Browsing by Author "Guerrero Setas, David"

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    PublicationOpen Access
    Absence of nuclear p16 is a diagnostic and independent prognostic biomarker in squamous cell carcinoma of the cervix
    (MDPI, 2020) Mendaza Lainez, Saioa; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Zudaire, Tamara; Guarch, Rosa; Guerrero Setas, David; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix –SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.
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    PublicationOpen Access
    Análisis del valor clínico de nuevos marcadores moleculares e inmunohistoquímicos según el subtipo de expresión del cáncer de colon
    (2024) Fernández de los Reyes, Irene; Gómez Dorronsoro, María Luisa; Guerrero Setas, David; Ciencias de la Salud; Osasun Zientziak
    La presente tesis doctoral, ha pretendido identificar biomarcadores pronósticos aplicables en la práctica clínica en CC en estadio II-III, mediante el estudio de metilación aberrante en los cuatro genes diferencialmente expresados en CMS4 y utilizados como marcadores subrogados: CDX2, HTR2B, FRMD6 y ZEB1. Para ello, se han reclutado un total de 144 pacientes con CC esporádico en estadio II-III. En primer lugar, se ha realizado la clasificación molecular subrogada por inmunohistoquímica de los pacientes a partir de TMAs (microarrays de tejidos), sobre los que se han realizado las técnicas inmunohistoquímicas (IHQ) MLH1, MSH2, MSH6, PMS2, CDX2, HTR2B, FRMD6 y ZEB1. Se identificaron 18 pacientes con alteración de proteínas reparadoras (12,5% CMS-IHQ1), 117 pacientes (80,6%) se clasificaron en el grupo CMS-IHQ2/3 y 9 pacientes (6,3%) en el subtipo CMS-IHQ4. El valor clínico de los subtipos valorado mediante la evaluación de supervivencia mostró que CMS-IHQ1 presentaba el mejor pronóstico, siendo significativamente distinto a CMS-IHQ4, grupo con peor pronóstico, observando un pronóstico intermedio en el grupo CMSIHQ2/3. De esta manera, pueden detectarse subtipos subrogados con distinto pronóstico (CMSIHQ1vs CMS-IHQ4). En segundo lugar, se realizó la pirosecuenciación de los genes subrogados CDX2, FRMD6 y ZEB1, a partir de ADN modificado con bisulfito de 144 bloques de tejido tumoral fijado en formol e incluido en parafina (FFPE) y de 40 bloques de tejido no tumoral FFPE control. No fue posible diseñar cebadores para analizar el patrón de metilación del promotor del gen HTR2B debido a la elevada densidad de CpGs. En tercer lugar, se analizó la expresión de ZEB1 mediante qRT-PCR a partir de ARN de 20 bloques de tejido tumoral FFPE y de 20 bloques de tejido no tumoral FFPE control, en la que se observó una diferencia de expresión estadísticamente significativa, observándose en los casos hipermetilados una menor expresión de ZEB1 respecto a los casos no metilados (p=0,035). Esta expresión no tuvo concordancia con el estudio IHQ de ZEB1 en el TMA, ni en el estudio IHQ de cortes completos del tumor. Posteriormente, se realizó la pirosecuenciación del gen ZEB1 a partir de ADN modificado con bisulfito de las líneas celulares HCT116, HT29, LoVo, RKO, SW480, SW837, T84, DLD1 y SW620. El gen ZEB1 se encontró hipermetilado en las líneas celulares DLD1, HCT116, HT29 y SW837 (94.5%, 80,5%, 96 % y 49%, respectivamente). Por el contrario, ZEB1 estaba completamente desmetilado en las células de la línea RKO (0%) y con baja metilación en las células LoVo, SW480, SW620, y T84 (3%, 5,5%, 2% y 4,5% respectivamente). La presencia o ausencia de hipermetilación del promotor de ZEB1 aporta un nuevo dato a las características propias de cada línea celular. Por último, se realizó el estudio de expresión de ZEB1 mediante qRT-PCR en las líneas con mayor nivel de metilación antes y después realizar un tratamiento de desmetilación con el agente desmetilante 5-aza-2ʹ-desoxicitidina (AZA), con el inhibidor de histonas desacetilasa tricostatina A (TSA) y con una combinación de ambos (AZA+TSA). El efecto del tratamiento con AZA, TSA o AZA+TSA sobre los niveles de expresión de ZEB1 en las líneas HCT116 y HT29 (ambas con elevado porcentaje de metilación y alta tasa de crecimiento), reveló una mayor reexpresión del gen ZEB1 en la línea celular HCT116 tratada con AZA (p < 0,0001) y en la línea celular HT29 tratada con AZA-TSA (p = 0,003) de forma transitoria.
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    PublicationOpen Access
    Approaching the epigenome of triple negative breast cancer to identify new biomarkers
    (2020) Mendaza Lainez, Saioa; Guerrero Setas, David; Martín Sánchez, Esperanza; Ciencias de la Salud; Osasun Zientziak
    El cáncer de mama (CM) es la neoplasia más frecuente en todo el mundo y la primera causa de muerte por cáncer en mujeres. El cáncer de mama triple negativo (CMTN) representa el 10-20% de todos los CM diagnosticados y es el subgrupo más agresivo puesto que carece tratamiento dirigido. Por lo tanto, el descubrimiento de nuevos biomarcadores y dianas terapéuticas es necesario. Dado que las alteraciones epigenéticas están involucradas en la tumorigénesis, la caracterización de la metilación del DNA y la acetilación de histonas puede ser útil para la identificación de nuevas firmas potencialmente diagnósticas o pronósticas, así como alteraciones destinatarias de fármacos dirigidos. En la presente tesis, se caracterizó la metilación de DNA del genoma completo de 8 CMTN y seis tejidos mamarios no neoplásicos mediante Illumina Human Methylation 450K BeadChip. Estos datos se analizaron desde dos enfoques bioinformáticos diferentes. Concluimos que el patrón epigenético (metiloma de DNA y acetiloma de histonas) está alterado en CMTN respecto a tejido mamario no neoplásico. Así como que la hipometilación de ADAM12 y la acetilación de H4K5 son potenciales biomarcadores de peor pronóstico y que ADAM12 es una potencial diana terapéutica en CMTN. También sugerimos la utilidad diagnóstica de una nueva firma basada en metilación de DNA y las diferencias entre los genes regulados por la acetilación de H4K6ac según la naturaleza de las líneas (no neoplásicas o TNBC).
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    PublicationOpen Access
    Búsqueda de nuevos biomarcadores epigenéticos en cáncer de mama triple negativo
    (2016) Ulazia Garmendia, Ane; Barajas Vélez, Miguel Ángel; Guerrero Setas, David; Martín Sánchez, Esperanza; Facultad de Ciencias de la Salud; Osasun Zientzien Fakultatea
    Introducción El estudio de las alteraciones epigenéticas contribuye a la caracterización del subtipo triple negativo (TN) de cáncer de mama (CM). Objetivos Identificar genes diferencialmente metilados entre CM TN y tejido no neoplásico. Material y Métodos El estado de metilación de DLEU7 y NKAPL se estudió mediante pirosecuenciación en tumores TN, su tejido adyacente, tejido no neoplásico y líneas celulares. La expresión proteica se analizó por IHQ en tejidos y por PCR a tiempo real y Western Blot en líneas celulares. Resultados Se encontraron niveles más elevados de metilación de DLEU7 y NKAPL en tejido tumoral comparado con tejido no neoplásico. Esta alteración epigenética, también se encuentra en el tejido adyacente al tumor, estando correlacionada con el grado de expresión proteica. Hay una tendencia hacia un peor pronóstico de las pacientes con hipermetilación. Conclusiones Esta es la primera descripción sobre la hipermetilación de DLEU7 y la NKAPL en CM TN.
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    PublicationOpen Access
    CDH22 hypermethylation is an independent prognostic biomarker in breast cancer
    (BioMed Central, 2017) Martín Sánchez, Esperanza; Mendaza Lainez, Saioa; Ulazia Garmendia, Ane; Monreal Santesteban, Iñaki; Córdoba Iturriagagoitia, Alicia; Vicente García, Francisco; Blanco Luquin, Idoia; Cruz, Susana de la; Aramendia, Ana; Guerrero Setas, David; Ciencias de la Salud; Osasun Zientziak
    Background: Cadherin-like protein 22 (CDH22) is a transmembrane glycoprotein involved in cell-cell adhesion and metastasis. Its role in cancer is controversial because it has been described as being upregulated in colorectal cancer, whereas it is downregulated in metastatic melanoma. However, its status in breast cancer (BC) is unknown. The purpose of our study was to determine the molecular status and clinical value of CDH22 in BC. Results: We observed by immunohistochemistry that the level of CDH22 expression was lower in BC tissues than in their matched adjacent-to-tumour and non-neoplastic tissues from reduction mammoplasties. Since epigenetic alteration is one of the main causes of gene silencing, we analysed the hypermethylation of 3 CpG sites in the CDH22 promoter by pyrosequencing in a series of 142 infiltrating duct BC cases. CDH22 was found to be hypermethylated in tumoral tissues relative to non-neoplastic mammary tissues. Importantly, this epigenetic alteration was already present in adjacent-to-tumour tissues, although to a lesser extent than in tumoral samples. Furthermore, CDH22 gene regulation was dynamically modulated in vitro by epigenetic drugs. Interestingly, CDH22 hypermethylation in all 3 CpG sites simultaneously, but not expression, was significantly associated with shorter progression-free survival (p = 0.015) and overall survival (p = 0.021) in our patient series. Importantly, CDH22 hypermethylation was an independent factor that predicts poor progression-free survival regardless of age and stage (p = 0.006). Conclusions: Our results are the first evidence that CDH22 is hypermethylated in BC and that this alteration is an independent prognostic factor in BC. Thus, CDH22 hypermethylation could be a potential biomarker of poor prognosis in BC.
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    PublicationOpen Access
    A DNA methylation-based gene signature can predict triple-negative breast cancer diagnosis
    (MDPI, 2021) Mendaza Lainez, Saioa; Guerrero Setas, David; Monreal Santesteban, Iñaki; Ulazia Garmendia, Ane; Córdoba Iturriagagoitia, Alicia; Cruz, Susana de la; Martín Sánchez, Esperanza; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype and lacks targeted treatment. It is diagnosed by the absence of immunohistochemical expression of several biomarkers, but this method still displays some interlaboratory variability. DNA methylome aberrations are common in BC, thereby methylation profiling could provide the identification of accurate TNBC diagnosis biomarkers. Here, we generated a signature of differentially methylated probes with class prediction ability between 5 non-neoplastic breast and 7 TNBC tissues (error rate = 0.083). The robustness of this signature was corroborated in larger cohorts of additional 58 non-neoplastic breast, 93 TNBC, and 150 BC samples from the Gene Expression Omnibus repository, where it yielded an error rate of 0.006. Furthermore, we validated by pyrosequencing the hypo-methylation of three out of 34 selected probes (FLJ43663, PBX Homeobox 1 (PBX1), and RAS P21 protein activator 3 (RASA3) in 51 TNBC, even at early stages of the disease. Finally, we found significantly lower methylation levels of FLJ43663 in cell free-DNA from the plasma of six TNBC patients than in 15 healthy donors. In conclusion, we report a novel DNA methylation signature with potential predictive value for TNBC diagnosis.
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    PublicationOpen Access
    Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures
    (BMJ Publishing Group, 2021) Arechederra, María; Rullán Iriarte, María; Amat Villegas, Irene; Oyón, Daniel; Zabalza, Lucía; Elizalde, María; Latasa, Maria Ujue; Mercado Gutiérrez, María R.; Ruiz-Clavijo, Daniel; Saldaña, Cristina; Fernández-Urién Sainz, Ignacio; Carrascosa, Juan; Jusué, Vanesa; Guerrero Setas, David; Zazpe, Cruz; González Borja, Iranzu; Sangro, Bruno; Herranz, José M.; Purroy, Ana; Gil, Isabel; Nelson, Leonard J.; Vila, Juan J.; Krawczyk, Marcin; Zieniewicz, Krzysztof; Patkowski, Waldemar; Milkiewicz, Piotr; Cubero, Francisco Javier; Alkorta Aranburu, Gorka; Fernández-Barrena, Maite G.; Urman Fernández, Jesús María; Berasain, Carmen; Ávila, Matías A.; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
    Objective: despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). Design: a prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. Results: an initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. Conclusion: implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.
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    PublicationOpen Access
    A novel prognostic biomarker panel for early‐stage colon carcinoma
    (MDPI, 2021) Azcue Sanromán, Pablo; Guerrero Setas, David; Encío Martínez, Ignacio; Ibáñez Beroiz, Berta; Mercado Gutiérrez, María R.; Vera García, Ruth; Gómez Dorronsoro, María Luisa; Ciencias de la Salud; Osasun Zientziak
    Molecular characterization of colorectal cancer has helped us understand better the biology of the disease. However, previous efforts have yet to provide significant clinical value in order to be integrated into clinical practice for patients with early‐stage colon cancer (CC). The purpose of this study was to assess PD‐L1, GLUT‐1, e‐cadherin, MUC2, CDX2, and microsatellite instability (dMMR) and to propose a risk‐panel with prognostic capabilities. Biomarkers were immunohistochemically assessed through tissue microarrays in a cohort of 144 patients with stage II/III colon cancer. A biomarker panel consisting of PD‐L1, GLUT‐1, dMMR, and potentially CDX2 was constructed that divided patients into low, medium, and high risk of overall survival or disease-free survival (DFS) in equally sized groups. Compared with low‐risk patients, medium‐risk patients have almost twice the risk of death (HR = 2.10 (0.99–4.46), p = 0.054), while high‐risk patients have almost four times the risk (HR = 3.79 (1.77–8.11), p = 0.001). The multivariate goodness of fit was 0.756 and was correlated with Kaplan–Meier curves (p = 0.002). Consistent results were found for DFS. This study provides a critical basis for the future development of an immunohistochemical assessment capable of discerning early‐stage CC patients as a function of their prognosis. This tool may aid with treatment personalization in daily clinical practice and improve survival outcomes.
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    PublicationOpen Access
    PD-L1 as a prognostic factor in early-stage colon carcinoma within the immunohistochemical molecular subtype classification
    (MDPI, 2021) Azcue Sanromán, Pablo; Encío Martínez, Ignacio; Guerrero Setas, David; Suárez Alecha, Javier; Galbete Jiménez, Arkaitz; Mercado Gutiérrez, María R.; Vera García, Ruth; Gómez Dorronsoro, María Luisa; Ciencias de la Salud; Osasun Zientziak
    Colorectal cancer (CRC) is a very heterogeneous disease. Efforts to characterize and search for biomarkers for these patients are currently ongoing in the hope of establishing a more targeted therapeutic approach. The role of PD-1 ligand (PD-L1) expression as a biomarker has not yet been fully elucidated. The Consensus Molecular Subtype classification has been delineated, but although already acknowledged in the most recent international guidelines, it has yet to be implemented in clinical practice. We investigate PD-L1 expression as a biomarker of prognosis in the early-stage setting and integrate it with the Consensus Molecular Subtype (CMS), in an effort to differentiate those patients with a worse prognosis who could potentially benefit from an early, more aggressive treatment. Our results suggest PD-L1 as an independent prognostic factor in early stage setting when assessed by immunohistochemistry. Additionally, PD-L1 expression appears to be a viable biomarker to differentiate patients in the CMS (CMS2/CMS3) who lack a clear prognosis.
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    PublicationOpen Access
    ZEB1 hypermethylation is associated with better prognosis in patients with colon cancer
    (BMC, 2023) Fernández de los Reyes, Irene; Gómez Dorronsoro, María Luisa; Monreal Santesteban, Iñaki; Fernández-Fernández, Agustín; Fraga, Mario; Azcue Sanromán, Pablo; Alonso, Laura; Fernández-Marlasca, Beatriz; Suárez Alecha, Javier; Córdoba Iturriagagoitia, Alicia; Guerrero Setas, David; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    Background: Colon cancer (CC) is a heterogeneous disease that is categorized into four Consensus Molecular Subtypes (CMS) according to gene expression. Patients with loco-regional CC (stages II/III) lack prognostic factors, making it essential to analyze new molecular markers that can delineate more aggressive tumors. Aberrant methylation of genes that are essential in crucial mechanisms such as epithelial mesenchymal transition (EMT) contributes to tumor progression in CC. We evaluate the presence of hyper- and hypomethylation in subrogate IHC markers used for CMS classification (CDX2, FRMD6, HTR2B, ZEB1) of 144 stage II/III patients and CC cell lines by pyrosequencing. ZEB1 expression was also studied in control and shRNA-silenced CC cell lines and in paired normal tissue/tumors by quantitative PCR. The pattern of ZEB1 staining was also analyzed in methylated/unmethylated tumors by immunohistochemistry. Results: We describe for the first time the hypermethylation of ZEB1 gene and the hypomethylation of the FRMD6 gene in 32.6% and 50.9% of tumors, respectively. Additionally, we confirm the ZEB1 re-expression by epigenetic drugs in methylated cell lines. ZEB1 hypermethylation was more frequent in CMS1 patients and, more importantly, was a good prognostic factor related to disease-free survival (p = 0.015) and overall survival (p = 0.006) in our patient series, independently of other significant clinical parameters such as patient age, stage, lymph node involvement, and blood vessel and perineural invasión. Conclusions: Aberrant methylation is present in the subrogate genes used for CMS classification. Our results are the first evidence that ZEB1 is hypermethylated in CC and that this alteration is an independent factor of good prognosis.