Tesis por compendio / [ES] El melanoma es el tipo de cáncer de piel más mortífero y peligroso, ya que tumores de pequeño tamaño pueden generar metástasis. Hasta la fecha, se ha tratado de clasificar desde el punto de vista clínico, epidemiológico y molecular, empleándose actualmente el nivel de exposición solar y la localización del tumor como criterios principales para dividir en distintos grupos a los pacientes de melanoma. En 1998, David Whiteman y colaboradores propusieron un "modelo de vías divergentes" para el desarrollo del melanoma. Este presentaba dos vías: una vinculada a la proliferación melanocítica (nevogénica) y otra relacionada con la exposición solar crónica (CSD). Corroborado desde el punto de vista clínico y epidemiológico, todavía no se ha aportado una caracterización molecular en profundidad. A nivel general se habían identificado genes cuyas mutaciones eran relevantes para el desarrollo del melanoma, como por ejemplo KIT. Sin embargo, todavía se había de estudiar con más detalle la distribución de estas mutaciones entre los distintos subgrupos de melanoma, así como su posible valor pronóstico. En esta tesis se han empleado técnicas de secuenciación - masiva y tradicional - para caracterizar los perfiles mutacionales de las poblaciones del modelo de vías divergentes. Encontramos diferencias tanto en el número de mutaciones como en los genes afectados. También hemos visto cómo los melanomas con mutaciones en KIT parecen desarrollarse por una vía independiente de la etiopatogenia conocida, careciendo el estatus mutacional de este gen de valor pronóstico para la supervivencia de los pacientes. / [CA] El melanoma és el tipus de càncer de pell més mortífer i perillós, ja que fins els tumors de menor mida poden acabar generant metàstasi. Al llarg dels anys, s'ha tractat de classificar des del punt de vista clínic, epidemiològic i molecular. Les classificacions actuals utilitzen el nivell d'exposició solar i la localització tumoral per dividir en diferents grups als pacients de melanoma. Al 1998, David Whiteman i col·laboradors proposaren un model de desenvolupament del melanoma que anomenaren "model de vies divergents". Aquest presentava dos vies per la melanomagènesi: una vinculada a la proliferació melanocítica (nevogènica) i l'altra relacionada amb l'exposició solar crònica (CSD). Malgrat aquest model fou corroborat des del punt de vista clínic i epidemiològic, encara no s'ha aportat una caracterització molecular en profunditat. A nivell general s'havien identificat gens les mutacions dels quals eren rellevants per al desenvolupament del melanoma, com el gen KIT. Però, encara s'havia d'estudiar amb més cura la distribució d'estes mutacions entre els distints subgrups de melanoma, així com el seu possible valor pronòstic. En aquesta tesi s'han emprat tècniques de seqüenciació -massiva i tradicional- per caracteritzar els perfils mutacionals de les dues poblacions proposades pel model de vies divergents, trobant diferències tant al nombre de mutacions com als gens afectats. També hem vist com els melanomes mutats en KIT semblen desenvolupar-se per una via independent de l'etiopatogènia coneguda, mancant l'estatus mutacional d'aquest gen de valor pronòstic per la supervivència dels pacients. / [EN] Melanoma is the deadliest and most dangerous type of skin cancer, given that a small tumor can spread and result in metastasis. Over the years, classifications have been made either from a clinical, epidemiological or molecular point of view. Current classifications use the degree of solar exposure and tumoral location to divide into different melanoma groups. In 1998, David Whiteman and collaborators proposed the divergent pathway model for melanoma development. This presented two pathways to melanomagenesis: one related to melanocytic proliferation (nevogenic) and the other related to chronic sun exposure (CSD). Despite corroborations of this model from the clinic and epidemiology, it is yet to be molecularly characterized in depth. At a general level, different genes had been identified with relevant mutations for the development of melanoma, as is the gene KIT. However, there was a lack of knowledge on how these mutations were distributed among different melanoma subgroups, as well as the potential prognostic value. In this thesis we have implemented sequencing techniques - both massive and traditional - to characterize the mutational profile of the two populations proposed by the divergent pathways model. We found differences both in the number of mutations and in the genes carrying the mutations. We have also seen how melanomas harboring KIT mutations seem to develop in a way which is independent from the known etiology, and how the mutational status of this gene lacks prognostic value on the outcome of the patients. / Millán Esteban, D. (2022). Next-Generation Sequencing in the Identification of Biomarkers in Cutaneous Melanoma According to the Etiopathogenic Development Pathway and their Potential Clinical Relevance [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182977 / TESIS / Compendio

ABSTRACT Prioritizing Diseases, Disorders and Disabilities and the Relative Importance of Skin Cancer: A Public Health Faculty Survey By James Thomas Sandwich, MD April 21, 2016 INTRODUCTION: Academic faculty in public health have diverse career interests and occupy positions of considerable influence. They play an important role in setting curriculum and training the future public health workforce. However, there is little published scholarly information regarding which public health diseases, disorders, and disabilities are most important to them. Skin cancer is a major public health problem that has been declared an epidemic. AIM: The Aim of this study is to discover which public health disorders are of highest concern and to determine the relative priority of skin cancer to public health faculty. METHODS: The primary design of the study was that of a non-experimental opinion based survey. Subjects were faculty members of national academic, public health programs. To obtain the broadest distribution, primary and secondary faculty as defined by the ASPPH were included. A 19 question survey document was administered electronically through Qualtrics. There were 15 questions on the importance of specific disorders and five questions on skin cancer. Responses were categorized ranked and compared. RESULTS: Obesity ranked the highest among all concerns with cardiovascular disease and cancer also receiving high priority. Cancer, diabetes, and cardiovascular disease led in secondary outcomes. Tertiary outcomes were nearly evenly split between cardiovascular disease, cancer, and mental health. Priorities varied by regions, age, gender and race. The majority placed skin cancer in the second quartile of importance and believed it to be appropriately ranked. CONCLUSION: Public health faculty prioritize disorders similarly in spite of diverse interests with minor differences across regions and demographics. National Funding as a proxy for importance does not cleanly align with faculty priorities. Public health faculty express familiarity with skin cancer, however, do not generally considered it of highest priority compared to other disorders. Increased faculty emphasis on interventions that prevent skin cancer may improve awareness and reduce negative sequela.

Metastasis is one of the main causes of death in cancer patients. Metastatic melanoma is a death sentence, as chemotherapeutic agents have a 5% success rate or do not extend survival beyond 10 months. The lack of effective chemotherapeutic agents for treating metastatic melanoma indicates a dire need to identify new drug targets and develop new therapies. Our lab has previously shown that the kinase activity of Abelson family of non-receptor tyrosine kinases (c-Abl and Arg) is elevated in invasive breast cancer cell lines as compared to non-invasive cell lines. Previous studies from our lab have shown that Abl kinases are convergent point of ErbB2 and Src Kinases in melanoma cells and Abl kinases promote invasion by an undefined mechanism. Although Abl kinases promote invasion, it is not known whether they are important for metastastic potential. For the first time, we report that Abl kinases promote melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. To investigate the mechanism by which Abl kinases promote invasion, we found out that active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3àMMP-1 pathway. In contrast, active Arg drives invasion in a STAT3-independent manner, and upregulates the expression of MMP-3 and MT1-MMP, in addition to MMP-1. We also found that Abl kinases promote invasion via lysosomal degradation of a metastasis suppressor, NM23-H1 by activating lysosomal cathepsins B and L, which directly cleave and degrade NM23-H1. Furthermore, c-Abl and Arg are activated in primary melanomas and cAbl/Arg activity is inversely correlated with NM23-H1 expression both in primary melanoma and human melanoma cells. We also demonstrate, for the first time that active Abl kinases promote metastasis in vivo, as inhibition of c-Abl/Arg with nilotinib, dramatically inhibits lung colonization/metastasis in a mouse model using two different melanoma cell lines. In summary, we identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and our data indicate that nilotinib may be useful in preventing metastasis in a select group of patients, harboring active Abl kinases.

The purpose of this study was to test a theoretical model which predicted subjective well-being in patients who had been diagnosed with malignant melanoma. The theoretical model was developed from empirical findings based on a review of the literature in which health locus of control, social support and self-esteem were identified as significant predictors of well-being. The specific aim of this study was to examine the strength of the predicted relationships between selected psychosocial variables and subjective well-being. The study utilized a nonexperimental correlational design with a causal modeling approach. The convenience sample was composed of 75 individuals (x age = 52.5) who had been diagnosed with malignant melanoma. Subjects completed four instruments which measured the theoretical concepts under study. Two additional instruments were administered which indexed the variables of search for meaning and concern of recurrence. Descriptive statistics were used in examining the demographic and situational characteristics of the sample. Multiple regression techniques were utilized to empirically test the predicted theoretical relationships and to estimate predictive validity for the theoretical concepts. Graphic residual analysis was performed to assess for violations in the statistical and causal model assumptions. Study findings revealed that social support had a direct positive impact on self-esteem (B =.27, R² =.06) and that self-esteem had a direct positive impact on well-being (B =.49, R² =.37). The two demographic variables of employment and income were found to have a direct positive impact on well-being (B =.22 and B =.26, respectively), and resulted in a 10% increase in the total explained variance in well-being. The theoretical model, which was generated to predict subjective well-being in malignant melanoma patients, explained 47% of the total variance in well-being. Research into the variables which influence patient well-being during the cancer experience is vital if nursing is to implement therapeutic interventions which will promote an improved life quality. By intervening with nursing actions that focus on a positive self-esteem, a greater sense of well-being could be attained by individuals diagnosed with cancer.

In order to study the process by which human melanoma cells achieve invasion of basement membranes, a modification of the Membrane Invasion Culture System was developed to allow the in vitro collection of invasive tumor cells from heterogeneous tumor cell populations. A significant increase in the number of double minute chromosomes was observed in metaphase nuclei of the low metastatic A375P human melanoma cells which had invaded 2 consecutive amniotic membranes over that of cells in the control groups. After 25 days in culture, the incidence of double minutes had dropped below the control range. These data indicate that an unstable gene amplification event may be part of the process by which melanoma cells execute invasion through basement membranes. A375P cells which had invaded 1, 3 and 5 consecutive basement membrane-coated filters were established and compared with the parental cell line and a highly metastatic subclonal line for the following characteristics: (a) in vitro invasive potential, (b) mRNA expression of several oncogenes, (c) expression of laminin receptor, at the (cell surface) protein and mRNA levels, and, (d) secretion of endogenous laminin. There was a progressive increase in invasive potential and expression of endogenous laminin and laminin receptor which correlated with the number of membrane-coated filters through which the A375P cells had been selected. There were significant increases in the steady-state mRNA expression of c-myc and c-fos, a decrease in c-jun, and no change in Ha-ras, that correlated with increases in the invasive and metastatic potential of the cells. A novel in vitro adhesion assay was developed to study the interaction of tumor cells with lymphatic endothelium, the first step of extravasation from the lymphatic vessel. Human tumor cells from: one primary Ewing sarcoma, two melanoma, two colon and two breast carcinomas were assayed for their ability to attach to monolayers of lymphatic endothelium. There was a clear positive correlation between the metastatic potential and attachment potential of the melanoma cell lines. Overall, these data suggested that highly fibroblastic established tumor cell lines were more adaptive in rapid adhesion than primary tumor cell cultures with a more rounded morphology.

No description available.

This thesis is concerned with developing and implementing a method for modelling and projecting cancer incidence data. The burden of cancer is an increasing problem for society and therefore, the ability to analyse and predict trends in large scale populations is vital. These predictions based on incidence and mortality data collected by cancer registries, can be used for estimation of current and future rates, which is helpful for public health planning. A large body of work already exists on the use of various modelling strategies, methods and fitting techniques. A multilevel method of preparing the data is proposed, fitted to historical data using regression modelling, to predict future rates of incidence for a given population. The proposed model starts with a model for the total incidence of the population, with each successive level stratifying the data into progressively more specific groupings, based on age. Each grouping is partitioned into subgroups, and each subgroup is expressed as a proportion of the parent group. Models are fitted to each of the proportional age-groups, and a combination of these models produces a model that predicts incidence for a specific age. A simple, efficient implementation of the modelling procedure is described, including key algorithms and measures of performance. The method is applied to data from populations that have very different melanoma incidence (the USA and Australia). The proportional structure reveals that the proportional age trends present in both populations are remarkably similar, indicating that there are links between causative factors in both populations. The method is applied fully to data from a variety of populations, and compared with results from existing models. The method is shown to be able to produce results that are reliable and stable, and are generally significantly more accurate than those of other models.

<p>The gene BRAF is mutated to remain aberrantly activated in a large number of human malignancies, most prominently in melanoma. The most common mutation in BRAF is a missense mutation that substitutes glutamic acid for valine at codon 600 (V600E) that leads to constitutive activation of this kinase. In this active state, BRAFV600E phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate the ERK1 and ERK2 kinases of the MAPK pathway to promote tumorigenesis. Targeting this pathway is a well-validated strategy to treat BRAF-mutant cancer. Inhibitors of both BRAFV600E and the MEK1/2 kinases are used to treat BRAF-mutant melanoma and are being evaluated in other cancers as well. However, the duration of response to these targeted therapies is limited by innate and acquired resistance, which is often mediated through reactivation of the MAPK pathway. Thus, new targeted therapies to inhibit MAPK signaling in BRAF-mutant malignancies are required. To this end, MEK1/2 kinases require copper (Cu) for enzymatic activity and signaling. We therefore tested whether the dependency of these validated targets on Cu could be leveraged for the treatment of BRAF-mutant cancer.</p><p>We report that genetic reduction of Cu import through disruption of the gene encoding the high affinity Cu transporter CTR1 or pharmacological chelation of Cu with the drug tetrathiomolybdate (TTM) suppressed MAPK signaling in both in vitro and in vivo models of BRAF-mutant tumorigenesis. This reduction in MAPK signaling correlated with a reduced potential for tumorigenic growth and an increase in survival of tumor-bearing mice. Finally, TTM reduced the transformed growth of a number of human melanoma cell lines engineered to be resistant to current MAPK pathway inhibitors. As such, Cu chelation holds promise as a novel treatment for BRAF-mutant cancers and may find value in targeting resistance to current MAPK pathway inhibitors.</p> / Dissertation

Intermittent sun exposure and sun sensitivity factors are the most well described risk factors for the development of cutaneous melanoma (CM). Other potential environmental risks for CM, such as arsenic, are rarely examined. Total sun exposure has not been a consistent risk factor for CM, but recall bias in self-reporting sun exposure throughout life may limit the ability to detect a true association. Objective measures of sun exposure including remotely sensed ambient ultra-violet radiation (UVR) may allow for better capture of total sun exposure. In three chapters, spatially derived factors (ambient UVR, environmental soil arsenic, drinking water arsenic) were observed to determine their relevance in exposure assessment and subsequent risk for CM.UVR trends were investigated using available satellite data (1978-2014) to generate inferences for UVB changes over time in the United States. We found that UVB changed across the study area, but these changes lack biological relevance based on the magnitude of changes observed. Thus, a more objective measure of lifetime ambient sun exposure may be estimated using 30-year average UVR by month in future studies. The spatial correlation between environmental soil arsenic and drinking water across the state of Iowa was investigated. Arsenic concentrations in soil were not significantly spatially correlated with either municipal public water source or non-municipal water source arsenic concentrations. Based on these findings, soil arsenic may not serve as a valid surrogate marker for arsenic in drinking water.In Chapter 5, we assessed the relationship of spatially derived estimates of lifetime ambient UVR, environmental arsenic exposure from soil and drinking water, and CM in a population-based case-control study. Our findings suggest that total sun exposure is positively associated with CM, while arsenic concentration in environmental soil and drinking water were not associated. Sun exposure measured through ambient UVR exposure may allow for better understanding of the association between cumulative or total sun exposure and CM. Additionally, more studies need to be completed to estimate the potential risks for CM in regions where high arsenic concentrations may not be endemic.

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.