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.

Orientador: Albina Messias de Almeida Milani Altemani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T21:50:24Z (GMT). No. of bitstreams: 1 Costa_AnaFlaviadeMattos_M.pdf: 3298163 bytes, checksum: 3fb6838b3da137877a94a25afd98a7e4 (MD5) Previous issue date: 2009 / Resumo: Para investigar se carcinomas salivares com e sem diferenciação mioepitelial poderiam apresentar diferenças em relação ao nível de angiogênese, nós comparamos a vascularização tumoral entre os carcinomas adenóide cístico (31 casos) e epitelial mioepitelial (14 casos) versos carcinoma mucoepidermóide (37 casos). A expressão da peroxiredoxina I foi também estudada para verificar a relação potencial entre o metabolismo celular e a densidade microvascular. A densidade microvascular para o CD34 e CD105 foi significantemente menor em carcinomas com diferenciação mioepitelial. Porém, nenhuma relação foi encontrada entre a angiogênese e a quantidade de células mioepiteliaís. A forte expressão de peroxiredoxina I foi encontrada em 73.7% de carcinomas mucoepidermóides enquanto que 85.1% dos carcinomas com diferenciação mioepitelial apresentaram fraca expressão. Em conclusão, carcinomas com diferenciação mioepitelial, independente da quantidade de células mioepiteliaís, estão associados com significante baixa densidade microvascular. As razões para essa baixa atividade angiogênica ainda deve ser esclarecida, mas pode ser relacionada com as características metabólicas das células cancerosas. / Abstract: To investigate whether salivary carcinomas with and without myoepithelial differentiation could present differences regarding degree of angiogenesis, we compared tumor vascularization between adenoid cystic (31 cases) and epithelial- myoepithelial carcinomas (14) versus mucoepidermoid (37) carcinoma. The expression of peroxiredoxin I was also studied to verify the potential relationship between cellular metabolism and microvascular density. Microvascular density for CD34 and CD105 were significantly lower in carcinomas with myoepithelial differentiation. However, no correlation was found between degree of angiogenesis and amounts of myoepithelial cells. High-grade peroxiredoxin I expression was found in 73.7% of mucoepidermoid carcinomas whereas 85.1 of carcinomas with myoepithelial differentiation presented low-grade expression. In conclusion, carcinomas with myoepithelial differentiation, regardless of the amounts of myoepithelial cells, are associated to a significantly lower vascular density. The reasons for this lower angiogenic activity remain to be determined, but could be related to metabolic characteristics of the cancer cells. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

A thesis on Head and neck cancer focusing on dose planning, salivary gland carcinoma and Single nucleotide polymorphism. For dose planning PET/CT (Positron emissions tomography/computed tomography) with tracer gave more precise information in comparison dose planning with CT. More primary tumours and metastases were found with the acetate tracer than with glucose tracer. Acetate PET/CT also showed larger volume of tumours attributed to lipid metabolism. In a retrospective study salivary gland cancer 5-year overall survival (OS) was 53 %. Salivary gland carcinoma consists of many histopathological groups, the two largest groups being mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ASCC). For ACC, having the best 5-year OS, it was 70 percent. Facial palsy, advanced stage disease, lymph node metastases worsened prognosis. ACC and polymorphous low grade carcinoma (PLGA) expressed c-myc and cyclin D1 to a larger extent than MEC. In squamous cell carcinoma of the head and neck we examined the occurrence of Single Nucleotide polymorphism, SNP. We found that the SNPs in male and female patients differed from each other. In male patients the SNPs were associated with immune response while in female patients the association was to SNPs concerning inflammation. This means that different pathways were engaged in cancer development for men and women. We also found that the SNPs in patients were different from those expressed in the healthy controls.