101 DNA methylation testing for endometrial cancer detection in patient-friendly sample types SUPPLEMENTARY INFORMATION Table of contents Supplementary Figure 1 Flowchart illustrating excluded endometrial cancer patients (n=55) from the total study population (n=158) due to various reasons: incomplete sample-set (n=40), invalid test-result for DNA methylation analysis (n=6), no residual cancer during sample collection (n=4), minimal residual cancer (i.e. < 5 mm) during sample collection, and diagnosed with synchronous lung cancer (n=1). Supplementary Figure 2 DNA methylation levels of ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 in endometrial cancer (n=33) and normal endometrial tissue (n=15). DNA methylation levels are shown by the 2log-transformed Ct ratios. Boxplots illustrate medians with lower and upper quartile and range whiskers. Outliers are indicated with black squares. A p-value of < 0.05 was considered statistically significant. **: p < 0.01; ****: p < 0.0001. EC = endometrial cancer. Supplementary Figure 3 The Spearman’s rank correlation coefficients (r) of methylation markers ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 between paired samples of patients diagnosed with endometrial cancer. The Spearman’s rank correlation coefficient was calculated based on 2log-transformed Ct ratios. Dot color and size indicate the degree of correlation (i.e. the larger and darker the dot, the more correlation). Supplementary Table 1 Youden’s Index ( J) thresholds for maximal sensitivity and specificity calculations of individual markers and marker panels. Supplementary Table 2 LOOCV univariable logistic regression analysis on the diagnostic performance of the nine individual markers and LOOCV multivariable logistic regression analysis on the diagnostic performance of the optimal marker combinations for endometrial cancer detection in the different sample types. Individual methylation markers with the highest AUC value per sample type are depicted in bold. Supplementary Table 3 Diagnostic performance of the nine individual methylation markers (univariable logistic regression) and optimal three-marker panels (multivariable logistic regression) for early-stage endometrial cancer detection (n=72) in the different sample types. 4
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