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Publicaciones científicas más recientes (desde 2010)

Autores: Macias, Monica; Cañada, Eva María; Alegre, Estíbaliz; et al.
ISSN 1434-6621  Vol. 58  Nº 8  2020  págs. 1341-1348
Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Next-generation sequencing (NGS) technology provides the most complete mutational analysis, but nowadays limited data are available related to the comparison of results reported by different platforms. Here we compare two NGS panels for cfDNA: Oncomine¿ Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360® (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory. Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360® (G360) was requested as part of their clinical assistance. Blood samples were sent to be analyzed with G360 panel, and an additional blood sample was drawn to obtain and analyze cfDNA with Oncomine¿ Pan-Cancer (OM) panel in an Ion GeneStudio S5¿ System.
Autores: Macias, Monica; Rebmann, V.; Mateos, B.; et al.
ISSN 1434-6621  Vol. 57  Nº 10  2019  págs. 1539 - 1545
Background: Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods: Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results: The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions: Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.
Autores: Macias, Monica; Alegre, Estíbaliz; Alkorta-Aranburu, Gorka; et al.
ISSN 0278-0240  Vol. 2019  Nº 7954921  2019  págs. 1 - 7
Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median = 11, IQR = 9.5-13) to the best response (median = 0, IQR = 0-0, p < 0.01), followed by a significant increase at progression (median = 11, IQR = 11-15, p < 0.01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received.
Autores: Sendino Miguel, T; Sandua, Amaia; et al.
ISSN 0009-8981  Vol. 493  Nº Supl. 1  2019  págs. S137
Autores: Macias, Monica; Alegre, Estíbaliz; et al.
ISSN 0065-2423  Vol. 83  2018  págs. 73 - 119
Liquid biopsy refers to the molecular analysis in biological fluids of nucleic acids, subcellular structures, especially exosomes, and, in the context of cancer, circulating tumor cells. In the last 10 years, there has been an intensive research in liquid biopsy to achieve a less invasive and more precise personalized medicine. Molecular assessment of these circulating biomarkers can complement or even surrogate tissue biopsy. Because of this research, liquid biopsy has been introduced in clinical practice, especially in oncology, prenatal screening, and transplantation. Here we review the biology, methodological approaches, and clinical applications of the main biomarkers involved in liquid biopsy.
Autores: Alegre, Estíbaliz; Martínez, Débora; Macias, Monica; et al.
ISSN 2305-5839  Vol. 4  Nº 24  2016  págs. 504
Autores: Alegre, Estíbaliz; Restituto, Patricia; et al.
ISSN 1010-4283  Vol. 37  Nº 10  2016  págs. 13687 - 13694
Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p < 0.01). There was high agreement for activating mutations between baseline cfDNA and tumor samples, especially for L858R mutation (kappa index = 0.679; p = 0.001). In 34 % of advanced NSCLC patients, we detected mutations in cfDNA not previously detected in tumor samples and double mutations in 17 %. Patients with baseline total EGFR copy levels above the median presented decreased overall survival (OS) (341 vs. 870 days, p < 0.05) and progression-free survival (PFS) (238 vs. 783 days; p < 0.05) compared with those with total EGFR copy levels below the median. Patients with baseline concentrations of activating mutations above the median (94 copies/mL) had lower OS (317 vs. 805 days; p < 0.05) and PFS (195 vs. 724 days; p < 0.05). During follow-up, T790M resistance mutation was detected in 53 % of patients. Total and mutated EGFR analysis in cfDNA seems a relevant tool to characterize the molecular profile and prognosis of NSCLC patients harboring EGFR mutations.
Autores: Alegre, Estíbaliz; Pérez, José Luis; et al.
ISSN 0009-8981  Vol. 454  2016  págs. 28 - 32
Background: Malignant melanoma is an aggressive cancer with an increasing incidence. Exosomes are actively secreted microvesicles, whose characteristics reflect those of the cell they are originated in. The aim of this study was to identify and evaluate the presence of the melanoma biomarlcers MIA, S100B and tyrosinase-related protein 2 (TYRP2) in exosomes and their potential clinical utility. Methods: Serum samples were obtained from stage IV melanoma patients, melanoma-free patients and healthy controls. Exosomes were precipitated and TYRP2, MIA and S100B concentrations were quantified in serum, exosomes, and exosome-free serum. Results: Both MIA and S100B were detected in exosomes and correlated significantly with serum concentrations (S100B: r = 0.968; MIA: r = 0.799; p < 0.001). MIA and S100B concentrations in exosomes were significantly higher in melanoma patients than in healthy controls and disease-free patients. However, TYRP2 concentrations in exosomes did not differ between these three groups. ROC curves analysis rendered AUCs for MIA of 0.883 (p < 0.01) and of 0.840 for S100B (p < 0.01). Patients with exosome MIA concentration higher than 2.5 mu g/L showed shorter median survival related to those with lower level (4 versus 11 months; p < 0.05). Conclusions: MIA and S100B can be detected in exosomes from melanoma patients and their quantification presents diagnostic and prognostic utility.
Autores: Alegre, Estíbaliz; Fernández, Sara; et al.
ISSN 0065-2423  Vol. 69  2015  págs. 47 - 89
Melanoma is an aggressive tumor with increasing incidence worldwide. Biomarkers are valuable tools to minimize the cost and improve efficacy of treatment of this deadly disease. Serological markers have not widely been introduced in routine clinical practice due to their insufficient diagnostic sensitivity and specificity. It is likely that the lack of objective responses with traditional treatment hinder biomarker research and development in melanoma. Recently, new drugs and therapies have, however, emerged in advanced melanoma with noticeable objective response ratio and survival. In this new scenario, serological tumor markers should be revisited. In addition, other potential circulating biomarkers such as cell-free DNA, exosomes, microRNA, and circulating tumor cells have also been identified. In this review, we summarize classical and emerging tumor markers and discuss their possible roles in emerging therapeutics.
Autores: Alegre, Estíbaliz; Rizzo, R.; Bortolotti, D.; et al.
ISSN 2314-8861  Vol. 2014  2014  págs. 657625
Human leukocyte antigen-G (HLA-G) is a low polymorphic nonclassical HLA-I molecule restrictively expressed and with suppressive functions. HLA-G gene products are quite complex, with seven HLA-G isoforms, four membrane bound, and other three soluble isoforms that can suffer different posttranslational modifications or even complex formations. In addition, HLA-G has been described included in exosomes. In this review we will focus on HLA-G biochemistry with special emphasis to the mechanisms that regulate its expression and how the protein modifications affect the quantification in biological fluids.
Autores: Fernández, Sara; et al.
ISSN 0009-8981  Vol. 429  2014  págs. 168 - 174
BRAF V600 mutation has been reported in more than 50% of melanoma cases and its presence predicts clinical activity of BRAF inhibitors (iBRAF). We evaluated the rote of MIA, S100 and LDH to monitor iBRAF efficiency in advanced melanoma patients presenting BRAF V600 mutations. This was a prospective study of melanoma patients harboring the BRAF V600 mutation and treated with iBRAF within a clinical trial (dabrafenib) or as part of an expanded access program (vemurafenib). MIA, S100 and LDH were analyzed in serum at baseline, and every 4-6 weeks during treatment. Eighteen patients with melanoma stages IIIc-IV were enrolled with 88.8% of response rate to iBRAF. Baseline concentrations of all the tumor markers correlated with tumor burden. MIA and S100 concentrations decreased significantly one month after the beginning of treatment and, upon progression, their concentrations increased significantly above the minimum levels previously achieved. MIA levels lower than 9 mu g/L one month after the beginning of treatment and S100 concentrations lower than 0.1 mu g/L at the moment of best response were associated With improved progression-free survival. In conclusion, MIA and S100 are useful to monitor response in melanoma patients treated with iBRAF.
Autores: Alegre, Estíbaliz; et al.
ISSN 0003-9985  Vol. 138  Nº 6  2014  págs. 828 - 832
CONTEXT: Malignant melanoma is an aggressive tumor that produces exosomes, which contain microRNAs (miRNAs) that could be of utility in following tumoral cell dysregulation. MicroR-125b is a miRNA whose down-regulation seems to be implicated in melanoma progression. OBJECTIVE: To analyze miR-125b levels in serum, and in exosomes obtained from serum, from patients with advanced melanoma. DESIGN: Serum samples were obtained from 21 patients with advanced melanoma, from 16 disease-free patients with melanoma, and from 19 healthy volunteers. Exosomes were isolated from serum by precipitation, and miR-16 and miR-125b levels were quantified by real-time polymerase chain reaction. RESULTS: MicroR-16, but not miR-125b, was detected in all samples, and miR-16 levels were significantly higher in serum than they were in exosomes. MicroR-16 expression levels did not differ significantly between the 2 groups (patients with melanoma and healthy donors). There was a significant relationship between miR-125b and miR-16 levels in exosomes. Additionally, miR-125b levels in exosomes were significantly lower in patients with melanoma compared with disease-free patients with melanoma and healthy controls. CONCLUSIONS: Exosomes can provide a suitable material to measure circulating miRNA in melanoma, and miR-16 can be used as an endogenous normalizer. Lower levels of miR-125b in exosomes obtained from serum are associated with advanced melanoma disease, probably reflecting the tumoral cell dysregulation.
Autores: Alegre, Estíbaliz; Rebmann, V.; LeMaoult, J.; et al.
ISSN 0014-2980  Vol. 43  Nº 7  2013  págs. 1933-9
The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis
Autores: HowangYin, K.Y.; Loustau, M.; Wu, J.; et al.
ISSN 1420-682X  Vol. 69  Nº 23  2012  págs. 4041 - 4049
The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains alpha(1)-alpha(2)-alpha(3) non-covalently bound to beta-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G alpha(1)-alpha(3) structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the alpha(1)-alpha(3)-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.
Autores: González, Álvaro; Rebmann, V.; LeMaoult, J.; et al.
ISSN 1040-8363  Vol. 49  Nº 3  2012  págs. 63 - 84
Autores: González, Álvaro; Alegre, Estíbaliz; et al.
Revista: Clinical Chemistry (Baltimore)
ISSN 0009-9147  Vol. 57  Nº 7  2011  págs. 1013 - 1022
Autores: Alegre, Estíbaliz; et al.
ISSN 1010-4283  Vol. 32  Nº 6  2011  págs. 1155 - 1161
Autores: González, Álvaro; Alegre, Estíbaliz; Torres, María Isabel; et al.
Revista: American Journal of Reproductive Immunology
ISSN 1046-7408  Vol. 64  Nº 5  2010  págs. 367 - 74
Autores: HoWangYin, K.Y.; Alegre, Estíbaliz; Daouya, M.; et al.
ISSN 1420-682X  Vol. 67  Nº 7  2010  págs. 1133 - 1145
Trogocytosis is the uptake of membranes from one cell by another. Trogocytosis has been demonstrated for monocytes, B cells, T cells, and NK cells. The acquisition of the tolerogenic molecule HLA-G by T cells and NK cells makes them behave as regulatory cells. We investigated here whether HLA-G, which is expressed by tumor cells in vivo, could be acquired by monocytes and if this transfer could have functional consequences. We demonstrate that resting, and even more so, activated monocytes efficiently acquire membrane-bound HLA-G from HLA-G tumor cells by trogocytosis. However, we demonstrate that HLA-G quickly disappears from the surface of the monocytes in contrast to the HLA-G acquired by T cells. Consequently, HLA-G(acq+) monocytes do not reliably inhibit the on-going proliferation of autologous activated T cells and do not inhibit their cytokine production. Thus, we show that the acquirer cell may control the functional outcome of trogocytosis.
Autores: Alegre, Estíbaliz; HoWangYin, Kiave-Yune; Favier, Benoit; et al.
Revista: Cell Research
ISSN 1001-0602  Vol. 20  Nº 11  2010  págs. 1239 - 1251
Autores: Martínez, Débora; Ramirez, Maria Carla; González, Álvaro; et al.
Libro:  Principios de bioquímica clínica y patología molecular
Vol. 3  2019  págs. 325-332
Autores: Ramirez, Maria Carla; González, Álvaro; Alegre, Estíbaliz; et al.
Libro:  Principios de bioquímica clínica y patología molecular
2019  págs. 151 - 164
Autores: Martínez, Débora; González, Álvaro; Alegre, Estíbaliz; et al.
Título: Elementos traza
Libro:  Principios de Bioquímica Clínica y patología molecular
Vol. 3  2019  págs. 143-152
Autores: Ramirez, Maria Carla; González, Álvaro; Alegre, Estíbaliz; et al.
Libro:  Principios de bioquímica clínica y patología molecular
2019  págs. 69 - 78
Autores: Martínez, Débora; González, Álvaro; Alegre, Estíbaliz; et al.
Título: Hormonas sexuales
Libro:  Principios de Bioquímica Clínica y Patología Molecular
Vol. 3  2019  págs. 313-324
Autores: Alegre, Estíbaliz; et al.
Libro:  Liquid biopsy in cancer patients : the hand lens for tumor evolution
2017  págs. 161-193
Autores: Salgado, Josefa; González, Álvaro; Alegre, Estíbaliz; et al.
Libro:  Principios de bioquímica clínica y patología molecular
2014  págs. 341-346
Complejo de glucoproteínas asociadas con la distrofina, distrofias musculares de Duchenne y Becker, técnicas bioquímicas diagnósticas de las distrofias musculares de Duchenne y Becker, otras distrofias.
Autores: Salgado, Josefa; González, Álvaro; Alegre, Estíbaliz; et al.
Libro:  Principios de bioquímica clínica y patología molecular
2014  págs. 275 - 285
Desarrollo tumoral, marcadores tumorales, determinación de los marcadores tumorales, principales marcadores tumorales séricos, aplicación de los marcadores tumorales farmacogenómica y farmacogenética.
Autores: González, Álvaro; Alegre, Estíbaliz; Monreal, José Ignacio; et al.
Libro:  Principios de bioquímica clínica y patología molecular
2014  págs. 347-353
Cadena respiratoria mitocondrial y fosforilación oxidativa, ADN mitocondrial, origen de las proteínas mitocondriales, alteraciones del ADN mitocondrial y envejecimiento, enfermedades mitocondriales o citopatías mitocondriales, estudio bioquímico general de las citopatías mitocondriales.
Autores: González, Álvaro; Alegre, Estíbaliz;
Libro:  Fundamentos de Nutrición y Dietética. Bases metodológicas y aplicaciones
2011  págs. 257-261