Background: Early detection and diagnosis of head and neck squamous cell carcinoma (HNSCC) will allow for the identification of patients at an earlier stage of disease. Here we evaluate the use of methylation markers with digital droplet PCR in the diagnosis of HNSCC.
Methods: Methylation of the tumour suppressor genes PAX5, EDNRB, DCC, MGMT, DAPK and P16 in prospectively collected HNSCC patient tumour tissues, HNSCC oral rinses, normal control tissues and normal oral rinses were analyzed using digital droplet PCR. The data was analysed by Mann-Whitney U test, Student’s t-test comparison of means and ROC analysis of the samples for sensitivity and specificity with MedCalc Statistical Software version 17.6 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2017)
Results: 36 HNSCC patients and 25 control patients were analysed. Using digital droplet PCR there was significant difference in the methylation density of PAX5 (P<0.001), EDNRB (P<0.001), DCC (P<0.001), MGMT (P<0.007), DAPK (P<0.03) and P16 (P = 0.03) when comparing HNSCC with paired normal tissues (PAX5 methylation is shown in figure 1). PAX5(P<0.001), EDNRB(P<0.001), DCC(P<0.001) showed aberrant methylation when compared with control patient tissues. A further analysis of oral rinses between HNSCC and control patients for PAX5 showed a sensitivity of 91.7% and specificity of 75% as shown in figure 2. EDNRB demonstrated a lower sensitivity of 37.5% and specificity of 93.7% for oral rinses when compared between HNSCC and control patients.
Conclusions: Using digital droplet PCR may improve the sensitivity in utilizing methylation markers, in particular PAX5 for the detection of HNSCC in oral rinses in patients with HNSCC.