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Proteinanalyse klinisch relevanter Signalmoleküle aus Formalin-fixierten Tumorgeweben.

Protein analysis of clinically relevant signaling molecules from formalin-fixed tumor tissues.

München, Technische Universität, Fakultät Wissenschaftszentrum Weihenstephan, Diss., 2012, 175 S.
Verlagsversion Volltext
Bei neuen Ansätzen zur Behandlung von Tumorpatienten nimmt die umfassende Charakterisierung deregulierter Signalproteine im erkrankten Gewebe eine Schlüsselrolle ein. Damit lassen sich zum einen Zielstrukturen für effektivere Therapien entwickeln, zum anderen stellen abnormal aktivierte Signalmoleküle Biomarker dar, um Patienten für diese neuen Therapieoptionen auswählen zu können. In meiner Arbeit habe ich zunächst die Proteinextraktion aus klinischen Tumorproben (Formalin-fixierte Brustkrebsgewebe) optimiert. Durch den nachfolgenden Einsatz der Hochdurchsatztechnik des „Reverse Phase Protein Arrays“ konnten in den Tumorzelllysaten erstmals Therapie-relevante Markerproteine (u.a. HER2, uPA und PAI-1) und deren assoziierte Netzwerke quantitativ dargestellt werden. Die Ergebnisse dieser Arbeit liefern ein besseres Verständnis für die Tumorbiologie und sind Ausgangspunkt zur Translation der funktionellen Signalwegskartierung in die molekulare in-vitro-Diagnostik.
In oncology a clear trend away from systemic therapies towards targeted treatment was recognizable in recent years. For some tumor types, like for example breast cancer, several molecular markers are available to support both treatment decision and personalised therapy. However problems still occur when it comes to the quantitative determination of these markers in clinical relevant, formalin-fixed tissues. Moreover in many entities only very little is known about the tumor biology. This knowledge however would be necessary to systematically identify novel molecular marker. The extraction of proteins from clinical tissues which was developed during the last years is an important step to overcome the mentioned obstacles. However, the current protocols are stretched to their limits if long-time stored or over-fixed samples are used for protein extraction. To deal with this issue an enhanced extraction protocol was developed in this thesis. Applying the optimised method the determination of three molecular breast cancer markers (uPA, PAI-1, HER2) should be enabled. For the two invasion-associated proteins uPA and PAI-1 this should be achieved first by using the clinically approved Femtelle-ELISA. Yet, this assay turned out to be incompatible with denatured proteins from formalin-fixed tissues. Not until the application of the Reverse Phase Protein Microarry (RPPA) approach it was possible to determine uPA and PAI-1 together with their associated protein networks. This high-throughput technology allows the parallel screening of a plurality of tumors for the expression and activation of whole protein networks. This method was also used for an absolute HER2 quantification from fixed tissue which was reached for the first time in this study. Furthermore capitalising the mentioned advantages of the RPPA, p-HER2 could be identified as a potential biomarker in breast cancer patients and p-HSP27(Ser15) and HER2 as markers in oesophageal adenocarcinoma. In conclusion, the results of this thesis show that the RPPA in combination with the optimised protein extraction method may contribute to the advancement of personalised medicine – both at research level and in the clinical routine.
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Schlagwörter FFPE-Gewebe; Reverse-Phase Protein Mikroarray (RPPA); Krebs; personalisierte Medizin; FFPE tissue; Reverse Phase Protein Microarray (RPPA); cancer; personalized medicine
Quellenangaben Band: , Heft: , Seiten: 175 S. Artikelnummer: , Supplement: ,
Hochschule Technische Universität
Hochschulort München
Fakultät Fakultät Wissenschaftszentrum Weihenstephan