Dokument: Genomische Analyse disseminierter Tumorzellen (DTCs) bei Ösophaguskarzinompatienten - Identifizierung einer prognostisch relevanten DTC-Subpopulation und Etablierung der aCGH zur hochaufgelösten genomischen Einzelzellanalyse von Chromosom 17
| Titel: | Genomische Analyse disseminierter Tumorzellen (DTCs) bei Ösophaguskarzinompatienten - Identifizierung einer prognostisch relevanten DTC-Subpopulation und Etablierung der aCGH zur hochaufgelösten genomischen Einzelzellanalyse von Chromosom 17 | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=19936 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20111129-100512-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Will, Daniel [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Knoefel, Wolfram Trudo [Betreuer/Doktorvater] Prof. Dr. Schmitt, Lutz [Gutachter] | |||||||
| Stichwörter: | Ösophaguskarzinom, DTCs, EpCAM, CK, aCGH, mCGH, | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibungen: | Ösophaguskarzinompatienten, die nach vollständiger Entfernung des Tumors als potenziell geheilt beurteilt werden, entwickeln dennoch häufig Metastasen, welche meist zum Versterben führen. Die Ursache für die Metastasenbildung stellen dabei einzelne gestreute Vorläuferzellen dar, die durch derzeitig zur Verfügung stehende Therapien nicht entfernt werden können. Die Untersuchung von disseminierten Tumorzellen (DTCs) könnte dabei zur Identifizierung von therapeutisch relevanten Zielstrukturen beitragen.
Am häufigsten werden solche disseminierten Tumorzellen (DTCs) im Knochenmark anhand von Zytokeratinen (CK) und im Lymphknoten anhand des Epithelialen Zelladhäsionsmoleküls (EpCAM) detektiert. Stoecklein et al. beschrieb dabei unterschiedliche genomische Veränderungen zwischen CK+-DTCs aus dem Knochenmark und EpCAM+-DTCs aus dem Lymphknoten. Ob die beobachteten Unterschiede dadurch zustande kommen, dass die DTCs in verschiedenen Organen auftreten oder ob anhand EpCAM und CK generell unterschiedliche Subpopulationen von DTCs detektiert werden, sollte in der vorliegenden Arbeit zunächst aufgeklärt werden. Eine Doppelimmunfluoreszenzfärbung konnte alle drei denkbaren DTC-Populationen (CK+/EpCAM-; CK+/EpCAM+; CK-/EpCAM+) sowohl im Knochenmark als auch im Lymphknoten identifizieren, wobei DTCs jedoch seltener EpCAM als CK exprimieren. Durch CGH-Analysen wurden analog zu den publizierten Daten von Stoecklein et al. generell mehr genomische Aberrationen in DTCs aus Lymphknoten als in DTCs aus dem Knochenmark detektiert. Umfassende CGH-Analysen zeigten dabei, dass die beobachteten Unterschiede zwischen den DTC-Populationen mit dem Disseminierungsort assoziiert und unabhängig von der exprimierten Markerkombination sind. Weiterhin konnte in der vorliegenden Arbeit die Prävalenz von EpCAM+-DTCs als unabhängiger Faktor für eine verkürzte Überlebensdauer bei Ösophaguskarzinompatienten identifiziert werden. Ferner gehörten Amplifikationen auf Chromosom 17 zu den häufigsten Veränderungen bei ösophagealen DTCs aus den Lymphknoten bzw. aus dem Knochenmark. Um nun potenzielle therapeutische Zielgene auf Chromosom 17 identifizieren zu können, sollte ein Hochdurchsatzverfahren zur hochauflösenden genomischen Analyse dieses Chromosoms mittels aCGH etabliert werden. Hierfür wurde zunächst die Isolation von BAC-DNA für die Herstellung von BAC-DNA-Microarrays semiautomatisiert. Durch den Einbau einer terminalen Aminogruppe in die BAC-DNA konnte diese durch eine kontrollierte kovalente Verknüpfung auf epoxidbeschichtete Chipoberflächen immobilisiert werden, was letztendlich die Reproduzierbarkeit des Array-Verfahrens enorm erhöhte. Nach Etablierung und Validierung eines Protokolls zur Hybridisierung von Einzelzell-DNA auf die hergestellten BAC-DNA-Testmicroarrays wurde ein BAC-DNA-Microarray zur hochaufgelösten Analyse des Chromosoms 17 von Einzelzell-DNA hergestellt. Im Zuge der Validierung wurde eine 1,37 Mbp große Amplifikation nachgewiesen, welche für Einzelzell-DNA bisher noch nicht gezeigt wurde. Zudem konnte auch reamplifizierte BAC-DNA verwendet werden, was die maximal herstellbare Menge an Microarrays mit einmalig aufgereinigter BAC-DNA in etwa um den Faktor 50 erhöht und somit Zeit und Kosten spart. Da zwei der sechs HER2-positiven DTCs (HER2-Status durch qPCR bestimmt) durch die aCGH fälschlicherweise als HER2-negativ befunden wurden, eignet sich der hergestellte Microarray bislang noch nicht zur Bestimmung von spezifischen Aberrationen für den individuellen Patienten, um darauf basierend Therapieentscheidungen zu treffen. Allerdings kann die etablierte Methode zur Untersuchung von DTC-Kollektiven verwendete werden, um häufige Aberrationen in disseminierten Tumorzellen zu detektieren, da durch die aCGH-Analysen erfolgreich zwischen prognostisch relevanten (HER2+) und prognostisch irrelevanten (HER2-) DTC-Populationen unterschieden werden konnte. Da sich unter den DTCs die Vorläuferzellen der Metastasen befinden, kann somit durch die Identifikation häufiger Aberrationen in DTCs ein Beitrag zur Aufklärung des Prozesses der Metastasierung geleistet werden. Des Weiteren können möglicherweise die durch aCGH identifizierten häufigen Aberrationen therapeutische Zielstrukturen für systemische Therapien darstellen, mit deren Hilfe die prognostisch relevanten DTCs eliminiert werden können.Esophageal cancer is often completely resected during surgery in order to cure the patient. Although many patients are diagnosed afterwards as tumor free, they frequently develop metastases to which they often succumb. Single disseminated tumor cells (DTCs), which can not be eradicated by the available therapies, account for the formation of metastasis. The analysis of these DTCs could help to identify therapeutically relevant target molecules. DTCs of the bone marrow are mostly detected via cytokeratins (CK) while DTCs from the lymph node are usually detected via the epithelial cell adhesion molecule (EpCAM). Stoecklein et al. have described different genomic aberrations for CK+-DTCs of the bone marrow and EpCAM+-DTCs of the lymph node. This thesis aimed to find out whether the detected differences are due to the ocurrence of the DTCs in different organs or due to the fact that EpCAM and CK characterize different DTC subpopulations. A double immunofluorescence staining was able to detect all three possible DTC populations (CK+/EpCAM-; CK+/EpCAM+; CK-/EpCAM+) in the bone marrow as well as in the lymph node. However, DTCs express CK more often than EpCAM. CGH analysis elicited more genomic abarrations in DTCs of lymph nodes than in DTCs of the bone marrow. Detailed CGH analysis indicated that the detected differences between the DTC populations are associated with the organ to which they have disseminated and are independent of the expressed marker combination. Furthermore, the prevalence of EpCAM+-DTCs was demonstrated to be an independent prognostic factor for overall survival of patients with esophageal cancer. Moreover, amplifications on chromosome 17 were among the most frequent aberrations in esophageal DTCs of the lymph node as well as the bone marrow. To identify potential therapeutic target genes on chromosome 17, a high throughput method for highly resolved genomic analysis of this chromosome via aCGH was required. Therefore the isolation of BAC-DNA for BAC-DNA microarrays was semiautomatized prior to any other steps. The modification of the BAC-DNA with terminal aminogroups enabled the immobilization of the BAC-DNA onto epoxycoated chip surfaces and an increased reproducibility of the arrays. After establishment and validation of a protocol for hybridization of single cell DNA on the generated BAC-DNA-test-microarrays, a BAC-DNA microarray for high resolution analysis of chromosome 17 of single cell DNA was generated. In the course of validation an amplification of 1.37 Mbp was detected which has not been visualized in single cells so far. Furthermore it was demonstrated that reamplified BAC-DNA is also suitable for aCGH. Consequently, approximately 50 times more microarrays can be produced with one batch of BAC-DNA which saves time and funds. The generated microarray cannot be used for the detection of specific aberrations for individual patients to facilitate decisions upon treatent as two out of six HER2-positive DTC (DTC status determined by qPCR) were wrongly classified as HER2-negative. However, the established method can be utilized for the analysis of DTC collectives to identify frequent aberrations in disseminated tumor cells as distinguishment between prognostic relevant (HER2+) and pognostic irrelevant (HER2-) DTC populations was feasible with the aCGH analysis. As the progenitor cells of metastases are assumed to be among the DTCs, the identification of frequent aberrations within DTCs could contribute to the elucidation of the process of metastasis. Furthermore, frequent aberrations identified by aCGH analysis might represent targets for systemic therapy with which prognostic relevant DTC could be eliminated. | |||||||
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| Lizenz: |  Urheberrechtsschutz | |||||||
| Bezug: | Düsseldorf/Regensburg 3/2006-11/2011 | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie | |||||||
| Dokument erstellt am: | 29.11.2011 | |||||||
| Dateien geändert am: | 29.11.2011 | |||||||
| Promotionsantrag am: | 16.08.2011 | |||||||
| Datum der Promotion: | 08.11.2011 |
