (PxxP) region. One of the surprising findings was the enhanced binding affinity of the tyrosine to phenylalanine p85-SH3 domain mutants in comparison to the wild-type p85-SH3. Based on these results we speculate on the capability of p85-SH3 to interact with BCR/ABL and on the p85-SH3 conformational requirements necessary for this reaction. PxxP - binding appears to be required for the interaction of p85-SH3 with BCR/ABL protein complex and activation of the catalytic activity of PI-3k, whereas subsequent BCR/ABL-dependent phosphorylation of the tyrosines may facilitate the release of activated PI-3k from the complex.
Cooperative Anti-Leukemic Effects of Imatinib and Mcl-1 Antisense: Identification of Mcl-1 as a Novel Target in CML.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Skvara Hans , Florian Stefan , Sonneck Karoline , Akgul Cahit , Derdak Sophia , Pickl Winfried F. , Wacheck Volker , Selzer Edgar , Monia Brett P. , Moriggl Richard , Valent Peter , Sillaber Christian 
Published: April 2020

Abstract
The BCR-ABL tyrosine kinase inhibitor STI571 (Imatinib) has successfully been introduced in the treatment of patients with CML. However, despite encouraging initial data and high expectations long term results are not available yet, and recent data suggest that resistance against STI571 can occur. Therefore, current studies focus on novel potential drug targets in CML cells. Recently, several anti-apoptotic members of the Bcl-2-family including Mcl-1 have been implicated in the regulation of survival of BCR/ABL+ cell lines and therefore proposed as potential targets. We have examined expression of Mcl-1 in primary CML cells and various BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner. The BCR/ABL-inhibitor STI571 decreased the expression of Mcl-1 in these cells. Correspondingly, BCR/ABL enhanced Mcl-1 promoter activity, Mcl-1 mRNA, and Mcl-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of Mcl-1 in Ba/F3 cells was counteracted by the MEK-inhibitor PD98059, but not by the PI3-kinase inhibitor LY294002. Identical results were obtained for constitutive expression of Mcl-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of Mcl-1 as a survival-related target gene in CML cells, Mcl-1 siRNA or an Mcl-1 antisense oligonucleotide (ASO) were applied. Both the ASO and the siRNA-induced downregulation of Mcl-1 were found to be associated with a substantial decrease in viability of K562 cells. Moreover, the Mcl-1 ASO was found to cooperate with STI571 in producing growth-inhibition in both STI571-sensitive and STI571-resistant K562 cells. Together, our data identify Mcl-1 as a survival factor and novel target in CML. Whether this concept is of clinical significance remains to be determined in forthcoming clinical trials.
CEBPα Mutations in Childhood Acute Myeloid Leukemia.
Authors: Liang Der-Cherng , Shih Lee-Yung , Huang Chein-Fuang , Hung Iou-Jih , Yang Chao-Ping , Liu Hsi-Che , Jaing Tang-Her , Wang Lin-Yen , Chang Wan-Hui 
Published: April 2020

Abstract
Transcription factor CCAAT/enhancer binding protein alpha(C/EBPα) is essential for granulocyte differentiation. CEBPα mutations have been described in 7–10% of adult patients with acute myeloid leukemia (AML) at initial diagnosis and conferred a favorable prognosis. CEBPα mutations in childhood AML has not been reported yet. In this study, we sought to assess the frequency of CEBPα mutration, its clinicohematologic correlation and to analyze the cooperating mutations, including FLT3 and N-ras mutations in childhood AML. CEBPα mutations were analyzed in 117 children (age one day to 17 years, median 5 years) with de novo AML. CEBPα mutation status was examined by performing DNA polymerase chain reaction (PCR) followed by direct sequencing for each PCR product. CEBPα mutations were detected in 7 (6.0%) of 117 patients. Of the 7 patients with CEBPα mutations, 4 had FAB subtype of M2, 2 M1 and 1 M4. All had intermediate cytogenetics and comprised 11.5% (7/61) of the intermediate cytogenetic risk group. Five of the 7 mutations occurred in both N-terminal part and bZIP domain, one had N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Clonal analysis using expand long template PCR assay with a primer pair covering entire coding sequence was performed in all 5 patients carrying two different mutations. The results demonstrated that one had homozygous combined mutation and 4 had heterozygous biallelic mutations with the cloned alleles carrying single mutations at N-terminal or bZIP domain in the different alleles or harboring combined mutations in the same alleles. None of the remission marrow samples obtained from 5 patients who had CEBPα mutations at diagnosis carried the mutations. FLT3-ITD mutations were detected in 2 of the 7 patients with CEBPα mutations compared to 15 of the 109 patients without CEBPα mutations. None of the patients carrying CEBPα mutations had FLT3-TKD mutations. Two of the 7 CEBPα(+) patients also harbored N-ras mutations compared to 10 of the 110 patients without CEBPα mutations. Taken together, 4 of 7 CEBPα(+) patients had cooperating mutations including FLT3-ITD or N-ras as compared to 27 in 109 CEBPα(−) patients (P=0.081). There were no statistical differences in age, WBC count, LDH level, 5-year overall survival and event-free survival between patients with and without CEBPα mutations; similar results were obtained if we only analyzed patients with intermediate cytogenetics. The present data showed that the frequency of CEBPα mutations in our series of childhood AML was similar to that in adults. CEBPα mutations were frequently associated with FLT3-ITD or N-ras mutations supporting the two-hit hypothesis for the pathogenesis of AML.
Internal Tandem Duplication and Kinase Domain Mutation of FLT3 in Pediatric Acute Myeloid Leukemia Treated on Japan Cooperative AML 99 Protocol.
Authors: Shimada Akira , Taketani Takeshi , Taki Tomohiko , Tabuchi Ken , Hanada Ryoji , Tawa Akio , Tsuchida Masahiro , Horibe Keizo , Tsukimoto Ichiro , Hayashi Yasuhide 
Published: April 2020

Abstract
FLT3 is a type III receptor tyrosine kinase and this kinase mutation is most frequently observed in cases of acute myeloid leukemia (AML). Approximately 30% of adult AML patients have internal tandem duplication (ITD) of the juxtamembrane domain or point mutation of kinase domain (D835Mt) leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. We examined FLT3-ITD and D835Mt in 160 pediatric AML patients treated on Japan pediatric AML cooperative treatment protocol, AML 99. This protocol is consisted of intensification of multi-drug chemotherapy and early application of stem cell transplantation (SCT) for the high risk patients (monosomy 7, Ph1, or delayed remission etc.). FLT3-ITD was found in 22 of 160 (13.8%) patients, including 5 of 15 (33.3%) patients with FAB-M3, 5 of 25 (20.0%) with M5, and 4 of 24 (16.7%) with M1. No specific chromosomal abnormalities were associated with ITD; 9 of 17 (52.9%) patients with ITD showed normal karyotype except for t(15;17). The D835Mt was found in 12 of 160 (7.5%) patients, including 4 of 15 (26.7%) patients with M3. The initial WBC count and the age at diagnosis in ITD or D835Mt patients were significantly higher than those in patients with wild type (WT) of FLT3 (p&lt; 0.05). Except for M3, 12 of 17 (70.6%) patients with ITD received SCT (3 patients in 1st CR, 1 patient in 2nd CR, 3 patients in 1st relapse, although 4 patients in non-CR). Eight patients showed early relapse after SCT and died. No patients with D835Mt showed the induction failure or relapse. In M3, only one patient with FLT3-WT died in early induction phase. The overall survivals (OS) for patients with ITD, D835Mt and WT by Kaplan-Meier method were 33%, 100% and 80%, respectively (p&lt;0.05). These results suggest that patients with ITD are associated with poor outcome despite of receiving SCT, while those with D835Mt are associated with good outcome.
MLL/AF9 Fusion Transcript Quantification in t(9;11)-Positive AML Identify Patients with High Risk of Relapse.
Authors: Scholl Claudia , Eiwen Karina , Frohling Stefan , Schlenk Richard F. , Dohner Hartmut , Dohner Konstanze 
Published: April 2020

Abstract
Detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) with specific gene fusions is an important tool for the assessment of response to treatment and the individual risk of relapse. We evaluated the predictive value of MLL/AF9 fusion-transcript quantification using real-time RT-PCR for disease relapse and verified the results with nested RT-PCR. A t(9;11) was identified in 27 younger AML patients (16–60 years) entered into the multicenter trials AML HD93 and AML HD98-A of the AML Study Group Ulm. One hundred seventy-eight samples (bone marrow [BM], n=84; peripheral blood [PB], n=79; leukapheresis product [LP], n=15) from 22 patients were available for real-time RT-PCR analysis. In 6 of these 22 patients, only samples from diagnosis were obtained, the remaining 16 patients were analyzed during and after therapy. The sensitivity of the real-time RT-PCR was 10−3 and of the nested RT-PCR 10−4 to 10−5. The MLL/AF9 copy number at diagnosis was not predictive of subsequent clinical outcome. Eight of the 16 evaluable patients became real-time RT-PCR-negative after double induction therapy. Six of these 8 patients are in continuous complete remission (CR), 1 died in CR from graft-versus-host disease, and 1 relapsed 11 months after diagnosis. Eight of 16 patients had detectable fusion-transcript levels after achieving complete hematologic remission. Six of these 8 patients relapsed after a median of 8.6 months (range, 3.6 to 13.5) from diagnosis, one patient died in CR 3 weeks after autologous BM transplantation, and one patient underwent allogeneic BM transplantation and is in continuous CR 16 months after diagnosis. Of 3 patients, who relapsed, we were able to analyze bone marrow 1 months before hematologic relapse occurred. Two of these 3 patients were real-time RT-PCR-negative after therapy and became positive 1 month before relapse, 1 patient was still real-time RT-PCR-positive after therapy and had increased MLL/AF9 copy numbers 1 month before relapse occurred. Five Patients, who were in continuous CR, were real-time RT-PCR-negative in all analyzed samples during follow up (median, 56 months; range, 13.8 to 68.9 months). One hundred seventy-two patient samples were examined by nested RT-PCR. Real-time RT-PCR-negative samples were also negative with nested RT-PCR, therefore nested RT-PCR did not enhance sensitivity of real-time PCR. We conclude that real-time RT-PCR is convenient for the detection of MRD in t(9;11)-positive AML and real-time RT-PCR positivity in at least one sample during therapy correlates with a high risk of relapse.
Effect of Sample Preservation Method on Gene Expression and RNA Integrity in Acute Myelogenous Leukemia.
Authors: Ben-Ezra Jonathan , Ladd Amy C. , Dumur Catherine I. , Archer Kellie J. , Chesney Alden , Ferreira-Gonzalez Andrea , Wilkinson David S. , Garrett Carleton T. 
Published: April 2020

Abstract
Cryopreservation of patient bone marrow specimens allows for future purification of tumor cells for research or diagnostic purposes. It has not been determined whether this practice causes significant changes in gene expression. In order to evaluate this, we performed microarray analysis (Affymetrix U133A and U133A 2.0 gene chips) on mononuclear cells from 5 acute myelogenous leukemia (AML) patient samples (&gt;70% blast count) that had been either a) cryopreserved, b) snap frozen, or c) resuspended in TRIzol and stored at −80°C. Samples remained frozen for a period of 1–4 weeks. While gene expression changes between groups were minor, there were some differences. Thirteen probe sets between TRIzol and cryopreserved samples, and 60 probe sets between TRIzol and snap frozen samples, varied significantly (&gt;2-fold difference and p&lt;0.01; t-test). While both non-TRIzol samples lost transcripts associated with mature, contaminating granulocytes, the snap frozen samples also lost transcripts associated with cell growth and metabolism. To determine whether the observed changes were enough to cause a misclassification of AML subtypes, we performed an unsupervised cluster analysis on the 15 original samples plus 15 new AML samples (13 unique cases), all of varying subtypes. RNA from all of these samples was undegraded. All samples passed strict quality control parameters, not limited to but including tumor content, % rRNA, cDNA and cRNA synthesis product size, and GAPDH 3′-5′ ratios. Differentially preserved samples originating from the same patient segregated together during the clustering process regardless of preservation method. Three main clusters emerged; cluster #1 (FAB-M0 and -M1, n=3), cluster #2 (FAB-M3, n=5), and cluster #3 (FAB-M2, -M4 and -M5b, n=22). In cluster #3, there were three sub-groupings. One group contained only M4 and M5b subtypes, another contained only the M2 subtype, and the third contained both M2 and M4 subtypes. RNA isolated from snap frozen samples was sometimes moderately to severely degraded. When we examined three snap frozen samples, not part of the above data set, exhibiting moderate RNA degradation, they all clustered incorrectly according to the above established groups. Given these results, we conclude that cryopreservation is an acceptable method of cell preservation for gene expression analysis, but snap frozen samples should be closely evaluated for RNA degradation before using in microarray analyses.
Characterization of Autoantibodies to PF4/Heparin in a Rat Model of Cardiopulmonary Bypass.
Authors: Arepally Gowthami M. , Homi H. Mayumi , De Lange Fellery , McCracken Emily K.E. , Goss Tina M. , Suvarna Shayela , Grocott Hilary P. 
Published: April 2020

Abstract
Patients undergoing cardiopulmonary bypass (CPB) represent a high-risk population for developing life-threatening complications of Heparin-Induced Thrombocytopenia and Thrombosis (HITT). Approximately 30-70% of patients undergoing CPB develop heparin dependent autoantibodies to PF4/heparin, which can provoke disease in susceptible individuals. To address the clinical relevance of bypass associated anti-PF4/heparin, we have created a rat model of autoimmune PF4/heparin in the setting of CPB. Sprague-Dawley rats were prepared for CPB using cannulae in the right external jugular vein for venous return to the CPB circuit and the ventral tail artery for arterial inflow. Rats were administered 0.3–0.4U/g heparin i.v. after cannulation, and were then connected to a miniaturized CPB system complete with a membrane oxygenator primed with 40 ml of blood from 2 syngeneic donors (each receiving 0.3U/g i.v. heparin). CPB was maintained for 75 minutes with a flow between 160–180 mls/kg/min (normal cardiac output in the rat) after which animals were decannulated and sacrificed for study over the ensuing 6 weeks. Control rats were subjected to a sham operation for which they received the same dose of heparin, as well as arterial and venous catheterization, but were not placed on CPB. Blood was collected from control animals or rats after CPB and tested for autoantibodies to rat PF4/heparin (rP+H) by ELISA. Rats were studied either after sham operation (n=6) or after CPB at the following time points: 15 minutes (n=15), 3 days (n=6), 16 days (n=25), and at 6 weeks (n=8). Rats undergoing CPB had markedly elevated anti-rP+H levels (as gauged by mean A450 nm ± SEM) 16 days after the procedure as compared to rats receiving sham operation (0.211 ± 04, 16 day CPB vs. 0.01 ± 0.01sham; P = 0&lt;.0001 by ANOVA). High titer antibody responders from day 16 {Rat 77 (A450nm=0.84), Rat 80 (A450nm=0.81) and Rat 70 (A450nm=0.46)} were characterized for reactivity to rPF4 alone, rP+H or rP+H in the presence of excess heparin. Anti-rP+H from all three animals showed maximal reactivity to rP+H, but reduced binding to rPF4 alone or rat P+H in the presence of excess heparin. We additionally tested functional effects of anti-rP+H on rat platelets and human platelets, using a 14C-SRA. Rat platelets were not activated by anti-rP+H or a human HIT-like monoclonal antibody, KKO confirming the absence of activating FcγRII on rat platelets. Human platelets were activated by KKO (60% release), but not rat anti-P+H (&lt;5% release). In conclusion, we demonstrate that autoantibodies occur in heparinized animals subjected to the platelet activating effects of CPB. This animal model will be critical for studying the many facets of bypass- related heparin sensitization, including: the immunobiology of HITT, non-FcR mediated thrombotic effects of anti-P+H, and for developing alternative therapies for HITT patients undergoing CPB.