De-restricted 15/11/2013

Nos últimos anos, o desenvolvimento da computação tem auxiliado profissionais da saúde no tratamento, prevenção e diagnóstico de doenças. Um dos grandes desafios do campo tem sido o desenvolvimento de métodos para a discriminação do câncer de pele do tipo melanoma maligno em relação à outras lesões benignas. Para tal, pesquisadores usam técnicas de processamento e análise de imagens, explorando métricas baseadas na regra ABCD, para o desenvolvimento de métodos de diagnóstico de melanoma através de imagens. Enquanto diversos estudos abordam a coloração e textura do melanoma, um tratamento sistemático da irregularidade dos melanomas ainda não foi relatado. O presente trabalho traz um estudo dos fatores que influenciam a discriminação de lesões melanocíticas malignas e benignas, tomando como referência a assimetria das lesões e as irregularidades contidas em sua borda. Foram coletadas 143 imagens de casos clínicos de melanoma maligno, nevos regulares e nevos atípicos. Após tratamentos iniciais das imagens e posterior segmentação das lesões, extraiu-se 52 métricas referentes ao propósito do trabalho. A visualização da projeção LDA das três categorias revelou boa discriminação entre as categorias melanoma com relação as demais, reforçando a premissa original da acentuada irregularidade dos melanomas. Este resultado foi confirmado pela validação cruzada da projeção, com acertos da ordem de 75% para o grupo melanoma e 54% e 40% para os respectivos grupos nevo regular e nevo atípico. Deste resultado prevê-se uma das aplicações do sistema, na discriminação geral entre melanoma dos demais nevos. Para tal, uniu-se os grupos benignos em uma única categoria para a validação cruzada, gerando cálculos de sensibilidade e especificidade da ordem de 90% e 73% respectivamente. Outro importante resultado foi a comparação desses valores com as informações sobre o diâmetro das lesões. Conclui-se que ambas medidas (borda e tamanho) são igualmente relevantes no diagnóstico do melanoma, reflexo da própria patologia do melanoma, na qual acentuam-se ambas características em relação as demais lesões. Porém, a junção de ambas informações num único processamento não melhora a qualidade do diagnóstico, que nos permite prever que possa ser mais vantajoso proceder duas validações distintas com pesos iguais para o diagnóstico final. Por fim, um teste realizado com clínicos gerais e especialistas em melanoma revelou uma importante contribuição do método no auxílio de triagens ambulatoriais de casos suspeitos, principalmente para médicos com baixa ou nenhuma experiência em diagnóstico de melanoma. / Recent development in computer science have helped health professionals in the heath area in the treatment, prevention and diagnostic of illnesses. One of the leading challenges in this field has been the development of methods for the skin cancer discrimination between the types of malignant melanoma in relation to other benign lesions. For this, researchers have been using techniques of image processing and analysis, exploring metrics based on the ABCD rule, to the development of methods of diagnostic of melanoma through images. While plenty of studies are about the color and texture of the melanoma, a systematic treatment of the irregularity of the melanoma has not been reported yet. The present work presents a study of the factors which influence the discrimination among the malignant melanocytic and benign lesions, having as a reference the asymmetry of the lesions and the irregularities in their edges. It was collected 143 images of clinic cases of malignant melanoma, regular nevus and atypical nevus. After initial treatment of the images, followed by a segmentation of the lesions, it was extracted 52 metrics referent to the purpose of this study. The visualization of the projection LDA in three categories revealed a good discrimination among the categories of melanoma in relation to the others, reinforcing the original premise of the melanomas´ sharp irregularity. This result was confirmed by the cross-validation of the projection, with successes of 75 % to the melanoma group and 54 %-40 % to the regular nevus group and atypical nevus group, respectively. This result predicts one of the applications of the system in the general discrimination between the melanoma and the other nevus. In this regard, it was joined together the benign groups in one category to the cross-validation, generating calculations of sensibility and specificity of 90 % and 73 % respectively. Another important result was the comparison among these metrics with the information about the diameter of the lesions. It was concluded that both measurements (edges and size) are equally relevant in the diagnostic of the melanoma, reflection of the melanoma´s own pathology, in which both characteristics are enhanced in relation to the other lesions. However, the combination of both measurement in only one processing does not improve the quality of the diagnostic, which allows the prediction that it can be more advantageous to produce two distinct validations with the same weighs to the final diagnostic. To sum up, an accomplished test with doctors who are general practioner and specialists in melanoma revealed a significant contribution in the method to help in the triage of medical consultations in suspicious cases, mainly to doctors with little or no experience in the diagnostic of melanoma.

Varje år drabbas 4000 personer av malignt melanom i Sverige. Prognosen är god vid tidig upptäckt. Ändå dör ca 20 % av de som drabbas. Ett förbättringsarbete initierades för att utveckla vårdprocesser för dessa patienter. Denna studie har syftat till att utveckla kunskaper om situationsbaserade erfarenheter av vad processen i förbättringsarbetet har inneburit i relation till sjuksköterskors, läkares och medicinska sekreterares egna insatser för projektmålen och en personcentrerad vård. För studien av förbättringsarbetet har Critical Incident Technique (CIT) används som metod. 84 situationer samlades in från projektdeltagare genom korta återkommande intervjuer under projekttiden. Resultatet visar att deltagarna befinner sig i situationer av ambassadörskap, självrannsakan och fristad. Situationer av ambassadörskap är viktiga för projektets riktning framåt men dessa situationer är inte oberoende av situationer av självrannsakan och situationer av ”fristad”. Det är snarare relationen dem emellan som har en viktig dynamisk kraft. Slutsatsen i studien av förbättringsarbetet är att när deltagare i ett förbättringsarbete får möjlighet att ändra på logistik och struktur som bygger på ”stuprörstänk”, professionsorientering och funktion så får det positiva konsekvenser för ett personcentrerat arbetssätt, i arbete med patienter och i arbete med projekt. / Every year 4,000 persons are diagnosed with malignant melanoma in Sweden. The prognosis is good if it is detected early. Nevertheless, about 20% of those who are diagnosed die of the disease. An improvement project was initiated for developing care processes for these patients at a local clinic in central Sweden. This study aimed to describe the knowledge derived from participants in the project regarding their own situated efforts to achieve a person-centered care for these patients. Critical Incident Technique was used as a method. 84 incidents were gathered from the participants through recurrent interviews during the project process. The results show that participants find themselves in situations of being ambassadors for the project where everything flows forward in the project, being in situations of self-examination and doubt about own achievements in the project and finally being in situations of sanctuary as a temporary moral refuge and stress release concerning the project. Situations of ambassadorship are important for the project's direction forward toward the project goal but these situations are not independent of situations of self-examination and situations of sanctuary. The relation between these situations has an important dynamic force that need to be further explored.

Οι φυλετικές ορμόνες επηρεάζουν τη βιολογική συμπεριφορά του μελανώματος. Μετά την ανακάλυψη των οιστρογονικών υποδοχέων (ER) και ιδίως του υποδοχέα βήτα, η άποψη πως τα μελανώματα εκφράζουν χαμηλές συγκεντρώσεις ER αναθεωρήθηκε. Ο σκοπός της παρούσας μελέτης ήταν να διερευνήσει το βαθμό έκφρασης των ER στο πρωτοπαθές μελάνωμα αλλά και στο λεμφαδένα φρουρό αυτού που παριστά ένα σημαντικό σταθμό στη μεταστατική διαδικασία και να εκτιμήσει πιθανή συσχέτιση με την πρόγνωση και τη συνολική επιβίωση. Πραγματοποιήθηκε ανάλυση δεδομένων 60 ασθενών, μέσης ηλικίας 54,4 ± 14,5 ετών, με διάγνωση μελανώματος κατά την περίοδο 2001-2012. Όλοι οι ασθενείς υποβλήθηκαν σε βιοψία λεμφαδένα φρουρού, μετά την αναγνώρισή του με ραδιοϊσοτοπική λεμφαγγειογραφία. Εάν η ιστοπαθολογική εξέταση αναδείκνυε διήθηση του λεμφαδένα από το νεόπλασμα, διενεργείτο συμπληρωματικός λεμφαδενικός καθαρισμός, σε δεύτερο χρόνο. Με ανοσοϊστοχημική μέθοδο εκτιμήθηκε η έκφραση των οιστρογονικών υποδοχέων (ERα και ERβ) σε όλες τις δυνατές περιπτώσεις. Η έκφραση του ERα ήταν εξαιρετικά ασθενής, αναδεικνύοντας τον ERβ σαν τον επικρατούντα οιστρογονικό υποδοχέα, τόσο στον πρωτοπαθή όγκο, όσο και στο λεμφαδένα φρουρό. H εντονότερη ανοσοέκφραση του ERβ καταδείχθηκε σε λεπτά, λιγότερο διηθητικά μελανώματα με αρνητικούς λεμφαδένες φρουρούς. Επιπρόσθετα, η έκφραση του ERβ στον πρωτοπαθή όγκο σχετιζόταν σαφώς με το κυτταρικό μικροπεριβάλλον του, πιθανά επηρεάζοντας τη λεμφογενή διασπορά. Τα επίπεδα έκφρασης του ERβ είναι ελαττωμένα σταθερά σε επιθετικά μελανώματα, μεγάλου πάχους με μεταστατική νόσο στο λεμφαδένα φρουρό. Η αξιολόγησή τους υποδεικνύει έναν πιθανό δείκτη του μεταστατικού δυναμικού των μελανωμάτων και κατ’ επέκταση της πρόγνωσης της νόσου. / Steroid hormones seem to affect the biological behavior of melanoma. Prior to the discovery of estrogen receptor beta (ERb), melanomas were considered to contain low estrogen receptor concentrations. Furthermore, no studies have examined the role of estrogen receptor expression in sentinel lymph nodes (SLNs) of melanomas. The purpose of this study was to investigate the immunoexpression of estrogen receptors in malignant melanomas and SLNs and to examine any possible association with prognosis and overall survival. A retrospective analysis of prospectively collected data was conducted during a 12-year period (2001-2012). Sixty patients with mean age of 54.4 ± 14.5 years old diagnosed with melanomas after excision biopsy of pre-existing melanocytic lesions, were included in the study. All patients underwent wide excision of the primary tumor and SLN identification. Therapeutic lymph node dissection was conducted in cases where the final pathological report was indicative of SLN tumor invasion. Determination of estrogen receptor alpha (ERa) and beta (ERb) status by immunohistochemistry on tumor and nodal paraffin blocks was performed in all feasible cases. ERb but not ERa was the predominant estrogen receptor found in all primary tumors and SLNs examined. The most intense ERb immunostaining was seen in negative SLNs associated with thinner, less invading melanomas. ERb expression in the primary tumor seems to correlate with the cellular microenvironment, possibly altering the process of SLN invasion. ERb expression is down-regulated in aggressive melanomas with sentinel nodal disease, suggesting its possible usefulness as a surrogate marker for metastatic potential and prognosis in malignant melanoma.

No description available.

Thesis on the topic "Search of New biomakers suitable for screening and early diagnostics of cancer" is dedicated to the issue of cancer, a cancer of the breast, prostate, ovarian and after the new findings out as melanom determination of tumor markers and their implications for clinical practice. Their aim is skouted up biomaker suitable for screening and early diagnostics of carcinoma( cancer ) of the breast, ovarian, prostate and malignant melanoma with utilization of various kinds of immunoassay. The theoretical part gives a brief and comprehensive overview of the epidemiology of selected tumors and tumor markers used and their qualities. In the methodological part are given principles applied iminoanalytics methods a list of the statistical methods and a detailed description of the groups of patients. Results section contains tables with results of biomarkers diveded by sort of avalueted tumors. In the discussion are then analyzed facts that follow from the results. It discussed the process and actualy status of searching convenients biomekers and their combinations that can distinguish a population of benign tumors of the population with malignant tumors and markers which are able to distinguish healthy population from the population with malignant tumors of the breast, ovary, prostate and melanoma. This work may serve as a source of knowledge and results to continue in search of such marker that could be used for screening and early diagnosis of cancer and so to improve the prevention of these illneses in the population.

Nos últimos anos, o desenvolvimento da computação tem auxiliado profissionais da saúde no tratamento, prevenção e diagnóstico de doenças. Um dos grandes desafios do campo tem sido o desenvolvimento de métodos para a discriminação do câncer de pele do tipo melanoma maligno em relação à outras lesões benignas. Para tal, pesquisadores usam técnicas de processamento e análise de imagens, explorando métricas baseadas na regra ABCD, para o desenvolvimento de métodos de diagnóstico de melanoma através de imagens. Enquanto diversos estudos abordam a coloração e textura do melanoma, um tratamento sistemático da irregularidade dos melanomas ainda não foi relatado. O presente trabalho traz um estudo dos fatores que influenciam a discriminação de lesões melanocíticas malignas e benignas, tomando como referência a assimetria das lesões e as irregularidades contidas em sua borda. Foram coletadas 143 imagens de casos clínicos de melanoma maligno, nevos regulares e nevos atípicos. Após tratamentos iniciais das imagens e posterior segmentação das lesões, extraiu-se 52 métricas referentes ao propósito do trabalho. A visualização da projeção LDA das três categorias revelou boa discriminação entre as categorias melanoma com relação as demais, reforçando a premissa original da acentuada irregularidade dos melanomas. Este resultado foi confirmado pela validação cruzada da projeção, com acertos da ordem de 75% para o grupo melanoma e 54% e 40% para os respectivos grupos nevo regular e nevo atípico. Deste resultado prevê-se uma das aplicações do sistema, na discriminação geral entre melanoma dos demais nevos. Para tal, uniu-se os grupos benignos em uma única categoria para a validação cruzada, gerando cálculos de sensibilidade e especificidade da ordem de 90% e 73% respectivamente. Outro importante resultado foi a comparação desses valores com as informações sobre o diâmetro das lesões. Conclui-se que ambas medidas (borda e tamanho) são igualmente relevantes no diagnóstico do melanoma, reflexo da própria patologia do melanoma, na qual acentuam-se ambas características em relação as demais lesões. Porém, a junção de ambas informações num único processamento não melhora a qualidade do diagnóstico, que nos permite prever que possa ser mais vantajoso proceder duas validações distintas com pesos iguais para o diagnóstico final. Por fim, um teste realizado com clínicos gerais e especialistas em melanoma revelou uma importante contribuição do método no auxílio de triagens ambulatoriais de casos suspeitos, principalmente para médicos com baixa ou nenhuma experiência em diagnóstico de melanoma. / Recent development in computer science have helped health professionals in the heath area in the treatment, prevention and diagnostic of illnesses. One of the leading challenges in this field has been the development of methods for the skin cancer discrimination between the types of malignant melanoma in relation to other benign lesions. For this, researchers have been using techniques of image processing and analysis, exploring metrics based on the ABCD rule, to the development of methods of diagnostic of melanoma through images. While plenty of studies are about the color and texture of the melanoma, a systematic treatment of the irregularity of the melanoma has not been reported yet. The present work presents a study of the factors which influence the discrimination among the malignant melanocytic and benign lesions, having as a reference the asymmetry of the lesions and the irregularities in their edges. It was collected 143 images of clinic cases of malignant melanoma, regular nevus and atypical nevus. After initial treatment of the images, followed by a segmentation of the lesions, it was extracted 52 metrics referent to the purpose of this study. The visualization of the projection LDA in three categories revealed a good discrimination among the categories of melanoma in relation to the others, reinforcing the original premise of the melanomas´ sharp irregularity. This result was confirmed by the cross-validation of the projection, with successes of 75 % to the melanoma group and 54 %-40 % to the regular nevus group and atypical nevus group, respectively. This result predicts one of the applications of the system in the general discrimination between the melanoma and the other nevus. In this regard, it was joined together the benign groups in one category to the cross-validation, generating calculations of sensibility and specificity of 90 % and 73 % respectively. Another important result was the comparison among these metrics with the information about the diameter of the lesions. It was concluded that both measurements (edges and size) are equally relevant in the diagnostic of the melanoma, reflection of the melanoma´s own pathology, in which both characteristics are enhanced in relation to the other lesions. However, the combination of both measurement in only one processing does not improve the quality of the diagnostic, which allows the prediction that it can be more advantageous to produce two distinct validations with the same weighs to the final diagnostic. To sum up, an accomplished test with doctors who are general practioner and specialists in melanoma revealed a significant contribution in the method to help in the triage of medical consultations in suspicious cases, mainly to doctors with little or no experience in the diagnostic of melanoma.

Malignant melanoma (MM) is the deadliest skin cancer with an ever-increasing incidence. New treatments have improved the prognosis for patients with advanced MM. Still, most patients do not respond, and the side effects can be severe, underlining the need for better therapies. The overall aim of this thesis was to evaluate new means to improve the treatment for patients with advanced MM. Immunostimulatory gene therapy (AdCD40L) was evaluated in a clinical study and BRAF-inhibitory treatment in rare cases of BRAF-mutated MM. Due to its immunogenicity, MM is an attractive target for immunostimulatory gene therapy. AdCD40L is an adenovirus carrying the human gene for CD40 ligand, which in different ways can stimulate the immune system to combat cancer. We conducted a Phase I/IIa study with AdCD40L in patients with metastatic MM having received established treatments. In cohort 1 (n=6), four weekly, intratumoural AdCD40L injections were given. In cohort 2 (n=9), low dose cyclophosphamide was added to increase the immune response. Since irradiation may act synergistically with immunotherapy, patients in cohort 3 (n=9) also received a single fraction of radiotherapy (8 Gy). This fraction was given towards the lesion selected for injections. The primary objectives were to assess the feasibility and safety of AdCD40L-treatment and secondarily its anti-tumour effects. Patients were thoroughly assessed for toxicity. The anti-tumour response was evaluated by imaging techniques (FDG-PET/CT, DW-MRI scans), tumour biopsies and blood tests. Plasma protein markers were measured with a multiplex platform. Another objective was to evaluate the potential of DW-MRI and FDG-PET/CT for prediction of AdCD40L treatment response, in terms of overall survival (OS). AdCD40L was well tolerated with mild transient reactions. Local and distant responses in PET/CT scans along with a significantly better 6-month survival in the cohorts that received cyclophosphamide conditioning were observed. Effector lymphocyte responses were elicited. All patients had an increased T effector/T regulatory-cell ratio and death receptors were significantly up-regulated post therapy. Inflammatory cytokines and other plasma proteins were altered in favourable ways by the AdCD40L treatment. The analyses support that the functional DWI parameters may be better early predictors of OS than the established metabolic and morphologic criteria of FDG-PET/CT and CT/MRI, respectively. In conclusion, the stimulation of the CD40 pathway to initiate anti-tumour immunity is a promising treatment alternative for MM patients. However, further studies with developed treatment schemes are warranted. In the first report ever on treatment of a pregnant patient with a BRAF-inhibitor, the therapy was initiated in the second trimester. The treatment with vemurafenib enabled prolonged gestation, hence reducing the risk of immaturity-related complications. Further, we report the first case worldwide of a patient with metastatic conjunctival melanoma who benefitted from treatment with vemurafenib. Additional studies are needed to assess the efficacy of BRAF -inhibitors in the different subtypes of ocular melanoma.

Die vorliegende Arbeit befasst sich dem Glykosaminoglykan (GAG) Hyaluronan (HA) und dessen Einfluss auf die Entwicklung des malignen Melanoms (MM). Das in der extrazellulären Matrix (ECM) reichlich vorkommende HA wird schon seit dem späten 20. Jahrhundert genauerer im Zusammenhang mit der Tumorentwicklung untersucht. Gegensätzliche Eigenschaften von HA, die zum einen Tumore fördern und zum anderen ihnen entgegenwirken, wurden seither veröffentlicht. Allgemeiner Konsens ist, dass das HA-Molekulargewicht über die verschiedenen Effekte von HA entscheidet. Momentan ist jedoch nicht ausreichend belegt, wie HA auf das Tumorwachstum einwirkt und welche HA-Größen dies speziell begünstigen. Diese Untersuchung basiert auf einem in vivo Knockout von der Hyaluronan Synthase 2 (Has2) in der Maus, der die Produktion von hoch-molekulargewichtigen HA (HMW-HA) weitestgehend einschränkte. Das Wachstum vom experimentellem MM wurde in Abwesenheit der meisten HMW-HA untersucht. Diese Arbeit versuchte den Beweis zu erbringen, dass das lokale Wachstum und die Metastasenbildung der MM-Zellen abhängig von der vorhandenen HMW-HA in der Nähe des Tumors ist. Der Has2-knockout in der Mauslinie C57BL6 wurde verifiziert und nach einem veränderten Phänotyp der Mäuse untersucht. Obwohl nahezu dreiviertel der absoluten HA Menge durch den Knockout fehlte, zeigten die Mäuse keinen offensichtlichen Veränderungen. Erst eine Messung der Haut-Permeabilität deutete auf eine womöglich verstärkte Ausbildung der Stratum corneum hin. Eine direkte Auswirkung vom fehlendem HA auf das Tumorwachstum und der Metastasierungsrate konnte nicht ausreichend belegt werden. Das verwendete Mausmodell sowie die Wahl des experimentellen Tumors werden in dieser Arbeit kritisch diskutiert. Parallel durchgeführte in vitro Versuche mit teilweise artifiziellen 3D Matrizen, die unterschiedliche Mengen von HA enthielten, zeigten einen stärkeren Einfluss von niedermolekulare HA (LMW-HA) auf die Proliferation und Invasion von MM Zellen. Diese Beobachtungen stimmen mit dem derzeitigen Konsens überein, dass LMW-HA aktivierende Signaltransduktion auslöst und HMW-HA eher homöostatisch wirkt.:TABLE OF CONTENT Zusammenfassung Summary Table of content List of figures List of tables Abbreviations 1 Introduction 1.1 Structures of the skin 1.2 The malignant melanoma 1.3 The tumor microenvironment (TME) 1.4 Hyaluronan 1.4.1 HA Structure 1.4.2 HA anabolism 1.4.3 HA catabolism 1.4.4 HA binding proteins 1.4.5 HA cell surface receptors 1.5 Hyaluronan in malignant melanoma 1.6 Aim of the thesis 2 Methods 2.1 Murine malignant melanoma cell lines 2.2 Inducible Has2-knockout mice 2.2.1 The Cre/loxP System 2.2.2 Genetical modification for the Has2-knockout 2.2.3 4-Hydroxytamoxifen induction of knockout 2.2.4 Experimental tumor model in mouse 2.2.5 Intravenous tumor injection 2.3 Primary Has2-knockout fibroblasts for in vitro experiments 2.3.1 Isolation of primary fibroblast from mouse skin tissue 2.3.2 Induction of Has2-ko fibroblasts 2.4 Molecular analysis 2.4.1 PCR analysis of genome DNA 2.4.2 Quantification of gene expression 2.4.3 Microarray analysis 2.4.4 Quantification of Has2 protein levels 2.4.5 Quantification of HA 2.4.6 HA size analysis by agarose gel electrophoresis 2.5 Physical analysis of the skin 2.5.1 Skin elasticity measurement 2.5.2 Skin permeability measurement 2.5.3 Skin hydration and TEWL measurement 2.6 Histological analysis 2.6.1 Cryo-fixed samples 2.6.2 Immunofluorescence staining 2.6.3 FFPE samples 2.6.4 HA staining 2.6.5 Histochemical images 2.7 Physiological analysis of MM cell proliferation with BrdU-Assay 2.8 Statistical analysis 3 Materials 3.1 Mouse lines 3.2 Cell lines 3.3 Buffer and solution recipes 3.4 Chemicals 3.5 Molecular-biological reagents and enzymes 3.6 Primers 3.7 Antibodies 3.8 Kits 3.9 Devices and tools 3.10 Disposables 3.11 Software 4 Results 4.1 The Has2-knockout mouse model 4.1.1 Has2-knockout characterization 4.1.2 Incomplete Has2 deletion 4.1.3 Effects of the HA knockdown 4.1.4 Has2-knockout efficiency in other organs 4.1.5 Effects of Has2-knockout on gene expression 4.2 In vitro Has2-knockout and effect on MM cells 4.2.1 In vitro Has2- and HA-knockdown 4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation 4.2.3 MM conditioned media influenced fibroblast’s HA secretion 4.2.4 The transition towards in vivo tumor experiments 4.3 In vivo Tumor experiments 4.3.1 HA threshold, correlation, and exclusions 4.3.2 Effects of HA knockdown on primary tumor volume and weight 4.3.3 Tumor histology and HA localization 4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples 4.3.5 Metastasis formation 5 Discussion 5.1 HA knockdown 5.2 HA knockdown phenotype 5.2.1 Skin stiffness 5.2.2 Skin water homeostasis 5.3 Paracrine interactions between MM and fibroblasts 5.4 HA thresholding 5.5 Tumor readouts 5.6 In vitro ECM models 5.7 Metastasis 5.8 Alternative tumor models with stronger stromal interaction 5.9 The presented results considering current HA-Tumor research 6 Conclusion 7 Literature Danksagung Lebenslauf Akademischer Werdegang Stipendium und Auszeichnung Publikation und Posterpräsentation Publikationen Vorträge und Posterpräsentationen Eigenständigkeitserklärung / The present work addresses the glycosaminoglycan (GAG) hyaluronan (HA) and its influence on the development of malignant melanoma (MM). HA, which is abundant in the extracellular matrix (ECM), has been studied closely in relation to tumor development since the late 20th century. Opposing properties of HA, on the one hand promoting tumors and the other hand counteracting them, have since been published. The general consensus is that HA molecular weight determines the various effects of HA. However, there is insufficient evidence on how HA affects tumor growth and which HA sizes specifically promote it. This study is based on an in vivo knockout of hyaluronan synthase 2 (Has2) in mice, which largely restricted the production of high molecular weight HA (HMW-HA). Growth from experimental MM was examined in the absence of most HMW-HA. This work sought to provide evidence that local growth and metastasis of MM cells is dependent on the presence of HMW-HA in the vicinity of the tumor. Has2 knockout in the mouse line C57BL6 was verified and examined for an altered phenotype of the mice. Although nearly three-quarters of the absolute HA amount was absent due to the knockout, the mice showed no obvious change. Only a measurement of skin permeability indicated a possible increased barrier function of the stratum corneum. A direct effect of the lack of HA on tumor growth and metastasis rate could not be sufficiently demonstrated. The applied mouse model and the choice of experimental tumor are critically discussed in this work. Parallel in vitro experiments with partially artificial 3D matrices containing different amounts of HA showed a stronger influence of low molecular weight HA (LMW-HA) on proliferation and invasion of MM cells. These observations are consistent with the emerging consensus that LMW-HA triggers activating signal transduction and HMW-HA is more homeostatic.:TABLE OF CONTENT Zusammenfassung Summary Table of content List of figures List of tables Abbreviations 1 Introduction 1.1 Structures of the skin 1.2 The malignant melanoma 1.3 The tumor microenvironment (TME) 1.4 Hyaluronan 1.4.1 HA Structure 1.4.2 HA anabolism 1.4.3 HA catabolism 1.4.4 HA binding proteins 1.4.5 HA cell surface receptors 1.5 Hyaluronan in malignant melanoma 1.6 Aim of the thesis 2 Methods 2.1 Murine malignant melanoma cell lines 2.2 Inducible Has2-knockout mice 2.2.1 The Cre/loxP System 2.2.2 Genetical modification for the Has2-knockout 2.2.3 4-Hydroxytamoxifen induction of knockout 2.2.4 Experimental tumor model in mouse 2.2.5 Intravenous tumor injection 2.3 Primary Has2-knockout fibroblasts for in vitro experiments 2.3.1 Isolation of primary fibroblast from mouse skin tissue 2.3.2 Induction of Has2-ko fibroblasts 2.4 Molecular analysis 2.4.1 PCR analysis of genome DNA 2.4.2 Quantification of gene expression 2.4.3 Microarray analysis 2.4.4 Quantification of Has2 protein levels 2.4.5 Quantification of HA 2.4.6 HA size analysis by agarose gel electrophoresis 2.5 Physical analysis of the skin 2.5.1 Skin elasticity measurement 2.5.2 Skin permeability measurement 2.5.3 Skin hydration and TEWL measurement 2.6 Histological analysis 2.6.1 Cryo-fixed samples 2.6.2 Immunofluorescence staining 2.6.3 FFPE samples 2.6.4 HA staining 2.6.5 Histochemical images 2.7 Physiological analysis of MM cell proliferation with BrdU-Assay 2.8 Statistical analysis 3 Materials 3.1 Mouse lines 3.2 Cell lines 3.3 Buffer and solution recipes 3.4 Chemicals 3.5 Molecular-biological reagents and enzymes 3.6 Primers 3.7 Antibodies 3.8 Kits 3.9 Devices and tools 3.10 Disposables 3.11 Software 4 Results 4.1 The Has2-knockout mouse model 4.1.1 Has2-knockout characterization 4.1.2 Incomplete Has2 deletion 4.1.3 Effects of the HA knockdown 4.1.4 Has2-knockout efficiency in other organs 4.1.5 Effects of Has2-knockout on gene expression 4.2 In vitro Has2-knockout and effect on MM cells 4.2.1 In vitro Has2- and HA-knockdown 4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation 4.2.3 MM conditioned media influenced fibroblast’s HA secretion 4.2.4 The transition towards in vivo tumor experiments 4.3 In vivo Tumor experiments 4.3.1 HA threshold, correlation, and exclusions 4.3.2 Effects of HA knockdown on primary tumor volume and weight 4.3.3 Tumor histology and HA localization 4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples 4.3.5 Metastasis formation 5 Discussion 5.1 HA knockdown 5.2 HA knockdown phenotype 5.2.1 Skin stiffness 5.2.2 Skin water homeostasis 5.3 Paracrine interactions between MM and fibroblasts 5.4 HA thresholding 5.5 Tumor readouts 5.6 In vitro ECM models 5.7 Metastasis 5.8 Alternative tumor models with stronger stromal interaction 5.9 The presented results considering current HA-Tumor research 6 Conclusion 7 Literature Danksagung Lebenslauf Akademischer Werdegang Stipendium und Auszeichnung Publikation und Posterpräsentation Publikationen Vorträge und Posterpräsentationen Eigenständigkeitserklärung

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1–2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.