1,720,984 research outputs found
Ultra-deep mutational analysis of NPM-ALK and possible implications on target therapy in anaplastic large cell lymphoma of childhood
Full text linkAnaplastic Large Cell Lymphoma (ALCL) represents a distinct subset of aggressive T-cell non-Hodgkin lymphoma (NHL) accounting for about 3% of adult NHL and 10 to 15% of childhood lymphomas. In the vast majority of the cases, ALCL is associated to chromosomal translocations, the most frequent being the t(2;5)(p23;q35), involving the Anaplastic Lymphoma Kinase (ALK) gene, which lead to aberrant NPM-ALK protein expression and kinase activity. It has been extensively demonstrated that aberrant
NPM-ALK expression contributes to the pathogenesis of ALK-positive ALCL, as it causes cell transformation through activation of several biological pathways related to cell proliferation, cell-cycle control and apoptosis. Although ALK-positive ALCL have a rather benign prognosis when treated with standard chemotherapy, the failure rate at two years is almost 30% for most of these regimens. Notably, most of relapses occur within the first year from the start of therapy, and long-term survival for relapsed disease is less than 50%. Aberrant ALK activity is one of the major oncogenic events not only in ALK-positive ALCL, but also in neuroblastoma, non-small cell lung cancer (NSCLC) and inflammatory myofibroblastic tumour (IMT) bearing ALK activating mutations/rearrangements, and the inhibition of ALK kinase activity was proven to substantially reduce cancer cell proliferation and invasiveness both in vitro and in vivo. Successful clinical experience with crizotinib further support the concept of ALK-specific inhibition as a valuable treatment strategy in ALK-positive ALCL, as well as in other
ALK-addicted tumours. However, similarly to other inhibitors selectively targeting oncogenic kinases, data on relapse to crizotinib due to newly acquired secondary mutations were reported. In this context, although a robust clinical response of ALCL patients to an ALK inhibitor is expected, some of those patients are also anticipated to develop resistance, making the knowledge of NPM-ALK kinase domain (KD) mutational status a valuable and mandatory information to the rational design of ALK-targeted therapies.
To detect somatic tumour mutations with potential utility for predicting treatment response in ALK-positive ALCL patients, we performed ultra-deep sequencing analysis on ALK exons 22-25, corresponding to the entire KD coding region, in 37 ALCL pediatric patients. Two low frequent point mutations were identified in two distinct cases, corresponding to the R1275Q and R1231Q amino acid changes. The R1275Q mutation has been already reported as one of the most frequent activating mutations in neuroblastoma, while the R1231Q amino acid substitution represents a novel ALK point mutation, which to our knowledge has never been reported neither in ALK receptor nor in other ALK-translocated kinases. The molecular implications of R1275Q and R1231Q point substitutions on NPM-ALK function and sensitivity to ALK-specific inhibition are still under our investigation. In addition to point mutation, oncokinase alternative spliced transcripts have been previously reported in patients with Bcr-Abl positive chronic myeloid leukemia and more recently ALK receptor isoforms were described in neuroblastoma. To our knowledge, however, NPM-ALK alternative splicing events have never been described. For the first time, we identified and characterized 9 NPM-ALK INDEL mutations, resulting from KD whole exons skipping or alternative canonical splicing sites recognition. To investigate the effect of INDEL mutations on the structure and activity of NPM-ALK, we performed molecular homology modelling and in vitro functional analysis. While all these mutants were shown to be kinase dead, we demonstrated that, when coespressed with wild-type NPM-ALK, these INDELs do interact with wild-type monomers and are likely to inhibit ALK kinase activity and increase sensitivity to treatment with crizotinib. This work demonstrates that NPM-ALK KD point mutations are extremely rare in newly diagnosed ALCL patients, but positive selection of mutated cells could not be excluded in case of an ALK-targeting therapy. Conversely, our results suggest that alternative splicing in NPM-ALK may represent a common event. A clear correlation between the presence of these variants and outcome could not be detected, possibly because of the restricted cohort of patients analyzed. However, we hypothesize that a significant impact of these mutations could be observed if an ALK-specific treatment is used. Patients bearing a consistent level of inactive NPM-ALK are therefore expected to respond differently to ALK kinase inhibitors; whether such a response will be increased or decreased, it remains to be elucidated
Small RNAs in Circulating Exosomes of Cancer Patients: A Minireview
Full text linkExtracellular vesicles (EVs) secreted from many cell types play important roles in intercellular communication, both as paracrine and endocrine factors, as they can circulate in biological fluids, including plasma. Amid EVs, exosomes are actively secreted vesicles that contain proteins, lipids, soluble factors, and nucleic acids, including microRNAs (miRNAs) and other classes of small RNAs (sRNA). miRNAs are prominent post‐transcriptional regulators of gene expression and epigenetic silencers of transcription. We concisely review the roles of miRNAs in cell‐fate determination and development and their regulatory activity on almost all the processes and pathways controlling tumor formation and progression. Next, we consider the evidence linking exosomes to tumor progression, particularly to the setting‐up of permissive pre‐metastatic niches. The study of exosomes in patients with different survival and therapy response can inform on the possible correlations between exosomal cargo and disease features. Moreover, the exploration of circulating exosomes as possible sources of non‐invasive biomarkers could give new implements for anti‐cancer therapy and metastasis prevention. Since the characterization of sRNAs in exosomes of cancer patients sparks opportunities to better understand their roles in cancer, we briefly present current experimental and computational protocols for sRNAs analysis in circulating exosomes by RNA‐seq
Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors.
Full text linkReceptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs.In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling.We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways.These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK a major therapeutic target in this malignancy, particularly if expression and activity cannot be accurately determined
IGH and IGK gene rearrangements as PCR targets for pediatric Burkitt's lymphoma and mature B-ALL MRD analysis
No full textWe recently reported that minimal residual disease (MRD) and minimal disseminated disease (MDD), assessed by long-distance PCR (LD-PCR) for t(8; 14), are negative prognostic factors in mature B-cell acute lymphoblastic leukemia (B-ALL) and in Burkitt's lymphoma (BL). However, t(8; 14) is detectable in only about 70% of patients, thus preventing MRD studies by this approach in the remaining patients. At present, no molecular assays have been reported for MRD and MDD analysis in t(8; 14)-negative patients. The aim of our study was to evaluate the characteristics of patient-specific immunoglobulin (Ig) gene rearrangements as RQ-PCR targets for MRD analysis, in order to extend MRD studies to those patients who are not eligible for the LD-PCR assay. The study was performed according to the guidelines of the European Study Group on MRD detection in ALL (ESG-MRD-ALL). Overall, 36 B-ALL and 19 BL cases were analyzed. Multiple PCR reactions were performed for each sample to identify heavy and kappa light-chain rearrangements. A total of 97 RQ-PCR targets (62 for B-ALL, 35 for BL) were analyzed for sensitivity. The rearrangement pattern identified was similar to that reported for normal peripheral blood lymphocytes. In 88% of the targets, a sensitivity of at least 10(-4) was achieved. In 87% of patients (84% of B-ALLs, 95% of BLs) at least one sensitive target was available. All PCR targets identified at diagnosis were preserved at relapse. Our results suggest that MDD and MRD can be successfully studied using a single sensitive Ig target in the great majority of B-ALL and BL cases. The combination of LD-PCR and Ig-based assays will allow MRD analysis in virtually all of the patients. Laboratory Investigation (2009) 89, 1182-1186; doi:10.1038/labinvest.2009.81; published online 10 August 200
TCR-Based RQ-PCR Assay for MDD and MRD Assessment in T-Cell Lymphoblastic Lymphoma of childhood
No full textComparative analysis of MRD by LD-PCR and Ig gene rearrangements in mature B-ALL of childhood
No full textIgGene rearrangements as PCR Targest for MRD analysis in pediatric B-all and Burkitt's lymphoma and comparison with T(8;14) detection by LD-PCR
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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
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