CHAPTER 5 112 practice. Despite their prognostic relevance for LNM and survival in EC, none of these were studied in relation to the postoperative ESMO-ESGO-ESTRO risk classification groups or to LN status13, 15-19. Therefore, our primary aim was to investigate the added prognostic relevance of preoperative IHC biomarkers, p53/L1CAM/ER/PR, to the postoperative ESMO-ESGOESTRO risk classification groups in EC. Secondary, the added prognostic relevance of these IHC biomarkers to LN status in EC. MATERIALS AND METHODS Study cohort Within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), a retrospective multicenter cohort study was performed. The patients were surgically treated between February 1995 and August 2013 at one of the 10 participating ENITEC centers and were identified from a previously published cohort9, 20. Only patients diagnosed by an expert gynecological pathologist with complete clinical and pathological data and follow-up of at least 36 months were included, yielding 1199 patients out of ten European hospitals. Pathological characteristics Preoperative tumor grade and histology were used for analysis, combined with IHC staining of p53, L1CAM, ER and PR according to ENDORISK9. Detailed information on tissue processing and IHC analysis is shown in Supplementary S1 method. Scoring of the IHC was performed twice by assessors blinded to pathological and clinical characteristics (N.V., H.K., J.B., K.v.d.V., C.R.). Disagreements in scoring were solved in a consensus meeting with all assessors. For p53, staining was considered abnormal/aberrant (p53-abn) when more than 80% of tumor cell nuclei showed strong expression (overexpression) or when there was complete absence of nuclear staining (null-expression). For L1CAM, the number of tumor cells showing membranous expression was scored and dichotomized, using 10% as a cut-off value. For ER and PR, the number of stained tumor nuclei was scored. Cases were also dichotomized, using 10% as a cut-off value. L1CAM expression was considered abnormal when >10% of tumor cells were positive (L1CAM+), ER and/or PR expression was considered abnormal when <10% nuclear staining was present (ER/PR-). Postoperative ESMO-ESGO-ESTRO risk classification Five subgroups were identified based on postoperative tumor stage, tumor histology, grade, myometrial invasion (MI) and presence of lymphovascular space invasion (LVSI): low, intermediate, high-intermediate, high and advanced/metastatic risk group5.
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