1,721,065 research outputs found
La mutazione di tipo A della nucleofosmina nella diagnosi e nel follow-up della leucemia mieloide acuta
Full text linkAcute myeloid leukaemias (AML), which account for about 80% of the acute leukaemias in adult, represent a phenotypically and genetically heterogeneous group of clonal haematopoietic diseases in which, failure to differentiate and over proliferation in the stem cell/progenitor compartment, result in accumulation of non-functional myeloblasts. Nucleophosmin-1 (NPM1) mutations is the most frequent gene alteration in AML (30%), and are particularly frequent in AML with normal karyotype (AML-NK) (60%). These alterations have been shown to carry prognostic significance because they seem to identify patients with better response to chemotherapy. As a consequence, the analysis of NPM1 mutational status is now recommended for inclusion in the genetic routine characterization of AML (Gorello P. et al., Leukemia 2006; Ammatuna E. et al., Clin Chem 2005; Nelida I.N. et al., Leukemia 2005; Scholl S. et al., Leuk Res 2007; Lin LI et al., Leukemia 2006).
Finally, given their high prevalence and stability over the course of the disease, NPM1 mutations may serve as an ideal target for minimal residual disease (MRD) assessment, particularly for patients with AML-NK (Gorello P. et al., Leukemia 2006).
In early 2005, Falini et al. (Falini B. et al., New Engl J Med 2005) reported that NPM1 gene mutations, mainly consisting of small nucleotide insertion/deletions, occur frequently in AML and are strongly associated with NK. Over 40 different types of mutations in the NPM1 (NPM1m) locus have been described so far, which result in the formation of different mutant proteins. This alterations consist of the insertion/deletion of short nucleotide stretches (4 or 10 bps) after nucleotide position 956 in NPM1 exon 12 that lead to an ORF shift resulting in different protein variants containig novel C-terminus portions. The most common NPM1 mutation is the type A (NPM1 mut-A), accounting for 75% - 85% of cases, and consist of duplication of a TCTG tetranucleotide at position 956 to 959 of the reference sequence (GenBank accession number NM_002520).
Available methods to detect nucleophosmin mutations are costly, require sophisticated equipment, and are often less sensitive. The aim of this work was to develop two assays that enable rapid and sensitive detection of NPM1 mut-A (Ottone T. et al., Genes Chromosomes Cancer 2009): a fragment analysis method and an RT-ASO-PCR (Reverse Trascriptase Allele Specific Oligonucleotide Polymerase Chain Reaction) method. Furthermore a semi-nested ASO-PCR method was also designed in order to increase the sensitivity of our assay for the monitoring of MRD.
After informed consent, bone marrow and peripheral blood cells were collected at presentation from 107 patients with AML diagnosed at the Institute of Hematology, Department of Biopathology of the University Tor Vergata of Rome. For each patient reverse trascribed cDNA was divided into three aliquotes used for capillary elettrophoresis, sequencing and ASO-PCR. Semi-nested ASO-PCR experiments were carried out using RT-ASO-PCR products. For capillary electrophoresis fluorescent labeled PCR products were used to screen for the presence of NPM1 mutations using the CEQTM 8000 Genetic Analysis Sistem (Beckman Coulter) instrument. Electroferogram showed 348 bp and 352 bp fragment size, corresponding to NPM1wt and NPM1m, respectively. OCI-Aml3 cell line was used as a positive control. The sensitivity of method was tested using the following serial dilutions of the mutated with wild type allele: 0.01, 0.10, 0.25, 0.50, 0.75, 1.00 (mut/wt). To confirm fragment analysis results, 17 NPM1m and 10 NPM1wt samples were sequenced: cDNA were amplified and the products, after purification, were prepared for sequencing using the CEQTM 8000 instrument.
With the aim of analyzing the presence of type A mutation in NPM1 exon 12, an RT-ASO-PCR strategy was used. A forward primer was designed to specifically amplify NPM1 exon 12 only if the mutation A is present; this primer in fact contains an intentional mismatch at third nucleotide from 3’ end to improve specificity. The amplified region includes the insertion of a TCTG tetranucleotide and corresponds to a 320 bp amplified product. As internal PCR control the ABL housekeeping gene was also amplified in each sample.
In order to increase the sensitivity for the assay for MRD monitoring, we set up a semi-nested ASO-PCR; for this purpose an internal forward primer NPM-AN for NPM1 mut-A was designed, corresponding to a 319 bp amplified fragment.
Fragment analysis allowed to identify 17 NPM1m samples. All the NPM1m samples were heterozygous confirming that NPM1m allele is negative dominant. As concerning sensitivity method enabled to detect the NPM1 mutations present in concentration of 10-2.
Sequencing analysis confirmed in all instances NPM1m and NPM1wt cases, showing that 12 samples harboured the most frequent type A mutation, the remaining mutated samples showing type B (3 cases), type D (1 case) and type K mutation (1 case).
RT-ASO-PCR assay confirmed the capillary electrophoresis and sequencing results, showing a concordance in 100% of cases; in fact this method identified the 12 out of 17 NPM1m samples as NPM1 mut-A, with an overall frequency of 70%.
The semi-nested ASO-PCR assay was carried out to increase the sensitivity of this assay for the monitoring of MRD. The sensitivity calculated for both methods, RT-ASO-PCR and semi-nested ASO-PCR, indicated that this assays could detect the NPM1 mut-A present in concentration of 10-2 and 10-5, respectively.
Out of 107 patients, cytogenetic data were available for 99 patients, divided into 2 groups: a group (n=15) consisting of NPM1m and a group (n=84) with NPMwt. Twenty seven out of 99 had normal karyotype. Amongst twenty seven, 13 were NPM1m with an incidence of 48%.
The capillary electrophoresis assay developed in this work proved to be a fast, reproducible and easy method applicable to NPM1m diagnosis, capable to discriminate 1 mutated cell out of 100. RT-ASO-PCR and the more sensitive semi-nested ASO-PCR were developed for specifically detect NPM1mut-A, the most frequent NPM1 mutation. These methods were capable to detect 1 mutated cell out of 100 and out of 100.000, respectively.
Monitoring more patients by these new assays in MRD longitudinal studies could be relevant for better assess molecular remission and the risk of relapse in AML-NK
Advances in the pathogenesis of FLT3 -mutated acute myeloid leukemia and targeted treatments
No full textpurpose of reviewFLT3 mutations are among the most common myeloid drivers identified in adult acute myeloid leukemia (AML). their identification is crucial for the precise risk assessment because of the strong prognostic significance of the most recurrent type of FLT3 alterations, namely internal tandem duplications (ITDs). recent advances in the pathogenesis and biology of FLT3-mutated AML have opened an opportunity for development and application of selective inhibition of FLT3 pathway. recent findingsIn the last decade, at least three targeted treatments have been approved by regulatory agencies and several others are currently under investigations. here, we review the latest advance in the role of FLT3 mutations in AML, providing an outline of the available therapeutic strategies, their mechanisms of actions and of resistance, as well as routes for potential improvement. summary the availability of FLT3 inhibitors has improved outcomes in AML harboring such mutations, currently also reflected in disease stratification and recommendations. newer inhibitors are under investigations, and combinations with chemotherapy or other targeted treatments are being explored to further improve disease outcomes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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