Interpreting p53 IHC-TP53 NGS discordance in endometrial cancer: a mechanism-based diagnostic framework and clinical implications.

OBJECTIVE: To analyse discordances between p53 immunohistochemistry (IHC) and TP53 next-generation sequencing (NGS) in endometrial cancer, with a focus on underlying mechanisms and their clinical implications. Despite increasing recognition of IHC-NGS discordance, a structured approach to its interpretation in routine practice remains lacking. METHODS: A retrospective analysis was performed in 614 patients with endometrial cancer treated at two oncological centres between 2022 and 2025. All patients underwent both p53 IHC and TP53 NGS. Molecular classification followed the ProMisE hierarchy (POLEmut, MMRd/MSI-H, p53abn, NSMP). The subgroup of 110 patients with p53 abnormalities identified by p53 IHC and/or TP53 NGS (defined as an abnormal p53 IHC pattern, a TP53 mutation detected by NGS, or both) was analysed to identify and mechanistically classify IHC-NGS discordant cases, as discordance carries the most direct therapeutic implications within this subtype. Associations between molecular subtype and disease stage were evaluated using logistic regression. RESULTS: Among 110 patients with p53 abnormalities identified by p53 IHC and/or TP53 NGS, 23 IHC-NGS discordances were identified (20.9%). The most frequent source of discordance was difficulty in interpreting IHC expression thresholds (14/23; 60.9%), particularly around the 1% and 80% cut-offs. Other mechanisms included intratumoral heterogeneity with subclonal p53 expression (n=6), low-VAF TP53 mutations (n=2), and TP53-independent p53 stabilisation (n=1). Discordant cases were systematically organised into seven mechanistic groups forming a structured interpretative framework. In the full cohort, p53abn was the only subtype significantly associated with advanced-stage disease (OR 1.79; 95% CI 1.06-3.03; p=0.031). CONCLUSIONS: IHC-NGS discordance in p53 assessment represents a multimechanistic phenomenon observed in over 20% of p53-abnormal cases. The proposed mechanism-based interpretative framework may support consistent evaluation of borderline cases and improve diagnostic decision-making in clinical practice. In clinically significant scenarios, particularly near diagnostic thresholds, pathological re-evaluation and supplementary NGS testing should be considered prior to treatment planning.