Nuestros investigadores

Álvaro González Hernández

Laboratorio de Bioquímica
Clínica Universidad de Navarra. Clínica Universidad de Navarra
Bioquímica y Genética
Facultad de Ciencias. Universidad de Navarra
Líneas de investigación
Biopsia líquida; marcadores tumorales; DNA circulante; exosomas
Índice H
32, (Google Scholar, 01/02/2020)

Publicaciones científicas más recientes (desde 2010)

Autores: Ajona, Daniel; et al.
ISSN 2662-1347  Vol. 1  2020  págs. 75 - 85
Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1¿programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.
Autores: Macias, Monica; Cañada, Eva María; Alegre, Estíbaliz; et al.
ISSN 1434-6621  Vol. 58  Nº 8  2020  págs. 1341 - 1348
Background: Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Nextgeneration 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: OncomineT Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360 (R) (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory. Methods: Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360 (R) (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 OncomineT Pan-Cancer (OM) panel in an Ion GeneStudio S5T System. Results: cfDNA analysis globally rendered 101 mutations. Regarding the 55/101 mutations claimed to be included by manufacturers in both panels, 17 mutations were reported only by G360, 10 only by OM and 28 by both. In those coincident cases, there was a high correlation between the variant allele fractions (VAFs) calculated with each panel (r = 0.979, p < 0.01). Regarding the six actionable mutations with an FDA-approved therapy reported by G360, one was missed with OM. Also, 12 mutations with clinical trials available were reported by G360 but not by OM. Conclusions: In summary, G360 and OM can produce different mutational profile in the same sample, even in genes included in both panels, which is especially important if these mutations are potentially druggable.
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: González, Álvaro, (Autor de correspondencia); Pérez-Barrios, C.; Macher, H.; et al.
ISSN 1888-4008  Vol. 12  Nº 3  2019  págs. e40 - e46
El análisis de ADN circulante a partir de sangre periférica ha demostrado ser de utilidad en campos clínicos tan diferentes como la oncología, los trasplantes y el cribado prenatal. Para su incorporación al laboratorio clínico es necesario asegurar protocolos preanalíticos adecuados, reproducibles y estandarizados. En este documento se pretenden dar unas recomendaciones preanalíticas para la obtención de ADN circulante a partir de sangre periférica. Incluyen el tipo de espécimen, el tipo de tubo de extracción, el modo de centrifugación de la muestra, la extracción del ADN circulante y cuantificación, así como su conservación.
Autores: Gaspar-Blázquez, M. J., (Autor de correspondencia); Trapé-Pujol, J.; Augé-Fradera, J. M.; et al.
ISSN 1888-4008  Vol. 12  Nº 1  2019  págs. 38 - 52
Este documento describe las causas de error más frecuentes en la medición de marcadores tumorales séricos proteicos en sus diferentes fases: preanalítica, analítica y postanalítica y recomendaciones para detectar y solventar problemas, así como la interpretación de los resultados de los marcadores tumorales en la práctica clínica.
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: Pita, G.; Pajares, María Josefa; et al.
ISSN 2045-7634  Vol. 7  Nº 7  2018  págs. 3474 - 3483
Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n=3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (p(combined)=5.66x10(-5); ORcombined=2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (p(combined)=1.02x10(-4); ORcombined=2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10D mRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p=0.01 and p<0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p=0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.
Autores: Alfaro, Carlos; Rodríguez, María Esperanza; et al.
ISSN 0305-7372  Vol. 60  2017  págs. 24 - 31
Interleukin-8 (CXCL8) was originally described asa chemokine whose main function is the attraction of a polymorphonuclear inflammatory leukocyte infiltrate acting on CXCR1/2. Recently, it has been found that tumors very frequently coopt the production of this chemokine, which in this malignant context exerts different pro-tumoral functions. Reportedly, these include angiogenesis, survival signaling for cancer stem cells and attraction of myeloid cells endowed with the ability to immunosuppress and locally provide growth factors. Given the fact that in cancer patients IL-8 is mainly produced by tumor cells themselves, its serum concentration has been shown to correlate with tumor burden. Thus, IL-8 serum concentrations have been shown to be useful asa pharmacodynamic biomarker to early detect response to immunotherapy. Finally, because of the roles that IL-8 plays in favoring tumor progression, several therapeutic strategies are being developed to interfere with its functions. Such interventions hold promise, especially for therapeutic combinations in the field of cancer immunotherapy.
Autores: Pérez, José Luis; Sanmamed, M. F.; Bosch, A.; et al.
ISSN 0305-7372  Vol. 53  2017  págs. 79 - 97
The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework the DESIGN guidelines-to-standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field. (C) 2017 The Authors. Published by Elsevier Ltd.
Autores: Whale, A. S.; Devonshire, A. S.; Karlin-Neumann, G.; et al.
ISSN 0003-2700  Vol. 89  Nº 3  2017  págs. 1724 - 1733
This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.
Autores: Sanmamed, M. F. ; Pérez, José Luis; Schalper, K. A. ; et al.
ISSN 0923-7534  Vol. 28  Nº 8  2017  págs. 1988 - 1995
Background: Surrogate biomarkers of efficacy are needed for anti-PD1/PD-L1 therapy, given the existence of delayed responses and pseudo-progressions. We evaluated changes in serum IL-8 levels as a biomarker of response to anti-PD-1 blockade in melanoma and non-small-cell lung cancer (NSCLC) patients. Patients and methods: Metastatic melanoma and NSCLC patients treated with nivolumab or pembrolizumab alone or nivolumab plus ipilimumab were studied. Serum was collected at baseline; at 2-4 weeks after the first dose; and at the time-points of response evaluation. Serum IL-8 levels were determined by sandwich ELISA. Changes in serum IL-8 levels were compared with the Wilcoxon test and their strength of association with response was assessed with the Mann-Whitney test. Accuracy of changes in IL-8 levels to predict response was estimated using receiver operation characteristics curves. Results: Twenty-nine melanoma patients treated with nivolumab or pembrolizumab were studied. In responding patients, serum IL-8 levels significantly decreased between baseline and best response (P < 0.001), and significantly increased upon progression (P = 0.004). In non-responders, IL-8 levels significantly increased between baseline and progression (P = 0.013). Early changes in serum IL-8 levels (2-4 weeks after treatment initiation) were strongly associated with response (P < 0.001). These observations were validated in 19 NSCLC patients treated with nivolumab or pembrolizumab (P = 0.001), and in 15 melanoma patients treated with nivolumab plus ipilimumab (P < 0.001). Early decreases in serum IL-8 levels were associated with longer overall survival in melanoma (P = 0.001) and NSCLC (P = 0.015) patients. Serum IL-8 levels also correctly reflected true response in three cancer patients presenting pseudoprogression. Conclusions: Changes in serum IL-8 levels could be used to monitor and predict clinical benefit from immune checkpoint blockade in melanoma and NSCLC patients.
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: Pérez, José Luis; et al.
ISSN 0923-7534  Vol. 27  Nº Supl. 6  2016  págs. 1055O
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: Gonzalez-Cao, M.; Mayo-de-las-Casas, C.; Molina-Vila, M. A.; et al.
ISSN 1473-5636  Vol. 25  Nº 6  2015  págs. 486 - 495
BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5-nuclease PCR (Taqman) in the presence of a peptide-nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1:20000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.
Autores: Fernández, Sara; Rodriguez, C.; et al.
ISSN 0009-9147  Vol. 61  Nº 1  2015  págs. 297 - 304
BACKGROUND: Around 50% of cutaneous melanomas harbor the BRAF(V600E) mutation and can be treated with BRAF inhibitors. DNA carrying this mutation can be released into circulation as cell-free BRAF(V600E) (cfBRAF(V600E)). Droplet digital PCR (ddPCR) is an analytically sensitive technique for quantifying small concentrations of DNA. We studied the plasma concentrations of cfBRAF(V600E) by ddPCR in patients with melanoma during therapy with BRAF inhibitors. METHODS: Plasma concentrations of cfBRAF(V600E) were measured in 8 controls and 20 patients with advanced melanoma having the BRAF(V600E) mutation during treatment with BRAF inhibitors at baseline, first month, best response, and progression. RESULTS: The BRAF(V600E) mutation was detected by ddPCR even at a fractional abundance of 0.005% in the wild-type gene. Agreement between tumor tissue BRAF(V600E) and plasma cfBRAF(V600E) was 84.3%. Baseline cfBRAF(V600E) correlated with tumor burden (r = 0.742, P < 0.001). cfBRAF(V600E) concentrations decreased significantly at the first month of therapy (basal median, 216 copies/mL; Q1-Q3, 27-647 copies/mL; first response median, 0 copies/mL; Q1-Q3, 0-49 copies/mL; P < 0.01) and at the moment of best response (median, 0 copies/mL; Q1-Q3, 0-33 copies/mL; P < 0.01). At progression, there was a significant increase in the concentration of cfBRAF(V600E) compared with best response (median, 115 copies/mL; Q1-Q3, 3-707 copies/mL; P = 0.013). Lower concentrations of basal cfBRAF(V600E) were significantly associated with longer overall survival and progression-free survival (27.7 months and 9 months, respectively) than higher basal concentrations (8.6 months and 3 months, P < 0.001 and P = 0.024, respectively). CONCLUSIONS: cfBRAF(V600E) quantification in plasma by ddPCR is useful as a follow-up to treatment response in patients with advanced melanoma.
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: Prior, Celia; Pérez, José Luis; Garcia-Donas J; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 9  Nº 1  2014 
Purpose: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance. Methods: We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance. Results: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance. Conclusions: We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.
Autores: Alfaro, Carlos; et al.
ISSN 1078-0432  Vol. 20  Nº 22  2014  págs. 5697-5707
IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance.
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: Pastrana, Juan Carlos; Campo, Arantza; González, Álvaro; et al.
ISSN 0014-2565  Vol. 214  Nº Esp. Congr.  2014  págs. 1185
Objetivos: Las pruebas ECOE (Evaluación clínica objetiva y estructurada) permiten conocer el nivel de adquisición de competencias clínicas en la formación médica. El proyecto de troncalidad plantea la conveniencia de valorar el grado de adquisición de estas competencias tras los primeros años de especialización. El objetivo de nuestro estudio, fue valorar la viabilidad y resultados de un ECOE sobre competencias clínicas troncales básicas, realizado en residentes de segundo año de especialidades médicas.
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: González, Álvaro; Rebmann, V.; LeMaoult, J.; et al.
ISSN 1040-8363  Vol. 49  Nº 3  2012  págs. 63 - 84
Autores: Alfaro, Carlos; Pérez, José Luis; et al.
Revista: The Journal of Immunology
ISSN 0022-1767  Vol. 187  Nº 11  2011  págs. 6130 - 6142
Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-alpha, TNF-alpha, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-gamma-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.
Autores: Alfaro, Carlos; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 6  Nº 3  2011  págs. e17922
IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.
Autores: González, Álvaro; Alegre, Estíbaliz; et al.
Revista: Clinical Chemistry (Baltimore)
ISSN 0009-9147  Vol. 57  Nº 7  2011  págs. 1013 - 1022
Autores: Alfaro, Carlos; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 6  Nº 12  2011  págs. e229300
Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d)) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d) PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b) DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d)) are coinjected in the footpad of mice with autologous DC (H-2(b)). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.
Autores: Suárez Fuentetaja, N.; Alfaro Alegría, C.; Dubrot Armendáriz, J.; et al.
Revista: International Journal of Cancer (Print)
ISSN 0020-7136  Vol. 129  Nº 2  2011  págs. 374 - 386
The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2R¿(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion
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: Alegre, Estíbaliz; HoWangYin, Kiave-Yune; Favier, Benoit; et al.
Revista: Cell Research
ISSN 1001-0602  Vol. 20  Nº 11  2010  págs. 1239 - 1251
Autores: Izaguirre-Ascargorta, M. ; Fernández, Sara; Mugueta, Carmen; et al.
Libro:  Principios de Bioquímica Clínica y Patología Molecular
Vol. 3  2019  págs. 33-40
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: González, Álvaro; García-del-Barrio, MA; Arbea, Leire; et al.
Libro:  Guía inmunotoxicidad: diagnóstico y manejo de los efectos secundarios asociados a inmunoterapia en oncología
2019  págs. 204 - 210
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: 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: 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;
Libro:  Fundamentos de Nutrición y Dietética. Bases metodológicas y aplicaciones
2011  págs. 257-261
Autores: González, Álvaro, (Editor)
Nueva edición de la obra en el campo de la bioquímica clínica que año tras año se está convirtiendo en una herramienta fundamental para la toma de decisiones del clínico, ya que gran parte de ellas se basan en los datos proporcionados por el laboratorio que necesitan por una interpretación adecuada teniendo en cuenta los diferentes factores preanalíticos y analíticos que puedan influir en ellos. Uno de las novedades más importantes de la bioquímica clínica es que ha ido incorporando progresivamente técnicas propias de la biología molecular y la proteómica.
Autores: González, Álvaro;
2ª edición 2014
Autores: González, Álvaro;