The Gleason Score (GS) is a powerful predictor of outcome among prostate cancer patients. Patients with tumours graded with a GS of 2 to 6 have a much greater chance of survival compared to those with a GS of 8 to 10. A significant proportion (~40%) of men present with early stage GS 7 tumours (indicating intermediate risk) for whom prognosis is highly variable. Three gene signatures were derived from publicly available gene expression profiles of prostate cancers from the Swedish Watchful Waiting cohort: 1) The Genomic Grade Index consisted of the top 24 genes discriminating between high (8, 9 & 10) and low (≤ 6) GS tumours, 2) The Lethal Gene Score consisted of the top 24 genes discriminating between lethal and indolent disease within GS 7 tumours only, and 3) The network-based gene signature consisted of 88 genes. When these gene signatures were tested in silico on the gene expression profiles of GS 7 patients in both the SWW and the Mayo cohort, patients were stratified into high and low risk for recurrence. These results demonstrate that gene signatures are capable of differentiating low risk and high risk patients within GS 7 tumours. The prognostic capacity of our gene signature will be tested prospectively in a retrospective collection of archived prostate cancer tissue blocks from a phase 3 clinical trial, and it is hypothesized that the patients can be stratified into good and poor outcome groups. NanoString Technology will be used to quantify mRNA values for the signature genes on selected paraffin blocks. Expression values of candidate genes will be correlated with patients’ long-term follow-up information to derive a clinically meaningful signature. Outcome will be defined as biochemical recurrence or metastatic event. The goal of this study is to identify multiple genes whose expression could be formulated into a clinically applicable assay, the implementation of which could serve to better stratify intermediate risk prostate cancer patients for appropriate treatment. / Thesis / Master of Science (MSc) / The over-treatment of prostate cancer patients is a significant concern, as recent clinical trials has shown that it can lead to significant patient morbidity. Although the Gleason Scoring system is a powerful predictor of lethal or indolent disease, a significant proportion of men who present with early stage Gleason Score 7 tumours experience poorer prognosis than expected. The goal of this study is to develop and optimize a gene signature that can be utilized on Gleason Score 7, intermediate risk prostate cancer patients to differentiate them into good and poor outcome groups. We hypothesize that this signature will be able to accurately predict outcome in a separate retrospective cohort of prostate cancer patients. In short, our study hopes to provide proof-of-principle that through the use of gene signatures, it is possible to better differentiate prostate cancer patients into different outcome groups so that they may receive more appropriate treatment specific to their disease type.

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

Os astrocitomas são os tumores cerebrais primários mais frequentes, dentre os quais, o glioblastoma multiforme (GBM) é o tipo mais agressivo, sendo classificado como astrocitoma de grau IV. O tratamento envolve remoção cirúrgica seguida de quimio e radioterapias, porém essa abordagem não é eficaz devido à alta resistência das células tumorais. Em um trabalho anterior, buscando caracterizar os mecanismos associados à esta característica, investigamos a expressão de genes de reparo de DNA em astrocitomas de diferentes graus. Foram encontradas alterações em 19 genes. Através da combinação destas alterações em todos os arranjos possíveis, definimos um grande conjunto de assinaturas de expressão gênica que foi utilizado como filtro para a busca de correlação em bancos de dados públicos. No total, 421 assinaturas foram associadas à redução na sobrevida dos pacientes, sendo que cinco dos genes (EXO1, NEIL3, BRCA2, BRIP1 e DDB2) foram isoladamente relacionados ao pior prognóstico. Notavelmente, DDB2 foi o único gene subexpresso a apresentar esta correlação, levando a um risco de morte aproximadamente três vezes maior. No presente estudo in vitro, após radiação ionizante, observamos que células com baixos níveis de DDB2 reparam mais rapidamente as quebras induzidas no DNA, saem mais facilmente da parada de ciclo na fase G2/M e se tornam ainda mais resistentes a este tratamento. Além disso, o silenciamento de DDB2 induziu o aumento dos níveis de Zeb1, um importante promotor da transição epitélio-mesênquima, bem como dos índices de invasão e migração celular. Estes dados mostram que a redução nos níveis de DDB2 induz um fenótipo mais agressivo, corroborando a correlação com pior prognóstico dos pacientes observado anteriormente. Tomados em conjunto, esses resultados sugerem que a função de DDB2 não está limitada ao âmbito do reparo de DNA, apontam uma potencial relação com o controle da transição epitélio-mesênquima e mostram que este gene possui papel fundamental no estabelecimento da agressividade e resistência de GBM. / Astrocytomas are the most common primary brain tumors. Glioblastoma multiforme (GBM) is the most aggressive type and is classified as grade IV astrocytoma. The treatment involves surgical resection followed by chemo and radiotherapies, however this approach is not effective due to the high resistance of tumor cells. In a previous work, to characterize the cellular mechanisms associated to this characteristic, we investigated the expression of DNA repair genes in samples of different astrocytoma grades. We found alterations in 19 genes. By combining these expression changes in all possible arrangements, a large set of gene expression signatures was defined and used as a filter to seek correlations in public databases. As a result, 421 signatures were associated with reduced patient survival. Among them, five genes (EXO1, NEIL3, BRCA2, BRIP1 and DDB2) were individually related to worse prognosis. Notably, DDB2 was the only down regulated gene to exhibit this correlation, making the risk of death approximately three times higher. In the present in vitro study, after ionizing radiation, we observed that cells with low levels of DDB2 are capable of faster DNA breaks repair, easily exit the G2/M arrest and become even more resistant to this treatment. Furthermore, DDB2 silencing enhanced the levels of Zeb1, an important promoter of the epithelial-mesenchymal transition, as well as the rates of cell invasion and migration. These data indicate that DDB2 knockdown leads to a more aggressive cell behavior, corroborating the association with worse prognosis previously observed. Taken together, these results suggest that DDB2 function is not limited to DNA repair, they point out its potential participation in epithelial-mesenchymal transition control and show that this gene might play a key role in GBM\'s aggressiveness and resistance.

Submitted by Talita Mariana Morata Raposo null (talita_raposo@yahoo.com.br) on 2016-08-26T23:53:52Z No. of bitstreams: 1 tese.pdf: 7349096 bytes, checksum: 15c4a9aa3753843c66b8604dad27c6e8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-30T18:12:04Z (GMT) No. of bitstreams: 1 ferreira_tmmr_dr_jabo.pdf: 7349096 bytes, checksum: 15c4a9aa3753843c66b8604dad27c6e8 (MD5) / Made available in DSpace on 2016-08-30T18:12:04Z (GMT). No. of bitstreams: 1 ferreira_tmmr_dr_jabo.pdf: 7349096 bytes, checksum: 15c4a9aa3753843c66b8604dad27c6e8 (MD5) Previous issue date: 2016-08-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os tumores mamários são os principais tumores que acometem as cadelas, sendo também os tumores mais frequentes em mulheres e, entre ambas as espécies, são observadas semelhanças relacionadas ao comportamento biológico e molecular dessa neoplasia. Assim, as cadelas podem ser um excelente modelo comparativo para o entendimento do processo carcinogênico desta neoplasia. A metástase é uma consequência comum e a principal causa de mortalidade em decorrência dessa enfermidade, em ambas as espécies. O perfil de expressão gênica global permite o melhor entendimento do processo de carcinogênese e de metástases, por permitir a identificação de inúmeros genes que possam estar envolvidos com esses processos. Assim, o objetivo desse estudo foi a realização do estudo em larga escala, através da técnica de microarray, em amostras de tecidos mamários caninos, considerando amostras de glândulas mamárias normais, tumores mamários benignos e tumores mamários malignos (carcinomas simples e mistos), além da avaliação de carcinomas mamários metastáticos e não metastáticos, para identificação de genes diferencialmente expressos entre esses grupos e relacionados com a tumorigênese e o desenvolvimento de metástases dos tumores mamários caninos. Observou-se, aproximadamente 1000 genes diferencialmente expressos entre as amostras tumorais e as glândulas mamárias normais e 465 genes diferencialmente expressos entre os tumores mamários benignos e malignos, sendo observados genes relacionados com o ciclo celular, desenvolvimento da glândula mamária e supressores tumorais. Em relação aos carcinomas mamários metastáticos e não metastáticos, verificou-se 633 genes diferencialmente expressos. Os genes supressores tumorais, como pertencentes a via de sinalização do ATM e do BRCA1, sugeriram importante papel tanto na progressão tumoral quanto no desenvolvimento de metástases. Outras vias observadas foram as relacionadas com angiogênese e de organização da matriz extracelular. Portanto, a análise do perfil gênico em larga escala permitiu a identificação de inúmeros genes e vias envolvidas tanto no processo de progressão tumoral como envolvidas com o desenvolvimento de metástases, permitindo a realização de estudos futuros em busca de marcadores prognósticos específicos. / Mammary tumors are the main tumors that affect female dogs, as well as in women and in both species, it is observed similarity related to the biological and molecular behavior of this neoplasia. So, female dogs are considered an excellent model for the understanding of carcinogenesis process from mammary tumors. Metastasis occurs frequently and it is the main responsible for the mortality by this neoplasia in both species. Global gene expression profile allows the better understanding of carcinogenesis and metastasis process by the identification of several genes that may be involved with these processes. Therefore, the aim of study was to perform a large-scale study by microarray technique using samples from canine mammary tissues, as normal mammary gland, benign tumors and malignant tumors (simple and mixed carcinomas), beyond metastatic and non-metastatic mammary carcinomas for the identification of differentially expression genes among these groups and related to canine mammary tumors tumorigenesis and metastasis. It was observed next to 1000 differentially expression genes between tumors and normal mammary glands samples and 465 differentially expression genes between benign and malignant mammary tumors mostly related to cell cycle, mammary gland development and tumor suppressors. Related to metastatic and non-metastatic mammary carcinomas was identified 633 differentially expression genes. ATM and BRCA1, associated with tumor suppressor gene pathway, showed to play an important role in both tumor progression and metastasis development. Other networks observed were related to angiogenesis and extracellular matrix organization. Thus, large-scale gene profile analysis allowed the identification of numerous genes and networks involved with both tumor progression and metastasis, allowing new studies in search of specific prognostic markers. / FAPESP: 2013/03940-4 / FAPESP: 2013/25220-3

Os astrocitomas são os tumores cerebrais primários mais frequentes, dentre os quais, o glioblastoma multiforme (GBM) é o tipo mais agressivo, sendo classificado como astrocitoma de grau IV. O tratamento envolve remoção cirúrgica seguida de quimio e radioterapias, porém essa abordagem não é eficaz devido à alta resistência das células tumorais. Em um trabalho anterior, buscando caracterizar os mecanismos associados à esta característica, investigamos a expressão de genes de reparo de DNA em astrocitomas de diferentes graus. Foram encontradas alterações em 19 genes. Através da combinação destas alterações em todos os arranjos possíveis, definimos um grande conjunto de assinaturas de expressão gênica que foi utilizado como filtro para a busca de correlação em bancos de dados públicos. No total, 421 assinaturas foram associadas à redução na sobrevida dos pacientes, sendo que cinco dos genes (EXO1, NEIL3, BRCA2, BRIP1 e DDB2) foram isoladamente relacionados ao pior prognóstico. Notavelmente, DDB2 foi o único gene subexpresso a apresentar esta correlação, levando a um risco de morte aproximadamente três vezes maior. No presente estudo in vitro, após radiação ionizante, observamos que células com baixos níveis de DDB2 reparam mais rapidamente as quebras induzidas no DNA, saem mais facilmente da parada de ciclo na fase G2/M e se tornam ainda mais resistentes a este tratamento. Além disso, o silenciamento de DDB2 induziu o aumento dos níveis de Zeb1, um importante promotor da transição epitélio-mesênquima, bem como dos índices de invasão e migração celular. Estes dados mostram que a redução nos níveis de DDB2 induz um fenótipo mais agressivo, corroborando a correlação com pior prognóstico dos pacientes observado anteriormente. Tomados em conjunto, esses resultados sugerem que a função de DDB2 não está limitada ao âmbito do reparo de DNA, apontam uma potencial relação com o controle da transição epitélio-mesênquima e mostram que este gene possui papel fundamental no estabelecimento da agressividade e resistência de GBM. / Astrocytomas are the most common primary brain tumors. Glioblastoma multiforme (GBM) is the most aggressive type and is classified as grade IV astrocytoma. The treatment involves surgical resection followed by chemo and radiotherapies, however this approach is not effective due to the high resistance of tumor cells. In a previous work, to characterize the cellular mechanisms associated to this characteristic, we investigated the expression of DNA repair genes in samples of different astrocytoma grades. We found alterations in 19 genes. By combining these expression changes in all possible arrangements, a large set of gene expression signatures was defined and used as a filter to seek correlations in public databases. As a result, 421 signatures were associated with reduced patient survival. Among them, five genes (EXO1, NEIL3, BRCA2, BRIP1 and DDB2) were individually related to worse prognosis. Notably, DDB2 was the only down regulated gene to exhibit this correlation, making the risk of death approximately three times higher. In the present in vitro study, after ionizing radiation, we observed that cells with low levels of DDB2 are capable of faster DNA breaks repair, easily exit the G2/M arrest and become even more resistant to this treatment. Furthermore, DDB2 silencing enhanced the levels of Zeb1, an important promoter of the epithelial-mesenchymal transition, as well as the rates of cell invasion and migration. These data indicate that DDB2 knockdown leads to a more aggressive cell behavior, corroborating the association with worse prognosis previously observed. Taken together, these results suggest that DDB2 function is not limited to DNA repair, they point out its potential participation in epithelial-mesenchymal transition control and show that this gene might play a key role in GBM\'s aggressiveness and resistance.

Soft tissue sarcomas (STS), a group of rare malignant tumours with high tissue heterogeneity, still lack effective clinical stratification and prognostic models. Therefore, we conducted this study to establish a reliable prognostic gene signature. Using 189 STS patients’ data from The Cancer Genome Atlas database, a four-gene signature including DHRS3, JRK, TARDBP and TTC3 was established. A risk score based on this gene signature was able to divide STS patients into a low-risk and a high-risk group. The latter had significantly worse overall survival (OS) and relapse free survival (RFS), and Cox regression analyses showed that the risk score is an independent prognostic factor. Nomograms containing the four-gene signature have also been established and have been verified through calibration curves. In addition, the predictive ability of this four-gene signature for STS metastasis free survival was verified in an independent cohort (309 STS patients from the Gene Expression Omnibus database). Finally, Gene Set Enrichment Analysis indicated that the four-gene signature may be related to some pathways associated with tumorigenesis, growth, and metastasis. In conclusion, our study establishes a novel four-gene signature and clinically feasible nomograms to predict the OS and RFS. This can help personalized treatment decisions, long-term patient management, and possible future development of targeted therapy.

Background: Head and neck squamous cell carcinomas (HNSCCs) are complex and highly aggressive tumours that develop in the mouth, throat, salivary glands and nose. HNSCCs account for more than half a million cases annually and are the sixth most common cancer worldwide. Alcohol, tobacco and human papilloma virus (HPV) infection are the well-known causes for HNSCC. The current options for treatment are surgery, radiotherapy, chemotherapy or a combination of therapies. Locally advanced HNSCC patients show heterogenous response to standard treatments and the survival after 5 years is about 50%. Therefore, there is a need to identify biomarkers to predict outcome and improve personalised therapies. The recent advancement in next generation sequencing technologies has allowed for understanding the molecular characteristics of the tumour and identify patients at high risk that are unresponsive to the standard treatment. HPV-associated oropharyngeal carcinoma have shown a very high rate of loco-regional control (LRC) and overall survival (OS) after postoperative radio- chemotherapy (PORT-C) and are being assessed for treatment de-escalation strategies to reduce toxicity in clinical trials. The treatment response of HPV-negative HNSCC, however, is still heterogeneous and novel biomarkers are required to identify subgroups of patients for treatment adaptation. Objectives: The overall aim of the thesis is to develop biomarkers to identify patients at high risk for future treatment adaptations and improve personalised radiotherapy based on the biological differences in HNSCC patients. For this purpose, novel gene signatures were developed and validated using machine learning approaches and biological information in order to predict LRC in patients with locally advanced HNSCC. The novel gene signatures will help to identify patients at high risk that do not respond to standard treatments and to further understand the molecular mechanisms involved in heterogenous treatment response. Materials and methods: The data from a total of 504 locally advanced HNSCC patients of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG) treated with postoperative radiotherapy (PORT) or postoperative radiochemotherapy (PORT-C) were evaluated. Data from 60 mice bearing xenografts of ten established human HNSCC cell lines were also evaluated. Gene expression analyses was performed using the GeneChip Human Transcriptome Array 2.0 and nanoString analyses. Differential gene expression analysis, Cox regression analysis and machine learning algorithms were used to develop gene signatures. Models were built on the training cohort and then applied on an independent validation cohort. Results: The patients with HPV-negative HNSCC that were treated with PORT-C were classified into the four molecular subtypes basal, mesenchymal, atypical and classical that were previously reported for HNSCC patients treated with primary radio(chemo)therapy or surgery and were related to LRC. The mesenchymal subtype had the worst prognosis as compared to the other subtypes. These tumours were associated with overexpression of epithelial-mesenchymal transition genes and DNA repair genes. A novel 6-gene signature was developed and validated based on full-transcriptome data using machine-learning approaches that was prognostic for LRC in patients with HPV-negative HNSCC treated with PORT-C. The 6-gene signature consisted of four individual genes CAV1, GPX8, IGLV3-25, TGFBI and one metagene combining the highly correlated genes, INHBA and SERPINE1. The identified gene signature was combined with the clinical parameters, T stage and tumour localisation as well as the stem-cell marker CD44 and the 15-gene hypoxia- associated classifier and this improved the performance of the model. Previously identified prognostic gene signatures and molecular-subtype classification were back-translated from HNSCC patients to pre-clinical tumour models. The tumour models were classified into the four subtypes basal, mesenchymal, atypical and classical, similar to the patients. The mesenchymal tumours were significantly associated with a higher TCD50 as compared to other subtypes. A novel 2-gene signature consisting of FN1 and SERPINE1 was developed based on tumour models and patient data using differential gene expression analysis. The 2-gene signature was prognostic for the TCD50 in tumour models and was successfully validated on an independent PORT-C patient cohort for LRC. A matched-pair analysis was performed between patients that were treated with postoperative radiochemotherapy and patients that were treated with postoperative radiotherapy. A 2- metagene signature, consisting of KRT6A, KRT6B, KRT6C forming one metagene and SPRR1A, SPRR1B, SPRR2A, SPRR2C forming the second metagene, was identified. The novel predictive signature stratified patients into high and low risk groups. The high-risk group patients that received PORT-C showed higher LRC as compared to the high-risk patients that received PORT. Thus, the predictive gene signature identified patients that were considered to be at intermediate risk according to clinical factors but were at biologically high risk for the development of loco-regional recurrences after PORT. These patients might benefit from PORT-C treatment. Conclusions: In this thesis, novel gene signatures were identified by combining machine learning and biological information to stratify locally advanced HNSCC patients into high and low risk groups for loco-regional control. This information could be used in the future, e.g. to adjust radiotherapy doses based on the risk group. The developed gene signatures could be combined with other gene signatures or the molecular subtype stratification to develop potential combined treatment approaches. Within the DKTK-ROG framework, the gene signatures will be incorporated with biomarkers developed on the same cohort at the other DKTK-ROG partner sites using the data from different omics platforms in the future. This would help to better understand the molecular basis of heterogenous treatment response in HNSCC patients and uncover novel targets for therapies. The thesis also provides a valuable insight into the applicability of preclinical tumour models to study the efficacy of personalised radiotherapy treatments. Overall, the gene signatures identified in this thesis were from retrospective studies and have to be validated in prospective studies before their application in interventional clinical trials to improve personalised radiotherapy treatments. Additionally, the methods used in the thesis to identify the gene signatures could be used and applied across different cancer datasets for identification of biomarkers. Therefore, this thesis has provided a basis for future studies on personalized treatment of HNSCC based on their genetic profile.:Content Abbreviations VII Tables XII 1 Introduction 1 2 Biological and statistical background 6 2.1 Head and neck squamous cell carcinoma 6 2.1.1 Tumourigenesis 6 2.1.2 Biomarkers: clinical and genomics 9 2.2 Statistics 12 General statistical analyses 17 2.3 Gene expression analyses 18 3 Molecular subtypes and mechanisms of radioresistance 20 3.1 Introduction and motivation 20 3.2 Patient cohort and experimental design 21 3.2.1 Patient cohort 21 3.2.2 Clinical endpoints and statistical analysis 23 3.2.3 Experimental design 23 3.3 Results 26 3.3.1 Prognostic factors for LRC and OS 26 3.3.2 Death as competing risk 26 3.3.3 Multivariable Cox regression for improved prognosis 29 3.3.4 Molecular subtypes in HPV-negative HNSCC patients 31 3.3.5 Molecular subtypes are prognostic for LRC after PORT-C 33 3.4 Discussion 36 4 A novel 6-gene signature for LRC prognosis 39 4.1 Introduction and motivation 39 4.2 Patient cohort and experimental design 40 4.2.1 Patient cohorts 40 4.2.2 Clinical endpoints and statistical analysis 41 4.2.3 Experimental design 41 4.3 Results 44 4.3.1 Characteristics of the patient cohorts 44 4.3.2 Development of the 6-gene signature prognostic for LRC 45 4.3.3 Combination of the 6-gene signature and clinical parameters 47 4.3.4 Extension with CD44 and the 15-gene hypoxia signature 48 4.3.5 Prognostic for secondary endpoints 49 4.3.6 Technical validation using nanoString technology 52 4.3.7 Death as competing risk 56 4.4 Discussion 58 5 Biomarker development in preclinical tumour models and HNSCC patients 62 5.1 Introduction and motivation 62 5.2 Patient cohort and experimental design 64 5.2.1 Patient derived xenograft tumour models 64 5.2.2 Patient cohorts 64 5.2.3 Clinical endpoints and statistical analysis 65 5.2.4 Experimental design 65 5.3 Results 68 5.3.1 Molecular subtypes 68 5.3.2 Development of the 2-gene signature 70 5.3.3 Technical validation using the nanoString technology 71 5.3.4 Back-translation of gene signatures in xenograft models 75 5.4 Discussion 79 6 PORT-C improves LRC in intermediate-risk patients 82 6.1 Introduction and motivation 82 6.2 Patient cohort and experimental design 83 6.2.1 Patient cohorts 84 6.2.2 Clinical endpoints and statistical analysis 84 6.2.3 Experimental design 84 6.3 Results 87 6.3.1 Characteristics of the patient cohorts 87 6.3.2 Propensity score matching analysis 88 6.3.3 Development of the predictive 2-metagene signature 90 6.4 Discussion 93 7 Conclusion and future perspectives 96 8 Summary 99 9 Zusammenfassung 102 Appendix 105 A. Supplementary Figures 105 B. Supplementary Tables 110 Bibliography 116 Erklärungen 149

Colorectal cancer is the third most common cancer and the leading cause of cancer deaths in Western industrialised countries. Despite recent advances in the screening, diagnosis, and treatment of colorectal cancer, an estimated 608,000 people die every year due to colon cancer. Our current knowledge of colorectal carcinogenesis indicates a multifactorial and multi-step process that involves various genetic alterations and several biological pathways. The identification of molecular markers with early diagnostic and precise clinical outcome in colon cancer is a challenging task because of tumour heterogeneity. This Ph.D.-thesis presents the molecular and cellular mechanisms leading to colorectal cancer. A systematical review of the literature is conducted on Microarray Gene expression profiling, gene ontology enrichment analysis, microRNA and system Biology and various bioinformatics tools. We aimed this study to stratify a colon tumour into molecular distinct subtypes, identification of novel diagnostic targets and prediction of reliable prognostic signatures for clinical practice using microarray expression datasets. We performed an integrated analysis of gene expression data based on genetic, epigenetic and extensive clinical information using unsupervised learning, correlation and functional network analysis. As results, we identified 267-gene and 124-gene signatures that can distinguish normal, primary and metastatic tissues, and also involved in important regulatory functions such as immune-response, lipid metabolism and peroxisome proliferator-activated receptors (PPARs) signalling pathways. For the first time, we also identify miRNAs that can differentiate between primary colon from metastatic and a prognostic signature of grade and stage levels, which can be a major contributor to complex transcriptional phenotypes in a colon tumour.

No description available.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.