CHAPTER 3 54 DNA analysis Representative areas of EC in the surgical specimen were marked and selected for formalinfixed paraffin-embedded (FFPE) 20 μm thick sections. Slides were cut from these FFPE section and stained with hematoxylin and eosin (H&E). Tumor areas were marked on these slides and the tumor cell percentage was estimated. These specimens were digested overnight at 56°C in TET-lysis buffer (10mmol/L Tris/HCL pH 8.5, 1 mmol/L EDTA pH 8.0, 0.01% Tween-20) with 5% Chelex-100 (Bio-Rad, Hercules, CA) and 0.2% proteinase K, with subsequent inactivation at 95°C for 10 min. After this was centrifugated, the supernatant was transferred into a clean tube. DNA concentration was determined using the Qubit Broad Range Kit (Thermo Fisher Scientific, Waltham, MA). smMIP design and library preparation Samples were analyzed with single-molecule Molecular Inversion Probes (smMIPs). The design (Integrated DNA Technologies Leuven, Belgium) as well as the library preparation were previously published.16 eMethod in the supplement shows further detailed information on smMIP design, library preparation and sequencing. Immunohistochemical staining and scoring Detailed information about the immunohistochemical staining for p53, PMS2 and MSH6 can be found in the eMethod in the supplement and original published studies.9, 10 In brief, staining for p53 was considered abnormal when more than 80% of tumor cell nuclei showed strong expression (overexpression) or when there was complete absence of nuclear staining (null-expression). Mismatch repair deficiency (MMR-D) was defined as total loss of nuclear staining of PMS2 and/or MSH6, in the presence of a positive internal control. Statistical analysis Early-stage was defined as FIGO stage I-II and advanced-stage as FIGO III-IV. Low-grade EC was defined as grade 1 and grade 2 EC and high-grade as grade 3 EEC and non-EEC (NEEC), according to the latest ESGO-ESTRO-ESP and WHO guideline.2, 17 The included patients in our retrospective cohort received either full lymphadenectomy or no lymphadenectomy, as sentinel lymph node procedure was not routinely incorporated yet. Statistical analyses were performed on SPSS version 25.0 (released 2017, Armonk, NY, United States) using χ2, Fisher’s exact test, Mann-Whitney U test, Kaplan-Meier survival analysis and univariable and multivariable Cox-regression analysis. SAS version 9.4 was used for survival curves including Hall-Wellner confidence bands. P<0.05 was considered statistically significant. The assumption of proportionality for the included variables was tested with log minus log curves and time-dependent covariate (time x covariate). Disease-
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