Differential Methylation Status of Tumor-associated Genes in Head and Neck Squamous Carcinoma: Incidence and Potential Implications

Purpose: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis.

Experimental Design: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated.

Results: In histologically normal squamous mucosa, no to low-level methylation (0-22%) was noted in some specimens at all genes except RARbeta2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RARbeta2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RARbeta, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas.

Conclusions: Methylation of p16, RARbeta, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.