bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.
Authors: Ye Qidong , Gu Long-Jun , Zhao Yanxia , Zhao Jincai , Chen Wengao , Zhang Bei , Jiang Limin 
Published: April 2020

Abstract
Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As4S4), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As4S4 in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As4S4 had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As4S4 had little effect on K562 cells. The effect of As4S4 on K562 was time and concentration dependent. After cultured with As4S4 at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As4S4 could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As4S4, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G1 phase decreased from 69.65% to 50.53%, whereas the percentage of G2/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-XL, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As4S4. The transcript and protein levels of BCR-ABL had no change after incubation with As4S4. These results indicated that As4S4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4S4 interferes with Akt pathway and down-regulate BCL-2, BCL-XL, BAD and BAX, which may be involved in the response of K562 to this agent. As4S4 could be beneficial for treatment of CML in combination with conventional drugs.
The Protein Kinase C beta (PKCβ) Inhibitor Enzastaurin HCl (LY317615) Augments Chemosensitivity of Multiple Myeloma (MM) Cells to Dexamethasone and the Proteasome Inhibitor Bortezomib.
Authors: Suvannasankha Attaya , Xu Dawei , Abonour Rafat 
Published: April 2020

Abstract
PKCβ isoforms are important in B-cell signaling and proliferation. PKCβ overexpression in diffuse large B-cell lymphoma relates with poor prognosis but the clinical significance of PKCβ in MM is not yet established. Herein, we report antimyeloma activity of the PKCβ inhibitor enzastaurin HCl (LY317615) both as a single agent and in combination with bortezomib and dexamethasone.
Methods: Six MM cell lines (NCI-H929, RPMI8226, IM9, U266, ARH-77 and HS Sultan) with varying degree of PKC activity were treated with graded dose of enzastaurin and studied for induction of apoptosis by flow cytometric analysis of annexin and propidium iodide stainings, and inhibition of proliferation by MTT assay.
Results: IC50 doses of both effects were low at 1-5 μM in all cell lines and did not correlate with the baseline PKC activity. Interestingly, the combination of enzastaurin at non-toxic concentrations with varying doses of dexamethasone and bortezomib yielded synergistic enhancement of cell death and inhibition of proliferation in all cell lines. Multiparameter immunoblottings of cell lysate after enzastaurin treatment showed that, at these functional doses, PI3/AKT, Erk and JNK pathways were unaffected. Additional studies are ongoing to evaluate the effect of enzastaurin on activities of apoptotic proteins, and verify its effect in primary samples.
Conclusions: Our preliminary data indicate that the PKCβ inhibitor enzastaurin demonstrates single-agent antimyeloma activity and can sensitize MM cells to bortezomib and dexamethasone. Enzastaurin is promising for further studies as an approach to increase activity and decrease toxicity of MM therapy.
Figure Figure
MHH-TALL1, a T-ALL Cell Line Expressing a Novel BCR-ABL Transcript.
Authors: Quentmeier Hilmar , Cools Jan , MacLeod Roderick A.F. , Marynen Peter , Uphoff Cord C. , Drexler Hans G. 
Published: April 2020

Abstract
The Philadelphia chromosome (Ph) is the result of the chromosomal translocation t(9;22)(q34;q11), leading to the BCR-ABL fusion gene. The Ph chromosome is the hallmark of chronic myeloid leukemia, but is also detected in acute lymphoblastic leukemia (ALL), particularly in adults. The majority of Ph-positive ALL cases belong to the category of B-cell precursor ALL, whereas Ph-positive T-ALL cases are rather rare. The MHH-TALL1 cell line was established from the peripheral blood of an 11-year-old boy with T-ALL in 1993. PCR analysis of primary tumor cells failed to reveal the existence of a BCR-ABL fusion. Interestingly however, conventional cytogenetics and fluorescence in situ hybridization performed on the cell line showed a 3-way t(1;9;22)(q32;q34;q11) rearrangement effecting Ph formation. As with the patient, standard RT-PCR of the various known BCR-ABL fusion transcripts was negative in the cell line. However, a weak band, about 600 bp larger than the usual e1-a2 BCR-ABL transcript was detected, and subsequently confirmed on reanalysis after optimizing PCR conditions. Sequencing of the RT-PCR product showed that MHH-TALL1 expressed an e6-a2 BCR-ABL fusion transcript. Northern and Western blot analyses demonstrated that the BCR-ABL gene products were expressed at very low levels only. It may be speculated that the presence of this novel BCR-ABL variant has been overlooked in previous analyses, because (i) PCR conditions used to screen for BCR-ABL fusion transcripts were not optimal to detect this variant and (ii) a weak signal running at the “wrong” size might have been neglected. In summary, we report a novel BCR-ABL fusion variant expressed in a T-ALL cell line. Our data raise the intriguing possibility that some BCR-ABL negative cases may express the novel e6-a2 transcript described herein. This might have an impact on treatment of the respective patients with ABL kinase inhibitors.
Aberrant Expression of the Chondroitin Sulfate Proteoglycan NG2 on Pediatric AML/abn(11q23) and ALL/t(4;11) Does Not Allow Flow Cytometric Detection of Leukemic Blasts with a Stem Cell-Like Immunophenotype.
Authors: Neudenberger Janna , Hotfilder Marc , Rosemann Annegret , Langebrake Claudia , Reinhardt Dirk , Vormoor Josef 
Published: April 2020

Abstract
Monitoring of minimal residual disease in childhood leukemia is widely used to determine treatment response and to detect persisting leukemic blasts for relapse prediction. In this context, it is increasingly recognized that only few leukemic blasts possess self-renewal capability and that only these self-renewing leukemic stem cells are able to initiate relapses. Therefore, the leukemic stem cells as the relapse-initiating cells should be the target cells of minimal residual disease detection. The human homologue of the rat chondroitin sulfate proteoglycan NG2 protein (detected by the monoclonal antibody 7.1) is aberrantly expressed in a high percentage of leukemias harboring 11q23 abnormalities and its expression has been proposed as a potential marker for MRD detection. To evaluate NG2 as a marker for leukemic stem cells, bone marrow or peripheral blood samples of 4 AML/t(9;11), 1 AML/t(10;11), 1 AML/t(?;11) and 2 ALL/t(4;11) patients with NG2-positive leukemia were analyzed by four color flow cytometry for NG2 expression on the cell surface of primitive cell populations. Immature cells were defined by the antigen profiles CD34+CD38− (CD33−) in AML and CD34+(CD117+)CD19− in ALL. AML stem cells are known to be CD34+CD38−. In the ALL patients, 32% resp. 81% of CD34+CD19− cells were leukemic by FISH analysis for MLL/AF4. For this rare event analysis, 5 x 10e6 cells were incubated with saturating amounts of the respective antibodies. Data acquisition and analysis was performed on a 2-laser FACSCalibur (BDIS). 1 x 10e6 events were acquired but only CD34+CD38− (in AML) or CD34+CD19− cells (in ALL) were stored (storage gate). Matched isotype controls were analyzed using the same storage gate to exclude amplification of unspecific events. In-between each acquisition, the cytometer was vigorously flushed to prevent carry-over of cells from previous acquisitions. In addition, 10.000 ungated events were acquired to compare the CD34+Lin− stem/progenitor cells with the dominant leukemic cell clone.
In all eight patients with NG2-positve leukemia, cells with a stem cell-like immunophenotype were shown to lack expression of NG2. Instead, flow cytometry analyses revealed a close correlation between expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing AML cells. Similarly, by LightCycler RT-PCR CD34+CD19+ cells showed higher expression of NG2-RNA compared with CD34+CD19− cell in both ALL samples. Our results demonstrate that NG2 is not expressed on primitive leukemic cells but is upregulated with differentiation within the leukemic cell clone. This hampers the use of NG2 expression as a sensitive marker for MRD detection as expression of NG2 does not allow detection of relapse-initiating leukemic stem cells.
Independent Prognostic Significance of Cytogenetics, Immunophenotype, Tumor Burden, and Age in Acute Myeloid Leukemia. Analysis of Patients Treated within PALG 1999 Prospective, Randomized Study.
Authors: Grosicki Sebastian , Holowiecki Jerzy , Giebel Sebastian , Haus Olga , Stella-Holowiecka Beata , Jagoda Krystyna , Bartkowska-Chrobok Aleksandra 
Published: April 2020

Abstract
Acute myeloid leukemia (AML) refers to a group of distinct diseases that differ with regard to their genetic charakteristics, clinical features, response to therapy, and prognosis. Patients require precise initial evaluation of risk factors including cytogenetics and immunophenotype to plan an optimal treatment program.
In this study we analyzed impact of cytogenetics, cell surface marker expression and clinical features on complete remission (CR) rate, leukemia-free survival (LFS) and overall survival (OS).
Four-hundred-forty-five AML patients, aged 18–60 years, treated within a multicentre study by the Polish Adult Leukemia Group (PALG 1999 Study) between 1999–2002 were included in the analysis. Induction therapy consisted of daunorubicine+cytarabine+/−cladribine, consolidation included two courses containing high-dose cytarabine (HAM, HD-AraC+/−cladribine).
In multivariate analysis the following factors were found to be associated with poor outcome:
For CR rate: unfavorable cytogenetics according to CALGB criteria (RR=5,5, p=0,00001), WBC ≥50 x109/L at diagnosis (RR=3,3, p=0,00007), and age ≥50 (RR=1,9, p=0,021). For LFS: unfavorable cytogenetics (RR=1,7, p=0,026), and lack of CD117 expression on leukemic cells (RR=1,8, p=0,026). For OS: unfavorable cytogenetics (RR=1,8, p=0,00002), WBC ≥50 x109/L at diagnosis (RR=1,1, p=0,0007), age ≥50 (RR=1,6, p=0,002), and lack of CD33 expression on leukemic cells (RR=1,7, p=0,014).
CONCLUSIONS: In this study we demonstrated that besides cytogenetics, other factors including particular phenotypic features, as well as initial tumor burden, and age independently influence outcome of AML patients. Results of the analysis provide practical information that could be taken into account when planning individualized treatment including indications for high-dose therapy followed by bone marrow transplantation.
High Expression Levels of Iaps Predict Poor Overall Survival in Childhood De Novo Acute Myeloid Leukemia.
Authors: Tamm Ingo , Richter Stephan , Oltersdorf Doreen , Creutzig Ursula , Harbott Jochen , Scholz Frank , Karawajew Leonid , Ludwig Wolf-Dieter , Christian Wuchter 
Published: April 2020

Abstract
Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. Using flow cytometry and western blot analysis, we therefore investigated 45 leukemic cell samples of children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins (CD95, Bcl-2, Bax, Bcl-xL, Procaspase-3, XIAP, cIAP-1, Survivin). XIAP (p&lt;0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by FAB morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (p&lt;0.01) and Bcl-xL (p&lt;0.01) expression was lower in patients with favorable than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP {30 (n=10) vs. 41 months (n=34); p&lt;0.05} and Survivin {27 (n=10) vs. 41 months (n=34); p&lt;0.05}. We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics and poor overall survival should be confirmed within prospective pediatric AML trials.
Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.
Authors: Sakhinia Ebrahim , Farahangpour Mahboubeh , Yin John A. Liu , Brady Gerard , Hoyland Judith A. , Byers Richard J. 
Published: April 2020

Abstract
Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.
Evaluation of MRD after Administration of Imatinib by Measurement of Real-Time Quantitative PCR-Based Major bcr/abl mRNA.
Authors: Takahata Mutsumi , Hashino Satoshi , Fujisawa Fumie , Kondo Takeshi , Ota Syuichi , Kobayashi Sumiko , Tanaka Junji , Imamura Masahiro , Asaka Masahiro 
Published: April 2020

Abstract
Evaluation of minimal residual disease (MRD) provides prognostic information on various hematological malignancies. We describe here the efficacy of real-time quantitative polymerase chain reaction (RQ-PCR)-based analysis of major bcr/abl mRNA in cases of chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ALL), especially in cases administered imatinib mesilate (imatinib) followed by stem cell transplantation (SCT). Twenty-one patients with CML or Ph+ALL were enrolled as subjects to determine the usefulness of RQ-PCR-based measurement of bcr-abl/abl ratios. Seven of the 21 patients were administered imatinib before allogeneic SCT. Hematological relapse or failure of treatment by SCT was observed in 2 out of those patients who showed bcr-abl/abl ratios of more than 0.002%, and 5 of the 7 patients showed both RQ-PCR and RT-PCR negativity immediately after SCT. All of the 5 patients who were not administered imatinib before allogeneic SCT showed RQ-PCR negativity immediately after SCT, but the results of RT-PCR were positive in 3 patients at the same time points, and the results became negative by donor lymphocyte infusion (DLI) or appearance of graft-versus-host disease (GVHD). Administration of imatinib before SCT was thought to result in an earlier remission.
On the other hand, 8 patients who were administered imatinib without SCT showed a remarkable decrease in bcr-abl/abl ratios. However, the ratio gradually rose in one patient with Ph+ALL, enabling prediction of hematological relapse preceding detection by fluorescence in situ hybridization (FISH) analysis. Standardization of RQ-PCR analysis of bcr-abl mRNA will help prediction of early hematological relapse in patients with MRD.
In conclusion, RQ-PCR positivity immediately after SCT in patients pre-treated with imatinib suggests risk of recurrence.
Significant Correlation between Immunophenotypings and Cytogenetic Features by Analysis of High Resolution Flow Cytometry with WHO Classification in Adult Acute Myeloid Leukemia in China.
Authors: Wang Xingbing , Zheng Jine , Yao Junxia , Liu Jun , Yang Jin , He Yanli , Yu Jingming , Wei Jun , Liu Zhongping , Zhao Hongzhou , Li Hongrui , Du Wen , Zhang Jiahua , Chen Xiangjun , Liu Wei , Huang Shiang 
Published: April 2020

Abstract
New WHO classification has been rapidly used in leukemic diagnosis. Based on the coexpression and correlation of lineage-associated antigens with multidimensional data analysis, high-resolution flow cytometry has been developed to precisely identify lineage characteristics of leukemia. In this study, 116 cases of acute myeloid leukemia at primary diagnosis with WHO classification were analyzed by high-resolution flow cytometry with a panel of 25 different monoclonal antibodies. The correlation of immunophenotypings with cytogenetic abnormal changes (88 available cases), clinical features and FAB classification were further investigated. The results demonstrated that CD33, CD38 and CD13 were the most commonly expressed antigens (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%)and CD2(15%)As compared with all other AMLs, some antigens including lymphoid lineage are significantly correlated with FAB subtypes, such as increasing expression of CD2 in M3; HLA-DR, CD34 and CD56 absent expressions in M3; increased frequency of CD19 in M2; increasing expressions of CD14 and CD56 in M5 and lack of MPO expression in M0. Cytogenetic abnormal features were detected in 48 cases (54.5%). CD22, CD56 and TdT expressions are remarkably associated with chromosomal abnormalities. All six cases with CD22 expression in AML demonstrated various cytogenetic abnormal changes. Significant associations between immunophenotypings and genetic features are observed: all cases with t (8; 21) highly expressed CD15, CD19, CD34 and CD56. No lymphoid lineage antigens were detected in AML-M3 with t (15; 17). In addition, the expression of both CD7 and CD14 was significantly correlated with higher WBC count; CD4 or TdT expression with increased age; CD4, CD14 or CD56 expression with higher platelet count. No significant correlation was found between hemoglobin level and markers. The relationships of immunophenotypings and genetic features with treatment and prognosis are currently being studied. Conclusion: 1. Significant correlation between immunophenotypings and cytogenetic features in AML strongly suggested that the abnormal antigen expression is tightly linked with aberrant genetic changes; 2. Analysis of high resolution flow cytometry with WHO classification is a powerful tool to be used in primary leukemia diagnosis as well as in study of understanding leukemia malignancy.
Overexpression of EVI1 in Translocation t(3;8)(q26;q24) in a Patient with Trilineage AML.
Authors: Wernli Martin , Fernandez Paula C. , Roethlisberger Benno , Heijnen Ingmar , Burger Johanna , Muehlematter Dominique , Jotterand Martine , Huber Andreas R. , Bargetzi Mario J. 
Published: April 2020

Abstract
EVI1 is a transcription factor that is targeted for de-regulation in 10% of patients with acute myeloid leukemia (AML). Overexpression of EVI1 is associated with a particularly poor prognosis. Various karyotypic abnormalities involving chromosomal region 3q26 have been shown to inappropriately activate expression of EVI1, the most frequent alterations being inv(3)(q21q26)and t(3;3)(q21;q26). Rearrangements of 3q are frequently associated with trilineage myelodysplasia, especially dysmegakaryocytosis. So far, translocation t(3;8)(q26;q24) has been reported in one patient with MDS and two cases of AML without further molecular analysis. No other recurrent chromosome aberration with a breakpoint in 8q24 has been shown in AML. We report a case of a 58 year old male patient with a multilineage AML featuring a t(3;8)(q26;q24) as the sole chromosomal aberration in 80% of bone marrow cells analysed by conventional cytogenetics. The normocellular bone marrow aspirate showed 25–50% atypical blasts, dyserythropoiesis, dysplastic myelopoiesis and hyperactivated megakaryopoiesis with many micromegakaryocytes. Immunophenotyping of the blast population by flow cytometry confirmed the myeloid nature of the cells with CD13+, CD33+, CD64+, CD117+, HLA−DR+ phenotype and variable expression of CD14 and CD34. Persisting pancytopenia occurred after two cycles of chemotherapy and a relapse was evident 4 months later. By real time quantitative RT-PCR (RQ-PCR) we showed that EVI1 is highly overexpressed (&gt;100:1) in this patients bone marrow cells as compared with an AML patient without 3q26 involvement. Fluorescence in situ hybridisation (FISH) is currently being performed to characterize the 8q24 and 3q26 breakpoints in more detail.
The chromosomal translocation t(3;8)(q26;q24) leads to overexpression of EVI1 and, as in other aberrations involving 3q, shows a poor prognosis in AML.