Search

[eng] Therapy development in cancer involves several consecutive steps including (1) the identification of the unmet medical need (disease or clinical question), (2) the establishment of clinically relevant resources or models to address such need, (3) the evaluation of therapies or experimental questions in the established models, and (4) the design and execution of clinical trials or protocols. The ambition of my PhD work was to follow the mentioned steps to build a body of results with potential clinical impact in the pediatric oncology field. Research at our institution, SJD, focuses on several types of cancers with bad prognosis, such as pediatric sarcomas. These cancers belong to a rare and heterogeneous group of skeletal and soft tissue malignancies accounting for approximately 12% of all childhood solid tumors. The most frequently occurring ones are Ewing sarcoma, osteosarcoma and rhabdomyosarcoma. With the up-to-date treatment modalities, based on surgery, radiation and chemotherapy, the 5-year survival rates of these patients have improved up to 70-80% in the last decades. However, a substantial proportion of patients relapse and do not respond consistently to rescue therapy. Whether the lack of response to therapy of these patients is due to drug delivery issues in resistant tumor cells is not completely understood. The main question I wanted to address in my work was whether patients whose tumors stop responding to anticancer medicines show diminished intratumoral drug distribution. Because this experimental question was challenging to address in the clinical setting, we needed to generate the adequate laboratory models. Thus, we established PDX models in immunodeficient mice. There is compelling evidence suggesting that PDX, at least at initial passages, reproduce the clonal heterogeneity, genetics and histology of several types of cancer. Whether this holds true for pediatric sarcomas was not sufficiently clear. Thus, we embraced an ambitious project to address (1) the identification of clinical and technical factors involved in pediatric sarcoma PDX engraftment, (2) the characterization of the stability of the PDX during initial passages, and (3) the relationship between successful PDX and patient prognosis. The main hypotheses of my work were: (1) PDX models faithfully represent pediatric sarcoma tumors established from patients at different disease stages. PDX conserve the main histology, molecular and functional properties of human tumors over successive passages in mice. (2) PDX engraftment correlates with patient prognosis. Successful establishment of PDX models helps identify patients with higher risk of recurrence and disease progression. (3) Intratumoral distribution of anticancer drugs becomes restricted due to the evolution of patient disease. Cancer cells in relapsed tumors displace chemotherapeutics from the intracellular to the extracellular tumor compartment. (4) The expression of multidrug resistance efflux transporters explains, at least partially, the shift of the intratumoral drug distribution towards the extracellular compartment upon tumor evolution in relapsed patients. To address my hypotheses, the main objectives of my thesis were: (1) To establish and characterize PDX models from biopsies and necropsies of pediatric patients diagnosed with Ewing sarcoma, osteosarcoma and rhabdomyosarcoma. To address this goal, we implanted patient samples obtained at Hospital Sant Joan de Déu in immunodeficient mice. Upon successful engraftment as PDX, we characterized the histology, genomics and response to model therapy (irinotecan) of the established tumor models over time in successive passages in mice. (2) To identify factors favoring PDX engraftment and to study the association between PDX engraftment and prognosis in pediatric patients with Ewing sarcoma, osteosarcoma and rhabdomyosarcoma. To address this goal, we collected clinical and technical data from the patients and the PDX, and we studied the relationship between PDX engraftment and several patient and sample factors, including disease stage (diagnosis or relapse) and patient outcome (event free survival and overall survival). (3) To detect and characterize changes in anticancer drug activity and intratumoral drug distribution during the evolution of Ewing sarcoma. To address this goal, we established pairs of PDX models from patients at diagnosis and late disease stages. In such paired PDX, we studied the activity and distribution of the model drug irinotecan and its active metabolite, SN-38. We applied the intratumoral microdialysis – tumor homogenate technique to obtain samples of PDX engrafted in mice receiving irinotecan infusions, and we calculated the unbound volume of distribution of SN-38 in the established PDX. We analyzed the expression and function of multidrug resistance efflux transporters, such as P-glycoprotein, as likely cause of the limited drug distribution and acquisition of resistance in relapsed tumors.

Burkitt-like lymphoma with 11q aberration is characterized by pathological features and gene expression profile resembling those of Burkitt lymphoma but lacks the MYC rearrangement and carries an 11q-arm aberration with proximal gains and telomeric losses. Whether this lymphoma is a distinct category or a particular variant of other recognized entities is controversial. To improve the understanding of Burkitt-like lymphoma with 11q aberration we performed an analysis of copy number alterations and targeted sequencing of a large panel of B-cell lymphomarelated genes in 11 cases. Most patients had localized nodal disease and a favorable outcome after therapy. Histologically, they were high grade B-cell lymphoma, not otherwise specified (8 cases), diffuse large B-cell lymphoma (2 cases) and only one was considered as atypical Burkitt lymphoma. All cases had a germinal center B-cell signature and phenotype with frequent LMO2 expression. The patients with Burkitt-like lymphoma with 11q aberration had frequent gains of 12q12-q21.1 and losses of 6q12.1-q21, and lacked common Burkitt lymphoma or diffuse large B-cell lymphoma alterations. Potential driver mutations were found in 27 genes, particularly involving BTG2, DDX3X, ETSI , EP300, and GNA13. However, ID3, TCF3, or CCND3 mutations were absent in all cases. These results suggest that Burkitt-like lymphoma with 11q aberration is a germinal center-derived lymphoma closer to high-grade B-cell lymphoma or diffuse large B-cell lymphoma than to Burkitt lymphoma. This work was supported by Asociacion Espanola Contra el Cancer (AECC CICPFI6025SALA), Fondo de Investigaciones Sanitarias Instituto de Salud Carlos III (Miguel Servet program CP13/00159 and PI15/00580, to IS), Spanish Ministerio de Economia y Competitividad, Grant SAF2015-64885-R (EC), Generalitat de Catalunya Suport Grups de Recerca (2017-SGR-1107 I.S. and 2017-SGR-1142 to EC), and the European Regional Development Fund "Una manera de fer Europa". JERZ was supported by a fellowship from the Generalitat de Catalunya AGAUR FI-DGR 2017 (2017 FI_B01004). EC is an Academia Researcher of the "Institucio Catalana de Recerca i Estudis Avancats" (ICREA) of the Generalitat de Catalunya. This work was developed at the Centro Esther Koplowitz, Barcelona, Spain. The group is supported by Accio Instrumental d'Incorporacio de Cientifics i Tecnolegs PERIS 2016 (SLT002/16/00336) from the Generalitat de Catalunya.

[eng] Pediatric RMS is a developmental tumor that affects patients from birth to late adolescence. RMS tumorigenesis involves the stalk of embryological myogenic processes. Classically, two main subgroups of RMS have been defined based on the histopathological features: Embryonal (ERMS) and Alveolar (ARMS). However, the molecular classification refines the subgrouping into Fusion Positive or Fusion Negative based on the occurrence of FOXO1 oncogenic translocations. The recent genetic and epigenetic landscapes of those subgroups have identified few potential druggable targets. Nowadays, the outcome of children with relapsed/refractory RMS remains very poor. In order to find new opportunities for these patients this thesis focuses on the identification of new therapeutic targets effective for RMS treatment as well as on characterizing biomarkers of response for the new targeted treatment. We initially performed a functional approach starting from IGF1R target therapies to main downstream pathways including PI3K/AKT/mTOR and MEK/ERK that we found active in primary samples as well as in patient derived models. The new IGF1/2 monoclonal antibody m708.5 was found active in a subset of RMS particularly dependent on the IGF/AKT/mTOR pathway. Interestingly, we found that the ATP-competitive pan-AKT inhibitor Ipatasertib was highly active causing tumor regression in a subset of patient derived models. We were able to classify RMS primary models and their parental samples into two subgroups: Ipatasertib- dependent and Ipatasertib non-dependent. Moreover, preclinical pharmacology corroborated the dose-dependent response of RMS to this inhibitor. Most interesting, those tumors particularly sensitive to Ipatasertib were shown to be resistant to the MEK inhibitor Trametinib. Surprisingly, the study of a mechanistically different AKT inhibitor did not recapitulate Ipatasertib efficacy in vivo. We discovered that Ipatasertib sensitivity correlated with high PRKG1 levels. We described for the first time PRKG1 gene expression as RMS specific and predictive of Ipatasertib response in vivo. The genetic signature of PRKG1 depleted RMS cells is enriched in the expression of myogenic genes, positioning for the first time PRKG1 in the landscape of RMS tumorigenesis. The potential of PRKG1 as a prognostic marker was evaluated. Ongoing studies will determine the mechanism of myogenic blocking dependent on PRKG1.

B-cell non-Hodgkin lymphoma (B-NHL) of pediatric and young adult population is a diverse group of neoplasms predominantly composed of aggressive B-cell lymphomas from the germinal center (GC). Molecular characterization of pediatric series has allowed the identification of several subtypes that predominantly occur in this subgroup of age. Despite of that, genomic features of these pediatric entities and their relationship to other B-NHL in this group of patients have not been extensively investigated. This thesis has aimed to address this gap of knowledge by performing a genetic and molecular characterization of large series of pediatric and young adult variants of GC-derived B-NHL including the Burkitt- like lymphoma with 11q aberration (BLL-11q) , pediatric type follicular lymphoma (PTFL) and large B-cell lymphomas (LBCL) such as diffuse large B-cell lymphomas (DLBCL) , high grade B- cell lymphomas, not otherwise specified (HGBCL, NOS) and large B-cell lymphoma with IRF4 rearrangement (LBCL-IRF4) entities. In the Study 1 we have molecularly characterized a series of 11 BLL-11q observing that BLL- 11q differed clinically, morphologically and immunophenotypically from conventional BL and instead showed features more consistent with HGBCL or DLBCL. Genomic profile was also different from that of BL and DLBCL with a mutational landscape characterized by the lack of typical BL mutations in ID3, TCF3, or CCND3 genes and recurrent specific BTG2 and ETS1 mutations, not present in BL but in germinal center B-cell (GCB) DLBCL subtype. All these observations suggest that BLL-11q is a neoplasm closer to other GC-derived lymphomas rather than BL. In Study 2, we expanded our knowledge on the genetic alterations associated to PTFL by verifying the presence of MAP2K1 and IRF8 mutations in a previously well characterized series of 43 PTFL. We demonstrate the activating effect of MAP2K1 mutations by immunohistochemical analysis observing phosphorylation of the MAP2K1 downstream target extracellular signal-regulated kinase in those mutated cases. Besides, we demonstrate the specificity of MAP2K1 and IRF8-K66R mutations since they are absent in conventional FL or in t(14;18)-negative FL. Finally, in the Study 3 we characterized a large series of LBCL including DLBCL, HGBCL, NOS and LBCL-IRF4 through an integrative analysis including targeted next generation sequencing, copy number, and transcriptome data. Results showed that each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B) whereas DLBCL, NOS was predominantly of GCB-DLBCL subtype and carried gene mutations similar to the adult counterpart (e.g., SOCS1 and KMT2D). A subset of HGBCL, NOS displayed recurrent alterations of BL-related genes such as MYC, ID3, CCND3 and SMARCA4, whereas other cases were genetically closer to GCB-DLBCL. Interestingly, we could identify age-related differences in pediatric DLBCL since pediatric and young adult cases were mainly of GCB subtype, displayed low genetic complexity and virtually lacked primary aberrations (BCL2, MYC and BCL6 rearrangements). Finally, we identify clinical and molecular features related to unfavorable outcome such as age >18 years, high LDH levels, activated B-cell (ABC) DLBCL profile, high genetic complexity, homozygous deletions of 19p13.3/TNFSF7/TNFSF9, gains of 1q21-q44/MDM4/MCL1 and TP53 mutations. Altogether, we conclude that GC-derived B-NHL of pediatric and young adult population is a heterogeneous group of tumors including different entities with specific molecular profiles and clinical behavior. This thesis has contributed to increase the knowledge of these lymphoma entities identifying biomarkers that might be helpful to improve their diagnosis and to design management strategies more adapted to their particular biological behavior.

Neuroblastoma (NB) is the most common extracraneal solid tumor diagnosed in the first 5 years of childhood and accounts for approximately 15% of all pediatric cancer-related deaths. At the time of diagnosis, about half of NB patients present disseminated disease, being the bone marrow (BM) the most common site of dissemination. The persistence of infiltrated BM during treatment or relapse is predictive of patient poor outcome. The BM microenvironment has unique biologic properties that favor progression of disseminated NB tumor cells. In this environment, the receptor CXCR4 has a pivotal role for BM homeostasis and is involved in metastatic dissemination in several cancers. In NB, CXCR4 is expressed in tumor cells; however, its oncogenic role in relation with its ligand CXCL12 has shown contradictory results. Using an in vitro model that recapitulates low oxygen levels and chemokine signaling present in the BM environment, we explored whether CXCR4 together with MIF, a second ligand, is critical for NB survival and proliferation in the niche. We also evaluated MIF inhibition as a therapeutic option for NB. To develop a BM-based in vitro model, NB cells were cultured in different conditioned media (CMs) derived from supernatants of patient-derived BM primary cells (CM-BM) and NB cell lines (CM-NB) cultured alone, or in combination (CM-BM/NB). To mimic BM oxygen levels, NB cells were cultured under hypoxia (1% O2), as compared to cell culture normoxia (21% O2). Expanded BM cultures used to generate CMs contained a heterogenic population with a predominant cellularity positive for mesenchymal stromal markers measured by flow cytometry. Cytokine arrays and ELISA assays of CMs revealed MIF as the highest NB released cytokine, whereas CXCL12 was not detected. The expression of MIF and CXCL12 signaling pathways was analyzed with different public NB databases. Among the analyzed genes, the high expression of CXCR4 and MIF was associated with patient poor outcome and high-risk disseminated staging. To further explore the role of CXCR4/MIF axis in high- risk disseminated NB, in vitro and in vivo functional studies were performed with or without the covalent MIF inhibitor 4-IPP and the CXCR4 antagonist AMD3100. When exposed to BM-derived CMs and hypoxia, BM-derived NB cell lines showed increased surface expression of CXCR4 by flow cytometry. The same culture condition increased phosphorylated levels of AKT/PI3K and ERK/MAPK measured by western blot. Cell viability assays showed that hypoxic conditions and BM-derived CMs enhanced NB cell proliferation at different time points. Similarly, in vivo, the co-injection of BM cells favored NB tumor progression by reducing engraftment times in contrast to NB injection alone. Using wound healing assays and Matrigel-coated Transwells, CM-BM/NB chemoattracted and enhanced migration and invasion of LAN-1 cells cultured under hypoxic conditions. These aggressive phenotypes promoted by the BM-based model were reverted by adding sub-lethal concentrations of the MIF inhibitor 4-IPP. We also explored whether MIF present in our CMs affected response to chemotherapy. After treatment with doxorubicin and etoposide at IC50 values LAN-1 cell viability increased in CM-BM/NB compared to control media. In both cases, chemo-sensitivity was restored when 4-IPP was added to CM-BM/NB. Finally, the administration of 4-IPP delayed the tumor progression and increased mice survival in a LAN- 1 subcutaneous xenograft model. In conclusion, our findings provide new understanding of the contribution of BM microenvironment to NB progression. Based on our BM-model, the relationship between the BM microenvironment and NB cells appears mediate, in part, by the autocrine CXCR4/MIF signaling axis. Furthermore, our results suggested that MIF could represent a therapeutic target for the treatment of patients with high-risk NB.

Introduction Cancer and blood disorders in children are rare. The progressive improvement in survival over the last decades largely relies on the development of international academic clinical trials that gather the sufficient number of patients globally to elaborate solid conclusions and drive changes in clinical practice. The participation of Spain into large international academic trials has traditionally lagged behind of other European countries, mainly due to the burden of administrative tasks to open new studies, lack of financial support and limited research infrastructure in our hospitals. Methods The objective of ECLIM-SEHOP platform (Ensayos Clínicos Internacionales Multicéntricos-SEHOP) is to overcome these difficulties and position Spain among the European countries leading the advances in cancer and blood disorders, facilitate the access of our patients to novel diagnostic and therapeutic approaches and, most importantly, continue to improve survival and reducing long-term sequelae. ECLIM-SEHOP provides to the Spanish clinical investigators with the necessary infrastructural support to open and implement academic clinical trials and registries. Results In less than 3 years from its inception, the platform has provided support to 20 clinical trials and 8 observational studies, including 8 trials and 4 observational studies where the platform performs all trial-related tasks (integral support: trial setup, monitoring, etc.) with more than 150 patients recruited since 2017 to these studies. In this manuscript, we provide baseline metrics for academic clinical trial performance that permit future comparisons. Conclusions ECLIM-SEHOP facilitates Spanish children and adolescents diagnosed with cancer and blood disorders to access state-of-the-art diagnostic and therapeutic strategies.

Background: The aim of this study was to test the feasibility and utility of developing patient-derived orthotopic xenograft (PDOX) models for patients with malignant peripheral nerve sheath tumors (MPNSTs) to aid therapeutic interventions in real time. Patient & Methods: A sporadic relapsed MPNST developed in a 14-year-old boy was engrafted in mice, generating a PDOX model for use in co-clinical trials after informed consent. SNP-array and exome sequencing was performed on the relapsed tumor. Genomics, drug availability, and published literature guided PDOX treatments. Results: A MPNST PDOX model was generated and expanded. Analysis of the patient’s relapsed tumor revealed mutations in the MAPK1, EED, and CDK2NA/B genes. First, the PDOX model was treated with the same therapeutic regimen as received by the patient (everolimus and trametinib); after observing partial response, tumors were left to regrow. Regrown tumors were treated based on mutations (palbociclib and JQ1), drug availability, and published literature (nab-paclitaxel; bevacizumab; sorafenib plus doxorubicin; and gemcitabine plus docetaxel). The patient had a lung metastatic relapse and was treated according to PDOX results, first with nab-paclitaxel, second with sorafenib plus doxorubicin after progression, although a complete response was not achieved and multiple metastasectomies were performed. The patient is currently disease free 46 months after first relapse. Conclusion: Our results indicate the feasibility of generating MPNST-PDOX and genomic characterization to guide treatment in real time. Although the treatment responses observed in our model did not fully recapitulate the patient’s response, this pilot study identify key aspects to improve our co-clinical testing approach in real time.

Colorectal cancer is one of the most prevalent, and one of the deadliest, types of cancer in the world. The colonic epithelium is positioned atop a subepithelial structure consisting of extracellular matrix and stromal cells, and colorectal tumors closely interact with this tissue. Myofibroblasts make up the primary cellular component of the subepithelial stroma in the colon, and play important roles in the maintenance of the normal colonic stem cell niche. Increased recruitment of local myofibroblasts into the tumor bulk is a feature of some primary and metastatic colorectal cancers, and is associated with poor prognosis. The first aim of this thesis was to probe the nature of the bidirectional interaction between colorectal cancer cells and colonic myofibroblasts. Two in vitro assays integrating myofibroblasts, colorectal cancer cells, and extracellular matrix were devised to mimic the in vivo arrangement and communication between these cells. Using these assays, it was shown that myofibroblast-derived hepatocyte growth factor reduces differentiation in a subset of colorectal cancer cell lines (Chapter 3). Additionally, colorectal cancer cells were shown to recruit myofibroblasts by secreting ligands of the platelet-derived growth factor family (Chapter 4). A secondary aim of the project was to develop an analytical technique to robustly categorize colorectal cancer gene expression profiles in an unsupervised manner. Gaussian Mixture Modeling was used to generate categorized expression data for an in-house colorectal cancer cell line panel, as well as a publically available dataset from primary tumors (Chapter 5). 2x2 statistical analyses on the categorized data confirmed known genetic associations in the cancer cells, and also uncovered characteristic gene expression signatures corresponding to intratumoral heterogeneity and stromal contamination (Chapter 6). This project identifies several mechanisms responsible for the heterogeneity observed in colorectal cancer behaviors, and provides tools to probe these characteristics further both experimentally and analytically.

[spa] INTRODUCCIÓN: Vivir la experiencia de tener un hijo con cáncer es una situación que genera una crisis en la familia que afecta no sólo a nivel estructural sino también emocional. Las enfermeras son los profesionales, miembros del equipo multidisciplinar, que interrelacionan de manera específica con estas familias, por lo que es de interés saber cómo evidencian esta experiencia y su impacto en las familias. Por tal de poder mejorar la practica asistencial que debe contemplar una orientación holística dentro de los cuidados de alta complejidad que precisa un niño con enfermedad oncológica. OBJETIVOS: El objetivo principal fue comprender y analizar en profundidad la vivencia en el momento del diagnóstico de la enfermedad oncológica infantil en las familias y la percepción del fenómeno en las enfermeras. Los objetivos específicos consistieron en analizar el impacto en las familias del debut de enfermedad oncológica infantil, explorar la percepción de las enfermeras sobre el impacto y la vivencia de las familias en el inicio de una enfermedad oncológica infantil, y comparar la percepción que tienen las familias con la percepción de las enfermeras respecto el debut de la enfermedad oncológica infantil. METODOLOGÍA: Estudio cualitativo siguiendo un enfoque basado en la fenomenología hermenéutica de Heidegger cuyo ámbito de estudio fueron las unidades de Oncología y Hospital de día del Hospital Sant Joan de Déu de Barcelona. Los informantes fueron las familias de niños con cáncer y las enfermeras dedicadas a su cuidado. En la selección de dichos informantes se usó la técnica de muestreo teórico o intencionado teniendo en cuenta variables sociodemográficas que ayudaron a definir el perfil del informante en ambos grupos de estudio hasta conseguir la saturación teórica de la información. Dicha saturación se consiguió antes de acabar con el proceso de recolección de información ya que interesaba poder disponer de la visión de los diferentes perfiles de informantes de cada grupo. Para la recogida de datos se utilizó la entrevista semiestructurada siguiendo un guión de tópicos preparado previamente y el diario de campo. El tipo de análisis utilizado fue temático bajo el método de análisis del contenido, siguiendo método de análisis Guía QUAGOL (Qualitative Analysis Guide of Leuven). Se utiliza como herramienta informática el programa de Nvivo10. Al ser el análisis un proceso circular y continuo, al ir profundizando en la información recibida, se fueron cambiando o añadiendo categorías y subcategorías. RESULTADOS: Las entrevistas realizadas fueron 14 en el grupo 1 (familias) y 17 en el grupo 2 (enfermeras).En el grupo 1 (familias) se obtuvieron 1060 unidades de significado que se agruparon en 11 categorías y 9 subcategorias. En el grupo 2(enfermeras) se obtuvieron 822 unidades de significado que se agruparon en 12 categorías y 7 subcategorias. En el grupo 1(familias), se pudo observar que la categoría que obtuvo un mayor número de referencias en unidades de significado fue repercusión de la enfermedad en la familia (REPERENFER) (n=121) y, la que menos, fue cuidado del hijo enfermo (CUIDADOHIJO) (n=10) que se referia a quién se centraba en cuidar al niño enfermo de cáncer. En el grupo 2 (enfermeras), se pudo observar que la categoría que obtuvo un mayor número de referencias en unidades de significado fue emociones observadas (EMOENFER) (n=134) y la que menos, presencia de fatiga por compasión (FATIGAENFERMERA) (n=1). Los resultados de este estudio describen el impacto y la experiencia de las familias ante el inicio de la enfermedad oncológica de sus hijos. Muestran que es una experiencia difícil de vivir y que afecta a todos los niveles (económico, organizativo, emocional, social) alterando los procesos familiares. Asimismo, existe un impacto emocional que les imposibilita procesar la situación que viven, dado que el primer sentimiento que sienten es dolor y miedo ante la información que reciben sobre el diagnóstico de cáncer. Muchas veces las reacciones vividas son rabia, enfado, negación, bloqueo emocional, culpa, tristeza. También los resultados permitieron detectar que las enfermeras no sólo expusieron el cómo perciben el impacto del inicio de la enfermedad oncológica en las familias sino que también se pudo evidenciar cómo afecta a las enfermeras la experiencia de cuidado de estas familias y sus hijos. CONCLUSIÓN: La enfermedad oncológica infantil provoca una situación de impacto emocional en las familias que se experimenta en todas las esferas de la vida. Contrariamente a la presunción adoptada en la premisa de investigación, las enfermeras perciben todas las vivencias descritas por las familias sobre el impacto de la enfermedad oncología infantil. Asimismo, fruto del cuidado humanizado y la relación terapéutica, las enfermeras muestran indicios de desgaste emocional que puede dificultar el cuidado., [eng] INTRODUCTION: Living the experience of having a child with cancer is a situation that creates a family crisis that affects not only structurally but also emotional. Nurses are professionals, who together with the multidisciplinary team, interact with these families, so it is of interest to know how to show this experience and its impact on families. For this to be able to improve health care practice that should include a holistic orientation with in the highly complex care that requires a child cancer. AIMS/OBJECTIVES: The main objective was to understand and analyze in profundity the experience at the time of diagnosis of childhood oncological disease in families and the perception of the phenomenon in nurses. The specific objectives were to analyze the impact on families of the debut of childhood cancer disease, to explore the perception of nurses about the impact and the experience of families in the onset of childhood cancer, and to compare the perception that families have with the perception of nurses regarding the onset of childhood cancer. METHODOLOGY: Qualitative study following an approach based on the hermeneutical phenomenology of Heidegger whose field of study were the Oncology and Day Hospital units of the Sant Joan de Deu Hospital in Barcelona. The informants were the families of children with cancer and the nurses dedicated to their care. In the selection of these informants the theoretical or intentional sampling technique was used, taking into account sociodemographic variables that helped to define the profile of the informant in both study groups. Until getting the theoretical saturation of the information. This saturation was achieved before ending the process of gathering information as it was interesting to be able to have the vision of the different informant profiles of each group. For the data collection, the semi-structured interview was used following a previously prepared topic script and the field diary. The type of analysis used was thematic under the content analysis method, following the QUAGOL (Qualitative Analysis Guide of Leuven) analysis method. The Nvivo10 program is used as a computer tool. As the analysis was a circular and continuous process, as we went deeper into the information received, we changed or added categories and subcategories. RESULTS: The interviews carried out were 14 in group 1 (families) and 17 in group 2 (nurses). In group 1 (families) 1060 units of meaning were obtained and grouped into 11 categories and 9 subcategories. In group 2 (nurses), 822 units of meaning were obtained, grouped into 12 categories and 7 subcategories. In group 1 (families), it was observed that the category that obtained the highest number of references in units of meaning was the repercussion of the disease in the family (REPERENFER) (n = 121) and, the least, it was taken care of sick child (CUIDADOHIJO) (n = 10) that referred to who was focused on caring for the child with cancer. In group 2 (nurses), it was observed that the category that obtained the highest number of references in meaning units was observed emotions (EMOENFER) (n = 134) and the least, presence of compassion fatigue (FATIGAENFERMERA) (n = 1). The results of this study describe the impact and experience of families before the oncological onset of their children. They show that it is a difficult experience to live and that affects all levels (economic, organizational, emotional, social) altering family processes. Also, there is an emotional impact that makes it impossible to process the situation they are living, given that the first feeling They feel it is pain and fear of the information they receive about the diagnosis of cancer. Many times the reactions experienced are anger, anger, denial, emotional block, guilt, sadness. The results also allowed us to detect that the nurses not only explained how they perceive the impact of the oncological disease onset in the families, but also how the nurses experience the care of these families and their children. CONCLUSIONS: Childhood oncology sickness causes a situation of emotional impact in families that is experienced in all spheres of life. Contrary to the presumption adopted in the research premise, the nurses perceive all the experiences described by the families about the impact of the childhood oncology disease. Likewise, as a result of humanized care and the therapeutic relationship, nurses evince signs of emotional exhaustion that can make care difficult.

[cat] ANTECEDENTS: els inhibidors d’histona deacetilasa (HDACi) han emergit els últims anys com potencials tractaments dirigits, amb la finalitat de revertir els canvis epigenètics associats a diferents tipus de càncer, incloent les neoplàsies hematològiques. La falta d’especificitat dels HDACi, però, dificulta la predicció dels seus efectes biològics i provoca una toxicitat no menyspreable, el que ha limitat el seu ús en la pràctica clínica. L’expressió de les HDACs no ha estat estudiada en les leucèmies agudes pediàtriques més que de manera parcial i en sèries amb un limitat número de pacients; per tant, són necessaris estudis que ajudin a aclarir el paper de cadascuna de les HDACs com a possibles biomarcadors i com a dianes terapèutiques, de cara a dirigir el tractament específic i personalitzat amb HDACi. HIPÒTESI: en aquest context, l’estudi global del perfil transcripcional de les HDACs en una sèrie àmplia i representativa de pacients pediàtrics amb diferents subtipus de leucèmia aguda permetria definir millor el seu impacte pronòstic i definir el seu rol com a dianes terapèutiques, ajudant a dirigir el tractament específic amb HDACi de forma més racional i individualitzada. OBJECTIUS: estudiar de forma global el perfil d’expressió de les HDAC1-11, SIRT1, SIRT7 i d’altres gens co-reguladors, MEF2C i MEF2D, en una sèrie de pacients pediàtrics amb leucèmia aguda diagnosticats i tractats en un sol centre. METODOLOGIA: anàlisi de l’expressió de l’mRNA de les HDAC1-11, SIRT1, SIRT7, MEF2C i MEF2D mitjançant PCR quantitativa en 211 pacients pediàtrics (0-18 anys), diagnosticats de leucèmia de novo des de l’any 2003 al 2017 en el nostre centre. Els pacients es van tractar uniformement d’acord als protocols consecutius de la Sociedad Española de Hematología y Oncología Pediátrica (SEHOP). Es va emprar un pool de pacients no-neoplàsics com a calibrador i també es va analitzar l’expressió dels diferents gens en cèl·lules CD34+ normals de moll d’os i a cèl·lules B CD19+ madures i limfòcits T madurs de sang perifèrica. RESULTATS: l’expressió de les HDACs va diferir en els diferents subtipus de cèl·lules hematopoètiques normals d’acord amb el grau de maduració i el llinatge. En els pacients amb leucèmia es va observar, en general, una sobreexpressió de les HDACs, amb perfils específics que correlacionaven amb característiques clíniques i biològiques i, fins i tot en algunes ocasions, amb la supervivència dels pacients. Així, alguns perfils d’expressió d’HDAC i el perfil de MEF2C semblaven reflectir, probablement, el llinatge i la maduresa dels blasts, mentre que d’altres identificaven la via oncogènica activa en les cèl·lules leucèmiques. Concretament, es va identificar un perfil d’expressió distintiu pels pacients amb reordenament del gen KMT2A (MLL), amb nivells elevats d’HDAC9 i MEF2D, independentment de l’edat, el partner del gen de fusió i el llinatge. A més, es va observar en els pacients amb LLA-B un pronòstic advers de la sobreexpressió de l’HDAC9, independentment de l’estat del gen KMT2A. CONCLUSIONS: en aquest treball s’han observat diferents perfils d’expressió segons els diferents subtipus de leucèmia aguda pediàtrica. Concretament, s’ha identificat un perfil distintiu dels pacients amb reordenament del gen KMT2A, amb la sobreexpressió de l’ HDAC9 i MEF2D, independentment del llinatge, de l’edat i del partner del gen de fusió. Aquests resultats aporten una imatge global de l’expressió de les HDACs en els pacients pediàtrics amb leucèmia aguda i recolzen el tractament dirigit amb HDACi més específics en seleccionats grups de pacients d’alt risc, com els pacients amb reordenament del gen KMT2A., [eng] Histone deacetylase inhibitors (HDACi) emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. We found a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles pointed out specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with MLL rearrangement, with high HDAC9 and MEF2D expression, regardless of age, MLL-partner and lineage. Moreover, we observed an adverse prognostic value of overexpression of HDAC9, regardless of MLL rearrangement. Our results provide useful knowledge on the complex picture of HDACs expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Introducción: vivir la experiencia de tener un hijo con cáncer es una situación que genera una crisis en la familia que afecta no solo a nivel estructural, sino también emocional. Las enfermeras son las profesionales miembros del equipo multidisciplinar que se relacionan más tiempo con estas familias, por lo que es interesante saber cómo evidencian esta experiencia para poder mejorar la práctica asistencial, que debe contemplar una orientación holística dentro de los cuidados de alta complejidad que precisa un niño oncológico. Objetivos: analizar el impacto en las familias producido por el debut de una enfermedad oncológica infantil; describir la percepción de las enfermeras sobre el impacto y la vivencia de las familias en el inicio de una enfermedad oncológica infantil y comparar la percepción que tienen las familias con la percepción de las enfermeras con respecto al debut de la enfermedad oncológica infantil. Ámbito de estudio: Unidad de Oncología y Hospital de Día del Hospital Sant Joan de Déu de Barcelona. Metodología: estudio cualitativo siguiendo un enfoque basado en la fenomenología hermenéutica de Heidegger. Los participantes serán las familias de niños con cáncer y las enfermeras dedicadas a su cuidado. Para la recogida de datos se utilizará la entrevista en profundidad y, si se considerara pertinente, el grupo de discusión. El tipo de análisis utilizado será el de la temática bajo el método de análisis del contenido, siguiendo el método de análisis Guía QUAGOL (Qualitative Analysis Guide of Leuven).

Organized and hosted by the Children's Tumor Foundation (CTF), the Neurofibromatosis (NF) conference is the premier annual gathering for clinicians and researchers interested in neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis (SWN). The 2016 edition constituted a blend of clinical and basic aspects of NF research that helped in clarifying different advances in the field. The incorporation of next generation sequencing is changing the way genetic diagnostics is performed for NF and related disorders, providing solutions to problems like genetic heterogeneity, overlapping clinical manifestations, or the presence of mosaicism. The transformation from plexiform neurofibroma (PNF) to malignant peripheral nerve sheath tumor (MPNST) is being clarified, along with new management and treatments for benign and premalignant tumors. Promising new cellular and in vivo models for understanding the musculoskeletal abnormalities in NF1, the development of NF2 or SWN associated schwannomas, and clarifying the cells that give rise to NF1-associated optic pathway glioma were presented. The interaction of neurofibromin and SPRED1 was described comprehensively, providing functional insight that will help in the interpretation of pathogenicity of certain missense variants identified in NF1 and Legius syndrome patients. Novel promising imaging techniques are being developed, as well as new integrative and holistic management models for patients that take into account psychological, social, and biological factors. Importantly, new therapeutic approaches for schwannomas, meningiomas, ependymomas, PNF, and MPNST are being pursued. This report highlights the major advances that were presented at the 2016 CTF NF conference.

[spa] Evidencias recientes han mostrado que los tumores sólidos pediátricos, incluidos el neuroblastoma y el meduloblastoma, albergan una baja cantidad de mutaciones genéticas recurrentes, pero presentan una proporción significativa de alteraciones recurrentes en los mecanismos epigenéticos. Además, la epigenética es un mecanismo regulatorio fundamental durante el desarrollo en mamíferos. En este proyecto hemos estudiado los cambios epigenéticos que afectan a dos tumores neuronales pediátricos prototípicos, el neuroblastoma y meduloblastoma. Mediante el estudio de los cambios de metilación del ADN, los miRNA y la expresión génica, hemos identificado modificaciones genéticas y epigenéticas que subyacen a la base molecular de la patogénesis y tienen implicación clínica en la evolución de estos tumores neurales. El objetivo principal de nuestro proyecto era identificar marcadores moleculares y posibles dianas de interés para el desarrollo de nuevas estrategias terapéuticas. Este proyecto fue el primer estudio completo de metilación del ADN en neuroblastoma empleando microarrays de alta densidad. Detectamos por primera vez en neuroblastoma la presencia de metilación del ADN en citosinas no-CpG, asociada a tumores de bajo riesgo clínico. El estado de metilación detectado en el contexto no-CpG indica un papel potencial de esta metilación en la diferenciación y regulación transcripcional de genes clave el en desarrollo, como por ejemplo ALK. Observamos que la metilación del ADN en el neuroblastoma afecta no sólo a los promotores, sino también a las regiones intragénicas e intergénicas tanto en sitios CpG como no-CpG en dominios funcionales de la cromatina de genes implicados en desarrollo y cáncer. Los cambios de metilación del ADN en sitos CpG y no-CpG tanto dentro como fuera de islas CpG (CGI) y localizados en el cuerpo del gen podian estar asociados con el comportamiento clínico del neuroblastoma. Detectamos cambios en contexto no-CpG que sugieren que el neuroblastoma puede ser epigenéticamente diferente a lo largo de la terapia. Además, analizando citosinas fuera de las regiones promotoras, en el cuerpo del gen y de las regiones intergénicas, en sitios CpG asociados a CGI y en sitios no-CpG dentro y fuera de CGIs observamos una asociación de la metilación con el comportamiento clínico en neuroblastoma. La segunda parte del proyecto se centró en tratar de dar respuesta a la creciente necesidad de aplicar en la práctica clínica el sistema de clasificación del meduloblastoma en los subgrupos moleculares WNT, SHH, Grupo 3 y Grupo 4, recientemente identificados y propuestos mediante análisis (epi)genómicos. El reconocimiento de los subgrupos consenso ha cambiado la forma en que se estudia el meduloblastoma en el contexto de la investigación y cómo se diagnostica y trata en el contexto clínico. Estos subgrupos moleculares se definen actualmente mediante robustos sistemas de clasificación basados técnicas difícilmente incorporables a la rutina clínica de la mayoría de hospitales que tratan tumores cerebrales infantiles. Hemos desarrollado una metodología clínicamente aplicable, robusta, rápida y reproducible para la predicción exacta de subgrupos de meduloblastoma de muestras individuales en base a dos paneles compuestos por conjuntos reducidos de biomarcadores epigenéticos, y por tanto, potencialmente incorporables en la rutina de diagnóstico. Los biomarcadores epigenéticos identificados podían aplicarse a muestras individuales de ADN de meduloblastoma obtenidas a partir de tejido congelado o FFPE. El patrón bimodal de los paneles permitía el análisis mediante diversas técnicas moleculares, tanto cuantitativas como cualitativas. Nuestro enfoque era técnicamente más simple, rápido y coste efectivo en comparación con las actuales técnicas estándar de clasificación de subgrupos de meduloblastoma., [eng] Recent evidence has shown that pediatric solid tumors harbor a paucity of recurrent genetic mutations as compared to other tumors from adults, suggesting that additional mechanisms such as epigenetic alterations may play an important role in the molecular pathogenesis of developmental tumors. So far the great majority of studies that have investigated DNA methylation in developmental tumors were biased towards CpG sites and promoter regions. Recent genome- wide studies are starting to reveal a role for DNA methylation outside such genomic contexts. Furthermore, it has recently been described that cytosine methylation also occurs in sites other than CpG dinucleotides (mCHG and mCHH) in embryonic stem cells and during brain development. However, DNA methylation at non-CpG contexts has rarely been described in cancer. We have analyzed the DNA methylome of two prototypical developmental neural tumors, neuroblastoma and medulloblastoma, using high-density microarrays with the aim of detecting epigenetic modifications at a genome-wide level that may have clinical relevance in the pathogenesis of these pediatric tumors, to identify molecular markers and potential targets of interest for the development of new therapeutic strategies. Our DNA methylation studies in neuroblastoma using high-density microarrays have defined the epigenetic landscape of this pediatric tumor and its potential clinicopathological impact. Our results reveal that: - DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at CpG sites and, for the first time in neuroblastoma, at non-CpG sites. - These epigenetic changes show a non-random distribution relative to functional chromatin states, and frequently target development and cancer-related genes relevant for neuroblastoma, such as CCND1 and ALK. - DNA methylation patterns in non-CpG sites provide new insights into the differentiation stage of high and low-risk neuroblastomas. - DNA methylation changes at CpG and non-CpG sites are strongly associated with clinicopathological features of neuroblastoma and with patient outcome. Furthermore, we have developed a simplified and reproducible approach to classify medulloblastoma tumors into clinically relevant subgroups applying epigenetic markers. Using this strategy, MB patients can be accurately classified into the three consensus subgroups WNT, SHH and non-WNT/non-SHH. In addition, we propose a similar approach for the specific classification of Group 3 and Group 4 medulloblastoma tumors. The proposed strategies allows for classification of single DNA samples from biopsies both frozen as well as FFPE of primary, metastasis or relapse compartments, using diverse molecular approaches. Our results show that the proposed strategy is robust, accurate, and cost-effective, making it adequate for molecular subgrouping of medulloblastoma in daily diagnostic practice of most centers treating children with brain tumors.

[eng] A new combined microdialysis – tumor homogenate method for the determination of compartmental (vascular, extra- and intracellular) SN-38 distribution in patient-derived xenografts (PDX) generated from pediatric solid tumors from fresh tumor samples from patients of Sant Joan de Deu Barcelona Hospital was developed. SN-38 in late-stage (chemoresistant) tumors presented limited distribution into the intracellular compartment while drug distribution into this compartment was significantly higher in early-stage (sensitive) models when SN-38 was administered as its prodrug irinotecan. Furthermore, two polymeric drug delivery systems were developed for the local and systemic administration of SN-38. Poly(lactic) acid electrospun nanofiber matrices with microcrystals of SN-38 were developed for the local administration of SN-38. Matrices showed maintained release of SN-38 over 48 h with local distribution and efficacy delaying tumor growth over PDX models. Dialysates showed limited SN-38 diffusion from the matrices through the tumor tissue, suggesting this therapy could only be useful for the local tumor control after successful surgery of the tumor or where only microscopic tumor seeds are left. Systemic administration of SN-38 was possible by encapsulating the drug into poly(lactic-co-glycolic) acid with polyethylene glycol nanoparticles, which were decorated with 3F8 monoclonal antibody, an anti-GD2 antibody that recognizes the ganglioside GD2 overexpressed on the surface of neuroblastoma cells surface for active targeting. Nanoparticles released SN-38 over 2 days and tumor exposition to SN-38 was increased when compared with the administration of an equimolar dose of irinotecan, and that was correlated with improved efficacy over the conventional irinotecan where 10 administrations of the drug had reduced efficacy compared to the direct administration of SN-38 in the targeted nanoparticles. Both nanofiber matrices and nanoparticles showed to be good platforms for SN-38 administration reducing systemic exposition by localizing SN-38 at the tumor microenvironment and significantly delaying tumor growth as shown in the efficacy studies. Thus, polymeric local drug delivery systems strategy should be of high interest for the potential future treatment of chemoresistant tumors

Renal malignancies are common in children. While the majority of malignant renal masses are secondary to Wilms tumor, it can be challenging to distinguish from more aggressive renal masses. For suspicious renal lesions, it is crucial to ensure prompt diagnosis in order to select the appropriate surgical procedure and treatment. This review article will discuss the common differential diagnosis that can be encountered when evaluating a suspicious renal mass in the pediatric population. This includes clear cell sarcoma of the kidney, malignant rhabdoid tumor, renal medullary carcinoma and lymphoma. Copyright: The Authors.

Treballs Finals de Grau d'Educació Social, Facultat d'Educació, Universitat de Barcelona, Curs: 2014-2015, Tutor: Jordi Méndez, Esta investigación pretende conocer la importancia de las buenas prácticas socioeducativas que puede aportar el educador social en el ámbito hospitalario, centrándome concretamente en la oncología pediátrica, debido a que es un campo emergente para la educación social. Por otro lado, también pretende elaborar algunas propuestas de mejora en la formación de los voluntarios de AFANOC (Associació de Familiars i Amics de Nens Oncològics de Catalunya). Como principal conclusión, se ha extraído que el educador social puede ser muy competente en el ámbito de la oncología pediátrica y puede realizar diferentes funciones que requieren de su presencia para mejorar la atención socioeducativa a los niños y sus familias. Por otro lado, también se ha podido ver que en la formación de los voluntarios es muy importante la formación continuada y obligatoria para todos los voluntarios de la entidad y las reuniones quincenales para compartir la experiencia y algunas dudas, especialmente los voluntarios del hospital, ya que es una situación más compleja.

[spa] El sarcoma de Ewing (SE) es el segundo tumor óseo maligno infantil más frecuente y presenta una alta incidencia de enfermedad metastásica. Este tipo de tumores presentan una translocación génica característica que da origen a una proteína de fusión, normalmente EWS/FLI1. Esta proteína de fusión actúa como factor de transcripción aberrante, regulando la expresión de diferentes genes implicados en la iniciación, mantenimiento y progresión del tumor. Nuestro grupo describió como uno de estos genes diana a caveolina 1 (CAV1), describiendo además su papel determinante en el fenotipo maligno del SE, en la tumorigénesis, en la angiogénesis y en la resistencia a apoptosis inducida por quimioterapia. Para investigar el papel concreto de CAV1 en el proceso metastásico de este sarcoma, se creó un modelo de baja expresión de CAV1 en líneas celulares de SE y se determinaron cambios en su capacidad migratoria, invasiva y metastásica. En los ensayos in vitro se halló una menor capacidad migratoria de las células con silenciamiento de CAV1 y una reducción en la expresión de metaloproteinasa 9 (MMP-9) y en la actividad de MMP-2, ambas MMPs implicadas en el proceso de invasión. La regulación de la actividad de MMP-2 parece estar relacionada con la posible regulación que ejerce CAV1 en la función de la MMP de membrana tipo 1 (MT1-MMP), proteína fundamental para la activación de MMP-2. Por otro lado, en este estudio se propone que CAV1 promueve la expresión de MMP-9 transcripcionalmente a través de la regulación de la activación de la vía de señalización de ERK1/2 (Extracellular signal-regulated kinase 1/2). En nuestro modelo, ERK1/2 activo translocaría al núcleo donde activaría los factores de transcripción responsables de la activación del promotor de MMP-9. Por otro lado, la fosforilación de RSK2 (Ribosomal S6 kinase 2) por parte de ERK1/2 en el citoplasma, produciría la activación de RSK2 que a su vez activaría la proteína ribosomal rpS6, uno de los responsables de la iniciación de la traducción, por lo que también podría estar influyendo a la producción de MMP-9 a este nivel. Según nuestros resultados, CAV1 estaría influyendo en la capacidad migratoria de las células de SE mediante dos mecanismos: 1) a través de la activación de la vía de ERK1/2 y 2) mediante la unión a diferentes proteínas con dominio SH2 a través de la fosforilación de CAV1 en la tirosina 14. ERK1/2 influye en la regulación de la capacidad migratoria de una forma de pendiente o independiente de RSK1. Además, en los ensayos de metástasis experimental in vivo las células con inhibición de CAV1 presentaron una menor incidencia de metástasis pulmonar, hecho que correlacionó con una disminución en la expresión de SPARC (Secreted protein acidic and rich in cysteine), una proteína de adhesión importante en procesos metastásicos. En resumen, nuestros resultados evidencian la importancia de CAV1 en el proceso metastásico del SE., [eng] Ewing’s sarcoma (ES) is the second most common bone tumor in childhood and occurs with a high incidence of metastatic disease. Such tumors have a characteristic gene translocation that gives rise to a fusion protein, most commonly EWS/FLI1. This fusion protein acts as an aberrant transcription factor regulating the expression of different target genes involved in the initiation, maintenance and progression of the tumor. Our group described caveolin 1 (CAV1) as one of these target genes, describing its role in the malignant phenotype, tumorigenicity and resistance to chemotherapy-induced apoptosis of ES cell lines. To investigate the specific role of CAV1 in the metastatic process of this sarcoma, we established a model of low expression of CAV1 in cell lines of ES. Then, we measured changes in their migratory capacity, invasiveness and metastatic potential. In vitro, we found a lower migratory capability of CAV1 knockdown cells and a reduction in MMP-9 expression and MMP-2 activity. The regulation of MMP-2 activity seems to be related to the possible regulation that CAV1 exerts on the function of MT1- MMP, an essential protein for the activation of MMP-2. On the other hand, we suggest that CAV1 promotes the expression of MMP-9, both transcriptionaly and post-transcriptionaly, through regulating ERK1/2 signaling pathway. In our model, activated ERK1/2 would translocate to the nucleus where it would activate the transcription factors responsible for MMP-9 promoter activation. At the cytoplasm, activated ERK1/2 would phosphorylate and activate RSK2, which, in turn, would promote rpS6 activation, leading to protein translation initiation. Our results indicate that CAV1 is regulating migratory capability of ES cells by two different mechanisms; 1) through ERK1/2 pathway activation, 2) by linking several proteins bearing SH2 domains trough phosphorylated Tyr14 of CAV1. ERK1/2 seems to regulate cell migration in both RSK1-dependent and independent manner. In addition, experimental metastasis assays in vivo showed that, CAV1 knockdown cells had a lower incidence of pulmonary metastasis, a fact that correlated with a decrease in the expression of SPARC, a major adhesion protein in metastatic processes. In summary, our results demonstrate the importance of CAV1 in the metastatic process on ES tumors.

The development of resistance to chemotherapeutic drugs is the main obstacle to the successful treatment of many cancers, including childhood neuroblastoma, the most common solid tumour of infants. One factor that may play a role in determining response of neuroblastoma tumours to therapeutic agents is the p53 tumour suppressor gene. A number of previous studies have suggested that this tumour suppressor protein is inactive in neuroblastoma due to its cytoplasmic sequestration. This thesis therefore has examined the functionality of p53 and its role in determining drug response of neuroblastoma cells. An initial study was undertaken that characterised an unusually broad multidrug resistance (MDR) phenotype of a neuroblastoma cell line (IMR/KAT100). The results demonstrated that the MDR phenotype of the IMR/KAT100 cells was associated with the acquisition of mutant p53. To explore the role of p53 in drug resistance further, p53-deficient variants in cell lines with wild-type p53 were generated by transduction of p53-suppressive constructs encoding either shRNA or a dominant-negative p53 mutant. Analysis of these cells indicated that: (i) in contrast to previous reports, wild-type p53 was fully functional in all neuroblastoma lines tested, as evidenced by its activation and nuclear translocation in response to DNA damage, transactivation of target genes and control of cell cycle checkpoints; (ii) inactivation of p53 in neuroblastoma cells resulted in establishment of an MDR phenotype; (iii) knockdown of mutant p53 did not revert the drug resistance phenotype, suggesting it is determined by loss of wild-type function rather than gain of mutant function; (iv) p53-dependent cell senescence, the primary response of S-type neuroblastoma cells to DNA damage, is replaced, after p53 inactivation, by mitotic catastrophe and subsequent apoptosis. In contrast to neuroblastoma, p53 suppression had no effect or increased drug susceptibility in several other tumour cell types, indicating the importance of tissue context for p53- mediated modulation of tumour cell sensitivity to treatment. Taken together, these data provide strong evidence for p53 having a role in mediating drug resistance in neuroblastoma and suggest that p53 status may be an important prognostic marker of treatment response in this disease.

Las neoplasias infantiles más frecuentes son...