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El gangli sentinella o ganglis sentinelles (GS) és el gangli que recull el drenatge limfàtic del tumor i són de localització axil·lars i extraaxil·lars, com a la cadena mamària interna (CMI). Hi ha evidència a la literatura del pronòstic negatiu de les metàstasis del càncer de mama en la CMI, però ha estat controvertit i abandonat el seu tractament quirúrgic, donada la morbiditat i la manca d'evidència d'impacta en la supervivència. Les pacients, però, seleccionades per rebre el buidament de la CMI presentaven tumors detectats clínicament. Des dels estudis publicats l'any 1997, on es demostrava que la radioteràpia (RT) postmastectomia en pacients de risc tant post com menopàusiques aporta un increment de la supervivència i que en la planificació de la irradiació sempre s'incloïa la CMI, hi ha un interès renovat en el coneixement i inclusió d'aquesta cadena ganglionar en la planificació de la RT. El tractament de RT incloent la CMI és complexa i incrementa el risc de toxicitat. A més, la complexitat augmenta quan s'ha realitzat una cirurgia conservadora de la mama que és la tècnica més àmpliament utilitzada en tumors en Estadi I-II. L'estudi del GS aplicat a tumors en Estadis I-II amb la comprovació de l'afectació de la CMI i les repercussions tant d'indicació com de la planificació de la RT és l'eix d'aquesta tesi. La hipòtesis d'aquest treball és que la tècnica de la biòpsia selectiva del GS aplicada a la CMI pot tenir implicacions terapèutiques en la RT.S'han analitzat diversos grups de pacients amb estudi del GS per càncer de mama. Subgrup a. (n=225) des del juny'98 fins desembre'02, d'elles 105 en la fase primera de validació (subgrup a.i) de la tècnica (fins desembre'00 (tumors fins 4 cm i sense biòpsia de la CMI) i 120 en fase d'aplicació (subgrup a.ii) (tumors fins 2,5 cm). Des de l'inici de la fase d'aplicació fins maig'04 s'inclouen 220 pacients (subgrup b.ii) on s'hi valora la taxa de recaigudes axil·lars i falsos negatius (FN) i el subgrup c.ii amb 278 pacients en la fase d'aplicació fins juliol'04. En total 383 pacients entre juny'98 i juliol'04. Des del gener'03 es va incloure tumors fins 3 cm. En el subgrup a.ii la taxa de pacients amb identificació del GS en l'acte quirúrgic va ser del 96,6%, amb un 17,2% a la CMI. Exèresis CMI exitosa 14/20 (69%) i en 2 casos (14%) es va demostrar infiltració per carcinoma. Del total de les pacients en què es va identificat el GS durant l'acte quirúrgic el percentatge de metàstasi a la CMI és baix, un 1,7% i en ambdós casos el GS axil·lar també va ser positiu. En el subgrup c.ii les dades són similars (1,8%). FN: 8,3% subgrup a.i; 2,7% subgrup a.ii i 3,45% subgrup b.ii, que es correlaciona amb la mida màxima tumoral definida en cada subgrup. El drenatge a la CMI va ser més freqüent en tumors de quadrants inferiors (24,5%) i medials (23%), essent la localització més freqüent el quadrant ínfero-medial (31,7%). Tot i que el valor real de la inclusió de la CMI en la planificació de RT en el càncer de mama en estadis inicials es desconeix i pel baix percentatge de metàstasis a la CMI en aquest estudi, no es podrà planificar un assaig aleatoritzat. No es recomana la inclusió de la CMI en el volum de tractament de RT a no ser que l'exèresi del GS a la CMI sigui positiu i sempre planificant la RT amb tècniques 3D per reduir al màxim els efectes adversos a nivell de la unió de camps i reduir la dosi als òrgans en risc., "Incidence of Internal Mammary Node Metastases After a Sentinel Lymph Node Technique in Breast Cancer and its Implication in the Radiotherapy Plan"To analyze the frequency in determining pathologically-proven metastatic involvement of internal mammary nodes (IMN) after sentinel lymph node (SLN) technique in breast cancer and evaluate its implications for radiotherapy (RT) management of patients.METHODS AND MATERIALS: Three hundred and eighty three patients who underwent lymphatic mapping for early breast cancer from June 1998 to July 2004 were evaluated. There were different subgroups, subgroup a. 225 patients till December 2002 with two phases in that period: the validation phase till December 2000 (subgroup a.i) (105 patients, T1, T2  4 cm), and the application phase (subgroup a.ii) (120 patients, T1, T2 < 2.5cm). The application phase till May 2004 (220 patients) and till July 2004 (278 patients). Since January 2003 tumors were included till 3 cm. In the validation if a drainage pathway to the IMN was identified no biopsy was performed in this phase. In the application if the study showed metastases in the IMNs biopsy was performed. When histologically proven IMN metastases were detected, RT was included on the IMN chain planned with a 3D treatment system using conformal techniques. At the beginning of the study the injection site was subdermal and subsequently, the injection site was changed to peritumoral and intratumoral in order to search for IMN.RESULTS: In 31 patients of 225 (14%) hot spots were observed in the IMN (11.5% and 17.2% in the phases a.i and a.ii respectively). Sampling of IMN was successful in 69% of the cases (14/20) and revealed metastatic involvement in 14% (2/14). This represents incidence of only 1.7% (2/116) in early breast cancer patients. These results were similar in the subgroup c.ii (1.8%). False negative rate: 8.3% subgroup a.i; 2.7% subgroup a.ii and 3.45% subgroup b.ii. CONCLUSIONS: The percentage in pathologically-proven metastatic involvement of IMN after SLN technique in early breast cancer is low, but it is not negligible. Although the real value of IMN irradiation in early breast cancer is not known, including this chain in post conserving surgery is not recommended unless pathologically-proven IMNs have been produced by SLN technique. In order to avoid over dosage or under dosage in the joint between the medial tangential and IMN fields an individualized 3D dosimetry study is mandatory to enhance dose distribution and reduce the heart volume to lessen side-effects.

The aim of this study was to compare contrast-enhanced ultrasonography (CEUS) to baseline US and contrast-enhanced computed tomography (CT) in metastatic disease of the liver diagnosed or suspected by US during presurgical staging or postsurgical follow-up for primary malignancies.Two hundred-fifty-three patients considered suitable for US due to the complete explorability of the liver and with one to five proven or suspected liver metastases at baseline US were included. All patients underwent US before and after microbubble injection, and multiphase contrast-enhanced CT. Independent panels of readers reviewed US and CT scans and recorded liver metastases according to a 5-grade scale of diagnostic confidence. Sensitivity, specificity (diagnostic performance) and area under the receiver operating characteristics (ROC) curve (diagnostic confidence) were calculated.Reference standards revealed no metastases in 57/253, more than five in 59/253, and one to five in 137/253 patients. In patients with one to five metastases, CEUS versus baseline US revealed more metastases in 64/137 and the same number in 73/137 patients while CEUS versus CT revealed more metastases in 10/137, the same number in 99/137, and lower number in 28/137. Sensitivity, specificity, and area under ROC curve of CEUS (83%, 84%, 0.929, respectively) differed from baseline US (40%, 63%, 0.579, respectively; P0.01) while did not differ from CT (89%, 89%, 0.945, respectively; P0.05).CEUS improved liver metastases diagnosis in comparison with baseline US while it revealed similar diagnostic performance and confidence to contrast-enhanced CT in patients considered suitable for US and with proven or suspected liver metastases at baseline US.

Metastatic bone lesions are exceptional at diagnosis in germ cell tumors (GCTs). Bone involvement is usually a late event combined with synchronous metastasis in the retroperitoneal lymph nodes, lung and liver. Bone examination is not considered a standard procedure in the staging of GCTs, and this may contribute to underestimation of the real proportion of bone metastases. Here we report a case of nonseminomatous GCT of the testis with a synchronous, symptomatic, solitary pubic bone metastasis that was completely controlled by systemic chemotherapy and locoregional radiation therapy. Solitary bone metastases from GCTs seem to be chemosensitive and radiosensitive, but we do not know their prognostic value. We reviewed the literature where 3 similar cases have been reported. We propose individualized management for symptomatic GCT patients including bone scintigraphy and/or PET examination at diagnosis and a combined cytotoxic approach with chemotherapy and radiation therapy

Aquest informe tècnic mostra la classificació, incidència, característiques i diagnòstic dels tumors ossis primaris i secundaris metastàsics més freqüents a partir de 145 radiografies digitalitzades, This technical report introduces the classification, incidence and diagnosis of common primary bone tumors. A set of 145 digitized radiographs were characterized. The differences between healthy bone and bone tumors were studied by using image processing and analysis. This work shows the usefulness of our method to contribute in the differential diagnosis between healthy and pathological bone when the exploration is insufficient.

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.

BACKGROUND Investigation of the relation between primary tumor and metastatic disease is necessary for the identification of predictive factors for postrecurrence survival (PRS) in patients with recurrent osteosarcoma. METHODS Cellular levels of P-glycoprotein, ErbB-2, p53, and Bcl-2 expression were evaluated in primary tumor biopsy and metachronous pulmonary metastasis specimens from 19 patients with high-grade osteosarcoma. Results were analyzed for differences between primary tumor and pulmonary metastases and for correlations between expression patterns and survival. RESULTS Positive staining in lung metastases was noted in 68%, 53%, 32%, and 84% of patients for P-glycoprotein, ErbB-2, p53, and Bcl-2, respectively. These percentages were higher than those observed in primary tumor specimens for all genetic markers evaluated, with a significant difference in the percentage of patients with positive staining for P-glycoprotein (68% vs. 32%; P = 0.05) and a near-significant difference in the percentage of patients with positive staining for Bcl-2 (84% vs. 53%; P = 0.08). Patients with ErbB-2 expression in the primary tumor were more likely to have multiple metastases and shorter recurrence-free intervals compared with patients in whom ErbB-2 expression was not observed, whereas differences in P-glycoprotein, p53, and Bcl-2 expression were not related to differences in metastatic pattern. PRS was influenced by p53 expression levels in pulmonary metastases, with patients who had negative staining for p53 having a significantly better PRS rate relative to patients with positive staining for p53 (3-year PRS rate: p53-negative, 64%; p53-positive, 17%; P = 0.008). CONCLUSIONS In the current study of patients with high-grade osteosarcoma, most patients exhibited increased cellular expression of P-glycoprotein, ErbB-2, and Bcl-2 in recurrent pulmonary metastases compared with primary tumor. Further studies aimed at investigating the relation between altered p53 expression in lung metastases and postrecurrence survival are recommended. Cancer 2004. © 2004 American Cancer Society.

[cat] Introducción: Los tumores diagnosticados a partir de sus metástasis en los que no se identifica el tumor primario, a pesar de un estudio más o menos exhaustivo, se conocen con el nombre de cáncer metastásico de origen desconocido (CMOD). Aquellos casos en que, siendo el motivo de consulta una metástasis y el origen no es evidente antes de la visita inicial, los hemos etiquetado como cáncer de presentación metastásica (CPM). El estudio diagnóstico debe consistir en un programa de exploraciones dirigidas a la identificación de aquellos casos que se puedan beneficiar de un tratamiento específico, tanto por razón de su tumor primario, como por determinados factores pronósticos individuales.Material y métodos: Entre enero de 1992 y diciembre de 1997 se estudiaron de forma prospectiva aquellos pacientes (pts) ingresados con CPM. Se aplicó un Estudio Básico (EB) consistente en una historia clínica y exploración física completa, análisis estándar de sangre con marcadores tumorales y una Rx tórax. Se consideraron CMOD los pts con EB negativo, y a éstos se les aplicó el Estudio Adicional (EA): TAC abdominal y mamografía (mujeres). El resto de pts se sometieron a seguimiento. Se realizó un análisis uni y multivariado para la identificación de los factores pronósticos relacionados con la supervivencia.Resultados: Se incluyeron 234 pts. El EB resultó positivo en 140 pts (59.8%). El EA identificó el tumor primario en 24 casos más, 8 considerados tratables. El tumor primario fue identificado en 182 casos (77.8%). El cáncer de pulmón fue el más frecuente (40.1%), seguido por la próstata (6,4%), mama (5.1%) y ovario (4.7%). Por histologías: adenocarcinoma bien y moderádamente diferenciado (55.6%), seguido del carcinoma y adenocarcinoma indiferenciado (24.0%) y carcinoma neuroendocrino y de célula pequeña (10.7%).Los análisis uni y el multivariado identificaron al tumor primario como el factor pronóstico fundamental. Las otras cinco variables pronósticas independientes en el análisis multivariado fueron: la edad, el estado funcional, localización metastásica dominante, histología y la modalidad de tratamiento. Las afectaciones ganglionar, pleural, ósea o peritoneal obtuvieron también información unívoca de una mayor supervivencia.Conclusiones: Es necesario descartar mediante protocolos de diagnóstico rápidos, todos aquellos tumores primarios que se presentan de forma diseminada y que gozan de tratamientos específicos. Asimismo, se deben seguir identificando todas aquellas situaciones, en que, a pesar de no haberse diagnosticado el tumor primario, podrían recibir tratamientos eficaces, o cuanto menos ser candidatos a ensayos clínicos.

Els factors de transcripció de la família Snail són fonamentals en la "transició epiteli-mesènquima", procés morfogènic essencial en el desenvolupament embrionari i en els fenòmens metastàsics tumorals.En els mamífers l'activitat d'Snail és modulada per dos mecanismes. (i) En el promotor humà es troben regions definides de resposta a factors repressors, predominants en les cèl·lules epitelials, i elements diferenciats de resposta a inductors de la "transició epiteli-mesènquima". (ii) L'activitat d'Snail és condicionada també per la seva localització subcel·lular, modulada per mecanismes no transcripcionals: la fosforilació d'Snail determina si és o no exclós del nucli. Al citosol no pot actuar com a repressor transcripcional però pot interaccionar amb la xarxa microtubular, que estabilitza i en condiciona el dinamisme. Això coincideix amb l'activació de la GTPasa RhoA i la reorientació dels filaments de vimentina, fets associats a l'adquisició de capacitat migratòria. L'efecte com a repressor transcripcional i la modulació del dinamisme microtubular són possiblement esdeveniments coordinats necessaris per al rol biològic d'Snail en mamífers., Snail family of transcription factors is fundamental to the "epithelial-mesenchymal transition", morphogenic process essential to embryonic development and metastatic phenomena in tumors.Snail's activity is modulated in two ways in mammals. (i) The human promoter harbors definite regions that respond to repressor factors, which prevail in epithelial cells; and differentiated elements that respond to known inducers of the "epithelial-mesenchymal transition". (ii) Snail's activity is also conditioned by its subcellular localization, mechanism not dependent on its transcriptional control: Snail phosphorylation determines whether Snail is excluded or not from the nucleus. When in the cytosol, Snail is unable to act as a transcriptional repressor, but however binds to the microtubular meshwork, which becomes stabilized and whose dynamism is conditioned as a result. This fact coincides with the activation of the RhoA GTPase and reorientation of vimentin filaments, both phenomena being related to the acquisition of cell motility. The transcriptional repressor and the microtubule dynamics effects are probably two coordinated events necessary to Snail's biological role in mammals.

To determine whether a pathologic fracture in osteosarcoma of long bones has prognostic importance, and limb salvage can be safely performed in such cases, we reviewed the surgical treatment and oncologic results in 46 patients with nonmetastatic osteosarcoma of the extremity and pathologic fracture at presentation who had been treated in our Institution with neoadjuvant chemotherapy, between 1983 and 1999. Neoadjuvant chemotherapy was given according to 6 consecutive protocols. Surgery consisted of limb salvage (34 patients), amputation (11 patients) and rotationplasty (1 patient). The average follow-up was 11 (3-20) years. 28 patients remained continuously disease-free, 17 patients relapsed and 1 died of chemotherapy-related toxicity. Despite the high rate of limb salvage, only 2 local failures occurred, 1 after amputation and 1 after limb salvage. The 5-year disease-free survival and overall survival rates were 59% and 65%, respectively, with no differences between amputated and resected patients. These results are similar to those obtained in 689 contemporary patients having an osteosarcoma without a pathologic fracture treated in our Institution, and using the same protocols for chemotherapy. We conclude that with neoadjuvant chemotherapy, osteosarcoma patients presenting with a pathologic fracture can be surgically treated like those with no fracture, and that limb salvage procedures do not increase the risk of local recurrence or death of these patients.

En un grupo de tumores de mama se decidió estudiar si la pérdida de función de la proteína Bax podía ser debida a la existencia de mutaciones frameshift. Los resultados obtenidos sugieren que en cáncer de mama, las mutaciones del gen bax no serían críticas para la carcinogénesis y el proceso de progresión tumoral. Este tipo de mutaciones sólo se han detectado en tumores con inestabilidad de microsatélites, por lo tanto decidimos estudiar cual era la incidencia de este tipo de alteración en nuestro grupo de tumores, encontrando que un 11,6 % eran inestables. La inestabilidad detectada quedó delimitada a las secuencias que afectaban a dinucleótidos, no se encontró ninguna alteración en las secuencias de mononucleótidos analizadas. Se encontró una asociación estadísticamente significativa entre la existencia de inestabilidad de microsatélites y la pérdida de apoptosis observada en los tumores. La mayor supervivencia celular resultado de la pérdida de apoptosis podría favorecer la existencia de este tipo de alteración en los tumores.Para corroborar si la pérdida de apoptosis se podía asociar a la existencia de inestabilidad de forma genérica en los tumores de mama, se decidió estudiar si la sobreexpresión de las proteínas antiapoptóticas Bcl-2 y Bcl-xL era capaz de inducir un acúmulo de alteraciones genéticas, y el efecto que las mismas podían tener sobre el proceso de progresión tumoral.Al analizar el daño genómico en las líneas transfectadas con respecto a la línea parental, los transfectantes con genes antiapotóticos tenían un GDF mayor que los transfectantes con el gen control. Por lo tanto, la inhibición de la apoptosis era capaz de inducir un aumento en el número de alteraciones genéticas detectables por AP-PCR, contribuyendo así a la inestabilidad genética que caracteriza a las células tumorales. El análisis filogenético de las líneas transfectadas, con respecto a la línea parental indicaba que los transfectantes con genes antiapoptóticos tenían un parentesco más cercano, originado por una mayor similitud de las alteraciones genéticas, mientras que las alteraciones de la línea control presentaban un mayor grado de divergencia. El análisis de los resultados mediante un programa de clusters mostraba la presencia de 3 zonas donde el tipo de alteraciones presentes en las líneas transfectadas con los genes antiapoptóticos y con el gen control son divergentes.Al analizar el GDF de las diferentes variantes tumorales y metastáticas, tomando como referencia las líneas derivadas de la generación anterior los resultados indicaban que a medida que avanzaba el proceso de progresión tumoral, el número de alteraciones genéticas detectadas iba en aumento, tanto a nivel de tumores como de metástasis. Este aumento de inestabilidad se correlaciona con una mayor capacidad tumorigénica y metastática de las líneas celulares. Sin embargo, las variantes celulares con mayor capacidad tumorigénica y metastática no son las que presentan un mayor número de alteraciones. Estos resultados indicarían que sólo una parte de las alteraciones detectadas son responsables del fenotipo tumoral y metastático. Por lo tanto quizá las alteraciones detectadas en el control son más aleatorias y sin repercusión biológica, mientras que la sobreexpresión de moléculas antiapopotóticas da de entrada una ventaja de crecimiento, con lo cual la célula es capaz de progresar con un menor número de alteraciones genéticas, pero de mayor efectividad biológica.El daño genómico de las metástasis con diferente organoespecificidad mostraba un comportamiento diferencial, tanto en relación con el número como la naturaleza de las mismas, en función del órgano de origen. Finalmente, decidimos estudiar si la organoespecificidad de las metástasis podía ser debida a cambios en la expresión génica. Estos estudios se hicieron utilizando la técnica de microarrays. Una vez hecho el análisis estadístico se determinaron genes asociados a actividad metastática, que serían aquellos genes que aparecían diferencialmente expresados en las cuatro variantes metastáticas analizadas. También se identificaron los genes cuya expresión se modificaba conjuntamente en las dos metástasis ganglionares, y aquellos cuya expresión diferencial se asociaba a la presencia de metástasis ósea., We studied in a group of breast cancer samples if the loss of function of Bax protein was due to frameshift mutations. Our results show that in breast cancer, bax mutations are not relevant for tumoral progression. This kind of alterations was only found in tumors with microsatellite instability, so we decide to know the incidence of this kind of instability in our samples, the result was that 11,6 % of the tumors show instability. The instability was detected only in dinucleotide sequences, no alteration was found in the mononucleotides analyzed. There was an association, with stadistic significance, between the microsatellite instability and the loss of apoptosis detected in the tumors. Loss of apoptosis can promote survival and this could favour the induction of this kind of alteration in tumors.To confirm if there is an association between loss of apoptosis and genetic instability in breast cancer cells, we studied if the overexpression of the antiapoptotic proteins Bcl-2 and Bcl-xL was able to induce an accumulation of genetic alterations, and which will be the effect of this alterations in tumor progression.The analysis of the genomic damage in transfected cells with regard to the parental line, show that cell lines overexpressing antiapoptotic genes have a greater GDF than control cells. So, the inhibition of apoptosis can induce an increase in the amount of genetic alterations detectable by AP-PCR, and can contribute to the genetic instability of tumoral cells.The philogenetic analysis of the transfected cells with regard to the parental line show that cell lines overexpressing antiapoptotic genes have a nearest relationship, due to a greater similarity between the genetic alterations. On the other hand, the alterations of the control cells are more divergent. The analysis of these results with a cluster software shows the existence of at least 3 regions with different kind of genetic alterations between cells transfected with antiapoptotic genes and control cell.The GDF analysis in the tumoral and metastatic variants, taking as a reference the cell lines derived from the previous generation, show that with the advance of tumor progression, there was an increase in the number of genetic alterations, both, in tumors and in metastasis. This increase in the detected instability correlates with a greater tumorigenic and metastatic ability of the cell lines. Nevertheless, the cell variants with a greater tumorigenic and metastatic ability show less genetic alterations. These results indicate that only some of the genetic alterations detected are responsible of the tumoral and metastatic phenotype. So, perhaps the alterations detected in the control cells are more random, and without biological significance. On the other hand, the overexpression of antiapoptotic genes could give the cells and inicial advantage, so the tumoral cell will be able to progress with less alterations, but more eficient ones.The genomic damage in metastasis with different organospecificity show a differential behaviour with regard to the organ of origin both, with regard to the number and the nature of the genetic alterations detected.Finally, we studied if the organospecificity of metastasis could be due to changes at gene expression level. These experiments have been performed using microarrays. After the analysis of the data, we have determined wich genes were associated to the metastatic ability of the cells. These genes will be those whose expression was modified simultaneously in the 4 metastatic variants studied. We have also determined wich genes were differentially expressed in lymh node metastasis, and those that were associated to bone metastasis.

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").

A case of fibrosarcoma in a 4-year-old male child is reported. Primary fibrosarcomas of bone in the head and neck region are rare. The histological appearance of the tumour is related to its grade of differentiation. It could present a high level of cellularity and the amount of collagen is variable. The accepted treatment is radical surgery; however, metastases could occur in the lungs. This article is presented to highlight the rarity of fibrosarcomas in the jaws of children.

The effects of E-64 (Cathepsin B and L inhibitor) and Pepstatin A (Cathepsin D inhibitor) on spontaneous and experimental metastasis formation were investigated in mice with MCa mammary carcinoma, M5076 ovarian sarcoma and L1210 leukemia. Pepstatin induced a marked decrease in the number of spontaneous metastasis in MCa or M5076 tumor bearing mice. This phenomenon was also noted with E-64 but only in M5076 tumor bearing mice. On the other hand, both these agents were unable to prevent the formation of experimental metastasis in mice injected i.v. with L1210, MCa or M5076 tumor cells or with tumor cells in which Cathepsin B, L and D activities were inhibited by a 24 hour continuous exposure to high non-cytotoxic concentrations of E-64 and/or Pepstatin. These data suggest that Cathepsin B, L and D seem to be involved in the early steps of the metastatic process rather than in the hematogenous spread of tumor cells. However, other pharmacological activities which may account for the discrepant effects of E-64 or Pepstatin on experimental and spontaneous metastasis cannot be ruled out.

Cathepsin D, B and L activity levels were determined in colorectal cancer and correlated with a number of biological and clinical parameters. Our studies have evidenced significant higher activity levels of these lysosomal enzymes in tumor cytosol compared to paired normal mucosa as well as an evident increase of tumor specific cathepsin D activity in Dukes' stage A tumors compared to later stages (B, C and D). Furthermore, significant higher cathepsin B and L activity levels were observed in Dukes' stage A compared to Dukes' stage D tumors while significant higher cathepsin B activity levels were observed in tumors ≤5 cm than in those >5 cm as well as in moderately differentiated tumors (G2) than in well differentiated (G1) ones. No further correlation between tumor specific cathepsin B activity levels and other parameters examined i.e., anatomical site, nodal status, DNA ploidy, and proliferation rate (S-phase fraction) or between tumor specific cathepsin D and L and all these parameters were observed. These results indicate that cathepsin D, B and L may be involved in colorectal tumor progression by acting, probably, at different levels of this process and suggest that the altered tumor specific activity of these proteinases may be of interest as independent, prognostic factor of malignant progression of this neoplastic disease.

Background Chemokines and chemokine receptors are major actors of leukocytes trafficking and some have been shown to play an important role in cancer metastasis. Chemokines CCL19, CCL20 and CCL21 and their receptors CCR6 and CCR7, were assessed as potential biomarkers of metastatic dissemination in primary breast cancer. Methods Biomarker expression levels were evaluated using immunohistochemistry on paraffin-embedded tissue sections of breast cancer (n = 207). Results CCR6 was expressed by tumor cells in 35% of cases. CCR7 was expressed by spindle shaped stromal cells in 43% of cases but not by tumor cells in this series. CCL19 was the only chemokine found expressed in a significant number of breast cancers and was expressed by both tumor cells and dendritic cells (DC). CCR6, CCL19 and CCR7 expression correlated with histologic features of aggressive disease. CCR6 expression was associated with shorter relapse-free survival (RFS) in univariate and but not in multivariate analysis (p = 0.0316 and 0.055 respectively), and was not associated with shorter overall survival (OS). Expression of CCR7 was not significantly associated with shorter RFS or OS. The presence of CCL19-expressing DC was associated with shorter RFS in univariate and multivariate analysis (p = 0.042 and 0.020 respectively) but not with shorter OS. Conclusion These results suggest a contribution of CCR6 expression on tumor cells and CCL19-expressing DC in breast cancer dissemination. In our series, unlike what was previously published, CCR7 was exclusively expressed on stromal cells and was not associated with survival.

Ruthenium anticancer drugs have attracted an increasing interest in the last 20 years and two of them have entered clinical trials. Compared to platinum drugs, the complexes based on ruthenium are often identified as less toxic and capable of overcoming the resistance induced by platinum drugs in cancer cells. These activities were attributed to the transportation to tumour cells by transferrin and to the selective activation to more reactive species by the reducing environment of solid tumours as compared to healthy tissues. Ruthenium anticancer drugs have been almost always designed to mimic platinum drugs, particularly for targeting DNA. Indeed, none of the above properties has never been clearly demonstrated even for the ruthenium drugs that entered clinical trials. The suggestion for the future is to change the perspective when designing new chemical entities, abandoning the philosophy that guided the actual panel of ruthenium drugs and to look further into the fine mechanism by which the most relevant ruthenium complexes available kill the target tumour cells, then focusing on targets selective of tumour cells and responsible for cell growth and malignancy.

The antitumor and antimetastatic effects of a ruthenium(III) complex, Na[trans-RuCl4(DMSO)Im], have been examined in mice bearing MCa mammary carcinoma. Na[trans-RuCl4(DMSO)Im] is capable of reducing primary tumor growth and of prolonging the survival time with different schedules of administration. However, better effects were obtained (a) with treatments started soon after tumor implantation, (b) with daily administration rather than with treatments at 4-day intervals and (c) using relatively low daily doses. The prolongation of survival time in tumor-bearing hosts, greater than that obtained with cisplatin, cannot be simply related to the effect on primary tumor growth, always less than that of cisplatin. Rather, emphasis is placed on the reduction of lung metastasis formation, obtained either by i.v. injection of tumor cells (lung colonization or spontaneously from i.m. tumor implants, which is significantly greater than the reduction of primary tumor growth. The antimetastatic effect depends on the treatment schedule and is greater with low doses given daily than with high doses administrated with drug-free intervals. Metastasis reduction involved mainly large metastatic nodules, reducing the average weight of each metastasis by 8-fold for spontaneous metastases and by 5-fold for lung colonies. The antimetastatic action of Na[trans-RuCl4(DMSO)Im] thus indicates the possibility that related analogs can represent a new generation of antitumor compounds capable of selectively interacting with metastasis formation of solid tumors.

The antimetastatic activity of the prostacyclin analog Iloprost has been examined in mice bearing Lewis lung carcinoma. An inhibition of lung colony formation is observed when 100 or 200 micrograms/kg Iloprost are administered i.v. 1 h before i.v. injection of tumor cells, which is dependent on the size of tumor inoculum. The effects of 200 micrograms/kg Iloprost persist for 24 h, and are of the same magnitude as those obtained with 10 mg/kg prostacyclin, which last only for 30 min. When treatment with Iloprost is followed by surgical removal of primary tumor, spontaneous metastasis formation is reduced, and the survival time of the treated animals is significantly increased over controls treated with surgery only. The antimetastatic effects of Iloprost appear dissociated from drug's effects on the hemostatic system of the host as indicated by the clot retraction assay, performed after in vivo treatment, using ADP or tumor cells as platelet aggregating agents. Iloprost thus appears to reduce spontaneous metastasis formation and intraoperative tumor cell dissemination, with pharmacological properties more favourable to therapeutic use than those of prostacyclin.

Summary The effects of the ortho and para isomers of aryldimethyltriazeno carboxylic and benzensulfonic acids and amides have been examined in mice bearing subcutaneous Lewis lung carcinoma. The toxicity of these compounds varies widely and does not correlate with the effects on tumor progression. The growth of subcutaneous primary tumor is unaffected or only marginally inhibited by all the tested compounds, while a marked depression in the formation of spontaneous lung metastasis is observed, with the exception of the sodium salt of the ortho benzensulfonic acid. The compounds showing the greatest activity on metastasis are the hydrosoluble salts of the ortho and para carboxylic acid and the para sulfonamido derivative, indicating that, in addition to the activity reported for the potassium salt of the para carboxylic acid, also its ortho isomer and the para sulfonamido derivative possess selective and pronounced antimetastatic effects. No correlation can be found between the reported effects on tumor progression and physicochemical parameters of the triazenes tested.

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.

Las alteraciones morfológicas pueden considerarse, en un sentido amplio, como el primer marcador tumoral útil para el estudio de las neoplasias. En base a ello, desde hace muchos años los patólogos reconocen los tumores según las características citológicas y estructurales de los tejidos neoplásicos, que permiten distinguirlos de sus homólogos sanos.

Circulating tumor cells can be detected by means of flow cytometry in the blood of mice bearing i.m. Lewis lung carcinoma. This technique can be applied in the case of aneuploid tumors and does not require either concentration of nucleated cells or other processing of the blood samples. It offers the advantages of simplicity and speed and allows quantitative measurement of the number of circulating tumor cells. It can be applied to the study of the effects of drug treatment on the number of circulating tumor cells, for those drugs which do not cause alterations in the nuclear DNA content of normal diploid blood cells. The number of circulating tumor cells determined by flow cytometry is markedly reduced by treatment with ICRF-159, by dimethyltriazene DM-COOK, and also by its clinically used analog, DTIC.

Metastatic tumors in soft tissues of oral cavity are uncommon, but toungue metastasis are quite infrequent. If it occurs, the invasion is indicative of a drop in the whole organism defenses. We describe two cases of lung carcinoma spreaded to the tongue and the post-mortem examinations. Before oral manifestations we confirm the development of many metastasis located in various organs. - Le metastasi ai tessuti molii del cavo orale a partenza da tumori localizzati in altri distretti dell'organismo sono molto rare. Tra queste le metastasi alla lingua appaiono estremamente infrequenti ed indicano un notevole scadimento delle condizioni di resistenza dell'organismo. Vengono anche descritti due casi di carcinoma polmonare metastatizzato alla lingua completi di rilievo autoptico che evidenzia le reali capacità invasive del tumore primitivo nei confronti dell'intero organismo.

L'autore prende in esame gli aspetti eziopatogenetici e clinici delle metastasi ossee dei mascellari. Viene inoltre trattato un caso particolarmente significativo. Una diagnosi precoce, anche se non risolutiva del tumore, permette di nsparmiare notevoli pene al malato e consente l'attuazione di una terapia mirata.