84 Chapter 4 three-marker combinations were formed for each sample type using multivariable logistic regression analysis with backward selection. The performance of the individual markers and optimal three-marker panels was visualized using receiver operating characteristic (ROC) curves, including the area under the ROC curve (AUC) with corresponding 95% confidence intervals (CIs). Sensitivities and specificities were based on the Youden’s Index (J) threshold. Diagnostic performances of each marker and threemarker panels for the detection of early-stage endometrial cancer was evaluated in a sub-analysis in which only stage IA and IB cancers were taken along in the univariable and multivariable regression analyses which were performed as described above. The diagnostic performances of each marker and the three-marker panels were assessed outside the set by leave-one-out cross-validation (LOOCV). Predicted probabilities of the individual markers and optimal three-marker panels were also plotted individually in a heatmap format to illustrate differences between the sample types, histological subtypes and the potential added value of the marker combination. Data were collected using Castor EDC (31). Statistical analyses were performed in RStudio (v3.6.1) using the corrplot (v0.84), cowplot (v1.1.0), compareGroups (v4.5.1), dplyr (v1.0.2), ggplot2 (v3.3.5), MASS (v7.3-58) and pROC (v1.18.0) packages. Reported P-values are two-sided and considered statistically significant if P < .05. RESULTS Study population and characteristics A total of 158 patients with histologically confirmed endometrial cancer were included in the SOLUTION1 study. For various reasons, mostly because not all three sample types were available (n = 40), cases were excluded, resulting in a final study population of 103 endometrial cancer patients (Supplementary Figure 1). Within this group, a paired urine, cervicovaginal self-sample, and clinician-taken cervical scrape of each case were available for methylation analysis. Unpaired samples of control women (n = 317) were used for comparison. Clinical characteristics of endometrial cancer patients and controls with valid qMSP results are depicted in Table 1. Additionally, FFPE tissue was collected from endometrial cancer cases of various histological subtypes (n = 33) and healthy endometrium (n = 15). DNA methylation levels in minimally- and non-invasive samples Methylation levels of ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 were significantly higher (Mann–Whitney U test; all P < .01) in urine samples, cervicovaginal self-samples and clinician-taken cervical scrapes of endometrial cancer
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