Genome-wide and gene-specific epigenomic platforms for hepatocellular carcinoma biomarker development trials

The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91

Genome-Wide and Gene-Specific Epigenomic Platforms for Hepatocellular Carcinoma Biomarker Development Trials

The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91

Methylation study in greek colon cancer patients

The incidence of colorectal cancer is a challenge to study and understand. To date, research directed at identifying mutations (point or chromosomal abnormalities) in DNA gene growth regulators of the cell. In recent years the involvement of epigenetic alterations in DNA and the deregulation of expression of regulators of cell growth genes leads as well as mutations in tumor transformation, not only enriched the knowledge of causing cancer, but also offered therapeutic approach as epigenetic changes are reversible. It is therefore a priority to detect methylotypes, groups of genes methylated selectively in specific tissues. The investigation focused on methylotypes in colorectal cancer and selected tumor suppressor genes, DNA repair genes and genes related to cell adhesion. The result of hypermethylation of selected genes was validated at both, the expression with the study of mRNA, and at the level of translation by using immunohistochemical techniques. Proved correlation at all levels, confirming the role of hypermethylation in cancer transformation. The investigation spread to other places which open prospects of application in cancer prognosis by attempting detection of circulating tumor cells in the blood of patients before and after surgery and receiving chemotherapy. Finally, the study of methylation and demethylation in cancer cell lines using the corresponding methylating and demethylating substances and conclusions leading to an integrated approach to methylation and its effects and its use for prevention and treatment.Η συχνή εμφάνιση καρκίνου του παχέος εντέρου είναι μια πρόκληση για τη μελέτη και κατανόησή του. Μέχρι σήμερα η έρευνα στρεφόταν στον εντοπισμό μεταλλάξεων (σημειακών ή χρωμοσωμικών ανωμαλιών) στο DNA σε γονίδια ρυθμιστικά της ανάπτυξης του κυττάρου.Τα τελευταία χρόνια η εμπλοκή επιγενετικών αλλοιώσεων στο DNA και η απορρύθμιση της έκφρασης των ρυθμιστικών για την ανάπτυξη του κυττάρου γονιδίων που οδηγεί όπως και οι μεταλλάξεις σε καρκινικό μετασχηματισμό, όχι μόνο εμπλούτισαν τις γνώσεις για την πρόκληση καρκίνου, αλλά προσέφεραν και θεραπευτική προσέγγιση καθώς οι επιγενετικές αλλαγές είναι αντιστρεπτές.Κατά συνέπεια αποτελεί άμεση προτεραιότητα η ανίχνευση μεθυλότυπων, δηλαδή ομάδων γονιδίων που μεθυλιώνονται επιλεκτικά σε συγκεκριμένους ιστούς.Η έρευνα μεθυλότυπων εστιάστηκε στον καρκίνο του παχέος εντέρου και επιλέχθηκαν ογκοκατασταλτικά γονίδια, γονίδια επιδιόρθωσης του DNA καθώς και γονίδια που σχετίζονται με την κυτταρική προσκόλληση.Το αποτέλεσμα της υπερμεθυλίωσης των επιλεγμένων γονιδίων, ελέγχθηκε τόσο σε επίπεδο έκφρασης με τη μελέτη του mRNA, όσο και στο επίπεδο της μετάφρασης με χρήση ανοσοϊστοχημικών τεχνικών. Αποδείχτηκε συσχέτιση σε όλα τα επίπεδα επιβεβαιώνοντας το ρόλο της υπερμεθυλίωσης στον καρκινικό μετασχηματισμό.Η έρευνα επεκτάθηκε και σε άλλα σημεία που ανοίγουν προοπτικές εφαρμογής στην πρόγνωση του καρκίνου με την προσπάθεια ανίχνευσης κυκλοφορούντων καρκινικών κυττάρων στο αίμα ασθενών προ και μετά τη χειρουργική αντιμετώπιση και λήψη χημειοθεραπείας.Τέλος η μελέτη μεθυλίωσης και απομεθυλίωσης σε καρκινικές σειρές με χρήση αντίστοιχα μεθυλιωτικών και απομεθυλιωτικών ουσιών και τα συμπεράσματα οδηγούν σε μια ολοκληρωμένη προσέγγιση της μεθυλίωσης και των επιπτώσεών της καθώς και της χρήσης της για πρόληψη και θεραπευτική αντιμετώπιση

Expression and promoter methylation status of hMLH1, MGMT, APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis

FAK-Src-paxillin system expression and disease outcome in human neuroblastoma

<p><b>Background:</b> Neuroblastoma (NB) often presents with metastatic disease and poor survival. The need for new prognostic markers remains invaluable. The FAK-Src-Paxillin protein system is associated with aggressive phenotype in adult malignancies but is largely unexplored in pediatric NB. <b>Objective:</b> To assess FAK-Src-Paxillin protein expression in human NB cell lines and clinical cytology material and to delineate its association with survival. <b>Design/Methods:</b> Western blot and immunohistochemistry were applied for FAK-Src-Paxillin expression in NB cell lines and 23 human cytology specimens, respectively. Protein expression in human clinical samples was correlated with clinicopathological parameters, <i>MYCN</i> amplification and survival. <b>Results:</b> FAK, Src and Paxillin proteins are expressed in human NB cells lines, and can be detected in clinical cytology specimens from NB patients, (59%, 32% and 33% respectively). Simultaneous FAK-Src-Paxillin expression was noted in 30% of NB patients. Children with concomitant positivity FAK, Src, and Paxillin tumors, as well as <i>MYCN</i> amplification, had increased mortality compared to those without. <b>Conclusions:</b> FAK-Src-Paxillin system is a marker of unfavorable prognosis for human NB patients but also a promising therapeutic target.</p

An epigenetic marker panel for recurrence risk prediction of low grade papillary urothelial cell carcinoma (LGPUCC) and its potential use for surveillance after transurethral resection using urine

By a candidate gene approach, we analyzed the promoter methylation (PM) of 8 genes genes (ARF, TIMP3, RAR-beta2, NID2, CCNA1, AIM1, CALCA and CCND2) by quantitative methylation specific PCR (QMSP) in DNA of 17 non-recurrent and 19 recurrent noninvasive low grade papillary urothelial cell carcinoma (LGPUCC) archival tissues. Among the genes tested, by establishing an empiric cutoff value, CCND2, CCNA1, NID2, and CALCA showed higher frequency of methylation in recurrent than in non-recurrent LGPUCC: CCND2 10/19 (53%) vs. 2/17 (12%) (p=0.014); CCNA1 11/19 (58%) vs. 4/17 (23.5%) (p=0.048); NID2 13/19 (68%) vs. 3/17 (18%) (p=0.003) and CALCA 10/19 (53%) vs. 4/17 (23.5%) (p=0.097), respectively. We further analyzed PM of CCND2, CCNA1, and CALCA in urine DNA from UCC patients including LGPUCC and controls. The frequency of CCND2, CCNA1 and CALCA was significantly higher (p<0.0001) in urine of UCC cases [ 38/148 (26%), 50/73 (68%) and 94/148 (63.5%) respectively] than controls [0/56 (0%), 10/60 (17%) and 16/56 (28.5%), respectively)]. Most importantly we found any one of the 3 markers methylation positive in 25 out of 30 (83%) cytology negative LGPUCC cases. We also explored the biological function of CCNA1 in UCC. Prospective confirmatory studies are needed to develop a reliable tool for prediction of recurrence using primary LGPUCC tissues and/or urine

16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment

Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropharyngeal (OPSCC), Oral Cavity Squamous Cell Carcinoma (OCSCC) patients and normal epithelium controls, to characterize the HNSCC saliva microbiota and examine their abundance before and after surgical resection. The analyses identified a predominance of Firmicutes, Proteobacteria and Bacteroidetes, with less frequent presence of Actinobacteria and Fusobacteria before surgery. At lower taxonomic levels, the most abundant genera were Streptococcus, Prevotella, Haemophilus, Lactobacillus and Veillonella, with lower numbers of Citrobacter and Neisseraceae genus Kingella. HNSCC patients had a significant loss in richness and diversity of microbiota species (