Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma

Resection is the preferred treatment for oral squamous cell carcinoma, and pathological staging of the resected specimen is crucial. The role of molecular biology in the diagnosis of minimal residual disease has not been fully investigated and may improve staging. Multiple adjacent specimens were taken from the tumour, the invasive front, the surgical margin, and the lymph nodes of 20 specimens from patients with oral cancer. Bisulphite-treated DNA from these specimens was assayed quantitatively with pyrosequencing methylation assays (PMA) of CpG islands within the gene promoters of the p16 and CYGB genes. Results were recorded with histopathological results, and compared with clinical outcome. Biological and technical replicates confirmed the reliability of the techniques. PMA upgraded 13 of the 20 surgical margins, 6 of which subsequently had a recurrent tumour. Not all of these recurrences were predicted and the effects of adjuvant treatment make firm conclusions difficult.