Department of Chemistry
Library of the School of Science

2023-03-05

2023

Stergiopoulou Dimitra

Ευρύκλεια Λιανίδου, Καθηγήτρια Αναλυτικής Χημείας-Κλινικής Χημείας, Τμήμα Χημείας, ΕΚΠΑ
Χρίστος Παπαδημητρίου, Καθηγητής Παθολογίας-Ογκολογίας, Ιατρική Σχολή, ΕΚΠΑ
Αθηνά Μάρκου, Επίκουρη Καθηγήτρια Αναλυτικής Χημείας, Τμήμα Χημείας, ΕΚΠΑ
G. M. Makrigiorgos, Professor, Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, USA
Αμάντα Ψυρρή, Καθηγήτρια Παθολογίας-Ογκολογίας, Ιατρική Σχολή, ΕΚΠΑ
Γαλάτεια Καλλέργη, Επίκουρη Kαθηγήτρια Βιοχημείας, Τμήμα Βιολογίας, Πανεπιστήμιο Πατρών
Λεωνίδας Αλεξόπουλος, Καθηγητής, Εργαστήριο Βιοϊατρικών Συστημάτων, Εθνικό Μετσόβειο Πολυτεχνείο, Αθήνα

Υγρή Βιοψία-Μοριακή διάγνωση καρκίνου: Ανάπτυξη μεθόδων ανάλυσης κυκλοφορούντων καρκινικών κυττάρων και αντίστοιχου ctDNA και κλινική αξιολόγηση της ετερογένειάς τους στον καρκίνο μαστού

Greek

Liquid biopsy-Molecular diagnosis of cancer: Development of assays for the analysis of circulating tumor cells and ctDNA and clinical evaluation of their heterogeneity in breast cancer

Liquid biopsy is a minimally invasive approach that provides important information regarding the tumor progression through the analysis of peripheral blood allowing real-time monitoring of the patient. Liquid biopsy is mainly based on the analysis of circulating tumor cells (CTCs) and extracellular tumor DNA (ctDNA). The analysis of single CTCs provides the possibility to elucidate the various mechanisms underlying the heterogeneity of cancer cells within the patient as well as those of tumor progression. The detection of mutations in liquid biopsy samples (ctDNA and CTCs) requires highly sensitive techniques due to the low frequency of mutated alleles in the total wild-type background.
In the present PhD thesis, we initially developed and analytically validated a novel, highly sensitive and specific ESR1-NAPA assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G). The ESR1-NAPA assay is based on the combination of: (a) a NaME-PrO step to eliminate the wild-type DNA, (b) an ARMS-PCR step for detection of the four ESR1 mutations separately, and 3) a real-time PCR-melting curve analysis step. The assay was further applied in ER+ breast cancer (BrCa) primary tumour tissues (FFPEs), non-cancerous breast tissues (mammoplasties), and pairs of liquid biopsy samples (CTCs and paired plasma-ctDNA) obtained at different time points from 8 ER+ metastatic breast cancer patients. In the plasma-ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected during the follow-up period at five consecutive time points in one patient. In the CTCs, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1-NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%.
Furthermore, we performed for the first time a comprehensive liquid biopsy analysis for 13 patients all diagnosed with early breast cancer, based on a long-term follow-up. Our analysis was based on CTC enumeration, CTC phenotypic characterization and molecular monitoring of CTCs at the gene expression, DNA mutation and DNA methylation level, and on corresponding plasma cell-free DNA for DNA mutation and DNA methylation markers. Our results indicate that a comprehensive liquid biopsy analysis provides valuable and important information for the therapeutic management of breast cancer patients since it is a unique way to shed light into the black box that was sealed till now between the information taken from the primary tumor and from a distant metastatic site. Our results demonstrate that liquid biopsy analysis could reveal the presence of MRD years before the appearance of clinically detectable metastatic disease and progression of disease.
In addition, a novel drop-off ddPCR assay, the Dual-drop-off ddPCR methodology, was developed and analytically validated for the simultaneous screening of mutations in exons 9 and 20 of the PIK3CA gene. The analytical validation of the methodology was followed by its clinical application to plasma-cfDNA samples of patients with ER+ metastatic breast cancer. A comparison study was performed with previously developed methodologies based on the combination of allele-specific ARMS-PCR and melting curve analysis.
In the last part of the present PhD thesis, a novel multimarker methodology was developed and validated for the simultaneous detection of PIK3CA and ESR1 mutations based on the Multiplex PCR-coupled liquid bead array technology (Luminex system). This methodology was first validated using cancer cell lines and then was applied to single CTCs isolated from CellSearch® cartridges through the VyCAP punching system.

Science

liquid biopsy, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), single cell analysis, drop-off ddPCR, Luminex system, DNA mutations, PIK3CA, ESR1, CK-19, ESR1 methylation, minimal residual disease, breast cancer

Yes

8

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https://pergamos.lib.uoa.gr/uoa/dl/object/3284888