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.

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

Neoplasias malignas das glândulas salivares são lesões raras e o mecanismo pelo qual esses tumores progridem ainda não está completamente esclarecido na literatura. A galectina-3 é uma proteína multifuncional expressa em uma grande quantidade de tecidos normais, mas que também tem sido associada à progressão tumoral de neoplasias malignas da tireóide, próstata e neoplasias gástricas. Estudos prévios demonstraram que a galectina-3 está também expressa em algumas neoplasias malignas das glândulas salivares como carcinoma adenóide cístico e adenocarcinoma polimorfo de baixo grau de malignidade. Recentemente foi sugerido que a superexpressão da galectina-3 controla alterações nos níveis de expressão de alguns reguladores do ciclo celular, dentre eles a ciclina D1. Além disso, outros estudos revelaram que a ciclina D1 é ativada pela -catenina de uma maneira dependente da galectina-3. O objetivo desse trabalho foi comparar a marcação imunoistoquímica nuclear e / ou citoplasmática da galectina-3 no carcinoma adenóide cístico e adenocarcinoma polimorfo de baixo grau tentando relacioná-la à marcação da -catenina e ciclina D1. Foram realizadas reações de imunoistoquímica para as três proteínas em 15 casos de carcinoma adenóide cístico e em 15 casos de adenocarcinoma polimorfo de baixo grau utilizando-se material parafinado. Para a galectina-3 os carcinomas adenóides císticos apresentaram marcação imunoistoquímica apenas nas células luminais, predominantemente no núcleo. Todos os casos de adenocarcinoma polimorfo de baixo grau revelaram uma marcação predominantemente citoplasmática para essa proteína. Ambos os tumores exibiram intensa marcação citoplasmática e/ou nuclear para a -catenina na maioria dos casos. Não houve imunorreatividade para a ciclina D1 em 14/15 casos de adenocarcinoma polimorfo de baixo grau. Em contraste, os carcinomas adenóides císticos revelaram marcação nuclear específica para a ciclina D1 em 10 de 15 casos estudados em mais de 5% das células neoplásicas e essa marcação estava associada à marcação citoplasmática e nuclear da galectina-3 (p<0,05). Esses resultados sugerem que nos carcinomas adenóides císticos a expressão da galectina-3 pode exercer uma função de proliferação celular e parece estar relacionada à diferenciação celular no adenocarcinoma polimorfo de baixo grau. Além disso, a perda de expressão da galectina-3 no carcinoma adenóide cístico pode estar associada a um comportamento clínico mais agressivo dessa lesão. Embora a -catenina pareça exercer algum papel no mecanismo de carcinogênese dessas duas lesões, ela não parece se ligar à galectina-3 para ativar a ciclina D1. / Salivary gland tumors are uncommon and the mechanism by which malignant tumors progresses is still undefined. In a previous study it was shown that galectin-3 is expressed in malignant salivary gland neoplasms as adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Galectin-3 is a multifunctional protein of a group of galactoside-binding lectins expressed in a variety of normal cells, but also has been implicated in tumor progression of some malignancies as thyroid, prostate and gastric cancers. Recently, it has been suggested that galectin-3 may be an important mediator of the -catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, in the present study we aimed to correlate galectin-3 expression, either nuclear or cytoplasmic, with the expression of -catenin (nuclear/cytoplasmic) and cyclin D1 (nuclear) in 15 cases of adenoid cystic carcinoma and in 15 cases of polymorphous low-grade adenocarcinoma. For galectin-3, adenoid cystic carcinomas showed specific staining only in luminal cells, mainly in the nuclei. In the cases of polymorphous low-grade adenocarcinoma, all tumor cells revealed a positive, mostly cytoplasmic, reaction to galectin-3. Both tumors showed intense cytoplasmic/nuclear staining for -catenin in the majority of cases. Cyclin D1 immunoreactivity was not detected in 14 of the 15 polymorphous low-grade adenocarcinomas studied. In contrast, adenoid cystic carcinomas showed specific nuclear staining for cyclin D1 in 10 of 15 cases studied in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in adenoid cystic carcinomas (p<0,05). These results suggest that in adenoid cystic carcinoma galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In polymorphous low-grade adenocarcinoma gal-3 expression seems to be associated with cellular differentiation. In addition, loss of cytoplasmic expression of galectin-3 in adenoid cystic carcinomas may be related to a more aggressive behavior of these lesions. Although -catenin seems to play a role in carcinogenesis, in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasmas de glândulas salivares. O carcinoma adenóide cístico pode apresentar-se em glândulas salivares maiores e menores, porém o adenocarcinoma polimorfo de baixo grau de malignidade acomete principalmente as glândulas salivares menores distribuidas na cavidade oral. Ambos os tumores compartilham muitas características comuns, como a alta propensão de invasão perineural e o padrão de infiltracão: sólido, tubular e cribriforme. Mas o carcinoma adenoide cístico e o adenocarcinoma polimorfo de baixo grau são tipos distintos de adenocarcinomas com prognóstico diferente, que ocasionalmente podem resultar em um diágnóstico errado. As proteínas c-jun e junB são membros da familia JUN, capazes de homodimerizar ou heterodimerizar com c-fos ou com outras proteinas bzip. Evidências das funcões específicas das subunidades do AP-1 foram mostradas por cjun e junB, que atúam antagónicamente no controle da transformação celular, diferenciação e expressão do AP-1 dependente do gene alvo. Mas a função de ambos é complexa e pode depender do tipo celular. O objetivo deste estudo foi determinar a expressão imunoistoquímica das proteínas c-jun e junB em 13 casos de carcinoma adenoide cístico e 12 de adenocarcinoma polimorfo de baixo grau de malignidade de glándulas salivares. Espécimes de mucosa normal foram incluídos e evidenciaram forte marcação nuclear e citoplasmática para junB e c-jun respectivamente. No presente estudo, independente da arquitetura histológica, ambos tumores mostraram muitas células tumorais com marcação nuclear e citoplasmática para a proteína c-jun e ausente para a proteína junB. As lesões do adenocarcinoma polimorfo de baixo grau de malignidade expressaram um maior número de células com marcação nuclear quando comparados ao do carcinoma adenoide císticode de mais baixo grau. De acordo com este estudo e com alguns estudos publicados na literatura, a c-jun é expressa em tumores de baixo grau e parece estar mais relacionada à diferenciação celular do que à proliferação celular. / Adenoid cystic carcinoma and polymorphous low grade are salivary gland neoplasms. ACC can arise in both, major or minor salivary glands. However, Polymorphous lowgrade adenocarcinoma, occurs specifically in minor salivary glands dispersed in the oral cavity. They share many common histologic features, as infiltrating solid, tubular and cribiform patterns and also high propensity for perineural invasion. Nevertheless, adenoid cystic carcinoma and polymorphous low grade are distinct types of adenocarcinomas with different prognosis, which occasionally may result in a diagnostic pitfall. C-jun and junB are family JUN members that may form homodimerizes or heterodimerizes with c-fos or other bzip proteins. Evidence for specific functions of AP-1 subunits was shown for c-jun and junB, which act antagonistically to control cell transformation, differentiation and expression of AP-1 depending on the target genes. However, the role of both of them is complex and it depends on cell type. The aim of this study was to determine immunohistochemistry of c -jun and junB expression in 13 cases of adenoid cystic carcinoma and 12 cases of polymorphous carcinoma low-grade adenocarcinoma of salivary glands. Moreover slides of normal mucosa were included and there were strong nuclei and cytoplasmic for junB and c-jun respectly. In the present study, independent of the histologic architecture, in both tumors shown many tumoral cells presented nuclear and cytoplasmic staining of c-jun and were absent to the protein junB. In polymorphous low-grade adenocarcinoma lesions expressed in a greater number cells staining than in the adenoid cystic carcinoma of the most lowgrade. According with this study and with some studies of the literature, the c-jun is expressed in low-grade tumors and seems to be related to cell differentiation more than with cell proliferation.

Avaliamos a capacidade de indução de migração, invasão e secreção de protease pelo peptídeo derivado da laminina, C16 (KAFDITYVRLKF, cadeia g1) em linhagem celular (CAC2) de carcinoma adenóide cístico humano. Laminina g1 foi imunolocalizada no carcinoma adenóide in vivo e in vitro. Ensaio de ferida, em câmara bipartite e em vídeo microscopia (time-lapse) mostraram que C16 estimula migração em células CAC2. C16 também estimulou invasão em ensaio com câmaras bipartites cobertas com Matrigel. Invasão depende de atividade de protease. Zimografia mostrou que C16 aumentou secreção de MMPs 2 e 9. Diferentes vias de sinalização podem estar relacionadas com os efeitos de C16. Immunoblot revelou que C16 aumentou a fosforilação de AKT e ERK. Para o estudo de possíveis receptores do peptídeo, preparações de membrana foram passadas em colunas de afinidade com C16 acoplado. Banda de 40kDa foi eluída e analisada por espectrometria de massa (LC-MS/MS) que identificou a cadeia a1 do colágeno. O fragmento de colágeno eluído poderia ser parte de um complexo protéico envolvendo C16. Integrinas são receptores de colágeno e candidatas a fazerem parte desse complexo. Células CAC2 expressaram as integrinas av, a5, b3 and b1. Silenciamento dessas integrinas promoveu redução da migração e secreção de protease induzidas por C16. Sugerimos que C16 estimularia migração, invasão e secreção de protease em células de carcinoma adenóide cístico através de integrinas a5b1 e avb3. O sinal gerado por C16 seria transduzido pelas vias AKT e ERK1/2. / We studied induction of migration, invasion and protease activity by laminin-derived peptide C16 (KAFDITYVRLKF, g1 chain) in a cell line (CAC2) from adenoid cystic carcinoma. Laminin g1 was immunolocalized in adenoid cystic carcinoma in vivo and in vitro. C16 increased migratory activity of CAC2 cells, as shown by monolayer wound assay, Transwell migration assay and time-lapse video microscopy. This peptide also stimulated cell invasion in Transwell chambers coated with Matrigel. Invasion depends on protease activity. Zymograms showed that C16 increased secretion of MMPs 2 and 9. Different signaling pathways could be related to C16 regulation in CAC2 cells. Immunoblot showed that C16 increased phosphorylation of both AKT and ERK compared to controls. To study putative receptors of this peptide we used affinity chromatography. Membrane preparations were run through C16-affinity columns. A 40kDa band was eluted and analyzed by mass spectrometry (LC-MS/MS) identifying a collagen a1 chain. The collagen fragment eluted could be part of a protein complex involving C16. This protein complex may include integrins, which are collagen receptors. CAC2 cells exhibited av, a5, b3 and b1 integrins. siRNA knockdown of these integrins inhibited both C16-induced migration and protease activity. We propose that C16 increases migration, invasion and protease activity of a human salivary gland adenoid cystic carcinoma cell line through a5b1 and avb3 integrins. The signal generated by C16 is transduced by AKT and ERK1/2 signaling pathways.