Genetic alterations and field defect in head and neck squamous cell carcinoma

Abstract

A genetic progression model of head and neck squamous cell carcinoma (HNSCC) has not yet been elucidated, and the genetic basis for has also remained unclear. Most of the has been explained by the presence of cells with genetic alterations, however, involvement of epigenetic alterations in field cancerization was shown, too. In the present study, paired specimens of tumour and adjacent normal tissues were obtained from materials surgically resected from 25 patients with squamous cell carcinoma of the head and neck. After extraction of RNA, quantitative RT-PCR method using 7300 TaqMan (AppliedBiosystems) was used to analyze gene expression of MGMT, p16, TIMP3. Next, we examined the association between MGMT, p16, TIMP3 promoters methylation and genes expression. The studies demonstrated higher expression of p16 gene in tumour compared with adjacent normal tissues. Other, MGMT and TIMP3 showed no differences. Our results revealed no correlation between MGMT, p16, TIMP3 promoters methylation level and expression of these genes. Finally, we performed direct DNA sequence analysis of MGMT somatic mutations both in tumour and adjacent normal tissue. MGMT is a 300,000 bp-long gene that consist of six relatively short exons. The gene is transcribed as a 1265 bp-long mRNA, covering the 717 bp-long coding sequence. To facilitate the screening of somatic mutations in the coding region of the MGMT, a simple approach has been developed. As the gene is expressed in HNSCC, we decided to amplify the whole coding region of MGMT in two separate PCR reactions using its cDNA as a template. The amplicons of individual patients were labeled in PCR with MID identifiers and sequenced using a medium-scale next generation sequencing system, GS Junior (Roche/454). The method developed can accurately identify low-level mutations, down to a level of 5% of cells within the testing sample. The lack of progress in head and neck oncology emphasizes the importance of molecular genetic studies to define alterations that may correlate with tumor behavior. Further studies may help clarify this issue.