Identification of Biomarkers Involved in CDK4/6 Inhibitor Therapy Resistance and the Molecular Response to Treatment for Metastatic ER+/HER2- Breast Cancer

https://openworks.mdanderson.org/sumexp21/1217/thumbnail.jp

Διερεύνηση και κλινική αξιολόγηση νέων δεικτών μεθυλίωσης του DNA στον καρκίνο, στα πλαίσια της υγρής βιοψίας

Η «υγρή βιοψία», βασισμένη στην ανάλυση των κυκλοφορούντων καρκινικών κυττάρων (circulating tumor cells, CTCs) και του εξωκυττάριου καρκινικού DNA (circulating tumor DNA, ctDNA) παρέχει μία μη-επεμβατική παρακολούθηση της εξέλιξης της νόσου και της αποτελεσματικότητας της θεραπείας, σε πραγματικό χρόνο. Η μελέτη της μεθυλίωσης του DNA, αποτελεί τα τελευταία χρόνια σημαντικό πεδίο έρευνας παγκοσμίως, καθώς αποτελεί πρώιμο γεγονός κατά την καρκινογένεση, αλλά λαμβάνει χώρα και σε όλα τα στάδια της νόσου. Στην παρούσα διδακτορική διατριβή, αρχικά μελετήθηκαν οι προαναλυτικές παράμετροι σταθερότητας και πραγματοποιήθηκε αξιολόγηση της πιστότητας και ακρίβειας των αναλύσεων μεθυλίωσης του DNA στην υγρή βιοψία. Αποτελεί την πρώτη μελέτη αξιολόγησης της σταθερότητας της μεθυλίωσης του DNA στο πλάσμα και της μεθυλίωσης του χημικά τροποποιημένου DNA υπό διαφορετικές συνθήκες αποθήκευσης, Τα αποτελέσματά μας υποδεικνύουν ότι η πληροφορία της μεθυλίωσης δε διατηρείται εφόσον η απομόνωση του εξωκυττάριου καρκινικού DNA πραγματοποιηθεί από δείγματα πλάσματος συντηρημένα στους -70℃ για χρονική περίδο μεγαλύτερη των 8 μηνών. Αντίθετα, το χημικά τροποποιημένο DNA μπορεί να διατηρηθεί σε καταψύκτες και των -20℃ και των -70℃ για ένα έτος, χωρίς κανένα πρόβλημα στην ανάλυση μεθυλίωσης με real-time MSP. Αναφορικά με την ακρίβεια ενίσχυσης του χημικά επεξεργασμένου DNA, λάβαμε πανομοιότυπα αποτελέσματα πριν και μετά την αντίδραση ενίσχυσης. Εν συνεχεία, πραγματοποιήθηκε ανάπτυξη, επικύρωση και κλινική αξιολόγηση μεθοδολογίας για τη μεθυλίωση του γονιδίου ESR1, σε δείγματα CTCs και ctDNA, για την παρακολούθηση ασθενών με μεταστατικό καρκίνο μαστού που λαμβάνουν ενδοκρινή θεραπεία. Μέσω της αναπτυχθείσας μεθοδολογίας, αναφέρουμε για πρώτη φορά την ανίχνευση μεθυλίωσης του ESR1 στα CTCs και την υψηλή αντιστοιχία με τη μεθυλίωση στο ctDNA πλάσματος των ίδιων ασθενών. Επιπλέον, η μεθυλίωση του ESR1 στα CTCs ασθενών με ορμονοεξαρτώμενο καρκίνο μαστού, βρέθηκε να παρουσιάζει στατιστικά σημαντική συσχέτιση με την έλλειψη ανταπόκρισης στην ορμονοθεραπεία (everolimus/exemestane). Επιπρόσθετα, αναπτύξαμε, επικυρώσαμε και αξιολογήσαμε κλινικά μία νέα μέθοδο για τη μεθυλίωση του υποκινητή του γονιδίου MLL3. Με βάση τη μεθοδολογία αυτή, αξιολογήσαμε τη μεθυλίωση του MLL3 ως νέο επιγενετικό βιοδείκτη στο μη-μικροκυτταρικό καρκίνο του πνεύμονα. Τα αποτελέσματά μας δείχνουν για πρώτη φορά την ύπαρξης μεθυλίωσης του MLL3 και την προγνωστική αξία της ανίχνευσής της στο ctDNA από πλάσμα ασθενών με μη-μικροκυτταρικό καρκίνο του πνεύμονα. Τέλος, πραγματοποιήσαμε μελέτη μεθυλίωσης ογκοκατασταλτικών γονιδίων σε CTCs απομονωμένα με το σύστημα Parsortix, από ασθενείς με πλακώδες καρκίνωμα κεφαλής και τραχήλου. Τα γονίδια SOX17, RASSF1A και MLL3 βρέθηκαν μεθυλιωμένα σε διαφορετικά ποσοστά, επιβεβαιώνοντας την ετερογένεια των CTCs.Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides a non-invasive, real-time monitoring of tumor evolution and therapeutic efficacy. During the last few years, DNA methylation changes have been widely studied, as DNA methylation is considered as an early event in carcinogenesis but is also present at all stages of the disease. In the present PhD thesis, we initially evaluated the preanalytical parameters and the performance of whole genome amplification (WGA) protocols for reliable downstream DNA methylation analyses in liquid biopsy. The present study is the first to evaluate the stability of DNA methylation in plasma and sodium bisulfite (SB)-treated DNA under different storage conditions. Our results indicate that DNA methylation information is not preserved, when ctDNA extraction is performed in plasma samples stored for more than 8 months even at -70℃. On the contrary, SB-treated DNA can be safely stored for a long time at both -20℃ and -70℃, and DNA methylation analysis by real-time MSP can be safely performed in a total period of one year. Concerning the accuracy and precision of WGA of SB-treated DNA our results suggest that it is reliable since we get identical results before and after amplification using both SB-conversion kits. Furthermore, we developed and clinically validated a liquid biopsy-based assay for ESR1 methylation for the follow-up of metastatic breast cancer patients under endocrine treatment. We reported for the first time the presence of ESR1 methylation in CTCs and its high concordance with paired plasma ctDNA derived from the same patients. Moreover, according to our findings, ESR1 methylation in CTCs of hormone receptor-positive metastatic breast cancer patients was strongly associated with lack of response to everolimus/exemestane treatment. We additionally developed and clinically validated an assay for MLL3 promoter methylation as a novel circulating epigenetic biomarker in non-small cell lung cancer (NSCLC). Our data indicate for the first time that the detection of MLL3 promoter methylation in plasma-ctDNA provides important prognostic information for NSCLC patients. Finally, we evaluated the methylation status of tumor-suppressor genes in CTCs of head and neck cancer patients (HNSCC), isolated using the microfluidic-based Parsortix system. SOX17, RASSF1A and MLL3 were found to be methylated in CTCs at various percentages, confirming the presence of heterogeneity, even in CTCs isolated from the same patient

Tenebrio molitor larvae meal inclusion affects hepatic proteome and apoptosis and/or autophagy of three farmed fish species

Herein, the effect of dietary inclusion of insect (Tenebrio molitor) meal on hepatic pathways of apoptosis and autophagy in three farmed fish species, gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss), fed diets at 25%, 50% and 60% insect meal inclusion levels respectively, was investigated. Hepatic proteome was examined by liver protein profiles from the three fish species, obtained by two-dimensional gel electrophoresis. Although cellular stress was evident in the three teleost species following insect meal, inclusion by T. molitor, D. labrax and O. mykiss suppressed apoptosis through induction of hepatic autophagy, while in S. aurata both cellular procedures were activated. Protein abundance showed that a total of 30, 81 and 74 spots were altered significantly in seabream, European seabass and rainbow trout, respectively. Insect meal inclusion resulted in individual protein abundance changes, with less number of proteins altered in gilthead seabream compared to European seabass and rainbow trout. This is the first study demonstrating that insect meal in fish diets is causing changes in liver protein abundances. However, a species-specific response both in the above mentioned bioindicators, indicates the need to strategically manage fish meal replacement in fish diets per species

Tenebrio molitor larvae meal inclusion affects hepatic proteome and apoptosis and/or autophagy of three farmed fish species

Acknowledgements Financial support for the trial on European sea bass was provided by the AQUAEXEL Project PROINSECTLIFE (Ref. No. 0013/03/05/15B), the AQUAEXEL Project INDIFISH (Ref. No. 0125/08/05/15/TNA), and by the University of Turin (ex 60%) Grant (Es. fn. 2014). NP (Scholarship Code: 1752) has been fnancially supported by the General Secretariat for Research and Technology (GSRT) of Greece and the Hellenic Foundation for Research and Innovation (HFRI) and MM by the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432) as implemented by the State Scholarships Foundation (ΙΚΥ). Tanks to Evelyn Argo and Craig Pattinson (University of Aberdeen) for providing help with 2DE. EM was fnancially supported by Marine Alliance for Science and Technology Scotland (MASTS) visiting Fellowship.Peer reviewedPublisher PD

A Cross-Sectional Evaluation of the Virtual Outpatient Management of People With Mpox.

BACKGROUND: To report on the implementation and outcomes of a virtual ward established for the management of mpox during the 2022 outbreak, we conducted a 2-center, observational, cross-sectional study over a 3-month period. METHODS: All patients aged ≥17 years with laboratory polymerase chain reaction-confirmed monkeypox virus managed between 14 May and 15 August 2022, at the Hospital for Tropical Diseases at University College London Hospitals National Health Service (NHS) Foundation Trust and sexual health services at Central North and West London NHS Foundation Trust, were included. Main outcomes included the proportion of patients managed exclusively on the virtual ward, proportion of patients requiring inpatient admission, proportion of patients with human immunodeficiency virus, and duration of lesion reepithelialization. RESULTS: Among confirmed cases (N = 221), 86% (191/221) were managed exclusively on the virtual ward, while 14% (30/221) required admission. Treatment for concomitant sexually transmitted infections was provided to 25% (55/221) of patients, antibiotics for other infective complications to 16% (35/221), and symptomatic relief to 27% (60/221). The median time from onset to complete lesion reepithelialization and de-isolation was 18 days (range, 8-56 days). Eleven percent (24/221) of individuals disengaged from services within 4 days of testing. CONCLUSIONS: The virtual ward model facilitated safe and holistic outpatient management of mpox, while minimizing admissions. This approach could serve as a model for future outbreak responses

Determination of Therapeutic Equivalence of Generic Products of Gentamicin in the Neutropenic Mouse Thigh Infection Model

Background: Drug regulatory agencies (DRA) support prescription of generic products of intravenous antibiotics assuming therapeutic equivalence from pharmaceutical equivalence. Recent reports of deaths associated with generic heparin and metoprolol have raised concerns about the efficacy and safety of DRA-approved drugs. Methodology/Principal Findings: To challenge the assumption that pharmaceutical equivalence predicts therapeutic equivalence, we determined in vitro and in vivo the efficacy of the innovator product and 20 pharmaceutically equivalent generics of gentamicin. The data showed that, while only 1 generic product failed in vitro (MIC = 45.3 vs. 0.7 mg/L, P,0.05), 10 products (including gentamicin reference powder) failed in vivo against E. coli due to significantly inferior efficacy (E max = 4.81 to 5.32 vs. 5.99 log 10 CFU/g, P#0.043). Although the design lacked power to detect differences in survival after thigh infection with P. aeruginosa, dissemination to vital organs was significantly higher in animals treated with generic gentamicin despite 4 days of maximally effective treatment. Conclusion: Pharmaceutical equivalence does not predict therapeutic equivalence of generic gentamicin. Stricter criteri