HORMONAL BIOMARKERS AND MOLECULAR SUBGROUPS IN EC 139 6 DNA analysis The molecular subgroups included in this study were determined by either full NGS or according to ProMisE. Both methods have been described previously13, 21 and details are provided for the different cohorts in the Supplementary Method S1. Briefly, for molecular profiling by full NGS, DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor blocks. Next, DNA was sequenced by NGS with single-molecule Molecular Inversion Probes (smMIPs).22 For the detection of MSI, 55 MSI markers were tested according to the previously published design.23 Multiple-classifiers were classified as the molecular subgroup with the best prognosis.24 For the molecular subgroups determined according ProMisE criteria, POLEmut analysis was performed by MiSeq, Sanger or NGS.13, 19 Immunohistochemical analysis IHC was performed on 4 um FFPE tumor sections for the ENITEC centers and tissue microarrays (TMA) for Vancouver cohort, as described previously and detailed in the Supplementary method S1.10, 13, 17, 19 In brief, antibodies specific to MSH6, PMS2, p53, ER and PR were used. Staining for p53 was considered abnormal when more than 80% of tumor cell nuclei showed strong expression (overexpression) or when there was complete loss of nuclear staining (null-expression) with a positive internal control. Mismatch repair deficiency (MMRd) was defined as complete absence of nuclear staining of PMS2 and/or MSH6, in the presence of a positive internal control. For the TMAs, staining for individual MMR proteins and ER/PR was repeated on whole sections whenever there was equivocal, uninterpretable, or aberrant staining. ER and PR expression was determined by estimating the percentage of positive nuclei in the whole invasive tumor area by ‘eyeballing’. Scoring for ER and PR expression within the included cohorts was performed by two assessors (pathologists and researchers, who were trained by an expert gynecologic-pathologist), reviewing discrepancies in a consensus meeting.10, 19 The ER/PR risk groups were defined as: ER/PR 0-10% as high-risk, ERPR 20-80% as intermediate risk and 90-100% as low risk.10 Percentages were scored by the pathologist as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%. Some percentages were scored in between, e.g. 85%, these were rounded off into the nearest category (so 85% was categorized as 90%). When ER and PR were taken together, the subgroups were defined as ER+PR 0-10%, ER+PR 20-80%, ER+PR 90-100% and discordant. Patients were grouped ‘discordant’ if the ER and PR percentages were not aligned in the same risk group (e.g. ER 10% and PR 90%). Primary objective To study the prognostic relevance of the three-tiered ER/PR risk classification within the molecular subgroups in EC.
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