Yes / Background: Epithelial ovarian cancer is a common malignancy, with no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We evaluated the expression of EN2 in Epithelial ovarian cancer, and reviewed its role as a biomarker. Methods: We evaluated 8 Epithelial ovarian cancer cell lines, along with > 100 surgical specimens from the Royal Surrey County Hospital (2009–2014). In total, 108 tumours and 5 normal tissue specimens were collected. En2 mRNA was evaluated by semi-quantitative RT-PCR. Histological sub-type, and platinum-sensitive/−resistant status were compared. Protein expression was assessed in cell lines (immunofluorescence), and in > 150 tumours (immunohistochemistry). Results: En2 mRNA expression was elevated in serous ovarian tumours compared with normal ovary (p < 0.001), particularly in high-grade serous ovarian cancer (p < 0.0001) and in platinum-resistant tumours (p = 0.0232). Median Overall Survival and Progression-free Survival were reduced with high En2 expression (OS = 28 vs 42 months, p = 0.0329; PFS = 8 vs 27 months; p = 0.0004). Positive cytoplasmic EN2 staining was demonstrated in 78% of Epithelial ovarian cancers, with absence in normal ovary. EN2 positive high-grade serous ovarian cancer patients had a shorter PFS (10 vs 17.5 months; p = 0.0103). Conclusion: The EN2 transcription factor is a novel ovarian cancer biomarker. It demonstrates prognostic value, correlating with worse Overall Survival and Progression-free Survival. It is hoped that further work will validate its use as a biomarker, and provide insight into the role of EN2 in the development, progression and spread of ovarian cancer. / Oncology Research and Development Departments at the Royal Surrey County Hospital and the University of Surrey

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25159号 / 医博第5045号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 森田 智視, 教授 永井 洋士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

BACKGROUND:Recent advances suggest fallopian tube as the main cellular source for women's pelvic serous carcinoma (PSC). In addition to TP53 mutations, many other genetic changes are involved in pelvic serous carcinogenesis. IMP3 is an oncofetal protein which has recently been observed to be overexpressed in benign-looking tubal epithelia. Such findings prompted us to examine the relationship between IMP3 over-expression, patient age and the likelihood of development of PSC.METHODS:Fallopian tubes from three groups (low-risk, high-risk, and PSC) of patients with matched ages were studied. Age was recorded in 10years intervals ranging from age 20 to older than 80. The number of IMP3 signatures (defined by 10 or more tubal secretory cells stained positively and continuously in benign appearing tubal mucosa) from both tubal fimbria and ampulla segments was measured. The data was analyzed by standard contingency table and Poisson distribution methods after age adjustment. IMP3 overexpression was also examined in serous tubal intraepithelial carcinoma and PSC.RESULTS:The positive IMP3-stained cells are mainly tubal secretory cells. The absolute number of tubal IMP3 signatures increased significantly within each age group. Age remained a significant risk factor for serous neoplasia after age adjustment. IMP3 signatures were more frequent in the patients of both high-risk and PSC groups. The presence of IMP3 signatures in tubal mucosa was significantly associated with tubal or pelvic serous carcinogenesis (p<0.001).CONCLUSIONS:The findings suggest that tubal secretory cells with IMP3 signatures showing growth advantage could potentially serve as a latent precancer biomarker for tubal or pelvic serous carcinomas in women.

INTRODUÇÃO: O diagnóstico diferencial entre cistadenomas serosos (CAS) e neoplasias císticas mucinosas (NCM) é essencial, pois os CAS são benignos e o tratamento em geral é conservador, ao passo que as NCM são pré-malignas (ou malignas) e devem ser ressecadas. A sobreposição de características de imagem não é incomum, portanto métodos auxiliares não-invasivos podem ser úteis na diferenciação entre elas. OBJETIVOS: Apresentar casuística de CAS e NCM estudados por ressonância magnética aplicando critérios diagnósticos morfológicos tradicionais e avaliar a quantificação da intensidade de sinal (IS) nas ponderações T1 e T2 (ambas com supressão de gordura) e do coeficiente de difusão aparente (ADC) na diferenciação entre estes tipos de lesões císticas pancreáticas (LCP). MATERIAIS E MÉTODOS: As LCP foram avaliadas quanto à loculação, paredes e presença ou não de cicatriz central, septos grosseiros e componentes sólidos. Foram feitas, ainda, avaliações qualitativa e quantitativa da IS nas ponderações T1 e T2, comparando as LCP com o parênquima pancreático (PP), bem como avaliação quantitativa do ADC. RESULTADOS: Dois radiologistas estudaram 9 CAS e 8 NCM de forma independente, sendo observada correlação perfeita entre eles quanto às avaliações morfológica e qualitativa da IS. Não houve diferença significativa entre os grupos de CAS e NCM na comparação das variáveis quantitativas da IS nas ponderações T1 e T2, bem como da razão ISLCP/ISPP e dos valores de ADC. CONCLUSÃO: Quanto à morfologia, a classificação do aspecto cístico (microcística, macrocística ou unilocular) foi a variável que apresentou a maior associação com os grupos, sendo, portanto, importante na definição diagnóstica. A quantificação da IS nas ponderações T1 e T2, assim como a medida do ADC das LCP, não permitiram a diferenciação das neoplasias císticas pancreáticas serosas e mucinosas. / INTRODUCTION: Differential diagnosis between serous cystadenomas (SCA) and mucinous cystic neoplasms (MCN) is essential, since SCA is benign and treatment is generally conservative, whereas MCN is premalignant (or malignant) and should be resected. The overlapping of imaging features is not uncommon, so non-invasive aids may be useful in differentiating between them. OBJECTIVES: To present a SCA and MCN caseload using magnetic resonance imaging traditional morphological diagnostic criteria and to evaluate the quantification of signal intensity (SI) in T1 and T2 weights (both with fat suppression) and the apparent diffusion coefficient (ADC) in differentiation between these types of pancreatic cystic lesions (PCL). MATERIALS AND METHODS: PCL were evaluated for loculation, walls and presence of central scar, coarse septa and solid components. Qualitative and quantitative SI assessments were also performed in T1 and T2 weights, comparing PCL with pancreatic parenchyma (PP), as well as ADC quantitative evaluation. RESULTS: Two radiologists studied 9 SCA and 8 MCN independently, with a perfect correlation between morphological and qualitative IS scores. There was no significant difference between the SCA and MCN groups when comparing the quantitative IS variables in the T1 and T2 weights, as well as the SIPCL/SIPP ratio and the ADC values. CONCLUSION: As to morphology, the classification of the cystic aspect (microcystic, macrocystic or unilocular) was the variable that presented the greatest association with the groups, being therefore important in the diagnostic definition. The quantification of SI in the T1 and T2 weights, as well as the ADC measurement of the PCL, did not allow the differentiation of serous and mucinous pancreatic cystic neoplasms.

The aim of this thesis is to investigate the role of inflammatory infiltrates and chemokines in metastasis of high-grade serous ovarian cancer, HGSC, to the omentum using human tissue biopsies and a 3- dimensional (3D) cell culture model. In ten patients with metastatic HGSC, omental tumour deposits contained a prominent leukocyte infiltrate of CD3+ T cells (9% of total cells) and CD68+ macrophages (11% of total cells). The presence of CD68+ macrophages showed a significant positive correlation with tumour cell proliferation analysed by Ki67 expression. Four ovarian cancer cell lines were co-cultured on a 3D model mimicking the microenvironment of the omentum for two weeks. The model was composed of collagen embedded human fibroblasts covered in a confluent layer of human primary mesothelial cells. The mesothelial cells in the 3D model significantly increased the growth (p = 0.002) and invasion (p = 0.0004) of the ovarian cancer cells. CXCL12 is the macrophage chemoattractant and ligand for the major chemokine receptor expressed on ovarian cancer cells. An association between CXCL12 and extracellular matrix remodelling was identified in two independent gene expression microarrays of ovarian cancer biopsies. The expression of CXCL12 in the HGSC omental metastases measured by quantitative Real Time-PCR positively correlated with decorin expression. Antibody mediated neutralisation of CXCL12 reduced growth (p = 0.012) and invasion (p = 0.029) in the 3D model. Mimicking an infiltrate of CD68+ macrophages in this multicellular 3D in vitro system also produced measurable changes in inflammatory cytokine and chemokine expression. There is currently a demand for more accurate models of HGSC and a necessity to study its metastasis that presents itself as the major clinical problem in patients. Therefore the development of this 3D model to mimic tumour-promoting inflammation in HGSC metastasis will provide researchers with an essential tool for testing novel therapeutic strategies.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

INTRODUÇÃO: O diagnóstico diferencial entre cistadenomas serosos (CAS) e neoplasias císticas mucinosas (NCM) é essencial, pois os CAS são benignos e o tratamento em geral é conservador, ao passo que as NCM são pré-malignas (ou malignas) e devem ser ressecadas. A sobreposição de características de imagem não é incomum, portanto métodos auxiliares não-invasivos podem ser úteis na diferenciação entre elas. OBJETIVOS: Apresentar casuística de CAS e NCM estudados por ressonância magnética aplicando critérios diagnósticos morfológicos tradicionais e avaliar a quantificação da intensidade de sinal (IS) nas ponderações T1 e T2 (ambas com supressão de gordura) e do coeficiente de difusão aparente (ADC) na diferenciação entre estes tipos de lesões císticas pancreáticas (LCP). MATERIAIS E MÉTODOS: As LCP foram avaliadas quanto à loculação, paredes e presença ou não de cicatriz central, septos grosseiros e componentes sólidos. Foram feitas, ainda, avaliações qualitativa e quantitativa da IS nas ponderações T1 e T2, comparando as LCP com o parênquima pancreático (PP), bem como avaliação quantitativa do ADC. RESULTADOS: Dois radiologistas estudaram 9 CAS e 8 NCM de forma independente, sendo observada correlação perfeita entre eles quanto às avaliações morfológica e qualitativa da IS. Não houve diferença significativa entre os grupos de CAS e NCM na comparação das variáveis quantitativas da IS nas ponderações T1 e T2, bem como da razão ISLCP/ISPP e dos valores de ADC. CONCLUSÃO: Quanto à morfologia, a classificação do aspecto cístico (microcística, macrocística ou unilocular) foi a variável que apresentou a maior associação com os grupos, sendo, portanto, importante na definição diagnóstica. A quantificação da IS nas ponderações T1 e T2, assim como a medida do ADC das LCP, não permitiram a diferenciação das neoplasias císticas pancreáticas serosas e mucinosas. / INTRODUCTION: Differential diagnosis between serous cystadenomas (SCA) and mucinous cystic neoplasms (MCN) is essential, since SCA is benign and treatment is generally conservative, whereas MCN is premalignant (or malignant) and should be resected. The overlapping of imaging features is not uncommon, so non-invasive aids may be useful in differentiating between them. OBJECTIVES: To present a SCA and MCN caseload using magnetic resonance imaging traditional morphological diagnostic criteria and to evaluate the quantification of signal intensity (SI) in T1 and T2 weights (both with fat suppression) and the apparent diffusion coefficient (ADC) in differentiation between these types of pancreatic cystic lesions (PCL). MATERIALS AND METHODS: PCL were evaluated for loculation, walls and presence of central scar, coarse septa and solid components. Qualitative and quantitative SI assessments were also performed in T1 and T2 weights, comparing PCL with pancreatic parenchyma (PP), as well as ADC quantitative evaluation. RESULTS: Two radiologists studied 9 SCA and 8 MCN independently, with a perfect correlation between morphological and qualitative IS scores. There was no significant difference between the SCA and MCN groups when comparing the quantitative IS variables in the T1 and T2 weights, as well as the SIPCL/SIPP ratio and the ADC values. CONCLUSION: As to morphology, the classification of the cystic aspect (microcystic, macrocystic or unilocular) was the variable that presented the greatest association with the groups, being therefore important in the diagnostic definition. The quantification of SI in the T1 and T2 weights, as well as the ADC measurement of the PCL, did not allow the differentiation of serous and mucinous pancreatic cystic neoplasms.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Mucociliary epithelia are found in the human airways and act as the first line of defence against inhaled foreign agents. Mucus traps potentially damaging particles and the cilia transport the mucus away from the airways to remove the threat. Modelling mucociliary epithelia for research purposes is challenging. This is because the airways are enclosed and are thus difficult to study directly. Instead, tissue is extracted or in vitro techniques are employed. Whilst these systems are useful, there is a need for accessible in vivo models to complement them. In this thesis I assess a new model system for studying mucociliary epithelia. This system is the larval epidermis of the amphibian, Xenopus tropicalis. Its epidermis comprises multi-ciliated cells that beat in a polarised direction reminiscent of those found in the human airways. It is also proposed to have a number of other cell types including mucus-secreting cells, but very little is known about them. The epidermis is open and accessible to manipulation meaning that it has great potential to be used in the study of mucociliary epithelia in live, native conditions. Such a system would be a valuable addition to the current models employed. However, the epidermis has not been thoroughly characterized before so its utility as a model system remains speculative.To develop and evaluate this new model, I fully characterize the epidermis, showing that it has five distinguishable cell types. This includes a population of cells called ionocytes that are shown to be essential for the health and function of the epidermis. I also test for the presence of mucins, the structural component of mucus, secreted from the epidermis in order to evaluate the proposal that mucus-secreting cells are present in the epidermis. A mucin-like protein called otogelin is identified. After characterizing the epidermal cell types, I compare them to the human mucociliary epithelium and consider potential applications and future perspectives for this model.