P2-037: Estrogen and progesterone receptors in women with non-small-cell lung cancer: a potential therapeutic target?

BACKGROUND: Thyroid dysfunction and thyroid autoimmunity are prevalent among women of reproductive age and are associated with adverse pregnancy outcomes. Preconception or early pregnancy screening for thyroid dysfunction has been proposed but is not widely accepted. We conducted a systematic review of the literature on the clinical significance of thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy. METHODS: Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register. RESULTS: From a total of 14 208 primary selected titles, 43 articles were included for the systematic review and 38 were appropriate for meta-analyses. No articles about hyperthyroidism were selected. Subclinical hypothyroidism in early pregnancy, compared with normal thyroid function, was associated with the occurrence of pre-eclampsia [odds ratio (OR) 1.7, 95% confidence interval (CI) 1.1-2.6] and an increased risk of perinatal mortality (OR 2.7, 95% CI 1.6-4.7). In the meta-analyses, the presence of thyroid antibodies was associated with an increased risk of unexplained subfertility (OR 1.5, 95% CI 1.1-2.0), miscarriage (OR 3.73, 95% CI 1.8-7.6), recurrent miscarriage (OR 2.3, 95% CI 1.5-3.5), preterm birth (OR 1.9, 95% CI 1.1-3.5) and maternal post-partum thyroiditis (OR 11.5, 95% CI 5.6-24) when compared with the absence of thyroid antibodies. CONCLUSIONS: Pregnant women with subclinical hypothyroidism or thyroid antibodies have an increased risk of complications, especially pre-eclampsia, perinatal mortality and (recurrent) miscarriage. Future research, within the setting of clinical trials, should focus on the potential health gain of identification, and effect of treatment, of thyroid disease on pregnancy outcome

Synthetic, enzyme kinetic, and protein crystallographic studies of C-[béta]-D-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase

LBK

Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 β- D-glucopyranose-NH-CO-R putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a “consensus scoring” approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors

The evolving landscape of COVID-19 and post-COVID condition in patients with chronic lymphocytic leukemia: a study by ERIC, the European research initiative on CLL

In this retrospective international multicenter study, we describe the clinical characteristics and outcomes of patients with chronic lymphocytic leukemia (CLL) and related disorders (small lymphocytic lymphoma and high-count monoclonal B lymphocytosis) infected by SARS-CoV-2, including the development of post-COVID condition. Data from 1540 patients with CLL infected by SARS-CoV-2 from January 2020 to May 2022 were included in the analysis and assigned to four phases based on cases disposition and SARS-CoV-2 variants emergence. Post-COVID condition was defined according to the WHO criteria. Patients infected during the most recent phases of the pandemic, though carrying a higher comorbidity burden, were less often hospitalized, rarely needed intensive care unit admission, or died compared to patients infected during the initial phases. The 4-month overall survival (OS) improved through the phases, from 68% to 83%, p = .0015. Age, comorbidity, CLL-directed treatment, but not vaccination status, emerged as risk factors for mortality. Among survivors, 6.65% patients had a reinfection, usually milder than the initial one, and 16.5% developed post-COVID condition. The latter was characterized by fatigue, dyspnea, lasting cough, and impaired concentration. Infection severity was the only risk factor for developing post-COVID. The median time to resolution of the post-COVID condition was 4.7 months. OS in patients with CLL improved during the different phases of the pandemic, likely due to the improvement of prophylactic and therapeutic measures against SARS-CoV-2 as well as the emergence of milder variants. However, mortality remained relevant and a significant number of patients developed post-COVID conditions, warranting further investigationsAssociazione Italiana per la Ricerca sul Cancro, Grant/Award Number: IG-25024; Consellería de Innovacion, Universidades, Ciencia y Sociedad digital, Grant/Award Number: ACIF/2021/169; Fondazione Veronesi, Grant/Award Number: 1852164; General Secretariat of Research and Innovation of Greece; Ricerca per Credere nella vita” RCV odv, Grant/Award Number: 2022-DIMED

Evidence for Novel Action at the Cell-Binding Site of Human Angiogenin Revealed by Heteronuclear NMR Spectroscopy and in silico and in vivo Studies

A member of the ribonuclease A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced-fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity in vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell-binding sites, NMR studies indicate greater affinity for the cell-binding site than for the active site. Additionally, molecular dynamics simulations at 100 ns confirmed the stability of binding at the cell-binding site with the predicted protein–ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng in vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity

COVID-19 severity and mortality in patients with CLL: an update of the international ERIC and Campus CLL study

Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to Coronavirus disease 2019 (COVID-19) due to age, disease, and treatment-related immunosuppression. We aimed to assess risk factors of outcome and elucidate the impact of CLL-directed treatments on the course of COVID-19. We conducted a retrospective, international study, collectively including 941 patients with CLL and confirmed COVID-19. Data from the beginning of the pandemic until March 16, 2021, were collected from 91 centers. The risk factors of case fatality rate (CFR), disease severity, and overall survival (OS) were investigated. OS analysis was restricted to patients with severe COVID-19 (definition: hospitalization with need of oxygen or admission into an intensive care unit). CFR in patients with severe COVID-19 was 38.4%. OS was inferior for patients in all treatment categories compared to untreated (p < 0.001). Untreated patients had a lower risk of death (HR = 0.54, 95% CI:0.41–0.72). The risk of death was higher for older patients and those suffering from cardiac failure (HR = 1.03, 95% CI:1.02–1.04; HR = 1.79, 95% CI:1.04–3.07, respectively). Age, CLL-directed treatment, and cardiac failure were significant risk factors of OS. Untreated patients had a better chance of survival than those on treatment or recently treated

The evolving landscape of COVID‐19 and post‐COVID condition in patients with chronic lymphocytic leukemia: A study by ERIC, the European research initiative on CLL

In this retrospective international multicenter study, we describe the clinical characteristics and outcomes of patients with chronic lymphocytic leukemia (CLL) and related disorders (small lymphocytic lymphoma and high-count monoclonal B lymphocytosis) infected by SARS-CoV-2, including the development of post-COVID condition. Data from 1540 patients with CLL infected by SARS-CoV-2 from January 2020 to May 2022 were included in the analysis and assigned to four phases based on cases disposition and SARS-CoV-2 variants emergence. Post-COVID condition was defined according to the WHO criteria. Patients infected during the most recent phases of the pandemic, though carrying a higher comorbidity burden, were less often hospitalized, rarely needed intensive care unit admission, or died compared to patients infected during the initial phases. The 4-month overall survival (OS) improved through the phases, from 68% to 83%, p = .0015. Age, comorbidity, CLL-directed treatment, but not vaccination status, emerged as risk factors for mortality. Among survivors, 6.65% patients had a reinfection, usually milder than the initial one, and 16.5% developed post-COVID condition. The latter was characterized by fatigue, dyspnea, lasting cough, and impaired concentration. Infection severity was the only risk factor for developing post-COVID. The median time to resolution of the post-COVID condition was 4.7 months. OS in patients with CLL improved during the different phases of the pandemic, likely due to the improvement of prophylactic and therapeutic measures against SARS-CoV-2 as well as the emergence of milder variants. However, mortality remained relevant and a significant number of patients developed post-COVID conditions, warranting further investigations

Biochemical studies on human angiogenin

Human angiogenin (hAng) is a potent angiogenic factor, playing central roles in many physiological and pathological states including the growth and establishment of human tumors. hAng exerts many crucial biological functions, inducing cell growth and survival. hAng presents ribonucleolytic activity which is significantly lower than that of RNase A, but essential for its angiogenic activity. The extracellular hAng activates many signal transduction pathways, and it is also implicated in the extracellular matrix degradation, promoting thus cell migration and invasion. Under growth conditions hAng accumulates in the nucleus/nucleoli promoting the mRNA transcription and rRNA transcription/procession, while under stress conditions it accumulates in the cytoplasm where it forms tiRNAs leading to the inhibition of translational initiation. hAng has been validated as a pharmaceutical target inasmuch as the control of angiogenic activity offers a means to treat cancer.In the framework of the present Thesis, biochemical studies on the role of hAng were performed with the overarching goal to elucidate the biological functions of this protein. The crystal structure of hAng in complex with sulphate anions was determined to highlight the structure-function relationship. Kinetic studies revealed that ammonium sulphate shows a similar inhibitory potency for hAng, EDN and RNase A, however, the sulphate binding pattern is significantly different in hAng from that in RNase A and EDN, with the sulphate ions bound at the surface of the protein and not at the active site. This finding revealed a different structural mode for the inhibition of hAng compared to that of the other RNase A superfamily members. Structural analysis of the hAng-sulphate complex suggested a structural pattern for the binding of hAng to its natural RNA substrate.The specificity of the enzymatic active site of hAng was studied, using low molecular weight nucleosides, as a strategy for the development of new anticancer agents. Since binding to the active site of hAng is impeded by the C-terminal segment, and the architecture of the active sites of hAng, RNase A, and EDN is concerved, a plausible strategy for the development of specific hAng inhibitors might be the usage of RNase A and/or EDN inhibitors as lead compounds. The inhibitory action of two series of modified nucleosides to RNase A and EDN has been studied by biochemical studies and X-ray crystallography. The most potent compounds were also evaluated for their antiangiogenic potency in vivo by targeting hAng. One of the most potent inhibitors has been studied further for its ribonucleolytic inhibitory potency in vitro. However, the in vitro potency of the inhibitor did not seem to justify the strong in vivo inhibitory potency against hAng angiogenicity. NMR studies revealed that the primary binding site is the cell binding site of hAng, whereas the secondary is the active site of the enzyme. This is the first time that a dual inhibitor of hAng is reported, blocking both the entrance of hAng into the cell and the ribonucleolytic activity.At last, significant details about the implication of hAng in many signaling pathways were highlighted by immunoprecipitation coupled mass spectrometry (MS) analysis of the cytoplasmic and nuclear extracts from an endothelial cell line. The MS analysis of three different biological replicates identified many statistically significant potential hAng-interacting proteins in the two cellular compartments. The multiprotein complexes formed by the proteins identified, revealed significant aspects of hAng on many crucial molecular pathways. Proliferating cell nuclear antigen (PCNA) and hAng were the two most statistically significant proteins. PCNA was co-immunoprecipitated with the hAng specific antibody in the cytoplasmic fraction, but not in the nuclear. The interaction between hAng and PCNA was validated through further immunoprecipitation and immunoblot studies, and subsequently, through immunocytochemical analysis for the co-localization of these two proteins in the specific compartment. Further studies are needed for the determination of the biological role of this specific interaction in the cytoplasm.Η ανθρώπινη αγγειογενίνη (hAng) αποτελεί έναν ισχυρό αγγειογενετικό παράγοντα με σημαντικό ρόλο σε πληθώρα φυσιολογικών και παθολογικών καταστάσεων και στην εγκατάσταση και μετάσταση μεγάλης ποικιλίας όγκων. Εμφανίζει ποικίλους μηχανισμούς δράσης, οι οποίοι συμβάλλουν στην κυτταρική ανάπτυξη και την επιβίωση. Παρουσιάζει ριβονουκλεολυτική δραστικότητα, η οποία μολονότι είναι σημαντικά χαμηλότερη από αυτή της ομόλογής της RNase A, εντούτοις είναι απαραίτητη για την αγγειογενετική της δράση. Η εξωκυττάρια hAng ενεργοποιεί διάφορα μονοπάτια μεταγωγής σήματος και επιπλέον εμπλέκεται στην αποικοδόμηση του εξωκυττάριου ιστού επάγοντας την κυτταρική μετανάστευση και την εισβολή. Κάτω από φυσιολογικές συνθήκες ανάπτυξης, συσσωρεύεται στον πυρήνα/πυρηνίσκο προάγοντας τη μεταγραφή μορίων mRNA και τη μεταγραφή και επεξεργασία μορίων rRNA, ενώ κάτω από συνθήκες στρες, στο κυτταρόπλασμα, προάγοντας το σχηματισμό μορίων tiRNA αναστέλλοντας έτσι την έναρξη της μετάφρασης. Λόγω της εξαιρετικής αγγειογενετικής της δράσης η hAng αποτελεί φαρμακευτικό στόχο για την ανάπτυξη αντιαγγειογενετικών φαρμάκων για την αντιμετώπιση νεοπλασματικών παθήσεων.Στην παρούσα διατριβή, πραγματοποιήθηκαν βιοχημικές μελέτες της δράσης της hAng με απώτερο σκοπό την κατανόηση του βιολογικού της ρόλου. Αρχικά, με στόχο τη αποσαφήνιση της σχέσης δομής και λειτουργίας της hAng, διεξήχθη κρυσταλλογραφική μελέτη του συμπλόκου hAng-θειικών ανιόντων. Μελέτες ενζυμικής κινητικής έδειξαν πως αν και η ανασταλτική ισχύς του θειικού αμμωνίου στην hAng είναι παρόμοια με αυτήν σε δύο άλλες ομόλογες ριβονουκλεάσες (RNase A και EDN), η δομική βάση της αναστολής στην hAng διαφέρει. Σε αντίθεση με τις RNase A και EDN, όπου η αναστολή οφείλεται στην πρόσδεση των ανιόντων στο ενεργό κέντρο, στην hAng αποδίδεται στη σύνδεση των ανιόντων σε περιφερειακές περιοχές σύνδεσης του υποστρώματος στο ένζυμο. Αυτές oι θέσεις πρόσδεσης των θειικών ανιόντων στην hAng, οδήγησαν στη χαρτογράφηση κέντρων σύνδεσης εκτεταμένων ανιονικών πολυμερών, όπως τα μόρια RNA, στην πρωτεΐνη. Στη συνέχεια, μελετήθηκε η εξειδίκευση του ενεργού κέντρου της hAng με τη βοήθεια μικρών νουκλεοζιτών, με στόχο τον κατευθυνόμενο από τη δομή σχεδιασμό αναστολέων του ενζύμου, ως εν δυνάμει αντικαρκινικά φάρμακα. Λόγω της παρεμπόδισης του ενεργού κέντρου της hAng από τη διαμόρφωση του καρβοξυτελικού άκρου, ο σχεδιασμός αναστολέων βασίζεται σε δομές συμπλόκων της RNase A και της EDN. Κινητικές και κρυσταλλογραφικές μελέτες δύο σειρών τροποποιημένων νουκλεοζιτών στην RNase A και στην EDN, ανέδειξαν τους ισχυρότερους αναστολείς, οι οποίοι αξιολογήθηκαν εν συνεχεία στην hAng για την αντιαγγειογενετική τους δράση in vivo. Η ένωση με τη μεγαλύτερη ανασταλτική ισχύ in vivo, μελετήθηκε περαιτέρω για την ικανότητα αναστολής της ριβονουκλεολυτικής δραστικότητας της hAng in vitro. Εντούτοις, ο αναστολέας αυτός παρουσίασε χαμηλή αναστολή της ριβονουκλεολυτικής δραστικότητας της hAng in vitro. Η ανάλυση της σύνδεσης του αναστολέα αυτού στην hAng με φασματοσκοπία NMR αποκάλυψε πως ο αναστολέας συνδέεται πρωταρχικά στο κέντρο σύνδεσης της πρωτεΐνης στην κυτταρική μεμβράνη, παρεμποδίζοντας την είσοδο της hAng στο κύτταρο και δευτερευόντως στο καταλυτικό της κέντρο αναστέλλοντας τη ριβονουκλεολυτική της δραστικότητα. Ο αναστολέας αυτός είναι ο πρώτος που αναφέρεται με διττή δράση έναντι της hAng.Τέλος, μελέτες ανοσοκατακρήμνισης στον πυρήνα και στο κυτταρόπλασμα σε ενδοθηλιακά κύτταρα και πρωτεομική ανάλυση αποκάλυψαν σημαντικές πληροφορίες για τα κυτταρικά μονοπάτια που συμμετέχει η hAng. Έτσι, η φασματομετρία μάζας οδήγησε στην ταυτοποίηση ενός συνόλου πιθανών πρωτεϊνικών αλληλεπιδράσεων της hAng σε κάθε κυτταρικό διαμέρισμα. Η ομαδοποίηση των πρωτεϊνών με βάση τα σύμπλοκα που συγκροτούν, αποκάλυψε σημαντικές λεπτομέρειες των μονοπατιών στα οποία εμπλέκεται η hAng αλλά και νέους εν δυνάμει βιολογικούς ρόλους. Το πυρηνικό αντιγόνο πολλαπλασιαζόμενων κυττάρων (PCNA), ήταν η αμέσως επόμενη από την hAng στατιστικά σημαντική πρωτεΐνη που ταυτοποιήθηκε. Ο PCNA κατακρημνίστηκε με την hAng στο κυτταρόπλασμα, αλλά όχι στον πυρήνα. Η αλληλεπίδραση hAng-PCNA επιβεβαιώθηκε με πειράματα ανοσοκατακρήμνισης, ανοσοαποτύπωσης και ανοσοκυτταροχημείας. Περαιτέρω βιοχημικές μελέτες απαιτούνται για την αποσαφήνιση της βιολογικής σημασίας της ειδικής αυτής αλληλεπίδρασης στο κυτταρόπλασμα

NMR study of Met-1 human Angiogenin: 1H, 13C, 15N backbone and side-chain resonance assignment

Here, we report the high yield expression and preliminary structural analysis via solution hetero-nuclear NMR spectroscopy of the recombinant Met-1 human Angiogenin. The analysis reveals a well folded as well as, a monomeric polypeptide. Τhe sequence-specific assignment of its 1H, 15N and 13C resonances at high percentage was obtained. Also, using TALOS+ its secondary structure elements were determined. © 2016, Springer Science+Business Media Dordrecht

“Role of loco-regional treatments for patients with breast cancer liver metastases”

Breast cancer liver metastases (BCLM) are not uncommon (about 18\% of cases): although some patients have been reported as still living after 25 months, median survival after hormonal- or chemotherapy is 6-14 months. In recent years, new chemotherapy regimens and molecular targeted therapies have given medical oncologists reason to believe that metastatic disease can be eradicated, or at least controlled for prolonged periods. In an attempt to improve survival, consideration has also been given to loco-regional treatments such as hepatic resection and radio-frequency ablation, which have been associated with better outcomes in selected patients. This review considers the role of two loco-regional approaches in a multidisciplinary perspective in the treatment of single or multiple breast cancer metastases limited to the liver. An expanded role for hepatic resection and ablation is being investigated. We assessed available data in the literature to determine their role on survival outcomes. They suggest that loco-regional treatments might be of significant benefit in a selected group of women with BCLM, but the role of these local treatments in multimodality treatment of liver metastases remains controversial. It can generally be said that loco-regional treatments can improve overall survival, with no mortality and less than 20\% morbidity in patients at low surgical risk; however, they should only be considered cytoreductive treatments and, as such, always need to be integrated with systemic therapy