Colorectal carcinoma arises through sequential genetic changes whereby an adenoma develops from normal colonic epithelium and then becomes a carcinoma. Critical to this process is two forms of mutually exclusive genomic instability ?? chromosomal instability (CIN) and microsatellite instability (MSI). The colorectal tumours that develop from each of these pathways have distinct pathological and molecular differences. Most MSI+ colorectal carcinomas are associated with the CpG island methylator phenotype (CIMP) - an epigenetic phenomenon where a specific and consistent group of genes are silenced through promoter methylation. However, over half of fall CIMP+ colorectal tumours are microsatellite stable (MSS). It is well known that the WNT/β-catenin signalling pathway is instrumental in the initiation and development of CIN type tumours but it is less clear whether this pathway has any significant involvement in MSI+ or methylated tumours. The role of the PI3K1AKT signalling pathway in the development of solid human tumours has only recently been established and the affects of abnormal PI3K/AKT signalling in sporadic colorectal carcinomas is yet to be fully elucidated. The objective of this thesis was to investigate the involvement of the WNT/β-catenin and PI3K/AKT signalling pathways in the CIN, MSI+ and methylated subgroups of sporadic colorectal carcinoma. To achieve this, the expression patterns of β-catenin, p-AKT and PTEN were identified by immunohistochemistry on sections from tissue microarrays consisting of cores from a large group of sporadic colorectal carcinomas. Each of these proteins is an integral part of the constitutive activation of WNT/β-catenin or PI3K/AKT signalling and their expression patterns were correlated with the clinical, pathological and molecular characteristics of the different subgroups of colorectal carcinoma. Increased nuclear β-catenin expression, an indicator of activated WNT signalling, is associated with MSS and the pathological features of CIN type tumours and inversely associated with the pathological and molecular features of MSI+ and CIMP+ tumours. In all forms of sporadic colorectal carcinoma, nuclear β-catenin expression was not an indicator of overall survival. PTEN was not associated with any particular subgroup of sporadic colorectal carcinoma, but decreased cytoplasmic expression was indicative of overall worse outcome, especially in MSS or CIN type tumours. While the identification of nuclear β-catenin in sporadic colorectal carcinomas is not a satisfactory prognostic marker, the immunohistochemical detection of absent PTEN expression may prove useful in identifying poor outcome in individuals with sporadic MSS colorectal carcinoma.

Made available in DSpace on 2018-08-01T21:35:01Z (GMT). No. of bitstreams: 1 tese_10831_122ª Ata de Defesa - Paola Fernandes Pansini.PDF: 76 bytes, checksum: e777df9eb60d248d167e2edfa6e23421 (MD5) Previous issue date: 2017-02-15 / O carcinoma epidermóide de cabeça e pescoço (CECP) é o sexto tipo de câncer mais comum em todo o mundo. Nos últimos anos, tem sido sugerida a participação da survivina na progressão tumoral em CECP. Este estudo teve como objetivo avaliar a survivina como potencial biomarcador de progressão tumoral em CECB. Foram utilizados no estudo dados clínicos e amostras biológicas de 115 indivíduos com carcinoma epidermóide da cavidade bucal. Lâminas contendo tecidos tumorais coradas pelo método hematoxilina e eosina foram usadas para as análises histopatológicas para avaliar o infiltrado linfocitário tumoral, padrão de invasão tumoral, gradação tumoral, invasão vascular, linfática e perineural. Tissue Microarrays foram construídos para realizar a análise imunohistoquímica da expressão da proteína survivina utilizando o anticorpo primário monoclonal de coelho anti-survivina. Para avaliar as associações entre as variáveis estudadas foram utilizados os testes Qui-Quadrado e o Exato de Fisher. A comparação das médias dos segmentos foi obtida pelo teste T de amostras independentes. As curvas de sobrevida foram calculadas pelo modelo de Kaplan-Meier e confirmadas pelo modelo multivariado de Cox. Nossos resultados mostraram existir correlação entre o infiltrado linfocitário tumoral alto, tamanho do tumor primário T1/T2 (p = 0,001) e estadiamento clínico I e II (p = 0,005). O padrão de invasão tumoral tipo IV foi correlacionado com o tamanho do tumor primário T3/T4 (p = 0,006) e estadiamento clínico avançado (estádio III e IV) (p = 0,028). Invasão perineural foi associada com o tamanho do tumor primário T1/T2 (p = 0,035). A expressão nuclear da survivina na porção mediana do tumor mostrou associação com a metástase em linfonodos regionais (p = 0,004) e o estadiamento clínico (p = 0,041). A análise regressiva multivariada confirmou que as variáveis tamanho do tumor primário (p = 0,004) e acometimento linfonodal (p= 0,06) são fatores prognósticos independentes para sobrevida global, enquanto o etilismo influencia na sobrevida livre de doença (p = 0,048). Com este estudo pode-se concluir que a elevada expressão da survivina está correlacionada com o comportamento tumoral mais agressivo, estadiamento clínico avançado, presença de mestástase linfonodal, podendo ser considerada como indicador de prognóstico em pacientes com CECB. A variável histopatológica padrão de invasão tumoral mostrou que sua correlação com tamanho do tumor primário e estadiamento clínico avançado podendo estar relacionada ao pior prognóstico dos pacientes em CECB.

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.

Follicular lymphoma (FL) is an attractive model for discovering biomarkers and elucidating mechanisms of tumour progression. We hypothesized that alterations in the expression of proteins with known roles in cancer biology and hematological cells might correlate with clinical outcome and thereby shed light on biological mechanisms. Sections from a tissue microarray (TMA) containing FL samples from 67 patients were immunostained for candidate biomarkers, including p53, p16INK4a, Bcl-2, Bcl-6, MUM1, PML, phospho-ERK, and p27Kip1. The Kaplan-Meier method and log-rank test were used to identify markers that correlate significantly (p<0.05) with overall survival (OS). The chi-squared or Fisher exact test were used to examine associations between histological markers and baseline clinical features, including the Follicular Lymphoma International Prognostic Index (FLIPI) score. Expression of p16INK4a or p53, or absent CD10 expression correlated with poor survival. Patients with p16INK4a-negative tumours had a median OS of 13.4 years compared to 8.3 years for those with p16INK4a-positive tumours (p=0.006). Expression of p16INK4a was significantly associated with low hemoglobin, elevated serum lactate dehydrogenase (LDH), high histological grade, high cell proliferation index, presence of associated diffuse large B-cell lymphoma (DLBCL) and high-risk FLIPI classification. Our observation of a positive association between p16INK4a expression and indicators of tumour aggressiveness is novel and perhaps surprising since loss of the INK4a tumour suppressor gene is one of the most frequently observed lesions in human cancers, including lymphoma. Expression of p16INK4a may be part of a cellular response to unidentified pro-mitotic mutations, such as deleterious mutations of the RB tumour suppressor gene, associated with more aggressive instances of FL. Immunostaining FL diagnostic biopsies for expression of p16INK4a may serve as an informative prognostic biomarker to aid clinicians managing FL patients. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-07-04 15:55:16.121

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer. / Medicine, Faculty of / Graduate

Ce travail de thèse a pour objectif de développer les méthodes originales pour l'analyse quantitative des images de Tissue Microarrays (TMAs) acquises en fluorescence par des scanners dédiés. Nous avons proposé des contributions en traitement d'images portant sur la segmentation des objets d'intérêts (i.e. des échantillons de tissus sur la lame de TMA scannée), la correction des artefacts d'acquisition liés aux scanners en question ainsi que l'amélioration de la résolution spatiale des images acquises en tenant compte des modalités d'acquisition (imagerie en fluorescence) et la conception des scanners. Les développements permettent d'envisager une nouvelle plateforme d'analyse de TMAs automatisée, qui représente aujourd'hui une forte demande dans la recherche contre les cancers. Les TMAs (ou “puces à tissus”) sont les lames histologiques sur lesquelles de nombreux échantillons tissulaires venant de différents donneurs sont déposés selon une structure de grille afin de faciliter leur identification. Pour pouvoir établir le lien entre chaque échantillon et ses données cliniques correspondantes, on s'intéresse non seulement à segmenter ces échantillons mais encore à retrouver leur position théorique (les indices de ligne et de colonne) sur la grille TMA car cette dernière est souvent très déformée pendant la fabrication des lames. Au lieu de calculer directement les indices de ligne et de colonne (des échantillons), nous avons reformulé ce problème comme un problème d'estimation de la déformation de la grille de TMA théorique à partir du résultat de segmentation en utilisant l'interpolation par splines ''plaques minces''. Nous avons combiné les ondelettes et un modèle d'ellipses paramétriques pour éliminer les fausses alarmes, donc améliorer les résultats de segmentation. Selon la conception des scanners, les images sont acquises pixel par pixel le long de chaque ligne, avec un change de direction lors du balayage entre les deux lignes. Un problème fréquent est le mauvais positionnement des pixels dû à la mauvaise synchronisation des modules mécaniques et électroniques. Nous avons donc proposé une méthode variationnelle pour la correction de ces artefacts en estimant le décalage entre les pixels sur les lignes consécutives. Cette méthode, inspirée du calcul du flot optique, consiste à estimer un champ de vecteurs en minimisant une fonction d'énergie composée d'un terme d'attache aux données non convexe et d'un terme de régularisation convexe. La relaxation quadratique est ainsi utilisée pour découpler le problème original en deux sous-problèmes plus simples à résoudre. Enfin, pour améliorer la résolution spatiale des images acquises qui dépend de la PSF (point spread function) elle-même variant selon le faisceau laser d'excitation, nous avons introduit une méthode de déconvolution d'images en considérant une famille de régulariseurs convexes. Les régulariseurs considérés sont généralisés du concept de la variation parcimonieuses (Sparse Variation) combinant la norme L1 de l'image et la variation totale (Total Variation) pour rehausser les pixels dont l'intensité et le gradient sont non-nuls. Les expériences montrent que l'utilisation de cette régularisation produit des résultats déconvolution d'images très satisfaisants en comparaison avec d'autres approches telles que la variation totale ou la norme de Schatten de la matrice Hessienne. / This thesis aims at developing dedicated methods for quantitative analysis of Tissue Microarray (TMA) images acquired by fluorescence scanners. We addressed there issues in biomedical image processing, including segmentation of objects of interest (i.e. tissue samples), correction of acquisition artifacts during scanning process and improvement of acquired image resolution while taking into account imaging modality and scanner design. The developed algorithms allow to envisage a novel automated platform for TMA analysis, which is highly required in cancer research nowadays. On a TMA slide, multiple tissue samples which are collected from different donors are assembled according to a grid structure to facilitate their identification. In order to establish the link between each sample and its corresponding clinical data, we are not only interested in the localization of these samples but also in the computation of their array (row and column) coordinates according to the design grid because the latter is often very deformed during the manufacturing of TMA slides. However, instead of directly computing array coordinates as existing approach, we proposed to reformulate this problem as the approximation of the deformation of the theoretical TMA grid using “thin plate splines” given the result of tissue sample localization. We combined a wavelet-based detection and a ellipse-based segmentation to eliminate false alarms and thus improving the localization result of tissue samples. According to the scanner design, images are acquired pixel by pixel along each line, with a change of scan direction between two subsequent lines. Such scanning system often suffers from pixel mis-positioning (jitter) due to imperfect synchronization of mechanical and electronic components. To correct these scanning artifacts, we proposed a variational method based on the estimation of pixel displacements on subsequent lines. This method, inspired from optical flow methods, consists in estimating a dense displacement field by minimizing an energy function composed of a nonconvex data fidelity term and a convex regularization term. We used half-quadratic splitting technique to decouple the original problem into two small sub-problems: one is convex and can be solved by standard optimization algorithm, the other is non-convex but can be solved by a complete search. To improve the resolution of acquired fluorescence images, we introduced a method of image deconvolution by considering a family of convex regularizers. The considered regularizers are generalized from the concept of Sparse Variation which combines the L1 norm and Total Variation (TV) to favors the co-localization of high-intensity pixels and high-magnitude gradient. The experiments showed that the proposed regularization approach produces competitive deconvolution results on fluorescence images, compared to those obtained with other approaches such as TV or the Schatten norm of Hessian matrix.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

This thesis includes studies on N-, mucin type O-, and glycosaminoglycan (GAG)-linked glycosylation in human biospecimens. Glycosylation plays a central role in biological processes, including protein folding, immune surveillance, and regulation of cell growth. The structures of GAG are regulated in a tissue-specific manner. Heparan sulfate (HS) and chondroitin sulfate (CS) are the two types of GAGs targeted in this thesis. Human leukocytes express both CS and HS GAGs with CS being the more abundant type; however, little is known regarding the properties and structures of GAG chains, their ranges of variability among normal subjects, and changes in structure associated with disease conditions. We measured the relative and absolute disaccharides abundances of HS and CS for purified B, T, NK cells, monocytes, and polymorphonuclear leukocytes (PMNs) using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with levels of sulfation more similar to heparin than to organ-derived HS. In addition, CS abundances varied considerably in a leukocyte cell type specific manner. Therefore, our results established the ranges of GAG structures expressed on normal leukocytes as well as necessary for subsequent inquiry into disease conditions. Glioblastoma (GBM) accounts for 30% of human primary brain tumors. It is deadly and highly invasive. In past decades, most GBM research focused on pathophysiological changes in genome. There remains a dearth of knowledge regarding alterations in glycomics, glycoproteomics, and proteomics during GBM tumorigenesis. Therefore, we developed a comprehensive platform for high-throughput sample preparation with surface digestion for tissue microarrays, LC-MS/MS data dependent acquisition, and semi-automated data analysis to integrate glycomics, glycoproteomics, and proteomics for different grade of tumor and different subtypes of GBM. By analyzing GBM tissue microarrays, we found tumor grade and subtype specific changes to the expression of biomolecules. We also identified approximately 100 site-specific N- and mucin type O-glycosylations, the majority of which were previously unreported. Overall, our results improved the fundamental understandings about GBM pathogenesis. / 2018-11-02T00:00:00Z