Transduction of melanoma cells with the aim to induce avidine expression on tumor cell surface was studied. Subsequently the method enabling quantification of binding of ligands to the cell surface was developed.

The aim of this thesis was to study the mechanisms of innate imunity involved in the degradation of tumor cells on which the ligands of phagocytic receptors were installed. For this purpose both in vivo experiments using immunodeficient mice, and in vitro experiments based on monitoring the levels of inflammatory cytokines produced in the tumor tissue and on measuring the level of myeloperoxidase released during neutrophil degranulation were performed.

Innledning Malignt melanom er en alvorlig kreftsykdom som oppstår i hudenspigmentceller(melanocytter). Ved malignt melanom har kreften spredd seg eller erinoperabel. Sykdommen er svært alvorlig og har til nå hatt en veldig dårlig prognose medtanke på overlevelsen. Formål Å undersøke om PD-1 hemmere er kostnadseffektive legemidler ved behandling av maligntmelanom og om det finnes forskjeller i bedømmelser av klinisk effektivitet ogkostnadseffektivitet mellom Sverige og Norge. Metode Dette er en litteraturstudie basert på seks originalartikler som er hentet fra databasen PubMed etter søkning med disse søkeordene: pembrolizumab, nivolumab, cancer, programmedcell death 1 receptor/antagonist and inhibitors og effectiveness. Det er dessuten søkt påhjemmesider på forskjellige svenske og norske statlige myndigheter som Janusinfo,Andvords- och läkemedelsförmånsverket (TLV) og Statens legemiddelverk (SLV). Sistnevntesøket resulterte i to metodevurderinger fra hvert land som sammen med originalartiklene blebrukt som underlag for dette arbeidet. Resultat Både pembrolizumab og nivolumab er effektive legemidler ved malignt melanomsammenlignet med tradisjonell behandling med immunmodulerende legemidler ogkjemoterapi. Frekvensen av behandlingsrelaterte bivirkninger av grad 3-4 forekommer myesjeldnere med bruk av PD-1 hemmere og kan bidra til at pasientene får økt livskvalitet underbehandlingen og i sluttfasen av livet. Diskusjon Malignt melanom er en alvorlig kreftsykdom med svært dårlige leveutsikter. PD-1 hemmereer en av flere nye legemidler som er kommet på markedet de siste årene og har vist en bedreeffekt og bivirkningsprofil enn tradisjonell behandling av sykdommen. Til tross for begrensetevidensgrunnlag er legemidlene vurdert som kostnadseffektive og allerede tatt i bruk ibehandlingen i Sverige og Norge. Det finns dog fortsatt stor usikkerhet ikostnadseffektivitetsanalysene. Det finnes også forskjeller i bedømmelser av analysenemellom landene selv om myndighetene har kommet frem til omtrent samme konklusjon tilslutt. Konklusjon Malignt melanom har tradisjonelt vært en sykdom med veldig dårlig prognose og overlevelse,men med introduksjonen av nye behandlingsformer har situasjonen endret seg fundamentaltde siste årene. Mange nye legemidler med nye virkningsprinsipper har kommet på markedetog flere studier har vist at behandlingene gir bedre effekt og mindre bivirkninger ennkjemoterapi. Hos noen av legemidlene, som for eksempel BRAF hemmer, har man observertrelativt rask resistensutvikling og tilbakefall av sykdommen. Studier på effekten av PD-1hemmere gir begrunnet håp om forbedret overlevelse og lavere bivirkningsfrekvens, hvilketkan gi økt livskvalitet til pasientene i livets sluttfase. Til tross for høye kostnader, selv etterrabattavtaler, er PD-1 hemmere nå vurdert som kostnadseffektive og er tilgjengelig forbehandling av pasienter i Sverige og Norge. De helseøkonomiske analysene i Norge ogSverige er dog basert på veldig få studier og er derfor beheftet med stor usikkerhet iestimatene. Den største forskjellen mellom Sverige og Norge er at SLV i Norge har tatt høydefor at utvalgte pasienter kan tilbys behandling i opp til 3 år med PD-1 hemmere dersom de erprogresjonsfrie etter 2 års behandling. Dette førte til at de to myndigheter brukte andreberegningsmodeller. Dette resulterte i forskjeller i kostnadseffektivitetsberegninger i Sverigeog Norge.

Hodková, Gabriela - Metabolic Effects Of Hyperthermic Isolated Limb Perfusion (HILP) in Malignant Melanoma Patients First Faculty of Medicine, Charles University in Prague, Praha 2, Kateřinská 32 Head of the work: Doc. MUDr. Michal Semrád, CSc. Supervisor - consultant: MUDr. Miroslav Špaček, Ph. D. The aim of the study is to assess the metabolic consequences of mechanical isolation and hyperthermic cytostatic perfusion in a limb affected by malignant process. The theoretical part refers to a topic of malignant melanoma, its clinical evaluation and treatment. Methods based on conservative and surgical treatment are described. The isolated hyperthermic cytostatic limb perfusion is a consecutive local treatment indicated in cases of recurrent malignant lesions following surgical resection, when next surgery is impossible. In the practical part, the laboratory samples and clinical data were recorded in patients who had undergone hyperthermic cytostatic limb perfusion in the 2nd Surgical Department of The General Teaching Hospital and First Faculty of Medicine, Charles University Prague. The affected limb was flushed with a warm oxygenated blood containing cytostatic drugs using an extracorporeal circuit apparatus. Selected arterial blood gas, metabolic and hematologic parameters were studied intra and...

Up to date, only four cases of primary intradural extramedullary spinal cord melanoma (PIEM) have been reported. No previous reports have described a case of PIEM located in the lower thoracic spine with long-termfollow-up. Purpose. Demonstrating an unusual, extremely rare case of melanoma manifestation. Study Design. Case report. Methods. We report a case of a 57-year-old female suffering from increasing lower extremity pain, left-sided paresis, and paraesthesia due to spinal cord compression caused by PIEM in the lower thoracic spine. Results. Extensive investigation excluded other possible primary melanoma sites and metastases. For spinal cord decompression, the tumor at level T12 was resected, yet incompletely. Adjuvant radiotherapy was administered two weeks after surgery. The patient was recurrence-free at 104 weeks after radiotherapy but presents with unchanged neurological symptoms. Conclusion. Primary intradural extramedullary melanoma (PIEM) is extremely rare and its clinical course is unpredictable.

Im Rahmen der Dissertation wurde ein Verfahren zur Quantifizierung monosomer Zellpopulationen innerhalb eines disomen Normalgewebes auf Basis der Pyrosequenzierung von Einzelbasenmutationen etabliert und hinsichtlich seiner Genauigkeit untersucht. Dabei liegt ein besonderer Schwerpunkt auf der Entwicklung eines Verfahrens zur Festlegung von Grenzwerten für die Detektion monosomer Population sowie für genetisch heterogene Subpopulationen. Zur Bestimmung der Genauigkeit wurden Mischreihen von DNA zweier Genotypen angefertigt und das Allelverhältnis durch Pyrosequenzierung gemessen. Diese Ergebnisse wurden genutzt, um Grenzwerte für die Detektion von LOH3-positiven Zellen im UMM estzulegen. In diesen Vorversuchen konnte die Anwendbarkeit der Analysemethode für Proben aus UMM sowohl aus Enukleations wie auch aus Feinnadelaspirationspräparaten demonstriert werden. Es wurde dann in einem weiteren Schritt analysiert, wie viele differente Loci für eine korrekte Diskriminierung zweier Genotypen analysiert werden müssen. Hier wurde gezeigt, dass zum einen die Anzahl der untersuchten SNP aber auch das gemessene Allelverhältnis maßgeblichen Einfluss auf die Genauigkeit der Analyse haben. Basierend auf diesen Daten wurde ein Verfahren entwickelt, das aus der gewünschten Genauigkeit eine Berechnung des Umfangs eines zu etablierenden SNP-Panels ermöglichte.

Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...

Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...

52 7.2 Souhrn v anglickém jazyce Malignant melanoma is the most lethal skin cancer whose incidence has been worldwide increasing. The prognosis of the disease is dependent on many factors - the effected locality, age, form of the tumor and the growth stage at which it was surgically removed. There has been no reliable adjuvant treatment available so far; although cytokins have been recently administered to high-risk patients. The accent is therefore put mainly on the timely detection and the relevant extent of surgical intervention whose degree depends on the stage of the disease. The result of the above mentioned facts is the necessity of an early diagnosis of new cases and permanent follow-up of the patients in whom melanoma has been already confirmed. The patients in the follow-up care are regularly checked, for the rest of their lives, at by the so called melanoma commissions, usually at dermatological clinics. The permanent contact with fact of the possible presence of the disease (information about the diagnosis, repeated follow-ups, waiting for the results of the examination) is stressful for these patients, although they become, to a certain extent, gradually adapted to stress. A good adaptation is however dependent also on the extent of social support, depression, the severity of the disease and...

Background: Metastatic melanoma remains a life-threatening disease because most tumours develop resistance in response to conventional treatment. Even though targeted drugs represent promising therapeutics, the clinical outcome remains poor, with high relapse rates coinciding with pronounced metastatic outgrowth. Therefore, successful therapy is still challenging, and alternative treatment options are demanded as first or second line therapy to overcome acquired resistance. In this context, cell death induction by the tumour-selective death ligand TRAIL (Tumour necrosis factor-Related Apoptosis-Inducing Ligand) might serve as an alternative single or co-treatment option. Unfortunately, melanoma cells were shown to stay largely resistant against conventional TRAIL treatment and the first-generation TRAIL-based therapeutics failed in clinical trials due to the limited therapeutic activity in patients. To overcome this therapeutic limitation, a second-generation hexavalent TRAIL receptor agonist IZI1551 has been developed showing increased bioactivity thereby enhancing the cytotoxic effects on cancer cells. Questions and Hypothesis. We hypothesized that cells that do not receive the full but only a sublethal drug-dose may not only be responsible for drug resistance but may also confer tumour relapse and metastatic outgrowth. By switching the signal transduction from pro-apoptotic to anti-apoptotic and pro-survival, these cells may foster an aggressive phenotype with enhanced proliferation and migration. Therefore, an in depth understanding of the underlying mechanisms of emerging drug resistance that may additionally trigger secondary metastasis formation is required to identify new therapeutic targets and alternative treatment options. Methodology. Melanoma cell lines were conditioned to the TRAIL receptor agonist IZI1551 and the expression of members of the anti-apoptotic NFκB and MAPK pathways as well as of the TRAIL receptor-driven apoptotic pathway were investigated by semi-quantitative Western-blot analysis. Protein expression/activation data of parental IZI1551-sensitive versus conditioned IZI1551-resistant melanoma cells were implemented into a network topology derived from literature. A Dynamic Bayesian Network (DBN) model was combined with a sophisticated regularisation strategy resulting in sparse and context-sensitive networks to identify cell line-specific deregulations within the signalling network. Predictions of the model were confirmed by siRNA-mediated knock down. Enhanced proliferation and migration of resistant-cells were investigated by proliferation, clonogenic, and scratch assays. Following the 2D studies, migration and invasion were monitored by confocal microscopy in 3D migration/invasion assays and 3D spheroids models. Expression of pro-metastatic cell adhesion molecules was evaluated by flow cytometry. In order to identify potential regulators of the aggressive phenotype, quantitative transcriptome analysis (RNA-seq) was performed. The therapeutic outcome of the new identified treatment options with IZI1551 alone or in combination with Smac mimetics or bortezomib was evaluated by cell death detection ELISA. Results. In this thesis, IZI1551 was shown to induce pronounced apoptotic cell death in melanoma cells compared to mutation specific targeted kinase inhibitors, as being used in the clinic. Comparing IZI1551-sensitive to IZI1551-resistant melanoma cells, the DBN model accurately predicted activation of NFκB in concert with upregulation of the anti-apoptotic protein XIAP to be the key mediator of IZI1551 resistance. Moreover, XIAP was identified to serve as a potential biomarker for TRAIL responsiveness. According to these findings, human melanoma cell lines were re-sensitised to TRAIL in vitro by co-application of the IAP antagonists Smac mimetics as well as bortezomib, a proteasome inhibitor currently used in cancer treatment. In addition, by triggering survival instead of apoptotic signalling pathways, resistant cells caused an aggressive phenotype with enhanced proliferation and migration/invasion into 3D collagen matrices, coinciding with upregulation of the cell adhesion molecules MelCAM and αVβ3 integrin, which are known to promote tumour progression and metastasis. Combining in silico studies of RNA-seq and protein expression data, YAP, an intracellular transducer of mechanical stimuli, and its upstream regulator FAK, a component of the focal adhesion complex, were identified as key promoters of proliferation and migration. Conclusions. Identification of new biomolecular markers or targets combining experimental and computational approaches is a promising avenue to assess the effects of drug combinations and to identify responders to selected combination therapies. In this thesis, IZI1551 was identified as an alternative treatment option for metastatic melanoma. Our data also suggest that XIAP expression may serve as a potential predictive marker for the sensitivity of tumour cells to TRAIL-induced apoptosis. Above this, we demonstrated that three alternative treatment options with IZI1551 in combination with Smac mimetics, bortezomib or FAK inhibitors may represent a promising approach for the treatment of TRAIL-resistant melanomas and to prevent undesired metastatic outgrowth.