Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels comparable to those from kidney cortex, while VEGF165 was lower. In PRCC and ChRCC, VEGF189 levels correlated inversely with advancing tumour stage, and in PRCC, VEGF165 levels correlated inversely with increasing tumour size. VEGF189 was an independent prognostic factor for survival in patients with PRCC. Conclusions. S-VEGF has a stronger association to progression in RCC than platelet count. CRCC showed high levels of VEGF mRNA isoforms and VEGF-R1 mRNA compared to PRCC. VEGF mRNA isoforms expression decreased with advancing stage. IHC demonstrated VEGF expression in cell cytoplasm related to tumour growth, particular in CRCC. Different VEGF isoform patterns were found in different RCC types. Protein VEGF189 expression was associated with tumour stage and was an independent prognostic factor in PRCC. Protein VEGF165 expression was generally low and had no prognostic value. The trend for decreasing levels of VEGF isoforms in advanced tumour stages may indicate that angiogenic activity is an early event in tumour growth induced by VEGF, but that during later tumour progression the role of VEGF is less clear.

Background: Prostate cancer is the most common cancer in Sweden with around 10,000 new cases every year. Kidney and bladder cancer are less common with 1,000 and 2,000 new cases annually, respectively. The incidence of these cancer sites is higher in developed, than in developing countries, suggesting an association between lifestyle and cancer risk. The aims of this thesis were to investigate body mass index (BMI), blood pressure, and blood levels of glucose, total cholesterol, and triglycerides as risk factors for prostate, kidney, and bladder cancer. Furthermore, we aimed at assess probabilities of prostate cancer and competing events, all-cause death, for men with normal and high levels of metabolic factors. Material and methods: This thesis was conducted within the Metabolic Syndrome and Cancer project (Me-Can), a pooled cohort study with data from 578,700 participants from Norway, Sweden, and Austria. Data from metabolic factors were prospectively collected at health examinations and linked to the Cancer and Cause of Death registers in each country.  Results: High levels of metabolic factors were not associated with increased risk of prostate cancer, but high levels of BMI and blood pressure were associated with risk of prostate cancer death. The probability of prostate cancer was higher for men with normal levels of metabolic factors compared to men with high levels, but the probability of all-cause death, was higher for men with high levels than for those with normal levels. For both men and women, high levels of metabolic factors were associated with increased risk of kidney cancer (renal cell carcinoma). Furthermore, blood pressure for men and BMI for women were found as independent risk factors of kidney cancer. High blood pressure was associated with an increased risk of bladder cancer for men. Conclusions: High levels of metabolic factors were associated to risk of kidney and bladder cancer and to death from kidney, bladder, and prostate cancer. Compared to men with normal levels, men with high levels of metabolic factors had a decreased probability of prostate cancer but an increased probability of all-cause death. / <p>Ytterligare forskningsfinansiärer: World Cancer Research Fund (2007/09) och Wereld Kanker Onderzoek Fonds (R2010/247)</p> / Me-Can

Background: Pancreatic métastases from renal cell carcinoma (RCC) are clinically rare but highly resectable. The aim of this article is to identify patients who profit from pancreatic resection of RCC despite the invasiveness of the surgery. Methods: Between January 1996 and December 2007, data from 744 patients were collected in a prospective pancreatic surgery database, and patients with metastasis into the pancreas from RCC were identified. Results: Resective surgery was performed in 14 patients with metastasis to the pancreas from RCC. Most patients were clinically asymptomatic. The median interval between primary treatment of RCC and occurrence of pancreatic metastasis was 94 months (range 32–158). The morbidity rate was 42.8%. Patients with a metastasis size <2.5 cm had a much better survival after resection (100 months) than those with a metastasis size >2.5 cm (44 months). Moreover, the number of métastases predicts the survival after resection. Conclusions: In patients with pancreatic métastases from RCC who have only limited disease, complete resection of all lesions can be successfully performed with a low rate of complications. Thus, patients with a history of RCC should be monitored for more than 10 years after nephrectomy to detect recurrence. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

No description available.

ABSTRACT Renal cell carcinoma (RCC), the most common type of kidney cancer, is a highly metastatic disease. Late stage metastatic RCC is essentially incurable and lethal. The average survival of patients, following metastatic RCC, is about 4 months and only 10% of patients survive for one year. None of the currently available chemotherapy, radiotherapy, hormonal or biological therapies have a significant impact on the progression of the disease. Novel chemotherapeutics are urgently required for the treatment of this deadly disease. The mechanisms that pose the greatest challenges to chemotherapeutics are resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. Resistance to apoptosis may be mediated by the up-regulation of anti-apoptotic proteins, especially Bcl-2 and Bcl-XL, and/or by the down- regulation of pro-apoptotic proteins, particularly Bax. Angiogenesis is pivotal for tumour growth and metastasis. Of all identified pro-angiogenic molecules, vascular endothelial growth factor (VEGF) is considered to be a key molecule. Drug resistance is thought to be mediated by the up-regulation of multi-drug resistance molecules such as MDR-1 and MRP-1. Up-regulation of Bcl-2 also confers drug resistance to cancer cells. The main hypothesis of this thesis was that treatment targets of metastatic RCCs are likely to multifactorial and that inhibition of molecules that regulate the processes of apoptosis, angiogenesis and multidrug resistance are likely to be better targets than those that regulate only one of these processes. In this regard, the transcription factor nuclear factor kappaB (NF-kB) meets the criterion, regulating the apoptotic, angiogenic and multi-drug resistance pathways of cancer cells. Its inhibition appeared to be an attractive strategy for the treatment of metastatic RCC. Many studies have demonstrated an association between the over-expression NF-kB and RCC. Thus, the major aim of this thesis was to explore the anti-cancer effect of pyrrolidine dithiocarbamate (PDTC), a potent NF-kB inhibitor on human metastatic RCC cell lines. The thesis is divided into seven Chapters. In Chapter 1, the literature on RCC, NF-B and the role of NF-kB in RCC development and progression are reviewed. The rationale for the inhibition of NF-kB as a potential anti-RCC strategy using PDTC is established. During the course of this research, the use of PDTC as an anti-cancer agent has risen to prominence. Chapter 2 describes the materials and methods used in the project. In Chapter 3, the expression of NF-kB in human kidney and the RCC cell lines, ACHN and SN12K1, was established. The proof of hypothesis that NF-kB inhibition using PDTC is an effective anti-cancer strategy was demonstrated. PDTC was selectively toxic to the RCC cell lines, but not to normal human kidney cells. PDTC induced apoptosis and inhibited proliferation of the RCC cells. PDTC also inhibited NF-kB, its upstream regulatory molecules such as the inhibitory protein family of the IkBs, and the kinase IKK complex. PDTC also inhibited anti-apoptotic Bcl-2 and Bcl-XL, but not pro-apoptotic Bax. Chapter 4 demonstrated the in vitro and ex vivo anti-angiogenic and anti-metastatic effects of PDTC. Protein microarrays for angiogenic factors produced controversial results. PDTC inhibited epidermal growth factor (EGF) produced in endothelial cells. VEGF had neutral effect on angiogenesis under the experimental conditions used. In the RCC cell lines, several pro-angiogenic molecules were modulated. Interestingly, the pro-angiogenic molecule interleukin (IL)-8 was up-regulated in both RCC cell lines. The monocyte chemoattractant protein-1 (MCP-1) was decreased in ACHN cells, but increased in SN12K1 cells. The implications of these controversial findings are discussed. Chapter 5 demonstrated the ability of PDTC to overcome drug resistance in a synergism with cisplatin. Individual non-toxic concentrations of PDTC and cisplatin, when combined, induced significant toxicity of RCC cell lines. The synergistic effect was not mediated by the inhibition of NF-kB, but rather through the inhibition of transcriptional activation of NF-kB. Bcl-2 rather than MDR-1 or the regulatory protein MRP-1 may be important in overcoming drug resistance in RCC. Chapter 6 showed the anti-cancer effect of PDTC in an animal model of RCC. PDTC significantly decreased the growth of RCC implanted in the kidney of severe combined immunodeficiency (SCID) mice. PDTC inhibited NF-kB and was not toxic to normal cells. The expression of Bcl-2, Bcl-XL and Bax were contradictory to the in vitro findings and a theory about the spread of RCC based on these findings is discussed. In Chapter 7, the findings are summarised. A case for PDTC as a potential therapeutic agent for RCC is established. Under the experimental conditions used, PDTC was demonstrated to be an effective anti-RCC agent by targeting the three most important characteristics of RCC that pose the greatest challenges to chemotherapeutics: resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. PDTC was selectively toxic to RCC, but not to normal renal cells. Thus PDTC appears to be a promising anti-cancer agent. This is supported by the current increase in interest, and in the number of publications, on the use of PDTC in several cancers. Some future directions are also discussed in this Chapter. These include, but are not limited to, an investigation of what is protecting normal cells from the toxicity of PDTC, the creation of an Australian database on RCC, and the characterisation of RCC based on NF-kB expression.

Resumo: O tumor renal é a mais letal das doenças urológicas. É uma doença histologicamente heterogênea, sendo o carcinoma renal de células claras o subtipo histológico mais comum. Embora a nefrectomia e imunoterapia sejam tratamentos bem estabelecidos, aproximadamente 30% dos pacientes tratados são acometidos por metástases. Alterações na expressão gênica e na inativação transcricional, devido ao mecanismo de metilação, são evidentes em células cancerosas. A metilação do DNA é um evento epigenético intimamente relacionado com o silenciamento da expressão gênica, e está envolvida em vários processos, dentre eles, a carcinogênese. Dessa forma, este trabalho teve como objetivos investigar se os genes selecionados, GPC3, CRABP2, KTN1 e ADAM23 apresentam expressão alterada nas amostras tumorais, verificar se a expressão gênica está associada com a progressão tumoral, e analisar o padrão de metilação de ilhas CpGs. Os quatro genes selecionados foram validados pela técnica de PCR em Tempo Real. Para validação desses genes foram utilizadas 35 amostras de carcinoma renal de células claras e 35 amostras de córtex renal normal. Os genes GPC3, CRABP2, KTN1 e ADAM23 apresentaram redução de expressão significativa em amostras de carcinoma renal de células claras quando comparadas ao pool de amostras de córtex renal normal. Observou-se que a redução da expressão do gene ADAM23 está diretamente relacionada com o avanço do estadiamento tumoral. Foi observada uma freqüência elevada de hipermetilação do gene ADAM23, entretanto, não houve associação do padrão de metilação com os dados clínicos. A análise da expressão gênica e dos mecanismos responsáveis pela inativação transcricional dos genes CRABP2, KTN1 e ADAM23, estudados pela primeira vez em carcinoma renal, e GPC3, podem fornecer informações relevantes para o conhecimento e desenvolvimento do carcinoma renal de células claras. / Abstract: The renal tumor, which is the most lethal of urological diseases, is a histologically heterogeneous disease, and the clear cell renal cell carcinoma the most common histological subtype. Although the treatment of nephrectomy and immunotherapy are established, approximately 30% of patients are affected by metastases. Changes in gene expression and transcriptional inactivation, due to the methylation mechanism are evident in cancer cells. The DNA methylation is an epigenetic event closely related to the silencing of gene expression, and is involved in several cases, including the carcinogenesis. The aim of this study was to investigate the gene expression of GPC3, CRABP2, KTN1 and ADAM23, check if gene expression was associated with tumor progression and analyze methylation pattern of CpG island. The four selected genes were validated by the quantitative RT-PCR. Thirty five samples of clear cell renal cell carcinoma and 35 samples of normal renal cortex were used for validation. The genes GPC3, CRABP2, KTN1 and ADAM23 showed significant reduction of expression in samples of clear cell renal cell carcinoma when compared to a pool of samples of normal renal cortex. It was observed that the lower expression of ADAM23 is directly related to the advancement of the tumor staging. Despite the high frequency of hypermethylation of ADAM23, there was no association with the methylation pattern of the clinical data. The analysis of gene expression and the mechanisms responsible for the transcriptional inactivation of genes CRABP2, KTN1 and ADAM23, first studied in clear cell renal cell carcinoma, and GPC3, may provide relevant information for the clear cell renal cell carcinoma understanding and development. / Orientador: Paula Rahal / Coorientador: Paulo Peitl Júnior / Banca: Ana Elizabete Silva / Banca: Andréia Machado Leopoldino / Mestre

INTODUÇÃO: O carcinoma renal (CR) é uma doença agressiva, e sua incidência vem aumentando. A variante de células claras (CRCC) é a mais comum e apresenta comportamento biológico mais agressivo. Os recentes avanços no conhecimento da biologia molecular do tumor demonstram que a oncogênese dos diversos tipos histológicos é regida por mecanismos celulares diversos. Os modelos prognósticos atuais vêm procurando incorporar os recentes avanços da biologia molecular, com o intuito de melhorar sua capacidade de predizer a evolução e o desfecho destes pacientes. OBJETIVOS: Correlacionar a imunoexpressão dos marcadores selecionados com: 1) sobrevida global e, 2) com parâmetros prognósticos estabelecidos (estadio clínico TNM, tamanho tumoral, grau nuclear de Fuhrman, invasão microvascular e invasão de gordura perirrenal) em portadores de CRCC não metastático. MÉTODOS: Neste estudo de coorte retrospectivo, avaliamos 99 pacientes portadores de CRCC não metastático, quanto à expressão imunoistoquímica das seguintes proteínas: CA-IX, EGF-R, Ki-67, p53, PTEN, VEGF e VEGF-R. Os parâmetros analisados foram: Sobrevida global, estadio TNM, tamanho tumoral, grau nuclear de Fuhrman, invasão microvascular e invasão de gordura perirrenal. Utilizamos um tissue microarray construído exclusivamente para esta finalidade e realizamos a leitura da imunoexpressão por técnica digital utilizando o software Photoshop®. RESULTADOS: O tempo de seguimento médio foi de 7,9 anos. Com relação à sobrevida global, não observamos sua correlação com nenhum dos marcadores avaliados. Quanto à correlação da expressão dos marcadores com os parâmetros prognósticos convencionais, observamos que a expressão do EGF-R se correlacionou com estadio T (p= 0,049) e invasão da gordura perirrenal (p=0,020); e o VEGF-R se correlacionou com grau de Fuhrman (p=0,022) e invasão microvascular (p=0,005). Nos demais marcadores, não foi observada correlação significativa. CONCLUSÃO: Os fatores prognósticos moleculares EGF-R e VEGF-R apresentam-se como ferramentas úteis para avaliação do risco de prognóstico desfavorável em portadores de carcinoma renal de células claras não metastático / INTODUCTION: Renal cell carcinoma is an aggressive disease and its incidence is rising. The clear cell variant is the most common, and also the most aggressive. Recent advances in the understanding of the tumors molecular biology indicate that the oncogenesis of each histologic subtype is controlled by distinct cellular mechanisms. Current prognostic models are gradually incorporating the advances in molecular biology, in the hope to improve their predictive capacity. OBJECTIVES: To correlate the immunoexpression of selected markers with 1) overall survival, and 2) with established prognostic parameters (clinical TNM stage, tumor size, Fuhrman nuclear grade, microvascular invasion and perirenal fat invasion) in patients with non-metastatic ccRCC. METHODS: This is a retrospective cohort study, we evaluated 99 patients with non-metastatic clear cell renal cell carcinoma, as to the expression of the following proteins: CA-IX, EGF-R, Ki-67, p53, PTEN, VEGF e VEGF-R. The analyzed parameters where: overall survival, TNM stage, tumor size, Fuhrman nuclear grade, microvascular invasion, perirenal fat invasion. We utilized a custom built tissue microarray, and the immunoexpression was digitally quantified using the Photoshop® software. RESULTS: The mean follow-up time was 7,9 years. We found no correlation between the expression of the studied molecular markers and overall survival. As for the conventional prognostic parameters, we found the expression of EGF-R to correlate with T stage (p= 0,049) and perirenal fat invasion (p= 0,020), and VEGF-R to correlate with Fuhrman nuclear grade (p= 0,022) and microvascular invasion (p= 0,022). None of the other markers showed correlation with the studied parameters. CONCLUSIONS: The expression of EGF-R and VEGF-R may be useful tools in the prognostic evaluation of unfavorable risk in patients with non metastatic clear cell renal cell carcinoma

The development of checkpoint immunotherapy has been a paradigm shift in the treatment of cancer, leading to dramatic improvement in treatment outcomes across a broad range of tumor types. Nevertheless, our current understanding of the tumor immune microenvironment and mediators of resistance to therapy are limited. The recent development of high-throughput single-cell RNA-Sequencing (scRNA-Seq) technology has opened up an unprecedented window into the transcriptional states of distinct tumor-infiltrating immune and stromal cells. However, even this technology has its biological limitations, with very high levels of data dropout induced by low total mRNA molecules and capture efficiency. This thesis explores the application of a transcriptional regulatory protein activity inference approach to single-cell data in order to resolve gene dropout and more deeply characterize upstream drivers of cell state within the micro-environment of several distinct tumor types. To this end, algorithms for inference of protein activity, drug sensitivity, and cell-cell interaction have been adapted to scRNA-Seq data, along with an approach for querying enrichment of single-cell-derived population marker gene sets patient-by-patient in larger bulk-RNA-Seq cohorts. By applying these tools systematically, we have identified distinct cellular sub-populations associated with clinical outcome in different tumor types, including a novel population of C1Q+/TREM2+/APOE+ macrophages associated with post-surgical tumor recurrence in clear cell renal carcinoma, a sub-population of fibroblasts associated with improved response to immunotherapy in head and neck squamous cell carcinoma, tumor cell subpopulations with distinct inferred drug sensitivities in cholangiocarcinoma and prostate cancer, as well as tumor-specific regulatory T-cells (Tregs), active as a mechanism of immunotherapy resistance across a range of tumor types. In ongoing clinical trials from both primary and metastatic prostate cancer as well as clear cell renal carcinoma, we are able to assess which of these populations are enriched in non-responders to checkpoint immunotherapy. The proteomic master regulators of each of these single-cell types have direct utility as potential biomarkers for treatment response, but they may also be therapeutically modulated as novel targets for combination immunotherapy, potentially improving treatment response rates and treatment outcomes in future clinical trials. Finally, this thesis also presents a discovery-to-validation platform to accelerate micro-environment-directed drug repurposing in the context of immunotherapy resistance and rapid CRISPRko validation of novel therapeutic targets. This platform has been developed specifically to validate newly identified master regulators of tumor-specific immunosuppressive regulatory T-cells (Tregs), resulting in discovery of low-dose gemcitabine as a tumor-specific Treg-modulating drug synergistic with anti-PD1 checkpoint immunotherapy and TRPS1 as a proteomic master regulator with clinically significant effect on tumor Treg-infiltrating and tumor growth rate. However, the platform itself may be readily extended in future work to prioritize agents against immunosuppressive macrophage and fibroblast populations for clinical development and trials. As we have discovered, different cancers have different populations of cells driving therapy response and resistance. Taken together, the analytical and validation tools presented in this thesis represent an opportunity to tailor future immuno-therapies at the single-cell level to particular tumor types and to individual patients.

Locally recurrent renal cell carcinoma and multiple pulmonary metastases were successfully resected in a patient 20 years after nephrectomy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Background: Pancreatic métastases from renal cell carcinoma (RCC) are clinically rare but highly resectable. The aim of this article is to identify patients who profit from pancreatic resection of RCC despite the invasiveness of the surgery. Methods: Between January 1996 and December 2007, data from 744 patients were collected in a prospective pancreatic surgery database, and patients with metastasis into the pancreas from RCC were identified. Results: Resective surgery was performed in 14 patients with metastasis to the pancreas from RCC. Most patients were clinically asymptomatic. The median interval between primary treatment of RCC and occurrence of pancreatic metastasis was 94 months (range 32–158). The morbidity rate was 42.8%. Patients with a metastasis size <2.5 cm had a much better survival after resection (100 months) than those with a metastasis size >2.5 cm (44 months). Moreover, the number of métastases predicts the survival after resection. Conclusions: In patients with pancreatic métastases from RCC who have only limited disease, complete resection of all lesions can be successfully performed with a low rate of complications. Thus, patients with a history of RCC should be monitored for more than 10 years after nephrectomy to detect recurrence. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.