Estudio del papel en la resistencia a antiangiogénicos en cáncer renal de células claras (CRCC) de los polimorfismos de nucleótido único (SNPs)

Los polimorfismos de nucleótido único son pequeñas variaciones en la secuencia de nuestros genes que, aun careciendo de significado patológico, son reponsables de parte de las diferencias fenotípicas interindividuales. Estas variaciones sí pueden implicar cambios en la actividad de las proteínas resultantes que, aunque pasen desapercibidas en la vida cotidiana, desembocarían en importantes diferencias en situaciones de estrés, como por ejemplo la administración de fármacos con una estrecha ventana terapéutica. Dado que los antiangiogénicos actúan inhibiendo el crecimiento del endotelio vascular, constituido por células sanas carentes de mutaciones, los polimorfismos representan un mecanismo plausible de resistencia a dichos fármacos. En la presente tesis se recogen los resultados de una intensa línea de investigación desarrollada por nuestro grupo en el campo de los SNPs y la predicción de eficacia y toxicidad en cáncer renal. Para ello pusimos en marcha en 2007 un estudio observacional prospectivo, denominado SUT-REN-07, en el que recogimos de forma sistemática muestras de sangre periférica para la extracción de ADN germinal y tejido tumoral parafinado de más de 100 pacientes con cáncer renal de células claras metastásico que fueran a recibir sunitinib dentro de la práctica asistencial.. Gracias a dicho estudio hemos podido completar tres grandes trabajos, que constituyen el cuerpo de esta tesis doctoral. Como principal hallazgo, hemos identificado dos SNPs en el gen VEGFR3 asociados a resistencia a sunitinib y un tercero, en el enzima CYP3A5, asociado a un mayor riesgo de reducción de dosis. También estudiamos los niveles de expresión proteica, determinados por inmunohistoquímica, de diferentes genes relacionados con la hipoxia y la proteína VEGFR3. Interesantemente, la baja expresión de VEGFR3 se asoció con la presencia de los SNPs de resistencia previamente mencionados. Por ultimo, conseguimos validar en nuestra serie, los hallazgos realizados por un grupo independiente en los que determinados SNPs en el gen IL-8 condicionaban también resistencia a antiangiogénicos

LectoTEA: inicios de un método informatizado de lectoescritura en alumnos con trastornos del espectro del autismo

El proyecto se enmarca en una colaboración entre cuatro grupos de trabajo: la asociación Autismo Sevilla, un Centro de Educación Infantil y Primaria de la ciudad de Sevilla, la Escuela de Ingeniería Informática de la Universidad de Sevilla, y la Facultad de Psicología de la misma Universidad, estas últimas implicadas en el proyecto Sinergia de trabajos de fin de grado. Objetivos: 1. Diseñar una aplicación informática, basada en la estructura básica del método de Ventoso (2003), que sea adecuada a las características y capacidades de estos usuarios y que ofrezca un primer nivel de enseñanza de la lectura. Por tanto se centra en el método global de palabras completas y la asociación de estas a una imagen. 2. Realizar un posterior pilotaje del prototipo diseñado en una pequeña muestra de niños afectados con TEA, analizando la adecuación de la aplicación

Experience with Sunitinib in metastatic renal cell carcinoma (mRCC) patients: pooled analysis from 3 Spanish observational prospective studies

[Abstract] Background: A pivotal, randomized, phase III trial demonstrated a statistically significant superiority of sunitinib over interferon-α in metastatic renal cell carcinoma (mRCC) patients. Objective: To evaluate the effectiveness and safety of sunitinib in patients with advanced or mRCC in routine clinical practice. Methods: Retrospective pooled analysis of clinical data from three observational and prospective studies carried out between 2007 and 2011 in 33 Spanish hospitals. Tumor response, Progression-free survival (PFS) and overall survival (OS), and main sunitinib-related toxicities were registered. Results: 224 patients were analyzed. Median PFS 10.6 months (95% CI: 9.02–12.25), median OS 21.9 months (95% CI: 17.2–26.6). Objective response rate (ORR) 43.8% (95% CI: 36.8–50.7). Median time to PR was 3.8 months (95% CI: 3.86–5.99) and to CR 8.2 months (95% CI: 4.75–9.77). The most common ≥ grade-3 AEs were asthenia/fatigue (18.7%), hand-foot syndrome (6.2%), hypertension (5.8%) and neutropenia (4.8%). Hand-foot syndrome, diarrhea and mucositis were confirmed as independent predictors for PFS and/or OS in a multivariate analysis (p < 0.05) Conclusions: Outcomes with sunitinib in daily clinical practice resemble those obtained in clinical trials. Long-term benefit with sunitinib is possible in advanced RCC patients but the appropriate management of toxicities is mandatory to enable patients to remain on treatment

The NER-related gene GTF2H5 predicts survival in high-grade serous ovarian cancer patients

We aimed to evaluate the prognostic and predictive value of the nucleotide excision repair-related gene GTF2H5, which is localized at the 6q24.2-26 deletion previously reported by our group to predict longer survival of high-grade serous ovarian cancer patients. In order to test if protein levels of GTF2H5 are associated with patients' outcome, we performed GTF2H5 immunohistochemical staining in 139 high-grade serous ovarian carcinomas included in tissue microarrays. Upon stratification of cases into high- and low-GTF2H5 staining categories (> and ≤ median staining, respectively) Kaplan-Meier and log-rank test were used to estimate patients' survival and assess statistical differences. We also evaluated the association of GTF2H5 with survival at the transcriptional level by using the on-line Kaplan-Meier plotter tool, which includes gene expression and survival data of 855 high-grade serous ovarian cancer patients from 13 different datasets. Finally, we determined whether stable short hairpin RNA-mediated GTF2H5 downregulation modulates cisplatin sensitivity in the SKOV3 and COV504 cell lines by using cytotoxicity assays. Low expression of GTF2H5 was associated with longer 5-year survival of patients at the protein (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) in high-grade serous ovarian cancer patients. We confirmed the association with 5-year overall survival (HR, 0.55; 95% CI, 0.38 to 0.78; p=0.0007) and also found an association with progression-free survival (HR, 0.72; 95% CI, 0.54 to 0.96; p=0.026) in a homogenous group of 388 high-stage (stages III-IV using the International Federation of Gynecology and Obstetrics staging system), optimally debulked high-grade serous ovarian cancer patients. GTF2H5- silencing induced a decrease of the half maximal inhibitory concentration upon cisplatin treatment in GTF2H5 -silenced ovarian cancer cells. Low levels of GTF2H5 are associated with enhanced prognosis in high-grade serous ovarian cancer patients and may contribute to cisplatin sensitization

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE).

PURPOSE: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. EXPERIMENTAL DESIGN: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. RESULTS: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. CONCLUSIONS: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications.See related commentary by McMullen et al., p. 5271.Core funding for this project was provided by the National Institutes of Health (R01-CA172404, PI: S.J. Ramus; and R01-CA168758, PIs: J.A. Doherty and M.A.Rossing), the Canadian Institutes for Health Research (Proof-of-Principle I program, PIs: D.G.Huntsman and M.S. Anglesio), the United States Department of Defense Ovarian Cancer Research Program (OC110433, PI: D.D. Bowtell). A. Talhouk is funded through a Michael Smith Foundation for Health Research Scholar Award. M.S. Anglesio is funded through a Michael Smith Foundation for Health Research Scholar Award and the Janet D. Cottrelle Foundation Scholars program managed by the BC Cancer Foundation. J. George was partially supported by the NIH/National Cancer Institute award number P30CA034196. C. Wang was a Career Enhancement Awardee of the Mayo Clinic SPORE in Ovarian Cancer (P50 CA136393). D.G. Huntsman receives support from the Dr. Chew Wei Memorial Professorship in Gynecologic Oncology, and the Canada Research Chairs program (Research Chair in Molecular and Genomic Pathology). M. Widschwendter receives funding from the European Union’s Horizon 2020 European Research Council Programme, H2020 BRCA-ERC under Grant Agreement No. 742432 as well as the charity, The Eve Appeal (https://eveappeal.org.uk/), and support of the National Institute for Health Research (NIHR) and the University College London Hospitals (UCLH) Biomedical Research Centre. G.E. Konecny is supported by the Miriam and Sheldon Adelson Medical Research Foundation. B.Y. Karlan is funded by the American Cancer Society Early Detection Professorship (SIOP-06-258-01-COUN) and the National Center for Advancing Translational Sciences (NCATS), Grant UL1TR000124. H.R. Harris is 20 supported by the NIH/National Cancer Institute award number K22 CA193860. OVCARE (including the VAN study) receives support through the BC Cancer Foundation and The VGH+UBC Hospital Foundation (authors AT, BG, DGH, and MSA). The AOV study is supported by the Canadian Institutes of Health Research (MOP86727). The Gynaecological Oncology Biobank at Westmead, a member of the Australasian Biospecimen Network-Oncology group, was funded by the National Health and Medical Research Council Enabling Grants ID 310670 & ID 628903 and the Cancer Institute NSW Grants ID 12/RIG/1-17 & 15/RIG/1-16. The Australian Ovarian Cancer Study Group was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729, The Cancer Council Victoria, Queensland Cancer Fund, The Cancer Council New South Wales, The Cancer Council South Australia, The Cancer Council Tasmania and The Cancer Foundation of Western Australia (Multi-State Applications 191, 211 and 182) and the National Health and Medical Research Council of Australia (NHMRC; ID199600; ID400413 and ID400281). BriTROC-1 was funded by Ovarian Cancer Action (to IAM and JDB, grant number 006) and supported by Cancer Research UK (grant numbers A15973, A15601, A18072, A17197, A19274 and A19694) and the National Institute for Health Research Cambridge and Imperial Biomedical Research Centres. Samples from the Mayo Clinic were collected and provided with support of P50 CA136393 (E.L.G., G.L.K, S.H.K, M.E.S.)

Maintenance therapy with vinflunine plus best supportive care versus best supportive care alone in patients with advanced urothelial carcinoma with a response after first-line chemotherapy (MAJA; SOGUG 2011/02): a multicentre, randomised, controlled, open-label, phase 2 trial

BACKGROUND: Maintenance therapy improves outcomes in various tumour types, but cumulative toxic effects limit the choice of drugs. We investigated whether maintenance therapy with vinflunine would delay disease progression in patients with advanced urothelial carcinoma who had achieved disease control with first-line chemotherapy. METHODS: We did a randomised, controlled, open-label, phase 2 trial in 21 Spanish hospitals. Eligible patients had locally advanced, surgically unresectable, or metastatic transitional-cell carcinoma of the urothelial tract, adequate organ function, and disease control after four to six cycles of cisplatin and gemcitabine (carboplatin allowed after cycle four). Patients were randomly assigned (1:1) to receive vinflunine or best supportive care until disease progression. We initially used block randomisation with a block size of six. Four lists were created for the two stratification factors of starting dose of vinflunine and presence of liver metastases. After a protocol amendment, number of cisplatin and gemcitabine cycles was added as a stratification factor, and eight lists were created, still with a block size of six. Finally, we changed to a minimisation procedure to reduce the risk of imbalance between groups. Vinflunine was given every 21 days as a 20 min intravenous infusion at 320 mg/m2 or at 280 mg/m2 in patients with an Eastern Cooperative Oncology Group performance status score of 1, age 75 years or older, previous pelvic radiotherapy, or creatinine clearance lower than 60 mL/min. The primary endpoint was median progression-free survival longer than 5·3 months in the vinflunine group, assessed by modified intention to treat. Comparison of progression-free survival between treatment groups was a secondary endpoint. This trial is registered with ClinicalTrials.gov, number NCT01529411. FINDINGS: Between April 12, 2012, and Jan 29, 2015, we enrolled 88 patients, of whom 45 were assigned to receive vinflunine and 43 to receive best supportive care. One patient from the vinflunine group was lost to follow-up immediately after randomisation and was excluded from the analyses. One patient in the best supportive care group became ineligible for the study and did not receive treatment due to a delay in enrolment, but was included in the intention-to-treat efficacy analysis. After a median follow-up of 15·6 months (IQR 8·5-26·0), 29 (66%) of 44 patients in the vinflunine group had disease progression and 24 (55%) had died, compared with 36 (84%) of 43 patients with disease progression and 32 (74%) deaths in the best supportive care group. Median progression-free survival was 6·5 months (95% CI 2·0-11·1) in the vinflunine group and 4·2 months (2·1-6·3) in the best supportive care group (hazard ratio 0·59, 95% CI 0·37-0·96, p=0·031). The most common grade 3 or 4 adverse events were neutropenia (eight [18%] of 44 in the vinflunine group vs none of 42 in the best supportive care group), asthenia or fatigue (seven [16%] vs one [2%]), and constipation (six [14%] vs none). 18 serious adverse events were reported in the vinflunine group and 14 in the best supportive care group. One patient in the vinflunine group died from pneumonia that was deemed to be treatment related. INTERPRETATION: In patients with disease control after first-line chemotherapy, progression-free survival exceeded the acceptable threshold with vinflunine maintenance therapy. Moreover, progression-free survival was longer with vinflunine maintenance therapy than with best supportive care. Vinflunine maintenance had an acceptable safety profile. Further studies of the role of vinflunine are warranted

Dissecting the polyhydroxyalkanoate-binding domain of the PhaF phasin: Rational design of a minimized affinity tag

ACKNOWLEDGMENTS This work was supported by the European Union’s Horizon 2020 Research and Innovation Program, grant agreement no. 633962 (P4SB), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R, BIO2016-79323-R, and RTI2018-094564-B-I00), and the Community of Madrid (P2013/MIT2807 and P2018/NMT4389). Francisco Blanco is the recipient of a predoctoral grant from the State Program for the Promotion of Talent and Its Employability in R&D&I (PRE-2018-083859) from the Spanish Ministry of Science and Innovation. This research was conducted by A. M. Aguirre in partial fulfillment of the requirements for a Ph.D. from the Universidad Complutense de Madrid, Madrid, Spain, 2019.Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro. The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP–β-galactosidase and MinP-CueO from Escherichia coli.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Ministerio de Economía y Competitividad (MINECO)Comunidad de MadridDepto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEunpu

MicroRNAs as potential predictors of extreme response to tyrosine kinase inhibitors in renal cell cancer

Background: MicroRNAs play an important role as modulators of gene expression in several biological processes and are closely related to development and cell differentiation regulation. Previous works have revealed a potential predictive role for miRNAs in different tumor types. This study aims to analyze the ability of miRNAs in segregating metastatic renal cell carcinoma patients according to their responses to tyrosine kinase inhibitors (TKIs). Methods: Extreme responders were considered in the study and were defined as those patients that either had a long-term response (LR) (progression-free survival ˃11 months) or those that were primary refractory (PR) (progression as best response). The expression of 754 miRNAs was analyzed in tumor tissue of these 2 sets of patients. Results: In a study cohort (n = 15) 4 miRNAs were significantly associated with patient response and differentially expressed in PR vs. LR (up-regulated in PR vs. LR: miR-425-5p, down-regulated in PR vs. LR: miR-139-3p, let-7d and let-7e). Further analysis in a validation cohort (n = 36) revealed similar results. Conclusion: The present data strength the potential role of miRNAs as a tool to predict treatment outcomes in patients with metastatic renal cell carcinoma treated with TKIs