O presente estudo teve por objetivo estudar a participação da apoptose na carcinogênese hepatocelular, quantificando os corpos apoptóticos imunomarcados por caspase-3 clivada em amostras de carcinoma hepatocelular (CHC) em pacientes com ou sem cirrose, comparando também estes achados com amostras correspondentes de fígado não tumoral. Visou também à análise semi-quantitativa da imuno-expressão da proteína p53, Bax e Citocromo-C, relacionadas à via mitocondrial da apoptose em busca de eventuais relações com as variáveis clínicopatológicas dos carcinomas hepatocelulares. A análise comparativa da distribuição das diversas proteínas aqui estudadas foi ainda efetuada, com vistas à possível demonstração de sua interação no processo de apoptose em CHC. Amostras selecionadas de 79 casos de CHC foram distribuídas em micromatriz tecidual e submetidas a pesquisa imuno-histoquímica com amplificação por polímeros curtos de dextran ligados a peroxidase. IA foi maior nos CHC que nas amostras não-neoplásicas, mostrando ainda tendência a associação com o grau histológico do CHC .A imuno-expressão de p53 foi maior nos CHC em fígado cirrótico (CHC-C), em casos com invasão vascular, e nos graus histológicos altos. Houve maior imunoexpressão de citocromo c em CHC-C, sendo importante sua associação com p53. Bax mostrou apenas tendência a associação com o tamanho do CHC. Essas evidências contribuem para a compreensão da importância da via mitocondrial da apoptose mediada pela proteína p53 no CHC, destacando também prováveis diferenças do mecanismo carcinogenético na presença ou não de cirrose / This study aimed at the assesment of aspects of the role of apoptosis in hepatocellular carcinogenesis, quantifying apoptotic bodies immunomarked by cleaved caspase-3 in samples of hepatocellular carcinoma (HCC) in patients with or without cirrhosis, further comparing these findings to those from samples in non-tumoral areas of these livers. We also aimed herein to semiquantitate the immunoexpression of p53, Bax, Cytochrome-C, participants of the mitochondrial pathway of apoptosis, searching for possible relations with clinico-pathological variables in HCC. Samples from 79 cases of HCC were arranged in tissue microarrays were and submitted to immunohistochemical reaction with signal amplification achieved by the short-polymer-peroxidase system. Apoptotic index measured by immunoexpression of cleaved-caspase 3 was higher in HCC than in samples from non-neoplastic areas. p53 immunoexpression was higher in HCC occurring in cirrhotic livers, (HCC-C), in cases with vascular invasion and in higher histological grades. Cytochrome-c immunoexpression was also higher in HCC-C and, interestingly, was directly related to p53. Bax immunoreactivity showed only a trend for a relation with the size of HCC. The evidences from the present study further demonstrate the importance of p53-mediated pathway of apoptosis in HCC, and also point for possible differences in carcinogenesis in cirrhotic versus non-cirrhotic livers

O presente estudo teve por objetivo estudar a participação da apoptose na carcinogênese hepatocelular, quantificando os corpos apoptóticos imunomarcados por caspase-3 clivada em amostras de carcinoma hepatocelular (CHC) em pacientes com ou sem cirrose, comparando também estes achados com amostras correspondentes de fígado não tumoral. Visou também à análise semi-quantitativa da imuno-expressão da proteína p53, Bax e Citocromo-C, relacionadas à via mitocondrial da apoptose em busca de eventuais relações com as variáveis clínicopatológicas dos carcinomas hepatocelulares. A análise comparativa da distribuição das diversas proteínas aqui estudadas foi ainda efetuada, com vistas à possível demonstração de sua interação no processo de apoptose em CHC. Amostras selecionadas de 79 casos de CHC foram distribuídas em micromatriz tecidual e submetidas a pesquisa imuno-histoquímica com amplificação por polímeros curtos de dextran ligados a peroxidase. IA foi maior nos CHC que nas amostras não-neoplásicas, mostrando ainda tendência a associação com o grau histológico do CHC .A imuno-expressão de p53 foi maior nos CHC em fígado cirrótico (CHC-C), em casos com invasão vascular, e nos graus histológicos altos. Houve maior imunoexpressão de citocromo c em CHC-C, sendo importante sua associação com p53. Bax mostrou apenas tendência a associação com o tamanho do CHC. Essas evidências contribuem para a compreensão da importância da via mitocondrial da apoptose mediada pela proteína p53 no CHC, destacando também prováveis diferenças do mecanismo carcinogenético na presença ou não de cirrose / This study aimed at the assesment of aspects of the role of apoptosis in hepatocellular carcinogenesis, quantifying apoptotic bodies immunomarked by cleaved caspase-3 in samples of hepatocellular carcinoma (HCC) in patients with or without cirrhosis, further comparing these findings to those from samples in non-tumoral areas of these livers. We also aimed herein to semiquantitate the immunoexpression of p53, Bax, Cytochrome-C, participants of the mitochondrial pathway of apoptosis, searching for possible relations with clinico-pathological variables in HCC. Samples from 79 cases of HCC were arranged in tissue microarrays were and submitted to immunohistochemical reaction with signal amplification achieved by the short-polymer-peroxidase system. Apoptotic index measured by immunoexpression of cleaved-caspase 3 was higher in HCC than in samples from non-neoplastic areas. p53 immunoexpression was higher in HCC occurring in cirrhotic livers, (HCC-C), in cases with vascular invasion and in higher histological grades. Cytochrome-c immunoexpression was also higher in HCC-C and, interestingly, was directly related to p53. Bax immunoreactivity showed only a trend for a relation with the size of HCC. The evidences from the present study further demonstrate the importance of p53-mediated pathway of apoptosis in HCC, and also point for possible differences in carcinogenesis in cirrhotic versus non-cirrhotic livers

O transplante autólogo de tecido ovariano constitui alternativa relevante na preservação da fertilidade e da função hormonal ovariana em mulheres sujeitas à falência ovariana prematura e infertilidade, por causas malignas, tratamentos adjuvantes ou cirurgias. É a única opção para crianças, fase pré-puberal e para mulheres que não podem retardar a quimioterapia ou não podem ser submetidas à estimulação do ciclo. O transplante ovariano autólogo pode ser, quanto ao local de reimplantação, ortotópico ou heterotópico e, quanto à conservação, a fresco ou após o período de criopreservação. As várias etapas envolvidas neste transplante são estudadas mundialmente na atualidade, como a retirada e preservação do tecido ovariano, as técnicas de criopreservação, o local apropriado para o reimplante e as possibilidades de redução da perda folicular. A avaliação da apoptose - morte celular programada - é útil na avaliação da rejeição e viabilidade dos enxertos de transplantes estabelecidos na prática clínica, tanto autólogos como heterólogos. Com o intuito de utilizar animais de maior porte, conseguir seguimento de médio prazo e realizar os procedimentos cirúrgicos por via laparoscópica, padrão ouro em humanos, o presente estudo utilizou como modelo experimental fêmeas suínas, em idade reprodutiva, da raça Minipig. Este projeto teve como propósito avaliar a influência da criopreservação e do local de implante na qualidade e na viabilidade do transplante autólogo de ovário, a fresco e após criopreservação, no tecido celular subcutâneo e na região intraperitoneal peri-infundibular. Foram avaliados a quantidade e a densidade folicular dos implantes e os aspectos morfológicos e histomorfométricos, bem como a apoptose, por meio da imunoexpressão de proteínas proapoptóticas- Bax e antiapoptóticas-Bcl-2, além da Caspase 3-clivada, fase final das vias extrínseca e intrínseca dos mecanismos de apoptose.Quarenta animais foram divididos em cinco grupos: Controle com ooforectomia (Grupo I), ooforectomia e transplante a fresco subcutâneo (Grupo II), a ooforectomia e transplante fresco intraperitoneal (Grupo III), ooforectomia e transplante criopreservado subcutâneo (Grupo IV) e ooforectomia e transplante criopreservado intraperitoneal (GrupoV). Os resultados mostraram que independente da técnica empregada, havia folículos em desenvolvimento e corpos lúteos em todos os tecidos ovarianos transplantados; que a contagem de folículos antrais não degenerados foi menor nos grupos após criopreservação em relação ao grupo controle e que a imunoexpressão sugestiva de apoptose ocorreu em todos os grupos transplantados, sendo maior nos transplantes intraperitoneais. Concluiu-se que a técnica utilizada para o transplante de ovário e criopreservação foi viável no modelo suíno, em tecido celular subcutâneo e na região intraperitoneal peri-infundibular. O transplante autólogo heterotópico subcutâneo apresentou melhores taxas de apoptose que o transplante ortotópico. / Autotransplantation of ovarian tissue is an important alternative to preserve fertility and hormonal ovarian function in women undergoing ovarian failure and premature infertilidade, because of cancer or surgery. It is the only option for infants, pre-pubertal patients and for women who can not delay chemotherapy or not may be subjected to stimulation of the cycle. The various steps involved in the transplant are studied worldwide today, as the removal and preservation of ovarian tissue, the techniques of cryopreservation, the appropriate site and mechanisms to reduce follicular loss. Assessment of apoptosis - programmed cell death-is useful in the study of the viability of the grafts and rejection of transplants established in clinical practice, both autologous and heterologous. In order to use larger animals, getting following medium term (over 21 days) and to perform surgical procedures by laparoscopy (gold standard in humans), this study used an experimental model sows, reproductive age, Minipig race. This project aims to evaluate the influence of cryopreservation and implantation site of the quality and viability of ovarian autografts, fresh and after cryopreservation, at subcutaneous site and at intraperitoneal site. We analyzed the quantity and density of follicular implants and the morphological and histomorphometric as well as apoptosis, by proteins immunoexpression antiapoptotic and proapoptotic. Forty animals were divided into five groups: Control with oophorectomy (Group I), oophorectomy and fresh transplantation to subcutaneous site (Group II), oophorectomy and fresh transplantation to intraperitoneal site (Group III), oophorectomy and transplantation of cryopreserved ovarian tissue to subcutaneous site (Group IV) and oophorectomy and transplantation of cryopreserved ovarian tissue to intraperitoneal site (Group V). We concluded that the autologous ovarian transplantation was feasible in the technical proposals, in subcutaneous and intraperitoneal site in the porcine model; that regardless of the technique, there was developing follicles and corpora lutea in all ovarian tissue transplanted; that antral non-degenerate follicle count was lower in groups after cryopreservation that in the control group and that the immunoexpression of apotposis occurred in all transplanted groups, more evident in intraperitoneal transplants

Prostatacancer är den sjätte mest förekommande cancertypen i världen, och den tredje vanligaste cancertypen bland män. De olika typer av behandlingar som finns idag botar oftast inte sjukdomen. Det är därför viktigt att utveckla bättre behandlingsmetoder. Det är sedan tidigare känt att mangan kan orsaka apoptos i olika celltyper. Det ger en möjlighet att använda mangan för att hämma cancer och det är därför viktigt att veta vilka apoptotiska markörer som är involverade. Syftet med projektet var att undersöka om de sker en ökning av de apoptotiska markörerna kluven Kaspas-3 och kluven PARP efter manganbehandling av prostatacancerceller. Därefter kunna avgöra om manganbehandlingen har orsakat celldöd genom att inducera apoptos. Under projektet odlades prostatacancerceller (PC3) som sedan behandlades med 200 µM mangan under 6, 24 och 48 timmar. Därefter mättes mängden av proteinerna kluven Kaspas-3 och kluven PARP med hjälp av ett sandwich-ELISA kit. En tydlig stegvis ökning av apoptosmarkörerna med inkuberingstid hade förväntats. Det förväntade resultatet erhölls inte, för kluven Kaspase-3 ledde manganbehandlingen t.o.m. till en sänkning av koncentrationen. Det kan ha uppstått problem vid analysen som t.ex dålig lysering av cellerna eller ojämn tillväxt av dem. Det behövs fler studier för att utreda detta och för att undersöka andar potentiella apoptosmarkörer. / Prostate cancer is the sixth most common cancer type in the world, and the third most common cancer type among men. The different types of treatments that are available today do not usually cure the disease. It is therefore important to develop better treatment methods. It has previously been stated that manganese can cause apoptosis in different cell types. It provides an opportunity to use manganese to inhibit cancer and it is therefore important to know which apoptotic markers that are involved. The purpose of the project was to investigate whether an increase in the apoptotic markers cleaved Kaspas-3 and cleaved PARP after manganese treatment of prostate cancer cells. Thereafter, it can determine whether manganese treatment has caused cell death by inducing apoptosis. During the project prostate cancer cells (PC3-cells) were cultivated, then treated with 200 µM manganese for 6, 24 and 48 hours. The amount of the proteins cleaved Kaspas-3 and cleaved PARP was measured by using a sandwich ELISA kit. A clear gradual increase of apoptotic markers with incubation time was expected. The expected result was not obtained, for cleaved Kaspase-3 manganese treatment even decreased the concentrations. There may have been problems with the performance of the analysis such as poor lysis of the cells or uneven growth of the cells. More studies are required to investigate this and other potential apoptotic markers.

O transplante autólogo de tecido ovariano constitui alternativa relevante na preservação da fertilidade e da função hormonal ovariana em mulheres sujeitas à falência ovariana prematura e infertilidade, por causas malignas, tratamentos adjuvantes ou cirurgias. É a única opção para crianças, fase pré-puberal e para mulheres que não podem retardar a quimioterapia ou não podem ser submetidas à estimulação do ciclo. O transplante ovariano autólogo pode ser, quanto ao local de reimplantação, ortotópico ou heterotópico e, quanto à conservação, a fresco ou após o período de criopreservação. As várias etapas envolvidas neste transplante são estudadas mundialmente na atualidade, como a retirada e preservação do tecido ovariano, as técnicas de criopreservação, o local apropriado para o reimplante e as possibilidades de redução da perda folicular. A avaliação da apoptose - morte celular programada - é útil na avaliação da rejeição e viabilidade dos enxertos de transplantes estabelecidos na prática clínica, tanto autólogos como heterólogos. Com o intuito de utilizar animais de maior porte, conseguir seguimento de médio prazo e realizar os procedimentos cirúrgicos por via laparoscópica, padrão ouro em humanos, o presente estudo utilizou como modelo experimental fêmeas suínas, em idade reprodutiva, da raça Minipig. Este projeto teve como propósito avaliar a influência da criopreservação e do local de implante na qualidade e na viabilidade do transplante autólogo de ovário, a fresco e após criopreservação, no tecido celular subcutâneo e na região intraperitoneal peri-infundibular. Foram avaliados a quantidade e a densidade folicular dos implantes e os aspectos morfológicos e histomorfométricos, bem como a apoptose, por meio da imunoexpressão de proteínas proapoptóticas- Bax e antiapoptóticas-Bcl-2, além da Caspase 3-clivada, fase final das vias extrínseca e intrínseca dos mecanismos de apoptose.Quarenta animais foram divididos em cinco grupos: Controle com ooforectomia (Grupo I), ooforectomia e transplante a fresco subcutâneo (Grupo II), a ooforectomia e transplante fresco intraperitoneal (Grupo III), ooforectomia e transplante criopreservado subcutâneo (Grupo IV) e ooforectomia e transplante criopreservado intraperitoneal (GrupoV). Os resultados mostraram que independente da técnica empregada, havia folículos em desenvolvimento e corpos lúteos em todos os tecidos ovarianos transplantados; que a contagem de folículos antrais não degenerados foi menor nos grupos após criopreservação em relação ao grupo controle e que a imunoexpressão sugestiva de apoptose ocorreu em todos os grupos transplantados, sendo maior nos transplantes intraperitoneais. Concluiu-se que a técnica utilizada para o transplante de ovário e criopreservação foi viável no modelo suíno, em tecido celular subcutâneo e na região intraperitoneal peri-infundibular. O transplante autólogo heterotópico subcutâneo apresentou melhores taxas de apoptose que o transplante ortotópico. / Autotransplantation of ovarian tissue is an important alternative to preserve fertility and hormonal ovarian function in women undergoing ovarian failure and premature infertilidade, because of cancer or surgery. It is the only option for infants, pre-pubertal patients and for women who can not delay chemotherapy or not may be subjected to stimulation of the cycle. The various steps involved in the transplant are studied worldwide today, as the removal and preservation of ovarian tissue, the techniques of cryopreservation, the appropriate site and mechanisms to reduce follicular loss. Assessment of apoptosis - programmed cell death-is useful in the study of the viability of the grafts and rejection of transplants established in clinical practice, both autologous and heterologous. In order to use larger animals, getting following medium term (over 21 days) and to perform surgical procedures by laparoscopy (gold standard in humans), this study used an experimental model sows, reproductive age, Minipig race. This project aims to evaluate the influence of cryopreservation and implantation site of the quality and viability of ovarian autografts, fresh and after cryopreservation, at subcutaneous site and at intraperitoneal site. We analyzed the quantity and density of follicular implants and the morphological and histomorphometric as well as apoptosis, by proteins immunoexpression antiapoptotic and proapoptotic. Forty animals were divided into five groups: Control with oophorectomy (Group I), oophorectomy and fresh transplantation to subcutaneous site (Group II), oophorectomy and fresh transplantation to intraperitoneal site (Group III), oophorectomy and transplantation of cryopreserved ovarian tissue to subcutaneous site (Group IV) and oophorectomy and transplantation of cryopreserved ovarian tissue to intraperitoneal site (Group V). We concluded that the autologous ovarian transplantation was feasible in the technical proposals, in subcutaneous and intraperitoneal site in the porcine model; that regardless of the technique, there was developing follicles and corpora lutea in all ovarian tissue transplanted; that antral non-degenerate follicle count was lower in groups after cryopreservation that in the control group and that the immunoexpression of apotposis occurred in all transplanted groups, more evident in intraperitoneal transplants

Einleitung: Nasenhöhlentumoren stellen mit bis zu 47 % die Hauptursache für Nasenausfluss beim Hund dar. Sie sind überwiegend maligne, zu 60 % Karzinome und zu 34 % Sarkome. Die mediane Überlebenszeit (MÜZ) liegt ohne Therapie bei etwa drei Monaten. Nach einer Bestrahlungstherapie beträgt sie etwa 8-20 Monate. Eine Therapie mit klassischen Chemotherapeutika oder eine Tumorablation über eine offen-chirurgische Rhinotomie führen nicht zu einer Verlängerung der Überlebenszeit. Durch ein neues Therapieverfahren, die endoskopisch interventionelle Zytoreduktion (EIZ), werden bei deutlich weniger Nebenwirkungen und Sitzungen in Allgemeinanästhesie ähnliche Überlebenszeiten erreicht wie durch eine Radiotherapie. Da es sich bei der EIZ um eine Zytoreduktion handelt, bei der die Nasenhöhlentumoren nicht mit Sicherheitsabstand im gesunden Gewebe entfernt werden können, stellt sich die Frage, ob die Überlebenszeit zusätzlich durch adjuvante Therapeutika verlängert werden kann. Als solche kommen beispielsweise Tyrosinkinase-Inhibitoren (TKI) und Cyclooxygenase-2 (COX-2)-Inhibitoren infrage, deren Zielstruktur-Expression besonders in kaninen nasalen Sarkomen noch unbekannt ist. Ziele der Untersuchungen: Ziel dieser Arbeit ist, anhand von Bioptaten von kaninen nasalen Karzinomen und Sarkomen eine immunhistochemische Charakterisierung durchzuführen. Hierzu wurden 10 Marker ausgewählt. Besonders im Fokus standen die Wachstumsfaktorrezeptoren vascular endothelial growth factor receptor-2 (VEGFR-2) und epidermal growth factor receptor (EGFR) sowie COX-2, die ersten beiden als Zielstrukturen von TKI und der letztere als Zielstruktur von COX-2-Inhibitoren. So soll eine Wirksamkeit dieser Medikamente bei kaninen nasalen Karzinomen und Sarkomen evaluiert werden. Weiterhin soll eine Korrelation zwischen der Expression von bestimmten Markern (p53, Ki-67, aktivierte Caspase-3, Survivin, E-Cadherin) mit den klinischen Daten zur Tumorkategorie und Überlebenszeit der Patienten untersucht werden. Außerdem soll als Grundlage für den Einsatz neuartiger Medikamente analysiert werden, ob EGFR, VEGFR-2 oder COX-2 in Karzinomen und Sarkomen unterschiedlich stark exprimiert werden. Tiere, Material und Methoden: Es wurden 19 Karzinome, sieben Sarkome und drei andere Tumorarten (ein malignes Melanom, zwei undifferenzierte maligne Tumoren unklarer Histogenese) retrospektiv immunhistochemisch auf die Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin, E-Cadherin, Zytokeratinen und Vimentin untersucht. Von drei Patienten wurden insgesamt vier Rezidivbioptate entnommen und ebenfalls immunhistochemisch untersucht. Beidseitige Nasenschleim-hautbioptate von neun gesunden Beagles dienten als Kontrollgruppe (genehmigungspflichtiger Tierversuch: TVV 02/18, Landesdirektion Sachsen). Alle Bioptate wurden während einer standardisierten Diagnostik mit computer-/ magnetresonanztomographischer Untersuchung - bei der auch ein Staging in vier Tumor-Kategorien (T1-T4) durchgeführt wurde - und Rhinoskopie zwischen Jan. 2015 und Dez. 2018 entnommen. Die immunhistochemische Untersuchung der in Formalin fixierten und in Paraffin eingebetteten Gewebeschnitte wurde mit der Avidin-Biotin-Komplex-Methode durchgeführt, nachdem die histopathologischen Diagnosen an Hämatoxylin-Eosin-gefärbten Schnitten gestellt wurden. Die immun-histochemischen Färbungen wurden entweder quantitativ oder semiquantitativ ausgewertet. Die Ergebnisse wurden auf Normalverteilung getestet und u.a. mit One-way Anova oder Kruskal-Wallis Test analysiert. Die MÜZ wurde mit der Kaplan Meier Methode berechnet und mit Log-Rank Test und Gehan-Breslow-Wilcoxon Test verglichen (Signifikanzniveau alpha = 5 %). Ergebnisse: 29 Hunde haben die Einschlusskriterien erfüllt. 14 Hunde wurden unmittelbar nach der Diagnostik euthanasiert; 15 Hunde wurden mit einer EIZ behandelt. Die MÜZ der Patienten in T1 (n = 3) nach EIZ betrug 1362 Tage und war signifikant länger als die MÜZ der Patienten in T2 (n = 1) mit 379 Tagen, in T3 (n = 8) mit 250 Tagen und in T4 (n = 1) mit 75 Tagen (p = 0,0062). Von den nasalen Karzinomen zeigten 68 % für EGFR, 100 % für VEGFR-2, 63 % für COX-2, 100 % für Survivin und 100 % für E-Cadherin eine immunhistochemisch positive Reaktion. Von den nasalen Sarkomen reagierten 100 % für VEGFR-2, 57 % für COX-2 und 86 % für Survivin positiv. Die Proteine EGFR und E-Cadherin werden ausschließlich von epithelialen Zellen exprimiert. Die Expression lag somit bei den vorliegenden Sarkomen bei 0 %. Unter den anderen Tumoren waren 33 % für EGFR, 100 % für VEGFR-2, 67 % für COX-2, 67 % für Survivin und 67 % für E-Cadherin positiv. Die mediane Expression von p53 lag bei 0,9 %, von Ki-67 bei 25 % und von aktivierter Caspase-3 bei 0,7 %. Die Unterschiede in der Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin und E-Cadherin zwischen den einzelnen histogenetischen Gruppen sowie zwischen den vier Tumor-Kategorien und in der MÜZ waren nicht signifikant. Eine Korrelation der VEGFR-2-Expression mit der MÜZ oder den T-Kategorien konnte nicht untersucht werden, da alle Tumoren der drei histogenetischen Gruppen VEGFR-2-positiv waren. 100 % der Karzinome zeigten eine Zytokeratin-Expression, 0 % eine Vimentin-Expression. Sarkome verhielten sich dazu konträr. In den anderen Tumoren konnten weder Zytokeratine noch Vimentin immunhistochemisch nachgewiesen werden. In den Rezidivbioptaten war ein Anstieg der COX-2 und aktivierte-Caspase-3-Expression zu beobachten, der aufgrund der geringen Fallzahl nicht statistisch untersucht werden konnte. Schlussfolgerungen: In der vorliegenden Studie wurden erstmalig kanine nasale Karzinome und Sarkome vergleichend immunhistochemisch untersucht. Weiterhin konnte erstmalig gezeigt werden, dass auch mesenchymale und andere Tumoren in vergleichbarer Häufigkeit wie Karzinome der Nase COX-2 exprimieren, wodurch ein Einsatz von COX-2-Inhibitoren nach einer Zytoreduktion bei Nasenhöhlentumoren allgemein von Nutzen sein könnte. Da alle Tumoren VEGFR-2 und die Mehrzahl der Karzinome (68 %) EGFR exprimierten, könnte eine adjuvante Therapie nach EIZ durch einen TKI mit VEGFR-2 oder EGFR als Zielstruktur einen positiven Einfluss auf die Überlebenszeit der erkrankten Hunde haben. Durch die Expression von E-Cadherin und Zytokeratinen in 100 % der Karzinome und 0 % der Sarkome sowie der Expression von Vimentin in 0 % der Karzinome und 100 % der Sarkome konnte die histopathologische Diagnose im Hinblick auf die Histogenese der Tumoren bestätigt werden. Auf der Grundlage der Ergebnisse der vorliegenden Studie sollte eine klinische Studie zur Anwendung von TKI und COX-2-Inhibitoren zur Untersuchung der klinischen Wirksamkeit und Sicherheit bei nasalen Tumoren von Hunden durchgeführt werden.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 125 / Introduction: Tumours of the nasal cavity are the main cause of nasal discharge in dogs (up to 47 %). They are almost always malignant, 60 % are carcinomas and 34 % are sarcomas. Without performing any treatment, the median survival time (MST) is about three months. After radiation therapy, the MST is about 8-20 months. A therapy with conventional chemotherapeutics or tumour ablation via open surgical rhinotomy does not prolong the survival time. A new treatment method, the endoscopic interventional cytoreduction (EIC), achieves survival times similar to radiotherapy with considerably fewer sessions under general anaesthesia and fewer potential adverse events. As EIC is a cytoreduction procedure in which the intranasal tumours cannot be removed with safety margins in surrounding healthy tissue, the question arises whether survival could be prolonged by an additional application of adjuvant therapeutics. As such, for example, tyrosine kinase inhibitors (TKIs) and cyclooxygenase-2 (COX-2) inhibitors may be considered, whose target expression is still unknown, especially in canine nasal sarcomas. Aims of the study: One aim of this study is to perform an immunohistochemical characterisation on the basis of biopsy specimens from canine nasal carcinomas and sarcomas. For this purpose, 10 markers were selected. We particularly focused on the growth factor receptors vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) as well as COX-2, the first two being targets of TKIs and the latter one being the target of COX-2-inhibitors. Thus, a possible efficacy of these drugs in canine nasal carcinomas and sarcomas should be evaluated. Furthermore, a correlation between the expression of specific markers (p53, Ki-67, cleaved caspase-3, survivin, E-cadherin) with clinical data of the tumour stage and patient survival time should be investigated. Moreover, it will be analysed as a basis for the use of novel drugs whether EGFR, VEGFR-2 or COX-2 are differentially expressed in carcinomas and sarcomas. Animals, Material and Methods: A total of 19 carcinomas, seven sarcomas and three other tumour types (one malignant melanoma, two undifferentiated malignant tumorus of unclear histogenesis) were retrospectively examined by immunohistochemistry for the expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin, E-cadherin, cytokeratins and vimentin. A total of four biopsies from recurrent tumours were obtained from three patients and also examined by immunohistochemistry. Bilateral nasal mucosal samples from nine healthy beagles served as a control group (animal experiment requiring approval: TVV 02/18, Directorate of the Federal State of Saxony, Germany). All biopsy specimens were collected during a standardised diagnostic procedure with computer /magnetic resonance tomography, which also included a staging into four tumour stages (T1-T4) and rhinoscopy between Jan 2015 and Dec 2018. The immunohistochemical examination of tissue sections fixed in formalin and embedded in paraffin was performed using the avidin-biotin complex method after histopathological diagnoses had been made on haematoxylin-eosin stained sections. The immunohistochemically stained sections were evaluated either quantitatively or semiquantitatively. Results were tested for normal distribution and analysed by the one-way anova or Kruskal-Wallis test, among others. MST was calculated using the Kaplan Meier method and compared with the log-rank test and Gehan-Breslow-Wilcoxon test (significance level alpha = 5 %). Results: A total of 29 dogs met the inclusion criteria. A total of 14 dogs were euthanised immediately after diagnosis; 15 dogs were treated with an EIC. The MST of patients in T1 (n = 3) after EIC was 1362 days and was significantly longer than the MST of those patients in T2 (n = 1) at 379 days, T3 (n = 8) at 250 days and T4 (n = 1) at 75 days (p = 0.0062). Of the nasal carcinomas, 68 % were immunohistochemically positive for EGFR, 100 % for VEGFR-2, 63 % for COX-2, 100 % for survivin and 100 % for E-cadherin. Of the nasal sarcomas, 100 % reacted positively for VEGFR-2, 57 % for COX-2 and 86 % for survivin. The proteins EGFR and E-cadherin were expressed exclusively by epithelial cells and, therefore, the expression was 0 % in the present sarcomas. Amongst the other tumours, 33 % were positive for EGFR, 100 % for VEGFR-2, 67 % for COX-2, 67 % for survivin and 67 % for E-cadherin. The median expression of p53 was 0.9 %, that of Ki-67 25 % and that of cleaved caspase-3 0.7 %. Differences in expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin and E-cadherin among the histogenetic groups, the four tumour stages and in MST were not significant. However, a correlation of VEGFR-2 expression with MST or the tumour stages could not be investigated because all tumours in the three histogenetic groups were VEGFR-2 positive. A total of 100 % of carcinomas showed cytokeratin expression, and 0 % showed vimentin expression. Sarcomas behaved in a contrary manner. In the other tumours, neither cytokeratins nor vimentin could be detected by immunohistochemistry. An increase in COX-2 and cleaved caspase-3 expression was observed in the recurrent tumour biopsies, which could not be statistically investigated due to the small number of cases. Conclusions: In the present study, canine nasal carcinomas and sarcomas were investigated comparatively by immunohistochemistry for the first time. Again, it was shown for the first time that mesenchymal and other tumours also express COX-2 at comparable frequencies to carcinomas of the nose, suggesting that the use of COX-2 inhibitors after cytoreduction may be of general benefit in nasal cavity tumours. As all tumours expressed VEGFR-2 and the majority of carcinomas (68 %) expressed EGFR, the adjuvant therapy after EIC by a TKI targeting VEGFR-2 or EGFR could have a beneficial effect on the survival of diseased dogs. The expression of E-cadherin and cytokeratins in 100 % of the carcinomas and 0 % of the sarcomas as well as the expression of vimentin in 0 % of the carcinomas and 100 % of the sarcomas confirmed the histopathological diagnosis with regard to the histogenesis of the tumours. Based on these results of the present study, a clinical trial should be performed on the use of TKIs and COX-2 inhibitors to investigate the clinical efficacy and safety of canine nasal tumours.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 125