Amer, Wafa (2016). Comprehensive Analysis of Disease Related Nuclear and Mitochondrial Genes in Hepatocellular Carcinoma. PhD thesis, Universität zu Köln.

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Abstract

Hepatocellular carcinoma (HCC) is the third most lethal cancer due to late detection, high recurrence and limited therapeutics. Although genetic alterations have been recently studied by whole genome or whole exome next generation sequencing, a comprehensive analysis of HCC relevant genes on lesions with low and high grade of dedifferentiation is missing. Herein, I aimed to study the nuclear and mitochondrial genomic alterations in order to characterize HCC development and clonality. First, for target enrichment of HCC relevant genes and mt-genome, gene loci of relevance had to be selected and primer sets designed. Subsequent ultra-deep sequencing revealed that in accordance with previous studies, the β-catenin gene (CTNNB1) was shown to be the most frequently mutated oncogene, whereas the TP53 and AXIN1 genes were the most frequently mutated tumour suppressor genes. Interestingly, CTNNB1 mutations were detected in lesions with early as well as advanced HCC stage, confirming its role in hepatocarcinogenesis. In contrast, TP53 mutations were only detected in nodules with advanced HCC stage. In addition, non- protein damaging mutations in the AXIN1 gene were detected in higher frequency in non-tumour than in tumour lesions; indicating the loss of heterozygosity (LOH) in some tumour samples. Since AXIN1 alterations effect as well as β-catenin mutations the WNT signalling, my study supports new therapeutical strategies targeting components of the WNT pathway. Furthermore, this study addressed the intra-tumour clonal structure by ultra-deep sequencing of the entire mitochondrial (mt) genome for better understanding how HCC originate, develop and progress. Since, the mt-genome is highly susceptible to DNA alterations due to the lack of protective histones and a limited DNA repair system, the mutations of the mtDNA are ideal targets to be used for follow up of tumour progression and analysis of tumour clonality. Therefore, I established a NGS approach for a rapid and sensitive mutation screening analysis of the entire mt- genome as a novel tool for tumour cell tracking. Beside a high mt-mutation rate in tumour areas, also frequent mt-mutations were observed in peri-tumour area suggesting that mt-genome is susceptible at earliest stage of hepatocarcinogenesis. Furthermore, most HCC nodules of individual sample have identical mt-mutations, indicates the monoclonal HCC origin. Interestingly, the increasing numbers and frequency of a particular mt-hotspot mutation refer to the progression of the HCC dedifferentiation. In summary, I established a fast pipeline of mutation analysis for simultaneous testing of a tumour specific, hotspot gene panel covering diagnostic relevant loci of HCC, and could assist in the selection of currently available treatments likely to be most effective for HCC patients. Notably, effective targeting of the Wnt signalling pathway is considered as a potential target for pharmacological therapy that is eagerly awaited. Additionally, our mt-genome screening based approach representing rapid and sensitive molecular tool and provide novel insights in cancer diagnostics and therapeutic strategies.

Item Type: Thesis (PhD thesis)
Creators:
CreatorsEmailORCID
Amer, Wafaw.amer85@yahoo.comUNSPECIFIED
URN: urn:nbn:de:hbz:38-93667
Subjects: Natural sciences and mathematics
Uncontrolled Keywords:
KeywordsLanguage
Tumor heterogeneity, clonality, mt-DNA alteration, formalin fixed and paraffin- embedded (FFPE), next generation sequencing, hepatocellulr carcinoma (HCC)English
Faculty: Faculty of Mathematics and Natural Sciences
Divisions: Faculty of Mathematics and Natural Sciences
Language: English
Date: 2016
Date of oral exam: 28 October 2016
Referee:
NameAcademic Title
Odenthal, MargareteProf. Dr.
Nürnberg, PeterProf. Dr.
Wunderlich, ThomasPriv-Doz.
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/9366

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