Supplementary Appendix

Part 1

PM value and its applications

PM (percentage of methylation) values calculated according to the following formula served as an alternative measurement of methylation level of AP-2E in our study.

Melting profile of the target sequence is determined by the ratio of shorter methylated domains within the sequence, according to the principles of MS-HRM analysis. The sequence of AP-2E amplified in our study displayed two independent melting peaks at 77.5 °C and 82.5 °C, respectively (Fig. S B). The partial methylation sequences presented two melting peaks whereas the 100% and 0% methylated DNA standards displayed only one melting peak at 82.5 °C and 77.5 °C, respectively. The ratio of -(d/dt)Fluorescence value corresponding to the melting peaks at 82.5 °C and 77.5 °C indirectly represented the ratio of shorter methylated domains and unmethylated domains within the full sequence amplified.

We assessed the consistency of PM values and results calculated by comparing curves of samples to be tested with curves of standard substances (Fig. S A), which was the recommended method to calculate the methylation level in MS-HRM analysis, and performed spearman rank correlation analyses. It showed that the consistency was perfect (R = 0.88, P 0.0001). However, there was no reference that had PM values calculated by our method serve as an alternative measurement of the methylation level of DNA sequence. PM values were only used to calculate the cut-off value to distinguish aberrant hypermethylation from hypomethylation of AP-2E in tumor tissues.

Fig. S Normalized Melting Curves (A) and the corresponding Melting Peaks (B) of AP-2E by MS-HRM analysis.

Part 2 Supplement Figures and legends

Fig. S1 Normalized Melting curves (A) and corresponding Melting Peaks (B) analyzed using the Gene Scanning Software.

Fig. S2 Kaplan-Meier curves for cumulative survival stratified by TNM staging.

Fig. S3 Kaplan-Meier curves for cumulative survival stratified by Tumor invasion status.

Fig. S4 Kaplan-Meier curves for cumulative survival stratified by Lymph nodes involved.

Fig. S5 Kaplan-Meier curves for cumulative survival stratified by metastasis status.

Fig. S6 Kaplan-Meier curves for cumulative survival stratified by histological grade.