Nouvelles méthodes de détection de l'ADN tumoral circulant par PCR digitale en gouttelettes : application au suivi des patients

Abstract : Circulating tumor DNA (ctDNA) carries tumor-specific alterations that are detectable by minimally invasive sampling. It represents a highly pertinent marker for cancer monitoring during patients’ follow-up. CtDNA detection requires a highly sensitive and quantitative technique. In this context, this project focused on ctDNA quantification and monitoring by picoliter-droplet digital PCR. Thanks to the compartmentalization in millions of picoliter droplets, this tool allowed the detection of single DNA molecule with a sensitivity reaching 0.001%. Testing of ctDNA was performed through the evaluation of different potential biomarkers: specific mutations, ctDNA fragmentation, and hypermethylation of target sequences. On one hand, we observed in cancer patients that ctDNA is more fragmented than wild-type DNA, and, globally more fragmented than circulating DNA in healthy individuals. On the other hand, a strong correlation between percentages of hypermethylated and mutated DNA was observed during the follow-up of patients. Such results suggest the feasibility to precisely and quantitatively monitor ctDNA by the evaluation of hypermethylation as an alternative to the determination of mutational status. We have applied such ctDNA detection strategies in the context of two clinical studies. The PLACOL study, enrolling 82 metastatic colorectal cancer patients, allowed to highlight two prognostic factors: a ctDNA concentration threshold of 0.1 ng / mL, and the evaluation of ctDNA decreasing slope. In the second study, ctDNA was monitored in 11 melanoma patients in the context of a targeted therapy (vemurafenib). An inverse correlation between the concentrations of vemurafenib and ctDNA was demonstrated. These results suggest the clinical relevancy of ctDNA in advanced cancer patients, for the optimization of therapeutic management.
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Fanny Garlan. Nouvelles méthodes de détection de l'ADN tumoral circulant par PCR digitale en gouttelettes : application au suivi des patients. Cancer. Université Sorbonne Paris Cité, 2016. Français. ⟨NNT : 2016USPCB108⟩. ⟨tel-02145652⟩

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