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Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. 18F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with 18F-FDG-PET/CT status, higher risk and lower response to PRRT.MethodsWhole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between 18F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models.ResultsWhile no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with 18F-FDG-PET/CT in PanNETs (p-value:18F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:68Gallium-DOTATOC captation values (p-value:SSTR2 when ectopically expressed in PanNET cells (p-value:Conclusionshsa-miR-5096 well performs as a biomarker for 18F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 23-06-2017, Esta tesis tiene embargado el acceso al texto completo hasta el 23-12-2018, Polycomb protein RING1B is part of the E3 ligase that makes the core component of Polycomb Repressive Complex 1 complexes responsible for monoubiquitination of histone H2A at lysine 119. RING1B has been described as transcriptional repressor and chromatin modifier, indispensable for a proper embryonic development and lineage specification in cellular differentiation. Previous work in our laboratory has revealed additional, nontranscriptional functions for RING1B i.e in S-phase progression. Using unperturbed neural stem cells (NSCs) derived from a murine conditional model of loss-of-function of RING1B, we unveil roles of RING1B in cell proliferation, DNA damage and redox homeostasis, independently of its activity as transcriptional repressor. RING1B deficiency caused p21/CDKN1A upregulation, the principal mediator of the proliferative defect. This is mostly due to activation of DNA damage response (DDR). Upregulation of p21 followed the known ATM/P53/p21 DDR axis, as shown by restoration of proliferation rate in p21/Cdkn1a and P53 knock out NSCs, or in the presence of ATM inhibitor. Concurrent with proliferation arrest of RING1B-depleted NSCs, accumulation of doublestrand breaks (DSBs) originated, at least in part, by an increase in endogenous Reactive Oxygen Species (ROS). Consistently, treatment with antioxidant was able to decrease DNA damage and recover normal proliferation. This essential function, preventing accumulation of ROS in NSCs was fulfilled by RING1B, but not its paralog RING1A through stabilization of Polycomb cofactor BMI-1. In summary, we have identified a novel function of RING1B promoting proliferation of multipotent progenitors through the maintenance of physiological levels of oxidative stress, avoiding and managing a response to DNA damage through mechanisms independent, at least partially, of its better known as transcriptional repressor and instead assuring the stability of its own cofactor in NSCs., La proteína RING1B es el componente principal del Complejo Represor Polycomb 1 (Polycomb Repressive Complex , PRC1) y cataliza la monoubiquitinación de la lisina 119 de la histona H2A. RING1B se ha descrito como un represor transcripcional y modificador de la cromatina y se conoce para su papel clave en el correcto desarrollo del embrión y especificación celular en la diferenciación celular. Recientemente, en nuestro laboratorio se han descrito para RING1B funciones adicionales a las transcripcionales, tales como la correcta progresión por la fase S del ciclo celular. En este trabajo hemos utilizado como modelo experimental células madre neurales crecidas en condiciones de proliferación, y hemos podido desvelar la participación de RING1B en la proliferación celular, daño al DNA y control de la homeostasis oxidativa. La deficiencia de RING1B causa una mayor expresión del inhibidor del ciclo celular p21/CDKN1A que ha resultado ser el principal mediador de la parada proliferativa. La expresión de p21/CDKN1A es consecuencia de la activación de una respuesta a daño al DNA y no simplemente de la ausencia de una represión transcripcional. En la respuesta a daño a DNA participan la quinasa ATM y la proteína P53, como demuestra la recuperación de la proliferación en ausencia de p21/Cdkn1a o P53, y también por tratamiento con un inhibidor de la actividad quinasa de ATM. En este trabajo se ha demostrado como RING1B es importante para prevenir la formación de rupturas dobles de cadena del DNA que se originan, principalmente, por el incremento de Especies Reactivas del Oxigeno (Reactive Oxygen Species, ROS) en las células mutantes, y que finalmente llevan a una parada de la proliferación. En consonancia, el tratamiento con el antioxidante N-acetil-cisteína previene la formación de daño a DNA y permite rescatar la proliferación. Los datos aquí presentados sugieren que el papel de RING1B, y no de RING1A, se desarrolla garantizando la estabilidad de su co-factor BMI-1, ya descrito como regulador de la homeostasis oxidativa. En conjunto, en este trabajo se han identificado nuevas funciones de RING1B en prevenir el estrés oxidativo y la consecuente activación de una respuesta de daño al ADN de manera independiente de su función como represor transcripcional, y que implica la estabilización de su co-factor BMI-1 en células madre neurales., El desarrollo del trabajo ha sido posible gracias a la financiación concedida por el Campus Excelencia Internacional-Universidad Autónoma de Madrid (CEI-UAM) a través de una beca de Formación de Personal Investigador (Resolución del Programa de Posgrado en Biociencias Moleculares de 26 de septiembre de 2012), así como a través de la Comunidad Autónoma de Madrid gracias al proyecto S2010/BMD-2470 del programa ONCOCYCLE y al proyecto SAF2013-47997-P (MINECO).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. One open question is whether genetics could influence the severity of symptoms. Considering the limited data on cancer patients, we analyzed public data repositories limited to investigate angiotensin-converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) expressions and genetic variants to identify the basis of individual susceptibility to SARS-CoV-2. Gene expression and variant data were retrieved from Tissue Cancer Genome Atlas, Genotype-Tissue Expression, and gnomAD. Differences in gene expression were tested with Mann-Whitney U-test. Allele frequencies of germline variants were explored in different ethnicities, with a special focus on ACE2 variants located in the binding site to SARS-CoV-2 spike protein. The analysis of ACE2 and TMPRSS2 expressions in healthy tissues showed a higher expression in the age class 20 to 59 years (false discovery rate [FDR] < 0.0001) regardless of gender. ACE2 and TMPRSS2 were more expressed in tumors from males than females (both FDR < 0.0001) and, opposite to the regulation in tissues from healthy individuals, more expressed in elderly patients (FDR = 0.005; FDR < 0.0001, respectively). ACE2 and TMPRSS2 expressions were higher in cancers of elderly patients compared with healthy individuals (FDR < 0.0001). Variants were present at low frequency (range 0% to 3%) and among those with the highest frequency, the variant S19P belongs to the SARS-CoV-2 spike protein binding site and it was exclusively present in Africans with a frequency of 0.2%. The mechanisms of ACE2 and TMPRSS2 regulation could be targeted for preventive and therapeutic purposes in the whole population and especially in cancer patients. Further studies are needed to show a direct correlation of ACE2 and TMPRSS2 expressions in cancer patients and the incidence of COVID-19.

The interplay between the immune system and the pleural mesothelium is crucial both for the development of malignant pleural mesothelioma (MPM) and for the response of MPM patients to therapy. MPM is heavily infiltrated by several immune cell types which affect the progression of the disease. The presence of organized tertiary lymphoid structures (TLSs) witness the attempt to fight the disease in situ by adaptive immunity which is often suppressed by tumor expressed factors. In rare patients physiological, pharmacological or vaccine-induced immune response is efficient, rendering their plasma a valuable resource of anti-tumor immune cells and molecules. Of particular interest are human antibodies targeting antigens at the tumor cell surface. Here we review current knowledge regarding MPM immune infiltration, MPM immunotherapy and the harnessing of this response to identify novel biologics as biomarkers and therapeutics through innovative screening strategies.

T cells are involved in the early identification and clearance of viral infections and also support the development of antibodies by B cells. This central role for T cells makes them a desirable target for assessing the immune response to SARS-CoV-2 infection. Here, we combined two high-throughput immune profiling methods to create a quantitative picture of the T-cell response to SARS-CoV-2. First, at the individual level, we deeply characterized 3 acutely infected and 58 recovered COVID-19 subjects by experimentally mapping their CD8 T-cell response through antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I presented viral peptides (class II data in a forthcoming study). Then, at the population level, we performed T-cell repertoire sequencing on 1,815 samples (from 1,521 COVID-19 subjects) as well as 3,500 controls to identify shared "public" T-cell receptors (TCRs) associated with SARS-CoV-2 infection from both CD8 and CD4 T cells. Collectively, our data reveal that CD8 T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the T-cell response to SARS-CoV-2 peaks about one to two weeks after infection and is detectable for at least several months after recovery. As an application of these data, we trained a classifier to diagnose SARS-CoV-2 infection based solely on TCR sequencing from blood samples, and observed, at 99.8% specificity, high early sensitivity soon after diagnosis (Day 3-7 = 85.1% [95% CI = 79.9-89.7]; Day 8-14 = 94.8% [90.7-98.4]) as well as lasting sensitivity after recovery (Day 29+/convalescent = 95.4% [92.1-98.3]). These results demonstrate an approach to reliably assess the adaptive immune response both soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points. This blood-based molecular approach to characterizing the cellular immune response has applications in clinical diagnostics as well as in vaccine development and monitoring.