published_or_final_version / Surgery / Master / Master of Surgery

Referat: Bei der folgenden Arbeit handelt es sich um eine retrospektive Untersuchung der im Zeitraum von 1993 bis 2009 an der Klinik für Hals-Nasen-Ohrenheilkunde (HNO) der Universität Leipzig behandelten Larynx- und Hypopharynxkarzinome. Die Arbeit hatte zum Ziel, die Prävalenz, Risikofaktoren und Therapieergebnisse des fortgeschrittenen Larynx- und Hypopharynxkarzinoms im Spiegel geänderter Therapiestandards im genannten Untersuchungszeitraum zu analysieren. Im Vordergrund steht der ab 2004 eingeleitete Paradigmenwechsel in der adjuvanten Therapie dieser Tumoren aufgrund einer geänderten Studien- bzw. Datenlage. Inhaltlich handelt es sich um die evidenzbasierte Einführung multimodaler adjuvanter Konzepte, die sich durch die zusätzliche Applikation der Chemotherapie (Cisplatin/5-Fluorouracil) als simultaner Partner zur adjuvanten Strahlentherapie in definierten Risikosituationen auszeichnet. Die Arbeit untersucht die definitiven Therapieänderungen und einhergehenden Effektivitätsunterschiede im Untersuchungszeitraum. Hierbei blieben die Prävalenz der Larynx- und Hypopharynxkarzinome sowie der Konsum von Tabak und Alkohol, die als wesentliche Risikofaktoren gewertet werden, neben unwesentlichen Schwankungen über die genannten Jahre unverändert. Erwartungsgemäß zeigte sich eine Zunahme der adjuvanten platinbasierten Radiochemotherapie bei unverändert primär operativer Indikation, jedoch geänderter Risikoprofile (knappe bzw. tumorinfiltrierte Randschnitte [R1, R0 < 5mm] und kapseldurchbrechende Halslymphknotenmetastasen [ECS]) und der primären platinbasierten Radiochemotherapie bei nicht sinnvoll resektablen Tumoren. Diese Entwicklung führte insbesondere bei den fortgeschrittenen Larynx- und Hypopharynxkarzinomen zu einer signifikanten Verbesserung der Überlebensraten ab dem Jahr 2004. Dieser Zusammenhang konnte in einer multivariaten Betrachtung überzeugend herausgearbeitet werden und belegt die positiven Effekte einer systematischen Standardisierung von onkologischen Konzepten auf dem Boden sich ändernder Evidenz.

Dissertation submitted in fulfilment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Apoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance. / National Research Foundation

Referat: Bei der folgenden Arbeit handelt es sich um eine retrospektive Untersuchung der im Zeitraum von 1993 bis 2009 an der Klinik für Hals-Nasen-Ohrenheilkunde (HNO) der Universität Leipzig behandelten Larynx- und Hypopharynxkarzinome. Die Arbeit hatte zum Ziel, die Prävalenz, Risikofaktoren und Therapieergebnisse des fortgeschrittenen Larynx- und Hypopharynxkarzinoms im Spiegel geänderter Therapiestandards im genannten Untersuchungszeitraum zu analysieren. Im Vordergrund steht der ab 2004 eingeleitete Paradigmenwechsel in der adjuvanten Therapie dieser Tumoren aufgrund einer geänderten Studien- bzw. Datenlage. Inhaltlich handelt es sich um die evidenzbasierte Einführung multimodaler adjuvanter Konzepte, die sich durch die zusätzliche Applikation der Chemotherapie (Cisplatin/5-Fluorouracil) als simultaner Partner zur adjuvanten Strahlentherapie in definierten Risikosituationen auszeichnet. Die Arbeit untersucht die definitiven Therapieänderungen und einhergehenden Effektivitätsunterschiede im Untersuchungszeitraum. Hierbei blieben die Prävalenz der Larynx- und Hypopharynxkarzinome sowie der Konsum von Tabak und Alkohol, die als wesentliche Risikofaktoren gewertet werden, neben unwesentlichen Schwankungen über die genannten Jahre unverändert. Erwartungsgemäß zeigte sich eine Zunahme der adjuvanten platinbasierten Radiochemotherapie bei unverändert primär operativer Indikation, jedoch geänderter Risikoprofile (knappe bzw. tumorinfiltrierte Randschnitte [R1, R0 < 5mm] und kapseldurchbrechende Halslymphknotenmetastasen [ECS]) und der primären platinbasierten Radiochemotherapie bei nicht sinnvoll resektablen Tumoren. Diese Entwicklung führte insbesondere bei den fortgeschrittenen Larynx- und Hypopharynxkarzinomen zu einer signifikanten Verbesserung der Überlebensraten ab dem Jahr 2004. Dieser Zusammenhang konnte in einer multivariaten Betrachtung überzeugend herausgearbeitet werden und belegt die positiven Effekte einer systematischen Standardisierung von onkologischen Konzepten auf dem Boden sich ändernder Evidenz.

La cyclooxygénase-2 (COX-2) a été associée à un mauvais pronostic dans plusieurs cancers. L'objectif de cette étude est d'évaluer si la COX-2 est un facteur pronostique dans le cancer glottique. Cette étude a été effectuée sur des tissus de patients ayant participé à un essai clinique qui évaluait l'efficacité de l'alpha-tocophérol pour réduire la survenue de . seconds cancers primaires (SCP) chez des patients avec un cancer de la tête et du cou. Des analyses immunohistochimiques ont été effectuées sur les biopsies pré-traitement de 301 patients avec un cancer glottique de stade précoce traité par radiothérapie. Les rapports d'incidence (RI) et leurs intervalles de confiance à 95% (le) associés à une surexpression de COX-2 sont de 0,94 (le: 0,55-1,62) pour la récidive et de 1,57 (le: 1,01-2,45) pour la mortalité globale. Pour les SCP, les RI sont de 2,63 (IC : 1,32-5,23) pour les premières 3,5 années de suivi et de 0,55 (IC : 0,22-1,32) pour les 3,5 années subséquentes. En conclusion, la surexpresssion de laCOX-2 dans le cancer du larynx glottique est associée à une augmentation de .la mortalité globale et confère une augmentation du risque de SCP durant les 3,5 premières années de suivi. Des études subséquentes sont nécessaires pour expliquer les effets observés au niveau des SCP. L'expression de la COX-2 pourrait s'avérer utile pour prédire le pronostic d'un patient sur une base individuelle.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The work presented in this thesis deals with the association between cancers of the upper gastro-intestinal tract and the tumor suppressor gene, TP53, and can be divided into three parts: (i) the analysis of the mutational spectrum of TP53 with respect to laryngeal cancer, (ii) the analysis of the mutational spectrum of TP53 with respect to esophageal cancer and (iii) the analysis of TP53 transcriptional levels in esophageal cancer. Laryngeal cancer (LC) is the 6th most common cancer in the world and the 2nd most common respiratory cancer, with approximately 500 000 new cases per annum detected worldwide. Over the last few years, LC has become increasingly prevalent within the Coloured Community of the Western Cape. The mechanisms of tumorigenesis in LC remain unknown, although smoking and alcohol consumption are considered to be major risk factors. Mutations within the gene TP53 have been strongly implicated as playing a role in cancer development, as they are frequently found in several cancer types. We therefore screened exons 5 - 8 of TP53 for mutations in DNA from tumor biopsies (n=44) and blood samples (n=42) from Coloured LC patients, using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Blood samples from a healthy, matched control group (n=40) were included in the study as controls. Significant correlations were found between the occurrence of LC and age and smoking, whereas daily meat consumption was a possible protective factor. In tumor-derived samples, mutations were found in 3 of the exons under investigation, representing 25% of the samples. The mutations were unique to the tumor biopsies, indicating a somatic origin for mutations. The data confirms that the region between codons 175 and 273 of TP53 is a mutational hotspot for cancers in general. This study reports 6 novel mutations within this same region. Esophageal cancer (EC) has a very high incidence in South Africa, relative to the rest of the world, and is particularly common amongst the Black Transkei population. The goal of this study was to determine whether there are differences in the TP53 mutational pattern observed in the Coloured Western Cape community as compared to that observed in the Black Transkei community. This required the analysis of the molecular structure of TP53, specifically exons 5 - 8, in a group of Coloured EC patients (n=44) treated at Tygerberg Hospital, Cape Town, South Africa. DNA obtained from tumor biopsies and blood (from patients) as well as from apparently healthy surrounding tissue was screened via PCR-SSCP and direct sequencing analysis. Only 4 nucleotide changes were observed from a total of 124 sequences obtained, of which two were novel to esophageal squamous cell carcinoma. These 4 nucleotide alterations were found only within the tumor biopsy sample set, representing 9% of the tumors investigated. This study revealed that the mutational spectrum of TP53 within the Coloured population of the Western Cape greatly differs from that of the Black community of the Transkei. This suggests that a different set of etiological factors are involved in the tumorigenic process for each of these distinct geographical communities, which is the subject of an epidemiological study undertaken by the MRC. The final part of this thesis deals with the quantification and comparison of TP53 transcription levels in esophageal cancer tumor tissue to the TP53 levels in healthy esophageal tissue obtained from patients from a unique geographical and ethnic background. The cohort used in this study consisted of Coloured patients (n=2) treated at Tygerberg Hospital. The LightCycler system was implemented in order to try to accurately quantify TP53 mRNA levels. Unfortunately, the desired results were unattainable due to unforeseen difficulties encountered during the study. These difficulties included the insufficient preservation of samples for RNA based studies. Several recommendations were made concerning future similar studies, including an improved planning strategy as well as the employment of an RNA stabilizing agent. Additionally, a few important contributions were made through this study, including the design and optimization of TP53 primers specifically intended for future RNA studies. These primers would enable the identification of the presence of TP53 RNA species as well as the absence of DNA contamination in a single PCR amplification step. Other contributions include the development of a well-optimized RNA extraction method for the extraction of RNA from tough tissues (such as the human esophageal tissue used in this study). This method makes the extraction of large quantities of RNA from small amounts of tough tissue types possible. In conclusion, this study has made a significant contribution to the field of cancer research, by shedding light on the TP53 mutational spectrum with regards to laryngeal as well as esophageal cancer in a population unique to the Western Cape. The first part of this thesis has been published in Cancer Genetics and Cytogenetics (Barnard, D., K. Lehmann, E.G. Haal, P.O. van Heiden, and l.C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarities to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145:126-132), of which a copy can be found in Appendix I. This work has also been presented (by D. Barnard) at an international conference entitled "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", held in Amsterdam, the Netherlands during the period 13 - 15 December 2002. / AFRIKAANSE OPSOMMING: Die werk wat in hierdie tesis voorgelê word handel oor die assosiasie tussen kankers van die boonste gastrointestinale weg en die tumor suppressor geen, TP53, en kan in 3 dele gedeel word, (i) die analise van die mutasiespektrum van TP53 in laringiale kanker (LK), (ii) die analise van die mutasiespektrum van TP53 in slukderm kanker (SK) en (iii) die analise van die transkripsievlakke van TP53 in SK. Laringeal kanker (LK) is die 6de algemeenste kanker in die wêreld en die 2de algemeenste respiratoriese kanker, met "n benaderde 500 000 nuwe gevalle jaarliks wêreldwyd. Oor die afgelope paar jare het LK "n toenemende probleem geraak, veral in die Kleurling gemeenskap van die Wes Kaap. Die meganismes van die tumorvorming in LK is onbekend, alhoewel rook-en alkoholgebruik vername risiko faktore is. Die voorkoms van mutasies in TP53 is verskeie kere aangetoon in verskillende kanker tipes en daar word vermoed dat dit "n rol speel in tumorvorming. In hierdie studie is dus na mutasies in eksons 5 - 8 van TP53 gesoek in tumor biopsie weefsel (n=44) en bloed isolate (n=42) van Kleurling LK pasiënte d.m.v. polimerase ketting reaksie - enkelstring konformasie polimorfisme (PKR-ESKP) analisering en direkte volgorde bepaling. Bloed monsters van "n vergelykbare groep (n=40) is ook in die studie ingesluit as "n kontrole. Betekenisvolle positiewe korrelasies is gevind tussen die voorkoms van LK en ouderdom sowel as rook. Daarmee saam is daaglikse vleisinname as potensiële beskermende faktor gevind. In tumor biopsies is mutasies in 3 van die ondersoekte eksons gevind, wat 25% van die biopsie monsters verteenwoordig. Hierdie mutasies is uniek aan die tumor biopsie weefsels en dui op "n somatiese oorsprong van mutasies. Hierdie bevindinge bevestig dat die gedeelte tussen kodons 173 - 273 van TP53 "n hipermuteerbare gebied geassosieer met kankers is. Hierdie studie bevestig 6 nuwe mutasies. Daar is 'n hoë insidensie van slukderm kanker (SK) in Suid Afrika relatief tot die res van die wêreld. Hierdie soort kanker word veral gevind by die Swart populasie van die Transkei. Die doel van hierdie studie was om verskille tussen die TP53 mutasie patroon van die Kleurling gemeenskap van die Wes Kaap en die Swart gemeenskap van die Transkei te vergelyk. Hiervoor is die molekulêre struktuur van TP53, veral eksons 5 - 8, in 'n groep Kleurling SK pasiënte (n=42) wat behandel is by Tygerberg Hospitaal, Kaapstad, Suid Afrika, geanaliseer. Analisering is gedoen deur DNS van tumor, bloed en ook oënskynlike gesonde aangrensende weefsel van dieselfde pasiënte te onderwerp aan PKR-ESKP analise en direkte volgorde bepaling. Slegs 4 nukleotied veranderings is gevind in 124 volgorde bepalings, waarvan 2 nuwe veranderings is in SK. Hierdie 4 nukleotied veranderinge verteenwoordig 9% van al die tumors wat ondersoek is in die studie. Hierdie studie bewys dat die mutasiespektrum van TP53 in die Kleurling gemeenskap van die Wes Kaap grootliks verskil van die Swart gemeenskap van die Transkei. Dit impliseer dat verskillende etiologiese faktore moontlik 'n rol mag speel op die tumorvormingsproses in die 2 afsonderlike geografiese gemeenskappe. Hierdie is die onderwerp van 'n epidemiologiese studie wat deur die MNR onderneem word. Die laaste deel van hierdie tesis handel oor die kwantifisering en vergelyking van TP53 transkripsievlakke in SK tumor weefsel teenoor TP53 vlakke in gesonde slukderm weefsel van pasiënte in 'n unieke geografiese en etniese agtergrond. Die studie populasie in hierdie projek het bestaan uit Kleurling pasiënte (n=2) wat by Tygerberg hospitaal behandel is. Die "LightCycler" sisteem is gebruik vir die akkurate kwantifisering van TP53 boodskapper RNS vlakke. Ongelukkig is die verlangde resultate nie gekry nie as gevolg van onvoorsiene probleme wat ondervind is tydens die studie. Hierdie probleme sluit in die onvoldoende preserv RNS studies. Hierdie inleiers maak dit nou moontlik om die teenwoordigheid van TP53 RNS spesies sowel as die afwesigheid van DNS kontaminasie in een PKR amplifikasie stap te kan identifiseer. 'n Ander belangrike bydrae is die ontwikkeling van 'n goed geoptimaliseerde RNS ekstraksie metode vir moeilike starre weelfsel tipes (soos menslike slukderm weefsel in hierdie studie) en maak die ekstraksie van groot hoeveelhede RNS uit klein hoeveelhede van moeilik hanteerbare weefsel tipes moontlik. Om saam te vat, hierdie studie het betekenisvolle bydraes gemaak tot die veld van kankernavorsing deur die ontrafeling van die TP53 mutasiespektrum in beide laringeale sowel as slukderm kanker, in 'n populasie uniek aan die Wes Kaap. Die eerste deel van hierdie tesis is gepubliseer in Cancer Geneties and Cytogenetics (Barnard, D., K. Lehmann, E. G. Hoal, P. D. van Heiden, and T. C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarites to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145: 126-132) en 'n afskrif van die artikel is ingesluit in Appendix I. Hierdie werk is ook voorgedra (deur D. Barnard) by 'n internasionale kongres getiteld "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", wat in Amsterdam, Nederland gehou is gedurende 13 - 15 Desember 2002

Made available in DSpace on 2016-04-04T18:27:47Z (GMT). No. of bitstreams: 1 Taisa Borges Grun.pdf: 713408 bytes, checksum: 77588a2820092fa9e412dae8803c2312 (MD5) Previous issue date: 2009-02-16 / Head and Neck cancer are responsible for about 5% of the new cases of cancer. Surveys indicate that prolonged stress can contribute to its development and can damage patient s treatment and quality of life. Appropriate social relationships promote better health conditions and moderate stress. The purpose of this essay was to verify the existence of stress and deficit of social skills in patients suffering from larynx cancer in one of Curitiba s cancer hospital. Twenty and one patients took part on the study. The instruments used were identification form, Inventory of Symptoms of Stress for Adults of Lipp and questionnaire of social skills. The results revealed 12 participants with stress. Results gotten from social skills questionnaire has showed high average. It was not verified statistics associations between stress and social skills values. The data did not support the initial hypothesis, since it was expected a higher number of participants with stress and deficit of social skills. Regarding the stress of the sample, a discussion is the probable intervening variables that might have helped to avoid the disease became an aversive situation for those patients until the time of the interview. As for social skills, a discussion is the adequacy of the instrument used for this sample. It is suggested replication of this research with larger samples and using another tool for evaluation of social skills. It was concluded that the data is valuable for a starting point for future researches with similar samples. / C?nceres de cabe?a e pesco?o s?o respons?veis por cerca de 5% dos novos casos de c?ncer. Pesquisas apontam que o stress prolongado pode contribuir para seu desenvolvimento e prejudicar o tratamento e a qualidade de vida do paciente. Rela??es sociais adequadas promovem melhores condi??es de sa?de e moderam o stress. O objetivo do presente trabalho foi verificar a exist?ncia de stress e d?ficit em habilidades sociais em pacientes com c?ncer de laringe de um hospital de c?ncer em Curitiba. Participaram do estudo vinte e um pacientes. Os instrumentos utilizados foram ficha de identifica??o, Invent?rio de Sintomas de Stress para Adultos de Lipp e question?rio de habilidades sociais. Os resultados revelaram 12 participantes com stress. Resultados obtidos com o question?rio de habilidades sociais n?o mostraram altas m?dias. N?o foram verificadas associa??es estat?sticas entre os valores referentes ao stress e ?s habilidades sociais. Os dados n?o apoiaram as hip?teses iniciais, uma vez que se esperava um maior n?mero de participantes apresentando stress e d?ficit em habilidades sociais. Com rela??o ao stress da amostra, discutiram-se prov?veis vari?veis intervenientes, que talvez tenham contribu?do para evitar que a doen?a se tornasse uma situa??o aversiva para esses pacientes at? o momento da entrevista. Quanto ?s habilidades sociais, discutiu-se a adequa??o do instrumento utilizado para essa amostra. Sugere-se a replica??o da presente pesquisa em amostras maiores e utilizando outro instrumento de avalia??o das habilidades sociais. Conclui-se que os dados obtidos s?o de valia como ponto de partida para futuras pesquisas com amostras semelhantes.

In addition, we investigated the cytotoxic effect of a widely used chemotherapeutic agent 5Fu on laryngeal squamous cell cancer cell lines and evaluated the role of p53 in 5Fu treatment. We found that the apoptosis and G1/S cell arrest mediated by 5Fu in laryngeal cancers is p53-independent but p21 WAF1/CIP1-dependent. We further demonstrated the effect of 5Fu on HPV16-associated laryngeal cancer cells. Using cytotoxicity assay and Annexin V staining, we proved that 5Fu induces apoptosis in all of the transfected cells in a dose- and time-dependent manner, suggesting that the process was not prevented by HPV16 E6 or E7. 5Fu induced the accumulation of active pRb and cyclin dependent kinase inhibitor p21WAF1/CIP1 together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. In addition, G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu in all cell lines. Through RT-PCR, 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in transfected UMSCC 12 cells. / Our results suggest that HPV16 E6 and E7 oncoproteins do not prevent 5Fu medicated apoptosis and G1/S cell arrest in laryngeal cancers. The anti-cancer effect of 5Fu is probably decided by the level of p21 WAF1/CIP1 while the sensitivity of laryngeal cancer cells responded to 5Fu treatment is associated with the increase of Bak or/and the decrease in Bcl-2, not with the HPV16 viral proteins and p53 status. 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer. However, the anti-viral effect of 5Fu still needs further investigation. / Our study indicated that (1) the evasion of apoptosis mediated by HPV16 E6 and E7 plays a critical role in laryngeal carcinogenesis; (2) HPV16 E6 or E7 plays an important role in regulating the expression of Bak, Bax and Bcl-2; (3) The degradation of Bak by HPV16 E6 is not caused by interacting with the promoter of Bak; (4) The induction of Bcl-2 is mediated through HPV16 E7; (5) HPV16 transfection does not interfere with the apoptosis and cell cycle arrest mediated by 5Fu in human laryngeal squamous cancer cells. / There is a growing body of evidence that human papillomavirus type 16 (HPV16) is involved in the development of human laryngeal cancer, especially in Chinese population. The two oncoproteins, HPV16 E6 and E7 that target host cell tumor suppressor proteins p53 and Rb respectively, may generate antiapoptotic effects and induce cell immortalization. However, the effect of both oncoproteins on apoptosis in laryngeal cancers is not completely clear. In this study, we demonstrated the possible mechanism of high risk HPV16 in laryngeal carcinogenesis and evaluated the effect of 5Fu on HPV16-positive laryngeal cancer cells. / We employed two human laryngeal cancer cell lines---UMSCC12 (with truncated p53) and UMSCC11A (with mutant but functional p53) in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1, which provided a good foundation for further study on the carcinogenic mechanism of HPV16 E6 or E7 in human laryngeal cancers. Through Annexin V staining and protein stability assay, we found that the transfection of HPV16 E6 and E7 induced fewer spontaneous apoptosis in both UMSCC11A and UMSCC12 cells accompanied with enhanced protein stability of Bcl-2 and increased protein degradation of Bak. Similar results were obtained when E6- and E7-transfected cells exposed to apoptosis stimuli---TNF-alpha/CHX. These results indicate that stable transfection of E6 and E7 in human laryngeal cancer cells on one hand shortened the half-life of Bak protein, and on the other hand, enhanced the steady-state levels of Bcl-2 protein. In order to gain insight into the role of Bak and Bcl-2 in regulating apoptosis in HPV-associated laryngeal cancer cells, we performed transient transfection of Bcl-2 into E6- and E7-transfected cells. It is found that HPV16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer, indicating that the induction of Bcl-2 require the transfection of HPV16 E7. Furthermore, Luciferase assay was performed to investigate whether the viral proteins E6 and E7 altered the stability of Bak through interaction with the promoter of Bak. Negative results were obtained, suggesting that E6 or E7 do not alter the transcription activity of Bak, indicating the degradation of Bak by E6 or E7 may be mediated through other mechanisms. / Liu Han-ching. / "August 2006." / Advisers: C. A. van Hasselt; George G. Chen. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1569. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 245-274). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Introdução: O prognóstico dos carcinomas epidermóides de laringe é limitado e a taxa de sobrevida em cinco anos é menor que 70%. A relação de características clínicas e epidemiológicas tem sido investigada na sobrevida de pacientes com tumores de laringe, mas pouco se conhece sobre o efeito dos polimorfismos genéticos no prognóstico da doença. Objetivo: Estudar o papel dos polimorfismos genéticos de genes relacionados aos processos de invasão e metástase (MMP1 e MMP3), de inflamação (Interleucina 2, Interleucina 6, LTA) e reparo de DNA(XRCC1) no prognóstico do carcinoma epidermóide de laringe. Material e métodos: Coorte com 170 pacientes com carcinoma epidermóide de laringe,confirmados por exame anátomo-patológico. Os casos tiveram origem em estudo caso-controle conduzido em cinco hospitais de São Paulo, um hospital em Porto Alegre e outro em Goiânia. As informações sobre o status vital dos pacientes foram levantadas dos prontuários médicos e dos bancos de óbitos municipais e estaduais. A extração do DNA das amostras de sangue dos pacientes foi realizada pelo Instituto de Medicina Tropical da USP e a genotipagem dos polimorfismos genéticos pela Fundação Hemocentro de Ribeirão Preto da Faculdade de Medicina da USP. Resultados: Os polimorfismos genéticos estudados (MMP1 1607, MMP1 -519,MMP3 -1171, IL2 -384, IL2 114, IL6 -174, LTA 252 e XRCC1) não apresentaram efeitos com significância estatística na sobrevida global ou específica pela doença quando analisados isoladamente. Para a sobrevida global, o consumo excessivo de álcool, em g/L/dia, reduziu a sobrevida dos pacientes (80-119 g/L/dia: hazard ratio(HR)=4,0 intervalos com 95% de confiança (IC95%)=1,10-14,53; _120 g/L/dia: HR=5,6 IC95%=1,71-18,24). No modelo de Cox múltiplo, quando ajustados pelo polimorfismo genético MMP3 -1171, a sobrevida piorou para esses pacientes (80-119 g/L/dia: HR=4,9 IC95%=1,07-22,91; _120 g/L/dia: HR=6,3 IC95%=1,49-26,84). Para a sobrevida específica pela doença, o estadiamento clínico IV reduziu a sobrevida dos pacientes (HR=3,5 IC95%=1,67-7,28). No modelo de Cox múltiplo,com ajuste pelos polimorfismos genéticos IL6 -174 e MMP1 1607, a sobrevidaespecífica pela doença piorou para esses pacientes (HR=4,7 IC95%=1,38-16,25).Conclusões: Na coorte examinada, somente três dos oito polimorfismos genéticos estudados relacionaram-se com a sobrevida global e específica pela doença, porém apenas alterando o efeito dos valores dos HR brutos dos fatores consumo de álcool e estadiamento clínico, respectivamente na sobrevida global e sobrevida específica pela doença. Isoladamente, nenhum polimorfismo genético estudado interferiu na sobrevida dos pacientes com câncer de laringe. / Introduction: The prognosis of laryngeal squamous cell carcinoma is limited and survival rate is lower than 70%. The relationships between clinical and epidemiological characteristics have been fully investigated on the survival of patients with laryngeal tumors, but the effect of genetics polymorphisms on squamous cell carcinoma of larynx is not well-known. Objective: To study the role of genetic polymorphisms of genes related to the processes of invasion and metastasis (MMP1 and MMP3), inflammation (Interleukin 2, Interleukin 6, and LTA) and repair of DNA (XRCC1) in the prognosis of laryngeal squamous cell carcinoma. Material and methods: Cohort with 170 laryngeal squamous cell carcinoma patients with histological confirmation. The cases have their origin in a case-control study carried out in hospitals of Sao Paulo, Porto Alegre and Goiania. The information about vital status of patients had been raised from medical records. The extraction of DNA was carried out by Institute of Tropical Medicine of USP and genotyping was carried out by the Center of Cellular Therapy of the Hemocentro of Ribeirao Preto of Medical School of USP. Results: The studied genetic polymorphisms (MMP1 1607, MMP1 -519, MMP3 -1171, IL2 -384, IL2 114, IL6 -174, LTA 252 and XRCC1), separately analyzed, did not have any statistical significant effect on the overall and cause-specific survival. High levels of alcohol consumption (g/L/day) reduced the overall survival (80-119 g/L/day: hazard ratio(HR)=4.0 intervals with 95% of confidence (95%CI)=1.10-14.53; _120 g/L/day:HR=5.6 95%CI=1.71-18.24). Multiple Cox model revealed, when adjusted for MMP3 -1171 genetic polymorphism, lower survival for those patients (80-119g/L/day: HR=4.9 95%CI=1.07-22.91; _120 g/L/day: HR=6.3 95%CI=1.49-26.84). The clinical staging (CS) IV was a factor for low cause-specific survival (CS IV:HR=3.5 95%CI 1.67-7.28). In the multiple Cox model, adjusted for genetic polymorphism IL6 -174 and MMP1 1607, the survival of those patients droppe(HR=4.7 95%CI=1.38-16.25). Conclusions: In this cohort, only three of eight genetic polymorphisms studied were showed to be related with overall and causespecific survival, however only modifying the effect of unadjusted HR of alcohol consumption and tumor clinical staging in the overall and cause-specific survival respectively. None of the studied genetic polymorphisms, when analyzed separately,affected the survival of laryngeal cancer patients.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-09-03T19:05:12Z No. of bitstreams: 2 Análise epidemiológica dos cânceres de pulmão e da laringe em 30 anos.pdf: 985787 bytes, checksum: 31e7777e04381a2403335ed5d4f54552 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-03T19:05:12Z (GMT). No. of bitstreams: 2 Análise epidemiológica dos cânceres de pulmão e da laringe em 30 anos.pdf: 985787 bytes, checksum: 31e7777e04381a2403335ed5d4f54552 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-06-26 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Introduction: Studies have shown a relationship between smoking and various cancers, especially in the respiratory tract, causing major impact on public health. Lung cancer is the leading cause of cancer death in the world, accounting for 20 %, while Laryngeal cancer represents 3 %. Objective: To analyze trends in incidence and mortality from lung and larynx cancer in Goiania in 20 years and to assess trends in mortality for these cancers, over the past 30 years for the Brazilian regions. Methods: All incident cases of lung cancer and larynx were obtained from the database registered in Goiania RCBP/ACCG and deaths were extracted from the Mortality Information System (SIM/MS) for both, Goiania as for other Brazilian regions. Incidence rates and standardized mortality rates were calculated using the world standard population and calculating the trend used the Poisson regression model using Joinpoint Regression Program, Version 4.0.4. Graphs and tables were made using the Excel 2013 software. Results: Two points were analyzed: the first showed that the trend of incidence for lung and larynx cancers in both sexes and age groups in Goiania, were in contrast to trend mortality for lung cancer in women over 50 increased by 2.5% per year, during the study period in Goiania. The second article assessed the trends in national mortality for cancers of the lung and larynx showing that in Brazil, the mortality rates for lung cancer ranged from 14.30 to 15.52/100,000 in men and 3.99 to 7.37/100,000 in women from 1980 to 2009. Laryngeal cancer in this variation was from 3.76 to 3.59/100,000 for males and 0.47 to 0.38/100,000 in the female. The analysis of trends showed increased mortality for lung cancer in both male and female genders respectively (APC 1.7 %; 95% CI 1.6 to 1.8, p < 0.001, and APC 4.1%; 95% CI 3.9, the 4.2, p < 0.000). Mortality rates for laryngeal cancer in men the increase was 1.4 % (95% CI 1.2 to 1.5 p < 0.0001), the rate for women increased by 1.3 % (95 % 0.9 -1.6, p < 0.0001) during the study period, so there were similar for both neoplasms. Conclusion: Incidence trends were stable for lung and larynx cancer in both sexes and age groups, in contrast, for women aged over 50 years there has been increasing trends in mortality in Goiania. When Abstract xviii analyzing mortality at the national level, there was decline for lung cancer in south and southeast whereas for cancer of the larynx, there was a decrease only in southeastern Brazil. / Introdução: Estudos têm mostrado a relação entre fumo e diversos cânceres, especialmente do trato respiratório, causando grande impacto na saúde pública. O câncer de pulmão é a principal causa de morte por câncer representando 20% no mundo, enquanto que o câncer da laringe representa 3% destas mortes. Objetivo: Analisar as tendências da incidência e mortalidade por câncer de pulmão e laringe, em Goiânia, em 20 anos, bem como avaliar a tendência da mortalidade para esses cânceres nos últimos 30 anos para as regiões brasileiras. Métodos: Os casos incidentes de câncer de pulmão e laringe foram obtidos no banco de dados registrados no Registro de Câncer de base Populacional (RCBP-Goiânia) da Associação de Combate ao Câncer em Goiás (ACCG) e os óbitos foram extraídos do Sistema de Informações de Mortalidade (SIM) do Ministério da Saúde (MS), tanto para Goiânia quanto para as demais regiões brasileiras. Foram calculadas taxas de incidência e mortalidade padronizadas utilizando a população padrão mundial, e para o cálculo da tendência utilizou-se o modelo de regressão de Poisson, utilizando o software Joinpoint Regression Program, Version 4.0.4. Gráficos e tabelas foram confeccionadas utilizando o software excel 2013. Resultados: Foram analisados dois pontos: o primeiro verificou a tendência de incidência para o câncer de pulmão, e da laringe em ambos os sexos e grupos etários em Goiânia, cujo resultado demonstrou estabilidade no período estudado. A tendência da mortalidade para o câncer de pulmão, em mulheres acima de 50, aumentou em 2,5% ao ano, em Goiânia. As tendências da mortalidade no Brasil, para os cânceres de pulmão e da laringe, mostraram que as taxas de mortalidade variaram de 14,30 a 15,52/100000, em homens e 3,99 a 7,37/100000, em mulheres, para o câncer de pulmão, entre 1980 a 2009. Para o câncer da laringe essa variação foi 3,76 a 3,59/100000 no sexo masculino e 0,47 a 0,38/100000 no feminino. A análise de tendências evidenciou aumento da mortalidade para o câncer de pulmão em ambos os sexos, masculino e feminino, respectivamente (Média Percentual Anual (MPA) 1,7% IC95% 1,6 a 1,8; p<0,001; e MPA 4,1% IC95% 3,9 a 4,2; p<0,000). As taxas de mortalidade Resumo xvi para câncer da laringe para os homens aumentou 1,4% ao ano (IC95% 1,2- 1,5 p<0,0001), para as mulheres, esse aumento foi 1,3% (IC95% 0,9 -1,6; p<0,0001), portanto foram semelhantes para ambos os sexos. Conclusão: As tendências de incidência se mantiveram estáveis para o câncer de laringe e do pulmão, em ambos os sexos e grupos de idade, em contrapartida, para mulheres com idade acima de 50 anos houve tendências de aumento da mortalidade em Goiânia. Ao analisar-se a mortalidade a nível nacional, observou-se declínio para o câncer de pulmão no sul e sudeste enquanto que para o câncer da laringe, houve declínio apenas na região sudeste do Brasil.