Oral scrapings were collected from 65 normal oral mucosa subjects, 107 oral precancer patients, and 95 oral squamous cell carcinoma patients. Methylation levels of the five genes were quantified by real-time methylation-specific PCR after bisulfite conversion.
Among the five tested genes, methylated ZNF582 (ZNF582m) and PAX1 (PAX1m) were found to be appropriate biomarkers for oral dysplasia and oral cancers. ZNF582m could detect mild dysplasia or worse oral lesions with the sensitivity and specificity being 0.85 and 0.87, respectively. PAX1m performed better in identifying moderate dysplasia or worse oral lesions with the sensitivity and specificity being 0.72 and 0.86, respectively. Moreover, the methylation levels and positive rates for ZNF582m and PAX1m were increased when disease severity increased. Thus, they may be applicable as a triage tool for patients with abnormal visual oral examinations. After cancer excision, both ZNF582m and PAX1m levels decreased. However, their levels increased again at the subsequently recurrent sites in some patients approximately 3-4 months before cancer recurrence. Finally, areca-quid chewing alone and in combination with cigarette smoking or alcohol drinking were found to be correlated with ZNF582 and PAX1 hypermethylation.
We conclude that hypermethylated ZNF582 and PAX1 are effective biomarkers for the detection of oral dysplasia and oral cancer and for the prediction of oral cancer recurrence.