Search

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line–based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo. Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.

Data de publicació electrònica: 22-12-2021 Objective: to assess the economic impact of introducing biosimilars of bevacizumab for the management of cancer patients receiving systemic bevacizumab in the National Health System (SNHS) of Spain. Methods: a 3-year budget impact analysis model was adapted to estimate the cost of introducing biosimilars of bevacizumab in the SNHS for the adult population who were candidates to receive treatment with bevacizumab. Values for the estimation of the population were obtained from the literature and were validated by an expert panel. In this analysis only pharmaceutical costs (€, year 2021) obtained from official databases were considered. A sensitivity analysis was performed to examine the robustness of the model. Results: the introduction of bevacizumab biosimilars would generate an annual cost saving of €11 558 268 (-5.1%) for the first year with a penetration share of biosimilars from 30.0%, €29 126 373 (-8.5%) for the second year with a share of 50.0% and €52 361 778 (-13.6%) for the third year with a share of 80.0%. The total pharmaceutical costs of the scenario without biosimilars are €227 033 352 for the first year, €342 663 209 for the second year and €385 013 076 for the third year. In contrast, the pharmaceutical costs of the scenario with bevacizumab biosimilars are €215 475 084, €313 536 836 and €332 651 297 for years 1, 2 and 3, respectively. Conclusions: the introduction of biosimilars in the Spanish Health System would generate saving costs in the pharmacological budget to boost biological drugs from the first year.

Cancer treatment involves the participation of multiple medical specialties and, as our knowledge of the disease increases, this fact becomes even more apparent. The degree of multidisciplinarity is determined by several factors, which include the severity and type of disease, the increasing diversity in the available pharmacological and non-pharmacological therapies, and the range of specialists involved in cancer therapy, such as medical oncologists, radiotherapists, gynecologists, gastroenterologists, urologists, surgeons, and pneumologists, among others. Across Europe, the situation of cancer care can be variable due to the diversity of health systems, differences in drug reimbursement, and the degree of establishment of Medical Oncology as a medical specialty in the European Union states.

Las células malignas deben adquirir un potencial de replicación ilimitado (inmortalidad celular) para la formación de tumores avanzados. La adquisición de inmortalidad celular depende de la reactivación de la telomerasa, una enzima que permite compensar el problema de replicación terminal del ADN asociado a cada división celular. En una serie de estudios presentados en esta tesis, se demuestra:1. La telomerasa es una enzima regulable en células tumorales humanas.2. Los tumores humanos expresan actividad telomerasa y su expresión se asocia a estadío tumoral, índice proliferativo y diferenciación3. La telomerasa es una diana antitumoral atractiva., Malignant cells have to acquire a limitless replicative potential (immortality) to be able to form an advanced tumor. The acquisition of cellular imortality is linked to the reactivation of telomerase, an enzyme that has the potential to overcome the DNA end replication problem that occurs during each round of cell división. In a series of studies presented in this thesis, it has been shown:1. Telomerase is a regulable enzyme in human tumor cells.2. Human tumors express telomerase activity and this expresión is asóciate to tumor stage, proliferative index and maturation.3. Telomerase is an attractive anticancer targe._

L'anàlisi molecular de l'tumor s'ha convertit en un procediment fonamental per a definir el maneig dels pacients amb càncer de pulmó. Encara que l'anàlisi de el teixit tumoral es considera la tècnica d'elecció per a l'estudi molecular, aquesta aproximació té una sèrie de limitacions, com l'absència de material tumoral suficient o la impossibilitat per capturar l'heterogeneïtat tumoral. L'ús de la biòpsia líquida podria ajudar a superar aquestes limitacions. En aquest treball analitzem la capacitat de la biòpsia líquida de detectar mutacions de EGFR o KRAS en plasma. Utilitzant la biòpsia líquida hem detectat mutacions de EGFR en un 83% dels casos (100% en casos amb disseminació extratorácica) i en un 82% dels casos KRAS mutats (96% en casos amb més d'una localització metastàtica). A més, el nostre treball demostra la capacitat de l'anàlisi de la dinàmica de la càrrega mutacional circulant de preveure la resposta o progressió amb antelació. En els casos EGFR mutats, la dinàmica de la càrrega mutacional va permetre predir la resposta en un 83% de els casos i la progressió en un 89% amb una antelació mitjana de 38 i 80 dies respectivament. En els casos amb mutacions de KRAS, la taxa de predicció de la resposta i progressió radiològiques va ser de el 93% i el 63% respectivament amb una antelació mitjana de 33 i 50 dies. D'altra banda, tant en els casos EGFR com en els KRAS mutats, la supervivència lliure de progressió va ser significativament superior en els casos en què, després del inici de tractament, les mutacions van deixar de ser detectables a ADN tumoral circulant. En resum, el nostre treball demostra, en un entorn de pràctica clínica habitual, la utilitat de la biòpsia líquida per al maneig de pacients amb càncer de pulmó. El análisis molecular del tumor se ha convertido en un procedimiento fundamental para definir el manejo de los pacientes con cáncer de pulmón. Aunque el análisis del tejido tumoral se considera la técnica de elección para el estudio molecular, esta aproximación tiene una serie de limitaciones, como la ausencia de material tumoral suficiente o la imposibilidad para capturar la heterogeneidad tumoral. El uso de la biopsia líquida podría ayudar a superar estas limitaciones. En este trabajo analizamos la capacidad de la biopsia líquida de detectar mutaciones de EGFR o KRAS en plasma. Utilizando la biopsia líquida hemos detectado mutaciones de EGFR en un 83% de los casos (100% en casos con diseminación extratorácica) y en un 82% de los casos KRAS mutados (96% en casos con más de una localización metastática). Además, nuestro trabajo demuestra la capacidad del análisis de la dinámica de la carga mutacional circulante de prever la respuesta o progresión con antelación. En los casos EGFR mutados, la dinámica de la carga mutacional permitió predecir la respuesta en un 83% de los casos y la progresión en un 89% con una antelación mediana de 38 y 80 días respectivamente. En los casos con mutaciones de KRAS, la tasa de predicción de la respuesta y progresión radiológicas fue del 93% y 63% respectivamente con una antelación mediana de 33 y 50 días. Por otro lado, tanto en los casos EGFR como en los KRAS mutados, la supervivencia libre de progresión fue significativamente superior en los casos en los que, tras el inicio de tratamiento, las mutaciones dejaron de ser detectables en ADN tumoral circulante. En resumen, nuestro trabajo demuestra, en un entorno de práctica clínica habitual, la utilidad de la biopsia líquida para el manejo de pacientes con cáncer de pulmón. Molecular profiling of the tumor has become a crucial procedure in the management of patients with lung cancer. Although the analysis of the tumor tissue is considered the gold standard for the molecular profiling, this approach has some limitations, such as the absence of the required amount of tissue or the inability to capture the tumor heterogeneity. The use of liquid biopsy may help to overcome these limitations. In this work we analyze the ability of the liquid biopsy to detect EGFR or KRAS mutations in plasma. Using liquid biopsy, we detected EGFR mutations in 83% of cases (100% in cases with extrathoracic metastases) and in 82% of mutated KRAS cases (96% in cases with more than one metastatic location). Furthermore, our work demonstrates the ability of the analysis of the circulating mutational load dynamics to predict the response or progression well in advance. In the EGFR mutated cases, the dynamics of the mutational load allowed predicting the response in 83% of the cases and the progression in 89% with a median advance of 38 and 80 days respectively. In the cases with KRAS mutations, the radiological response and progression prediction rate was 93% and 63%, respectively, with a median lead time of 33 and 50 days. On the other hand, in both EGFR and KRAS mutated cases, progression-free survival was significantly higher in cases in which, after starting treatment, the mutations became undetectable in circulating tumor DNA. In summary, our work demonstrates, in a routine clinical practice setting, the utility of liquid biopsy for the management of patients with lung cancer. Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

El cáncer de mama HER2-positivo es un subtipo muy agresivo. El desarrollo de terapias anti-diana HER2, particularmente el anticuerpo monoclonal (Mab) trastuzumab, supuso una mejora significativa en el pronóstico de esta enfermedad. De manera más reciente, otro Mab llamado pertuzumab, ha mejorado todavía más la eficiencia de trastuzumab. Aún y así, no todas las pacientes se beneficiarán de esta combinación de mAbs. Una proporción de estas pacientes no se beneficiarán de estas terapias anti-HER2, y fallecerán a causa de la presencia o el desarrollo de mecanismos de resistencia. Los tumores consisten no únicamente de una población heterogénea de células tumorales; si no también del conocido como rnicroentorno tumoral (IME por sus siglas en inglés). En los últimos años, se ha evidenciado que los fibroblastos asociados al tumor (CAFs por sus siglas en inglés) (una población estroma! muy abundante dentro del TME), directamente promueven los procesos tutnorogénicos así cotno la resistencia a los fármacos. No obstante, al inicio de este proyecto de tesis doctoral, los estudios relativos sobre el papel de los CAFs en la resistencia a la terapia anti-HER2 eran escasos. En resumen, este proyecto de tesis doctoral evidencia el papel de los fibroblastos asociado al tumor en la resistencia a la terapia anti-HER2 a través de la secreción de factores solubles que promueven la activación de mecanismos moleculares que promueven la supervivencia tumoral así como la resistencia terapéutica. HER2-positive breast cancer (BC) is an aggressive subtype of this disease. The development of anti-HER2 targeted therapies, particularly the monoclonal antibody (Mab) trastuzumab, significantly improved its otherwise poor prognosis. More recently, another Mab called pertuzumab, has further improved the efficacy of trastuzumab, yet no all patients benefit from the combination of the two Mabs. A proportion of HER2-breast cancer patients will not benefit from anti-HER2 agents, and will ultimately die as a consequence of innate or acquired drug resistance mechanisms. Tumours consist not only of heterogeneous populations of cancer cells, but also of the tumour microenvironment (TME). In recent years, increasing evidence has shown that cancer-associated fibroblasts (CAFs; an abundant stromal cell population within the TME), directly support tumorigenesis and promote therapy resistance. However, at the beginning of this PhD study, there was little published work on the role of CAFs on anti-HER2 targeted therapy resistance. The work presented in this doctoral thesis supported a role of CAFs in tumour resistance to anti-HER2 targeted therapies in HER2+ breast cancer through paracrine secretion of soluble molecules that ultimately will promote breast cancer survival and therapy resistance.

Targeted therapies against the Epidermal Growth Factor Receptor (EGFR) are useful to treat many human cancers such as non-small cell lung cancer, colorectal cancer and head and neck cancer. However, the efficacy of such treatments is always compromised by resistance. This doctoral thesis has focused in the mechanisms of acquired resistance to targeted therapies against the EGFR (such as the small tyrosine kinase inhibitors gefitinib and erlotinib, or the monoclonal antibody cetuximab) in squamous cell carcinomas. In the first part of the thesis, preclinical studies with cellular and xenograft models were developed to elucidate the molecular mechanisms of resistance; the second part of the thesis was performed in tumor samples from patients with advanced squamous cell carcinomas of the head and neck. The main finding from the preclinical analysis was that the activation of the insulin-like growth factor receptor 1 system, mainly through downregulation of insulin-like growth factor binding proteins, is responsible for the acquired resistance to anti-EGFR therapies. However, these results could not be validated in a small sample set of advanced head and neck cancer patients., Els tractaments dirigits contra el receptor del factor de creixement epidèrmic (EGFR) són útils en diversos càncers en l’home, com el càncer de pulmó de cèl·lula no petita, el càncer colorrectal o els tumors de cap i coll. Però l’eficàcia d’aquests tractaments sempre està limitada per l’aparició de resistències. Aquesta tesi doctoral s’ha centrat en investigar els mecanismes de resistència adquirida a tractaments dirigits contra l’EGFR (com els inhibidors tirosina quinasa gefitinib i erlotinib o l’anticòs monoclonal cetuximab) en carcinomes escamosos. En la primera part de la tesi s’han desenvolupat estudis preclínics amb models cel·lulars i xenoinjerts per desxifrar els mecanismes moleculars de resistència; la segona part de la tesi ha inclòs estudis en mostres de carcinomes escatosos de cap i coll de pacients amb tumors avançats. La troballa principal dels estudis preclínics ha estat que l’activació del sistema del receptor del factor de creixement semblant a la insulina, principalment a través de la disminució dels nivells de les proteïnes d’unió als factors de creixement semblants a la insulina, és la responsable de l’aparició de resistència adquirida als tractaments anti-EGFR. Posteriorment, però, aquests resultats no han estat validats en una petita cohort de pacients amb tumors avançats de cap i coll.

Trastuzumab-emtansine (T-DM1) is an antibody-cytotoxic agent (DM1) conjugated drug. DM1 delivery by trastuzumab inside the HER2 positive cells affects microtubule polymerization, cell cycle arrest and finally cell death. Although T-DM1 is approved for the treatment of HER2 positive metastatic breast cancer patients, primary and acquired resistance towards this drug is still a main challenge. Looking for the mechanisms of resistance is necessary to improve patient selection and to develop novel treatment strategies. Here, we focused on finding mechanisms of acquired resistance to T-DM1 in a panel of HER2 positive breast cancer cell lines (HCC1954, HCC1419 and SKBR3 parental vs. resistant cells) generated by an established protocol of T-DM1 exposure, increasing the concentration of T-DM1[1-4µg/mL], 3days on/3days off, for 54 days overall. We generated acquired resistant cells with different level of resistance to T-DM1 evaluated by 3, 7 and 10 days proliferation assay, using automated cell counting in SKBR3, HCC1419 and HCC1954 parental and the acquired resistant cells. Analysis of T-DM1 effects on cell cycle showed a significant induction of G2/M arrest in the parental cells, while this effect was not observed in the resistant cells. Expression/activity analysis of cyclin B1/CDK1 complex, the main apparatus involve in G2/M cell cycle arrest induction, showed a remarkable decrease in the basal level of cyclin B1 in the resistant cells. Cyclin B1 accumulation induced by T-DM1 in the parental cells was not observed in the resistant cells. CDK1 activity assay was also correlated with cyclin B1 expression, increasing following T-DM1 treatment in the parental cells, but not in the resistant cells. Functional analysis revealed that cyclin B1 knock down in the parental cells induced a significant T-DM1 resistance. Furthermore, the silencing of cdc20, a protein mainly involved in APC complex related cyclin B1 degradation, could sensitize the resistant cells to T-DM1. Finally, cyclin B1 induction by T-DM1 was confirmed in in vivo and ex vivo xenograft animal model and patients’ explants, respectively. By cyclin B1 induction pattern, we could categorize T-DM1 responsive/non-responsive in fresh breast cancer explants from HER2 positive breast cancer patients. Our results showed that T-DM1 induced G2/M cell cycle arrest in a cyclin B1/CDK1 dependent-manner. Lack of these effects appeared in acquired T-DM1 resistant cells. Besides, similar pattern in G2/M and cyclin B1 was verified in vivo and in patients explants. These data strongly suggest that induction of cyclin B1 is necessary for T-DM1 antitumor effects and emerges as a potential pharmacodynamic marker. Our finding also raises the question on what are the mechanisms leading to cyclin B1 dysregulation in resistant cells.

[eng] Prostate cancer is the second most frequently diagnosed cancer worldwide and the fifth leading cause of cancer death in men. Despite, the above mentioned incidence and mortality, the existence of a small proportion of patients who have indolent prostate cancers has led to the widely held belief that prostate cancer has low lethality. The widespread use of PSA screening in asymptomatic patients in some countries might partially explain this belief. Overdiagnosis of indolent prostate cancers with PSA screening might have diluted the real mortality rates of more aggressive and advanced prostate cancers. Epidemiological studies analyzing real prostate cancer mortality and causes of death in countries were early PSA screening is uncommon are therefore urgently needed to provide a real picture of the impact of prostate cancer on mortality. Several second-generation androgen receptor pathway inhibitors such as enzalutamide or abiraterone have been successfully tested in patients with metastatic castration-resistant prostate cancer (CRPC). Although the individual efficacy of abiraterone and enzalutamide in prostate cancer is well established, the therapeutic benefit of targeting AR signaling axis by sequential administration of these agents is not clear, since cross-resistance could occur. Similarly, while an antiandrogen withdrawal syndrome (AAWS) has been reported for almost all first-generation antiandrogens such as bicalutamide, there is no evidence of any withdrawal effect with second-generation antiandrogen enzalutamida. The main objectives of this thesis was to investigate mortality and causes of death in men with prostate cancer diagnosed in London (UK) where routine screening testing for PSA amongst asymptomatic men is low; to analyze the antitumour activity of enzalutamide in metastatic castration-resistant prostate cancer patients previously treated with abiraterone and to examine PSA levels following enzalutamide discontinuation in metastatic castration-resistant prostate cancer patients, in order to assess whether an AAWS exists with enzalutamida. As main methods, we analyzed prostate cancer mortality on 53.081 men diagnosed with prostate cancer between January 1st 1997 and December 31st 2006 extracted from death certificates from the Thames Cancer Registry (TCR) database. The TCR is a population-based epidemiological registry, covering around 12 million people resident in an area of South East England. The enzalutamida cross-resistance and potential AAWS was analyzed retrospectively in patients with metastatic CRPC treated with enzalutamide in the UK between 2012 and 2013. For the Enzalutamide Cross-Resistance study, we identified 39 patients previously treated with both abiraterone and docetaxel and subsequently treated with enzalutamida. For the Enzalutamide Withdrawal study, we identified patients with evidence of disease progression on enzalutamide for whom at least 1 available PSA post-enzalutamide discontinuation was available. 30 patients were initially identified. A subsequent updated analysis increased the patient sample to 49 patients. The main results of this thesis are that the mortality of prostate cancer remains significant, challenging the traditional belief that prostate cancer is an indolent disease and is not an important cause of death in men. We showed that prostate cancer was the most frequent cause of death in the whole study population as well as in all the subgroups, regardless of age, stage of disease and treatment received. We also showed that the antitumour activity of enzalutamide is significantly reduced when it is given to heavily pre-treated metastatic CRPC patients previously treated with abiraterone acetate. All the efficacy endpoints analyzed in our cohort were significantly poorer compared to the data reported in the AFFIRM pivotal study in abiraterone-naïve patients, including PSA response rates, radiological response rates and survival. This indicates a phenomenon of cross-resistance between these 2 hormonal drugs targeting the AR signaling pathway. Our results also showed for the first time that enzalutamide is associated with an antiandrogen withdrawal syndrome in a minority of patients (2%)., [spa] El cáncer de próstata es el segundo cáncer diagnosticado con mayor frecuencia en todo el mundo y la quinta causa de muerte por cáncer en varones. Cuando la enfermedad se halla en fase metastásica no existen tratamientos curativos y la supervivencia de los pacientes es limitada. Los objetivos principales de esta tesis eran investigar la mortalidad y las causas de muerte en varones con cáncer de próstata diagnosticado en Londres (Reino Unido), así como analizar la posible existencia de resistencia cruzada entre los inhibidores del receptor de andrógenos enzalutamida y abiraterona en pacientes con cáncer de próstata resistente a castración y la posible existencia de un síndrome de retirada del antiandrógeno con enzalutamida. Los principales resultados de la tesis son, en primer lugar, que la mortalidad debida al cáncer de próstata en nuestro estudio fue significativa, independientemente de los factores basales y que, en un entorno epidemiológico donde la detección precoz del PSA es bajo, como es el caso del Reino Unido, nuestros resultados desafían la creencia de que el cáncer de próstata no es una causa importante de muerte en varones. En segundo lugar, vimos que la actividad antitumoral de enzalutamida de nuestro estudio en paciente previamente pretratados con abiraterona es significativamente inferior a los publicados en los ensayos aleatorizados de enzalutamida en pacientes no previamente tratados con abiraterona. Estos datos sugieren una actividad antitumoral reducida de enzalutamida en los pacientes previamente tratados con abiraterona, indicando una posible resistencia cruzada entre dichos fármacos que comparten un similar mecanismo de acción. Finalmente, nuestro estudio proporcionó la primera evidencia de que enzalutamida puede tener un síndrome de retirada del antiandrógeno en una minoría de pacientes con cáncer de próstata metastásico resistente a la castración.

L’èxit de de la medicina de precisió en oncologia depèn, en gran mesura, d’una adequada selecció dels pacients que rebran teràpies dirigides contra dianes específiques del seu tumor. Per poder seleccionar els pacients, és indispensable disposar de biomarcadors amb valor predictiu que informin les decisions terapèutiques. MET i PD-L1 són dos receptors de membrana rellevants en la biologia del carcinoma pulmonar no microcític (CPNM). MET és un oncogen i l’activació de la seva via es troba relacionada amb múltiples processos pro-tumorals com són la proliferació i la motilitat cel·lulars, així com la invasió d’estructures veïnes. PD-L1 és una molècula clau en la resposta immunitàries, i la seva sobre-expressió en els tumors està relacionada amb la capacitat de les cèl·lules tumorals d’evitar el seu reconeixement i destrucció per part del sistema immunitari. Actualment, existeixen teràpies específiques dirigides contra aquestes molècules. L’estratègia més emprada per seleccionar els pacients que se’n poden beneficiar és la determinació de l’expressió d’ambdues molècules en teixit tumoral. Tanmateix, el valor de MET i de PD-L1 com a biomarcadors predictius i el mètode pel qual s’han de determinar és subjecte de debat. Estudis recents han detectat un alt grau d’heterogeneïtat genòmica en mostres tumorals en CPNM. Aquesta heterogeneïtat podria afectar de forma rellevant la classificació de pacients basada en l’expressió de biomarcadors. A més, aquest fet seria especialment rellevant en el cas del CPNM, ja que l’estudi de biomarcadors es fa generalment en mostres petites de teixit, provinents de biòpsies o citologies obtingudes mitjançant tècniques mínimament invasives. L’objectiu principal dels treballs presentats en aquesta tesi és estudiar l’heterogeneïtat de l’expressió de MET i PD-L1 en mostres de CPNM. Amb aquesta finalitat, hem analitzat mostres tumorals procedents de pacients tractats quirúrgicament de CPNM a l’Hospital del Mar. De cada tumor, hem seleccionat múltiples àrees geogràficament separades, les quals hem analitzat de forma independent. En l’estudi en que hem avaluat MET hem seleccionat quatre àrees per cada pacient, mentre que en l’estudi de PD-L1 n’hem seleccionat dues. En cada àrea tumoral, hem mesurat l’expressió de MET i de PD-L1 mitjançant mètodes d’immunohistoquímica i d’hibridació in situ fluorescent (FISH). Finalment, hem comparat l’expressió de MET i de PD-L1 entre diferents àrees tumorals. En el cas de MET, hem trobat discordances entre diferents àrees tumorals en un 20-40% per immunohistoquímica i en un 25-50% per FISH. En el cas de PD-L1, aquesta discordança ha estat major si es valora només l’expressió en limfòcits infiltrants de tumor (17-27%) que si es valora en cèl·lules tumorals (10-19%). A més, un 36% dels casos amb amplificació del gen que codifica PD-L1 determinada per FISH presenten aquesta amplificació només en una de les dues àrees analitzades. En conjunt, els nostres resultats suggereixen que l’expressió d’ambdós biomarcadors és heterogènia, tant si es mesura mitjançant immunohistoqumímica com mitjançant FISH. Aquesta heterogeneïtat pot tenir un impacte potencial en la classificació de tumors basada en l’expressió de biomarcadors i per tant, pot suposar una dificultat afegida a l’hora de desenvolupar teràpies dirigides per pacients amb CPNM., The success of precision medicine in oncology is dependent to a large extent on an adequate selection of patients who will receive targeted therapies aimed at specific molecular traits of their tumor. In order to be able conduct such patient selection, predictive biomarkers that can inform therapeutic decisions are essential. MET and PD-L1 are two relevant membrane receptors for non-small cell lung cancer (NSCLC) biology. MET is an oncogene the activation of which is involved in multiple pro-tumorigenic processes such as cell proliferation, motility and invasion. PD-L1 is a key molecule that acts during the immune response, and its overexpression in tumors is thought to mediate the ability of tumor cells to avoid immune cell recognition and destruction. Currently, there are specific therapies directed against these molecules. The most commonly used strategy to select the patients that will benefit from such drugs is the analysis of the expression of both molecules in tumor tissue. However, the value of MET and PD-L1 as predictive biomarkers and the method by which it should be determined is a subject of debate. Recent studies have detected a high degree of genomic heterogeneity in NSCLC tumor samples. This heterogeneity could significantly affect biomarker-based patient classification especially in the case of NSCLC, since biomarker studies are usually performed in small biopsies or cytology samples obtained through minimally invasive techniques. The main objective of the work presented in this thesis is to study the heterogeneity of the expression of MET and PD-L1 in NSCLC samples. For this purpose, we have analyzed tumor samples from NSCLC patients that had undergone surgical treatment at Hospital del Mar. Of each tumor, we have selected multiple geographically separate areas, which we analyzed independently. In the study evaluating MET, we selected four tumor areas per patient, while in the study evaluating PD-L1 we selected two areas. In each tumor area, we measured the expression MET and PD-L1 using immunohistochemical and fluorescence in situ hybridization methods (FISH). Finally, we compared the expression of MET and PD-L1 in different tumor areas. Regarding MET, we have found discordances between different tumor areas in 20-40% of cases using immunohistochemistry and in 25-50% of cases using FISH. Regarding PD-L1, this discrepancy was greater if we evaluated PD-L1 expression in tumor infiltrating lymphocytes (17-27%) than if we did so only in tumor cells (10-19%). Moreover, 36% of the cases with amplification of the gene coding for PD-L1 determined by FISH presented gene amplification only in one of the two areas analyzed. Overall, our results suggest that the expression of both biomarkers is heterogeneous, whether measured by immunohistochemistry or by FISH. This heterogeneity can have a potential impact on the classification of tumors based on the expression of biomarkers and, therefore, could represent a hurdle for the development of targeted therapies for NSCLC patients.

INTRODUCCIÓN: La quimioterapia neoadyuvante (QTN) ofrece una oportunidad única para evaluar la respuesta al tratamiento en estadios iniciales de cáncer de mama (CM). La valoración de marcadores inmunohistoquímicos define unos fenotipos intrínsecos clínicos (FIC) para el establecimiento de un plan terapéutico, con impacto en la respuesta patológica completa (RPC) y la supervivencia libre de enfermedad (SLE) y global (SG). MÉTODOS: Estudio de cohortes retrospectivo multicéntrico de mujeres diagnosticadas de CM candidatas a QTN. Se ha definido el FIC según la clasificación subrrogada del Consenso de St. Gallen de 2013. Se ha realizado la estadificación loco-regional según la 7ª edición del sistema TNM, la respuesta al tratamiento mediante resonancia magnética (RM) según los criterios RECIST 1.1 y la valoración de la respuesta patológica según el sistema de clasificación de Miller-Payne (M-P). El cálculo estadístico se ha realizado aplicando el test χ2, la supervivencia según la escala logarítmica de Kaplan-Meier y el análisis multivariado según riesgos proporcionales de Cox. Se ha valorado la relación entre los FIC y el valor de p53 (valor positivo cuando es superior al 50%). RESULTADOS: Entre 01-2006 y 06-2013, 239 pacientes con CM cumplieron los criterios de elegibilidad. El FIC ha sido: 43 luminal A (18,0%), 82 luminal B-Her2 negativo (34,3%), 32 luminal B-Her2 positivo (13,4%), 31 Her2 (13,0%) y 51 triple negativo (21,3%). La pauta de tratamiento más frecuente de QTN fue con antraciclinas y taxanos (83,7%). Todas las pacientes con sobreexpresión y/o amplificación de Her2 (20,5%) recibieron tratamiento con el anticuerpo anti-Her2 trastuzumab. La cirugía conservadora de la mama se ha realizado en el 69,9% de los casos. En todos los casos se realizó linfadenectomía axilar de niveles I y II de Berg, excepto en 34 (14,2%) en que se realizó biopsia selectiva del ganglio centinela antes de la QTN y el resultado fue negativo. La mayor tasa de respuesta completa por RM fue en lesiones Her2 y triple negativas. Según M-P la respuesta grado 5 en la mama se obtuvo en el 18,4% de los casos, y la D en la axila en el 18,0%, siendo las lesiones Her2 y triple negativas las de mayor respuesta (54,8% - 33,3% en la mama (p, INTRODUCTION: Neoadjuvant chemotherapy (NCT) offers a unique opportunity to evaluate the response to treatment of patients diagnosed with early-stage breast cancer (BC). Evaluation of immunohistochemistry markers define a variety of clinical intrinsic phenotypes (CIP) in order to establish a therapeutic plan with an impact on complete pathologic response (CPR), free-disease survival (DFS) and global survival (GS). METHODS: A retrospective multicenter cohort study of patients with BC candidates to NCT. CIP was defined following the subrogate classification of the St Gallen meeting in 2013. Loco-regional staging was classified according to the 7th edition of the TNM system. The response to treatment was evaluated by magnetic resonance imaging (MRI) following RECIST 1.1 criteria, and CPR was evaluated with Miller-Payne classification (M-P). A chi-square test was applied to measure the quantitative differences between groups, Kaplan-Meier curves were used for the survival analysis and a cox proportional hazard regression was used to assess multivariate analysis. Whenever the proportion was over 50%, the association between CIP and p53 value was assessed. RESULTS: From 01/2006 to 06/2013, 239 patients met the eligibility criteria. The results of CIP were: 43 luminal A (18.0%), 82 luminal B-Her2 negative (34.3%, 32 luminal B-Her2 positive (13.4%), 31 Her2 positive (13.0%) and 51 triple negative (21.3%). The most frequent treatment protocol used was anthracyclines and taxans (83.7%). All patients with overexpression and/or Her2 amplification (20.5%) received treatment with anti-Her2 antibody trastuzumab. Conservative surgical treatment of the breast was performed on 69.9% of the cases. Level I and II Berg axillary lymphadenectomy was performed in all cases except for 34 (14.2%) patients who had a negative result for centinel node biopsy before NCT. The greatest proportion of complete response evaluated by MRI was in patients with Her2 positive and triple negative lesions. Following M-P, grade 5 response for the breast was obtained in 18.4% of cases, and grade D response for the axilla in 18.0% of cases. Of them, Her2 positive and triple negative were the type of lesions with a greater response (54.8% - 33.3% for the breast (p < 0.001), and 35.5% - 23.5% for the axilla (p 0.025), respectively). Triple negative phenotype was associated with the worst results for FDS and GS (p < 0.001). Only grade of the response and CIP was associated to DFS and GS at the multivariate analysis as independent prognostic factors (p=0.031, p=0.001 respectively). P53 positivity was greater for lesions triple negative and luminal B-Her2 negative (p

[spa] El cáncer colorrectal es uno de los principales problemas de salud en nuestro medio. Los pacientes presentan una evolución muy heterogénea y no es posible predecir de manera fidedigna la evolución clínica o el pronóstico de un paciente individual con los biomarcadores disponibles en la actualidad. La presente tesis doctoral evaluó dos estrategias diferentes para determinar el valor de nuevos biomarcadores en cáncer colorrectal. La primera es la evaluación pronóstica de la determinación en sangre periférica de Metaloproteinasa de Matriz 7 (MMP-7). La segunda es una extensa descripción de eventos genéticos relacionados con la aparición de resistencia adquirida al tratamiento con el anticuerpo anti-EGFR cetuximab. MMP-7 es un enzima que degrada proteínas extracelulares, y su expresión se ha relacionado con peor pronóstico en varias enfermedades oncológicas. Este sub-proyecto plateó como hipótesis que la concentración sanguínea de MMP-7 podría ser un biomarcador pronóstico en los pacientes diagnosticados de cáncer colorrectal que serán sometidos a cirugía curativa. Se determinó la concentración de MMP-7 mediante técnica de ELISA en 175 pacientes previa a la intervención quirúrgica. Los resultados mostraron que MMP-7 era un factor pronóstico independiente tanto para supervivencia libre de enfermedad (HR= 1.119, IC95% 1.055-2.0, p, [eng] INTRODUCTION: Colorectal cancer is a main health problem. New biomarkers to define prognosis or predict drug effectivity are warranted. The aim of this thesis is define new biomarkers using two different approaches: Evaluation of serum Matrilysine (MMP-7) as a prognostic marker in localized colorectal cancer and determination of the molecular events related with acquired resistance to the anti-EGFR antibody cetuximab in advanced disease. MATHERIAL AND METHODS: We evaluated MMP-7 blood concentration by ELISA in 175 patients before surgery with curative intention. On the other hand, 37 patients with acquired resistance to cetuximab were biopsied in order to analyze KRAS, NRAS, BRAF, PIK3CA and EGFR mutations. We compared these alterations with the mutational profile previous the cetuximab treatment RESULTS: MMP-7 blood concentration is an independent prognostic marker for overall survival and disease- free survival. Combining MMP-7 and nodal status we defined three different prognostic groups. Mutational analysis in tumors after acquired resistance to cetuximab shows that 66% of patients had almost one of the mutations analyzed and 63% of these mutations were not present before the treatment. KRAS and NRAS genes were the most frequently affected. 14% of patients presented mutations at EGFR gene: S492R, K467T and R451C. Biopsies form three patients treated with biological therapies after cetuximab failure were also available, detecting dynamic changes in the mutational profile. Mutations were also detected in 62% of serum circulating DNA. CONCLUSSION: MMP-7 is a potential prognostic biomarker for colorectal cancer and its determination is technically applicable in clinical practice. KRAS and NRAS are the genes most frequently mutated after resistance to cetuximab. We also define two new mutations affecting EGFR (K467T and R451C). Tumor mutational profile is dynamic, and changes after the acquired resistance to each therapy. Blood DNA-sequencing could effective to monitor resistance to cetuximab even before clinical and radiological progression.

La implementación de programas de cribaje y/o de prevención junto a terapias innovadoras está disminuyendo la mortalidad por cáncer de mama. Ahora bien, más de 120.000 muertes anuales ocurren en Estados Unidos y en Europa por esta causa. Una explicación de este hecho es que aún nos falta obtener una fotografía completa de la heterogeneidad biológica del cáncer de mama con respeto a sus alteraciones moleculares, sensibilidad al tratamiento, y composición celular. De hecho, esta complejidad no se halla reflejada por los principales parámetros clínicos (edad, afectación ganglionar, tamaño tumoral, grado histológico) o los marcadores patológicos (receptor de estrógeno [ER], progesterona [PR] y el receptor del factor de crecimiento epidérmico tipo 2 [HER2]), los cuales están siendo utilizados hoy en día en la clínica para estratificar las pacientes en grupos pronósticos y/o terapéuticos. Los estudios basados en expresión global de genes han proporcionado una mayor comprensión de este escenario tan complejo. Durante los últimos 10 años, seis subtipos moleculares de cáncer de mama (Luminal A, Luminal B, HER2-enriched, Claudin-low, Normal Breast-like and Basal-like) han sido identificados y estudiados extensamente. Conocidos como los subtipos intrínsecos, este grupo de tumores ha revelado diferencias críticas en incidencia, supervivencia y respuesta a los tratamientos. De forma muy importante, la información proporcionada por los subtipos intrínsecos complementa y expande la información proporcionada por las variables clínico-patológicas clásicas. En esta tesis doctoral, presento los resultados de tres proyectos independientes sobre la caracterización molecular de los subtipos intrínsecos de cancer de mama y la implementación clínica de test basados en expresión génica. En global, los resultados sugieren que la información que proporciona los subtipos intrínsecos, combinados con los datos clínico-patológicos, nos ayuda a entender mejor la complejidad biológica del cáncer de mama, mejora la eficacia de los tratamientos actuales y los tratamientos novedosos, y finalmente mejora la supervivencia de las pacientes con cáncer de mama., Implementation of screening/prevention programs and novel treatment strategies is decreasing breast cancer mortality. However, more than 120,000 estimated deaths due to breast cancer are expected annually in the US and Europe combined. A plausible explanation for this scenario is, in part, that we still lack a complete enough picture of the biologic heterogeneity of breast cancers with respect to molecular alterations, treatment sensitivity, and cellular composition. Importantly, this complexity is not entirely reflected by the main clinical parameters (age, node status, tumor size, histological grade) and pathological markers (estrogen receptor [ER], progesterone receptor [PR] and human epidermal growth factor receptor 2 [HER2]), all of which are routinely used in the clinic to stratify patients for prognostic predictions and to select treatments. Studies based on global gene expression analyses have provided additional insights into this complex scenario. During the last 10 years, four molecular ‘intrinsic’ subtypes of breast cancer (Luminal A, Luminal B, HER2-enriched, and Basal-like) and a Normal Breast-like group have been identified and intensively studied. Known as the ‘intrinsic subtypes of breast cancer’, these groups of tumors have revealed critical differences in incidence, survival, and response to treatment. Importantly, the information provided by the intrinsic subtypes complements and expands the information provided by classical clinical-pathological markers. In this doctoral thesis, I am presenting the results of three independent projects regarding the molecular characterization of the intrinsic subtypes of breast cancer and the implementation of gene expression-based tests in the clinical setting. Overall, the results suggest that the information provided by the intrinsic subtypes, when combined with the current clinical-pathological markers, helps to further explain the biological complexity of breast cancer, increases the efficacy of current and novel therapies, and ultimately improves outcomes for breast cancer patients.

Small cell lung carcinoma (SCLC) is a highly lethal disease due to its chemorefractory nature after first line treatment. The mechanisms to overcome this resistance have remained elusive to tackle up to date. MET is a transmembrane tyrosine kinase receptor and its activation is associated with increased motility, migration and invasion in cancer cells. MET is activated in several tumour types and has been linked to patient prognosis. MET activation by its natural ligand Hepatocyte Growth Factor (HGF) has been involved in Epithelial to Mesenchymal Transition (EMT), a process by which cells decrease adhesion, lose polarity, acquire the ability to migrate and invade surrounding tissue. EMT is also associated with resistance to anticancer agents. We hypothesized that HGF-induced EMT would explain resistance to chemotherapy in SCLC and that MET inhibitors could revert this phenomenon. In our work we demonstrated that HGF-induced MET activation in SCLC cells resulted in a more aggressive phenotype. In human SCLC we demonstrated in two independent series that MET phosphorylation was associated with poor prognosis. In preclinical models we observed that MET activation by HGF induced EMT that resulted in chemoresistance in vitro and in vivo. MET inhibition was able to block or reverse this process and resensitized cells to chemotherapy. Mesenchymal markers in human SCLC specimens were associated with MET activation, predicted a worse survival and were upregulated in chemorefractory disease. Finally, increased HGF serum levels in SCLC patients correlated with higher risk of death. These results suggest that the use of MET inhibitors in combination with chemotherapy as a therapeutic approach in the MET-activated subpopulation of SCLC merits further investigation., El Cáncer de Pulmón de Célula Pequeña (CPCP) es una enfermedad altamente letal debido a su naturaleza quimiorefractaria después del tratamiento de primera línea. Los mecanismos para derrotar estar quimioresistencia han fracasado hasta la fecha. MET es un receptor de membrana tirosina cinasa y su activación está asociada a una incrementada motilidad, migración e invasión en células tumorales. MET está activado en diversos tipos tumorales y está asociado al pronóstico de los pacientes. La activación de MET mediante su ligando natural “Factor de Crecimiento Hepatocitario” (HGF) ha estado involucrado en la Transición Epitelio- Mesenquimal (EMT), un proceso mediante el cual las células disminuyen su adhesión, pierden la polaridad, adquieren la habilidad de migrar e invaden los tejidos adyacentes. La EMT está también asociada a la resistencia a agentes antitumorales. Nuestra hipótesis es que la EMT inducida por HGF explicaría la resistencia a la quimioterapia en el CPCP y que los inhibidores de MET podrían revertir este fenómeno. En nuestro trabajo demostramos que la activación de MET inducida por HGF en líneas celulares de CPCP dio lugar a un fenotipo más agresivo. En dos series independientes de CPCP humano demostramos que la fosforilación de MET estaba asociada a un peor pronóstico. En modelos preclínicos observamos que la activación de MET mediante HGF indujo la EMT, dando lugar a quimioresistencia in vitro e in vivo. La inhibición de MET fue capaz de bloquear o revertir este proceso, sensibilizando las células tumorales a la quimioterapia. Los marcadores mesenquimales en muestras humanas de CPCP se asociaron a activación de MET, prediciendo una peor supervivencia. Además, la expresión de estos marcadores estaba incrementada en la enfermedad quimiorefractaria. Estos resultados sugieren que el uso de inhibidores de MET en combinación con quimioterapia como una aproximación terapéutica en la subpoblación de pacientes de CPCP con activación de MET merece una investigación más extensa.

Objetivos: La fosfatasa 1 de las protein-quinasas activadas por mitógenos (Mitogen-activated protein kinase (MAPK) phosphatase-1, MKP-1) regula por defosforilación la actividad de las vías de señalización de las protein-quinasas activadas por mitógenos, la quinasa regulada por señales extracelulares (extracellular signal-regulated kinase, ERK), la quinasa terminal c-Jun (c-Jun NH2-terminal kinase, JNK) y p38. MKP-1 también media en los mecanismos de quimioresistencia en cáncer de mama y se inhibe por la actividad de la doxorrubucina en modelos celulares de cáncer de mama. Este trabajo aquí presentado tiene como objetivo caracterizar los efectos de la doxorrubicina sobre MKP-1 y sobre las MAPK en cáncer de mama e investigar su relevancia como factor pronóstico en esta enfermedad. Experimental Design: Los efectos de la doxorubicin en MKP-1, la forma fosforilada (p) de ERK1/2 (p-ERK1/2), de p-JNK y de p-p38 se estudiaron en un panel de líneas celulares de cáncer de mama humano por técnicas de Western blot y en tumores humanos cultivados ex vivo por inmunohistoquímica. De forma adicional, MKP-1 se estudió en una serie de tejidos mamarios desde parénquima normal hasta carcinoma infiltrante de mama, y en una cohorte amplia de pacientes de cáncer de mama con seguimiento clínico. Results: MKP-1 se expresaba en niveles bajos en el tejido mamario normal y en la hiperplasia ductal, detectándose sobreexpresión en el carcinoma ductal in situ. MKP-1 también se sobreexpresaba en casi el 50% de los tumores mamarios. De forma similar en las líneas de cáncer de mama y en los tumores cultivados ex vivo, de forma general la doxorrubicina redujo la expresión de MKP-1 e incrementó los niveles de p-ERK1/2 y JNK. Aquellos casos, sin embargo, que sobreexpresaban MKP-1, la doxorrubicina no afectó la expresión de MKP-1 ni de las formas fosforiladas de las MAPK. Por último, la sobreexpresión de MKP-1 se reveló como un factor de mal pronóstico en los análisis uni- y multivariados para la supervivencia libre de enfermedad en las pacientes con cáncer de mama estudiadas. Conclusions: MKP-1 se sobreexpresa en la transformación maligna de la mama y se asocia a una peor supervivencia en las pacientes con cáncer de mama. Además, MKP-1 es inhibida por la doxorrubicina en esta enfermedad., Purpose: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) regulates by dephosphorylation the activity of the major intracellular signaling pathways mediated by mitogen-activated protein kinase [extracellular signalregulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38]. MKP-1 mediates breast cancer chemoresistance and is repressible by doxorubicin in breast cancer cells. This work is aimed to characterize doxorubicin effects on MKP- 1 and phospho-MAPKs in human breast cancers and to study the clinical relevance of MKP-1 expression in this tumor. Experimental Design: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a broad panel of human breast cancer cells by Western blotting and in human breast cancer were assayed ex vivo by immunohistochemistry. MKP-1 expression was also assayed in a spectrum of normal to malignant breast lesions and in a large series of breast cancer patients with clinical follow-up. Results: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in approximately 50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin downregulated MKP-1, and upregulated activation of ERK1/2 and JNK, in most of studied cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or activation levels of MAPKs. With regard to patient outcome, MKP-1 overexpression was a significant adverse prognostic factor for relapse both by univariate and multivariate analysis. Conclusions: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in human breast cancer.

EGFR is a transmembrane tyrosine kinase receptor from the HER family which, upon ligand stimulation, activates different signaling pathways involved in tumorogenesis. EGFR can be targeted by monoclonal antibodies, as cetuximab and panitumumab, which bind to EGFR preventing ligand stimulation of the receptor. Cetuximab and panitumumab are approved for colorectal cancer treatment. However, its clinical success is uniformily limited by the development of acquired drug resistance. We describe a new mechanism of acquired resistance to cetuximab in colorectal cancer that was due to a missense mutation in the EGFR ectodomain (S492R mutation). Upon chronic exposure to cetuximab, colorectal cancer cell lines acquired S492R mutation and became resistant to the treatment. We observed that cetuximab was not able to bind mutant EGFR. Notably, this amino acid change did not affect the ability of panitumumab to bind to EGFR, and panitumumab effectively suppressed growth of mutant cells. EGFRS492R mutation was detected in 2 out of 10 tumor specimens from patients following progression on cetuximab. One of these patients was subsequently treated with single agent panitumumab yielding a partial response. The S492R mutation defines a novel biomarker of resistance to cetuximab but not to panitumumab in colorectal cancer, EGFR és un receptor transmembrana tirosina cinasa de la família HER el qual, després de l’estimulació mitjançant lligands, activa vies de senyalització involucrades en processos tumorogènics. L’EGFR es pot inhibir amb anticossos monoclonals, com cetuximab i panitumumab, que s’uneixen al receptor prevenint-ne l’activació per part dels lligands. Cetuximab i panitumumab estan aprovats per al tractament del càncer colorectal, però el seu ús es veu limitat per el desenvolupament de resistència adquirida al tractament. Nosaltres describim un mecanisme de resistència adquirida a cetuximab en càncer colorectal degut a l’adquisió d’una mutació en el domini extracel•lular de l’EGFR, la mutació S492R. Durant l’exposició crònica a cetuximab, linies cel•lulars de càncer colorectal van adquirir la mutació S492R tornat-se resistents al tractament. Cetuximab no era capaç d’unir-se a l’EGFR mutat. Aquests canvi d’aminoàcid no afectava a l’habilitat que té panitumumab a unir-se al EGFR, pertant, panitumumab suprimia el creixement de les cèl•lules tumorals mutades. Vam detectar la mutació EGFRS492R en 2 de 10 mostres tumorals de pacients que havien recaigut al tractament amb cetuximab. Un d’aquest pacients va ser posteriorment tractat amb panitumumab obtenint-ne una resposta tumoral parcial. La mutació S492R defineix un nou mecanisme de resistència a cetuximab però no a panitumumab en el tractament del càncer colorectal.

Las infecciones nosocomiales son una causa frecuente de incremento de estancia hospitalaria, costos sanitarios y mortalidad. En la actualidad, el enterococo es uno de los microorganismos implicados con mayor frecuencia en infecciones nosocomiales. Con frecuencia estas infecciones son difíciles de tratar debido la resistencia múltiple de estos microorganismos frente a los antibióticos convencionales. La literatura disponible sobre las infecciones producidas por este microorganismo es limitada. Este problema está más acentuado cuando se trata de conocer las características de infecciones producidas en localizaciones concretas, como es el caso de las bacteriemias. Es ampliamente conocido que E. faecalis tiene una mayor susceptibilidad a la ampicilina que E. faecium. Debido a que la diferencia en la susceptibilidad a los antimicrobianos entre las diferentes especies del género Enterococcus puede tener un impacto en la práctica asistencial, se ha realizado un estudio comparativo entre las características clínicas y microbiológicas y el pronóstico de los pacientes con bacteriemias por E. faecalis frente a E. faecium ya que se desconoce si estas características son coincidentes con lo reportado en la literatura médica. Se realizó así mismo un estudio similar centrado en los pacientes con cáncer con aislamiento de enterococos sensibles a vancomicina. Éste último es el único estudio que se ha realizado hasta la fecha en esta población analizando específicamente bacteriemias por Enterococcus sensibles a vancomicina, los más predominantes en España y Europa. El conocimiento de estos factores puede ser utilizado como mecanismo de alerta para la sospecha de bacteriemia por este microorganismo y así poder iniciar un tratamiento empírico adecuado temprano. El tratamiento empírico adecuado está directamente relacionado con el aumento de supervivencia en este tipo de infecciones. Para responder a estos objetivos se realizó un estudio retrospectivo observacional de cohortes realizado en el Hospital del Mar entre el 1 de enero del 2000 y el 31 de diciembre de 2006. Se incluyeron todos los episodios de bacteriemia con aislamiento de E.faecalis o E.faecium en hemocultivo. De cada paciente se recogieron datos demográficos, clínicos y bioquímicos al ingreso y en la fecha de realización del hemocultivo, historia de ingresos previos, unidad de hospitalización (médica, quirúrgica, UCI), posibles factores desencadenantes, enfermedades de base, exposición previa a antibióticos durante el ingreso actual, características microbiológicas y mortalidad. Al ingreso se clasificó la gravedad de los pacientes mediante la puntuación del Simplified Acute Physiology Score (SAPS II) Se estudiaron un total de 228 episodios de bacteriemia: 168 de E.faecalis y 60 de E.faecium. Todos los aislamientos de E.faecalis fueron sensibles a ampicilina y sólo el 25% lo fue en el grupo de E.faecium. Hubo un único caso anecdótico de resistencia a vancomicina que se trataba de una cepa de E.faecium. Las variables que se asociaron de manera independiente a la adquisición de bacteriemia por E.faecium cuando se compararon con aquéllas producidas por E.faecalis fueron el ingreso en una unidad quirúrgica (OR: 4,223; p=0,001), más de cinco días de tratamiento con cefalosporinas o carbapenémicos (OR: 2,564; p=0,013 y OR: 2,652; p=0,027 respectivamente), la administración previa de penicilinas (OR: 2,008; p=0,044); SAPS II >30 al ingreso (OR=3,530; p=0,001) y enfermedad hepática de base (OR: 3,754; p34 (OR:6,896; p=0,009). Los resultados de ambos estudios son de vital importancia a la hora de elegir que antibiótico utilizar en primera línea y de manera empírica en pacientes ingresados en nuestro hospital y han dado lugar a la modificación de los protocolos actuales de tratamiento empírico de pacientes con infección nosocomial en nuestro hospital. Además, se trata de una línea de investigación que está abierta a la inclusión de futuros estudios que se complementen con los efectuados en este proyecto de tesis doctoral., Nosocomial infections are a common cause of increased hospital stay, health care costs and mortality. Currently, the Enterococcus spp. is one of the most common microorganisms involved in nosocomial infections. Often these infections are difficult to treat because of the multiple resistances of these microorganisms to conventional antibiotics. The literature on infections caused by this microorganism is limited. This problem is more pronounced when it comes to know the characteristics of infections in specific locations, as is the case of bacteraemia. It is widely known that E. faecalis have an increased susceptibility to ampicillin compared to E. faecium. Because the difference in antimicrobial susceptibility among different species of the genus Enterococcus can have an impact on clinical practice, it was performed a comparative study of clinical and microbiological characteristics and prognosis of patients with bacteraemia due to E. faecalis against E. faecium and it is unknown whether these features are consistent with those reported in the literature. A similar study focused on cancer patients with bacteraemia of vancomycin-susceptible enterococci was also carried out. To our knowledge, this is the first study evaluating this population with enterococcal bacteraemia susceptible to vancomycin, the most prevalent susceptible pattern in Spain and Europe. Previous knowledge of these factors can be employed as a warning mechanism for suspected bacteraemia in order to initiate early appropriate empiric therapy. Appropriate empirical treatment is directly related to increased survival in these infections. It was performed a retrospective observational cohort study conducted at the Hospital del Mar between 1 January 2000 and December 31, 2006. We included all episodes of bacteraemia with E.faecalis or E.faecium isolation in blood culture. It was collected demographic, clinical and biochemical data at admission date and the date of positive blood culture for Enterococcus spp., history of previous admissions, hospital ward (medical, surgical, ICU), other possible risk factors, underlying diseases, previous antibiotic treatment, microbiological characteristics, and mortality. On admission, severity of illness of each patient was classified by the Simplified Acute Physiology Score (SAPS II) It was assessed a total of 228 episodes of bacteraemia, 168 corresponding to E.faecalis and 60 to E.faecium. All isolates of E.faecalis were susceptible to ampicillin and only 25% was in the group E.faecium. There was one anecdotal isolated of E.faecium resistant to vancomycin. The independent factors associated with the acquisition of bacteriaemia of E.faecium when compared to those produced by E.faecalis were the surgical ward admission (OR: 4.223, p = 0.001), more than five days of previous treatment with cephalosporin or with carbapenem (OR: 2.564, P = 0.013 and OR: 2.652, p = 0.027 respectively), prior administration of penicillin (OR: 2.008, p = 0.044), SAPS II> 30 at admission (OR = 3.530, p = 0.001) and liver disease at baseline(OR: 3.754, p 34 (OR: 6.896, p = 0.009). The results of both studies are of upmost importance to decide empirical antibiotic treatment in patients admitted to our hospital. In addition, these conclusions led to the modification of current protocols for empirical treatment of patients with nosocomial infections in our hospital. Furthermore, this line of research is open to the inclusion of future studies that complement those performed in this PhD project.

Breast cancer is the main cause of cancer death in women. Improved treatments, prevention programs and earlier detection are reducing the rate of death; however, there is still a high percentage of mortality by this cancer. Identification of novel targets to predict response to specific treatments is a key goal for personalizing breast cancer therapy and to improve survival. Few years ago, PARP inhibitors appeared as a promising therapy, particularly in BRCA-mutated cancers. However, there was a clear need to conduct further preclinical and translational work to improve the rational development of PARP inhibition in breast cancer. In this work we described PARP1 expression in breast tumour samples and characterized the effects of its inhibition in preclinical models. We found that nuclear PARP1 protein overexpression was associated with malignant transformation and poor prognosis in breast cancer. PARP1 overexpression was more common in triple negative subtype, but was also detectable in small subsets of estrogen receptor positive and HER2 positive breast cancers. In preclinical models, PARP1 played distinct roles in different molecular subtypes of breast cancer. Moreover, we described that olaparib (novel PARP inhibitor) had antitumour effects in different breast cancer subtypes, and its combination with trastuzumab (anti-HER2 antibody) enhanced the antitumour effects of this therapy., El càncer de mama és la principal causa de mort per càncer en dones. La millora dels tractaments i la detecció precoç estan reduint la taxa de mort, però segueix sent elevada. Identificar noves dianes per predir la resposta a tractaments és clau per millorar les teràpies contra aquest càncer i la supervivència. Els inhibidors de PARP van aparèixer com una teràpia prometedora, particularment en càncers BRCA-mutants, però, cal dur a terme més estudis preclínics i translacionals per fomentar un desenvolupament racional d’aquesta teràpia en càncer de mama. Aquest treball descriu l’expressió de PARP1 en mostres de tumors mamaris i caracteritza els efectes de la seva inhibició a models preclínics. Vam observar que la sobreexpressió nuclear de la proteïna PARP1 fou associada amb: la transformació maligna; mal pronòstic en càncer de mama; i fou més freqüent al subtipus triple-negatiu, però també es va detectar en un subgrup de càncers de mama receptors d’estrogen positius i HER2 positius. En models preclínics, PARP1 va exercir rols diferents als diferents subtipus de càncer de mama. Per altra banda, vam descriure que olaparib (inhibidor de PARP) té efectes antitumorals en els diversos subtipus, i combinat amb trastuzumab (anticòs anti-HER2) potencia els efectes antitumorals d’aquesta teràpia.