Dissertation committee:
Evi Lianidou, Professor, Analytical Chemistry-Clinical Chemistry, Department of Chemistry, N.K.U.A.
Christos Papadimitriou, Professor, School of Medicine, N.K.U.A.
Athina Markou, Assistant Professor, Analytical Chemistry, Department of Chemistry, N.K.U.A.
Nikolaos Thomaidis, Professor, Analytical Chemistry, Department of Chemistry, N.K.U.A.
Alexandros Stratigos, Professor, Department of Dermatology-Venereology, Medical School, Andreas Syggros Hospital, N.K.U.A.
Galatea Kallergi, Associate Professor, Biochemistry, Department of Biology, University of Patras
Mike Makrigiorgos, PhD, Professor and Director, Medical Physics and Biophysics, Dana-Farber Cancer Institute, Harvard Medical School
Summary:
Molecular analysis in liquid biopsy samples requires the development of methodologies with high specificity and sensitivity in order to overcome the limitations and challenges. Here, in this PhD thesis, new methodologies with highly sensitive technologies were developed and validated in liquid biopsy samples. 1) We evaluated the performance of a novel multiplex assay for the detection of ten ESR1 mutations and AKT1 E17K in plasma-cell free DNA (cfDNA) based on Crystal Digital PCR®. Towards this, we analyzed in parallel identical plasma-cfDNA samples for ESR1 mutations with the multiplex Crystal Digital PCR® assay and the ESR1-NAPA assay and compared directly the results. 2) We investigated whether analysis of ESR1 mutations in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) provide complementary information on resistance to endocrine therapy. Using a highly sensitive multiplex ddPCR assay, we detected a higher percentage of mutations in CTC-derived genomic (gDNAs) than in paired ctDNA in patients with metastatic breast cancer. 3) We conducted a study to directly compare our ultrasensitive real-time PCR assay based on the combination of ARMS-PCR for the detection of PIK3CA p.E545K, p.E542K, p.H1047R mutations with ddPCR in primary tumors and plasma-cfDNA, and directly compare the PIK3CA mutational status in CTC-derived gDNAs and paired plasma-cfDNA samples. 4) A new method was evaluated that uses ultraviolet light to eliminate wild-type (wt-DNA) alleles and enables improved detection of minor genetic or epigenetic changes. PD-UVME employed oligonucleotide probes that incorporated a photosensitive 3-cyanovinylcarbazole nucleoside (CNVK), placed directly opposite interrogated pyrimidines, such as thymine or cytosine in wt-DNA. PD-UVME was combined with ddPCR to detect BRAF V600E mutation in model systems, thyroid patient cancer tissue samples, and ctDNA from melanoma patients. 5) We conducted a study to develop and validate a novel RT-qPCR assay for CTLA-4 transcripts and study the expression of CTLA-4 in CTCs of patients with metastatic cancer under immunotherapy.
Keywords:
Liquid biopsy, CTCs, ctDNA, DNA mutations, gene expression
