Συμβολή στην μελέτη επιγενετικών μεταβολών στο ακανθοκυτταρικό καρκίνωμα της στοματικής κοιλότητας

Abstract

INTRODUCTION Oral squamous cell carcinoma (OSCC) accounts for more than 90% of oral malignancies and it is the most common type of oral cancer. Oral leukoplakia (OL) is the most frequent oral potentially malignant disorder with an annual risk of malignant transformation, which depends on the subtype of OL, ranging from 3% to 17,5%. Lichen planus (LP) is a chronic inflammatory mucocutaneous disease that often affects oral mucosa, but the malignant transformation of LP remains controversial. Among the most common risk factors associated with the development of OSCC are tobacco smoke, alcohol, oncogenic viruses like HPV etc. In the last years there has been a great interest about the potential role of epigenetic alterations in the oral carcinogenesis. Epigenetics are heritable modifications in gene expression without alterations of the DNA sequences. They occur more frequently than gene mutations and are potentially reversible. There are three types of epigenetic changes: DNA-hypermethylation, histones modifications and micro RNAs. DNA-hypermethylation is the most common and best studied of all epigenetics and refers to the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA. Hypermethylation can lead to the transcriptional silencing of tumor suppressor genes (TSG) and this may play a key role to the initiation and progression of OSCC DCC gene is located in chromosome 18 (18q 21.2) and encodes the production of a transmembrane protein which belongs to the family of dependence receptors. These receptors are active both with ligand bound and unbound, but they transmit different signals depending on the presence of their ligand. The DCC ligand is called netrin 1. In its absence DCC gene induces, through the caspase pathway, cellular apoptosis, whereas in the presence of netrin 1 the gene becomes antiapoptotic. These interactions of DCC with its ligand suggest that it might act as a putative tumor suppressor gene. Hypermethylation of DCC gene has been observed in various types of cancers, including OSCC, leading to its functional loss. METHODS In the present study, DCC hypermethylation was evaluated in 10 μm paraffin sections using bisulfate conversion and Methylation specific PCR-MSP techniques. The following samples were included: 20 samples of lichen planus-LP subdivided into reticular (10) and erosive (10) forms 62 samples of oral leukoplakias-OLs, subdivided into groups: hyperkeratosis (21), mild dysplasia (22), and moderated to severe dysplasia (19) 39 samples of OSCCs subdivided into groups: well (7) moderate (30) and poorly differentiated (2) 12 normal mucosa specimens. Besides hypermethylation, DCC molecules’ tissue distribution in LP, OLs, OSCCs and normal tissues was evaluated using semiquantitative immunohistochemistry. All the samples were provided by the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki and the divisions of Maxillofacial Surgery of General Hospital G. Papanikolaou and St. Loukas Hospital of Thessaloniki, Greece during the period 2004-2019. RESULTS Regarding LP, DCC hypermethylation was observed in 11 of 20 LP samples. Comparing with OSCC, there was a statistically significant difference (p<0,001 Fisher’s exact Test). Regarding OL, it was also observed that 33 of 62 OL samples (53 %) were DCC methylated and there was a significant statistical difference between OSCC and OL methylation (p<0,0001 chi-square test) Moreover, DCC gene was methylated in 15 of 19 OL samples with moderated and severe dysplasia (79%) Comparing the various degrees of dysplasia in OL, it was also found that there was a statistical significance between: OL with moderated-severe dysplasia and normal tissues (p=0,022 Fisher’s exact test) OL with hyperkeratosis and OL with moderated-severe dysplasia (p= 0,041 chi square) OL with mild dysplasia and OL with moderated-severe dysplasia (p= 0,011 Fisher’s exact test) Regarding OSCC, it was observed that 37 out of 39 OSCCs samples (95%) presented DCC promoter hypermethylation, whereas only 4 of 12 normal tissues samples (33%) showed DCC promoter hypermethylation and there was a significant statistical difference. (p<0.001Fisher’s exact test) Comparing OSCC and OLs with various degrees of dysplasia, there was also a statistical significant difference between OSCC samples and OL No dysplasia (p<0,001 Fisher’s exact test) and between OSCC and OL with mild dysplasia (p<0,001 chi square test) According to immunohistochemical analysis of the DCC expression, no significant difference in DCC staining was found between normal tissues, LP, OL lesions with no dysplasia, OLs of any degree of dysplasia and OSCC samples. Interestingly, DCC staining was more intense in all epithelial layers except basal and suprabasal cell layers in OLs of various degrees of dysplasia as well as OSCCs. Moreover, a characteristic pattern of expression of DCC in OSCCs was observed with positive neoplastic cells in the center of neoplastic epithelial islands and especially around keratin pearls. CONCLUSION This study showed that DCC methylation was present in ~95% of OSCC samples with a significant statistical difference in DCC hypermethylation between OSSC and normal tissues. This finding supports the hypothesis that hypermethylation is a possible mechanism of DCC inactivation in OSCC. The fact that there was not a correlation between DCC hypomethylation and DCC immunohistochemistry expression could be explained because DCC as a protein of cell cycle suppressor was absent in the basal cell layer where cells have an increased potential of proliferation. Instead DCC expression was more intense in spinous and granular layers where cells are clearly differentiated without proliferation capacity. The present study is one of the few that also evaluated the DCC hypermethylation status in OL samples. It was found that there was a significant statistical difference in DCC hypermethylation between OSCCs and OLs, particularly in OL hyperkeratosis and OL with mild dysplasia. These results also support the significance of hypermethylation as a mechanism of DCC inactivation in the procedure of oral tumorigenesis. Finally, there was a significant statistical difference in DCC methylation of OL with moderated - severe dysplasia in comparison with normal tissues and OL samples with hyperkeratosis or mild dysplasia suggesting that as the degree of dysplasia in OLs becomes more severe, the DCC promoter methylation is increased statistical significance. This finding supports the possible use of DCC methylation as a biomarker to predict the biological behavior of OLs. Epigenetics represent a new and very exciting field and since they occur early in carcinogenesis and are potentially reversible, they could be used as disease biomarkers for diagnosis, prognosis and prediction, as well as therapeutic targets in human cancer with reference to OLs and OSCCs.

Η μεθυλίωση του DNA έχει αποδειχτεί ότι διαδραματίζει σημαντικό ρόλο στην ανάπτυξη διαφόρων τύπων καρκίνου, μέσω μηχανισμών όπως η απενεργοποίηση ογοκατασταλτικών γονιδίων. Το DCC(18q21.2) είναι ένα δυνητικά ογκοκατασταλτικό γονίδιο και η μεθυλίωση του έχει συσχετιστεί με διάφορα νεοπλάσματα, όπως το καρκίνωμα από πλακώδη κύτταρα του στόματος (ΚΠΕ). Στην παρούσα μελέτη, διερευνήθηκε η DCC μεθυλίωση σε 20 περιστατικά Ομαλού λειχήνα, 62 λευκοπλακίας, 39 ΚΠΕ καθώς και 12 φυσιολογικού ιστού. Εφαρμόστηκε η τεχνική της δισουλφιδικής τροποποίησης/κατεργασίας του DNA σε συνδυασμό με την αλυσιδωτή αντίδραση πολυμεράσης ειδική στη μεθυλίωση. Επιπλέον εκτιμήθηκε ανοσοιστοχημικά και η έκφραση του DCC μορίου. Η DCC μεθυλίωση βρέθηκε αυξημένη στα περιστατικά ΚΠΕ εν συγκρίσει με τη λευκοπλακία και τους φυσιολογικούς ιστούς με στατιστική σημαντικότητα. Αντιθέτως δεν διαπιστώθηκε στατιστικά σημαντική διαφορά στη γονιδιακή έκφραση του DCC στα διάφορα περιστατικά. Τα αποτελέσματα επιβεβαιώνουν το ρόλο του DCC ως ογκοκατασταλτικού γονιδίου που αδρανοποιείται μέσω μεθυλίωσης στις περιπτώσεις ΚΠΕ, εύρημα που θα μπορούσε να συσχετιστεί με τη διάγνωση και πρόγνωση του καρκινώματος.