83 DNA methylation testing for endometrial cancer detection in patient-friendly sample types DNA extraction and bisulfite treatment For DNA isolation of full void urine of cases and controls (30 mL; one-third of the original sample), the Quick DNA urine kit (Zymo Research, Irvine, CA) was used. DNA was isolated from the cervicovaginal self-samples and the clinician-taken cervical scrapes of cases and controls (each one-sixth of the original sample) using the NucleoMag 96 Tissue kit (Machery-Nagel) and a Microlab Star robotic system (Hamilton, Germany). DNA of the tissue samples was isolated using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). The NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA, US) was used to measure the DNA concentration. Bisulfite conversion of isolated DNA was done using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, US). All procedures were performed according to manufacturer’s guidelines. DNA methylation analysis using quantitative methylation specific PCR Promoter hypermethylation of the ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 genes was tested in three multiplex assays by quantitative methylation-specific PCR (qMSP) using 50 ng of bisulfite-converted DNA. Primer and probe sequences were described before (26, 29), or are available upon request. Each assay also targets the reference gene ACTB for quantification and quality control. To ensure sufficient sample quality, samples with a Cycle threshold (Ct) value for ACTB ≥ 32 were excluded from further analysis. Methylation levels were determined using the comparative Ct method using the following formula: 2-(Ct marker – Ct ACTB) x 100. The discriminatory power of the qMSP assays was verified by testing tissue specimens of a subset of endometrial cancer patients included in the SOLUTION1 study and normal endometrial tissue specimens as controls. Data analysis Only complete sample sets with valid DNA methylation test results (ACTB < 32) from endometrial cancer patients were included (e.g. of cases with an invalid urine sample, also the self-sample and scrape were removed from the analysis). Methylation levels were expressed as 2log-transformed Ct ratios. Differences in DNA methylation levels between endometrial cancer patients and controls were visualized using boxplots and tested for statistical significance using the Mann-Whitney U test. To assess the correlation of DNA methylation levels between paired sample types, the Spearman’s rank correlation was used. The Spearman’s rank correlation coefficient (r) was interpreted as poor (r ≤ .19), fair (r = .20 - .39), moderate (r = .40 - .59), strong (r = .60 - .79), and very strong (r ≥ .80) (30). The diagnostic performance of individual methylation markers was evaluated by univariable logistic regression analysis in which the predicted probability was calculated for each sample. The predicted probability (a value ranging from 0 to 1) represents the probability for the presence of endometrial cancer. Optimal 4
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