In the first part of the theses, I studied therapeutical effect of intratumoral application of various types of bacteria on melanoma B16-F10 bearing mice alone or in combination with another immunostimulatory compounds. Tumour size, metastasis and survival were monitored. The second part was focused on study of cytotoxic effect of neutrophils on melanoma cells and the role of neutrophil extracellular traps formation.

The aim of this thesis was to investigate the possible role of neutrophil granulocytes in antitumor reactions. Most of our experiments were focused on in vitro studies assessing the cytotoxic effect of mouse neutrophils on B16-F10 melanoma cells labelled with PAMPs. We put an emphasis on activation and generating a prime state of neutrophils. Moreover, a release rate of enzyme myeloperoxidase from azurophil granules was detected as a marker of neutrophil degranulation. We also attempted to attract neutrophils into tumor microenvironment using thioglycolate medium and its main compound casein.

The aim of thesis was to study murine melanoma B16- F10 therapy based on the use of TLR agonists combinedwith activation of phagocytosis. Mechanisms of this therapy were studied on the bases of analysis of tumor infiltrating immune cells and evaluationof thein effect on tumor cells.

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...