Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk

Promoter Methylation of p16INK4A, hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence

Cervical cancer is a common cancer inflicting women worldwide. Even though, persistent infection with oncogenic Human Papillomavirus (HPV) types is considered the most important risk factor for cervical cancer development, less than 5% of women with HPV will eventually develop cervical cancer supporting that other molecular events, like methylation-dependent inactivation of tumor suppressor genes, may cocontribute in cervical carcinogenesis. We analyzed promoter methylation of three candidate genes (p16, MGMT, and hMLH1) in 403 liquid-based cytology samples. Methylation was commonly identified in both benign and pathologic samples and correlated with higher lesion grade determined by cytological, colposcopical, or histological findings, with HPV DNA and mRNA positivity of specific HPV types and p16INK4A protein expression. Overall accuracy of methylation is much lower than traditional diagnostic tests ranking it as an ancillary technique with more data needed to identify the exact value of methylation status in cervical carcinogenesis

Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE)

Introduction Emphysema and idiopathic pulmonary fibrosis (IPF) present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE). Serum surfactant proteins (SPs) A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival. Methods 122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE) and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year. Results SP-A and SP-D levels differed between groups (p = 0.006 and p= 26 ng/mL) presented a weak association with reduced survival (p = 0.05). Conclusion In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials

Molecular analysis of sporadic colorectal carcinomas in liquid based cytology medium ThinPrep

The aim of this study was the molecular characterization of colon carcinomas collected and preserved in liquid based cytology ThinPrep® focused on identification of molecular fingerprints that could aid in decision-making of chemotherapy scheme. A total of 251 samples were collected. 107 paired samples from carcinomas and normal neighboring mucosa, 3 polyps, 9 adenomas and 25 carcinomas. Extracted nucleic-acids were quantified and checked for their integrity. Three TYMS polymorphisms that are connected to its protein expression were analyzed with PCR and RFLP. Promoter methylation status of the CDK2A, MLH1 and MGMT genes was evaluated with MSP Real-Time PCR with Scorpion primers was used for mutation detection of KRAS and EGFR genes. For BRAF and to verify the KRAS results samples were sent for sequencing. mRNA gene expression of EGFR, HER2 TOP1 and TOP2A genes normalized with three house-keeping genes was measured in selected samples with qPCR with dual labelled probe chemistry. Immunocytochemistry and western blot was recruited for validation of selected results. Finally, aneuploidies were detected with flow cytometry. The results clearly supported that ThinPrep® is an excellent choice of medium for collection and storage of samples that are to be analyzed with molecular techniques as amplification of large products from DNA and mRNA and aneuploidy detection was possible. Results for TYMS polymorphisms identified 18 different genotypes suggesting a much larger sample should be used to study their significance. Promoter DNA methylation was common finding for CKDN2A and MGMT less common for MLH1 and resulted in reduction or total loss of protein expression. KRAS and BRAF mutation were identified in accordance to international frequencies. Rare EGFR mutations were found though they seemed to be present only in a small portion of tumor cells. mRNA up-regulation was found in cancer samples for the products of TOP1 and TOP2A genes. In conclusion, ThinPrep® brushings allow the thorough analysis of molecular characteristics of colorectal carcinomas. In each patient's sample at least one molecular characteristic that could be used to group the patient as more likely to benefit from specific chemotherapeutic schemes. Due to the adding layers of complexity with each new characteristic further multi-centered studies are needed to collect and manage the vast amount of information.Σκοπός της παρούσας εργασίας αποτέλεσε η μοριακή ανάλυση καρκινωμάτων παχέος έντερου σε υλικό κυτταρολογία υγρής φάσης ThinPrep®, Ειδικότερα, η εφαρμογή μοριακών τεχνικών για την ανίχνευση συγκεκριμένων χαρακτηριστικών του κάθε καρκινώματος που μπορούν χρησιμοποιηθούν στην απόφαση λήψης συγκεκριμένων χημειοθεραπευτικών. Συλλέχθηκαν 251 δείγματα, 107 ζεύξη δειγμάτων από καρκίνωμα και παρακείμενο φυσιολογικό βλεννογόνο, 3 πολύποδες, 9 αδενώματα και 25 καρκινώματα. Σε αυτά αφού πραγματοποιήθηκε έλεγχος των νουκλεϊκών οξέων που απομονώθηκαν ακολούθησε ανάλυση γενετικών πολυμορφισμών που καθορίζουν την έκφραση της θυμιδιλικής συνθάσης (TYMS) και χαρακτηρισμός της κατάστασης μεθυλίωσης του υποκινητή των γονίδιων MLH1, MGMT και CDKN2A (p16ΙΝΚ4Α). Σε καρκινώματα πραγματοποιήθηκε έλεγχος για την ύπαρξη μεταλλάξεων στα γονίδια KRAS, BRAF και EGFR. Σε επιλεγμένα ζεύγη δειγμάτων αναλύθηκε η γονιδιακή έκφραση των γονίδιων EGFR, HER2, ΤΟΡ1 και ΤΟΡ2Α κανονικοποιημένη ως προς γονίδια αναφοράς. Επιπλέον, ανοσοκυτταροχημεία και western πραγματοποιήθηκαν για την επιβεβαίωση των αποτελεσμάτων. Τέλος, διερευνήθηκε η ύπαρξη ανευπλοειδίων με κυτταρομετρία ροής. Τα αποτελέσματα υποστηρίζουν πως το ThinPrep® αποτελεί εξαιρετική επιλογή για την λήψη και αποθήκευση δειγμάτων που προορίζονται για ανάλυση με τεχνικές μοριακής βιολογίας καθώς είναι εφικτός ο προσδιορισμός ανευπλοειδίων και ο πολλαπλασιασμός μεγάλων τμημάτων DNA και mRNA. Οι πολυμορφισμοί της TYMS κατατάσσουν τους ασθενείς σε 18 διαφορετικούς γονότυπους προτείνοντας την ανάγκη ανάλυση μεγαλυτέρου δείγματος για την ανάδειξη της σημασίας τους. Επιπλέον, η μεθυλίωση των υποκινητών των υπό μελέτη γονίδιων είναι πολύ συχνή για τα MGMT και CDKN2A και σπανιότερη για το MLH1. Η μεθυλίωση συνοδεύτηκε από απώλεια της πρωτεΐνης. Τα ποσοστά των μεταλλαγών του KRAS και BRAF συμβάδισαν με τα διεθνή πρότυπα. Επίσης, αν και σε λίγα κύτταρα του όγκου φαίνεται να υπάρχουν σπάνια μεταλλαγές στον EGFR. Αύξηση της μεταγραφής παρατηρείται για τα ΤΟΡ1 και ΤΟΡ2Α σε καρκινικά δείγματα. Συμπερασματικά, η λήψη υλικού από καρκινώματα του παχέος έντερου σε ThinPrep® καθιστά εφικτή την διεξοδική ανάλυση των μοριακών χαρακτηριστικών τους. Σε όλα τα δείγματα σχεδόν υπήρχε κάποιο μοριακό χαρακτηριστικό που κατέτασσε τον ασθενή σε κατηγορία που θα είχε όφελος από λήψη συγκεκριμένου χημειοθεραπευτικού πρωτοκόλλου. Λόγω της αυξανόμενης πολυπλοκότητας με την προσθήκη κάθε νέου χαρακτηριστικού είναι απαραίτητη οργάνωση πολυκεντρικών μελετών για την συγκέντρωση και διαχείριση όλων των πληροφοριών

CxCaDSS: A Web-Based Clinical Decision Support System for Cervical Cancer

Data from countries with well-organized screening programs and cancer registries indicate that the vast majority of participants who developed cervical cancer could be explained as underestimation of cases that had at least one abnormal Pap test. Nowadays, there are ancillary molecular biology techniques available that provide important information related to cervical cancer and the HPV natural history, including DNA micro-arrays identifying HPV subtypes, mRNA techniques such as nucleic acid based amplification or flow cytometry identifying E6/E7 oncogenes, and immunocytochemistry techniques such as overexpression of p16. However, each one of these techniques has its own performance, advantages and limitations, thus a combinatorial approach via artificial intelligence methods could exploit the benefits of each method and produce more accurate results. In this paper we present a novel web-based clinical decision support system and its integration with underlying artificial neural networks, for the combination of the results of classic and ancillary techniques in order to increase the accuracy of diagnosis and thus identify women at true risk of developing cervical cancer. The presented system follows the MVC approach enabling it to easily adapt to any underlying data and structure to support clinical decisions for other domains as well

Promoter Methylation of p16 INK4A , hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence

Cervical cancer is a common cancer inflicting women worldwide. Even though, persistent infection with oncogenic Human Papillomavirus (HPV) types is considered the most important risk factor for cervical cancer development, less than 5% of women with HPV will eventually develop cervical cancer supporting that other molecular events, like methylation-dependent inactivation of tumor suppressor genes, may cocontribute in cervical carcinogenesis. We analyzed promoter methylation of three candidate genes (p16, MGMT, and hMLH1) in 403 liquid-based cytology samples. Methylation was commonly identified in both benign and pathologic samples and correlated with higher lesion grade determined by cytological, colposcopical, or histological findings, with HPV DNA and mRNA positivity of specific HPV types and p16 INK4A protein expression. Overall accuracy of methylation is much lower than traditional diagnostic tests ranking it as an ancillary technique with more data needed to identify the exact value of methylation status in cervical carcinogenesis

Alterations of HPV-Related Biomarkers after Prophylactic HPV Vaccination. A Prospective Pilot Observational Study in Greek Women

The objective of this study was to investigate the hypothesis that HPV vaccination administered in patients with low-grade (LG) cytology shortly after an initial colposcopic assessment could prospectively alter HPV-related biomarkers. This was a prospective pilot observational study involving women attending a colposcopy clinic for evaluation of abnormal LG cytology that were advised to undergo HPV vaccination and proceeded accordingly. These women were compared with a matched unvaccinated group. Women requiring cervical biopsies or CIN treatment were excluded. Intervention: A full three-dose HPV vaccination was undertaken with either the 2-valent or the 4-valent anti-HPV VLP vaccine. LBC samples were obtained prior and after the completion of the vaccination regimen and tested for HPV DNA genotyping (CLART-2 HPV test) and E6 and E7 mRNA (NASBA technique). Results: Alterations of HPV-related biomarkers at a colposcopy reassessment appointment 12 months later. Analysis: The p-values, relative risk (RR), absolute relative risk (ARR), number needed to treat (NNT) and 95% confidence intervals for each biomarker in each group were assessed. Results: A total of 309 women were included in the analysis. One hundred fifty-two women received the vaccine. HPV vaccination reduced in a statistically significant manner (p < 0.05) HPV DNA positivity rates for genotypes 16, 18, and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18, and 31 genotypes, respectively, prior to vaccination. A less pronounced, statistically insignificant reduction was shown for women who tested positive for both HPV DNA and mRNA E6 and E7 expression for HPV16, 18, and 33 subtypes. Statistically significant reduction in HPV mRNA positivity was solely documented for genotype 31 (p = 0.0411). Conclusions: HPV vaccination appears to significantly affect the rates of HPV16, 18, and 31 DNA-positive infections in the population testing HPV DNA-positive for the aforementioned genotypes. The above findings deserve verification in larger cohorts

Palatine tonsil metastasis of cecal mixed neuroendocrine‐non‐neuroendocrine neoplasm (MiNEN): A unique case

Abstract This case demonstrates the importance of understanding that patients with malignant neoplasms may exhibit metastases in unexpected sites and illustrates the necessity of a thorough clinical examination and pathologic correlation

Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk