Methylation status of TFPI-2 determined by pyrosequencing assay. (A) Box plots of TFPI-2 methylation level determined by the pyrosequencing assay. TFPI-2 methylation level is expressed as a percentage of that in IVD (given as 100%). The median methylation percentage across all 11 CpG sites was 3.82% in normal oral tissues, and 18.26%, 12.17%, 21.03%, and 33.10% in P1-P4 tumors, respectively. In each box plot, the whiskers represent the 10th and 90th percentiles, the lower and upper limits of the box indicate the 25th and 75th percentiles, and the red line is the mean. (B) Differential pattern of TFPI-2 methylation at each individual CpG site between early-stage (P1 and P2) and late-stage (P4) tissues. Numbers at the x-axis indicate nucleotide positions of CpG site relative to the transcription start site. Statistically significant differences in methylation levels are indicated by asterisks (*). (C) ROC curve obtained using TFPI-2 methylation to detect OSCC. The AUROC was 0.91. (D) ROC curve obtained using TFPI-2 methylation to detect early-stage OSCC (P1 and P2). The AUROC was 0.94.