Dose selection trial of metronomic oral vinorelbine monotherapy in patients with metastatic cancer: a hellenic cooperative oncology group clinical translational study

BACKGROUND: Metronomic chemotherapy is considered an anti-angiogenic therapy that involves chronic administration of low-dose chemotherapy at regular short intervals. We investigated the optimal metronomic dose of oral vinorelbine when given as monotherapy in patients with metastatic cancer. METHODS: Patients with recurrent metastatic breast (BC), prostate (PC) or non-small cell lung cancer (NSCLC) and adequate organ functions were randomly assigned to 30, 40 or 50 mg vinorelbine, taken orally three times a week. Treatment continued until disease progression, unacceptable toxicity, withdrawal of consent or maximum 24 months. Primary endpoint was time-to-treatment failure (TTF) and secondary were progression-free survival (PFS), toxicity, changes in blood concentrations of angiogenesis-associated biomarkers and pharmacokinetics. RESULTS: Seventy-three patients were enrolled. Four-month TTF rate did not differ between the three arms: 25.9% (11.1%-46.2% 95% Confidence Interval), 33.3% (15.6%-55.3%) and 18.2% (5.2%-40.3%) for the 30 mg, 40 mg and 50 mg arms (p-value = 0.56). Objective response was seen in 2 patients with NSCLC (treated at 30 and 50 mg respectively), one with BC (at 40 m g) and one with PC (at 50 mg) and lasted from 4 to 100 weeks, with maximum response duration achieved at 50 mg. Adverse events were mild and negligible and did not differ between the three arms. Blood levels of vinorelbine reached steady state from the second week of treatment and mean values for the 30, 40 and 50 mg were respectively 1.8 ng/ml (SD 1.10), 2.2 ng/ml (SD 1.87) and 2.6 ng/ml (SD 0.69). Low pre-treatment blood concentrations of FGF2 and IL8 predicted favorable response to therapy (p values 0.02 and 0.006, respectively), while high levels of TEK gene transcript predicted treatment resistance. CONCLUSIONS: Considering the antitumor activity and response duration, the negligible toxicity of the highest dose investigated and the lack of drug accumulation over time, we suggest that 50 mg given three times a week is the optimal dose for metronomic oral vinorelbine. Further investigation of metronomic oral vinorelbine (MOVIN) at this dose is warranted in combination with conventional chemotherapy regimens and targeted therapies. TRIAL REGISTRATION: Clinicaltrials.gov NCT0027807

Cellural and molecular effects of cytostatic drugs on human endothelial cells in an in vitro simulation model of metronomic chemotherapy: in vitro translation study of clinical application of metronomic vinorelbine

A chemotherapy dosing strategy that attracts growing clinical interest is low dose metronomic chemotherapy. It involves the close, regular administration of conventional chemotherapeutic drugs without prolonged drug free intervals over extended periods of time. In contrast with conventional chemotherapy of maximum tolerated dose (MTD), the primary target of metronomic chemotherapy is thought to be tumor’s neovasculature. Angiogenesis, as the pioneer in the field Judah Folkman suggested in one of his latest papers, plays in cancer the role of ‘organizing principle’ because of its critical role in malignant tumor development. In this context angiogenesis is understood as a tumor supporting process, which is kept up aberrantly active through the influence of counterbalancing proteins that are produced by cancer cells and the microenvironment and drive endothelial cells functional and proliferating. In view of that, antiangiogenic cancer therapy optimally aims to inhibit proliferation and function of activated endothelial cells and thereof tackle cancer progression on a chronic basis. Among different classes of cytotoxics, microtubule disruptors are considered most appropriate for metronomic low-dose antiangiogenic chemotherapy because of their potency to inhibit proliferation and also function of endothelial cells at very low concentrations. Recently, availability of oral formulation of microtubule disrupting vinorelbine (Navelbine Soft Capsules®) led to clinical investigation of this drug at a metronomic dosing schedule [NCT00278070]. In this context we investigated functional and molecular effects of vinorelbine (VRL) against proliferating human umbilical vein endothelial cells (HUVEC) in an experimental in vitro simulation of conventional and metronomic chemotherapy.In regard to induced changes in angiogenesis signaling genes under the influence of protracted and short exposure to VRL we studied interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), thrombospondin-1 (TSP-1), the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) and the cytoplasmic receptors CD36, and finally the transcription factors NF-κB and HIF-1α. Also, we studied the antiproliferative and the molecular effects of rosiglitazone in combination with metronomic vinorelbine on proliferating endothelial cells and investigate the role of PPARγ in the metronomic model.In this study, we used primary human umbilical cord vein (HUVEC) cells, which were isolated from human umbilical cord veins. HUVECs were plated to sub-confluence in plates coated with collagen and treated with VRL, RSG or the combination for 4, 24 and 96hrs. In the 96hr experiment, to simulate a metronomic dosing schedule we replaced the drug-enriched medium every 24hrs. In contrast, the treatment with VRL for 4hrs with a wash out period with drug-free medium for the consecutive 92hrs, and the 24hr time point simulate the classic schedule of chemotherapy with the Maximum Tolerated Dose (MTD). Initially, with the MTS Assay method we investigated the inhibition of cell proliferation and calculated IC50. By qRT-PCR, ELISA and Western blot we measured gene and protein expression of markers of angiogenesis, while with flow cytometry we studied apoptosis and cell cycle of HUVECs.The metronomic administration of vinorelbine was significantly more active in inhibiting proliferation of HUVEC compared to the short exposure with comparative difference of four orders of magnitude (IC50: 1,23nM vs. 32mM). Still, it did not affect cell cycle progression or induced cell apoptosis. Moreover, metronomic vinorelbine was shown to induce the expression of PPARγ and CD36 and inhibit the expression of angiogenic factors such as IL-8 and COX-2.Co-administration of metronomic vinorelbine with rosiglitazone was found to improve the antiangiogenic effects and increase the expression of PPAR even at concentrations previously inhibited his expression. Furthermore, coadministration of the two drugs conserved angiogenic factors in low, basic levels. Finally, synergistic effect of the two drugs was observed in inhibiting proliferation of the endothelial cells.In conclusion, metronomic vinorelbine inhibits proliferation of endothelial cells and suppresses molecular mechanisms of angiogenesis, mainly affecting the expression of PPARγ and IL-8 in enthothelial cells. IL-8 seems to be a metronomic vinorelbine therapy mediator. The activation of the nuclear receptor PPARγ by metronomic chemotherapy opens the way for the creation of co-administration studies of metronomic chemotherapy with activators of PPARγ in order to identify his exact role. Generally, the metronomic chemotherapy emerges as a promising alternative in the treatment of cancer, with satisfactory results. However, many studies are still needed to reveal the mechanisms involved in its anticancer actions.Η θεραπευτική αντιμετώπιση του καρκίνου βασίζεται στη χορήγηση κυτταροστατικών φαρμάκων σε μέγιστες ανεκτές δόσεις ανά τακτά χρονικά διαστήματα, με στόχο την αναστολή του πολλαπλασιασμού των ταχέως αναπτυσσόμενων καρκινικών κυττάρων. Όμως, εκτός από τη καταστροφή των καρκινικών κυττάρων, δημιουργούνται φθορές και στα φυσιολογικά κύτταρα του οργανισμού, με αποτέλεσμα την εμφάνιση αρκετών παρενεργειών. Επιπρόσθετα, οι μέχρι τώρα θεραπευτικές επιλογές δεν έχουν προσφέρει σημαντικό κλινικό όφελος στην αντιμετώπιση των περισσοτέρων τύπων προχωρημένου ή μεταστατικού καρκίνου. Έτσι, η επιστημονική κοινότητα στράφηκε σε νεότερα πεδία έρευνας όπως η αγγειογένεση και η μετρονομική χημειοθεραπεία.Ο όρος Μετρονομική Χημειοθεραπεία χαρακτηρίζει δοσολογικά σχήματα χημειοθεραπείας σε συχνή, αδιάλειπτη χορήγηση υποτοξικών δόσεων κυτταροστατικών φαρμάκων σε μακροχρόνια βάση. Η στρατηγική αυτή στοχεύει κατά κύριο λόγο στα ενεργοποιημένα, πολλαπλασιαζόμενα ενδοθηλιακά κύτταρα των αγγείων των όγκων παρά στα καρκινικά κύτταρα. Σύμφωνα λοιπόν, με επιστημονικά δεδομένα η μετρονομική χημειοθεραπεία ενεργεί μέσω μηχανισμών αναστολής του πολλαπλασιασμού των ενδοθηλιακών κυττάρων των αγγείων των όγκων και ρύθμισης της ισορροπίας των αγγειογενετικών παραγόντων.Σκοπό της παρούσας ερευνητικής διατριβής απετέλεσε η διερεύνηση της κυτταρικής και μοριακής δράσης της VRL (Navelbine®) επί πολλαπλασιαζόμενων ανθρώπινων ενδοθηλιακών κυττάρων σε σχήματα που προσομοιάζουν με αυτά της μετρονομικής θεραπείας στην κλινική της εφαρμογή. Επίσης, η επίδραση της VRL σε κύριους μηχανισμούς που διέπουν την αγγειογένεση στο παραπάνω in vitro μοντέλο αποτέλεσε σημαντικό ερευνητικό στόχο. Επιπλέον στόχοι του ερευνητικού έργου ήταν η μελέτη του ρόλου του υποδοχέα PPARγ στο μοντέλο της in vitro μετρονομικής χημειοθεραπείας ενδοθηλιακών κυττάρων, και οι αλλαγές που προκαλεί στο μετρονομικό προφίλ της VRL η συγχορήγηση με ενεργοποιητές του PPARγ (ροσιγλιταζόνη) ως προς την αναστολή του πολλαπλασιασμού και της αγγειογένεσης.Υλικό της συγκεκριμένης μελέτης αποτέλεσαν τα ανθρώπινα ενδοθηλιακά κύτταρα (HUVEC) που απομονώθηκαν από την ομφαλική φλέβα ομφάλιου λώρου και καλλιεργήθηκαν σε κατάλληλο θρεπτικό υλικό που περιείχε βινορελμπίνη, τον μεταβολίτη ή ροσιγλιταζόνη ή και τον συνδυασμό τους, σε συγκεντρώσεις που κυμαίνονταν από 0,001nM μέχρι 100μΜ για 4, 24 και 96 ώρες. Αρχικά, με τη μέθοδο MTS Assay διερευνήθηκε η αναστολή του κυτταρικού πολλαπλασιασμού και υπολογίστηκε η μεσομέγιστη ανασταλτική συγκέντρωση (IC50). Με την μέθοδο qRT-PCR, ELISA και Western blot μετρήθηκε η γονιδιακή και η πρωτεϊνική έκφραση δεικτών αγγειογένεσης, ενώ με κυτταρομετρία ροής μελετήθηκε η απόπτωση και ο κυτταρικός κύκλος.Η μετρονομική χορήγηση VRL βρέθηκε σημαντικά πιο δραστική στην αναστολή πολλαπλασιασμού των HUVEC σε σχέση με την βραχεία έκθεση με συγκριτική διαφορά τεσσάρων τάξεων μεγέθους (IC50: 1,23nM vs. 32μΜ). Παρόλα αυτά δεν επηρέασε την εξέλιξη του κυτταρικού κύκλου και δεν προκάλεσε απόπτωση των HUVEC. Επιπλέον, σε μετρονομικούς χρόνους και συγκεντρώσεις η VRL έδειξε να επάγει την έκφραση των αγγειοκατασταλτικών γονιδίων PPARγ και CD36 ενώ ταυτόχρονα αναστέλλει την έκφραση των αγγειογενετικών παραγόντων IL-8 και COX-2. Η IL-8 αποτελεί σημαντικό ρυθμιστή της μετρονομικής VRL in vitro αλλά και in vivo καθώς τα χαμηλά επίπεδα πριν τη μετρονομική θεραπεία με VRL καθορίζουν τους ασθενείς που θα ωφεληθούν από την συγκεκριμένη θεραπεία.Η συγχορήγηση της μετρονομικής VRL με τη ροσιγλιταζόνη βρέθηκε να βελτιώνει τα αντιαγγειογενετικά αποτελέσματα, καθώς αυξάνει την έκφραση του PPARγ ακόμα και στις συγκεντρώσεις που προηγουμένως ανέστειλαν την έκφρασή του. Επιπλέον, με τη συγχορήγηση έχουμε διατήρηση των αγγειογενετικών παραγόντων σε βασικά, χαμηλά επίπεδα. Τέλος συνεργική δράση των δυο φαρμάκων παρατηρήθηκε και στην αναστολή του πολλαπλασιασμού.Συμπερασματικά σε ένα in vitro μοντέλο μετρονομικής χημειοθεραπείας η VRL αναστέλλει τον πολλαπλασιασμό των ενδοθηλιακών κυττάρων και επηρεάζει κατασταλτικά τους μοριακούς μηχανισμούς της αγγειογένεσης με σημαντικότερη δράση στο μονοπάτι του PPARγ και της IL-8. Η ενεργοποίηση του πυρηνικού υποδοχέα PPARγ από τη μετρονομική χημειοθεραπεία ανοίγει το δρόμο για τη δημιουργία μελετών συγχορήγησης μετρονομικής χημειοθεραπείας με ενεργοποιητές του PPARγ, έτσι ώστε να διερευνηθεί καλύτερα κ εις βάθος ο ρόλος του. Γενικότερα, η μετρονομική χημειοθεραπεία προβάλλει ως μια υποσχόμενη εναλλακτική στη θεραπευτική αντιμετώπιση του καρκίνου, με ικανοποιητικά αποτελέσματα. Όμως, χρειάζονται ακόμα πολλές μελέτες για να αποκαλυφθούν οι μηχανισμοί που εμπλέκονται σε αυτήν

Methylation Status of Corticotropin-Releasing Factor (CRF) Receptor Genes in Colorectal Cancer

The corticotropin-releasing factor (CRF) system has been strongly associated with gastrointestinal pathophysiology, including colorectal cancer (CRC). We previously showed that altered expression of CRF receptors (CRFRs) in the colon critically affects CRC progression and aggressiveness through regulation of colonic inflammation. Here, we aimed to assess the potential of CRFR methylation levels as putative biomarkers in CRC. In silico methylation analysis of CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2) was performed using methylome data derived by CRC and Crohn’s disease (CD) tissues and CRC-derived circulating cell-free DNAs (ccfDNAs). In total, 32 and 33 differentially methylated sites of CpGs (DMCs) emerged in CRFR1 and CRFR2, respectively, between healthy and diseased tissues. The methylation patterns were verified in patient-derived ccfDNA samples by qMSP and associated with clinicopathological characteristics. An automated machine learning (AutoML) technology was applied to ccfDNA samples for classification analysis. In silico analysis revealed increased methylation of both CRFRs in CRC tissue and ccfDNA-derived datasets. CRFR1 hypermethylation was also noticed in gene body DMCs of CD patients. CRFR1 hypermethylation was further validated in CRC adjuvant-derived ccfDNA samples, whereas CRFR1 hypomethylation, observed in metastasis-derived ccfDNAs, was correlated to disease aggressiveness and adverse prognostic characteristics. AutoML analysis based on CRFRs methylation status revealed a three-feature high-performing biosignature for CRC diagnosis with an estimated AUC of 0.929. Monitoring of CRFRs methylation-based signature in CRC tissues and ccfDNAs may be of high diagnostic and prognostic significance in CRC

Implementing pharmacogenetic testing in fluoropyrimidine-treated cancer patients: DPYD genotyping to guide chemotherapy dosing in Greece

Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FP-associated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients.Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), C__11985548_10 (rs55886062), C__27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160).Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (β = −0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007).Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy

Dose selection trial of metronomic oral vinorelbine monotherapy in patients with metastatic cancer: a hellenic cooperative oncology group clinical translational study

Background: Metronomic chemotherapy is considered an anti-angiogenic therapy that involves chronic administration of low-dose chemotherapy at regular short intervals. We investigated the optimal metronomic dose of oral vinorelbine when given as monotherapy in patients with metastatic cancer. Methods: Patients with recurrent metastatic breast (BC), prostate (PC) or non-small cell lung cancer (NSCLC) and adequate organ functions were randomly assigned to 30, 40 or 50 mg vinorelbine, taken orally three times a week. Treatment continued until disease progression, unacceptable toxicity, withdrawal of consent or maximum 24 months. Primary endpoint was time-to-treatment failure (TTF) and secondary were progression-free survival (PFS), toxicity, changes in blood concentrations of angiogenesis-associated biomarkers and pharmacokinetics. Results: Seventy-three patients were enrolled. Four-month TTF rate did not differ between the three arms: 25.9% (11.1%-46.2% 95% Confidence Interval), 33.3% (15.6%-55.3%) and 18.2% (5.2%-40.3%) for the 30 mg, 40 mg and 50 mg arms (p-value = 0.56). Objective response was seen in 2 patients with NSCLC (treated at 30 and 50 mg respectively), one with BC (at 40 m g) and one with PC (at 50 mg) and lasted from 4 to 100 weeks, with maximum response duration achieved at 50 mg. Adverse events were mild and negligible and did not differ between the three arms. Blood levels of vinorelbine reached steady state from the second week of treatment and mean values for the 30, 40 and 50 mg were respectively 1.8 ng/ml (SD 1.10), 2.2 ng/ml (SD 1.87) and 2.6 ng/ml (SD 0.69). Low pre-treatment blood concentrations of FGF2 and IL8 predicted favorable response to therapy (p values 0.02 and 0.006, respectively), while high levels of TEK gene transcript predicted treatment resistance. Conclusions: Considering the antitumor activity and response duration, the negligible toxicity of the highest dose investigated and the lack of drug accumulation over time, we suggest that 50 mg given three times a week is the optimal dose for metronomic oral vinorelbine. Further investigation of metronomic oral vinorelbine (MOVIN) at this dose is warranted in combination with conventional chemotherapy regimens and targeted therapies. Trial registration: Clinicaltrials.gov NCT0027807