Estimating Time-Varying Effects of Prognostic Factors for Stomach Cancer Patients within a Dynamic Grouped Cox Model

We describe the identification of prognostic factors in the framework of a completely resected stomach cancer survival-study. For the analysis the dynamic grouped Cox-Model was used allowing for time-varying covariate effects. Therefore the hazard rate might be non-proportional. As estimation concept we applied the posterior mode, computed by iteratively weighted Kalman filtering and smoothing steps. The medical study and questions are described, the statistical method is illustrated, the results are given and interpreted and the method is discussed

Design and Synthesis of Peptide-Based Nanofibers for Imaging and Therapy of Cancer

Nanotechnology has been the subject of significant scientific and biomedical development efforts over the past decades. Improvement in biomarker discovery, targeting approaches and conjugation chemistries has led to the development of many novel nanomaterials for individualized therapy. In this thesis, we investigate a new class of nanomaterial called “nanofiber precursor” (NFP). The NFP is composed of multiple self-assembling peptides via electrostatic and non-covalent interactions. Each peptide consisted of β-sheet sequence attached to a methoxypolyethylene glycol (mPEG) via a linker. By conjugating either near infrared fluorophore or therapeutic antibodies, we demonstrate the application of NFP in diagnosis and therapy of cancer respectively. The main objectives of this thesis are: (1) To design and synthesize a near infrared nanofiber for imaging urokinase plasminogen activator (uPA) activity (2) To develop a Herceptin-conjugated nanofiber as multivalent targeted system for increasing therapeutic efficacy of Herceptin, a monoclonal antibody used for breast cancer treatment. We were successful in conjugating near infrared dye NIR664 to the nanofiber as well as Herceptin on the surface of nanofiber. (1) The NIR-NFP conjugate could detect recombinant uPA activity with sensitivity of 3 ng. (2) The Herceptin-conjugated nanofiber (HER-NFP) was more than two fold effective in inhibiting growth of HER-2 positive cells. In the second half of the thesis, we have also investigated tumorigenic role of 15-LOX-1, a lipid peroxidizing enzyme in prostate cancer. The aim of this study was: (3) To investigate the role of 15-lipoxygenase-1 (15-LOX-1) in upregulation of uPA in PC-3 prostate cancer cells. As a whole, the research presented in this thesis is aimed at designing new strategies and understanding molecular mechanisms that lead to prevention and treatment of cancer

The detection of occult metastatic disease in patients with cutaneous melanoma

The ability to identify melanoma patients with progressive disease is central to efficient management. The challenge therefore, is to develop prognostic markers and techniques which will allow the identification of those patients whom, at the time of primary tumor diagnosis, already have micrometastases (occult or clinically undetectable metastases). The use of the reverse transcription-PCR (RT-PCR) technique for the detection of circulating melanoma cells (CMCs) is potentially a powerful tool for identifying those patients at risk for developing metastases. The first aim of this study was to develop a more sensitive, reproducible, cost effective and clinically applicable assay and to eliminate the problem of false positives. A combined RT-PCR assay for tyrosinase mRNA, a marker specific for melanoma cells, was developed and tested. It was shown that the assay can reproducibly detect a single, viable melanoma cell in 10-15 ml of peripheral blood. Furthermore, a simple but effective procedure was developed to prevent carryover contamination. It was found that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling was only 2% and that illegitimate transcription does not contribute to sporadic false positives. The second aim of this study was to determine whether the early detection of CMCs is of any clinical value to monitor melanoma progression. Peripheral blood samples from 143 patients with primary melanoma (PM) were analysed by RT-PCR for the presence of tyrosinase mRNA. Seven percent (10/143) of the patients with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) compared to stage I (5.3%) but the difference was not significant. A significantly higher percentage (P < 0.05) PCR-positive patients were found to have tumors greater than 1.5 mm thick and with ulceration present. Although this finding supports the notion that tumor thickness and ulceration are the two most significant prognostic factors, it was not possible at this stage, to link this directly to a poor prognosis since the majority of the PCR-positive patients have not yet (within four years) developed metastatic disease. However, the data does indicate that cells from tumors greater than 1.5 mm thick and with ulceration have a greater propensity to enter the circulation but that these cells do not necessarily have the ability to establish metastases. The results suggest that the detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16/67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, one week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P = 0.3485). The other nine PCR-positive patients had not yet developed metastases after a median follow-up period of four years. It is concluded that the current technique for the detection of CMCs is of limited clinical value to predict the likelihood of metastasis in patients with PM. It is suggested that other anatomic compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases. The third aim of this study was to determine whether high or low plasma levels and/or activity of plasminogen activator inhibitor type 1 (PAI1) correlate with the presence of metastatic disease in patients with melanoma. PAI1 is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key enzyme in the metastasis and vascularization of solid tumors. A unique enzyme-linked immunosorbant assay was developed to measure both the total amount of PAI1 in plasma as well as the active fraction of the inhibitor. This novel assay was then used to analyse and compare the plasmatic PAI1 levels and activity of a group of patients with advanced melanoma (AM) with a group of patients with primary disease and a control population. There was no statistical difference in the total plasmatic PAI1 levels between the controls and patients with PM and AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. This means that a melanoma patient with a plasmatic PAI1 activity value less than 44% will have a 50% chance of harbouring metastases. Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity levels might be predictive of metastatic disease. The false positive rate was 2.6%. It is speculated that this reduction in the active fraction of PAI1 for patients with AM might be attributed to tumor-derived tissue plasminogen activator and/or other melanoma-derived proteases or factors. The last section of this study describes several monoclonal antibodies (Mabs) that were developed against PAI1 in order to obtain useful reagents to study the regulatory functions of PAI1 in the metastasis and vascularization of solid tumors. The baculovirus expression system was used to express human PAI1 in insect cells and the crude infected cell population was used as the immunogen in mice. This approach was followed since the Escherichia coli-derived recombinant molecule elicited a poor immune response. A unique panel of anti-PAI1 Mabs was developed that were characterized with regard to their use for immunoblotting, immunofluorescence and immunocytochemistry. One of these antibodies blocked the binding of PAI1 to vitronectin and inhibited the activity of the inhibitor. Finally, two of these Mabs turned out to be extremely valuable and were used to develop a novel microtiter plate assay for measuring the active fraction of PAI1 in biological fluids by making use of Mabs against different epitopes of PAI1

The role of the urokinase family in invasion by breast cancer

Breast cancer is the leading cause of cancer mortality in women in the Western world. Its growth and development is directly governed by endogenous steroids and growth factors. Endocrine therapy is the most effective form of treatment for a proportion of breast cancer patients. Work from several laboratories has shown that breast cancer cells metastasise via a pathway that involves the plasminogen activator system and metalloproteinases. Thus, the plasminogen activator system, which consists of urokinase (uPA), its inhibitor (PAI-1) and receptor (uPAR), is central to the process of invasion and metastasis. Given their important role, it is hardly surprising that these three proteins have been used as biological markers of patient prognosis. uPA and PAI-1 has been significantly implicated as markers of aggressive breast cancer. The first step of invasion involves the conversion of plasminogen into plasmin by uPA. uPA is localised on the cell surface (bound to its receptor uPAR) and is principally regulated by two inhibitors, plasminogen activators type-1 and type-2 (PAI-1 and PAI-2). Possessing a wide substrate specificity, plasmin in turn activates a variety of enzymes which degrade different components of the extracellular matrix such as laminin, fibronectin, collagenase(s), proteoglycans etc. The present study was conducted to further understand the effect(s) of different growth factors and steroid hormones on the proliferation and invasive potential of two well characterised human breast cancer cell lines, MCF-7 (hormone sensitive) and MDA-MB-231 (hormone insensitive). Furthermore, extracellular matrix (ECM) was used to determine the effect, if any that support substrate might have on the levels of plasminogen activators and growth rate. Plasminogen activators were quantified both at the mRNA and protein levels, using RT-PCR and ELISA, respectively. uPA activity was determined by chromogenic assay and the invasive properties of the two cell lines were determined using the Boyden invasion chamber. In the initial phase, proliferation of each cell line on both substrata, plastic and EHS, was studied after treatment with growth factors (TGFalpha, TGFbeta and EGF), steroid hormones (E2 and Pg) and anti-oestrogen, tamoxifen. Different amounts of each growth factor and hormone were used and cell numbers were obtained after 24, 48 and 72 hours. These studies showed that growth of MCF-7 cells was stimulated significantly by exogenous TGFalpha and EGF, on both EHS and plastic with cells being more sensitive to growth factors on EHS. Proliferation of MDA cells was only slightly increased by the same growth factors on either substrate. By contrast, TGFbeta inhibited the growth of MDA cells, on both surfaces. However, the concentrations required for inhibition on the two substrata were enormously different. E2, Pg, and TAM (an anti-oestrogen) also stimulated the growth of MCF-7 cells on both plastic and EHS. For quantification of uPA, uPAR and PAI-1, a combination of Western blotting and ELISA, using specific monoclonal antibodies, was employed to monitor the relative amounts of these proteins after treating the cell lines with growth factors and hormones. These studies showed that the MDA cell line expressed uPA and PAI-1 proteins whereas uPA was hardly delectable in the MCF-7 cell line. Moreover, qualitative RT-PCR analysis clearly showed that uPAR and PAI-1 transcripts were present in both MDA and MCF-7 cells. These results clearly show that various growth factors and/or hormones not only have an effect on the proliferative properties of cell lines but they also change the relative amounts of uPA, PAI-1 and uPAR and that these changes can be reflected in invasive activity. The ECM contributes to the overall response of these cells. (Abstract shortened by ProQuest.)

Novel Biomarkers of Gastrointestinal Cancer

Gastrointestinal (GI) cancer is a major cause of morbidity and mortality in the world. Since early diagnosis and optimal treatment selection are crucial to improving the prognosis of these diseases, the discovery of useful biomarkers has the potential to greatly reduce their burden. Recent technical and mechanical developments have allowed for the detection of tiny differences in various factors modified in physical conditions, which could contribute to the discovery of novel biomarkers for some diseases.In this Special Issue, we aim to focus on novel biomarkers for GI cancers, including esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer and biliary cancer. In addition, any samples (tissue, blood, urine and feces) are useful as biomarker sources, although body-fluid-based biomarkers are promising as diagnostic biomarkers due to their noninvasiveness. This Special Issue aims to collect novel insights clarifying the current situation and future perspective in this field

The Shaping of Cancer by the Tumour Microenvironment and Its Relevance for Cancer Therapy

In this book, we present a compilation of original research articles as well as review articles that are focused on improving our understanding of the molecular and cellular mechanisms by which cancer cells adapt to their microenvironment. These include the interplay between cancer cells and the surrounding microenvironmental cells (e.g., macrophages, tumor-infiltrating lymphocytes and myeloid cells) and microenvironmental environments (e.g., oxidative stress, pH, hypoxia) and the implications of this dynamic interaction to tumor radioresistance, chemoresistance, invasion and metastasis. Finally, the importance and relevance of these findings are translated to cancer therapy