hTERT promoter methylation status in peripheral blood leukocytes as a molecular marker of head and neck cancer progression

Abstract

Cancer cells, including head and neck cancer cell carcinoma (HNSCC), are characterized by an increased telomerase activity. This enzymatic complex is active in approximately 80-90% of all malignancies, and is regulated by various factors, including methylation status of hTERT gene promoter. hTERT methylation pattern has been thoroughly studied so far. It was proved that hTERT is aberrantly methylated in tumor tissue versus healthy counterparts. However, such effect has not yet been investigated in PBLs (peripheral blood leukocytes) of cancer patients. The aim of this study was to analyze the hTERT gene promoter methylation status in blood leukocytes. DNA was extracted from PBL of 92 patients with histologically diagnosed HNSCC and 53 healthy controls. Methylation status of whole hTERT promoter fragment with independent analysis of each 19 CpG sites was performed using bisulfide conversion technique followed by sequencing of PCR products. Not significant (p = 0.0532) differences in the general frequency of hTERT CpG sites methylation were detected between patients and healthy controls. However, it was discovered that some of analyzed positions (CpG islands: 1 [p = 0.0235], 5 [p = 0.0462], 8 [p = 0.0343]) are significantly more often methylated in HNSCC patients than in controls. The opposite finding was observed in case of CpG position 2 (p = 0.0210). Furthermore, closer analysis of single CpG positions revealed differences in methylation status dependent on anatomical site and TNM classification. To conclude, hTERT promoter methylation status (general or single CpG sites) would be considered as a molecular markers of HNSCC diagnostics.

Intron 4–5 hTERT DNA Hypermethylation in Merkel Cell Carcinoma: Frequency, Association with Other Clinico-pathological Features and Prognostic Relevance

“…Although this result needs to be validated in larger case series, it suggests that mhTERT could be one important epigenetic mechanism involved in hTERT regulation, cooperating with other ones to justify the high levels of hTERT mRNA and telomerase activity found in these patients. The second main finding is the prognostic impact of mhTERT high , as just observed in other tumors [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. In the multivariable model, we found that only mhTERT high and p63 affected OS, with marginal significance for angioinvasion and absence of MCPyV.…”

“…For the majority of the genes, hypo-methylation of the promoter is fundamental to permit an active transcription [13][14][15]. Nevertheless, several studies showed that hypermethylation of hTERT was paradoxically associated with higher hTERT mRNA levels and telomerase activity depending on the region of interest investigated [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. Although several mechanisms cooperate in determining the final telomerase activity, promoter methylation proved to be one of the most relevant ones, singly affecting survival, recurrence, and progression in pediatric brain tumors, meningiomas, head and neck squamous cell carcinoma, leukemias, prostatic adenocarcinoma, and several other tumors [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32].…”

“…We previously characterized DNA methylation alterations in this locus in a large cohort of oral squamous cell carcinoma, identifying the most correlated CpG located at Chr5:1279643-1279644, with an area under the curve (AUC) of 0.92 [18,19]. Since mhTERT could be a relevant epigenetic mechanism in hTERT expression and an independent prognostic factor in several tumors, we investigated whether mhTERT is associated with overall mortality and with the most relevant clinical-pathological features in a series of MCC patients [9,[13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32].…”

<p>A Study of hTERT Promoter Methylation in Circulating Tumour DNAs of Patients with Ovarian Magnificent Tumour</p>

“…It has been validated that hTERT can be detected in free DNAs in serums, and the methylation of hTERT promoter can be a predictor of malignant tumours. 7,8 Hence, this study aims to verify the significance of hTERT promoter methylation in OC diagnosis.…”

“…An analysis of the CPG loci of 92 patients with head and neck cancer (HNSCC) and 53 healthy controls indicated that some sites in HNSCC patients presented a higher frequency of methylation than those in the controls, whilst a contrary finding was observed at another location of CPG. 7 By probing the relationship between gene loci and activity of the hTERT promoter, it was found that the first exon, the first intron and the second exon of 37bp in front of the ATG upstream of the promoter of malignant tumour cells were active, whilst their expression in benign cells was relatively low. 20 Therefore, in this study, the upstream region of the hTERT core promoter was taken as the methylation detection target area.…”