bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Molecular Expression of the Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia Cells as a Possible Marker for Minimal Residual Disease Detection.
Authors: Aluigi Michela , Curti Antonio , Ottaviani Emanuela , Lucchetti Giovanna , Salvestrini Valentina , D’Addio Alessandra , Trabanelli Sara , Fiore Francesca , Massaia Massimo , Martinelli Giovanni , Baccarani Michele , Lemoli Roberto M. 
Published: October 2019

Abstract
The expression of the catalytic enzyme of tryptophan, indoleamine 2,3 dioxygenase (IDO) has been identified as a T-cell inhibitor effector pathway in different normal and neoplastic cells. We have recently shown that normal bone marrow (BM) cells, including hematopoietic CD34+ cells, express IDO mRNA only upon IFN-γ stimulation, whereas in a subset of human acute myeloid leukemia cells (AML) IDO is constitutively expressed both at molecular and protein level and induces immunological escape by promoting the generation of T-reg cells. To investigate whether the IDO transcript can be used as a marker for minimal residual disease (MRD) detection, we used a sensitive real-time reverse transcription-polymerase reaction assay (qRT-PCR) to quantify IDO mRNA levels in peripheral blood (PB) and BM samples of newly diagnosed AML patients. The level of IDO transcript was evaluated as IDO copy number/104 ABL copies. As control samples, we used normal PB and BM mononuclear cells (MNCs). Our data showed that normal BM and PB cells expressed minimal amount of IDO mRNA (range 100–1000). Among AML samples, we identified three subsets of patients according to IDO mRNA expression: 1) 33/74 (44.6%) IDO negative (i.e. range < 100), 28/74 (37.84%) IDO low (range 100–1000) and 13/74 (17.6%) IDO high (i.e. range > 1000). BM and PB AML blasts gave similar results. Assessment of protein expression and enzymatic activity was in accordance with molecular results. Some patients were evaluated for IDO mRNA expression before and after induction chemotherapy and the IDO levels were found to correlate with the reduction of BM blasts. Taken together, our qRT-PCR data demonstrate that
normal PB and BM cells are negative for IDO mRNA expression, which, in turn, is significantly up-regulated in a subset of AML patients (IDO high) and IDO mRNA expression correlates with tumor burden,
thus suggesting its possible role in the detection of MRD in IDO high patients.
Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.
Authors: Faderl Stefan H. , Van Quin , Koch Patricia E. , Harris David M. , Hallevi Inbal , Liu Zhiming , Wang Yongtao , Ferrajoli Alessandra , Estrov Zeev 
Published: October 2019

Abstract
Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.
Acute Myeloid Leukemia with Normal Karyotype: Analysis of FLT3, NPM1, MLL and CEBPA Gene Abnormalities.
Authors: Trigo Fernanda , Torres Fatima , Guimaraes Patricia , Silva-Coelho Pedro , Carvalhais Ines , Santo Ana Espirito , Melo Teresa , Castro Miriam , Fernandes Alexandra , Tavares Purificacao , Guimaraes Jose E. 
Published: October 2019

Abstract
Acute myeloid leukemia with normal karyotype (AML-NK) is markedly heterogeneous depending on the presence or absence of certain genetic alterations. We studied the prevalence of different abnormalities, namely of the FLT3, NPM1, CEBPA and MLL genes, in AML-NK patients and tried to correlate them with clinicopathological findings. Techniques used were as follows: FLT3-ITD (internal tandem duplications), PCR using fluorescent primers, analysis in automatic sequencer; FLT3-TKD (D385), PCR, fragment cut with EcoRV restriction enzyme run in agarose gel; NPM1, RT-PCR and automatic sequencing of a fragment containing exon 12; CEBPA, PCR and automatic sequencing of the entire coding region; MLL-PTD, RT-PCR with analysis in agarose gel. The following data from patients were recorded: sex, age, FAB classification, WBC and platelet count, hemoglobin and LDH level, response to induction, survival time and cause of death. The incidence of FLT3 alterations (all ITDs) was 10/23 (43.5%), varying from 1 to 10 ITDs; NPM1 was mutated in 12/24 cases (50.0%) and MLL-PTD in 2/24 patients (8.3%); for CEBPA, of the 10 cases in which all 3 amplicons (1A, 2B, 3C) were analysed, 6 had a mutation mostly in amplicon 2B (5 cases). More than one genetic abnormality was present in 9 cases: six showed coexistence of FLT3-ITD and NPM1 mutation, one of MLL and CEBPA mutations, one of FLT3-ITD and CEBPA mutation and one of FLT3-ITD, NPM1 and CEBPA. Regardind correlation with clinical characteristics, we observed that 72.7% of nucleophosmin mutated (NPM1+) cases were CD34 negative and 91.7% of CD34+ patients were NPM1 negative (p=0.003). There is a tendency, although not significant, for CEBPA+ cases to correlate with low WBC counts and for patients with high WBC count to be NPM1+ and FLT3-ITD+ (odds ratio of 3.0 and 3.33, respectively). All NPM1+ patients treated with appropriate induction therapy achieved complete remission; 22.2% of NPM1 negative patients were refractory to therapy. No significant correlation was observed between survival and presence or absence of any of the genetic alterations. This work was funded by an unrestricted grant from Roche Farmaceutica - Portugal.
Abnormal Expression of Receptor Tyrosine Kinase Mer in Adult Acute Leukemia.
Authors: Fan Lei , Xie Liqian , Shen Fei , Dai Lan , Xu Wei , Jianyong LI , Ruan Changgeng 
Published: October 2019

Abstract
BACKGROUND & OBJECTIVE Mer was found a proto-oncogene in some solid tumor. it belongs to Axl receptor tyrosine kinase family. This study was to detect Mer expression in 57 adult acute leukemia patients’ bone marrow (32 AML and 25 ALL patients) and normal control.
Methods Flow cytometry with anti-CD15, CD3, CD19 and CD14 antibody were used to detect Mer expression on granulocytes, T-lymphocytes, B-lymphocytes and monocytes in normal bone marrow. Mer expression on leukemia cells of patients’ bone marrow samples were also analysed by flow cytometry, and RT-PCR was used to verify parts of Mer mRNA level expreesion in mononuclearcell of bone marrow samples.
Results Flow cytometry showed that in normal bone marrow, there was no Mer expressed on granulocytes, T-lymphocytes and B-lymphocytes; and middle-expression on monocytes(37.5%). In all 32 AML patients, there were 23 Mer positive(71.8%), Mer expression level was high in 3 AML patients, middle in 8 AML patients, low in 12 AML patients and very low/negative in left 9 AML patients. Among 3 Mer high Expression AML patients, the FAB typping were all M1, and all of them had the high expression level of CD34 and CD117. In all 25 ALL patients, there were 16 Mer positive(64.0%), Mer expression level was high in 1 ALL patients, middle in 6 ALL patients, low in 9 ALL patients and very low/negative in left 9 ALL patients. The results of RT-PCR was similar to flow cytometry
Conclusion Mer was ectopic expression parts of adults acute leukemia patients, and may related to genesis and development of acute leukemia.
Molecular Remission in MLL/AF4-Positive Infant Leukemia Treated with the All Trans-Retinoic Acid Based MLL-Baby Protocol.
Authors: Tsaur Grigory , Semenikhina Elizaveta , Ivanova Anna , Nasedkina Tatiana , Popov Alexander , Shorikov Egor , Saveliev Leonid , Fechina Larisa 
Published: October 2019

Abstract
Statement. We recently developed a new treatment approach for infant leukemia with MLL rearrangements (MLL-Baby) that consists of intermittent pulses of all-trans retinoic acid (ATRA) and conventional chemotherapy. Here, we present data on the molecular remission status of patients with the MLL/AF4 rearrangement enrolled in this study.
Materials and methods. Six patients with MLL/AF4 rearrangement have been enrolled in the MLL-Baby study in Ekaterinburg and Moscow since September 2003. Median age was 6.5 months (range 1.5–10 months). All have had the BI immunophenotype. Leucocytes from bone marrow (BM) and peripheral blood (PB) were obtained. Quality and integrity of RNA was estimated using RNA 6000 Nano LabChip kit and 2100 Bioanalyzer (Agilent Technologies). Only RNA with RNA integrity number more than 4.5 was taken further. Qualitative PCR for MLL/AF4 fusion gene (FG) was performed either by nested PCR (4 patients) (A. Borkhardt et al, 1994) or by low-density biochips (2 patients). Conditions of qualitative real-time PCR (qRT-PCR) for MLL-AF4 FG detection were previously described (J. Gabert et al., 2003). β2-microglobulin was used as the control gene. Normalized copy number (NCN) of MLL/AF4 was calculated. Sensitivity of qRT-PCR for BM specimens was 1×10−4 – 6×10−5, for PB was 1×10−4 – 7×10−4. In accordance with the treatment design all patients were enrolled on the high risk (HR) schedule as approved by Local and Federal Ethics committees. Parents’ informed consent was obtained.
Results. We found 2 types of MLL/AF4 FG clearance. In 4 patients the elimination rate of FG transcripts was very high. In this group FG could not be detected after 1 course of ATRA that followed induction therapy or after HR 1 (I); in 1 patient with MLL/AF4, FG total clearance was achieved after HR block 3 (VI) respectively; 1 patient currently under treatment has few copies of MLL/AF4 after HR block 2 (V). There was no correlation between the rate of elimination of MLL/AF4 FG with age, initial WBC count or type of MLL/AF4 transcript. Once achieved, the molecular remission in 5 patients has remained PCR negative.
Conclusions. Achievement of molecular remission is one of the most important steps toward long-term survival and cure. Despite differences in the rate of elimination of MLL/AF4 FG, 5 of 6 patients treated on the MLL-Baby protocol have remained in hematological and molecular remission with a median follow-up 18 months (range 6–45 months). None have relapsed. Moreover, the youngest patient, a girl who was 2 months old at the time of diagnosis, has had the longest continuous molecular remission of up to 45 months. The 6th patient has single copies of MLL/AF4 after HR block 2 (V) and is currently receiving therapy.
Frequent Molecular Aberrations in Normal Karyotype (NC) Precursor B-Cell Acute Lymphoblastic Leukemia (pB-ALL) Detected by Comparative Genomic Hybridization.
Authors: Zeidan Amer , Gaile Dan , Conroy Jeffrey M. , McQuaid Devin E. , Wang Eunice S. , Deeb George , Wallace Paul K. , Sait Sheila N.J. , Nowak Norma J. , Wetzler Meir 
Published: October 2019

Abstract
The prognosis of adult NC pB-ALL has not improved over the last decade mainly because separation into distinct molecular subsets has been lacking. We conducted a proof-of-principle experiment to screen the genome of blasts from adult NC pB-ALL patients for novel genomic alterations using 19,000 bacterial artificial chromosomes (BAC) by array CGH and verified our results with gene expression profiling (GEP, Agilent oligonucleotide expression arrays) and fluorescent in situ hybridization (FISH) using the same BAC probes or commercially available probes if available. The median age at diagnosis of the 6 males and 4 females was 55 years; all were treated with a 5-drug induction regimen followed by intensive consolidation and maintenance regimens. Five achieved CR of whom 3 relapsed with complex karyotype; samples were available on 2 of the patients at relapse. Samples with <60% blasts (2 at diagnosis, 1 at relapse) were enriched for >95% purity by sorting according to their immunophenotype pattern. Only one case had a normal CGH pattern. A decreased copy number of p16 was found in 4 of the 10 diagnostic samples; 3 on whom mRNA was available demonstrated a decreased p16 mRNA expression by GEP. In addition, a loss at 9p21.2 and a loss and a gain at 8q21.2 were detected in one sample each; those were verified by FISH. Copy number losses were also observed across multiple samples at 1p34–1p36, 4p16, 5p15.33, 8q24, 9q34, 10q26, 11q12–11q13, 12q24, 14q32, 17p, 19p13.3, 22q11, and 22q13. Copy number gains across multiple samples were observed at 15q11.2 and 18q21. Those are currently being verified by FISH. Comparison of samples at diagnosis and at relapse revealed a disparate molecular pattern. In summary, enriching samples by sorting from NC pB-ALL to be studied by CGH is feasible; normal chromosome pattern does not correlate with normal molecular pattern; and diagnostic and relapsed samples have a different molecular pattern. Our work on 10 patient samples at diagnosis and 2 at relapse demonstrates the feasibility to detect specific molecular aberrations in NC pB-ALL. Since attaining good quality metaphase cells in ALL is often suboptimal, this work suggests that array CGH can complement conventional cytogenetic analysis to better characterize NC pB-ALL by identifying cryptic copy number changes. Work on a larger number of samples is warranted to determine the prognostic significance of the aberrations detected herein.
Correlation between High Expression of Natural Killer Related-Genes (NCAM/CD94) and Early Death during Induction in Children with Acute Myeloid Leukemia.
Authors: Nigro Luca Lo , Mirabile Elena , Munda Silvana , Bottino Daria , Manuela Tumino , Gino Schiliro 
Published: October 2019

Abstract
Death early (DE) during the induction phase of therapy is a rare but dramatic event in children with acute myeloid leukemia (AML). Recent reports emphasize that DE in patients with leukemia is related to aggressive disease, signaled by hyperleukocytosis, and to the early administration of intensive chemotherapy. Many efforts have been made to better characterize subgroups of patients with AML by defining biologic markers associated with aggressive disease. Even if little is known about the molecular mechanisms that regulate blast migration into tissues, adhesion receptors are likely to play an important role in this process. We have attempted to identify the molecular characteristics of an aggressive subset of pediatric patients with AML through a prospective evaluation of the CD56+ neural cell adhesion molecule (NCAM) and CD94 expression. For this purpose we developed an RT-PCR semi-quantitative methodology, using specific primers from NCAM, CD94 and β-actin as an internal quality and quantity control. We analyzed a total population of 44 children with AML, distributed as follow: 5 with M3v (group A), 9 with APL (group B) and 30 with non-APL AML (group C). We found a dramatic correlation in group A between high expression of NK-related genes and occurrence of early death. In groups B and C, primarily used as controls, we found a strong correlations between a high level of NCAM-CD94 expression and two cases with APL, three with FAB M5, two with FAB M2 AML, one case with FAB M4 and a child with dendritic cell leukemia (DCL). We primarily found that 9 out of 13 cases presenting with a high level of NCAM-CD94 expression died of disease-related events during the early days of induction. Our data strongly suggest that doses and timing of chemotherapy induction in children with AML need to be modulated because of the high risk of hemorrhagic events. These appear to be related to the presence of high concentration of adhesion molecules secondary to acute blast lysis and to the associated severe coagulopathy. We know of no pharmacological correctives for this phenomenon. It would be important to develop specifically designed molecules in order to disrupt the interactions with adhesion molecules, and thus to improve the clinical course of both children and adults with AML during the early days of induction.
The mTOR Inhibitor Rapamycin Demonstrates Activity Against AML in Combination with Imatinib Mesylate and with 5-Azacytidine.
Authors: Liesveld Jane L. , Rosell Karen , Lu Chaohui , Mulford Deborah , Walker Alison 
Published: October 2019

Abstract
The serine/threonine kinase mTOR (mammalian target of rapamycin) integrates pathways involved in cell cycle regulation and apoptosis and has been shown to be aberrantly activated in AML. mTOR (mammalian target of rapamycin) serine threonine kinase is involved in cell cycle regulation and apoptosis. mTOR inhibitors are a class of signal transduction inhibitors, originally developed as immunosuppressive agents, with antineoplastic activity in solid tumor models. Acute myelogenous leukemia blasts and stem cells have been reported previously to be inhibited by rapamycin, an mTOR inhibitor. As a single agent, rapamycin has minimal activity, however, suggesting that for meaningful clinical efficacy, it would require combination with other agents. We have therefore begun to examine its effects in combination with other signal transduction inhibitors and demethylating agents. Rapamycin by itself had minimal inhibitory effect on AML cell lines or primary AML samples. However, in combination with the c-kit inhibitor, imatinib mesylate, 2.5–25 uM, rapamycin demonstrated synergistic inhibition of AML cell lines such as MV411 as measured in a standard MTT assay (combination index 0.32 to 0.576 as determined with the Calcusyn program),, indicating strong synergism. In this cell line, rapamycin by itself demonstrated no significant inhibitory activity. Synergism with imatinib mesylate was also noted in the U937 and HL60 cell lines but not in the KG1a cell line. The combination of rapamycin and imatinib inhibited AKT phosphorylation to a greater extent than either agent alone in both the MV411 and U937 cell lines, but the combination did not effectively inhibit ERK phosphorylation. Inhibition of phosphorylation of p70S6 kinase by the combination of 20 uM imatinib mesylate and 80uM rapamycin did not occur to a greater extent than that seen with rapamycin alone, although phosphorylation of 4EBP1 was inhibited to a greater extent when both agents were added concurrently. When AML cell lines were exposed to 5-azacytidine alone, 1 to 16 uM, the IC50 was 4–8uM. 5-azacytidine increased apoptosis above control whereas rapamycin by itself had minimal effect on apoptosis as assessed in an Annexin V flow cytometric assay. In the U937 cell line, the combination index for the U937 cell line was 0.465 (10 uM 5-azacytidine and 100 nM rapamycin), indicating synergism. For MV411, the combination index was 0.736, for KG1a, 0.118, and for HL60, no synergism was noted. Exposure to the combination of 5-azacytidine and rapamycin resulted in greater suppression of AKT and 4EBP1 phosphorylation than did either agent singly. These data suggest that simultaneous targeting of more than one signaling pathway may improve inhibition of leukemic cell growth. Furthermore, combining signal transduction inhibitors with hypomethylating agents may also result in effective suppression of AML proliferation.
Discrimination and Quantification of Spectrally Similar Near-Infrared Probes by Time-Domain (TD) Optical Imaging in Acute Myeloid Leukemia Mouse Models.
Authors: McCormack Emmet , Belankov Alexandre , Mujic Maja , Couture Pierre , Gjertsen Bjorn T. 
Published: October 2019

Abstract
The use of whole-body optical imaging in the near-infrared (NIR) spectrum (650–1100 nm) employing fluorescently labelled reagents recognising cell-specific biomarkers of leukemia has become a standard modality in preclinical models of the human disease. A particular challenge is represented by leukemic infiltrates in liver and spleen, organs with high optical absorbance. While there are increasingly impressive arrays of fluorescently labelled biomolecules available for exploitation via optical imaging, the number of commmercially availible fluorophores for NIR imaging remain limited. In particular, simultaneous imaging of disease progression and functional imaging of more specific biological processes within the same sample is complicated by the requiste for multiple filtersets for fluorophores with similar spectral properties. Subsequent “bleeding” fluorescence through filtersets is unavoidable precluding ones ability to quantify specific fluorophores based on fluorescence. Similarly, descrimination of in vivo autofluorescence of similar spectral properties to commonly employed NIR dyes, consequent of ingested food comlicates contrast even further. More recently spectral imaging techniques have aided discrimination of fluorophores of similar spectral profiles however, these techniques attenuate much of the light reaching the detector. Time-domain (TD) optical imaging through the use of pulsed laser diodes and time resolved detector system, typically a photo-multiplier tube (PMT), has previously been demonstrated to distinguish between changes in physiological processes such as; tissue pH or calcuim concentration, based on changes in fluorescence lifetime of a fluorescently labelled probe. Here we demonstrate employing a single wave lenght TD optical imaging (eXplore Optix™, ART Inc) the potential to discriminate and quantify combinations of diverse NIR probes of spectrally similar properties but differing fluorescence lifetime on the basis of fluorescence lifetime in appropriate in vitro phantoms. Similarly, we illustrate the ability of this technique to discriminate between endogenous autofluorescence from administered fluorophores in vivo of leukemic cells in liver and spleen, and subsequent distinction of mixtures these fluorophores via their inherent fluorescent lifetimes in vivo.