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Liquid biopsy can be used as a tool to unveil resistance mechanisms in NSCLC. Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC whereas circulating tumor cells (CTCs) constitute a unique source of information at the cellular level. Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. The main goal of the present doctoral thesis was to evaluate molecular alterations in CTCs and ctDNA of NSCLC patients during osimertinib treatment at the gene expression level, DNA mutation level and DNA methylation level and further evaluate their heterogeneity and clinical significance. At first, we studied for the first time, changes in gene expression of CK-8, CK-18, CK-19, VIM, TWIST-1, AXL, ALDH-1, PD-L1, PIM-1 and CD-45 in CTC-enriched fractions of 30 NSCLC patients before initiation of osimertinib treatment (baseline), after one cycle of treatment and at progression of disease (PD). The high prevalence of VIM positive CTCs suggest a dynamic role of EMT during osimertinib treatment, while increased prevalence of PD-L1 expression at PD suggests a theoretical background for immunotherapy in EGFR-mutant NSCLC patients, an observation that merits further evaluation through prospective immunotherapy trials, Then, we quantified mutation allele frequencies (MAF) of EGFR mutations in plasma-cfDNA samples of these patients and in their matched CTC-derived genomic DNA samples using crystal digital PCR technology. EGFR mutation analysis was concurrently conducted using the FDA-cleared cobas® EGFR mutation test (Roche) in identical plasma samples and direct comparison with crystal digital PCR revealed high concordance. Besides, direct comparison of ctDNA with the paired CTC-derived gDNA and corresponding primary tissue revealed a high heterogeneity. Finally, for the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients was performed for nine selected genes

Η υγρή βιοψία αποτελεί πολύτιμο εργαλείο για τη μοριακή διάγνωση του μη-μικροκυτταρικού καρκίνου του πνεύμονα (NSCLC). Η κλινική σημασία των EGFR μεταλλάξεων στο κυκλοφορούν καρκινικό DNA (circulating tumor DNA, ctDNA) έχει ήδη αποδειχθεί ενώ τα κυκλοφορούντα καρκινικά κύτταρα (circulating tumor cells, CTCs) αποτελούν μια μοναδική πηγή πληροφοριών του όγκου. Η χορήγηση θεραπείας με osimertinib σε ασθενείς που φέρουν τη μετάλλαξη Τ790Μ του γονιδίου EGFR έχει θετικά αποτελέσματα, όμως η εμφάνιση αντίστασης στη θεραπεία είναι αναπόφευκτη. Ο κύριος στόχος της διδακτορικής διατριβής ήταν η αξιολόγηση των μοριακών αλλαγών που παρατηρούνται στα CTCs και ctDNA σε επίπεδο γονιδιακής έκφρασης, DNA μεταλλάξεων και μεθυλίωσης DNA πριν και μετά τη θεραπεία με osimertinib και η περαιτέρω αξιολόγηση της ετερογένειάς τους και της κλινικής της σημασίας. Αρχικά, μελετήθηκε για πρώτη φορά η έκφραση των γονιδίων CK-8, CK-18, CK-19, VIM, TWIST-1, AXL, ALDH-1, PD-L1, PIM-1 και CD-45 στα CTCs 30 ασθενών με NSCLC πριν τη θεραπεία με osimertinib, μετά τον πρώτο κύκλο και στη πρόοδο νόσου (PD). Τα αποτελέσματα έδειξαν ότι αυξημένη παρουσία της VIM συνδέεται με το ρόλο της επιθηλιο-μεσεγχυματικής μετάβασης κατά τη διάρκεια της θεραπείας ενώ η αυξημένη έκφραση του PD-L1 στο PD υποδεικνύει ένα θεωρητικό υπόβαθρο για περαιτέρω αντιμετώπιση των ασθενών αυτών με ανοσοθεραπεία. Η ανίχνευση και ο ποσοτικός προσδιορισμός των EGFR μεταλλάξεων στο ctDNA πλάσματος και στα CTCs στο περιφερικό αίμα των ασθενών αυτών με την τεχνολογία crystal digital PCR έδειξε την ετερογένεια του όγκου σε επίπεδο μεταλλάξεων EGFR. Παράλληλα, αναλύθηκαν ταυτόσημα δείγματα ctDNA με την εγκεκριμένη από τον FDA μεθοδολογία cobas (Roche) και από την άμεση σύγκριση προέκυψαν υψηλά ποσοστά συμφωνίας. Επιπλέον, η άμεση σύγκριση της παρουσίας EGFR μεταλλάξεων στο ctDNA πλάσματος με τα αντίστοιχα δείγματα CTCs και ιστών αποκάλυψε την ύπαρξη μεγάλης ετερογένειας. Τέλος, πραγματοποιήθηκε για πρώτη φορά μελέτη μεθυλίωσης εννέα γονιδίων (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARβ, SHISA3, and FOXA1) στο ctDNA πλάσματος και σε αντίστοιχα δείγματα CTCs πριν και μετά τη θεραπεία με osimertinib. Τα αυξημένα ποσοστά μεθυλίωσης στο PD θα μπορούσαν να συσχετιστούν με την αντίσταση στο osimertinib ενώ οι διαφορές μεταξύ ctDNA και CTCs υποδεικνύουν τη σημασία της συνδυαστικής τους ανάλυσης. Liquid biopsy can be used as a tool to unveil resistance mechanisms in NSCLC. Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC whereas circulating tumor cells (CTCs) constitute a unique source of information at the cellular level. Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. The main goal of the present doctoral thesis was to evaluate molecular alterations in CTCs and ctDNA of NSCLC patients during osimertinib treatment at the gene expression level, DNA mutation level and DNA methylation level and further evaluate their heterogeneity and clinical significance. At first, we studied for the first time, changes in gene expression of CK-8, CK-18, CK-19, VIM, TWIST-1, AXL, ALDH-1, PD-L1, PIM-1 and CD-45 in CTC-enriched fractions of 30 NSCLC patients before initiation of osimertinib treatment (baseline), after one cycle of treatment and at progression of disease (PD). The high prevalence of VIM positive CTCs suggest a dynamic role of EMT during osimertinib treatment, while increased prevalence of PD-L1 expression at PD suggests a theoretical background for immunotherapy in EGFR-mutant NSCLC patients, an observation that merits further evaluation through prospective immunotherapy trials, Then, we quantified mutation allele frequencies (MAF) of EGFR mutations in plasma-cfDNA samples of these patients and in their matched CTC-derived genomic DNA samples using crystal digital PCR technology. EGFR mutation analysis was concurrently conducted using the FDA-cleared cobas® EGFR mutation test (Roche) in identical plasma samples and direct comparison with crystal digital PCR revealed high concordance. Besides, direct comparison of ctDNA with the paired CTC-derived gDNA and corresponding primary tissue revealed a high heterogeneity. Finally, for the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients was performed for nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARβ, SHISA3, and FOXA1). Our results indicate that DNA methylation could be a possible resistance mechanism. Results from direct comparison of methylation status between ctDNA and CTCs indicated that the latter give complementary clinical information.

Simple Summary Liquid biopsy is a highly useful tool for the management of NSCLC patients and could provide valuable information on early detection of resistance to osimertinib. Epigenetic biomarkers are very promising for the early diagnosis, prognosis, and prediction of drug response in many types of cancer. We performed a DNA methylation analysis in plasma cell-free DNA and paired CTCs of NSCLC patients before osimertinib treatment and at progression of disease (PD). Our results revealed a significant increase in DNA methylation at PD. Epigenetic alterations should be further evaluated as a possible resistance mechanism to osimertinib and their detection in liquid biopsy samples can be valuable for the follow-up of patients in real time. Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. DNA methylation has been previously implicated in NSCLC progression and often in therapy resistance, however its distinct role in osimertinib resistance is not elucidated as yet. In the present study, we directly compared DNA methylation of nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RAR beta, SHISA3, and FOXA1) in plasma-cfDNA and paired CTCs of NSCLC patients who were longitudinally monitored during osimertinib treatment. Peripheral blood (PB) from 42 NSCLC patients was obtained at two time points: (a) baseline: before treatment with osimertinib and (b) at progression of disease (PD). DNA methylation of the selected genes was detected in plasma-cfDNA (n = 80) and in paired CTCs (n = 74). Direct comparison of DNA methylation of six genes between plasma-cfDNA and paired CTC samples (n = 70) revealed a low concordance, indicating that CTCs and cfDNA give complementary information. DNA methylation analysis of plasma-cfDNA and CTCs indicated that when at least one of these genes was methylated there was a statistically significant increase at PD compared to baseline (p = 0.031). For the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients during osimertinib therapy indicated that DNA methylation of these genes could be a possible resistance mechanism.