Rinovírus (RV) é freqüentemente detectado nos tecidos tonsilares e nas secreções de nasofaringe de pacientes com doença adenotonsilar crônica, sem sintomas de infecção respiratória aguda (IRA). O objetivo deste estudo foi investigar a infecção por rinovírus em tonsilas humanas, com base em dois aspectos: infecção in vivo de células linfóides de tonsilas humanas naturalmente infectadas; e infecção ex vivo de células dissociadas desses tecidos inoculadas com rinovírus, visando a contribuir para elucidar possíveis mecanismos de infecção em amígdalas palatinas e adenóides humanas. De um total de 104 pacientes com doenças adenotonsilar crônicas, 21.1% (22/104) e 42.3% (44/104) apresentaram respectivamente amígdalas palatinas e adenóides positivas para RV por PCR. A replicação viral foi confirmada por hibridização in situ com sonda para intermediário replicativo nas regiões internas e externas aos folículos linfóides de amígdalas e adenóides, bem como em porções do epitélio ciliado de adenoides, e apenas raramente nas células epiteliais escamosas de tonsilas palatinas. A presença e distribuição de proteína estrutural do capsídeo viral foi detectada por imunohistoquímica (IHQ), utilizando anticorpos contra proteínas estruturais virais VP1 e VP2 nas tonsilas positivas para RV por qPCR. Os resultados indicaram marcação positiva tanto na superfície (epitélio), quanto em regiões extrafoliculares e centros germinativos. Em seguida, foi possível verificar a co-localização da marcação positiva da proteína estrutural VP2 de RV com marcadores linfocitários de membrana. Células CD4 + e CD20 + apresentaram marcação positiva para VP2 verificada usando estratégia de \'sequential immuno-peroxidase labelling and erasing\' (SIMPLE). Culturas primárias de células linfomononucleares (CLMN) de tonsilas sabidamente negativas para RV por PCR, foram infectadas in vitro, com RV (MOI=1). A replicação de RV foi titulada por TCID50, mostrando aumento inicial (24 h) e subsequente queda após 48 horas. Por IF observamos que os fenótipos de CLMN infectadas com RV in vitro foram células T CD4 + e B, mas também com células CD8 +, CD56 + e CD33 +. RV não infectou células CD123 +. RV foi isolado em WI- 38 e HeLa a partir de tecidos e secreções de nasofaringe de pacientes com hipertrofia tonsilar sem sintomas de infecção respiratória aguda. Nossos resultados confirmam que tonsilas de pacientes sem sintomas respiratórios agudos podem ser reservatórios de RV, que infecta não somente epitélio, mas também CLMN (frequentemente linfócitos T CD4 + e linfócitos B). A detecção de RNA intermediário replicativo e proteínas estruturais VP1 e VP2 nas tonsilas hipertróficas, além do isolamento de vírus infeccioso a partir de tecidos e secreções nasofaríngeas, classificam tonsilas hipertróficas como sítios de infecção e replicação de RV, e sugerem que esses indivíduos hipertróficos são portadores assintomáticos de RV persistente, e podem ser importantes fontes de transmissão de RV na comunidade. / The chronic adenotonsillar diseases are frequent otorhinolaryngologic conditions caused by chronic inflammation of adenoids and palatine tonsils. Rhinovirus (RV) is highly frequently detected in secretions, and tonsillar tissues from patients experience chronic tonsillar hypertrophy without symptoms of ARI, and our goal is to full understanding of viral infections in hypertrophic tonsillar tissues by RV. Of 104 enrolled patients with adenotonsillar chronic diseases, 21.1% (22/104) and 42.3% (44/104) had palatine tonsils and adenoids positive for RV by qPCR, respectively. RV Viral replication was confirmed by in situ hybridizations. Minus-strand RNA were detected in all tested samples (7 tonsils and 9 adenoids), and positive reactions were seen inside and outside of lymphoid follicles from tonsils and adenoids, in the ciliated epithelium of the adenoids and rarely in positive squamous epithelium cells from tonsils. The presence of viral structural protein VP1 and VP2 was detected within and outside of the lymphoid follicles from tonsils and adenoids, and also in epithelial cells from adenoids by immunohistochemistry (IHC). Later, by sequential immuno-peroxidase labelling and erasing protocol (SIMPLE), we saw co-localization of RV VP2 capsid protein staining with CD4 positive cells and CD20 positive cells. We confirmed that RV could infect primary culture of tonsilar mononuclear cells (TMNCs). Additionally, intracellular replication of RV in TMNCs, measured by TCID50 in HeLa cells, had an initial increase in the first 24 hours, and dropped at 48 hours post infection. Immunolocalization staining with anti-RV and TMNCs surface markers indicated that phenotypes of susceptible cells were T-cells both CD4+ and CD20+, but also, we saw co-localization of VP-2 protein with CD8+ cells, CD56+ cells and CD33+ cells. RV-16 couldn\'t infect CD123+ cell in our experiments. Finally, we were able to recover 4 rhinoviruses by inoculating WI-38 fibroblast cells and HeLa cells, confirming by the cytopathic effect and immunofluorescence positive staining with anti-VP1 antibody. Taken together, our results indicate that tonsils and adenoids of patients without ARI may be reservoirs of replicating human rhinovirus, infecting manly Tcells CD4+ and CD20+ B-cells. The high-frequency detection of RNA (-) and VP1 expression in tissues from patients with chronic adenotonsillar diseases, plus the isolation of infectious viral particles, suggests that these detected agents replicate in the adenotonsillar tissues and this specific sites may be important sources of transmission of RV in the community.

Orientador : Marcio Ajudarte Lopes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T00:25:09Z (GMT). No. of bitstreams: 1 Ito_FabioAugusto_D.pdf: 9945598 bytes, checksum: 5d7825a97ef3dd77141a70dd22e719c3 (MD5) Previous issue date: 2006 / Resumo: Além de incomuns, os tumores de glândulas salivares despertam interesses por apresentarem uma grande diversidade histológica, morfológica e de comportamento biológico. Nas últimas décadas vários estudos têm mostrado que tais diversidades estão relacionadas ao acúmulo de alterações genéticas. As investigações de tais alterações são importantes para entender os mecanismos de oncogênese, avaliar o comportamento biológico e conseqüentemente aperfeiçoar terapêuticas favorecendo o prognóstico. O objetivo deste estudo foi analisar as características histopatológicas e imunohistoquímicas dos quatro tumores de glândulas salivares mais freqüentes: adenoma pleomórfico, carcinoma mucoepidermóide, tumor de Warthin e carcinoma adenóide cístico. Para isso foi realizada análise histológica do componente epitelial e mesenquimal dos adenomas pleomórficos, classificação histológica dos tumores de Warthin, carcinomas mucoepidermóides e carcinomas adenóide cístico e estudo imunohistoquímico para Ki-67, EGF, EGFR, ErbB-2, FAS, receptor de andrógeno, receptor de estrógeno e receptor de progesterona. Dos 189 casos de adenoma pleomórfico selecionados para o estudo histopatológico, 99 (52,4%) foram classificados como estroma-rico, 69 (36,5%) como celular e 21 (11,1%) como clássico. Dos 30 casos de tumor de Warthin, 17 casos (56,7%) foram classificados como típicos, 10 (33,3%) como estroma pobre e 3 (10%) como estroma-rico. Dos 30 casos de carcinoma mucoepidermóide, 15 casos (50%) foram classificados histologicamente como de baixo grau, 3 (10%) como grau intermediário e 12 (40%) como de alto grau. Dos 30 casos de carcinoma adenóide cístico, 15 casos (50%) foram classificados como cribriforme, 8 (26,7%) como tubular e 7 (23,3%) como sólido. Ki-67, EGFR e ErbB-2 foram mais freqüentemente encontrados em carcinomas mucoepidermóides, principalmente em tumores de alto grau de malignidade. EGF e FAZ foram encontrados mais freqüentemente em adenomas pleomórficos e carcinomas mucoepidermóides. Todos os casos estudados foram negativos para receptor de estrógeno e receptor de progesterona. Receptor de andrógeno foi positivo em apenas 2 casos de adenoma pleomórfico, 2 de carcinoma mucoepidermóide e 2 de carcinoma adenóide cístico. Concluindo, EGFR, ErbB-2 e FAS parecem desempenhar papel na tumorigênese de tumores de glândulas salivares, especialmente em carcinomas mucoepidermóides, e devem ser mais extensivamente estudados; receptor de estrógeno e receptor de progesterona não desempenham papel na tumorigênese de adenomas pleomórficos, tumores de Warthin, carcinomas mucoepidermóides e carcinomas adenóide cístico; e apesar de poucos casos positivos em tumores de glândulas salivares, receptor de andrógeno deve ser melhor estudado e considerado como potencial alvo em tratamentos com drogas anti-andrógenos / Abstract: Beyond uncommon, salivary gland tumors are interesting because their great diversity of histological, morphological and biological behavior. In the last decades some studies have shown that such diversities are related to the accumulation of genetic alterations. The investigation of such alterations are important to understand the mechanisms of oncogenesis, to evaluate the biological behavior and consequently to improve therapeutics favoring the prognosis. The aim of this study was to analyze the histopathological and immunohistochemical characteristics of the four more frequent salivary gland tumors: pleomorphic adenoma, mucoepidermóide carcinoma, Warthin¿s tumor and adenoid cystic carcinoma. For this, it was realized the histological analysis of the epithelial and mesenchymal components of pleomorphic adenomas, histological classification of Warthin¿s tumors, mucoepidermoid carcinomas and adenoid cystic carcinoma and immunohistochemical study for Ki-67, EGF, EGFR, ErbB-2, FAS, androgen receptor, estrogen receptor and progesterone receptor. Of the 189 cases of pleomorphic adenoma selected for the histopathological study, 99 (52.4%) were classified as stroma-rich, 69 (36.5%) as cellular and 21 (11.1%) as classic. Of the 30 cases of Warthin¿s tumor, 17 cases (56.7%) were classified as typical, 10 (33.3%) as stroma-poor and 3 (10%) as stroma-rich. Of the 30 cases of mucoepidermoid carcinoma, 15 cases (50%) were classified as low grade, 3 (10%) as intermediate grade and 12 (40%) as high grade. Of the 30 cases of adenoid cystic carcinoma, 15 cases (50%) were classified as cribriform, 8 (26.7%) as tubular and 7 (23.3%) as solid. Ki-67, EGFR and ErbB-2 were more frequently found in mucoepidermoid carcinomas, particularly in high grade tumors. EGF and FAS were more frequently found in pleomorphic adenomas and mucoepidermoid carcinomas. All studied cases were negative for estrogen receptor and progesterone receptor. Androgen receptor was positive in only 2 cases of pleomorphic adenoma, 2 of mucoepidermoid carcinoma and 2 of adenoid cystic carcinoma. In conclusion, EGFR, ErbB-2 and FAS seem to play a role in the tumorigenesis of salivary gland tumors, especially in MEC, and they should be more extensively studied; estrogen receptor and progesterone receptor do not play a role in the tumorigenesis of pleomorphic adenomas, Warthin¿s tumors, mucoepidermoid carcinomas and adenoid cystic carcinomas; and regardless of few positive cases in salivary gland tumors, androgen receptor should be better studied and considered as potential targets for treatment with anti-androgens drugs / Doutorado / Patologia / Doutor em Estomatopatologia

Orientadores: Albina Messias de Almeida Milani Altemani, Marinus Antonius Jacobus Hermsen / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T20:49:21Z (GMT). No. of bitstreams: 1 Costa_AnaFlaviadeMattos_D.pdf: 73992592 bytes, checksum: 3b90deaed579b13c568eeda812a10ef2 (MD5) Previous issue date: 2014 / Resumo: O carcinoma adenóide cístico pode raramente sofrer desdiferenciação, um fenômeno também referido como transformação para alto grau. Contudo, alguns casos de carcinoma adenóide cístico foram descritos mostrando transformação para adenocarcinomas que não são pobremente diferenciados, indicando que a transformação para alto grau pode não refletir necessariamente um estágio mais avançado da progressão tumoral, mas sim uma transformação em uma outra forma histológica, que pode abranger um amplo espectro de carcinomas em termos de agressividade. Inicialmente, investigamos a expressão das proteínas reguladas pela hipóxia (HIF-1?, VEGF, GLUT-1 e CD105), dado que a hipóxia contribui para a agressividade tumoral e, pode, também, promover um fenótipo desdiferenciado em certos tipos de câncer. Em seguida, analisamos um importante ponto de interesse em relação ao carcinoma adenóide cístico com transformação para alto grau, o seu pior prognóstico, que é sugerido ser comparável ou até pior do que o subtipo sólido. Para isso, comparamos as alterações genéticas do carcinoma adenóide cístico com transformação para alto grau com o subtipo sólido e, com os aspectos clínicos e patológicos de ambos os tumores. Além disso, em outro trabalho, usamos a hibridização genômica comparativa em microarranjo para comparamos o perfil genético de ambos os componentes histológicos do carcinoma adenóide cístico com transformação para alto grau. Atenção especial foi dada à expressão da proteína e à translocação cromossomal do gene MYB, que está sendo considerado o maior evento precoce e oncogênico do carcinoma adenóide cístico clássico. Nossos resultados mostraram que o carcinoma adenóide cístico com transformação para alto grau pode apresentar uma complexidade genética similar ao subtipo sólido e, também, que o processo de transformação não é sempre acompanhado pelo acúmulo de alterações genéticas, o que indica uma progressão paralela de ambos os componentes do carcinoma adenóide cístico transformado. Em contrapartida à expressão da proteína MYB, a translocação entre MYB/NFIB não é necessariamente um evento precoce e, bem como a hipóxia, não são fundamentais para o desenvolvimento destes tumores. Finalmente, o estudo advindo do carcinoma adenóide cístico com transformação para alto grau, também nos permitiu fazer uma revisão sobre o assunto. Neste outro estudo fizemos um panorama sobre os recentes conceitos na classificação histopatológica dos tumores de glândula salivar com desdiferenciação/transformação para alto grau descritos na literatura. Destaque também foi dado aos achados imuno-histoquímicos e genéticos que podem ajudar no diagnóstico de cada um destes tumores / Abstract: Adenoid cystic carcinomas can occasionally undergo dedifferentiation, a phenomenon also referred to as high-grade transformation. However, cases of adenoid cystic carcinomas have been described showing transformation to adenocarcinomas that are not poorly differentiated, indicating that high-grade transformation may not necessarily reflect a more advanced stage of tumor progression, but rather a transformation to another histological form, which may encompass a wide spectrum of carcinomas in terms of aggressiveness. The aim of this study was to gain more insight in the biology of this pathological phenomenon. Firstly, we investigated expression of proteins regulated by hypoxia (HIF-1?, VEGF, GLUT-1 and CD105), given that hypoxia contributes to aggressive tumor behavior and can also promote a dedifferentiated phenotype in certain types of cancer. Hereafter, we analyzed an important point of interest of adenoid cystic carcinoma with high-grade transformation that is its proposed poor prognosis to be comparable to or even worse than solid subtype. Therefore, we compared the genetic changes of transformed and solid subtype adenoid cystic carcinomas and correlated the results to their clinico-pathological features. In addition, in another work, we used microarray comparative genomic hybridization to compare the genetic profiles of both histological components of adenoid cystic carcinomas with high-grade transformation. Special attention was given to chromosomal translocation and protein expression of MYB, recently being considered to be an early and major oncogenic event in adenoid cystic carcinomas. Our data showed that transformed adenoid cystic carcinoma with high-grade transformation may present a genetic complexity similar to the solid subtype and, also that the process of high-grade transformation is not always be accompanied by an accumulation of genetic alterations; which indicate a parallel progression of the two histological components of transformed adenoid cystic carcinoma. In contrast to MYB protein expression, MYB/NFIB translocation is not necessarily an early event and, as hypoxia, not fundamental for the development of these tumors. Finally, the study that comes from of adenoid cystic carcinoma with high-grade transformation also allowed us to do a review about it. In this study we made an overview of the latest concepts in histopathological classification of salivary gland tumors with dedifferentiation / high-grade transformation described in the literature. Highlight was also given to immunohistochemical and genetic findings that can help in the diagnosis of each of these tumors / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Hintergrund Das adenoidzystische Karzinom ist ein seltenes Malignom. Es macht weniger als 1% aller Malignome im Kopf-Hals Bereich aus und hat einen Anteil an allen malignen Speicheldrüsentumoren von etwa 20%. Nach Therapie durch chirurgische Resektion und/oder Radiotherapie rezidiviert das adenoidzystische Karzinom häufig. Fragestellung/Hypothese Welches sind die Attribute der aufgrund eines adenoidzystischen Karzinoms strahlentherapierten Patienten? Wie hoch sind die Gesamtüberlebensraten? Wie hoch sind krankheitsspezifische und krankheitsfreie Überlebensraten? Wie hoch sind lokoregionäre Kontrollraten und die Wahrscheinlichkeit für das Auftreten von Fernmetastasen nach Strahlentherapie beim adenoidzystischen Karzinom? Können Vergleiche zu ähnlichen Arbeiten gezogen werden? Was sind prognoserelevante Faktoren des adenoidzystischen Karzinoms? Material und Methode Es wurden 55 Patienten retrospektiv analysiert, welche aufgrund eines adenoidzystischen Karzinoms in der „Klinik und Poliklinik für Strahlentherapie und Radioonkologie der TU Dresden“ zwischen dem 30.03.1982 und 06.03.2007 bestrahlt wurden. Es fand kein Ausschluss von Patienten aufgrund von Erkrankungsschwere oder Therapiemodalität statt. Das letzte Follow up erfolgte 2009 durch Arztanfragen und Meldeamtsanfragen. Die Patienten hatten ein medianes Erkrankungsalter von 61 Jahren (28 - 82 Jahre). Bei 63,6% der Patienten fand sich ein lokales Tumorstadium von T3 bis T4, regionäre Lymphknoten waren zu 21,8% vom Tumor befallen und Fernmetastasen wiesen 9,1% der Patienten auf. Bei 18,2% der Patienten lag bereits vor Strahlentherapie ein postoperatives Lokalrezidiv vor. Primäre Radiotherapie ohne Operation erfolgte bei 16,4% der Patienten. Eine postoperative Radiatio wurde bei 83,6 % der Patienten durchgeführt, wobei 21,8% mikroskopisch tumorfreie Resektionsränder aufwiesen. In der ersten Bestrahlungsserie wurden zu 92,6% konventionell fraktionierte Teletherapien durchgeführt mit einer medianen Gesamtdosis von 60 Gy bei Behandlung der Primärtumorregion. Bei 34,4% der Patienten wurde nach der ersten strahlentherapeutischen Behandlung mindestens eine weitere Radiotherapie durchgeführt. Ergebnisse Die Gesamtüberlebensraten nach 5- und nach 10 Jahren betrugen 50,7% respektive 36,4%. Die Krankheitsspezifischen Überlebensraten nach 5- und nach 10 Jahren betrugen 57,2% respektive 42,3%. Die Krankheitsfreien Überlebensraten nach 5- und nach 10 Jahren betrugen 43,5% respektive 20,5 %. Bei 70,4% der Patienten beendeten Rezidive das Krankheitsfreie Überleben. Lokale Rezidive waren mit 63,2% aller Rezidive am Häufigsten, gefolgt von 18,4% Fernmetastasen sowie 10,5% regionären Lymphknotenmetastasen und 5,3% Fernmetastasierung bei gleichzeitigem Lokalrezidiv. Die Lokoregionären Kontrollraten nach 5- und nach 10 Jahren betrugen 49,1% respektive 26,7%. Die Raten des Fernmetastasenfreies Überlebens nach 5- und nach 10 Jahren betrugen 70% respektive 65%. In der univariaten Analyse zeigten sich folgende Eigenschaften als signifikante positive Einflussfaktoren auf den Endpunkt Gesamtüberleben: postoperative Strahlentherapie bei maximal mikroskopisch infiltrierten Resektionsgrenzen, geringe Tumorgröße T1 und T2, Abwesenheit von Schädelbasisinfiltration, Abwesenheit von Nerveninfiltration und Erkrankungsalter < 60 Jahre. Univariat signifikant wirkten sich die Eigenschaften: postoperative Strahlentherapie bei maximal mikroskopisch infiltrierten Resektionsgrenzen, Tumorgröße T1-T3, Abwesenheit von Knocheninfiltration und Abwesenheit von Schädelbasisinfiltration auf die lokoregionären Kontrollraten aus. Weiterhin zeigten Patienten mit Entwicklung eines lokoregionären Rezidives signifikant geringere Krankheitsspezifische Überlebensraten. In der multivariaten Analyse waren unabhängige negative Prädiktoren der Gesamtüberlebensraten: Schädelbasisinfiltration, Erkrankungsalter > 60 Jahre und makroskopischer unvollständige Resektion oder primäre Radiotherapie. Schlussfolgerung Ein großes Problem in der Therapie des adenoidzystischen Karzinoms sind lokale Rezidive nach Operation und adjuvanter Radiotherapie, sowie die auch Jahre später zu ungefähr einem Drittel auftretenden Fernmetastasen. Infiltration der Schädelbasis durch das Karzinom, Erkrankungsalter > 60 Jahre und makroskopisch unvollständige Resektion oder Inoperabilität stellen unabhängige, prognostisch ungünstige Merkmale dar. Die Ergebnisse der Überlebens- und Rezidivanalysen lassen sich mit Studien ähnlicher Patientenselektion vergleichen. Aufgrund geringer Fallzahlen und Retrospektivität aller zur adjuvanten Therapie des adenoidzystischen Karzinoms vorhanden Studien wäre die Durchführung prospektiver, multizentrischer, randomisierter Studien für weitere Evidenz in der stadiengerechten Behandlung des adenoidzystischen Karzinoms empfehlenswert.:II Abbildungsverzeichnis V III Tabellenverzeichnis VII IV Abkürzungsverzeichnis XI 1 Einleitung 1 2 Allgemeiner Teil 2 2.1 Häufigkeit und Manifestationen 2 2.2 Krankheitsentwicklung 3 2.3 Therapie beim adenoidzystischen Karzinom 4 2.4 Prognose des adenoidzystischen Karzinom 5 2.5 Geschichtliche Entwicklung der Strahlentherapie in Dresden 5 2.6 Ziel der vorliegenden Arbeit 7 3 Patienten und Methode 8 3.1 Patientenselektion und Datenerhebung 8 3.2 Nachbeobachtungszeiten und Follow up 10 3.3 Statistik und Darstellung 11 3.3.1 Deskriptive Statistik 11 3.3.2 Überlebenswahrscheinlichkeiten nach Kaplan-Meier 11 3.3.3 Univariate Analyse 12 3.3.4 Multivariate Analyse 13 3.4 Patientencharakteristika 14 3.4.1 Altersverteilung und Geschlechterverteilung 14 3.4.2 Symptomatik 15 3.4.3 Beginn der Symptomatik 15 3.4.4 Tumorlokalisationen 16 3.5 Verteilung der TNM-Klassifikation 17 3.5.1 Tumorgröße T 19 3.5.2 Lymphknotenstatus N 20 3.5.3 Fernmetastasen M 21 3.5.4 Histopathologisches Grading 21 3.5.5 Wachstumsmuster beim adenoidzystischen Karzinom 22 3.6 Chirurgische Vorbehandlung 22 3.7 Aufteilung in Bestrahlungsfälle und Patientenverläufe 24 3.7.1 Behandlungszeitraum und Bestrahlungstechniken 25 3.7.2 Bestrahlungsqualitäten und -gesamtdosen bei allen Fällen 26 3.7.2.1 Dosis pro Fraktion bei fraktionierten Teletherapien 27 3.7.2.2 Dosis pro Fraktion bei Brachytherapie 28 3.7.2.3 Dosis bei Radiochirurgie mittels Einzeitbestrahlung 29 3.7.2.4 Dosis pro Fraktion bei Teletherapien kombiniert mit Brachyboost 29 3.7.2.5 Das Linearquadratische Modell 31 3.7.3 Bestrahlungsqualitäten und -gesamtdosen bei der Erstbestrahlung 32 3.7.3.1 Teletherapie mit konventioneller Fraktionierung 33 3.7.3.2 Stereotaktische Bestrahlung am Linearbeschleuniger 35 3.7.3.3 Teletherapie mit Brachytherapieboost 35 3.7.3.4 Alleinige Brachytherapie 36 3.7.3.5 Latenzzeiten zwischen Diagnose und Strahlentherapiebeginn 36 3.7.4 Bestrahlungsqualitäten und -gesamtdosen bei Wiederbestrahlungsfällen 37 3.7.4.1 Wiederbestrahlungsfälle aufgrund von Rezidiven 38 3.7.4.2 Wiederbestrahlung aufgrund von inoperablen Lokalrezidiven 38 3.7.4.3 Wiederbestrahlung von Lokalrezidiven nach R1-Resektion 41 3.7.4.4 Stereotaktische Radiochirurgie durch Einzeitbestrahlung bei Fernmetastasen 41 3.7.4.5 Stereotaktische Mehrfeldbestrahlung am Linearbeschleuniger in der Wiederbestrahlung 42 3.7.4.6 Kontaktbestrahlung und Kombinationen in der Wiederbestrahlung 42 3.8 Bestrahlung wegen Fernmetastasen in der Erstbestrahlung und bei Wiederbestrahlungsfällen 43 4 Ergebnisse 44 4.1 Überlebensanalysen 44 4.1.1 Gesamtüberleben aller Patienten 44 4.1.1.1 Gesamtüberleben nach Resektionsrändern 45 4.1.2 Krankheitsspezifisches Überleben aller Patienten 47 4.1.2.1 Krankheitsspezifisches Überleben nach Resektionsrändern 48 4.1.3 Krankheitsfreies Überleben der Patienten 50 4.1.3.1 Krankheitsfreies Überleben nach Resektionsrändern 52 4.1.4. Lokoregionäre Kontrolle 54 4.1.4.1 Lokoregionäre Kontrolle nach Resektionsrändern 55 4.1.5 Krankheitsspezifisches Überleben bei Auftreten von lokoregionärem Rezidiv 57 4.1.6 Fernmetastasenfreie Zeit 58 4.1.7 Überleben nach Fernmetastasenbestrahlung 61 4.2 Univariate Analyse des Gesamtüberlebens nach erster Radiotherapie 64 4.2.1 Einfluss des T-Stadiums 64 4.2.2 Einfluss des N-Stadiums 65 4.2.3 Einfluss des M-Stadiums 65 4.2.4 Einfluss der Schädelbasisinfiltration 65 4.2.5 Einfluss der Knocheninfiltration 66 4.2.6 Einfluss der Nerveninfiltration 67 4.2.7 Einfluss der lokoregionären Rezidive nach der ersten Strahlentherapie 67 4.2.8 Einfluss der Latenzzeiten zwischen Primärtumordiagnose und Beginn einer Strahlentherapie 68 4.2.9 Einfluss der Gesamtdosen 69 4.2.10 Einfluss des Geschlechts 70 4.2.11 Einfluss des Alters bei Diagnose in Jahren 70 4.2.12 Einfluss der Behandlungszeiträume 71 4.3 Univariate Analyse der lokoregionären Kontrollraten nach der ersten Radiotherapieserie 71 4.3.1 Einfluss des T-Stadiums 71 4.3.2 Einfluss des N-Stadiums 72 4.3.3 Einfluss einer initialen Schädelbasisinfiltration 72 4.3.4 Einfluss einer initialen Knocheninfiltration 72 4.3.5 Einfluss einer initialen Nerveninfiltration 73 4.3.6 Einfluss des Alters bei Diagnose 73 4.3.7 Einfluss des Radiatiobeginns nach Diagnose 74 4.3.8 Einfluss der applizierten Gesamtdosis bei der ersten Bestrahlung 74 4.3.9 Einfluss der Behandlungszeiträume 75 4.4 Multivariate Analyse des Gesamtüberlebens 76 4.4.1 Schädelbasisinfiltration und Resektionsränder 76 4.4.2 Schädelbasisinfiltration, Resektionsränder und Alter > 60 Jahre 76 4.5 Strahlennebenwirkungen 77 4.5.1 Frühe Strahlenreaktionen der Haut 77 4.5.2 Frühe Strahlenreaktionen der Schleimhaut 78 4.5.3 Späte Strahlenreaktionen 79 5 Diskussion 81 6 Zusammenfassung 98 7 Summary 101 Erklärungen 103 Literaturverzeichnis 105 Danksagung 112

Salivary glands (SGs) are important exocrine glands of the craniofacial region, whose main role is to produce and secrete saliva, a seromucous solution necessary for a diverse spectrum of critical functions, such as the preliminary digestion and swallowing of solid food, the articulation of speech, the maintenance of dental enamel and the prevention of oral infections. The production and secretion of saliva is orchestrated by a large and diverse collection of epithelial cell populations. Although many of the cell types that form the SG epithelium can be recognized morphologically and investigated using histological assays, it is currently impossible to achieve their differential purification from primary tissues as live cells, due to the lack of surface markers known to be either selectively or preferentially expressed by various cell subsets. This critical gap in knowledge limits our capacity to conduct functional studies in many areas of SG biology, including studies aimed at elucidating the developmental relationships that link different cell types (e.g. testing whether selected cell types can act as progenitors for the generation of others), studies elucidating the roles played by different cell types during regeneration of the SG epithelium following injury (e.g. radiotherapy), and studies investigating the biology of SG malignancies characterized by a heterogeneous cell composition, such as Adenoid Cystic Carcinomas (ACCs). In this work, we aimed to advance our understanding of the cell composition of the salivary gland epithelium and to identify surface markers that enable the differential purification of its various cell types by fluorescence-activated cell sorting (FACS), in order to facilitate functional investigations of their individual capacity to act as stem/progenitor cells in prospective assays. In the first portion of our studies, we leveraged single-cell RNA sequencing (scRNA-seq) to dissect the transcriptional identities of various epithelial cell populations found in normal murine SGs, and discovered surface markers that allowed us to purify eight distinct cell types by FACS. We then used bulk RNA sequencing to generate high-resolution transcriptomic profiles of seven of these populations, and annotated their identity (e.g. acinar, ductal, basal, myoepithelial) in terms of anatomical location and differential expression of lineage-specific biomarkers. Furthermore, using a three-dimensional (3D) in vitro organoid tissue culture assay, we tested each of the newly identified SG populations for stem/progenitor properties, and demonstrated that organoid forming capacity is primarily restricted to only one of them, characterized by a basal phenotype, and able to function as a bipotent progenitor in vitro. Finally, we used FACS to examine the effects of radiotherapy on the cell composition of the mouse SG epithelium, and demonstrated that, of the eight newly identified populations, at least four display preferential sensitivity to radiation injury. In the second portion of our studies, we tested whether the surface markers that we identified as differentially expressed between different subtypes of SG epithelial cells could also be leveraged to achieve the purification of the two subsets of malignant cells known to co-exist in Adenoid Cystic Carcinoma (ACC), one of the most common and lethal forms of human SG malignancy. A defining feature of ACC is the presence of two distinct cell populations, resembling myoepithelial and ductal cell types found in the normal salivary gland epithelium. However, little is known about the developmental relationship linking these two cell populations, their individual capacity to sustain the growth of malignant tissues upon xeno-transplantation, as well as their distinct behavior in terms of responses to therapeutic manipulations. By utilizing cell surface markers identified as differentially expressed in the mouse SG epithelium, we developed a sorting strategy that enabled us to isolate the two major subtypes of malignant cells found in ACCs. By conducting prospective xeno-transplantation experiments in immunodeficient mice, we demonstrated that, contrary to common belief, myoepithelial-like cells are highly tumorigenic (i.e. do not represent an indolent component of the tumor) and can act as progenitors of ductal-like cells. Furthermore, by investigating differences in the transcriptional profiles of myoepithelial-like and ductal-like cells, we discovered that the two cell types differ in the expression of multiple components of the biochemical pathways that control retinoic acid (RA) signaling. We find that RA direct and inverse agonism have opposing effects on cell composition through distinct molecular mechanisms, whereby direct agonism facilitates differentiation of myoepithelial-like to ductal-like cells, and inverse agonism induces selective cell death of ductal-like cells. Finally, we demonstrate that inhibition of RA signaling with inverse agonists is able to profoundly impair in vivo growth of human ACCs implanted in immunodeficient mice. Overall, the findings reported in this study advance our understanding of the cellular composition of both normal and malignant SG epithelia, establish novel and robust analytical assays for the purification of multiple subtypes of SG epithelial cells, and reveal novel strategies for the therapeutic manipulation of differentiation programs in human ACCs.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasias malignas das glândulas salivares que apresentam semelhança nos padrões histológicos, porém com comportamento clínico, tratamento e prognóstico completamente diferentes. A galectina-3 é uma proteína multifuncional da família das lectinas que está envolvida em vários fenômenos biológicos como crescimento celular, adesão celular, diferenciação celular e apoptose. Além disso, tem sido estudada como um marcador de invasão tumoral e metástase. O objetivo deste trabalho foi estudar qualitativamente a expressão imunoistoquímica da galectina-3 em 14 casos de carcinoma adenóide cístico (2 do subtipo tubular, 4 do subtipo sólido e 8 do subtipo cribriforme) e em 12 casos de adenocarcinoma polimorfo de baixo grau de malignidade com padrões histológicos variados, incluindo os padrões lobular, tubular e cribriforme. Espécimes de glândula salivar normal foram também incluídos na amostra. Nas glândulas salivares normais houve forte marcação da galectina-3 no núcleo e no citoplasma das células luminais dos ductos. Nos carcinomas adenóides císticos houve uma maior marcação da galectina-3 no subtipo tubular, localizada apenas nas células luminais das estruturas tubulares. Nos subtipos sólido e cribriforme a marcação foi menor, mas sempre localizada nas células que circundavam espaços luminais. Em todos os casos de carcinomas adenóides císticos estudados a marcação foi predominantemente nuclear. Nos adenocarcinomas polimorfos de baixo grau de malignidade a marcação da galectina-3 foi predominantemente citoplasmática em praticamente todas as células neoplásicas. Diante disso podemos sugerir que, nas neoplasias estudadas, a expressão da galectina-3 parece estar mais relacionada à diferenciação celular do que à progressão tumoral e ao prognóstico. / Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are malignant neoplasms of salivary glands which are similar in histologic patterns but very different in clinical behavior, treatment and prognosis. Galectin-3 is a multifunctional protein of a growing family of beta-galactoside-binding animal lectins which is implicated in a variety of biological events such as tumor cell adhesion, proliferation, differentiation and angiogenesis. This protein was found to be implicated in cellular transformation, and a correlation between its expression and cancer progression and metastasis has been described. The aim of this study was to determine the galectin-3 immunoprofile in 14 cases of adenoid cystic carcinoma (2 cases of tubular subtype, 4 cases of solid subtype and 8 cases of cribriform subtype) and in 12 cases of polymorphous low-grade adenocarcinoma with different histologic patterns, included lobular, tubular and cribriform. Moreover, slides of normal salivary glands were included. In normal salivary glands there were strong nuclei and cytoplasmic staining for galectin-3 in ductal luminal cells. Adenoid cystic carcinomas showed specific staining in luminal cells mainly in the nuclei. In the tubular subtype of adenoid cystic carcinoma galectin-3 was strong in the luminal cells of the ductiform structures. The cribriform and solid subtypes showed a few positive cells for galectin-3 only in the luminal cells of small ducts presenting in the cribriform structures and in solid nests respectly. In the cases of polymorphous low-grade adenocarcinoma, independent of the histologic architecture, all tumor cells revealed a positive cytoplasmic reaction with the galectin-3 antibody. Galectin-3 expression seems to be related to cell differentiation more than tumor progression and prognosis in the neoplasms studied.

As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasias malignas das glândulas salivares que apresentam semelhança nos padrões histológicos, porém com comportamento clínico, tratamento e prognóstico completamente diferentes. A galectina-3 é uma proteína multifuncional da família das lectinas que está envolvida em vários fenômenos biológicos como crescimento celular, adesão celular, diferenciação celular e apoptose. Além disso, tem sido estudada como um marcador de invasão tumoral e metástase. O objetivo deste trabalho foi estudar qualitativamente a expressão imunoistoquímica da galectina-3 em 14 casos de carcinoma adenóide cístico (2 do subtipo tubular, 4 do subtipo sólido e 8 do subtipo cribriforme) e em 12 casos de adenocarcinoma polimorfo de baixo grau de malignidade com padrões histológicos variados, incluindo os padrões lobular, tubular e cribriforme. Espécimes de glândula salivar normal foram também incluídos na amostra. Nas glândulas salivares normais houve forte marcação da galectina-3 no núcleo e no citoplasma das células luminais dos ductos. Nos carcinomas adenóides císticos houve uma maior marcação da galectina-3 no subtipo tubular, localizada apenas nas células luminais das estruturas tubulares. Nos subtipos sólido e cribriforme a marcação foi menor, mas sempre localizada nas células que circundavam espaços luminais. Em todos os casos de carcinomas adenóides císticos estudados a marcação foi predominantemente nuclear. Nos adenocarcinomas polimorfos de baixo grau de malignidade a marcação da galectina-3 foi predominantemente citoplasmática em praticamente todas as células neoplásicas. Diante disso podemos sugerir que, nas neoplasias estudadas, a expressão da galectina-3 parece estar mais relacionada à diferenciação celular do que à progressão tumoral e ao prognóstico. / Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are malignant neoplasms of salivary glands which are similar in histologic patterns but very different in clinical behavior, treatment and prognosis. Galectin-3 is a multifunctional protein of a growing family of beta-galactoside-binding animal lectins which is implicated in a variety of biological events such as tumor cell adhesion, proliferation, differentiation and angiogenesis. This protein was found to be implicated in cellular transformation, and a correlation between its expression and cancer progression and metastasis has been described. The aim of this study was to determine the galectin-3 immunoprofile in 14 cases of adenoid cystic carcinoma (2 cases of tubular subtype, 4 cases of solid subtype and 8 cases of cribriform subtype) and in 12 cases of polymorphous low-grade adenocarcinoma with different histologic patterns, included lobular, tubular and cribriform. Moreover, slides of normal salivary glands were included. In normal salivary glands there were strong nuclei and cytoplasmic staining for galectin-3 in ductal luminal cells. Adenoid cystic carcinomas showed specific staining in luminal cells mainly in the nuclei. In the tubular subtype of adenoid cystic carcinoma galectin-3 was strong in the luminal cells of the ductiform structures. The cribriform and solid subtypes showed a few positive cells for galectin-3 only in the luminal cells of small ducts presenting in the cribriform structures and in solid nests respectly. In the cases of polymorphous low-grade adenocarcinoma, independent of the histologic architecture, all tumor cells revealed a positive cytoplasmic reaction with the galectin-3 antibody. Galectin-3 expression seems to be related to cell differentiation more than tumor progression and prognosis in the neoplasms studied.

We hypothesized that lymph node involvement in minor salivary gland cancers is associated with clinical and pathological factors commonly available to the clinician after a typical initial workup. Our aim was to identify these factors using a dataset that allowed us to compile the largest series of minor salivary gland cancers in the published literature. Using this dataset we also aimed to characterize the distribution of histological types by primary site, identify the predictors of the use of external beam radiation therapy and neck dissection, and examine the effect of lymph node involvement on survival. Using the SEER database, we identified 2667 minor salivary gland cancers with known lymph node status from 1988 to 2004. Univariate and multivariate analyses were conducted to identify factors associated with the use of neck dissection, the use of external beam radiation therapy, and the presence of cervical lymph node metastases. Kaplan Meier survival curves were constructed to examine the effect of lymph node involvement on survival. 426 (16.0%) patients had neck nodal involvement. Factors associated with neck nodal involvement on univariate analysis included increasing age, male gender, increasing tumor size, high tumor grade, T3-T4 stage, adenocarcinoma or mucoepidermoid carcinomas, and pharyngeal site of primary malignancy. On multivariate analysis, four statistically significant factors were identified, which included male gender, T3-T4 stage, pharyngeal site of primary malignancy, and high-grade adenocarcinoma or high-grade mucoepidermoid carcinomas. The proportions (and 95% confidence intervals) of patients with lymph node involvement for those with 0, 1, 2, 3 and 4 of these prognostic factors were 0.02 (0.01-0.03), 0.09 (0.07-0.11), 0.17 (0.14-0.21), 0.41 (0.33-0.49), and 0.70 (0.54-0.85) respectively. Grade was a significant predictor of metastasis for adenocarcinoma and mucoepidermoid carcinoma but not for adenoid cystic carcinoma. Overall survival was significantly worse at 5, 10, and 15 years for patients with lymph node involvement on presentation. A prognostic index using the four clinicopathological factors listed above can effectively differentiate patients into risk groups of nodal metastasis. The precision of this index is subject to the limitations of SEER data and it should be validated in further clinical studies.