Methylation of T-UCRs (Transcribed-Ultra Conserved Regions) in colorectal cancer patients

Colorectal cancer is the second leading cause of cancer related deaths for men and third for women worldwide. Although screening through colonoscopy has attributed to decline of cancer mortality, patients do not comply with the examination. Screening through analysis of circulating DNA and especially genetic and epinetic changes provide a minimally invasive method. Transcribed-Ultraconserved Regions (T-UCRs) are genomic regions longer than 200 bp, which are highly conserved among human, mouse and rat genomes. Their transcription is often deregulated in many cancer types and is regulated by epigenetic mechanisms.The aim of this study was the assession of expression and methylation levels of T-UCRs 160, 283 and 346 in colorectal adenocarcinomas and adjacent non neoplastic tissues, the investigation of their role in colorectal cancer progression and the possible diagnostic value of methylated T-UCRs in plasma or urine for colorectal cancer or adenoma detection.T-UCRs expression levels were assessed in cancer and adjacent non neoplastic tissues from 51 patients with colorectal cancer, with non neoplastic tissues having higher levels of Uc160 (p<0.001). T-UCRs methylation levels were assessed in cancer and adjacent non neoplastic tissues from 64 patients with colorectal cancer, as well as in 6 adenomas, with cancer tissues having higher methylation levels of all three T-UCRs (p<0.001, p=0.001 and p=0.004). Moreover, T-UCRs methylation levels seemed to have a prognostic value in specific subgroups of patients. More specifically, low methylation levels of Uc160 or high methylation levels of Uc283 were associated with longer time to disease progression for patients older than 66 yearls old (p=0.005 and p=0.050 respectively). In patients with positive lymph nodes, high methylation levels of Uc346 were associated with longer 5-year survival (p=0.016).T-UCRs methylation levels were also assessed in plasma samples from 50 colorectal cancer patients, 59 adenoma patients, 12 patients with bowel inflammation and 40 healthy volunteers and were associated with colorectal cancer or adenoma existence. Uc160 and Uc346 methylation levels differed significantly in the plasma samples of the four groups, with cancer patients having the highest levels (p<0.001 and p=0.039 respectively). Furhter investigating their diagnostic value, methylation of Uc160 and the combination of methylated T-UCRs were found to have the best sensitivity and specificity values for colorectal cancer (35% and 89% for Uc160, p=0.016 and 45% and 74.3% for all T-UCRs, p=0.013, respectively), while their diagnostic value for adenomas was low. The same analyses were performed in urine samples from 24 colorectal cancer patients, 26 adenoma patients, 10 patients with bowel inflammation, and 26 healthy volunteers, without a diagnostic value of T-UCRs for colorectal cancer or adenoma being revealed.Furthermore, the functional role of Uc160 and Uc346 were assessed in colon cancer cells. Uc160 and Uc346 were overexpressed in colon cancer cell lines Caco-2, DLD-1 and HT-29, and proliferation, motility and migration rates were assessed. Proliferation rates were higher in cells overexpressing Uc160 or Uc346 in all cell lines and all different time points studied (24h και 48h) (p=0.050 and p=0.002 respectively in Caco-2 cells in 24 h). Increased rates were also observed in motility of all cell lines in all differents time points in cells overexpressing Uc160 or Uc346 (p=0.017 and p=0.041, in 24 h and p=0.042 and p=0.043, in 48h in DLD-1 cells). Moreover, more DLD-1 cells overexpressing Uc160 or Uc346 migrated compared to control cells (p=0.005 for both T-UCRs).In conclusion, in the present study we showed that T-UCRs 160, 283 and 346 expression and methylation are deregulated in colorectal cancer, while Uc160 and Uc346 affect proliferation and migration rates of the cells. Furthermore, their methylation have prognostic value and is a minimally invasive test for colorectal cancer detection, provided that the sensitivity of the method will be improved.Ο κολοορθικός καρκίνος αποτελεί τη δεύτερη αιτία θανάτου σχετιζόμενου με καρκίνο στους άνδρες και την τρίτη στις γυναίκες παγκοσμίως. Παρόλο που ο έλεγχος με κολονοσκόπηση έχει συμβάλει στη μείωση της θνητότητας, η συμμόρφωση στις οδηγίες προληπτικού ελέγχου παραμένει πολύ χαμηλή. Λιγότερο επεμβατικό τρόπο αποτελεί η μελέτη του κυκλοφορούντος DNA και η ανεύρεση κατάλληλων βιοδεικτών, μεταξύ των οποίων συχνότεροι είναι αυτοί που σχετίζονται με γενετικές και επιγενετικές αλλαγές. Οι μεταγραφόμενες υπερσυντηρημένες περιοχές (Transcribed-Ultraconserved Regions, T-UCRs) είναι περιοχές του γονιδιώματος, οι οποίες είναι μεγαλύτερες των 200 bp και είναι απόλυτα συντηρημένες ανάμεσα στον άνθρωπο, τον μυ και τον επίμυ. Συχνά, η έκφρασή τους είναι απορρυθμισμένη σε διάφορους τύπους καρκίνου και ρυθμίζεται από επιγενετικούς μηχανισμούς.Αντικείμενο της παρούσας διατριβής ήταν η μελέτη της έκφρασης και της μεθυλίωσης των T-UCRs 160, 283 και 346 σε αδενοκαρκινώματα παχέος εντέρου και ορθού, όπως επίσης και σε παρακείμενους μη νεοπλασματικούς ιστούς, καθώς και η διερεύνηση του ρόλου τους στην εξέλιξη του καρκίνου του παχέος εντέρου. Επίσης, εκτιμήθηκε η πιθανή διαγνωστική αξία της μεθυλίωσης των T-UCRs στο πλάσμα και στα ούρα για τον κολοορθικό καρκίνο και τα αδενώματα του παχέος εντέρου.Τα επίπεδα έκφρασης των T-UCRs προσδιορίστηκαν σε καρκινικούς και παρακείμενους μη νεοπλασματικούς ιστούς 51 ασθενών, με τη Uc160 να εκφράζεται σε υψηλότερα επίπεδα στους παρακείμενους μη νεοπλασματικούς ιστούς (p<0,001). Τα επίπεδα μεθυλίωσης των T-UCRs προσδιορίστηκαν επίσης σε καρκινικούς και παρακείμενους μη νεοπλασματικούς ιστούς 64 ασθενών, καθώς επίσης και σε 6 αδενώματα, με τους καρκινικούς ιστούς να έχουν υψηλότερα επίπεδα μεθυλίωσης των τριών T-UCRs, σε σχέση με τους παρακείμενους μη νεοπλασματικούς ιστούς (p<0,001, p=0,001 και p=0,004). Επιπλέον, αναδείχθηκε η προγνωστική αξία των επιπέδων μεθυλίωσης των T-UCRs στους καρκινικούς ιστούς σε συγκεκριμένες ομάδες ασθενών. Πιο συγκεκριμένα, τα χαμηλά επίπεδα μεθυλίωσης της Uc160 ή τα υψηλά επίπεδα μεθυλίωσης της Uc283 συσχετίστηκαν με μεγαλύτερο διάστημα υποτροπής της νόσου των ασθενών άνω των 66 ετών (p=0,005 και p=0,050 αντίστοιχα). Σε ασθενείς, που είχαν διήθηση των επιχώριων λεμφαδένων, τα υψηλά επίπεδα μεθυλίωσης της Uc346 συσχετίστηκαν με μεγαλύτερη πενταετή επιβίωση (p=0,016).Τα επίπεδα μεθυλίωσης των T-UCRs προσδιορίστηκαν επίσης στο πλάσμα 50 ασθενών με κολοορθικό καρκίνο, 59 ασθενών με αδένωμα, 12 ασθενών με φλεγμονώδη νόσο του εντέρου και 40 υγιών ατόμων και στη συνέχεια διερευνήθηκε η διαγνωστική τους αξία. Τα επίπεδα μεθυλίωσης των Uc160 και Uc346 στο πλάσμα διέφεραν ανάμεσα στις τέσσερις ομάδες, με τους ασθενείς με καρκίνο να έχουν τα υψηλότερα επίπεδα (p<0,001 και p=0,039 αντίστοιχα). Διερευνώντας την πιθανή διαγνωστική τους αξία, βρέθηκε ότι η μεθυλίωση της Uc160 και ο συνδυασμός των τριών μεθυλιωμένων T-UCRs είχαν τις μεγαλύτερες τιμές ευαισθησίας και ειδικότητας (35% και 89% για τη Uc160, p=0,016 και 45% και 74,3% για τις τρεις T-UCRs, p=0,013 αντίστοιχα) για τον κολοορθικό καρκίνο, ενώ για το αδένωμα οι μεθυλιωμένες T-UCRs είχαν χαμηλή διαγνωστική αξία. Οι ίδιες αναλύσεις πραγματοποιήθηκαν σε ούρα από 24 ασθενείς με κολοορθικό καρκίνο, 26 ασθενείς με αδένωμα, 10 ασθενείς με φλεγμονώδη νόσο του εντέρου και 26 υγιή άτομα, χωρίς ωστόσο να αναδειχθεί διαγνωστική αξία των T-UCRs για τον κολοορθικό καρκίνο ή τα αδενώματα.Εν συνεχεία, διερευνήθηκε η λειτουργική σημασία της έκφρασης των ανωτέρω T-UCRs στις καρκινικές κυτταρικές σειρές παχέος εντέρου Caco-2, DLD-1 και HT-29. Αρχικά, οι Uc160 και Uc346 υπερεκφράστηκαν και προσδιορίστηκε ο ρυθμός πολλαπλασιασμού, μετακίνησης και μετανάστευσης. Σε όλες τις κυτταρικές σειρές που μελετήθηκαν και σε όλες τις χρονικές στιγμές (24h και 48h), οι ρυθμοί πολλαπλασιασμού ήταν υψηλότεροι στα κύτταρα, τα οποία υπερεξέφραζαν τις Uc160 ή Uc346 (p=0,050 και p=0,002 αντίστοιχα για τα Caco-2 κύτταρα στις 24 h). Αυξημένοι ρυθμοί παρατηρήθηκαν επίσης και στη μετακίνηση σε όλες τις κυτταρικές σειρές και σε όλες τις χρονικές στιγμές, στα κύτταρα που υπερεξέφραζαν τις Uc160 ή Uc346 (p=0,017 και p=0,041, στις 24 h και p=0,042 και p=0,043, στις 48h για τα DLD-1). Επιπλέον, περισσότερα κύτταρα DLD-1, τα οποία υπερεξέφραζαν τις Uc160 ή Uc346 μετανάστευσαν σε σχέση με τα κύτταρα μάρτυρες (p=0,005 και για τις δύο T-UCRs).Συμπερασματικά, στην παρούσα μελέτη δείξαμε, αφενός μεν, πως η έκφραση και η μεθυλίωση των T-UCRs Uc160, Uc283 και Uc346 στον κολοορθικό καρκίνο είναι απορρυθμισμένες, αφετέρου δε, πως επηρεάζουν θεμελιώδη χαρακτηριστικά του καρκινικού κυττάρου, όπως είναι ο πολλαπλασιασμός, η μετακίνηση και η μετανάστευση. Τέλος, βρέθηκε πως η μεθυλίωσή τους έχει προγνωστική αξία και αποτελεί μία υποσχόμενη ελάχιστα επεμβατική δοκιμασία για την ανίχνευση του κολοορθικού καρκίνου, η οποία όμως χρήζει περαιτέρω διερεύνησης και βελτίωσης

PIOS (Patras Immunotherapy Score) Score Is Associated with Best Overall Response, Progression-Free Survival, and Post-Immunotherapy Overall Survival in Patients with Advanced Non-Small-Cell Lung Cancer (NSCLC) Treated with Anti-Program Cell Death-1 (PD-1) Inhibitors

Immunotherapy with immune checkpoint inhibitors (ICIs) has changed the therapeutic management of advanced non-small cell lung cancer (aNSCLC) over the last decade. However, there is an unmet need for clinically useful biomarkers in this patient subgroup. The aim of this study was to combine baseline clinical characteristics of aNSCLC patients, in the form of a scoring system, and to investigate its predictive and prognostic value in NSCLC patients treated with ICIs. A total of 112 patients with advanced (stages IIIA to IV) NSCLC, treated with nivolumab or pembrolizumab, were enrolled in this study. Patras Immunotherapy Score (PIOS) was developed based on four of the studied parameters (performance status (PS), body mass index (BMI), age, and lines of treatment (LOT), which were incorporated into our formula (PS &times; BMI/ LOT &times; age). PIOS score was strongly associated with best overall responses (BOR), with those patients having benefit/good response (stable disease (SD) or partial (PR) or complete response (CR), achieving a higher score compared to patients who developed progressive disease (PD) (p &lt; 0.001). Furthermore, PIOS score was associated with progression-free survival (PFS), since high-score patients had longer PFS (p &lt; 0.001, hazard ratio (HR) = 0.469). Moreover, PIOS was associated with post-immunotherapy overall survival (OS), with high-score patients having improved OS (log-rank p = 0.019). This study suggests that a combination of baseline parameters, which give rise to PIOS score, may predict the best response of NSCLC patients treated with anti-program cell death -1 (PD-1) monotherapy as well as it may have a potent prognostic value for PFS and post immunotherapy OS

The Survivin −31 Snp in Human Colorectal Cancer Correlates with Survivin Splice Variant Expression and Improved Overall Survival

Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated

Expression of Immune System-Related Membrane Receptors CD40, RANK, BAFFR and LTβR is Associated with Clinical Outcome of Operated Non-Small-Cell Lung Cancer Patients

An increasing number of studies implicates the NF-&#954;B (Nuclear Factor of kappa light chain gene enhancer in B cells) alternative pathway in non-small-cell lung cancer (NSCLC). We assessed the clinical significance of CD40 (Tumor necrosis factor receptor superfamily member 5, TNFRSF5), BAFFR (B-cell activating factor receptor), RANK (Receptor activator of NF-&#954;B) and LT&#946;R (lymphotoxin &#946; receptor) receptors, which activate the alternative pathway of NF-&#954;B, in NSCLC. Evaluation of CD40, BAFFR, RANK and LT&#946;R expression was performed based on the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets, while protein expression was assessed by immunohistochemistry in specimens from 119 operated NSCLC patients. CD40 gene overexpression was correlated with improved five-year overall survival (OS) (p &lt; 0.001), while increased BAFFR and LT&#946;R mRNA levels were associated with worse OS in patients with adenocarcinomas (p &lt; 0.001 and p &lt; 0.001, respectively). Similarly, patients with adenocarcinomas exhibited a negative correlation between membranous BAFFR protein expression in carcinoma cells and three- and five-year survival (p = 0.021; HR, 4.977 and p = 0.030; HR, 3.358, respectively) as well as between BAFFR protein overexpression in cancer-associated fibroblasts (CAFs) and two-year survival (p = 0.036; HR, 1.983). Patients with increased LT&#946;R nuclear protein staining or stage II patients with lower cytoplasmic LT&#946;R protein expression had worse five-year OS (p = 0.039 and p = 0.008, respectively). Moreover, CD40 protein expression in tumor infiltrating lymphocytes (TILs) and CAFs was positively associated with metastatic spread while BAFFR protein expression in CAFs was negatively associated with bone metastasis (p = 0.041). Our data suggests that CD40, BAFFR, RANK and LT&#946;R play an important role in NSCLC and further supports the role of NF-&#954;B alternative pathway in NSCLC

Differentially Methylated Ultra-Conserved Regions Uc160 and Uc283 in Adenomas and Adenocarcinomas Are Associated with Overall Survival of Colorectal Cancer Patients

Deregulation of the transcribed ultra-conserved regions (T-UCRs) Uc160, Uc283, and Uc346 has been reported in colorectal cancer (CRC) recently. Here, we investigated promoter methylation of these T-UCRs during the adenoma&ndash;carcinoma sequence and their clinical significance in CRC patients. Methylation levels were assessed in CRC, adenomas, infiltrated lymph nodes, and metastatic tissue specimens. In situ hybridization was performed in representative tissue specimens. T-UCRs expression levels were also evaluated in HT-29 colon cancer cells before and after the acquired resistance to 5-fluorouracil (5-FU) and oxaliplatin. A gradual increase in T-UCRs methylation levels from hyperplastic polyps to adenomas and to in situ carcinomas (ISC) and a gradual decrease from ISC to infiltrative and metastatic carcinomas was observed (p &lt; 0.001 for Uc160 and Uc283, p = 0.018 for Uc346). Uc160 and Uc283 methylation was associated with the grade of dysplasia in adenoma specimens (p = 0.034 and p = 0.019, respectively). Furthermore, higher Uc160 methylation, mainly in stage III and IV patients, was related to improved overall survival (OS) in univariate (p = 0.009; HR, 0.366) and multivariate analysis (p = 0.005; HR, 0.240). Similarly, higher methylation of Uc283 was associated with longer OS (p = 0.030). Finally, T-UCRs expression was significantly reduced in HT-29 cells after resistance to chemotherapy. This study suggests that promoter methylation of Uc160, Uc283, and Uc346 is altered during CRC development and that Uc160 and Uc283 methylation may have prognostic significance for CRC patients