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Brain metastases arlsmg from lung tumours are relatively common. Metastasis formation involves events initiated by the tumour cell, including the release of proteolytic enzymes, cytokines and growth factors, and also the induction of angiogenesis Le. the formation of new blood vessels. Tumour cells migrate through the endothelial cell layer lining cerebral blood vessels and contact astrocytes (the main supporting cells of the brain). Characteristically a boundary forms around the metastasis restricting further brain invasion. The cellular processes involved in metastasis formation are largely unknown.Using an in vitro model this study investigated the cellular changes induced (', by human lung tumour cells on human astrocytes and endothelial cells. {\strocytic changes during direct cell:cell contact with lung tumour cells were examined as a model of boundary formation and a novel in vitro tubule formation assay was developed to examine the angiogenic potential of human lung tumour cells.

Small cell lung cancer (SCLC) accounts for around 15% of new cases and 25% of deaths from lung cancer, making it the most aggressive subtype. This is due to the fact that tumours quickly develop resistance to therapy and metastasise early to the brain, liver and bone marrow. SCLC is normally characterised as a neuroendocrine tumour which secretes hormone precursors. Pro-opiomelanocortin (POMC) is primarily produced as a component of the stress pathway but neuroendocrine lung tumours can secrete high levels of unprocessed POMC into the circulation. Given the well recognised neuroendocrine origin of SCLC, the prevalence of epithelial biomarkers highlights the ambiguity of the phenotype and origin of SCLC. We proposed that POMC could be a novel biomarker to identify patients with SCLC and those with more aggressive, metastatic tumours. Results from a panel of SCLC cell lines, show that 40% of cell lines secreted POMC. All cell lines were positive for cytokeratin 18 and there was distinct heterogeneous cytokeratin staining in cell lines. There was also heterogeneity when looking at other markers of both neuroendocrine and epithelial origin.In vivo, circulating POMC correlated strongly with tumour growth in a subcutaneous xenograft model. POMC was elevated 11 fold compared to a SCLC xenograft model derived from a non-POMC-secreting cell line (H526). POMC was also a sensitive biomarker of tumour response to varying doses of radiotherapy and of tumour relapse after complete regression post-radiotherapy. Unfortunately, metastases could not be detected in the brains and livers of these xenografted animals even when several alternative methods of tumour inoculation were used. However there was evidence of aggressive invasion of cells with a dual neuroendocrine and epithelial phenotype through the surrounding fibrotic sheath and muscle of all the primary tumours.Phenotypic characterisation of the xenograft tumour showed that cells appeared to homogeneously express both neuroendocrine and epithelial markers and did not express mesenchymal markers. However, post-irradiation the tumours appeared to have a distinct heterogeneous expression of POMC. These results suggest that irradiation treatment could cause a phenotypic shift which could have implications in tumour progression and metastasis.Epithelial to mesenchymal transition (EMT) has been linked to tumour metastasis in a number of epithelial cancers. When DMS 79 SCLC cells were treated with a known stimulus of EMT, transforming growth factor β (TGF-β), the mesenchymal marker N-cadherin was upregulated along with a decrease in cell proliferation. These changes are both characteristic of an EMT. However, there was no change in the epithelial nature of these cells and no obvious change in morphology. We suggest the cells are undergoing a partial transition and that this transition could possibly be involved in SCLC metastasis.In summary, the analysis of neuroendocrine features in SCLC, using POMC as a biomarker, has provided new evidence for a dual neuroendocrine and epithelial phenotype both in vitro and in vivo. Circulating POMC can also identify tumour development, progression and regression after irradiation treatment. In addition, we have highlighted the dual phenotype of invasive cells and the difficulty in studying metastasis of human SCLC in a mouse model.

LIM domain containing protein 1 (LIMD1) is a tumour suppressor located at 3p21.3, a region that harbours multiple tumour suppressor genes and is commonly subject to homozygous deletions and loss of heterozygosity in many cancers. The mechanism of LIMD1 tumour suppressive activities are not fully elucidated, however to date it has been shown to bind to the retinoblastoma protein (pRb) and repress E2F driven transcription as well as being a critical component of miRNA mediated gene silencing. Recent work has also identified LIMD1 as a possible negative regulator of hypoxia inducible factor α (HIF1α) and the hypoxic response. In lung cancer, LIMD1 protein expression is down regulated in up to 79% of tumours when compared to normal tissue with gene deletion and loss of heterozygosity accounting for 32 and 12% respectively, leaving 30% of tumours with unexplained mechanism of LIMD1 protein loss. In an aim to identify other possible mechanisms of LIMD1 loss, scrutinisation of the LIMD1 promoter identified a CpG Island in the 5’ promoter region, within which a small region was found to be critical for transcriptional activation. This region was methylated in the non-LIMD1 expressing MDA-MB435 cell line, but became hypomethylated and LIMD1 expressed following treatment with the DNA methylation inhibitor 5-Aza-2’-deoxycytidine. In primary lung tumours, analysis of genomic DNA also identified increased methylation of this region as well as a reduction in LIMD1 mRNA levels when compared to matched normal lung tissue. Furthermore, in silico analysis identified a conserved binding motif for the Ets transcription factor PU.1. Experimentally PU.1 was verified as binding to the LIMD1 promoter with siRNA mediated depletion of PU.1 significantly reducing endogenous LIMD1 protein levels, thus identifying two possible novel mechanisms of LIMD1 silencing. Transcription of LIMD1, like that of other HIF1α regulatory proteins, was enhanced when cells were exposed to hypoxia (1% O2), facilitated by HIF1α binding a hypoxic responsive element (HRE) within the promoter. At the molecular level, in vivo LIMD1 forms an endogenous complex with proline hydroxylase 2 (PHD2) and the von Hippel-Lindau (VHL) protein, with LIMD1 loss decreasing the efficiency of HIF1α degradation and impeding the resultant cellular adaptation to chronic hypoxia. In summary these studies identified epigenetic silencing of LIMD1 as a possible explanation for LIMD1 protein loss in transformed cells. Furthermore, LIMD1 transcription was identified as being activated by PU.1 and enhanced by HIF1α, and a revised, LIMD1 integrated, model of HIF1α regulation is proposed.

Introduction: Lung cancer is the commonest cause of cancer related death in men and women in England. In 2004 the National Lung Cancer Audit (NLCA) was created, as a national non-mandatory contemporary dataset of clinical features of individuals with lung cancer in part to identify variations in clinical practice and outcomes. The main aims of this dissertation are to determine the validity and representativeness of this dataset and then to investigate what factors influence access to surgery and chemotherapy and subsequent survival. In addition I have taken the opportunity afforded by this large dataset to describe the natural history of lung cancer in young adults (20-40 years). Methods: In order to establish if the dataset was representative, I created a measure of case ascertainment at the level of an NHS Trust, and examined the distribution of patient features and outcomes for varying levels of case ascertainment. I have then quantified the impact of patient and NHS Trust level features on access to surgery in people with non-small cell lung cancer and access to chemotherapy in people with small cell lung cancer using multivariate logistic regression. I have also conducted a series of survival analyses using Cox regression. Results: I have found no evidence that patient features vary systematically according to levels of case ascertainment in the NLCA. Age, sex, performance status, stage and co-morbidity all influenced the likelihood of having surgery for people with non-small cell lung cancer. Those patients first seen in a thoracic surgical centre where more likely to receive surgery than patients seen at peripheral centres (adjusted OR 1.51, 95% CI 1.16, 1.97), and surgery had a significant benefit on mortality (adjusted HR 0.41, 95% CI 0.39, 0.44). Although the resection rate was higher for patients first seen at a surgical centre (17% v 12%) these patients did equally well after surgery suggesting they were not a higher risk group. Individuals with small cell lung cancer first seen in a hospital with a high participation in clinical trials, (>5% of expected lung cancer patients being entered into clinical trials), were more likely to receive chemotherapy (adjusted OR 1.42, 95% CI 1.06, 1.90). Chemotherapy was associated with an improvement in survival (adjusted HR 0.51, 95% CI 0.46, 0.56), and amongst those patients receiving chemotherapy, mortality was not affected by the trial status of the hospital where they were first seen. In limited stage small cell disease, those patients who had chemo-radiotherapy had an improved survival compared with those patients who received chemotherapy alone (adjusted HR 0.72, 95% CI 0.62, 0.84). This dataset of English patients with lung cancer contains one of the largest cohorts of young adults (20-40 years) with lung cancer (N=583). I have been able to demonstrate that the majority present with a good performance status (0 or 1 in 80% of those with PS recorded), but advanced (stage IV) disease at diagnosis (55% of those with stage recorded). Those who have surgery have a survival profile similar to their older counterparts. Conclusion: The National Lung Cancer Audit is a representative, contemporary cohort of people with lung cancer, which can provide valuable information for health service research in lung cancer. I have found evidence that there is variation in access to treatment based on the facilities or the performance of individual NHS Trusts. My results suggest that by improving access to thoracic surgery for those individuals with non-small cell lung cancer we may be able to raise the resection rate and improve five year survival. The pattern is similar for people with small cell lung cancer and access to chemotherapy. What this research cannot explain is the aetiology for this variation, and where in the diagnostic pathway changes need to be made to improve the active management and access to potentially curative regimes. As the audit matures with more detailed information on NHS Trust level care, further analyses will be possible to try and determine more clearly what explains these variations, and how we might intervene to reduce them.

Various ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, migration and invasion. Expression of functional voltage-gated sodium (Nav) channels is implicated in the metastatic potential of breast, prostate, colon, cervical and lung cancer cells. However, the cellular mechanisms that regulate Nav channel expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, the role of epidermal growth factor receptor (EGFR) signalling and Nav channels in non-small cell lung cancer (NSCLC) cell lines have been examined. It is shown that functional expression of Nav1.7 promotes invasion in strongly metastatic H460 NSCLC cells. However, in non-invasive A549 NSCLC cells, Nav1.7 is completely absent. Inhibition of Nav1.7 either pharmacologically by tetrodotoxin (TTX) or genetically by small interfering RNA (siRNA) reduces H460 cell invasion by up to 50%. Whilst EGFR signalling enhances proliferation, migration and invasion of H460 cells, EGFR-mediated upregulation of Nav1.7 specifically, is required to promote invasive behaviour in these cells. Examination of Nav1.7 expression at the mRNA, protein and functional levels further reveals that EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of Nav1.7 expression to promote cellular invasion in NSCLC. The role of Nav channels in promoting cancer cell invasion is also unclear. Therefore, the effect of Nav channel activity on two likely downstream contributors to cellular invasion, intracellular calcium concentration, [Ca2+]i, and intracellular pH, pHi, have been examined. It is shown that functional expression of Nav1.7 likely drives H460 NSCLC cell invasion via H+ efflux from the cell in an uncharacterised mechanism potentially involving NHE1, resulting in extracellular acidification of the perimembrane space. However, much more work is needed to understand this Na+-dependent invasive mechanism. Immunohistochemistry (IHC) of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significant potential as a novel target for therapeutic intervention, possibly in conjunction with existing EGFR inhibitors, and/or as a diagnostic/prognostic marker in NSCLC.

Lung cancer is a disease affecting millions of people every year and poses a serious threat to global public health. Accurate lung cancer staging is crucial to choose an appropriate treatment protocol and to determine prognosis, this requires the acquisition of contrast-enhanced diagnostic CT (d-CT) that is usually followed by a PET/CT scan. Information from both d-CT and PET scan is used by the clinician in the staging process; however, these images are not intrinsically aligned because they are acquired on different days and on different scanners. Establishing anatomical correspondence, i.e., aligning the d-CT and the PET images is an inherently difficult task due to the absence of a direct relationship between the intensities of the images. The CT acquired during the PET/CT scan is used for attenuation correction (AC-CT) and is implicitly aligned with the PET image as they are acquired at the same time using a hybrid scanner. Patients are required to maintain shallow breathing for both scans. In contrast to that, the d-CT image is acquired after the injection of a contrast agent, and patients are required to maximally inhale, for better view of the lungs. Differences in the AC-CT and d-CT image volumes are thus due to differences in breathhold positions and image contrast. Nonetheless, both images are from the same modality. In this thesis, we present a new approach that aligns the d-CT with the PET image through an indirect registration process that uses the AC-CT. The deformation field obtained after the registration of the AC-CT to d-CT is used to align the PET image to the d-CT. Conventional image registration techniques deform the entire image using homogeneous regularization without taking into consideration the physical properties of the various anatomical structures. This homogeneous regularization may lead to physiologically and physically implausible deformations. To register the d-CT and AC-CT images, we developed a 3D registration framework based on a fluid transformation model including three physically motivated properties: (i) sliding motion of the lungs against the pleura; (ii) preservation of rigid structures; and (iii) preservation of topology. The sliding motion is modeled using a direction dependent regularization that decouples the tangential and the normal components of the external force term. The rigid shape of the bones is preserved using a spatially varying filter for the deformations. Finally, the topology is maintained using the concept of log-unbiased deformations. To solve the multi-modal registration problem due to the contrast injected for the d-CT, but lack thereof in the AC-CT, we use local cross correlation (LCC) as the similarity measure. To illustrate and validate the proposed registration framework, different intra-patient CT datasets are used, including the NCAT phantom, EMPIRE10 and POPI datasets. Results show that our proposed registration framework provides improved alignment and physically motivated deformations when compared to the classic elastic and fluid registration techniques. The final goal of our work was to demonstrate the clinical utility of our new approach that aligns d-CT and PET/AC-CT images for fusion. We apply our method to ten real patients. Our results show that the PET images have much improved alignment with the d-CT images using our proposed registration technique. Our method was successful in providing a good overlap of the lungs, improved alignment of the tumours and a lower target registration error for landmarks in comparison to the classic fluid registration. The main contribution of this thesis is the development of a comprehensive registration framework that integrates important physical properties into a state-of-the-art transformation model with application to lung imaging in cancer.

Orientadora: Profª Drª Nen Nalú Alves das Mercês / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação Mestrado Profissional em Enfermagem. Defesa: Curitiba, 13/12/2016 / Inclui referências : f.148-173 / Área de concentração: Prática profissional de enfermagem / Resumo: Conhecer o itinerário terapêutico revelado pelo familiar da pessoa com mesotelioma maligno. Trata-se de uma pesquisa qualitativa, de natureza descritiva, exploratória, utilizando o método de estudos de casos múltiplos. Aprovada pelo Comitê de Ética em Pesquisa em Seres Humanos, parecer favorável nº 677.015. Definiu-se a proposição do estudo com a questão norteadora: como ocorreu o itinerário terapêutico da pessoa com mesotelioma maligno revelado pelo familiar? O referencial teórico adotado foi o Sistema de Cuidado à Saúde de Arthur Kleinman, constituído por três subsistemas: o profissional, popular e folclórico. Participaram do estudo seis casos de pessoas que adoeceram e foram a óbito por mesotelioma, assistidas em uma Instituição de Saúde referência no tratamento do câncer e seus familiares, no estado do Paraná. A coleta de dados ocorreu de janeiro a junho de 2016.Os dados foram coletados do prontuário por meio de formulário estruturado, sobre o perfil sócio demográfico e clínico das pessoas com mesotelioma e da narrativa do familiar que acompanhou o itinerário terapêutico percorrido por essa pessoa, através da técnica de entrevista. Para análise dos dados documentais utilizou-se a análise comparativa proposta por Robert Yin e para as narrativas dos familiares a análise de conteúdo de Lawrence Bardin. Dos seis casos descritos nas unidades de análise, quatro eram do sexo masculino e dois do sexo feminino; quatro com diagnóstico de mesotelioma pleural, e dois com mesotelioma peritoneal. As idades variaram entre 44 a 69 anos. Os principais sintomas identificados foram: febre, dor nas costas, dor no estomago, emagrecimento e derrame pleural. A terapêutica instituída foi: quimioterapia e radioterapia. A exposição ao amianto confirmado em um caso. A sobrevida dos primeiros sintomas ao óbito ocorreu de cinco meses a oito anos; e, do diagnóstico de mesotelioma ao óbito variou de quatro meses a cinco anos. Da análise das narrativas dos familiares emergiram sete categorias: reconhecimento do adoecer; cuidados populares e a tentativa de escapar do adoecimento; subsistema popular direciona ao profissional; subsistema profissional: desvendar o mistério da doença; família: supremacia do cuidado; religião: esperança e alento; e, o adoecimento por mesotelioma pela lente do familiar. O inicio do itinerário terapêutico foi marcado pela identificação dos sintomas, com as primeiras práticas populares adotadas. A familia foi a unidade central do cuidado. O subsistema profissional carregou o desafio de desvendar o mistério da doença, com a dificuldade de um diagnóstico acertado. A religião foi a fonte de esperança para as pessoas durante o processo de adoecimento, e a familia carrega o fardo de enfrentar as dificuldades da agressidade da doença, do tratamento até optar por interromper a terapêutica. Os cuidados à saúde adotadas são representadas pela forte ação dos conceitos sócio culturais de cada pessoa. Palavras-chave: Mesotelioma; Atitude frente à saúde; Pacientes; Cuidados de saúde; Família e Enfermagem / Abstract: Knowing the therapeutic itinerary revealed by the relative of the person with malignant mesothelioma. It is a qualitative research, of descriptive nature, exploratory, using the method of multiple case studies. Approved by the Ethics Committee on Research on Human Beings, favorable opinion nº 677.015. The study proposition was defined with the guiding question: how did ocurr the therapeutic itinerary of the person with malignant mesothelioma revealed by the relative? The theoretical reference chosen was the Arthur Kleinman Health Care System, made up of three subsystems: the professional, popular and folkloric. Participated in the study six cases of people who fell ill and died due to mesothelioma, attended at a Health Care Institution which is a reference in the treatment of cancer and their relatives, in the state of Paraná. The data were collected from January to June 2016. The data were collected from the medical record by means of a structured form, about the socio demographic and clinical profile of people with mesothelioma and the narrative of the family member who accompanied the therapeutic itinerary lived by this person through the technique of interview. For the analysis of the documentary data was used the comparative analysis proposed by Robert Yin and for the narratives of the relatives the content analysis of Lawrence Bardin. Of the six cases described in the units of analysis, four were male and two female; Four with diagnosis of pleural mesothelioma and two with peritoneal mesothelioma. The ages ranged from 44 to 69 years. The main symptoms identified were: fever, back pain, stomach pain, weight loss and pleural effusion. The therapy instituted was: chemotherapy and radiotherapy. Exposure to asbestos confirmed in one case. The survival of the first symptoms at death occurred from five months to eight years; and from the diagnosis of mesothelioma to death varied from four months to five years. From the analysis of the family narratives emerged seven categories: recognition of the illness; popular care and the attempt to escape from illness; popular subsystem directed to the professional; professional subsystem: unveil the mystery of the disease; family: care supremacy; religion: hope and encouragement; and mesothelioma sickness by the relative view. The beginning of the therapeutic itinerary was marked by the identification of the symptoms with the first popular practices adopted. The family was the central care unit. The professional subsystem carried the challenge of unraveling the mystery of the disease with the difficulty of a correct diagnosis. The religion was the source of hope for people during the process of becoming ill and the family carries the burden of facing the difficulties of the aggression of the disease, from the treatment until to choose stopping the treatment. The health care adopted is represented by the strong action of the socio-cultural concepts of each person. Keywords: Mesothelioma; Attitude towards health; patient; health care; Family;Nursing