CHAPTER 4 80 MATERIAL AND METHODS Patient cohort The nationwide Dutch database of histopathology and cytopathology (PALGA) was used to search for all patients diagnosed with uterine CCC between January 1990 and December 2020 at the Radboud university medical center and the Canisius Wilhelmina Hospital Nijmegen, The Netherlands 21. Patients were excluded when having less than 10% clear cell component, when not receiving a surgical treatment and when no histological tumor tissue could be retrieved for IHC/molecular analysis. Data collection Clinicopathological data was collected regarding age at diagnosis, body mass index (BMI), cancer antigen 125 (CA-125), cervical cytology, preoperative endometrial sampling, extend of primary surgical approach, stage, adjuvant treatment, and follow-up data. Stage of disease was based on the 2009 International Federation of Gynecology and Obstetrics (FIGO) endometrial cancer criteria 22. Histopathological review Hematoxylin & eosin (H&E) slides of the hysterectomy specimens were systematically reviewed by two pathologists with special interest in gynecology (J.B., H.K.), being blinded to any clinical or histological data. Histological review included classification of tumor histology, an estimation of percentages of the different components if present, depth of myometrial invasion (MI) and the presence of cervical stromal invasion (CI). Slides were screened for the presence of lymphovascular space invasion (LVSI). Diagnosis was made according to the 2020 World Health Organization (WHO) guidelines and a tumor was classified as mixed when it contained at least 2 different histological components, regardless of component percentage 11. To support the diagnosis mixed uterine CCC, IHC stains were used in doubtful cases according to the 2020 WHO guidelines. For each case, an H&E slide with representative tumor tissue was selected and marked off for the purpose of DNA extraction in parallel unstained slides. In case of mixed uterine CCC, the different components were marked off separately, if possible. Immunohistochemical staining Immunohistochemical staining was performed for estrogen receptor (ER), progesterone receptor (PR), L1 Cell Adhesion Molecule (L1CAM), PMS2 and MSH6 (Supplementary A). For ER and PR, the number of stained tumor nuclei was scored. Cases were dichotomized, using 10% as a cut-off value. For L1CAM, the number of tumor cells showing membranous expression was scored and dichotomized, using 10% as a cut-off value. Mismatch repair
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