Identifizierung genetischer Biomarker für die Wirksamkeit von Oxaliplatin:Kandidatengen-bezogene und Genom-weite Analysen
Identification of genetic biomarkers for the efficacy of oxaliplatin - candidate gene and genome-wide approaches
von Sadik Saman
Datum der mündl. Prüfung:2014-12-02
Erschienen:2014-11-28
Betreuer:Prof. Dr. Jürgen Brockmöller
Gutachter:Prof. Dr. Jürgen Brockmöller
Gutachter:Prof. Dr. Tim Beißbarth
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-4792
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Zusammenfassung
Englisch
Oxaliplatin is an anticancer drug used to treat advanced and metastatic stages of colorectal carcinoma. In patients, different treatment responses can be observed suggesting an impact of germline genetic variability. Regarding platin compounds, cellular sensitivity in relation to genetic variability was ascertained by genome-wide as well as candidate related approaches mainly for the older platinum derivatives cisplatin and carboplatin but barely for oxaliplatin. The intention of this dissertation was to identify genetic markers for oxaliplatin sensitivity by genotype-phenotype analysis to provide candidates to test as biomarkers in clinical settings. As model system, 198 lymphoblastoid cell lines were used of which 104 were used as training set and 94 as test set. Phenotypic variability of oxaliplatin sensitivity was ascertained by dose-response effects measured by flow cytometry. Based on eight oxaliplatin concentrations and a drug-free control for each cell line the substrat concentration corresponding to the half maximum effect (EC50) of reduction in cell vitality and delay in cell proliferation was determined by mathematical curve fitting. For the training set, a genome-wide association analysis was conducted using the genotypes from the databases of HapMap and 1000 human genomes. The 20 strongest association signals from the training set were tested through genotyping by primer extension in the test set. The mRNA transcription of candidate genes for transport (SLC31A1 and ATP7A) and DNA damage repair (ERCC1, ERCC2 and ERCC5) of oxaliplatin were measured with and without exposure to oxaliplatin by quantitative real time PCR and tested for correlation with the aforementioned cytotoxicity parameters. The candidate gene approach was employed for these genes to evaluate the potential influence of genetic variability. The genome-wide analysis showed a reproducible modulation of oxaliplatin sensitivity by the single nucleotide polymorphism (SNP) rs74382821. The sensitivity towards oxaliplatin was increased by the variant allele which occurs at 8% in Caucasians. No genes or transcripts are yet annotated in the region harboring this SNP. Furthermore, nothing is known about the function of this SNP and there is also no hint by bioinformatic analysis. Concerning the investigated candidate genes, a consistent influence on the oxaliplatin sensitivity was only observed for the copper transporter SLC31A1: Higher baseline expression was associated with an increased sensitivity towards oxaliplatin. This relationship was not modulated by any of the comprehensively studied genetic polymorphism in the genomic region of SLC31A1. Furthermore, an obvious suppression of SLC31A1 transcription was observed upon exposure to oxaliplatin. A comparison with other genes showed that this suppression seems to be rather unspecific and might be due to the cytotoxic effects provoked by oxaliplatin. This oxaliplatin-induced SLC31A1 suppression was mitigated in presence of the variant allele at the SNP rs11793978 located in intron 1 of the SLC31A1 gene. There is no information about the functional role of this SNP in literature. However, this SNP has a moderate common heredity with another SNP, which was reported to impact the survivial of patients with lung cell carcinoma treated with cisplatin. In silico analysis revealed a binding of the micro-RNA hsa-miR-1277-3p only at the wild type allele of rs11793978. As this SNP is not part of the mRNA sequence it could not directly be involved in transcript stability regulation, the relevance of this finding has to be clarified. The other four candidate genes did not show any significant correlation with oxaliplatin sensitivity. Further functional and clinical studies should be carried out to confirm these results. The two herein identified SNPs should be evaluated in clinical cohorts of patients treated with oxaliplatin. Moreover, SLC31A1 expression status in tumor tissue and/or peripheral blood might serve as a promising parameter for oxaliplatin response or side-effects. This assumption has to be proven in the clinical situation. Furthermore, the observed down-regulation of SLC31A1 by oxaliplatin may represent an important resistance mechanism which should further be explored. If so, strategies to target this reaction should be considered. The identified genetic and expression markers for oxaliplatin sensitivity may be important findings for the establishment of biomarkers with the aim to individualize future therapy with higher response rates and less side effects.
Keywords: lymphoblastoid cell lines; ERCC1; SLC31A1; rs74382821; rs11793978; genome-wide; HapMap; 1000 human genomes; cisplatin; carboplatin; micro-RNA hsa-miR-1277-3p; colorectal carcinoma; ATP7A; ATP7B; Oxaliplatin; cytotoxocity; cell vitality; cell proliferation; Sytox; Vybrant Ruby; CFSE; counting beads; LCL; Intron 1 SLC31A1
Schlagwörter: Oxaliplatin; Kolorektales Karzinom; Cisplatin; Carboplatin; rs11793978; rs74382821; micro-RNA hsa-miR-1277-3p; individualisierte Therpaie; Genom-weite Analyse; ERCC1; ERCC2; ERCC5; Zytotoxizität; CFSE; Vybrant Ruby; Sytox; Zellproliferation; Zellvitalität; Proliferationshemmung; DACH-Ligand; SLC31A1; LCL; lymphoblastoide Zelllinien; 1000 human genomes; HapMap; Intron 1 SLC31A1