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// Alfons Navarro 1 , Rut Tejero 1 , Nuria Vinolas 2 , Anna Cordeiro 1 , Ramon M. Marrades 3 , Dolors Fuster 1 , Oriol Caritg 1 , Jorge Moises 3 , Carmen Munoz 1 , Laureano Molins 5 , Josep Ramirez 4 , Mariano Monzo 1 1 Molecular Oncology and Embryology Laboratory, Human Anatomy Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain 2 Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncologiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain 3 Department of Pneumology, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain 4 Department of Pathology, Centro de Diagnostico Biomedico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain 5 Department of Thoracic Surgery, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain Correspondence to: Alfons Navarro, e-mail: anavarroponz@ub.edu Keywords: PIWI proteins, piwiRNAs, PIWIL1, PIWIL4, NSCLC Received: November 14, 2014 Accepted: December 21, 2014 Published: January 23, 2015 ABSTRACT The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1 -positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) ( p = 0.006) and overall survival (OS) ( p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue ( p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR ( p = 0.048) and OS ( p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Background: Assessing of the costs of treating disease is necessary to demonstrate cost-effectiveness and to estimate the budget impact of new interventions and therapeutic innovations. However, there are few comprehensive studies on resource use and costs associated with lung cancer patients in clinical practice in Spain or internationally. The aim of this paper was to assess the hospital cost associated with lung cancer diagnosis and treatment by histology, type of cost and stage at diagnosis in the Spanish National Health Service. Methods: A retrospective, descriptive analysis on resource use and a direct medical cost analysis were performed. Resource utilisation data were collected by means of patient files from nine teaching hospitals. From a hospital budget impact perspective, the aggregate and mean costs per patient were calculated over the first three years following diagnosis or up to death. Both aggregate and mean costs per patient were analysed by histology, stage at diagnosis and cost type. Results: A total of 232 cases of lung cancer were analysed, of which 74.1% corresponded to non-small cell lung cancer (NSCLC) and 11.2% to small cell lung cancer (SCLC); 14.7% had no cytohistologic confirmation. The mean cost per patient in NSCLC ranged from 13,218 Euros in Stage III to 16,120 Euros in Stage II. The main cost components were chemotherapy (29.5%) and surgery (22.8%). Advanced disease stages were associated with a decrease in the relative weight of surgical and inpatient care costs but an increase in chemotherapy costs. In SCLC patients, the mean cost per patient was 15,418 Euros for limited disease and 12,482 Euros for extensive disease. The main cost components were chemotherapy (36.1%) and other inpatient costs (28.7%). In both groups, the Kruskall-Wallis test did not show statistically significant differences in mean cost per patient between stages. Conclusions: This study provides the costs of lung cancer treatment based on patient file reviews, with chemotherapy and surgery accounting for the major components of costs. This cost analysis is a baseline study that will provide a useful source of information for future studies on cost-effectiveness and on the budget impact of different therapeutic innovations in Spain.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Deltaex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

// Francisco J. Cimas 1, * , Juan L. Callejas-Valera 2, * , Raquel Pascual-Serra 1 , Jesus Garcia-Cano 1 , Elena Garcia-Gil 1 , Miguel De la Cruz-Morcillo 1 , Marta Ortega-Muelas 1 , Leticia Serrano-Oviedo 1 , J. Silvio Gutkind 2 , Ricardo Sanchez-Prieto 1 1 Unidad de Medicina Molecular, laboratorio de Oncologia, Centro Regional de Investigaciones Biomedicas, Universidad de Castilla-La Mancha, 02006, Albacete, Spain Unidad de Biomedicina UCLM-CSIC 2 Moores Cancer Center/UCSD, La Jolla, CA 92093-0819, USA * These authors contributed equally to this work Correspondence to: Ricardo Sanchez-Prieto, e-mail: ricardo.sanchez@uclm.es Juan L. Callejas-Valera, e-mail: jlcallejasvalera@ucsd.edu Keywords: E1a, MKP1, cisplatin, chemotherapy, lung cancer Received: September 02, 2015 Accepted: November 25, 2015 Published: December 12, 2015 ABSTRACT The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells. In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene.

El carcinoma broncogénico (CB) representa la primera causa de mortalidad por cáncer en ambos sexos en los países desarrollados. La evaluación prequirúrgica del mediastino es esencial para definir el pronóstico y guiar el tratamiento de estos pacientes. Las guías para la estadificación ganglionar mediastínica preoperatoria de la European Society of Thoracic Surgeons (ESTS) recomiendan una exploración con certificación cito-histológica, ya sea con técnicas endoscópicas de punción guiada por ecografía o mediante una exploración quirúrgica del mediastino, en las siguientes situaciones: cuando la tomografía computerizada detecta ganglios mediastínicos de tamaño patológico, en tumores de localización central o cuando la tomografía por emisión de positrones (PET) muestra una hipercaptación mediastínica o hiliar. Así mismo, estas guías recomiendan la exploración y biopsia de, como mínimo, las siguientes estaciones ganglionares mediastínicas: paratraqueales inferiores derechas e izquierdas, y subcarínica, cuyo estudio puede llevarse a cabo mediante técnicas de punción eco-endoscópicas. Cuando no se dispone de estas técnicas o su resultado es negativo, debe realizarse una exploración quirúrgica del mediastino (principalmente una mediastinoscopia cervical estándar), por presentar un mayor valor predictivo negativo. En el caso de los tumores izquierdos también se recomienda explorar las estaciones ganglionares paraórtica y subaórtica, que no son accesibles a través de las técnicas de punción eco-endoscópica. Para llevar a cabo su exploración es necesaria una técnica quirúrgica como la mediastinotomía paraesternal izquierda, la mediastinoscopia cervical extendida (MCE) o la videotoracoscopia izquierda. La MCE permite explorar las estaciones paraórtica y subaórtica, utilizando la misma incisión cervical realizada para la mediastinoscopia cervical estándar, y por lo tanto, la vía transcervical permite una exploración completa del mediastino superior y de la ventana aorto-pulmonar. Los objetivos de la presente tesis, que se basa en dos artículos publicados y relacionados entre sí, son: evaluar los valores de estadificación obtenidos tras la aplicación de un protocolo prequirúrgico de evaluación ganglionar mediastínica basado en las guías de estadificación de la ESTS, evaluar la rentabilidad de la MCE en la estadificación del CB izquierdo y, finalmente, evaluar la supervivencia de aquellos pacientes intervenidos quirúrgicamente con afectación ganglionar mediastínica insospechada N2p. El primer trabajo es un estudio retrospectivo (1998-2010) de 221 MCE de estadificación realizadas en pacientes con CB izquierdo. Se analizó la precisión diagnóstica de la técnica y del protocolo en 2 períodos: 1998-2003 con MCE sistemática y 2004-2010 con PET sistemática y MCE selectiva. Los resultados obtenidos mostraron que la MCE posee una elevada precisión diagnóstica (sensibilidad: 0.67; valor predictivo negativo: 0.94; exactitud diagnóstica: 0.95) para la detección clínica de N2. Su uso selectivo permite reducir la MCE en cerca del 30 % de los pacientes con sin disminuir el valor predictivo negativo del protocolo en ambos períodos. El segundo trabajo es un estudio retrospectivo (2004-2010) de 540 pacientes estadificados y operados siguiendo las guías de la ESTS. Se analizaron los valores de estadificación y la supervivencia del subgrupo de pacientes con resultado final N2p. Los resultados obtenidos mostraron que tras la aplicación de un protocolo prequirúrgico basado en las guías de la ESTS se consigue unos valores de estadificación elevados (valor predictivo negativo: 0.91) con una baja tasa de falsos negativos (5.5%). Los pacientes con resultado final N2p insospechado presentan una mejor supervivencia (supervivencia a los 5 años del 40%) que la esperada en comparación con la observada en los pacientes quirúrgicos de la base de datos de la International Association for the Study of Lung Cancer (IASLC) utilizada para la 7ª edición de la clasificación TNM. En conclusión, las guías de la ESTS deben implementarse en la práctica clínica a la luz de los buenos resultados de estadificación y supervivencia., Preoperative mediastinal staging is essential to predict prognosis and to guide treatment in patients with non-small cell lung cancer (NSCLC). The European Society of Thoracic Surgeons (ESTS) guidelines for preoperative mediastinal nodal staging recommend tissue confirmation when computed tomography shows enlarged nodes, in central tumors or when positron emission tomography (PET) shows an increased mediastinal or hilar uptake. The recommendation is to explore the right and left inferior paratracheal, and subcarinal stations. In left-sided tumors, the exploration of subaortic and paraortic nodal stations is also recommended. This can be done by extended cervical mediastinoscopy (ECM). This thesis is based on two published and related articles, the objectives of which are: to evaluate the staging values after applying a preoperative mediastinal staging protocol according to the ESTS guidelines, to evaluate the accuracy of ECM for staging left-sided NSCLC and to evaluate the survival of surgically treated patients with unsuspected mediastinal pathological (p) N2 disease. The first publication is a retrospective study that includes 221 ECM for staging left NSCLC. The accuracy of the technique and of the protocol were calculated in two periods: 1998-2003 with systematic ECM and 2004-2010 with systematic PET and selective ECM. The results of this study show a high diagnostic accuracy of ECM (sensitivity: 0.67; negative predictive value: 0.94; accuracy: 0.95). Its selective indication allows reducing ECM in near 30% of the patients without reducing the negative predictive value of the protocol. The second publication is a retrospective study (2004-2010) including 540 patients staged and operated following the ESTS guidelines. The results of this study show that after applying the ESTS guidelines, high staging values (negative predictive value: 0.91) and low false negative rate (5.5%) are observed. Survival rate in patients with unsuspected pN2 disease was higher (5-year survival rate of 40%) than expected, compared with survival of surgical patients with pN2 of the International Association for the Study of Lung Cancer (IASLC) database used to inform the 7th edition of TNM classification. Therefore, the ESTS guidelines on preoperative mediastinal nodal staging should be used in clinical practice.

El gen supresor tumoral LKB1/STK11b, codifica una serina/treonina quinasa. Las mutaciones de línea germinal en LKB1 están asociadas al síndrome de Peutz-Jeghers (PJS), trastorno dominante autosómico caracterizado por el crecimiento de pólipos en el tracto gastrointestinal, y depósitos de melanina mucocutánea, aumentando el riesgo de ciertos cánceres, incluido el de pulmón. El locus de PJS se encuentra situado en el cromosoma 19p13.3 y el gen responsable fue identificado como LKB1. Las mutaciones del gen LKB1 también están asociadas a los adenocarcinomas de pulmón, ocurren aproximadamente en un tercio de los tumores primarios y en la mitad de las líneas celulares de adenocarcinoma de pulmón, implicando a LKB1 en la patogénesis del cáncer de pulmón. Estudios recientes sugieren que la vía de señalización de LKB1 juega un papel en la protección de las células frente a la apoptosis, en respuesta a niveles de energía o nutrientes bajos, lográndose en parte a través de la supresión de la actividad de mTOR. Hay evidencias en la bibliografía que demuestran que LKB1 activa al complejo heterotrimerico AMPK, formado por tres subunidades, una catalítica (α) y dos reguladoras (β y γ). AMPK es un sensor de energía celular que contribuye a regular el balance energético y la ingesta calórica en la célula. Posteriormente, AMPK fosforila a TSC2. El heterodímero TSC1-TSC2 actúa para inhibir la actividad de mTOR, que es esencial para el control del crecimiento celular y la proliferación. Así, LKB1 funciona como un regulador negativo de mTOR. Está demostrado que LKB1 también regula 11 de las 12 quinasas de la subfamilia de quinasas AMPK in vitro, sugiriendo que una de las funciones del gen supresor LKB1 podría ser la regulación de la vía de señalización de AMPK. Por tanto, LKB1 podría ser un blanco potencial para el tratamiento de cáncer y trastornos metabólicos. Nuestros principales objetivos fueron: ▪ Determinar la frecuencia de alteraciones genéticas y epigenéticas en la vía de señalización LKB1/AMPK/TSC2 en CPCNP, en un panel de 23 líneas celulares (4 líneas celulares epiteliales normales bronquiales inmortalizadas con SV40 y 19 líneas celulares de CPCNP). ▪ Estudiar el estado de metilación de 4 quinasas de la subfamilia de quinasas AMPK: BRSK1, BRSK2, MARK1 MARK4 en 23 líneas celulares. ▪ Examinar la vía LKB1/BRSK2. ▪Analizar el estado de metilación de BRSK2 en 58 FFIP de pacientes con CPCNP. ▪ Determinar el impacto de la pérdida de LKB1 en líneas celulares de CPCNP. Las principales conclusiones fueron: ▪ Las mutaciones de LKB1 no se limitan a los adenocarcinomas, también ocurren en otro subtipo de CPCNP, los carcinomas de células grandes. No se encontró metilación en el promotor de LKB1 en ninguna de las líneas celulares de cáncer pulmón analizadas en este estudio. ▪ No se detectaron mutaciones ni ningún grado de metilación en los distintos componentes estudiados de AMPK y TSC en las 23 líneas celulares analizadas. ▪ De la subfamilia de quinasas de AMPK analizadas, el estado de metilación del promotor BRSK2 representa el de mayor porcentaje: ~ 26% de las líneas celulares y 25,8% del grupo de 58 FFIP de pacientes con CPCNP. ▪ El estado de metilación de BRSK2 se correlaciona significativamente con cuatro parámetros clinicopatológicos: estadio tumoral (P = 0, 025), histología (P = 0, 001), tamaño tumoral (P = 0, 073) y nódulos afectados (P = 0, 04), sugiriendo que el estado de metilación del promotor BRSK2 podría utilizarse como un nuevo biomarcador en la progresión del cáncer de pulmón. ▪ La interrupción de la vía LKB1/BRSK2 podría ser un importante mecanismo molecular, durante el desarrollo del cáncer de pulmón., LKB1, also known as STK11b, is a serine/threonine kinase that functions as a suppressor of tumor growth. Germ line mutations in LKB1 are associated with the Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by benign hemartomas of the gastrointestinal tract and mucocutaneous melanin deposits and an increased risk of certain cancers, including lung. The main PJS locus has been mapped to chromosome 19p13.3, and the gene responsible was identified as LKB1. Mutations in LKB1 gene are also associated with sporadic lung adenocarcinomas and occurs in approximately one-third of primary tumors and half of lung adenocarcinoma cells lines, implicating LKB1 in the pathogenesis of lung cancer. Recent evidence suggests that LKB1 signaling plays a role in protecting cells from apoptosis in response to energy or nutrient deprivation, which is achieved in part through the suppression of mTOR activity. Evidence from the literature supports a protein kinase cascade model in which LKB1 phosphorylates AMPK. AMPK is a heterotrimeric complex comprising a catalytic subunit (α) and two regulatory subunits (β and γ) that act as an intracellular energy sensor maintaining the energy balance within the cell. AMPK subsequently phosphorylates TSC2. Together the TSC1-TSC2 heterodimer acts to suppress the activity of mTOR, which is essential for the control of cell growth and proliferation. Thus, LKB1 functions as a negative regulator of mTOR. Additionally, LKB1 has been shown to regulate 11 of the 12 AMPK family members in vitro, suggesting that one of the tumour suppressor functions of LKB1 may be regulation of AMPK signaling. Therefore, LKB1 could be a potential target for treatment of both cancer and metabolic disorders. In this work we sought to study the signal transduction LKB1/AMPK/TSC2 pathway alterations that contribute to the pathogenesis NSCLC. Our major objectives were: ▪To determine the frequency of genetic and epigenetic alterations in the signal traduction LKB1/AMPK/TSC2 pathway in NSCLC in a panel of 23 cell lines (4 normal bronchial epithelial cell lines immortalized with SV40 and 19 NSCLC cell lines). ▪ To study some components of AMPK-related kinase family that could be activated downstream of LKB1, in particular we analyzed the methylation status of: BRSK1, BRSK2, MARK1, MARK4 in our panel of 23 cell lines. ▪ To examine LKB1/BRSK2 signaling.▪ To make a clinical validation of BRSK2 methylation status, in 58 FFPE of patients with NSCLC. ▪ To determinate the impact of LKB1 depletion on NSCLC cell lines. The major conclusions of this work were: ▪ LKB1 mutations are not confined to adenocarcinomas, they also occur in other NSCLC subtype such as large cell carcinomas. But we found no evidence of LKB1 methylation in any NSCLC cell lines used in this study. ▪ There are not sequence alterations or/and promoter methylation of AMPK and TSC components studied in our 23 panel cell lines. ▪ Of the four AMPK related kinases -BRSK1, BRSK2, MARK1 and MARK4- studied, BRSK2 represents the highest percentage of methylation; ~26 % of NSCLC cell lines and 25.8% in the 58 NSCLC samples. ▪ BRSK2 methylation is significantly correlated with four clinicopatological parameters: tumor stage (P=0,025), histology (P=0,001), tumor size (P=0,073) and nodules affected (P=0,04), suggesting that methylation of BRSK2 could provide a novel biomarker for disease progression in lung cancer. ▪ The disruption of LKB1/BRSK2 signaling could be important in the carcinogenesis of lung, as a molecular mechanism for the development of lung cancer.

El càncer de pulmó afecta 1,6 milions de persones a tot el món, i és la primera causa de mort per càncer. El promig de l’índex de supervivència als 5 anys per tots els càncers està al voltant del 50%, mentre que en el cas del càncer de pulmó no arriba a un 10%, és un dels més baixos. El mal pronòstic del càncer de pulmó es deu al diagnòstic en fases avançades i a la manca de teràpies específiques pels seus subtipus. Tot i així ja hi ha teràpies basades en alteracions genètiques que es donen específicament en un tipus histològic i que estan funcionant de manera exitosa. Aquests avenços posen en relleu la importància del coneixement de les bases moleculars del càncer des d’un punt de vista de la biologia del càncer i la necessitat de la classificació dels tumors segons les alteracions genètiques que pateixen. Per tot això, en aquesta tesi doctoral s’ha fet una cerca de nous gens supressors tumorals que estan implicats en càncer de pulmó. Partint de la informació de número de còpies de la base de dades de Sanger, s’han buscat línies cel•lulars de càncer de pulmó amb delecions homozigotes en possibles gens supressors tumorals. Després d’escollir alguns dels candidats i confirmar les delecions que patien, s’ha fet un screening de mutacions en els gens SMAD2, PARD3 i PIK3R1. El gen SMAD2 està alterat en càncer de pulmó però amb una freqüència molt baixa, doncs no s’han trobat mutacions addicionals a la deleció homozigota de partida (Sanger Database). El treball amb el gen PARD3 ha permès la identificació de delecions intragèniques i homozigotes i de mutacions puntuals en tumors primaris i línies cel•lulars de pulmó. S’ha detectat una major incidència d’inactivació de PARD3 en tumors del subtipus histològic escamós, on la freqüència d’inactivació és del 8%. Algunes de les mutacions s’han estudiat fenotípicament. Algunes proteïnes PAR3 mutants no permeten la formació dels contactes entre cèl•lules veïnes mitjançant la reducció de la capacitat de formar unions estretes i protrusions d’actina. La senyalització corrent avall de PAR3 també està afectada en alguns mutants, ja que perden la capacitat d’unió a aPKC i activació de RAC1. A més, també s’ha descobert que la proteïna PAR3 salvatge activa l'expressió de transcrits relacionats amb l'adhesió cel•lular i la polaritat; així com dianes transcripcionals de STAT3. S’ha vist que PAR3 activa STAT3 sota condicions de confluència cel•lular. Finalment, la restitució de PAR3 en models in vivo redueix significativament la formació de metàstasis. Pel que fa al gen PIK3R1, la freqüència mutacional trobada en càncer de pulmó ha estat del 3,5%. En aquest cas, s’ha vist que una manca de la proteïna codificada per aquest gen (p85alfa) en línies cel•lulars de càncer de pulmó no activa constitutivament la via de AKT. Contràriament a l’esperat, la manté menys activa comparant-se amb altres línies cel•lulars de càncer de pulmó sense aquesta alteració. Per altra banda, la reintroducció de la proteïna p85alfa en línies deficients disminueix l’expressió de transcrits de la via de PI3K que són protumorogènics. En conclusió, en aquesta tesi doctoral s’han descobert dos nous gens supressors tumorals implicats en la carcinogènesi pulmonar i s’han establert les bases moleculars per les quals potencien l’oncogènesi., Lung cancer is the leading cause of cancer death and has very bad prognosis. Despite that, nowadays, there are therapies based on genetic alterations that are running successfully. These developments highlight the importance of understanding the molecular basis of cancer and the need to classify tumors according to their genetic alterations. Therefore, the aim of this thesis has been the search for new tumor suppressor genes that are involved in lung cancer. Based on the copy number information from the Sanger database, we have selected genes with homozygous deletions in lung cancer cell lines to be candidate tumor suppressor genes. After choosing some candidates and confirming the deletions they suffer we made mutational screenings. We identified homozygous intragenic deletions and mutations in primary tumors and cell lines of lung cancer at PARD3 gene. We detected a higher incidence of PARD3 inactivation in the squamous histological subtype, where the inactivation rate is 8%. Some of the mutations found were studied phenotypically. Some PAR3 mutant proteins do not allow the formation of contacts between neighboring cells by reducing the ability to form tight junctions and actin protrusions. Signaling downstream of PAR3 is also affected in some mutants, as they lose the ability to bind aPKC and to activate RAC1. In addition, we have discovered that the wild type PAR3 protein activates the expression of transcripts related to cell adhesion and cell polarity, as well as transcriptional targets of STAT3. We have shown that PAR3 activates STAT3 especially under conditions of high cell density. Finally, the recovery of PAR3 in tumors produced in two mouse models significantly reduces metastasis formation. The mutation frequency found for PIK3R1 gene in lung cancer was 3.5%. The lack of the protein encoded by this gene (p85alpha) in lung cancer cell lines does not maintain constitutively active the AKT pathway. Contrary to the expectations, this pathway is less active in cell lines without p85alpha expression compared to other lung cancer cell lines without this alteration. Furthermore, the reintroduction of p85alpha protein in deficient lines decreases the expression of transcripts of the PI3K pathway that are protumorogenic. In conclusion, in this thesis we have discovered two new tumor suppressor genes involved in lung carcinogenesis.

El propósito de esta Tesis Doctoral es el de explorar nuevas posibilidades de tratamiento y de diagnóstico del cáncer de pulmón, y concretamente del cáncer de pulmón no microcítico de células con mutaciones activadoras de KRAS. Para conseguirlo, las finalidades de esta Tesis incluyen la determinación de la actividad biológica de nuevos compuestos organometálicos con actividad antitumoral, la caracterización de la reprogramación tumoral en líneas de cáncer de pulmón en la búsqueda de nuevas dianas terapéuticas de tipo metabólico y el desarrollo de un método diagnóstico no invasivo con la potencialidad de detectar el cáncer de pulmón en muestras de aire exhalado. En la realización de la misma se han llevado a cabo los siguientes objetivos: 1- Se ha determinado la actividad biológica de nuevos complejos metálicos sobre la viabilidad de células tumorales de pulmón. a) Se ha caracterizado la actividad antiproliferativa de nuevos compuestos cicloplatinados de siete miembros. b) Se ha caracterizado la actividad antiproliferativa de nuevos compuestos de platino con iminas polifuncionalizadas. c) Se ha caracterizado la actividad antiproliferativa de nuevos compuestos de platino y paladio derivados del pirazol. d) Se ha caracterizado la actividad antiproliferativa de nuevos complejos ferroceno-indol. e) Se ha caracterizado la actividad antiproliferativa de nuevos compuestos de platino(II) derivados del ferroceno. 2- Se ha determinado el mecanismo de acción del compuesto de platino(II) derivado del ferroceno 6a sobre líneas celulares de cáncer de pulmón, y se ha evaluado su potencial como agente antitumoral. 3- Se han caracterizado las adaptaciones metabólicas asociadas a células de cáncer de pulmón mutadas en KRAS, y se ha explotado el estudio de esta reprogramación en el desarrollo de nuevas terapias combinadas. 4- Se ha desarrolladoun nuevo sistema de análisis de aire exhalado mediante GC-MS, con potencial para diagnosticar el cáncer de pulmón y la enfermedad pulmonar obstructiva crónica. La consecución de estos objetivos permite abrir nuevos caminos en la lucha contra la elevada mortalidad del cáncer de pulmón, tanto desde el punto de vista terapéutico como desde el punto de vista diagnóstico., The purpose of this Thesis is to explore new possibilities in the treatment and diagnostic of lung cancer, and especially of non-small cell lung cáncer with activating mutations in KRAS. In order to do so, the objectives of this Thesis include the determination of the biological activity of new metallic compounds with antitumor activity, the characterization of lung cancer metabolic reprogramming in order to find new metabolic therapeutic targets, and the development of a non-invasive diagnostic method able to detect the presence of lung cancer in exhaled air samples. The following goals have been achieved in this Thesis: 1- The biological activity of new metalic compounds over lung cáncer cells viability has been determined. a) The antiproliferative activity of new seven-membered cycloplatinated complexes has been characterized. b) The antiproliferative activity of new platinum compounds with polyfunctional imines has been characterized. c) The antiproliferative activity of new platinum and palladium platinum compounds derived from pyrazol has been characterized. d) The antiproliferative activity of new ferrocene-indole hybrids has been characterized. e) The antiproliferative activity of new platin(II) compounds derived from ferrocene has been characterized. 2- The mechanism of action of the antiproliferative activity of new platin(II) compounds derived from ferrocene 6a over lung cancer cell lines has been characterized. 3- The metabolic reprogramming of KRAS-mutated lung cancer cell lines has been characterized, and the study of this reprogramming has been exploited in the development of new antiproliferative therapies. 4- A new system for the analysis of volatile metabolites in breath samples has been developed, in order to diagnose diseases like lung cáncer and COPD in a non-invasive way. The achievement of these goals allows the definition of new paths in the fight against lung cancer’s high mortality rate, from either a therapeutic or a diagnostic point of view.

We present a 53-year-old man with a vocal cord paralysis observed as a primary manifestation of lung carcinoma. Tc-99m MDP whole body bone scan was performed and resulted in a normal scintiscan. The bone scan did not reveal any suspicious foci of uptake. The possibility of bone metastasis was taken into consideration. A whole body F18-FDG-PET scan showed intense uptake in the left upper lung corresponding to the primary tumor. A bronchial biopsy confirmed infiltration by small cell lung carcinoma (SCLC). SCLC is composed of poorly differentiated, rapidly growing cells with diseases usually occurring centrally rather than peripherally. It metastasizes early. The whole-body F18-FDG-PET scan clearly demonstrated a focus of increased uptake in the second lumbar vertebral body suspicious for osteolytic metastasis. A lytic bone metastasis was confirmed by MRI. The patient then received therapy and underwent follow up abdominal CT. The scan showed blastic changes in the L2 vertebra suggesting response to treatment.

Background: Atypical carcinoids (AC) of the lung are rare intermediate-grade neuroendocrine neoplasms. Prognostic factors for these tumours are undefined. Methods: Our cooperative group retrieved data on 127 patients operated between 1980 and 2009 because of an AC. Several clinical and pathological features were studied. Results: In a univariable analysis, T-status (p=0.005), N-status (p=0.021), preoperative M-status (previously treated) (p=0.04), and distant recurrence developed during the outcome (p

Resumen En el ano 2011 se inicio un proyecto conjunto entre la Sociedad Espanola de Oncologia Medica (SEOM) y la Sociedad Espanola de Anatomia Patologica (SEAP) para establecer unas recomendaciones basadas en la evidencia actual con respecto a la determinacion de biomarcadores en pacientes con carcinoma de pulmon de celula no pequena (CPCNP) avanzado. La mayoria de estas recomendaciones siguen siendo validas; sin embargo, existen nuevas evidencias que hacen necesaria la actualizacion de algunos aspectos. En concreto, se modifica la recomendacion de que biomarcadores hay que analizar y en que pacientes, y se define el manejo optimo de la muestra tumoral asi como las caracteristicas del material minimo necesario para la determinacion de biomarcadores. Ademas, se revisan las tecnicas adecuadas para la determinacion de las mutaciones de EGFR y el reordenamiento de ALK , y se consensua en que situaciones se debe llevar a cabo una re-biopsia.

[spa]Durante los últimos años se ha visto que los microRNAs (miRNAs) han emergido como prometedores marcadores moleculares en múltiples tipos de cáncer, incluyendo el cáncer de pulmón de célula no pequeña (CPCNP) y el cáncer colorrectal (CRC), dos modelos tumorales de mismo origen endodérmico, que hemos estudiado en este trabajo. Los miRNAs son moléculas de RNA de 19 a 25 nucleótidos no codificantes, que inhiben la traducción a proteína. La familia miR-200 está formada por cinco miembros clasificados según su posición cromosómica en dos clúster: cluster 1 que incluye el miR-200a, miR-200b y miR-429 localizados en el cromosoma 1 y cluster 2 que incluye el miR-200c y miR-141, localizados en el cromosoma 12. Su expresión está asociada a la regulación de los procesos de la transición epitelio-mesénquima (TEM) y la transición mesénquima-epitelio (TME). El trabajo presentado nos ha permitido valorar la utilidad de esta familia como marcador pronóstico en dos modelos tumorales diferentes y conocer así el comportamiento de cada uno de los miembros que la componen. En el estudio de muestras de pacientes de CPCNP, obtuvimos que niveles altos de miR-200c se asociaban a una menor supervivencia global (SG). Esto se mantuvo con el grupo histológico adenocarcinoma, juntamente con niveles altos de miR-141. El estudio in vitro con tres líneas celulares de CPCNP, de subtipos adenocarcinoma, H23, A-549 y HCC-44, nos permitió observar que miR-200c inducía una menor migración celular y que miR-141 reducía los niveles de la proteína KLF6, promoviendo un incremento de la secreción de VEGFA, que correlaciona con una mayor densidad de vasos en el tejido tumoral de los pacientes. En el estudio de muestras de pacientes de CCR los niveles altos de miR-200a y miR-200c se asociaron a una mayor SG, mientras que los niveles altos de miR-429 correlacionaban con una mayor SG y supervivencia libre de enfermedad (SLF). En el grupo de pacientes tratados con fluoropirimidinas también observamos una mayor SG en aquellos casos con niveles altos de miR-200a/c/141 y miR-429. Estos resultados se mantuvieron en el grupo de pacientes con estadio II, en los que el beneficio del tratamiento con fluoropirimidinas aún no ha sido demostrado. Lo que nos permitiría determinar los pacientes susceptibles a recibir tratamiento. En este modelo tumoral se observó la participación de esta familia en la TEM y la influencia in vitro de 5-fluoracil, que se evaluó in vitro en la línea celular LOVO de CCR. Estos resultados indican que diferentes mecanismos pueden estar involucrados en la participación de esta familia de miRNAs en la tumorogénesis y como el estudio de esta familia así como de sus moléculas dianas nos puede llevar a una mejor comprensión de los modelos tumorales y probablemente la búsqueda de terapias contra esta familia de miRNAs y utilizar estos miRNAs como arma terapéutica contra el cáncer., [eng] MicroRNAs (miRNAs) have emerged as promising molecular markers in multiple cancers, including non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). The members of the miR-200 family of miRNAs (miR-200a/b/c, miR-141 and miR-429) are associated with the regulation of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). In the present study, we have examined the role of the miR-200 family in NSCLC and CRC. We have correlated the expression of the miR-200 family members in tumor samples with patient clinocopathological characteristics and outcome and have investigated the effect on tumor cells in vitro. In patients with NSCLC, high levels of miR-200c were associated with shorter overall survival (OS), and in the subset of patients with adenocarcinoma, high levels of miR-141 were also associated with shorter OS. In NSCLC cell lines, increased miR-200c induced less cell migration and overexpression of miR-141 led to a reduction of KLF6 and an increase of VEGF-A secretion, which correlates with a higher density of vessels in tumor tissue. In patients with CRC, high levels of miR-200a, miR-200c and miR-429 were associated with longer OS, but only high miR-429 levels were correlated with prolonged disease-free survival (DFS). However, in the subset of stage II patients treated with fluoropyrimidines, high levels of miR-200a/c/141/429 were associated with longer OS. The benefit of treatment with fluoropyrimidines has not yet been demonstrated in stage II CRC, and our findings may help identify patients likely to benefit from fluoropyrimidine treatment. Finally, in a CRC cell line, we found that these miRNAs played a role in EMT and modulated the effect of 5-fluorouracil. Our results indicate that different mechanisms are involved in the activity of the miR-200 family in tumorogenesis and can help in the development of new therapeutic targets and in the identification of patients most likely to benefit from treatment.

Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare clinical entity where tumor cell embolisms in pulmonary circulation induce thrombotic microangiopathy (TMA), respiratory failure, and subacute cor pulmonale. We describe 3 cases of PTTM that presented as the initial manifestation of metastatic gastric adenocarcinoma with TMA and pulmonary infiltrates. All 3 cases had similar clinical and laboratory features, which included moderate thrombocytopenia without renal failure, hemolysis with extremely high serum lactate dehydrogenase levels, leukoerythroblastosis in peripheral blood smear, altered coagulation tests, lymphadenopathies, and interstitial pulmonary infiltrates. All patients died within 2 weeks of diagnosis. Two cases were initially misdiagnosed as idiopathic thrombotic thrombocytopenic purpura and treated with plasma exchange with no response. One patient had bone marrow infiltration by malignant cells. Autopsies revealed PTTM associated with gastric disseminated adenocarcinoma (signet-ring cell type in 2 patients and poorly differentiated type in 1). PTTM should be considered in the differential diagnosis of patients with fulminant microangiopathic hemolytic anemia, such as atypical thrombotic thrombocytopenic purpura, mainly those with pulmonary infiltrates, disseminated intravascular coagulation, or Trousseau syndrome.

Fundacion Mutua Madrilena; Fondo de Investigaciones Sanitarias [PI11/02866], Conde, E., Suárez-Gauthier, A., Benito, A., Garrido, P., García-Campelo, R., Biscuola, M., Paz-Ares, L., Hardisson, D., De Castro, J., Camacho, M.C., Rodriguez-Abreu, D., Abdulkader, I., Ramirez, J., Reguart, N., Salido, M., Pijuán, L., Arriola, E., Sanz, J., Folgueras, V., Villanueva, N., Gómez-Román, J., Hidalgo, M., López-Ríos, F.

[cat] El desenvolupament del càncer de pulmó de cèl•lula no petita (CPCNP) és un procés amb múltiples etapes en el qual s’acumulen tant alteracions genètiques com epigenètiques. Com alteració epigenètica la metilació de “novo” dels promotors gènics es pot utilitzar com a eina de diagnòstic, factors pronòstics, i com a marcadors predictius de resposta al tractament. Per estudiar l’estat de metilació, els mètodes basats en la PCR són els més utilitzats en l’estudi d’un sol locus. No obstant això, la majoria d’aquests mètodes està subjecte a la conversió del DNA per Bisulfit sòdic, afectant al rendiment de l’amplificació i comportant una conversió incompleta del DNA i comportant falsos positius. Aquest fet fa que la translació a la pràctica clínica d’aquestes metodologies es vegi dificultada per la incertesa associada a la metodologia emprada. Però malgrat això, la prospecció de biomarcadors en aquest àmbit ajudarà a discernir el perquè alguns malalts amb adenocarcinoma pulmonar (ADC) estadi I es curen amb la cirurgia, mentre d’altres tenen una alta probabilitat de recidiva. En aquesta tesi hem realitzat la nostra tècnica d’anàlisi amb un anàlisis de PCR amb dues etapes combinant-la amb un disseny específic dels primers a utilitzar. Aquesta metodologia, que hem anomenat “Bisulfite Specific – Melting Curve Analysis (BS-MCA)”, ofereix una elevada sensibilitat sense comprometre la especificitat i permet eliminar el fals positiu que es produeix per conversions incomplertes mitjançant el Bisulfit sòdic. A través d’aquesta tècnica hem avaluat un panell de marcadors de metilació en l’ADC establert per diferents gens supressors de tumors (p16, CDH13, IGFBP3, RASSF1A, APC i TMS1) i de miRNAs (miR-148a i miR-34b/c) associats a l’aparició de metàstasis, sobre una sèrie retrospectiva de biòpsies d’ADC corresponents a diferents estadis de la malaltia. Demostrant arrel dels resultats obtinguts que la presència d’hipermetilació en els promotors de miR-34b/c i CDH13 s’associa a la agressivitat tumoral en ADC per al conjunt dels estadis i que l’estat d’hipermetilació de miR-34b/c sol o en combinació amb miR-148a és un marcador molecular pronòstic independent en els ADC d’estadis I i II., [eng]The non-small cell lung cancer (NSCLC) development is a multi-stage process where the genetic alterations accumulate as much as epigenetic. As an epigenetic alteration, the “de novo” methylation of the genes promoters can be useful as a diagnostic tool, prognosis factor and a predictive marker for treatment response. The PCR-based methods are the most used in order to assess the methylation status of a single locus. Despite this, most of these methods are subject to the DNA conversion by the Sodium bisulfite which affects the performance of the amplification and this fact can entail an incomplete DNA conversion and involves false positives. This means that the translation of these methods to the clinical practice is hampered by the associated uncertainty of the methodological approach. However, the biomarker research in this field would help to discern why some patients with lung adenocarcinoma (ADC) stage I are recovered with surgery while others have a high probability of relapse. We have based our assessment method on a two-steps PCR combined with a specific design of primers that offers a great sensitivity without compromising the specificity. This method, which we named "Bisulfite Specific – Melting Curve Analysis (BS-MCA)", avoids the false positive results which occur by the incompletes conversions for Sodium bisulfite. Through this technique we evaluated a panel of methylation markers in ADC conformed by different tumor suppressor genes (p16, CDH13, IGFBP3, RASSF1A, APC and TMS1) and miRNAs (miR-148a and miR-34b/c) related to the appearance of metastases. The sample set was comprised by a retrospective series of ADC biopsies that correspond to different stages of the disease. We have demonstrated across our assessment that the presence of hypermethylation in the promoters of miR-34b/c and CDH13 is associated with tumor aggressiveness in ADC for all the stages and that the hypermethylation status of miR-34b/c single or in combination with miR-148a is an independent prognostic marker in ADC on stages I and II.

Purpose Non–small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. Patients and Methods A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. Results Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). Conclusion The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Using non-small cell lung carcinoma (NSCLC) cells harboring the erlotinib-sensitizing Epidermal Growth Factor Receptor (EGFR) exon 19 mutation delE746-A750, we developed erlotinib-refractory derivatives in which hyperactive Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling associated with enrichment in epithelial-to-mesenchymal transition (EMT)-related morphological and transcriptional features. We then explored whether an IGF-1R/EMT crosstalk was sufficient to promote erlotinib refractoriness in the absence of second-site EGFR mutations, MET and AXL hyperactivation. Transforming Growth Factor-beta1 (TGF beta 1)-induced mesenchymal trans-differentiation was sufficient to impede erlotinib functioning in the presence of drug-sensitive delE746-A750 EGFR mutation. Pharmacological blockade of IGF-1R fully prevented the TGF beta 1's ability to activate an EMT protein signature [E-cadherin low/vimentin high]. The sole presence of erlotinib was capable of rapidly activate an IGF-1R-dependent, vimentin-enriched mesenchymal-like phenotype in delE746-A750-mutated epithelial cells. Even if transient, NSCLC cells' intrinsic plasticity to undergo crosstalk between IGF-1R and EMT signaling pathways can sufficiently eliminate the erlotinib-sensitizing effect of highly prevalent EGFR mutations and suggests the urgent need for dual IGF-1R/EMT-targeting strategies to circumvent erlotinib resistance.

The flavolignan silibinin was studied for its ability to restore drug sensitivity to EGFR-mutant NSCLC xenografts with epithelial-to-mesenchymal transition (EMT)-driven resistance to erlotinib. As a single agent, silibinin significantly decreased the tumor volumes of erlotinib-refractory NSCLC xenografts by approximately 50%. Furthermore, the complete abrogation of tumor growth was observed with the co-treatment of erlotinib and silibinin. Silibinin fully reversed the EMT-related high miR-21/low miR-200c microRNA signature and repressed the mesenchymal markers SNAIL, ZEB, and N-cadherin observed in erlotinib-refractory tumors. Silibinin was sufficient to fully activate a reciprocal mesenchymal-to-epithelial transition (MET) in erlotinib-refractory cells and prevent the highly migratogenic phenotype of erlotinib-resistant NSCLC cells. Given that the various mechanisms of resistance to erlotinib result from EMT, regardless of the EGFR mutation status, a water-soluble, silibinin-rich milk thistle extract might be a suitable candidate therapy for upcoming clinical trials aimed at preventing or reversing NSCLC progression following erlotinib treatment.

Johansson, Mattias Roberts, Angus Chen, Dan Li, Yaoyong Delahaye-Sourdeix, Manon Aswani, Niraj Greenwood, Mark A Benhamou, Simone Lagiou, Pagona Holcatova, Ivana Richiardi, Lorenzo Kjaerheim, Kristina Agudo, Antonio Castellsague, Xavier Macfarlane, Tatiana V Barzan, Luigi Canova, Cristina Thakker, Nalin S Conway, David I Znaor, Ariana Healy, Claire M Ahrens, Wolfgang Zaridze, David Szeszenia-Dabrowska, Neonilia Lissowska, Jolanta Fabianova, Eleonora Mates, Ioan Nicolae Bencko, Vladimir Foretova, Lenka Janout, Vladimir Curado, Maria Paula Koifman, Sergio Menezes, Ana Wunsch-Filho, Victor Eluf-Neto, Jose Boffetta, Paolo Franceschi, Silvia Herrero, Rolando Fernandez Garrote, Leticia Talamini, Renato Boccia, Stefania Galan, Pilar Vatten, Lars Thomson, Peter Zelenika, Diana Lathrop, Mark Byrnes, Graham Cunningham, Hamish Brennan, Paul Wakefield, Jon McKay, James D eng 1R03DE020116/DE/NIDCR NIH HHS/ R01 CA092039 05/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2012/06/05 06:00 PLoS One. 2012;7(5):e36888. doi: 10.1371/journal.pone.0036888. Epub 2012 May 25.; International audience; BACKGROUND: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. METHODS: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest―the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. RESULTS: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5x10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. CONCLUSION: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config).

Anaplastic lymphoma kinase (ALK) rearrangements represents a new driver oncogenic event in non-small cell lung cancer (NSCLC). ALK positive patients account for a 1-7% of NSCLC patients. The objective of this study is to know the prevalence and clinical characteristics of ALK positive patients in a cohort of NSCLC patients and to compare inmunohistochemistry with D5F3 monoclonal antibody with gold standard method fluorescence in situ hybridation Les translocacions del gen de la quinasa del linfoma anaplàssic (ALK) suposen una nova alteració oncogénica en el càncer de pulmó de cèl.lula no petita (CPNM). Els pacients ALK positius suposen un 1-7% dels pacients amb CPNM. L’objectiu d’aquest estudi és conèixer la prevalença y les característiques clíniques dels pacients ALK positius en una cohort de pacients amb CNMP i comparar la técnica de detecció per immunohistoquímica amb l’anticòs D5F3 amb el mètode diagnòstic habitual, la hibridació in situ amb fluorescència amb una sonda de break appart.

Objectives: We aimed at extending the Natural and Orthogonal Interaction (NOIA) framework, developed for modeling gene-gene interactions in the analysis of quantitative traits, to allow for reduced genetic models, dichotomous traits, and gene-environment interactions. We evaluate the performance of the NOIA statistical models using simulated data and lung cancer data. Methods: The NOIA statistical models are developed for additive, dominant, and recessive genetic models as well as for a binary environmental exposure. Using the Kronecker product rule, a NOIA statistical model is built to model gene-environment interactions. By treating the genotypic values as the logarithm of odds, the NOIA statistical models are extended to the analysis of case-control data. Results: Our simulations showed that power for testing associations while allowing for interaction using the NOIA statistical model is much higher than using functional models for most of the scenarios we simulated. When applied to lung cancer data, much smaller p values were obtained using the NOIA statistical model for either the main effects or the SNP-smoking interactions for some of the SNPs tested. Conclusion: The NOIA statistical models are usually more powerful than the functional models in detecting main effects and interaction effects for both quantitative traits and binary traits.

The objective of the present review is to study the relationship between flavonoids and lung cancer, proposing that their regular consumption in Western diets could be beneficial for protecting patients against lung cancer.An extensive search of the scientific literature was performed in the following electronic specialized databases (PubMed central (PMC)-NBCI, Elsevier Journal, SciELO Spain, Scirus, Science Direct), including studies in animals, cells, and humans, in order to establish the effect of flavonoids in the prevention and development of lung cancer.Although in vitro and animal studies show the potential ability of flavonoids to act against different types of cancers, especially against lung cancers, the diverse results reported within epidemiological studies, together with the lack of experiments in humans, are the major factors in limiting making dietary recommendations based on scientific evidence for the management of patients with lung cancer. Therefore, the authors of the present study recommend following the dietary health practice guidelines which promotes the consumption of food enriched in flavonoids and reflects the current state of knowledge of an effective and appropriate diet in lung cancer patients. El objetivo de la presente revisión es estudiar la relación entre los flavonoides y el cáncer de pulmón, postulando que su consumo habitual en la dieta occidental podría resultar beneficioso para proteger a los pacientes contra el cáncer de pulmón. Se realizó una extensa búsqueda de publicaciones científicas recientes en las siguientes bases de datos electrónicas especializadas: PubMed central (PMC)-NCBI, Elsevier Journal, SciELO España, Scirus y Science Direct, incluyendo estudios en células, animales y humanos sobre el efecto de los flavonoides en la prevención y el desarrollo de cáncer de pulmón. Pese a que los estudios in vitro y en modelos animales muestran la capacidad potencial de los flavonoides para actuar contra diferentes tipos de cáncer, y en especial contra el riesgo de padecer cáncer de pulmón, la presencia de datos contradictorios en los estudios epidemiológicos y la falta de estudios clínicos en humanos actualmente impiden realizar recomendaciones dietéticas basadas en la evidencia científica para los pacientes con cáncer de pulmón. Así, los autores del presente artículo recomiendan seguir los consejos de las guías de alimentación saludable, las cuales promocionan el consumo de alimentos ricos en flavonoides y reflejan el conocimiento necesario para tratar a los pacientes con cáncer de pulmón.

Among all inflammatory cells involved in COPD, those with a cytolytic or elastolytic activity are thought to play a key role in the pathogenesis of the disease. However, there is no data about the infiltration of cells expressing the CD57 marker in small airways and parenchyma of COPD patients. In this study, surgical specimens from 43 subjects undergoing lung resection due to lung cancer (9 non-smokers, 18 smokers without COPD and 16 smokers with moderate COPD) and 16 patients undergoing double lung transplantation for very severe COPD were examined. CD57+ cells, neutrophils, macrophages and mast cells infiltrating bronchioles (epithelium, smooth muscle and connective tissue) and parenchymal interstitium were localized and quantified by immunohistochemical analysis. Compared to the other groups, the small airways of very severe COPD patients showed a significantly higher density of CD57+ cells, mainly infiltrated in the connective tissue (p=0.001), and a significantly higher density of neutrophils located characteristically in the epithelium (p=0.037). Also, the density of neutrophils was significantly higher in parenchyma of very severe COPD patients compared with the rest of the groups (p=0.001). Finally, there were significant correlations between the bronchiolar density of CD57+ cells and the FEV1 values (R=-0.43, p=0.022), as well as between the parenchymal density of neutrophils and macroscopic emphysema degree (R=0.43, p=0.048) in COPD groups. These results show that CD57+ cells may be involved in COPD pathogenesis, especially in the most severe stages of the disease

El objetivo de la presente revisión es estudiar la relación entre los flavonoides y el cáncer de pulmón, postulando que su consumo habitual en la dieta occidental podría resultar beneficioso para la protección de los pacientes frente el cáncer de pulmón. L'objectiu d'aquesta revisió és estudiar la relació entre els flavonoides i el càncer de pulmó, postulant que el seu consum habitual en la dieta occidental podria ser beneficiós per a la protecció dels pacients davant el càncer de pulmó. The objective of the present review is to study the relationship between flavonoids and lung cancer, proposing that their regular consumption in Western diets could be beneficial for protecting patients against lung cancer.

[spa] El análisis de la expresión global y de número de copias de DNA en tumores primarios y líneas celulares de cáncer de pulmón reveló la presencia de amplificación génica en los cromosomas 11q12, 13q34 y 6p22. En los cromosomas 11q12 y 13q34 se identificaron ocho y tres genes sobre‐expresados, respectivamente; globalmente, la expresión de los transcritos del amplicón 13q34 era bastante superior a la del amplicón 11q12. Debido a su función biológica y a su relevancia en otros tipos de cáncer se seleccionaron los genes CTNND1 y TFDP1, en los cromosomas 11q12 y 13q34 respectivamente, para estudios posteriores. Estudiamos los niveles de expresión de CTNND1 y TFDP1 mediante inmunohistoquímica en tumores primarios de cáncer de pulmón no microcítico. El 1,4% y 2,7% de los tumores, todos de tipo escamoso, presentaron los niveles más altos de expresión de p120ctn y DP1 codificadas, por los genes CTNND1 y TFDP1 respectivamente. El 10% de los tumores tenían una localización citosólica aberrante de p120ctn. Por otra parte, el gen CTNND1 codifica diferentes isoformas de p120ctn por splicing alternativo. En general, los tumores están enriquecidos en la isoforma 3 de p120ctn. Se interfirió la expresión de DP1 mediante siRNA y se midió la proliferación celular mediante ensayos de MTT. Se observó que la inhibición de la expresión de DP1 reduce alrededor de un 50% la viabilidad de células con amplificación de TFDP1 mientras que no afecta a las células sin DP1 sobre‐expresado. El gen SOX4 fue el único sobre‐expresado respecto a tejido pulmonar normal en la línea celular con amplificación en 6p22. El 3,5% de las líneas celulares y el 6% de los tumores primarios tenían sobre‐expresión de SOX4. Se encontró que tanto SOX4 como SOX2 y SOX11 estaban sobre‐expresados específicamente en células de cáncer de pulmón microcítico. Dentro de los tumores de cáncer de pulmón no microcítico, SOX2 y SOX9 estaban sobre‐expresado en carcinomas escamosos respecto a adenocarcinomas y SOX4 presentaba un patrón contrario. El análisis de mutaciones de SOX4 en cáncer de pulmón reveló dos variantes germinales en tumores primarios de pulmón, una de ellas se trataba de un polimorfismo presente en la población española y una mutación somática que codifica para una proteína truncada. En las líneas celulares se encontraron dos variantes de cambio de aminoácido y una deleción en pauta. La capacidad de transactivación de SOX4 no cambió en las formas no truncadas respecto a la forma salvaje, mientras que la forma truncada estaba incapacitada para activar la transcripción. Mediante un ensayo de cooperación oncogénica con RHOA‐Q63L y HRAS‐G12V se observó que la expresión creciente de la forma salvaje y las formas no truncadas de SOX4 incrementaban significativamente la habilidad oncogénica de RHOA‐Q63L y no afectaba la capacidad transformante de HRAS‐G12V mientras que la forma truncada reducía el número de focos transformantes originados por HRAS‐G12V. Mediante la depleción de SOX4 se identificaron nuevos genes regulados directa o indirectamente por este factor de transcripción, muchos de ellos relacionados con el desarrollo neural. A su vez, estos genes se sobre‐expresaban tras la expresión ectópica de SOX4 en líneas celulares y en tumores primarios de pulmón con altos niveles de SOX4. Por otro lado, estos genes tenían una expresión reducida en fibroblastos embrionarios de ratones Sox4/. Finalmente, se demostró el reclutamiento de SOX4 en los promotores de los genes MYB, VASH2, TEAD2 y TUBB3 y en la región de control del cluster de PCDHBs. La firma de expresión génica de SOX4 resultó compartir muchas similitudes con la de cáncer de pulmón microcítico, de origen neuroendocrino, por lo que hay una alta implicación de SOX4 en el desarrollo de este tipo de cáncer., [eng] The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. To screen for amplicons, we aligned the gene expression data according to the position of transcripts and searched for clusters of over‐expressed genes. We confirmed the presence of two amplicons, at chromosomes 11q12 and 13q34. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q34 amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line with TFDP1 amplification and protein over‐expression reduced cell viability by 50%. Then, we analyzed DNA copy number and mRNA expression levels in lung cancer cell lines and identified the 6p22.3 amplicon. The SOX4 gene was overexpressed in cells with amplification relative to normal cells. SOX4 expression was also stronger in a fraction of lung primary tumours and lung cancer cell lines. We also found variants of SOX4 in lung primary tumours and cancer cell lines, including a somatic mutation that introduced a premature stop codon. Overexpression of the wildtype and of the non‐truncated variants in NIH3T3 cells significantly increased the transforming ability of the weakly oncogenic RHOA‐Q63L. Ablating SOX4 expression in SOX4‐amplified lung cancer cells revealed a gene expression signature that included genes involved in neuronal development such as PCDHB, MYB, RBP1 and TEAD2. Interestingly, we found that small cell lung cancer was generally characterized by high levels of SOX2, SOX4 and SOX11 along with the SOX4‐specific gene expression signature identified. In conclusion, we report the identification of three novel amplicons, at 13q34, 11q12 and 6p22.3. TFDP1 is a candidate oncogene at 13q34. Within the 6p22 amplicon, SOX4 is overexpressed in lung cancer and we provide evidence of its oncogenic properties. We also identify a functional role for SOX genes and several SOX4 novel targets in SCLC.

BACKGROUND: The Cancer Fast-track Programme's aim was to reduce the time that elapsed between well-founded suspicion of breast, colorectal and lung cancer and the start of initial treatment in Catalonia (Spain). We sought to analyse its implementation and overall effectiveness. METHODS: A quantitative analysis of the programme was performed using data generated by the hospitals on the basis of seven fast-track monitoring indicators for the period 2006-2009. In addition, we conducted a qualitative study, based on 83 semistructured interviews with primary and specialised health professionals and health administrators, to obtain their perception of the programme's implementation. RESULTS: About half of all new patients with breast, lung or colorectal cancer were diagnosed via the fast track, though the cancer detection rate declined across the period. Mean time from detection of suspected cancer in primary care to start of initial treatment was 32 days for breast, 30 for colorectal and 37 for lung cancer (2009). Professionals associated with the implementation of the programme showed that general practitioners faced with suspicion of cancer had changed their conduct with the aim of preventing lags. Furthermore, hospitals were found to have pursued three specific implementation strategies (top-down, consensus-based and participatory), which made for the cohesion and sustainability of the circuits. CONCLUSION: The programme has contributed to speeding up diagnostic assessment and treatment of patients with suspicion of cancer, and to clarifying the patient pathway between primary and specialised care.

[spa] 1) INTRODUCCIÓN El carcinoma pulmonar, en nuestro medio, es la neoplasia de mayor incidencia en varones y la tercera en mujeres, con una elevada letalidad y baja prevalencia, ya que tiene una tasa de mortalidad (49,2 casos/100.000), muy cercana a la de incidencia (55,8 casos/100.000). El factor pronóstico más relevante es el estadio patológico de la enfermedad (pTNM). Otros menos relevantes son los dependientes del propio paciente como el estado funcional, edad, sexo, parámetros biológicos sanguíneos o las mutaciones de oncogenes. Existen además otros factores, relacionados con el propio tumor, como es el grupo histológico, el tamaño tumoral, el grado de diferenciación celular del tumor y el Standerdized Uptatke Values (SUVmax) tumoral medido en la Tomografía de Emisión de Positrones (PET). En un intento de disponer de otros indicadores pronósticos, no evaluados en el TNM, han surgido estudios sobre el valor de los receptores de proliferación celular, concretamente el antígeno Ki-67. Se trata de una proteína nuclear sintetizada por la propia célula tumoral, valorada como un indicador de proliferación celular del carcinoma broncogénico y factor pronóstico del cáncer de pulmón. 2) OBJETIVOS Principal: Determinar la concordancia del antígeno Ki-67, con los factores de mayor valor pronóstico: grupo histológico, pTNM, tamaño, SUVmax, y el grado de diferenciación celular del tumor. Secundario: Determinar la relación entre el antígeno Ki-67 tumoral, la incidencia de la recidiva y mortalidad postquirúrgica. 3) MATERIAL Y MÉTODO Entre agosto de 2006 y junio de 2008, tras la aplicación de los criterios de exclusión, se han seleccionado consecutivamente 61 pacientes intervenidos de una neoplasia pulmonar con un seguimiento de 12 a 34 meses. Criterios de inclusión - Diagnostico de un carcinoma pulmonar. - Estadificación clínica (PET-TC). - Resección pulmonar mínima lobar y linfadenectomía. Criterios de exclusión - Tumores con componente inflamatorio/infeccioso. - Tumores menores de 1 centímetro. - Carcinomas pulmonares de célula pequeña. - Antecedente de neoplasia no tratada. - Resección pulmonar sublobar y/o sin linfadenectomía completa. - Resecciones pulmonares no radicales. - Pacientes con tratamiento neoadyuvante. - Pacientes con mortalidad perioperatoria. - Pacientes sin seguimiento postoperatorio. Variables a estudio - Edad. - Sexo. - Tabaquismo. - Antecedentes oncológicos. - Diagnóstico de otra neoplasia durante el seguimiento. - Localización anatómica del tumor. - Resección pulmonar prevista y realizada. - Índice del antígeno Ki-67. - Histología tumoral. - Grado de diferenciación celular del tumor. - cTNM y pTNM. - SUVmax tumoral y adenopatías. - Tamaño tumoral (PET). - Tamaño tumoral (pieza quirúrgica). - Tratamiento adyuvante. - Recurrencia de la enfermedad. - Supervivencia global. Metodologia estadistica Tras un análisis descriptivo de tendencia central y de dispersión, así como frecuencias, se ha realizado un análisis multivariado del índice Ki-67, incidencia de recidiva y mortalidad. 4) RESULTADOS Y CONCLUSIONES El antígeno Ki-67 tumoral, esta asociado de manera estadísticamente significativa, a los factores de mayor valor pronóstico del carcinoma pulmonar: histología del tumor, tamaño tumoral, SUVmax, grado de diferenciación celular del tumor y estadio patológico. El carcinoma escamoso de tamaño superior a 4 centímetros, pT2, bajo grado de diferenciación celular y SUVmax superior a 7 gr/ml, se asocia a un índice Ki-67 superior al 25%. El antígeno Ki-67, sin asociarse de forma estadísticamente significativa a la incidencia de recidiva, tiempo libre de enfermedad, mortalidad y tiempo de supervivencia, tiene significación clínica, puesto que con un índice Ki-67 superior al 25%, se registra el doble de recidivas y su aparición 6 meses antes, el triple de fallecimientos secundario a la progresión de la enfermedad, así como vivir 4 meses menos que los casos con un índice Ki-67 inferior al 25%., [eng] 1) INTRODUCTION Lung cancer has a high case-fatality rate. Prevalence is low because of the similarity between incidence and mortality rates (55,8 and 49,2 cases/100.000, respectively). Pathological stage (pTNM) is considered the most relevant prognostic factor for survival in this disease, although several other tumor-related factors, including histological group, tumor size, degree of cellular differentiation, and the Standardized Uptake Values (SUVmax) as measured by Positron Emission Tomography (PET), are also important. Attempts to identify new prognostic indicators have led to the discovery of cell proliferation receptors, in particular, the Ki-67 antigen. Ki-67 is a marker of cell proliferation in bronchogenic carcinoma and is considered a prognostic factor. 2) OBJECTIVES Main objectives: To determine the agreement between the Ki-67 antigen and the best prognostic factors: histologic group, pTNM, tumor size, tumor SUVmax, and degree of cellular differentiation. Secondary objectives: To determine the association between the Ki-67 antigen and rates of tumor recurrence and postoperative mortality. 3) MATERIAL AND METHODS Between August 2006 and June 2008, 173 patients with lung cancer underwent surgery at our institution. Of these, 61 fulfilled the inclusion criteria and were included in this study. These patients were followed for a period ranging from 12 to 34 months. Statistical analysis included a descriptive analysis of central tendency and dispersion, as well as frequencies. This was followed by a multivariate analysis of the Ki-67 index, and relapse and mortality rates. 4) RESULTS AND CONCLUSIONS The Ki-67 tumor antigen is significantly associated with the factors that have the greatest prognostic value in lung carcinoma: histology group, tumor size, tumor SUVmax, degree of cellular differentiation, and pathological stage. The Ki-67 antigen is not, however, significantly associated with relapse rates, disease-free interval, mortality, or survival time. Nevertheless, Ki-67 provides clinically relevant information when the index is above 25%; when compared to patients with a Ki-67 < 25%, relapse rates were twice as high, tumor recurrence occurred 6 months earlier, there were 3 times as many deaths secondary to disease progression, and survival was 4 months less.

Purpose: To investigate the hypothesis that non-steroidal anti-inflammatory drugs (NSAIDs) lower lung cancer risk. Methods: We analysed pooled individual-level data from seven case-control and one cohort study in the International Lung Cancer Consortium (ILCCO). Relative risks for lung cancer associated with self-reported history of aspirin and other NSAID use were estimated within individual studies using logistic regression or proportional hazards models, adjusted for packyears of smoking, age, calendar period, ethnicity and education and were combined using random effects meta-analysis. Results: A total of 4,309 lung cancer cases (mean age at diagnosis 65 years, 45% adenocarcinoma and 22% squamous-cell carcinoma) and 58,301 non-cases/controls were included. Amongst controls, 34% had used NSAIDs in the past (81% of them used aspirin). After adjustment for negative confounding by smoking, ever-NSAID use (affirmative answer to the study-specific question on NSAID use) was associated with a 26% reduction (95% confidence interval 8 to 41%) in lung cancer risk in men, but not in women (3% increase (-11% to 30%)). In men, the association was stronger in current and former smokers, and for squamous-cell carcinoma than for adenocarcinomas, but there was no trend with duration of use. No differences were found in the effects on lung cancer risk of aspirin and non-aspirin NSAIDs. Conclusions: Evidence from ILCCO suggests that NSAID use in men confers a modest protection for lung cancer, especially amongst ever-smokers. Additional investigation is needed regarding the possible effects of age, duration, dose and type of NSAID and whether effect modification by smoking status or sex exists. © 2011 Springer Science+Business Media B.V.

1) Hipòtesi El càncer de pulmó és la neoplàsia més freqüent en el moment actual i és la primera causa de mort per càncer en el món. Aproximadament el 85 % dels pacients presenten CPCNP. Inclús els casos identificats en estadis precoços i en què es pot realitzar un tractament quirúrgic curatiu, tenen un risc de recaiguda global del 60%. A pesar dels avenços en el tractament de la malaltia realitzats durant els darrers anys, la supervivència global continua essent molt baixa, al voltant del 15 % als 5 anys. És per aquest motiu que es fa necessària la troballa de biomarcadors que puguin predir el risc de recaiguda, una vegada realitzada la resecció quirúrgica. Aquests biomarcadors ens permetrien estratificar els pacients pel seu risc i individualitzar el seguiment i el tractament de la malaltia, especialment en els pacients en estadi I que no reben cap tractament adjuvant. La majoria de pacients amb CPNCP són fumadors i durant els últims anys s’ha observat que variants polimòrfiques d’enzims relacionats amb la metabolització de carcinògens del tabac, en gens de reparació del DNA o en gens relacionats amb l’expulsió de carcinògens de la cèl•lula s’han associat a un risc augmentat de càncer de pulmó. No obstant, l’impacte d’aquests SNPs en la recaiguda del CPCNP no ha estat establert. Si tenim en compte que els SNPs són característiques innates dels individus, podríem pensar que la seva presència podria tenir un efecte també en la recaiguda de pacients fumadors que han desenvolupat un càncer de pulmó, ja que les diferencies genotípiques es podrien traduir en diferències en el procés de malignització mediat pel tabac. Aquests SNPs doncs podrien seleccionar-nos individus que han tingut un risc incrementat de càncer de pulmó i que, una vegada realitzat un tractament local quirúrgic, podrien tenir un risc augmentat de recidivar. Per altre banda recentment els miRNAs han esdevingut com a elements claus en el procés de carcinogènesi. Els miRNAs són molècules que controlen la diferenciació i el creixement cel•lular i en diferents tipus tumorals s’associen a l’evolució de la malaltia. Polimorfismes en gens relacionats amb miRNAs implicats en càncer o en els propis miRNAs poden jugar un paper en la progressió tumoral. Més concretament, polimorfismes en gens de la via de processament dels miRNAs o en la seqüència del pri-miRNA o pre-miRNA poden afectar als nivells finals de determinats miRNAs. A més, polimorfismes en les seqüències d’anclatge de miRNAs poden reprimir la inhibició dels gens diana per aquests i per tant, afectar la seva funció. En definitiva, polimorfismes relacionats amb els miRNAs podrien modificar el risc de recaiguda després de la cirurgia en pacients amb CPCNP. Basant-nos en aquestes hipòtesis ens plantegem els següents objectius: 2) Objectiu general Trobar biomarcadors de recaiguda i supervivència global en pacients quirúrgics de CPCNP basant-nos en l’estudi de les variants polimòrfiques del DNA. 3) Objectius específics 1. Analitzar, en una sèrie retrospectiva de pacients amb CPCNP quirúrgics, polimorfismes en gens relacionats amb el metabolisme del tabac (fase I i fase II), gens de reparació del DNA i en el gen MDR1/ABCB1 en pacients amb hàbit tabàquic. 2. Analitzar polimorfismes presents en regions del DNA codificants per miRNAs relacionats amb proliferació, apoptosi, angiogènesi i cicle cel•lular en una sèrie retrospectiva de pacients amb CPCNP quirúrgics. 3. Analitzar polimorfismes presents en els gens que intervenen en el procés de maduració dels miRNAs en una sèrie retrospectiva de pacients amb CPCNP quirúrgics. 4. Analitzar polimorfismes localitzats al lloc d’anclatge del miRNA al mRNA en una sèrie retrospectiva de pacients amb CPCNP quirúrgics. 5. Establir si existeix relació entre els diferents genotips i el temps a la recaiguda o la supervivència global.

[spa] El cáncer de pulmón no célula pequeña es la neoplasia más frecuente en la actualidad en los países industrializados, siendo la primera causa de mortalidad por cáncer en el varón. Su incidencia continúa aumentando progresivamente, y su tratamiento, pese a los esfuerzos de investigación de los últimos años, sigue siendo poco efectivo en la mayor parte de los casos, situándose la supervivencia global a los cinco años alrededor del 13%. La alta tasa de recaída, incluso en estadios iniciales susceptibles de cirugías radicales, justifica el interés de estudiar marcadores pronósticos de supervivencia y recaída. Esto nos ayudaría a distinguir grupos de riesgo, cuyo interés radica en ayudarnos a conocer la necesidad y efecto de los tratamientos adyuvantes, con quimioterapia y radioterapia así como abrir la puerta a posibles nuevas armas terapéuticas. Dada la alta incidencia de alteraciones en la vía de p53 en el cáncer de pulmón no célula pequeña, y su probable valor pronóstico, nuestra hipótesis es que existen alteraciones en los niveles de expresión de los miRNAs directamente regulados por p53, así como en los reguladores intermedios de la función de p53, con posible valor pronóstico en recaída y supervivencia de pacientes operados de cáncer de pulmón no célula pequeña o no microcítico(CPNCP). Tratamos pues de identificar el papel que juegan los miRNAs como marcadores útiles en el pronóstico del CPNCP en estadios iniciales, tras cirugía radical. De forma más específica determinar el posible papel pronóstico en recaída y en supervivencia de los miRNAs de la familia de miR-34: miR-34a, miR-34b y miR-34c, cuya transcripción es activada por p53. También definir el posible papel pronóstico en recaída y en supervivencia de los miRNAs miR-16 y miR-143, cuyos niveles finales en la célula, están modulados por p53. Así como, conocer si existen posibles interacciones entre miR-34a, miR-34b, miR-34c, miR-16 y miR-143 a nivel pronóstico. Para ello, hemos planteado un estudio retrospectivo, con las muestras de tejido tumoral y normal pareado de 70 pacientes, diagnosticados y tratado, de cáncer de pulmón no microcítico estadios I-III, entre Febrero de 1996 y Septiembre de 2002, en el Hospital Clínic de Barcelona. Se realizaron la extracción y cuantificación de RNA. Determinación de los niveles de los microRNAs mencionados. Determinación del estado de metilación de la región promotora del gen MIR34A. Determinación de la presencia de mutaciones de P53 entre los exones 5-8. Tras estos estudios los resultado obtenidos se han publicado en dos artículos. El primero de ellos en la revista “Carcinogenesis” y el segundo en la revista “Journal of Surgical Oncology”. En el primer artículo, titulado “miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer”, relacionamos los niveles de miR-34a con la recaída tumoral, estableciendo tres grupos en función de esta tasa de recaída: el grupo con niveles bajos de miR34a, con un 67% de recaída, los que tenían niveles altos, con un 43% de recaída y los que tenían los niveles más altos con un 0% de recaída. Además, en el análisis univariado para riesgo de recaída, el estado mutacional de P53, los niveles de miR-34a y el estadio IA vs el resto, se correlacionaron con probabilidad de recaída. Se realizaron dos análisis mutilvariados, incluyendo o no el estado mutacional de P53, permaneciendo en ambos análisis la expresión de miR-34a, como factor independiente para recaída tumoral. Se observó que los pacientes con mutaciones de P53 presentaban una media de expresión de miR-34a más baja. Se observó que el subgrupo de pacientes en el que los niveles de expresión de miR-34a baja coincidían con presencia de mutaciones de P53, presentaban una alta tasa de recaída. En los pacientes sin mutaciones de de P53, existe una diferencia significativa en los niveles de expresión de miR-34a entre los pacientes que presentaban la región promotora de MIR34A metilada vs los que no. Por todo esto, encontramos que miR-34a se demuestra como un nuevo marcador biológico con significación en el pronóstico de la recaída de pacientes sometidos a cirugía del CPNCP, abriendo la posibilidad de una futura herramienta en el algoritmo de decisiones terapéuticas. En el segundo artículo, titulado “Prognostic Implications of miR-16 Expression Levels in Resected Non-Small-Cell Lung Cancer”, desde la hipótesis de que P53 activa la transcripción de la familia de miRNAs de miR-34 y regula la maduración de miR-16 y miR143, se estudiaron los niveles de expresión de miR-143 y miR-16 en el tejido normal y tumoral pareado de cada paciente de la serie, antes mencionada, de 70 pacientes. Así los pacientes se clasificaron de acuerdo a los niveles de expresión de miR-16(alto, normal y bajo). Aquellos pacientes con niveles normales tuvieron la mejor evolución, mientras que aquellos con niveles más altos tuvieron la peor. La supervivencia libre de enfermedad (SLE) era de 22,4 meses para los pacientes con niveles altos de miR-16, 71,8 meses para los de niveles normales y de 55,8 meses para los de niveles bajos. La supervivencia global (SG) fue de 23,9 meses para los que tenían niveles altos de miR-16, de 97,6 meses para los que tenían niveles normales y 63,5 meses para aquellos con niveles bajos. No se observó correlación entre los niveles de miR-143 y la evolución clínica de los pacientes. El análisis multivariado mostró a miR-16 como factor pronóstico independiente de SLE Y SG. En un análisis secundario, examinamos la correlación potencial entre la expresión de miR-16 y miR-34a y el estado mutacional de P53.No hubo correlación entre el estado mutacional de P53 y miR-16, pero si se observó una interacción entre miR-16 y miR-34a. En los pacientes con niveles altos de miR-34a, se observaron diferencias entre SLE Y SG, de acuerdo a los niveles de expresión de miR-16, mientras que en los pacientes con niveles bajos de miR-34a presentaban un pobre pronóstico, independientemente de los niveles de expresión de miR-16. Estos resultados nos indican el posible valor pronóstico de miR-16 en pacientes intervenidos de CPNCP, además del posible sinergismo entre miR-34a y miR16., [eng] Non small cell lung cancer is more common today, in industrialized countries and is the leading cause of cancer death in men. Its incidence continues to increase gradually, and their treatment, despite the research efforts of recent years, it remains ineffective in most cases, putting the overall survival at five years about 13%. The high rate of relapse, even in early stages susceptible to radical surgery, justifies the interest in studying prognostic markers of survival and relapse. This would help us distinguish risk groups, whose interest lies in helping to meet the need and effect of adjuvant treatment with chemotherapy and radiotherapy as well as opening the door to possible new therapeutic tools. Given the high incidence of alterations in the p53 pathway in non small cell lung cancer, and likely its prognostic role, our hypothesis is that there are alterations in the expression levels of miRNAs directly regulated by p53, as well as in intermediate regulators of p53 function with potential prognostic value on relapse and survival of patients undergoing non small cell lung cancer (NSCLC). Then, we try to identify the role of miRNAs as useful markers for the prognosis of NSCLC in initial stages, after radical surgery. More specifically determine the possible prognostic role in relapse and survival of the family miRNAs miR-34: miR-34a, miR-34b and miR-34c, whose transcription is activated by p53. Also define the possible prognostic role in relapse and survival of the miRNAs miR-16 and miR-143, whose final levels in the cell are modulated by p53. And, determine whether there are possible interactions between miR-34a, miR-34b, miR-34c, miR-16 and miR-143 as regards prognosis. To do this, we have proposed a retrospective study, with the tumor tissue and paired normal in 70 patients, diagnosed and treated of non small cell lung cancer stages I-III, between February 1996 and September 2002, at the Hospital Clínic, in Barcelona. We performed RNA extraction and quantification. Determining levels of microRNAs mentioned. Determining the status of methylation of the promoter region of the gene MIR34A. Determining the presence of p53 mutations between exons 5-8. Following, the results obtained from these studies have been published in two articles. The first in the journal "Carcinogenesis" and the second in the "Journal of Surgical Oncology." In the first article, entitled "miR-34a as a Prognostic marker of relapse in surgically resected non-small-cell lung cancer", we relate the levels of miR-34a with the tumor relapse, establishing three groups according to the rate of relapse: the group with low levels of miR34a, with 67% relapse, those with high levels, with 43% relapse and those with the highest levels with a 0% relapse. Furthermore, in univariate analysis for risk of relapse, the mutational status of p53, the levels of miR-34a and stage IA vs the rest, were correlated with likelihood of relapse. Two multivariate analyzes were performed, including or not the P53 mutational status, remaining in both analyzes the expression of miR-34a, as an independent factor for tumor relapse. It was observed that patients with p53 mutations had a mean expression of miR-34a lower. It was noted that the subgroup of patients in which the expression levels of miR-34a low coincided with the presence of p53 mutations, had a high rate of relapse. In patients without mutations of p53, there is a significant difference in expression levels of miR-34a between the patients with the methylated promoter region MIR34A vs. those without. For all this, we found that miR-34a is shown as a novel biomarker with prognostic significance in patients relapsed NSCLC undergoing surgery, opening the possibility of a future tool in the therapeutic decision algorithm. In the second article, entitled "Prognostic Implications of miR-16 Expression Levels in Resected Non-Small-Cell Lung Cancer", from the hypothesis that p53 activates transcription of the family of miRNAs miR-34 and regulates the maturation of miR-16 to miR-143, we studied the expression levels of miR-143 and miR-16 in the paired normal and tumor tissue of each patient in the series, mentioned above, in 70 patients. So patients were classified according to the expression levels of miR-16 (high, normal and low). Patients with normal levels had the best clinical course, while those with higher levels had the worst. The disease-free survival (DFS) was 22.4 months for patients with high levels of miR-16, 71.8 months for normal levels of 55.8 months for low levels. Overall survival (OS) was 23.9 months for those with high levels of miR-16, from 97.6 months for those with normal levels and 63.5 months for those with low levels. No correlation was observed between the levels of miR-143 and the clinical course of patients. Multivariate analysis showed miR-16 as an independent prognostic factor for DFS and OS. In a secondary analysis, we examined the potential correlation between the expression of miR-16 and miR-34a and the mutational status of P53. There was no correlation between p53 mutational status and miR-16, but observed an interaction between miR-16 and miR-34a. In patients with high levels of miR-34a, there were differences between DFS and OS, according to the expression levels of miR-16, whereas in patients with low levels of miR-34a had a poor prognosis, regardless of expression levels of miR-16. These results indicate the possible prognostic value of miR-16 in patients operated for NSCLC, as well as possible synergism between miR-34a and miR16

Objective: Accurate preoperative staging is essential to provide the best treatment for lung cancer. The objective of the present study was to determine agreement between preoperative and surgical—pathological staging and to analyse the impact of any disparity on treatment. Methods:Thisisadescriptivestudyofaseriesof176lungcancercasestreatedbysurgerybetween2005and2007.Preoperativestagingwasbased on clinical information and computed tomography (CT), positron emission tomography (PET), PET-CT, bronchoscopy and mediastinoscopy. In all cases, surgical—pathological staging was based on the analysis of surgical samples and the findings during surgery. Both preoperative and pathological stage determination were based on the TNM (tumour, node, metastasis) classification established in 1997. Concordance was measured by calculating agreement rates and the kappa value. Results: Preoperative and surgical—pathological staging agreed in 102 cases, an agreement rate of 58% and kappa value of 0.54 (95% confidence interval (CI) 0.44—0.63). The highest kappa value (0.68, 95% CI 0.53—0.82) was obtained in stage IA patients. Patients who underwent PETor PET-CT had a better kappa index (0.56, 95% CI 0.45—0.67, vs 0.39, 95% CI 0.21— 0.56). Surgical—pathological staging validated surgery in 145 cases (82%), while 21 (12%) were revised to stage IIIA N2 and 10 (6%) to non-surgical stages.Conclusions:Globalagreementbetweenpreoperativeandsurgical—pathological stagingwasmoderate.Thebestagreementwasfoundin stages IV and IA. # 2009 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

La punció aspirativa guiada per ultrasonografia endobronquial (PA-USEB) ha demostrat ser per a l’estadiatge del càncer de pulmó. L’objectiu d’aquesta tesi és estudiar la utilitat d’aquesta tècnica no només en l’estadiatge del càncer de pulmó, sinó també com a eina de diagnòstic inicial en pacients amb ocupació mediastínica, i també la possibilitat de detecció mutacions de l’epidermal growth factor receptor (EGFR) en mostres obtingudes mitjançant aquesta tècnica en pacients amb adenocarcinoma de pulmó, o carcinoma de pulmó de cèl·lula no petita (CPCNP). També es va investigar la seguretat de la tècnica, I la disminució de mediastinoscòpies que comportava la seva utilització. Aquesta tesi es base en tres treballs, que són: - Garcia-Olivé I, Monsó E, Andreo F, et al. Sensitivity of linear endobronchial ultrasonography and guided transbronchial needle aspiration for the identification of nodal metastasis in lung cancer staging. Ultrasound in Medicine and Biology 2009;35:1271-77. - Garcia-Olivé I, Valverde EX, Andreo F et al. La ultrasonografía endobronquial lineal como instrumento de diagnóstico inicial en el paciente con ocupación mediastínica. Arch Bronconeumol 2009;45:266-70. - Garcia-Olivé I, Monsó E, F Andreo F, et al. Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations. Eur Resp J 2010;35:391-5. La finalitat del primer estudi va ser determinar la sensibilitat de la PA-USEB en l’estadiatge del càncer de pulmó. Es varen mesurar els diàmetres major i menor durant els procediments en aquells pacients que havien estat remesos per a estadificació, i es va determinar la sensibilitat de la USEB per a la detecció de malignitat als ganglis. Es varen realitzar 315 puncions ganglionars en 161 pacients, i en 87 d’aquests pacients es va confirmar metàstasis a N2/N3, amb la qual cosa es va fer innecessària la pràctica d’una mediastinoscòpia. La mediana dels diàmetres menors (rang interquartílic) dels ganglis mediastínics i hiliars va ser, respectivament d’11 (8-15) i 8 (7-12) mm. La punció va proporcionar mostres satisfactòries en 269 ganglis (85.4%), i es va assolir una sensibilitat del 100% per a la identificació de ganglis malignes en la citologia de la punció (agafant com a referència el resultat de la citologia de la punció) per a aquells ganglis amb diàmetre menor de més de 5 mm i aquells amb un quocient diàmetre menor/diàmetre major per sobre de 0.5. La probabilitat que un gangli fos maligne era superior al 90% per ganglis amb un diàmetre menor de més de 20 mm, i del 55% per a ganglis rodons (quocient d’1). En 18 dels 50 pacients amb mediastí normal a la TC, la ultrasonografia endobronquial va identificar ganglis augmentats de mida (36%), especialment a regió subcarinal, i la punció en va confirmar la malignitat en 5 (10%). La PA-USEB obté mostres ganglionars satisfactòries en gairebé el 90% dels casos i millora la identificació de ganglis augmentats de tamany en pacients en què la TC mostra un mediastí normal. Si s’agafa com a patró de referència el resultat de la citologia de la punció, el fet de tenir un diàmetre menor ≥ 5 mm i un quocient ≥ 0.5 detecta la malignitat d’un gangli amb una sensibilitat del 100%. L’objectiu del segon treball va ser determinar la utilitat de la tècnica com a instrument diagnòstic en el pacient amb ocupació mediastínica de causa desconeguda després d’obtenir mostres amb exploracions no invasives i amb broncoscòpia amb llum blanca. Es varen incloure a l’estudi tots els pacients explorats per PA guiada per USEB lineal per al diagnòstic de massa i/o adenopatia/es mediastinica/ques. Es consideraren diagnòstics finals aquells obtinguts amb PA guiada per USEB lineal i els assolits amb qualsevol altra tècnica quirúrgica realitzada posteriorment a la USEB, quan aquesta no havia estat diagnòstica. Es varen estudiar 128 pacients (edat mitja ± desviació estàndard: 62.0 ± 11.2 anys) als quals es va realitzar 294 PA sobre 12 masses i 282 ganglis. A les masses mediastíniques es varen obtenir mostres valorables en 11 casos (91.7%) i als ganglis, en 233 casos (82.6%). La PA dirigida per USEB lineal va ser l’eina diagnòstica i va evitar la mediastinoscòpia en 115 pacients (sensibilitat diagnòstica: 89.8%). La tècnica va confirmar el diagnòstic en 85 dels 94% amb neoplàsia (90.4%), en 8 dels 10 amb tuberculosi (80.0%), i als 5 amb sarcoïdosi (100%). La PA guiada per USEB lineal és una eina de diagnòstic útil en els pacients amb afectació mediastínica, el diagnòstic anatomopatològic dels quals no s’assoleix mitjançant exploracions no invasives ni broncoscòpia amb llum blanca. Al tercer treball es va estudiar la utilitat de la USEB per obtenir mostres de ganglis malignes en les quals es pogués detectar mutacions de l’EGFR. Es varen incloure tots aquells pacients amb adenocarcinoma pulmonar o CPCNP remesos per a estadificació mitjançant USEB. Es varen prendre mostres de tots aquells ganglis amb diàmetre menor de més de 5 mm, i es va obtenir DNA genòmic de cèl·lules tumorals per a analitzar els exons 19 i 21. Es va estudiar l’impacte dels resultats en el maneig del pacient. L’anàlisi del gen de l’EGFR en mostres obtingudes per PTB-USEB va ser possible en 26 (72.2%) dels 36 pacients amb metàstasis ganglionars mediastíniques. Es varen detectar mutacions somàtiques del gen de l’EGFR en teixit obtingut per USEB en 2 (10%) dels 20 pacients amb adenocarcinoma pulmonar metastàtic. Les mostres de teixit maligne obtingudes per USEB dels pacients amb metàstasis ganglionars de CPCNP són útils per a la detecció de mutacions de l’EGFR en la majoria dels casos, i aquesta tècnica evidencia la presència de cèl·lules neoplàsiques mutades en una desena part dels pacients amb adenocarcinoma, als qual no va caldre fer-los una mediastinoscòpia No varen haver-hi complicacions derivades de la tècnica durant la realització de la mateixa o en les 48 hores posteriors en cap dels pacients inclosos als estudis. Com a conclusions, la PA-USEB ha demostrat ser útil i segura a l’hora d’estudiar masses i ganglis mediastínics i/o hiliars en pacients amb càncer de pulmó, fins i tot els que tenen TC normal. També pot ser útil com a eina diagnòstica en pacients amb afectació mediastínica sense diagnòstic. També permet obtenir mostres en què és possible detectar mutacions del gen de l’EGFR en pacients amb CPCNP. Linear endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has proven useful for staging lung cancer. The objective of this thesis is to assess the usefulness of this technique not only in lung cancer staging but also as a diagnostic tool in patients with mediastinal disease, and the possibility of detecting epidermal growth factor receptor (EGFR) mutations in samples obtained through this technique in patients with lung adenocarcinoma or non small cell lung cancer (NSCLC). The safety of the technique and the decrease in the need for mediastinoscopy were also assessed. This thesis is based on three studies, which are: - Garcia-Olivé I, Monsó E, Andreo F, et al. Sensitivity of linear endobronchial ultrasonography and guided transbronchial needle aspiration for the identification of nodal metastasis in lung cancer staging. Ultrasound in Medicine and Biology 2009;35:1271-77. - Garcia-Olivé I, Valverde EX, Andreo F et al. La ultrasonografía endobronquial lineal como instrumento de diagnóstico inicial en el paciente con ocupación mediastínica. Arch Bronconeumol 2009;45:266-70. - Garcia-Olivé I, Monsó E, F Andreo F, et al. Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations. Eur Resp J 2010;35:391-5. The aim of the first study was to determine the sensitivity of EBUS-TBNA in lung cancer staging. Short- and long-axis node diameters were measured during EBUS in patients referred for lung cancer staging and sensitivities for the identification of nodal malignancy at TBNA determined. Three hundred fifteen real-time EBUS-guided TBNA nodal sampling procedures were performed in 161 patients and in 87 of them, N2/N3 metastasis was confirmed (50.9%), eliminating the need for mediastinoscopy. The median (interquartile range [IQR]) short-axis diameters of the sampled mediastinal and lobar nodes were 11 (8–15) and 8 (7–12) mm, respectively. TBNA provided satisfactory samples from 269 nodes (85.4%) and a sensitivity of 100% for the identification of malignant TBNA samples was reached for a short-axis diameter cut-off of 5mmand a short- to long-axis ratio of 0.5. The probability of malignancy was over 90% for nodes with a short-axis diameter .20 mm and 55% for round nodes (short- to long-axis ratio of 1). In 18 out of 50 patients with a normal mediastinal computed tomography (CT) scan, EBUS identified enlarged nodes in the mediastinum (36%), mainly in the subcarinal region and confirmed mediastinal malignancy in 5 (10%). Real-time EBUS-guided TBNA obtains satisfactory node samples in almost 90% of cases and improves the identification of enlarged nodes in patients with a normal mediastinum at CT. If sampling all nodes with a short-axis diameter of >5 mm and a short- to long-axis ratio >0.5, a sensitivity of 100% for the cytological identification of malignant nodes can be expected. The aim of the second study was to assess the usefulness of this diagnostic tool in patients with indications of mediastinal disease that could not be diagnosed by non invasive methods or white light bronchoscopy. All patients undergoing linear EBUS for the diagnosis of mediastinal masses and/or adenopathy at our endoscopy unit were included in the study. Diagnoses obtained by linear EBUS or any surgical technique performed alter a non diagnostic EBUS were considered as final. In the study population of 128 patients with a mean (SD) age of 62.0 (11.2) years, a total of 294 TBNAs were performed on 12 masses and 282 nodes. Satisfactory samples were obtained in 11cases (91.7%) from masses and in 233 cases (82.6%) from nodes. Linear EBUS was diagnostic, obviating the need for mediastinoscopy in 115 patients (diagnostic sensitivity, 89.8%). The technique confirmed the diagnosis in 85 of the 94 patients with cancer (90.4%), in 8 of the 10 patients with tuberculosis (80.0%), and in the 5 patients with sarcoidosis. The usefulness EBUS for the detection of EGFR mutations in cells recovered from malignant mediastinal nodes in patients with NSCLC was assessed in the third article. All patients with lung adenocarcinoma or NSCLC referred for staging with EBUS were included. Nodes with a short-axis diameter of 5 mm were sampled, and genomic DNA from metastatic tumour cells was obtained for analysis of exons 19 and 21. The impact of sampling on management was assessed. EGFR gene analysis of the EBUS-TBNA sample was feasible in 26 (72.2%) out of the 36 patients with lymph node metastasis. Somatic mutations of the EGFR gene were detected in tissue obtained through EBUS-TBNA in two (10%) out of 20 patients with metastasic lung adenocarcinoma, eliminating the need for mediastinoscopy in these patients. Malignant tissue samples obtained by EBUS-TBNA from patients with nodal metastasis of NSCLC are suitable for the detection of EGFR mutations in most cases, and this technique demonstrates mutated neoplastic cells in a tenth of patients with adenocarcinoma. No complications related to the procedure developed during its performance or in the following 48 hours in any of the patients included in any of the three studies. To conclude, EBUS-TBNA has shown to be useful and safe in detecting metastatic mediastinal and/or hilar lymph nodes in patients with lung cancer, even in those with radiologically normal mediastinum. It can also be useful as a diagnostic tool in other mediastinal diseases. It is possible to detect EGFR gene mutations in samples obtained with this technique from patients with NSCLC.

6 páginas, 2 figuras, 3 tablas., Introduction While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples. Methods With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC. Results The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p, This work was partially funded by grants from Fundacion Mutua Madrileña to EC, FLR, and LPA; CIBER Respiratory Disease to ALE (ISCIII-CB06/06); and Red Temática de Investigacion Cooperativa en Cancer (RTICC) to MSC (RD06/0020/0062). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A mesura que l'esperança de vida augmenta, augmenta la incidència i prevalença dels malalts grans amb càncer. El càncer de pulmó està associat amb l'edat(1). Malgrat que quasi la meitat dels nous casos es donen en gent gran, baix és el percentatge de malalts grans inclòs en assaig clínic(2). La principal característica de l'envelliment és la seva heterogeneïtat. Per identificar millor el malalt gran, la geriatria ha desenvolupat eines d'aproximació a la població gran. Aquestes s'anomenen avaluació geriàtrica global(CGA). La CGA contribueix amb l'oncologia aportant informació addicional, que permet estratificar els malalts en funció del seu risc de deteriorament funcional front a situacions d'estrès, com ara una neoplàsia(2). Detecta aquells malalts que es beneficiarien d'una intervenció geriàtrica prèvia a una intervenció/tractament oncològic(2-4). L'ús de la CGA en oncologia és molt limitat. Per tal de descriure la població gran amb càncer de pulmó de la nostra consulta, apliquem de manera prospectiva instruments d'avaluació geriàtrica. L'objectiu del treball va ser la implementació d'aquestes eines en la pràctica diària. Es tracta d'un estudi epidemiològic descriptiu del malalt gran amb càncer de pulmó del Departament 14.Resultats: de Gener de 2006 a Febrer del 2008, 83 malalts van ser avaluats prospectivament. El 97,6% eren homes, amb una mitjana d'edat de 77 anys (70-91). El 96,4% tenien història de tabaquisme; 72,5% exfumadors. La relació entre hàbit tabàquic i supervivència va mostrar tendència a la significació estadística. El tabaquisme es va relacionar significativament amb la histologia. Un 21,7% tenien antecedents d'altres neoplàsies i forta història de tabaquisme a la família. L'estadi es va relacionar amb la supervivència. El 61,3% tenien histologia epidermoide, relacionada significativament amb la supervivència. 84% estaven simptomàtics al diagnòstic. Alguns dels símptomes es van relacionar amb la supervivència. El 70% presentaven PS 0-1 al diagnòstic, associat significativament amb la supervivència. Pel que fa a les variables geriàtriques, els malalts van presentar un ampli espectre de dependències en ADL i IADL. El 51,8% eren independents per ADL mentre sòls el 30,1% ho era per IADL. Destaca comorbiditat elevada, no relacionada amb la supervivència. El 26,4% tenien criteris de demència i 31,3% de depressió. El 41,4% pèrdua de pes al diagnòstic i 34,9% albúmina baixa. Els malalts presentaven un fort recolzament social, per part de la família. Un 48,2% tenia algun síndrome geriàtric i el 72,3% complia criteris de fragilitat. Respecte la informació, el 73,5% dels malalts sol·licitava informació activa. Un 56,6% acceptaria com a objectiu del tractament l'augment de la supervivència. En el moment de l'anàlisi, el 70,1% dels malalts havien mort. La supervivència global fou de 326 dies. La principal causa de mort fou la progressió tumoral. Els factors relacionats amb la supervivència foren principalment els tumorals: estadi (pConclusions: l'aplicació d'una aproximació geriàtrica és factible en la pràctica diària. Aquesta, aporta informació addicional, no detectada per l'avaluació oncològica. Tot i que alguns paràmetres de l'envelliment es van relacionar amb pitjor supervivència; el mal pronòstic del càncer de pulmó va ser superior al pronòstic intrínsec a l'envelliment. La principal causa de mort en el malalt gran amb càncer de pulmó del nostre treball fou la tumoral. La col·laboració conjunta de la geriatria i l'oncologia ens ha d'ajudar a optimitzar la balança risc/benefici en el tractament d'aquesta població heterogènia., As a result of an increasing life expectancy, incidence and prevalence of elderly cancer patients rises. Lung cancer is one of the most elderly related neoplasms. Despite almost 50% of new diagnoses of lung cancer occur in the elderly; elderly are underrepresented at clinical trials. Aging is a highly individualized process and all the changes that occur cannot be predicted on the basis of chronological age. A much more thorough method, such a comprehensive geriatric assessment (CGA), has been developed by geriatricians to be used when evaluating elderly cancer patients. The CGA has the ability to detect health problems in elderly patients, helping oncologist. It can identify patients that potentially benefit from an extensive evaluation. CGA use in oncology is limited; the best form of CGA for cancer patients remains to be defined. To describe oncology and geriatric profile of elderly lung cancer patients, we prospectively applied an evaluation form. The ultimate goal was to know if the application was feasible in an oncology outpatient unit. This is an epidemiologyc descriptive study of elderly lung cancer patients at Department 14.Results: Between January 2006 and February 2008, 83 patients > 70 years of age underwent prospectively assessment in our Department. 81 patients (97.6%) males. Median age 77 years (range 70-91). 96.4% had smoking history; 72.5% ex-smokers. Neither smoking habit (former versus exsmoker versus nonsmoker) was correlated with survival. 18 patients (21.7%) had a previous diagnosis of neoplastic disease. Staging: 19 patients (22.9%) had localized disease, 34 had regional spreading (41%) and 43 (36.1%) disseminated stage. The stage at diagnosis significantly correlated with survival (logrank p84% were symptomatic at diagnosis; some symptoms were related to survival. 70% had PS 0-1 at diagnosis; significantly correlated with survival. Roughly half of the patients (40; 48.2%) had ADL and 58 (69.9%) had IADL dependency. High comorbidity index, measured by Charlson and SCS scores weren't related to survival. Cognitive deficits were found in 22 (26.4%) patients and 26 (31.3%) had depression. An unintentional weight loss of more than 8.2% (range 1-21%) total body weight over the previous three months (range 1-8 months) was reported by 37 patients (44.6%). Mean albuminemia was 2.6 g/dl (range 2-5.9 g/dl). Albuminemia levels were below normal reference limits in 35% of patients. Almost all patients reported social support (98.8%) by family members. At least one geriatric syndrome was found in 40 (48.2%) patients. 72.3% had frailty criteria. 73.5% wanted to have diagnosis information. 56.6% if treated, shown their desire of improve survival.At the end of the study (December 2008), 59 patients had died (70.1%). Mean survival was 326 days (10.8 months; CI 95% 259-393). The main cause of death was lung cancer disease progression (69.5%, 57 patients), with 20 patients (25%) dying of other non-neoplastic causes. Neoplasic factors related to survival were: stage (pConclusions: A geriatric assessment could be applied to elderly lung cancer patients attended at an outpatient oncology department. Although most of the patients are frail, it seems that lung cancer prognosis is superior than aging factors. We have applied CGA and it seems that aging does not worsen the prognosis of lung cancer, except for certain aging-related parameters. Only by working together (medical oncologists, geriatrists) can we hope to offer a quality assistance in this increasing population.

A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT KEAP1, and LRP1B/LRP-DIP CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 1 1%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. (C) 2010 Wiley-Liss, Inc.

Existeix certa controvèrsia sobre els efectes del b-carotè com a promotor o protector del càncer de pulmó. A més, el b-carotè, com a precursor de l'àcid retinoic, podria estimular la termogènesi i regular l'adipositat corporal. La fura representa un bon model per estudiar els efectes de la ingesta de b-carotè ja que l'absorbeix de manera pràcticament intacta (semblant als humans). Hem demostrat que el b-carotè ingerit amb altres antioxidants no sembla tenir efectes inductors del càncer ja que no augmenta la proliferació cel·lular al pulmó i a més prevé els efectes del carcinogen benzo[a]pirè. D'altra banda, dosis farmacològiques de b-carotè fan a la fura efectes contraris als de l'àcid retinoic, ja que augmenten l'adipositat i resulten en una menor capacitat termogènica, principalment al teixit adipós retroperitoneal que, a la fura, presenta certes característiques de teixit adipós marró, ja que té un percentatge considerable d'adipòcits multiloculars i expressa quantitats significatives d'UCP1., Existe controversia sobre los efectos del b-caroteno como promotor o protector del cáncer de pulmón. Además, el b-caroteno, como precursor del ácido retinoico, podría estimular la termogénesis y regular la adiposidad corporal. El hurón representa un buen modelo para estudiar los efectos de la ingesta de b-caroteno pues lo absorbe de manera prácticamente intacta (similar a los humanos). Hemos demostrado que el b-caroteno ingerido con otros antioxidantes no parece tener efectos inductores del cáncer puesto que no aumenta la proliferación celular del pulmón y además previene los efectos del carcinógeno benzo[a]pireno. Por otra parte, dosis farmacológicas de b-caroteno producen en el hurón efectos contrarios a los del ácido retinoico, ya que aumentan la adiposidad y reducen la capacidad termogénica, principalmente del tejido adiposo retroperitoneal que, en el hurón, presenta ciertas características de tejido adiposo marrón, al contener un porcentaje considerable de adipocitos multiloculares y expresar cantidades significativas de UCP1., The effects of b-carotene promoting or protecting against lung cancer are unclear. Furthermore, b-carotene, as retinoic acid precursor, could induce thermogenesis and regulate body adiposity. The ferret represents a good model to study the effects of oral administration of b-carotene because it absorbs it almost intact (similarly to humans). We have shown that the intake of b-carotene together with other antioxidants does not seem to inducecancer as it does not increase cellular proliferation in lung and prevents the effects of the carcinogen benzo[a]pyrene. In addition, pharmacological b-carotene doses in ferrets have different effects from retinoic acid, increasing adiposity and decreasing thermogenic capacity, especially in the retroperitoneal adipose tissue which, in ferrets, resembles brown adipose tissue, since it has an important percentage of multilocular adipocytes and expresses significant amount of UCP1.

Aberrant proteins encoded from genes altered in tumors drive cancer development and may also be therapeutic targets. Here we derived a comprehensive gene-alteration profile of lung cancer cell lines. We tested 17 genes in a panel of 88 lung cancer cell lines and found the rates of alteration to be higher than previously thought. Nearly all cells feature inactivation at TP53 and CDKN2A or RB1, whereas BRAF, MET, ERBB2, and NRAS alterations were infrequent. A preferential accumulation of alterations among histopathological types and a mutually exclusive occurrence of alterations of CDKN2A and RB1 as well as of KRAS, epidermal growth factor receptor (EGFR), NRAS, and ERBB2 were seen. Moreover, in nonsmall- cell lung cancer (NSCLC), concomitant activation of signal transduction pathways known to converge in mammalian target of rapamycin (mTOR) was common. Cells with single activation of ERBB2, PTEN, or MET signaling showed greater sensitivity to cell-growth inhibition induced by erlotinib, LY294002, and PHA665752, respectively, than did cells featuring simultaneous activation of these pathways, underlining the need for combined therapeutic strategies in targeted cancer treatments. In conclusion, our gene-alteration landscape of lung cancer cell lines provides insights into how gene alterations accumulate and biological pathways interact in cancer. Hum Mutat 30, 1199–1206, 2009. & 2009Wiley-Liss, Inc.

La ultrasonografía endoscópica (USE) combina la endoscopia con la ecografía para conseguir imágenes ecográficas de 360º desde el interior del tubo digestivo. Ello permite una muy buena visualización de las diferentes capas de la pared y de las estructuras de vecindad. Por otro lado, existe un ecoendoscopio sectorial que permite introducir una aguja por el canal operativo y realizar la punción con aguja fina guiada por USE en tiempo real desde el interior del tubo digestivo y obtener material citológico para el diagnóstico (USE-PAAF). Asi pues, la USE y la USE-PAAF constituyen unas técnicas idóneas para estudiar el engrosamiento de los pliegues gástricos, explorar tanto las lesiones focales como la patología difusa del páncreas, y realizar la estadificación locoregional de los tumores digestivos asi como para la obtención de material para el diagnóstico citológico. A pesar de la conocida utilidad de la USE y USE-PAAF en Oncología, existen patologías de difícil manejo clínico en los que el rendimiento de esta técnica no ha sido evaluado adecuadamente. Además, tampoco se han analizado cuáles son las circunstancias que permiten obtener un mayor rendimiento de la USE-PAAF. Por otro lado, la combinación de una técnica diagnóstica novedosa como es la USE-PAAF con los métodos de biología molecular podría suponer un verdadero avance en el diagnóstico de las neoplasias digestivas y pulmonares.La presente Tesis Doctoral está constituida por cuatro trabajos originales encaminados a resolver estas cuestiones y cuyos resultados permiten extraer las siguientes conclusiones:1. El engrosamiento de las capas profundas de la pared gástrica (submucosa y/o muscular) es el único criterio endosonográfico independiente predictivo de malignidad en pacientes con pliegues gástricos engrosados y biopsias negativas. 2. Utilizando este criterio, la USE es una técnica muy precisa y con un importante impacto clínico en el manejo de este grupo de pacientes. 3. En los pacientes referidos para USE-PAAF, esta técnica aumenta significativamente el porcentaje de diagnósticos correctos en cuanto a benignidad y malignidad con respecto a la USE, en especial en adenopatías y lesiones quísticas. 4. La localización intraparietal de la lesión es el único factor asociado a una mayor probabilidad de obtener un diagnóstico incorrecto por USE-PAAF. 5. La presencia de un patólogo en la sala de exploración aumenta el rendimiento de la USE-PAAF al reducir el número de pases necesarios para obtener un diagnóstico correcto y es una estrategia coste-eficaz.6. Es posible efectuar el análisis mutacional del gen KRAS en el material obtenido por USE-PAAF, con una elevada rentabilidad. 7. La citología convencional es la estrategia más precisa para el diagnóstico del cáncer de páncreas, pudiendo el análisis molecular incrementar su rendimiento en los pocos casos en los que la primera no es diagnóstica. 8. Es posible detectar la presencia de micrometástasis ganglionares mediante determinación de patrones de metilación aberrante en material obtenido por USE-PAAF.9. La combinación de este análisis con la citología convencional puede ser una aproximación útil en el diagnóstico de extensión de los pacientes con neoplasias digestivas y pulmonares., Endoscopic ultrasound (EUS) consists in a high-frequency transducer placed at the tip of an endoscope. This technique permits to obtain high-resolution transmural sonographic images of the intestinal wall and surrounding structures. Moreover, it is possible to obtain cytological material from lesions located around the intestinal wall by means of FNA guided by EUS. EUS and EUS-FNA have become well-established procedures for diagnosing and staging both gastrointestinal and lung cancer as well as to study the pancreas. The aims of the present Doctoral Thesis were:1) to assess the predictive variables of malignancy in EUS, and the impact of EUS in patients with large gastric folds at endoscopy and endoscopic biopsies negative for malignancy; 2) To evaluate factors that permit to obtain a correct diagnosis by EUS-FNA and to establish the usefulness of disposing of an attendant pathologist; 3) To establish the usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by comparing this technique with conventional cytology in aspirates obtained by means of EUS-FNA; and, 4) to evaluate whether hypermethylation gene promoter analysis was feasible on samples obtained by EUS-FNA from lymph nodes, as well as to establish the usefulness of this strategy for the detection of micrometastases in patients with gastrointestinal and non-small cell lung cancer.Results obtained permitted to conclude that: 1) the enlargement of deep layers is the only independent predictive factor for malignancy in patients with large gastric folds at endoscopy and biopsies negative for malignancy and that EUS has a high clinical impact in these patients; 2) The availability of an attendant pathologist seems to increase the diagnostic yield of the FNA, minimizing the number of passes and resulting in a cost-effective strategy. In absence of on-site evaluation the number of passes to perform should be 3 to 4 depending on the type of lesion; 3) Cytology from aspirates obtained by EUS-FNA is the most precise single technique for the diagnosis of pancreatic adenocarcinoma. However, when adequate specimens are not available to reach a cytological diagnosis, the addition of KRAS mutational analysis represents the best strategy; and, 4) It is feasible to detect occult neoplastic cells in EUS-FNA samples by hypermethylation gene promoter analysis. Moreover, addition of methylation analysis to conventional cytology may increase its sensitivity at the expenses of a decrease in its specificity.

Delahaye-Sourdeix, Manon Oliver, Javier Timofeeva, Maria N Gaborieau, Valerie Johansson, Mattias Chabrier, Amelie Wozniak, Magdalena B Brenner, Darren R Vallee, Maxime P Anantharaman, Devasena Lagiou, Pagona Holcatova, Ivana Richiardi, Lorenzo Kjaerheim, Kristina Agudo, Antonio Castellsague, Xavier Macfarlane, Tatiana V Barzan, Luigi Canova, Cristina Thakker, Nalin S Conway, David I Znaor, Ariana Healy, Claire M Ahrens, Wolfgang Zaridze, David Szeszenia-Dabrowska, Neonilia Lissowska, Jolanta Fabianova, Eleonora Mates, Ioan Nicolae Bencko, Vladimir Foretova, Lenka Janout, Vladimir Curado, Maria Paula Koifman, Sergio Menezes, Ana Wunsch-Filho, Victor Eluf-Neto, Jose Boffetta, Paolo Garrote, Leticia Fernandez Serraino, Diego Lener, Marcin Jaworowska, Ewa Lubinski, Jan Boccia, Stefania Rajkumar, Thangarajan Samant, Tanuja A Mahimkar, Manoj B Matsuo, Keitaro Franceschi, Silvia Byrnes, Graham Brennan, Paul McKay, James D eng 1R03DE020116/DE/NIDCR NIH HHS/ R01 CA092039 05/05S1/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural 2015/03/21 06:00 PLoS One. 2015 Mar 20;10(3):e0117639. doi: 10.1371/journal.pone.0117639. eCollection 2015.; International audience; Genetic variants located within the 12p13.33/RAD52 locus have been associated with lung squamous cell carcinoma (LUSC). Here, within 5,947 UADT cancers and 7,789 controls from 9 different studies, we found rs10849605, a common intronic variant in RAD52, to be also associated with upper aerodigestive tract (UADT) squamous cell carcinoma cases (OR = 1.09, 95% CI: 1.04-1.15, p = 6x10(-4)). We additionally identified rs10849605 as a RAD52 cis-eQTL inUADT(p = 1x10(-3)) and LUSC (p = 9x10(-4)) tumours, with the UADT/LUSC risk allele correlated with increased RAD52 expression levels. The 12p13.33 locus, encompassing rs10849605/RAD52, was identified as a significant somatic focal copy number amplification in UADT(n = 374, q-value = 0.075) and LUSC (n = 464, q-value = 0.007) tumors and correlated with higher RAD52 tumor expression levels (p = 6x10(-48) and p = 3x10(-29) in UADT and LUSC, respectively). In combination, these results implicate increased RAD52 expression in both genetic susceptibility and tumorigenesis of UADT and LUSC tumors.

Introducció: El carcinoma de pulmó és un dels problemes de salut prioritari de la societat actual, amb una major presència en homes entre els 55 i 65 anys. És una neoplàsia molt agressiva i malgrat els esforços de crear noves teràpies de QT amb diferents combinacions de fàrmacs, o RT i QT conjuntament, s'han millorat poc les taxes de supervivència d'aquesta malaltia. Sabem, per altre banda, que el cicle cel·lular amb les seves diferents fases són objecte d'una regulació bioquímica molt complexa. En aquesta tesi ens vam centrar en l'estudi d'unes quantes proteïnes implicades en el cicle (PCNA, Ki-67, p53, ciclines D1 i E) i veure quin paper tenien. Objectius de la tesi: 1) Determinar mitjançant tècniques de citometria de flux (CMF) l'índex de DNA, la fase S i el percentatge de cèl·lules PCNA positives i també el grau d'heterogeneïtat per a cadascuna en el carcinoma de pulmó de cèl·lula no petita (CPNCP), tant en els tumors primaris com en les seves metàstasis ganglionars. 2) Investigar si existeix un patró o patrons d'índexs de DNA i/o de fase S i/o de percentatge de PCNA relacionables amb el fenomen metastàtic. 3) Determinar l'expressió de l'antígen de proliferació cel·lular PCNA a les diferents fases del cicle cel·lular i avaluar si hi ha alguna relació estadística amb altres paràmetres estudiats. 4) Quantificar per mitja de tècniques d'immunohistoquímica el grau d'expressió de marcadors de proliferació cel·lular (PCNA, Ki-67), els reguladors negatius (p53) i les ciclines (D i E). 5) Avaluar les possibles relacions entre les variables següents: clíniques, anatomopatològiques, variables de CMF i variables immunohistoquímiques. 6) Analitzar estadísticament les variables que puguin ser valorades com a factor pronòstic independent. Material i mètodes: Es varen seleccionar 99 mostres de CPCNP entre 1993-1997, 89 homes i 10 dones. Es van recollir 4 tipus de dades diferents i per separat, per garantir l'objectivitat en l'obtenció de les variables. dades clíniques, anatomopatològiques, immunohistoquímiques i de citometria de flux. Les dades clíniques es van recollir per calcular la supervivència lliure i la global de la malaltia. Les dades anatomopatològiques, immunohistoquímiques i de CMF s'obtingueren de les anàlisis de les recessions quirúrgiques remeses al laboratori durant la intervenció dels malalts. Cada mostra fou dividida en dues parts: una per a CMF per l'estudi del cicle cel·lular i expressió de l'antígen PCNA a les diferents fases del cicle i l'altre per a l'estudi histològic rutinari i per l'anàlisi amb tècniques d'immunohistoquímica. Resultats: Un 70% dels adenocarcinomes es van diagnosticar a l'estadi IIIA. Ki-67, PCNA i ciclina E estaven relacionades amb el tipus histològic de carcinoma indiferenciat de cèl·lula gran (CICG). La ciclina E també estava relacionada amb el CE. La ciclina D1 amb el grau de diferenciació. PCNA i ciclina E també estaven relacionades significativament amb Ki-67. A l'anàlisi univariable en relació amb la supervivència global de la malaltia, l'estadi, CV de l'íDNA i de la fase S assoleixen la significació estadística, com a variables relacionades amb l'evolució de la malaltia. Finalment a l'anàlisi multivariable per la supervivència global de la malaltia, l'estadi i el CV de la fase S assoleixen un valor pronòstic independent. Conclusions: Hi ha una relació directa entre l'estadi i la supervivència global dels malalts. Les variables determinades per immunohistoquímica no tenen implicacions pronostiques. Hi ha una relació entre el CICG i l'expressió augmentada de marcadors de proliferació. La sobreexpressió de ciclina D1 es relaciona directament amb els CPNCP pobrament diferenciats. La fase S es relaciona amb l'expressió augmentada del PCNA per CMF. L'heterogeneïtat en l'expressió del PCNA per CMF es relaciona amb el grau d'heterogeneïtat de l'íDNA i fase S., Introduction: Lung carcinoma is one of the priority health problems in our society. It shows mainly in men from 55 to 65 years. Is a very aggressive neoplasm and in spite of the efforts done in chemotherapy or combined with radiotherapy, the survival rate has not improved much. On the other hand, we know that the cellular cycle with its different phases is object of a very complex biochemical regulation. In this thesis we pay attention to the study of some cell cycle proteins (PCNA, KI-67, P53, cyclin D1 and E) and to try to see their functions. Objects of thesis: 1) To determine by flow cytometry (FCM) DNA index, S phase, percentage of positive PCNA cells and the heterogeneity degree of every one in non-small-cell-lung carcinomas (NSCLC), in primary tumors and ganglionary metastases. 2) To research the existence of a model or models of DNA index and/or S phase and/or a percentage of PCNA related with metastatic phenomenon. 3) To determine the proliferating cell nuclear antigen (PCNA) expression during the cell cycle and to evaluate if there are any other statistic relation with other study parameters. 4) To calculate by immunohistochemistry the degree of expression of cellular proliferating markers (PCNA, Ki-67), negative regulators (p53) and cyclins (D and E). 5) To assess the potential relations between the following values: clinics, anatomopathologics, FCM and immunohistochemistry. 6) To analyze statistically the values that might be important as an independent prognostic factor. Material and methods: We colleted 99 samples of NSCLC from 1993 to 1997, 89 men and 10 women. We collected four different types of data, in order to guarantee the objectivity when we obtain the values: clinical data, anatomopathologic, immunohistochemistry and FCM data. The clinical data was obtained to calculate the free survival and global survival of disease. The anatomopathologic, immunohistochemistry and FCM data was obtained by the analysis of surgically resected lung carcinomas that were sent to laboratory. Every sample was divided in two parts: one to study cell cycle and PCNA expression in different phases of cycle by FCM and the other to study the sample by histological study and immunohistochemical techniques. Results: 70% of adenocarcinomas were diagnosed as stage IIIA. Ki-67, PCNA and cyclin E were related with large cell carcinoma histological type, cyclin E was related too with squamous cell carcinoma type. Cyclin D1 was related with the degree of cell differentiation. PCNA and cyclin E were significantly correlated with Ki-67. A univariate analysis of global survival showed that study, variation coeficient of DNA index and variation coeficient of S phase were the most significant prognostic factors as variables related with disease evolution. Finally in a multivariate analysis to global survival disease, study and variability of S phase showed an independent prognostic value. Conclusions: There is a direct relationship between study and global survival of patients. The variables detected immunohistochemically do not have prognostic factors. There is a relationship between large cell carcinoma and high value of proliferative markers. Cyclin D1 over-expression is directly related with poorly differentiated NSCLC. S phase is related with a high PCNA expression by flow cytometry. Heterogeneity in PCNA expression by flow cytometry is related with heterogeneity degree of DNA index and S phase.

DE LA TESI:La metàstasi és un procés histopatològic especialment complex per al seu estudi. Aquesta complexitat es deriva tant de la implicación de tots els nivells d'organització de l'organisme afectat (molecular, cel.lular, tisular, orgànic i sistèmic) com de l'alta diversitat inherent a cadascun d'ells. Una eina essencial de treball és la disposició de models cel.lulars que permetin tant l'estudi específic i puntual d'alguns aspectes del desenvolupament de la patologia com la seva integración en el conjunt del procés. A partir d'aquests principis estratègics hem disposat, pel desenvolupament d'aquest estudi, de dues línies cel.lulars d'adenocarcinoma de pulmó humà derivades d'una mateixa població parental (Inufusa et al., 1991). Aquestes línies, caracteritzades per una alta (HAL-8) i nul.la (HAL-24) capacitat metastàsica, proporcionen un model molt sòlid per a la validació dels resultats diferencials. És així que ens varem plantejar com a objectiu general la caracterització d'expressions diferencials que, de manera constitutiva o regulada, presentessin ambdós tipus cel.lulars. Donat que les cèl.lules HAL-8 sobreexposen l'antigen sialil-Lewis(x) dimèric en relació a les cèl.lules HAL-24, ens varem proposar com a primer objectiu caracteritzar l'expressió d'altres oligossacàrids fucosilats més relacionats amb la metàstasi i determinar el seu paper a l'adhesió d'aquestes cèl.lules tumorals a l'endoteli. Donades les diferències d'expressió que varem observar en algunes d'aquestes mol.lècules, ens varem plantejar l'estudi de l'expressió dels gens de les Fucosiltransferases del tipus alfa(1,3), enzims responsables de la seva síntesi. (Capítol I: "Enhaced expresión of alpha(1-3)-Fucosyltransferase genes correlates with E-selectin-mediated adhesion and metastasic potencial of human lung adenocarcinoma cells"). Un cop demostrat el paper clau del sialil-Lewis(x) a l'adhesió de les cel.lules HAL-8 així com la relació entre l'expressió dels gens de les Fucosiltransferases del tipus alfa(1,3) i la capacitat metastàsica d'aquestes cèl.lules, el següent objectiu plantejat va ser determinat si la sobreexpressió de determinades Fucosiltransferases alfa(1,2) modifica les propietats adhesives i el potencial colonitzador de pulmó de les cèl.lules no metastàtiques HAL-24 (Capítol II: "Overexpression of alpha(1,3)-fucosyltransferase VII is sufficient for the acquisition of lung colonization phenotype in human lung adenocarcinoma HAL-24Luc cells)".Caracteritzat el model d'adhesió a l'endoteli de les cèl.lules HAL-8 i HAL-24, i demostrat el paper de l'alfa(1,3)-Fucosiltransferasa VII en aquesta etapa de la metàstcasi, ens varem proposar estudiar la implicación de les metal.loproteases en la migración transendotelial de les cèl.lules metastàsiques d'adenocarcinoma de pulmó així com la regulació de la seva expressió tant per integrines com per la matriu extracel.lular (Capítol III: "Expressió de les metal.loprotesases de matriu i la regulació de la seva activitat a cèl.lules d'adenocarcinoma de pulmó humà)".Donat el caracter multiènic del desenvolupament de les metàstasis i l'interès en trobar gens responsables d'aquest procés, com a objectiu final ens varem plantejar l'anàlisi de l'expressió gènica diferencial de les cèl.lules HAL-8 i HAL-24 (Capítol IV: "Identification of semaphorin E gene expresión in metastasic human lung adenocarcinoma cells by mRNA differential display").

Metastatic human lung adenocarcinoma HAL-8Luc cells display an enhanced expression of alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-Ts) compared with their non-metastatic counterpart HAL-24Luc cells. This correlates with an increased surface expression of Lewis(x) (Le(x))- and Lewis(a) (Le(a))-related molecules and an in vitro enhanced adhesive capacity to E-selectin-expressing endothelial cells (Martin-Satué et al (1998). Cancer Res 58: 1544-1550). In the present work we have stably transfected HAL-24Luc cells with the cDNAs for the alpha(1,3)-Fuc-TIV and VII enzymes and analysed by flow cytometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a) and sialyl-Le(a). Fuc-TVII transfectants exclusively overexpress sialyl-Le(x) while Fuc-TIV-transfected cells only overexpress the Le(x) oligosaccharide. We show that solely Fuc-TVII transfectants are able to adhere to interleukin-1beta-stimulated HUVEC monolayers. We also demonstrate that Fuc-TVII overexpression in HAL-24Luc cells is sufficient for the acquisition of the lung colonization phenotype. This is the first report directly showing the contribution of an alpha(1,3)-Fuc-T to the metastatic behaviour of human lung adenocarcinoma cells.

The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers and genetic alterations. We analyzed markers of DNA damage response (DDR), proliferative stress, and telomeric stress: gamma-H2AX, p16, p53, and TERT. Lung cancer-related epigenetic and genetic alterations, including promoter hypermethylation status of p16(CDKN2A), APC, CDH13, Rassf1, and Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, and c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase and p53 induction. p16 was also induced in early tumorigenic progression and was inactivated in bronchiolar dysplasias and tumors. Remarkably, lack of mutations of Ras and epidermal growth factor receptor, and a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A), CDH13, and APC, but not in Rassf1 and Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer.

Sr. Director: Si bien el 17 % de los pacientes afectos de neoplasia pulmonar presentan metástasis óseas en el momento de realizar el diagnóstico clínico, la afección ósea secundaria de los huesos de la mano o del pie (acrometástasis) es muy poco frecuente constituyendo un hecho excepcional el que sea la primera manifestación de una neoplasia primaria subyacente clínicamente silente. Presentamos a continuación un caso de acrometástasis como forma de presentación de una adenocarcinoma pulmonar que recientemente hemos tenido la oportunidad de observar.

Sr. Director: Existen descritos en la literatura una amplia variedad de trastornos musculosqueléticos paraneoplásicos. De ellos, la osteoartropatía hipertrófica (OH), la dermatomiositis y la poliartritis paraneoplásica son los que se citan con mayor frecuencia. El síndrome hombromano (SHM), variante clínica de la distrofia simpática refleja, es otra entidad reumatológica ocasionalmente asociada a neoplasia. El motivo de esta carta es comunicar el caso de un paciente afecto de adenocarcinoma pulmonar que presentó concomitantemente una OH y un SHM. Se trata de un varón de 61 años de edad, fumador de 60 paquetes/año y con criterios clínicos de bronquitis crónica, que ingresó en enero de 1988 en nuestro hospital por hemoptisis. En 1985, a raíz de ser ingresado en otro centro por un episodio de neumonía, se constató la existencia de un nódulo en LSD. Ocho meses antes de ingresar en nuestro centro el paciente inició de forma insidiosa algias en ambas regiones tibiales junto con dolor y tumefacción en rodillas. Tres meses antes del ingreso se añadió al cuadro dolor en hombro derecho junto con tumefacción difusa de la mano del mismo lado. En la semana previa al internamiento el paciente sufrió varios episodios de hemoptisis por lo que fue ingresado para estudio.

Lung cancer is a serious health problem and the leading cause of cancer death worldwide. The standard use of cell lines as in vitro pre-clinical models to study the molecular mechanisms that drive tumorigenesis and access drug sensitivity/effectiveness is of undisputable importance. Label-free mass spectrometry and bioinformatics were employed to study the proteomic profiles of two representative lung cancer cell lines and to unravel the specific biological processes. Adenocarcinoma A549 cells were enriched in proteins related to cellular respiration, ubiquitination, apoptosis and response to drug/hypoxia/oxidative stress. In turn, squamous carcinoma SW900 cells were enriched in proteins related to translation, apoptosis, response to inorganic/organic substances and cytoskeleton organization. Several proteins with differential expression were related to cancer transformation, tumor resistance, proliferation, migration, invasion and metastasis. Combined analysis of proteome and interactome data highlighted key proteins and suggested that adenocarcinoma might be more prone to PI3K/Akt/mTOR and topoisomerase IIα inhibitors, and squamous carcinoma to Ck2 inhibitors. Moreover, ILF3 overexpression in adenocarcinoma, and PCNA and NEDD8 in squamous carcinoma shows them as promising candidates for therapeutic purposes. This study highlights the functional proteomic differences of two main subtypes of lung cancer models and hints several targeted therapies that might assist in this type of cancer. published

Lung cancer is the most common cancer globally and has the highest mortality. Although this disease is not associated with a particular gender, its incidence is rising among women, who are diagnosed at an increasingly younger age compared with men. One of the main reasons for this rise is women taking up smoking. However, many non-smoking women also develop this disease. Other risk factors implicated in the differential development of lung cancer in women are genetic predisposition, tumour histology and molecular profile. Proportionally more women than men with lung cancer have a mutation in the EGFR gene. This consensus statement reviews the available evidence about the epidemiological, biological, diagnostic, therapeutic, social and psychological aspects of lung cancer in women.