Reliable Identification of Endometrial Precancers Through Combined Pax2, β-Catenin, and Pten Immunohistochemistry

Abstract

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably β-catenin and Ari-d1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n = 111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, β-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n = 79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), β-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥ 2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or β-catenin. A focused panel of only 3 markers (Pax2, Pten, and β-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity

“…However, Pten loss is a relatively poor driver of EMT, as evidenced by a lack of invasive phenotypes in Pten-alone contexts in this study, including live mice, cell lines, and organoids. This is also well-documented in aging human endometrium, where definitive Pten loss occurs in normal or hyperplastic endometrium (i.e., very early in EC progression, well before the acquisition of invasive phenotypes) ( 24 , 70 , 71 ). In contrast, in our studies, Foxa2 inhibited cell cycle progression via Myc, albeit modestly, confirming that it is a multitasking tumor suppressor with diverse biological roles, but with a much more potent effect as an EMT suppressor, as evidenced by upregulation of EMT factors and the striking pro-invasive/pro-metastatic phenotypes observed in the live animal and human/mouse cell line and organoid systems.…”

Morules But Not Squamous Differentiation are a Reliable Indicator of CTNNB1 (β-catenin) Mutations in Endometrial Carcinoma and Precancers

“…Briefly, when absence of staining was observed in ≥5% of lesional glands, PAX2 and PTEN were interpreted as lost. The cutoff is based on our previous observation that nonspecific PAX2 and PTEN loss in normal endometrium do not exceed 5% of the endometrial glands 29. The presence of strong, unequivocal nuclear staining with β-catenin in lesional glands, even when focal, was interpreted as positive/abnormal.…”

“…When tumor glands showed confluence, a diagnosis of EEC was rendered. Information regarding interpretation of β-catenin, PAX2 and PTEN was detailed and further reviewed in our recent publication 29,31. Briefly, when absence of staining was observed in ≥5% of lesional glands, PAX2 and PTEN were interpreted as lost.…”

“…IHC for PAX2, PTEN, and β-catenin was performed in the Parkland Hospital clinical laboratory on a DAKO Autostainer Link 48 instrument, following protocols previously validated for clinical testing. 29 The following primary antibodies were used: β-catenin (prediluted, clone β-catenin-1, #IR70261-2; Agilent), PAX2 (prediluted, clone EP235, #BSB2567; Cancer Diagnostics, Durham, NC), PTEN (prediluted, clone 6H2.1, #PM278AA; BioCare, Pacheco, CA), p40 (prediluted, clone Poly.Rb; BioCare, Pacheco, CA) and CD10 (prediluted, clone 56C6; Dako). High pH (50× Tris/ EDTA buffer, pH 9) solution was used for antigen retrieval.…”

“…To investigate the relationship between the presence of morules and β-catenin status in AH/EIN, we divided all cases into groups with and without morules, and studied the expression pattern of β-catenin in the glandular component of AH/EIN and/or EEC in those 2 groups. The status of 2 other markers shown to often aberrant in AH/ EIN, PAX2, and PTEN, 29 were also examined in comparison. The normal and aberrant expression of the 3 markers is illustrated in Figure 2.…”