FRET-Bildgebung zum Nachweis der Phopshorylierung der anaplastic lymhoma kinase (ALK) im nicht-kleinzelligen Bronchialkarzinom
FRET-Imaging to proof the phosphorylation of the anaplastic lymphoma kinase in NSCLC
Tabea Wagner
Doctoral thesis
Date of Examination:
2018-06-05
Date of issue:
2018-05-22
Advisor:
Kroemer, Heyo K. Prof. Dr.
Referee:
Ströbel, Philipp Prof. Dr.
Referee:
Wouters-Bunt, Freddy Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002E-E3F0-F
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Dissertation Tabea Wagner final SUB 1.pdf
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Abstract
English
Background: Anaplastic lymphoma kinase (ALK) is an oncogenic driver mutation in T-cell lymphoma and NSCLC. Small molecule inhibitors are available. Treatment decisions are mostly made on ALK expression but their efficacy widely varies from complete response to progression under therapy. I regard ALK activity, judged by its phosphorylation, a highly relevant factor in the evaluation of therapeutic sensitivity. Phosphorylation can be quantified by Förster Resonance Energy Transfer (FRET), here shown by confocal acceptor photobleaching micorscopy, and could be used to select patients for targeted therapies. Methods: Confocal laser microscopy, cell culture, FFPE-tissue, Immunocytofluorescence, Immunohistofluorescence, Westernblots, Immunoprecipitation, overall-survival-measurements Results: I show ALK phosphorylation in human cancer cell lines. FRET strongly correlates with the pharmacological ALK inhibition sensitivity of the cell lines, implying a promising role as companion diagnostic test for ALK activity drug. The same cell lines were subjected to cytospin and block preparation. I could closely reproduce the results, demonstrating the utility of the method in routine cytological work. Also I could implement Immunohistofluorescence to apply the method in formalin-fixed, paraffin-embedded (FFPE) human tissue. Conclusions: I present a quantitative cell ALK phosphorylation assay, based on confocal acceptor photobleaching FRET microscopy that holds promise for future diagnostic applications on FFPE samples of human cancer. This method provides both information on ALK expression, comparable to immunohistochemistry and FISH methods, and on ALK phosphorylation, relfecting its activity and likely predicting sensitivity to targeted therapies.
Keywords: EML4-ALK; FRET; Acceptor Photobleaching; Lung cancer; Confocal microscopy
Schlagwörter: EML4-ALK; Bronchialkarzinom; Konfokale Mikroskopie
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Human- und Zahnmedizin [2277]