Introdução: Os carcinomas vulvares são tumores raros com morbidade elevada associada ao tratamento cirúrgico padrão e altos índices de recorrência loco-regional. Os vasos linfáticos são importantes vias de disseminação regional e o estado linfonodal é o principal indicador prognóstico. A densidade linfática do tumor, assim como moléculas relacionadas à linfangiogênese tem sido avaliadas em vários tumores para prever metástase linfonodal e para identificar possíveis alvos terapêuticos. Entre as moléculas relacionadas ao controle da linfangiogênese destaca-se a podoplanina, cuja expressão por células neoplásicas de tipo escamoso pode inibir a disseminação linfática. Não identificamos até o momento nenhum estudo que tenha avaliado o papel da densidade de vasos linfáticos intratumorais ou da expressão de podoplanina pelas células neoplásicas no comportamento de carcinomas escamosos vulvares. Objetivos: Nossos objetivos foram estudar a densidade intratumoral dos vasos linfáticos (DVL) e a expressão de podoplanina pelas células neoplásicas em carcinomas escamosos vulvares no sentido de determinar sua relação com o desfecho e fatores prognósticos clássicos, incluindo metástase linfonodal. Métodos: Selecionamos 35 pacientes com carcinoma de células escamosas vulvares submetidas à tratamento cirúrgico primário incluindo vulvectomia e dissecção regional de nódulos linfáticos. Após revisão dos dados dos prontuários médicos (idade da paciente, estadiamento, tipo de tratamento e tamanho do tumor), todas as lâminas foram reexaminadas para determinar o grau histológico, invasão linfática peritumoral e profundidade da infiltração. Foram selecionadas áreas do tumor para a construção de blocos de parafina com microarranjos de tecidos e identificação imunoistoquímica de podoplanina pelo anticorpo D2-40. A DVL intratumoral foi quantificada pela contagem de vasos marcados nas áreas de maior densidade. O número de vasos foi contado em 10 campos microscópicos de grande aumento e a média foi o valor atribuído para a DVL em cada caso. A expressão de podoplanina pelas células neoplásicas foi considerada positiva quando mais de 10% das células apresentaram coloração citoplasmática de intensidade moderada a intensa. Investigamos a associação das duas variáveis com as características prognósticas clássicas (idade, estadiamento, tamanho do tumor, grau histológico, embolização vascular peritumoral, nível de infiltração do tumor, comprometimento linfonodal), assim como sua associação com desfecho. Resultados: Valores mais elevados de DVL intratumoral foram identificados em tumores de baixo grau, em estádios iniciais, em tumores sem invasão linfática e naqueles com menor infiltração estromal. Na análise univariada, DVL intratumoral elevada foi associada a maior sobrevida geral . A expressão de podoplanina pelas células neoplásicas não se relacionou a nenhuma das variáveis prognósticas, comprometimento linfonodal ou sobrevida, assim como não se associou à DVL. Conclusões: A DVL intratumoral em carcinomas escamosos vulvares associa-se a características prognósticas favoráveis. Já a expressão de podoplanina pelas células neoplásicas não parece interferir na apresentação e comportamento destes carcinomas. / Introduction: Vulvar carcinomas are rare tumors presenting high morbidity associated to the standard surgical treatment and high rates of locoregional recurrence. Lymphatic vessels serves as major routes for regional dissemination and the lymph node status is the main prognostic indicator. Tumor lymphatic density as well as lymphangiogenesis-related molecules has being studied in various tumors in order to predict lymph node metastasis and to identify a possible candidate to target therapy. Among the molecules related to lymphangiogenesis in the group of squamous cell carcinomas podoplanin expression by neoplastic cells emerges as an inhibitor of lymphatic dissemination. To our knowledge, no study evaluated the role of intratumoral lymphatic vessels or podoplanin expression by neoplastic cells in the behavior of vulvar squamous carcinomas. Objectives: Our aims were to study the intratumoral lymphatic vessel density (LVD) and podoplanin expression by neoplastic cells in vulvar squamous carcinoma according their relationship with outcome and classical prognostic factors, including lymph node metastasis. Methods: We selected 35 cases of patients with vulvar squamous cell carcinoma submitted to primary surgical treatment that included vulvectomy and regional lymph nodes dissection. After revision of medical records data (age of patient, stage, type of surgery and tumor size), all the slides were reviewed to achieve histological grade, peritumoral lymphatic invasion and depth of infiltration. Areas of the tumor were selected to construction of tissue microarrays paraffin blocs and immunohistochemical identification of podoplanin by the D2-40 antibody. Intratumoral LVD was quantified by counting the stained vessels in hotspots areas. The number of vessels was counted in 10 high power microscopic fields and the mean was the value of the LVD for each case. Podoplanin expression by neoplastic cells was considered positive when more than 10% of the cells showed moderate to strong cytoplasmatic stained. We investigated the association of the two variables with classical prognostic features (age of patient, stage, tumor size, histologic grade, vascular involvement, depth of infiltration, lymph node involvement as well outcome. Results: Higher values of intratumoral LVD were identified in low grade and low stage tumors, in tumors without lymphatic invasion and those with lesser stromal infiltration. In univariate analysis, high intratumoral LVD was associated to higher overall survival. Podoplanin expression by neoplastic cells did not show association with any of the prognostic variable, nor with lymph node involvement or outcome. Conclusions: Intratumoral LVD in vulvar squamous carcinoma is associated with prognostic favorable features. On the other hand, podoplanin expression by neoplastic cells did not seem to influence the behavior of these carcinomas

INTRODUÇÃO: A ressecção de metástases hepáticas (MH) do câncer colorretal (CCR) é considerada segura e potencialmente curativa. Apesar dos avanços no diagnóstico e nas estratégias cirúrgicas até 70% dos pacientes operados vão apresentar recidiva. Os critérios prognósticos clínico-patológicos disponíveis são insuficientes e a utilidade de modelos prognósticos é considerada inconsistente. Deste modo, torna-se necessário o desenvolvimento de novos critérios que se apoiam em outras variáveis biológicas. A disseminação intra-hepática de metástases de câncer colorretal pode ocorrer através de diferentes vias: linfática, sanguínea (vasos portais), através dos sinusóides ou de ductos biliares. Embora estas vias tenham sido bem descritas, o valor prognóstico de cada uma após a ressecção hepática não está completamente definido. O adenocarcinoma colorretal mucinoso (ACM) é um subtipo de carcinoma colorretal com importante produção mucina, e que está associado clinicamente a tumores proximais, estágio avançado no momento do diagnóstico, instabilidade de microssatélites e mutação do gene BRAF. Há controvérsias sobre o impacto prognóstico da histologia mucinosa nos tumores colorretais primitivos e o seu papel nas lesões metastáticas hepáticas permanece desconhecido. O objetivo do nosso estudo foi determinar a frequência e elucidar o impacto prognóstico de quatro vias diferentes de disseminação intra-hepática assim como da histologia mucinosa em uma série consecutiva de pacientes operados por MH de CCR. PACIENTES E MÉTODOS: Os prontuários de 132 pacientes submetidos à ressecção cirúrgica de MH de CCR entre dezembro de 1999 e janeiro de 2010 foram revisados. Os pacientes que faleceram por complicações pós-operatórias (n= 3), aqueles com ressecções incompletas (R2; n= 2), ou nos quais não havia material disponível para o estudo (n= 14) foram excluídos. Os 113 pacientes restantes tiveram suas variáveis clínico-patológicas e resultados (recidiva e sobrevida) avaliados. Os espécimes cirúrgicos foram submetidos à avaliação histológica de rotina e agrupados de acordo com o conteúdo mucinoso da maior lesão hepática da seguinte forma: ACM >50 %; adenocarcinoma mucinoso intermediário (AIM) com componente mucinoso < 50 %; não-MAC (NMA) se ausência de componente mucinoso. A disseminação intra-hepática foi analisada por hematoxilina e eosina e imuno-histoquímica através dos anticorpos anti-D2-40 (vasos linfáticos), anti-CD34 (vasos sanguíneos), anti-CK-7 (epitélio biliar) e anti-CK-20 (epitélio colorretal). RESULTADOS: O tempo médio de seguimento após a ressecção foi de 37 meses. Recidivas foram observadas em 76 pacientes, com um intervalo médio de 13 meses após a ressecção. As porcentagens de sobrevida global e de sobrevida livre de doença (SLD) após a hepatectomia em 3 e 5 anos foram de 62 e 56 %, e 26% e 24%, respectivamente. A disseminação intra-hepática foi classificada em quatro categorias distintas avaliadas separadamente: invasão da veia porta, invasão dos sinusóides, através dos ductos biliares, e através dos vasos linfáticos/espaço perineural. Encontrou-se invasão microscópica intravenosa portal em 49 pacientes, sinusoidal em 43, biliar em 20 e linfática em 33 pacientes. A disseminação intra-hepática através dos vasos linfáticos foi a única via de disseminação independentemente associada ao risco de recidiva hepática (OR= 2,75) e de menor SLD (p= 0,006). Os focos tumorais de invasão angiolinfática intra-hepática foram detectados em um raio de 2mm de distância da lesão principal. As lesões com componente mucinoso (MAC e AIM) estiveram relacionadas à localização proximal do tumor primário e ao sexo feminino. A análise multivariada revelou que as lesões de tipo ACM apresentavam prognóstico desfavorável (RR= 3,13; IC95% 1,30 - 6,68; p= 0,011) quando comparadas às lesões de componente mucinoso < 50% (NMA e AIM). CONCLUSÃO: A presença de invasão angiolinfática intra-hepática é um importante fator prognóstico após a ressecção de MH de CCR. As outras vias de disseminação intra-hepática, embora observadas com freqüência, não estão relacionadas à recidiva da doença ou sobrevida, sugerindo que o sistema linfático possa ser a principal via de disseminação de MH de CCR. Além disso, o uso da imuno-histoquímica na detecção da invasão angiolinfática intra-hepática parece trazer benefícios na prática clínica. As MH de CCR com componente mucinoso > 50% (ACM) estão associadas a um pior prognóstico quando comparadas às lesões sem componente mucinoso (NMA). A presença de mucina pode, em um futuro próximo, influenciar a estratégia terapêutica e deve se tornar um ponto importante para discussão e investigação futuras / BACKGROUND: Resection of colorectal cancer liver metastases (CRCLM) is generally accepted as a safe and potentially curative treatment. Despite advances in diagnosis and surgical strategies, up to 70% of patients will present recurrence of the disease after resection of CRCLM. Clinicopathological prognostic factors are inadequate to determine disease prognosis and the utility of prognostic models on general population is considered highly inconsistent. Herein, future attempts to develop prognostic scores may include additional biologic variables. Intrahepatic spread from CRLM may take place through four different routes: lymphatic vessels, portal vessels, sinusoids and biliary ducts. Although these routes have been well described, their prognostic value after hepatectomy is not completely understood. Colorectal mucinous adenocarcinoma (MAC) is a subtype of colorectal adenocarcinoma with prominent mucin production associated with tumor proximal location, advanced stage at diagnosis, microsatellite instability, and BRAF mutation. There are controversies about the prognostic impact of mucinous histology on colorectal cancer and the prognostic implication of MAC in CRCLM is unknown. The purpose of our study was to determine the frequency and elucidate the prognostic implication of four different routes for intrahepatic dissemination and the mucinous histology in a consecutive series of resected CRCLM. METHODS: Medical records of 132 patients who underwent a first resection of CRCLM between December 1999 and January 2010 were reviewed. Patients who died from postoperative complications (n=3), those with incomplete resections (R2; n=2), or no available tissue for the study (n=14) were excluded. The remaining 113 patients had their clinicopathologic variables and outcome parameters evaluated. Resected specimens were submitted to routine histological evaluation and were grouped according to metastasis mucinous content: > 50%, MAC; < 50%, adenocarcinoma with intermediated mucinous component (AIM); and without any mucinous component, non-MAC (NMA). Intrahepatic dissemination was analyzed by H&E and immunohistochemical staining with D2-40 (lymphatic vessels), CD34 (blood vessels), CK-7 (biliary epithelium), and CK-20 (CRC cells). RESULTS: Mean follow-up after resection was 37 months. Tumor recurrence was observed in 76 patients, with a median interval of 13 months after resection. Overall survival and disease-free survival (DFS) rates after hepatectomy were 62 and 56%, and 26 and 24% at 3 and 5 years, respectively. Intrahepatic spread was classified into discreet categories that were evaluated separately: invasion of the portal vein, sinusoids, bile duct, and lymphatic/perineural space. Intrahepatic microscopic invasion included portal vein in 49 patients, sinusoidal in 43 patients, biliary in 20 patients, and lymphatic in 33 patients. Intra-hepatic lymphatic invasion was the only route of dissemination independently associated with the risk of hepatic recurrence (OR=2.75) and shorter DFS (P= 0.006). All tumor foci of intrahepatic lymphatic invasion were detected within 2mm away from tumor edge. Tumors with mucinous component (AIM and MAC) were related to proximal location of the primary tumor and were more frequently observed in females. Multivariate analysis revealed that MAC was an independent negative prognostic factor (hazard ratio= 3.13; 95% CI, 1.30-6.68; P= 0.011) compared with non-MAC (NMA and AIM). CONCLUSION: Intrahepatic lymphatic invasion is a significant prognostic factor. Other mechanisms of invasion, although frequently observed, are not related to disease recurrence or survival, suggesting that the lymphatic system is the main route for dissemination of CRCLM. Furthermore, immunohistochemical detection of intrahepatic lymphatic invasion might be of value in clinical practice. MAC has an adverse prognostic impact compared with NMA, which may influence therapeutic strategy raising an important subject for discussion and future investigation

Introdução: Os carcinomas vulvares são tumores raros com morbidade elevada associada ao tratamento cirúrgico padrão e altos índices de recorrência loco-regional. Os vasos linfáticos são importantes vias de disseminação regional e o estado linfonodal é o principal indicador prognóstico. A densidade linfática do tumor, assim como moléculas relacionadas à linfangiogênese tem sido avaliadas em vários tumores para prever metástase linfonodal e para identificar possíveis alvos terapêuticos. Entre as moléculas relacionadas ao controle da linfangiogênese destaca-se a podoplanina, cuja expressão por células neoplásicas de tipo escamoso pode inibir a disseminação linfática. Não identificamos até o momento nenhum estudo que tenha avaliado o papel da densidade de vasos linfáticos intratumorais ou da expressão de podoplanina pelas células neoplásicas no comportamento de carcinomas escamosos vulvares. Objetivos: Nossos objetivos foram estudar a densidade intratumoral dos vasos linfáticos (DVL) e a expressão de podoplanina pelas células neoplásicas em carcinomas escamosos vulvares no sentido de determinar sua relação com o desfecho e fatores prognósticos clássicos, incluindo metástase linfonodal. Métodos: Selecionamos 35 pacientes com carcinoma de células escamosas vulvares submetidas à tratamento cirúrgico primário incluindo vulvectomia e dissecção regional de nódulos linfáticos. Após revisão dos dados dos prontuários médicos (idade da paciente, estadiamento, tipo de tratamento e tamanho do tumor), todas as lâminas foram reexaminadas para determinar o grau histológico, invasão linfática peritumoral e profundidade da infiltração. Foram selecionadas áreas do tumor para a construção de blocos de parafina com microarranjos de tecidos e identificação imunoistoquímica de podoplanina pelo anticorpo D2-40. A DVL intratumoral foi quantificada pela contagem de vasos marcados nas áreas de maior densidade. O número de vasos foi contado em 10 campos microscópicos de grande aumento e a média foi o valor atribuído para a DVL em cada caso. A expressão de podoplanina pelas células neoplásicas foi considerada positiva quando mais de 10% das células apresentaram coloração citoplasmática de intensidade moderada a intensa. Investigamos a associação das duas variáveis com as características prognósticas clássicas (idade, estadiamento, tamanho do tumor, grau histológico, embolização vascular peritumoral, nível de infiltração do tumor, comprometimento linfonodal), assim como sua associação com desfecho. Resultados: Valores mais elevados de DVL intratumoral foram identificados em tumores de baixo grau, em estádios iniciais, em tumores sem invasão linfática e naqueles com menor infiltração estromal. Na análise univariada, DVL intratumoral elevada foi associada a maior sobrevida geral . A expressão de podoplanina pelas células neoplásicas não se relacionou a nenhuma das variáveis prognósticas, comprometimento linfonodal ou sobrevida, assim como não se associou à DVL. Conclusões: A DVL intratumoral em carcinomas escamosos vulvares associa-se a características prognósticas favoráveis. Já a expressão de podoplanina pelas células neoplásicas não parece interferir na apresentação e comportamento destes carcinomas. / Introduction: Vulvar carcinomas are rare tumors presenting high morbidity associated to the standard surgical treatment and high rates of locoregional recurrence. Lymphatic vessels serves as major routes for regional dissemination and the lymph node status is the main prognostic indicator. Tumor lymphatic density as well as lymphangiogenesis-related molecules has being studied in various tumors in order to predict lymph node metastasis and to identify a possible candidate to target therapy. Among the molecules related to lymphangiogenesis in the group of squamous cell carcinomas podoplanin expression by neoplastic cells emerges as an inhibitor of lymphatic dissemination. To our knowledge, no study evaluated the role of intratumoral lymphatic vessels or podoplanin expression by neoplastic cells in the behavior of vulvar squamous carcinomas. Objectives: Our aims were to study the intratumoral lymphatic vessel density (LVD) and podoplanin expression by neoplastic cells in vulvar squamous carcinoma according their relationship with outcome and classical prognostic factors, including lymph node metastasis. Methods: We selected 35 cases of patients with vulvar squamous cell carcinoma submitted to primary surgical treatment that included vulvectomy and regional lymph nodes dissection. After revision of medical records data (age of patient, stage, type of surgery and tumor size), all the slides were reviewed to achieve histological grade, peritumoral lymphatic invasion and depth of infiltration. Areas of the tumor were selected to construction of tissue microarrays paraffin blocs and immunohistochemical identification of podoplanin by the D2-40 antibody. Intratumoral LVD was quantified by counting the stained vessels in hotspots areas. The number of vessels was counted in 10 high power microscopic fields and the mean was the value of the LVD for each case. Podoplanin expression by neoplastic cells was considered positive when more than 10% of the cells showed moderate to strong cytoplasmatic stained. We investigated the association of the two variables with classical prognostic features (age of patient, stage, tumor size, histologic grade, vascular involvement, depth of infiltration, lymph node involvement as well outcome. Results: Higher values of intratumoral LVD were identified in low grade and low stage tumors, in tumors without lymphatic invasion and those with lesser stromal infiltration. In univariate analysis, high intratumoral LVD was associated to higher overall survival. Podoplanin expression by neoplastic cells did not show association with any of the prognostic variable, nor with lymph node involvement or outcome. Conclusions: Intratumoral LVD in vulvar squamous carcinoma is associated with prognostic favorable features. On the other hand, podoplanin expression by neoplastic cells did not seem to influence the behavior of these carcinomas

Le récepteur activateur de NF-κB (RANK), membre de la famille des récepteurs au TNF, est connu pour son rôle dans l’homéostasie de l’os, mais joue aussi un rôle important dans le système immunitaire. J’ai tout d’abord étudié des outils permettant de cibler RANK/RANKL. J’ai caractérisé et comparé l’activité biologique de deux anticorps anti-RANK. J’ai également criblé une librairie de petites molécules pour identifier des inhibiteurs de l’interaction RANK/RANKL. Dans une deuxième partie, je me suis intéressée au rôle du ligand de RANK (RANKL) dans l’homéostasie du ganglion lymphatique. RANKL joue un rôle dans la différenciation des ostéoclastes mais son rôle dans la différenciation d’autres macrophages n’a pas été étudié. Nous avons étudié des souris déficientes pour RANKL dans les cellules marginales réticulaires (MRC) qui expriment RANKL de manière constitutive dans le ganglion adulte. Nous avons observé une diminution de la population de macrophages sous-capsulaires (SSM). Nous avons également montré que les cellules endothéliales lymphatiques (LEC) expriment l’intégrine alpha 2b (ITGA2b) et que cette expression est sensible à la présence de RANKL. / The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation. Firstly I looked for new molecular tools to target RANK/RANKL axis. I characterized and compared the biological activity of two anti-RANK antibodies. Moreover, I screened the Prestwick Chemical Library® of small molecules in order to identify inhibitors of RANK/RANKL interaction. Secondly, I studied the effect of the RANK/RANKL axis in lymph node homeostasis. RANKL is known to promote osteoclast differentiation but whether it also plays a role in the differentiation of other macrophage subsets is not known. We addressed this question by conditionally deleting RANKL from marginal reticular stromal cells (MRCs) that constitutively express RANKL in the lymph node. We observed impaired differentiation of the subcapsular sinus macrophages (SSMs). We also studied lymph node lymphatic endothelial cells (LECs) and showed that integrin alpha 2b (ITGA2b) is expressed by a lymph node subset of LECs and its expression is sensitive to RANKL.

Hokkaido University (北海道大学) / 博士 / 医学

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

We hypothesized that lymph node involvement in minor salivary gland cancers is associated with clinical and pathological factors commonly available to the clinician after a typical initial workup. Our aim was to identify these factors using a dataset that allowed us to compile the largest series of minor salivary gland cancers in the published literature. Using this dataset we also aimed to characterize the distribution of histological types by primary site, identify the predictors of the use of external beam radiation therapy and neck dissection, and examine the effect of lymph node involvement on survival. Using the SEER database, we identified 2667 minor salivary gland cancers with known lymph node status from 1988 to 2004. Univariate and multivariate analyses were conducted to identify factors associated with the use of neck dissection, the use of external beam radiation therapy, and the presence of cervical lymph node metastases. Kaplan Meier survival curves were constructed to examine the effect of lymph node involvement on survival. 426 (16.0%) patients had neck nodal involvement. Factors associated with neck nodal involvement on univariate analysis included increasing age, male gender, increasing tumor size, high tumor grade, T3-T4 stage, adenocarcinoma or mucoepidermoid carcinomas, and pharyngeal site of primary malignancy. On multivariate analysis, four statistically significant factors were identified, which included male gender, T3-T4 stage, pharyngeal site of primary malignancy, and high-grade adenocarcinoma or high-grade mucoepidermoid carcinomas. The proportions (and 95% confidence intervals) of patients with lymph node involvement for those with 0, 1, 2, 3 and 4 of these prognostic factors were 0.02 (0.01-0.03), 0.09 (0.07-0.11), 0.17 (0.14-0.21), 0.41 (0.33-0.49), and 0.70 (0.54-0.85) respectively. Grade was a significant predictor of metastasis for adenocarcinoma and mucoepidermoid carcinoma but not for adenoid cystic carcinoma. Overall survival was significantly worse at 5, 10, and 15 years for patients with lymph node involvement on presentation. A prognostic index using the four clinicopathological factors listed above can effectively differentiate patients into risk groups of nodal metastasis. The precision of this index is subject to the limitations of SEER data and it should be validated in further clinical studies.

Previous studies have indicated that the promoters of the human filarial parasite Brugia malayi are unusual in that they do not exhibit the CAAT or TATAA sequences usually found in the core domains of promoters of most eukaryotic organisms. Analysis of the promoters of the ribosomal proteins showed that the region flanking the splice leader (SL) addition site plays an important role in transcription and may function as the core promoter domain in B. malayi. To test the hypothesis that the SL addition domain is the most important essential region of the ribosomal protein promoters, the SL addition site of the BmRPL13 gene was replaced with the SL addition domains from other ribosomal protein genes from B. malayi. The promoter activity of the replacement constructs were tested using a transient transfection dual luciferase assay. Promoter activity with RPL13 replacement constructs was correlated with that seen in the wild type promoters, suggesting that roughly 80% of the variations seen in promoter activity among ribosomal protein promoters is due to variation in the SL core promoter domain.

The aim of this study was to determine whether functional ecdysone response elements (EcREs) exist within the genome of Brugia malayi, a parasitic nematode that causes lymphatic filariasis. The hypothesis that EcREs exist in B. malayi stemmed from previous demonstration of a functional ecdysone response system in B. malayi (Tzertzinis et al., 2010). Real-time PCR (qPCR) experiments were conducted to measure gene expression levels for twelve genes proximal to five putative EcREs in 20-OH ecdysone treated and untreated B. malayi embryos. Seven genes showed consistent upregulation with 20-OH ecdysone treatment. Each of the five putative EcREs had at least one proximal gene consistently upregulated, suggesting that all five might be functional EcREs. One of the genes consistently upregulated in the qPCR experiments, Bm1_48650, codes for a MIZ zinc finger family protein, a likely transcription factor. Transgenic ecdysone induction assay experiments were conducted using embryos transiently transfected with a reporter construct driven by the EcRE-containing promoter of Bm1_48650. Significantly higher mean reporter gene activity (~3.5-fold) was seen in 20-OH ecdysone treated versus untreated embryos. In another set of transgenic ecdysone induction assays, the EcRE motif in the Bm1_48650 promoter was completely mutated, and this construct was tested in 20-OH ecdysone treated and untreated embryos. The mean reporter gene activity for the treated and untreated embryos transfected with the mutant constructs did not differ significantly from the untreated embryos transfected with the native EcREcontaining promoter construct. These results showed that the EcRE in the promoter of Bm1_48650 is necessary for regulating gene expression in response to 20-OH ecdysone. This study substantiates previously discovered evidence of a functional ecdysone response system in B. malayi, which could potentially serve as a target for drug discovery for lymphatic filariasis.