DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization

Abstract

Background and Objectives: MGMT methylation is a well-described biomarker in several solid tumors and MLH1 seems to occur in the initial stages of oral carcinogenesis. The aims of this study were to evaluate MHL1 and MGMT methylation levels in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), and to integrate this information with The Cancer Genome Atlas (TCGA) database. Materials and Methods: To determine the percentage of gene methylation in MLH1 and MGMT, pyrosequencing analysis was conducted. Samples were divided as follows: (1) patients diagnosed with OSCC (N = 16); (2) patients with OPDM who developed OSCC in the same location (N = 47); and (3) patients with OPDM who developed OSCC in a different location (N = 22). As a validation cohort in this study, data from The Cancer Genomic Atlas (TCGA) database, particularly regarding Head and Neck Squamous Cell Carcinoma, was used. Results: Overall MLH1 methylation levels of 8.6 ± 11.5% and 8.1 ± 9.2% for MGMT were obtained. With regard to MHL1, the OSCC presented the highest degree of methylation with 9.3 ± 7.3% (95%CI 5.1–13.6), and with regards to MGMT, the simultaneous malignancy group presented the highest degree of methylation with 10 ± 13.5% (95%CI 6–10), although no significant differences were found between the groups (p = 0.934 and p = 0.515, respectively). The estimated survival was higher for MGMT methylated cases (19.1 months, 95%CI 19.1–19.1) than for unmethylated cases (9.4 months, 95%CI 6–12.8), but not statistically significant. Conclusions: Our results did not show a correlation between MGMT and MLH1 methylation and any clinicopathological feature or survival in our institutional cohort. MLH1 methylation was present mainly in OSCC, whilst MGMT in OPMD represented a modest contribution to field cancerization, with an overall consistency with the TCGA database.

Oral field cancerization: Genetic profiling for a prevention strategy for oral potentially malignant disorders

“…DNA aneuploidy 29 and chromosome aberrations30 are commonly used to detect field cancerization at the DNA level. Several markers (p53, Ki-67, 31 cytokeratin fragments 21-1, 28 variations in nucleolar organizer regions, 32 phosphatases and tensin homolog deleted on chromosome 10 allelic loss, 33 DEK overexpression, 34 micro RNA [hsa-miR-221, hsa-miR-21, hsa-miR-135b, and hsa-miR-29c] detection, 35 ATP-binding cassette subfamily G member 2, 36 MutL protein homolog 1, methylguanine-methyltransferase methylation, 37 interferonstimulated gene 15, 38 aldehyde dehydrogenase, Notch1, 39 and Bmi1 40 ) have been identified in pre-cancerization transformation into oral cancer, stimulating the cell cycle and promoting DNA replication (Figure 3).…”

Pyrosequencing Analysis of O-6-Methylguanine-DNA Methyltransferase Methylation at Different Cut-Offs of Positivity Associated with Treatment Response and Disease-Specific Survival in Isocitrate Dehydrogenase-Wildtype Grade 4 Glioblastoma

“…In the context of DGs and various other cancers, the MGMT gene takes center stage due to its role in chemotherapy resistance. When the MGMT gene is hypermethylated, its expression is silenced, rendering the tumor cells vulnerable to the effects of alkylating chemotherapy agents like temozolomide (TMZ) [ 13 , 14 , 15 , 16 ].…”

“…DNA methylation is an epigenetic phenomenon involving chemical modifications that instigate heritable genetic changes, yet it preserves the underlying DNA sequence intact. These modifications lead to epigenetic silencing and bear significant relevance to processes like cellular differentiation and gene expression regulation [ 15 ].…”