Unit:
Department of ChemistryLibrary of the School of Science
Dissertation committee:
Ευρύκλεια Λιανίδου, Καθηγήτρια, Τμήμα Χημείας, ΕΚΠΑ
Δημήτριος Μαυρουδής, Καθηγητής, Ιατρική Σχολή, Πανεπιστήμιο Κρήτης
Χρήστος Κρούπης, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Χρίστος Παπαδημητρίου, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Νικόλαος Θωμαϊδης, Καθηγητής,Τμήμα Χημείας, ΕΚΠΑ
Αμάντα Ψυρρή, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεωργία Σωτηροπούλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Πανεπιστήμιο Πατρών
Original Title:
ΥΓΡΗ ΒΙΟΨΙΑ: ΑΝΑΠΤΥΞΗ ΚΑΙ ΚΛΙΝΙΚΗ ΑΞΙΟΛΟΓΗΣΗ ΝΕΩΝ ΜΗ-ΕΠΕΜΒΑΤΙΚΩΝ ΜΕΘΟΔΩΝ ΜΟΡΙΑΚΗΣ ΔΙΑΓΝΩΣΤΙΚΗΣ ΓΙΑ ΤΗΝ ΠΑΡΑΚΟΛΟΥΘΗΣΗ ΑΣΘΕΝΩΝ ΜΕ ΚΑΡΚΙΝΟ
Translated title:
Liquid Biopsy: Development and clinical validation of new non-invasive molecular diagnostics methods for monitoring of cancer patients
Summary:
Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), circulating miRNAs and exosomes provides a non-invasive, real-time monitoring of tumor evolution and therapeutic efficacy.
In the present PhD thesis, we evaluated the effect of pre-analytical conditions on gene expression analysis in CTCs. For this purpose, we performed spiking experiments of 100 MCF7 cells into different peripheral blood collection tubes. Furthermore, CTCs were isolated using EpCAM-positive immunomagnetic enrichment at 0h, 24h and 48h and gene expression was quantified using RT-qPCR for CK-19 and B2M. At the same time, we performed a systematic quality control for all steps involved in the procedure for gene expression analysis in liquid biopsy. Our results indicate that CTC-analysis at the mRNA level is severely impeded by the preservatives or/and anticoagulants used in most peripheral blood collection tubes.
Furthermore, our next aim was to select the optimal isolation/enrichment system for CTC downstream molecular characterization in HNSCC. For this reason, we directly compared for the first time the performance of ParsortixTM device, a label-independent size-based microfluidic device versus an EpCAM-based CTC enrichment system using identical blood draws from patients with HNSCC, and downstream molecular characterization at the gene expression level. Our data clearly indicate that the CTCs population enriched using the ParsortixTM device is of higher purity than that of CTCs isolated using EpCAM positive immunomagnetic CTC enrichment.
We additionally developed a multiplex RT-qPCR for the simultaneous quantification of AR-full length (AR-FL), splice variants AR-V7, AR-567 and AR-Total with hybrolysis probes in Cobas z480 (Roche Diagnostics).The developed assay was applied and clinically validated in CTCs and exosomes of metastatic castration resistant prostate cancer. Our results clearly indicate that the developed assay can be applied both in CTCs and exosomes.
Finally, we evaluated the molecular profile of CTCs and exosomes of patients with metastatic castration resistant prostate cancer. More specifically, we evaluated gene expression profile for PD-L1, CK-19, splice variants AR-FL, AR-V7, AR-567 and AR-total, and the methylation profile of GSTP1 and RASSF1A.
This PhD thesis was funded by the European Program IMI-CANCER-ID (Contract No. 115749): “Cancer treatment and monitoring through identification of circulating tumour cells and tumour related nucleic acids in blood” (https://www.cancer-id.eu/)
Main subject category:
Science
Other subject categories:
Health Sciences
Keywords:
liquid biopsy, circulating tumor cells (CTCs), exosomes, preanalytical parameters, quality control, molecular characterization
Number of index pages:
XXIV
Number of references:
291
